Item D Number °5169 D Author Corporate Author Report/Article Tltto Effects of 2,4,5-T on Man and the Environment: Hearings Before the Subcommittee on Energy, Natural Resources, and the Environment of the Committee on Commerce, United States Senate, Ninety-First Congress, Second Session, on Effects of 2,4,5-T on Man and the Environment. Journal/Book Tltto Year 197 Month/Day A Color D Number of manes ° Descrtaton Notes ltems 475 612 629 795 8S5 1074 1142 and 2926 are ° P"' > - - - - each pieces of this full report. Friday, March 01, 2002 ' > Page 5169 of 5263 EFFECTS OF 2,4,5-T ON MAN AND THE ENVIRONMENT •> .._ -_*,*•,•' *. --.=sh;^e-s / . ^•.^ v-jl*"-., BEFORE THH ".'*?'•>'-. ' " "-"' ••' - " = - A :" _'' ""' - .-^ >-.-}-*£•£> » -'• SUBCOMMITTEE ON ENERGY, NATURAL RESOURCES, AND THE ENVIRONMENT OF THH ---"_•-., COMMITTEE ON COMMERCE - - . . - '^': UNITED STATIC SENATE; - NINETY-FIRST CONGRESS .- ^ , -^J EFFECTS OF 2,4,5-T ON MAN AND THE ENVIRONMENT , (i , , ^W' ^ : : - ; f^ ATRILT AND.>5, 1970_;-, /;^.^--; ^^^ v^^--''^J ^?,^ I -• -' T>h^/.-^r-.:^y^-T-» -• •.-.--_• ry^^^/.^gff^- •^^^ii^y^.^.'/^y •' § Serial 91-60 Printed for the use of She Committee on Commerce tJ.-y-;'. ~^ .^. CONTENTS Opening statement by the chairman 1 CHRONOLOGICAL LIST OF WITNESSES APRIL 7, 1970 Bayley, Dr., Ned D., director, Science and Education, Department of Agriculture; accompanied by Dr. T. C. Byerly, assistant director Letter of January 7, 1970 Prepared statement Kotin, Dr., Paul, director, National Institute of Environmental Health Sciences Turner, James, Center for Study of Responsive Law, Washington, D.C_. Letter of April 30. 1970 Wellford, Harrison, Center for Study of Responsive- Law, Washington, D.C Letter of April 30, 1970 Westing, Dr. Arthur II., chairman, Biology Department, Windham College, Putney, Vt COMMITTEE ON COMMERCE WARREN G. MAGNUSON, Washington, Chairman NORHIS COTTON, New Hampshire JOHN 0. PASTORE, Rhode Island HUGH SCOTT, Pennsylvania VANCE IIAKTKK, Indiana WINSTON rilOUTY, Vermont 1'IIILir A. HART, Michigan JAMES B. PEARSON, Kansas HOWARD W. CANNON, Nevada ROBERT P. GRIFFIN, Michigan RUSSELL B. LONG, Louisiana HOWARD H. BAKER, JR., Tennessee FRANK E. MOSS, Utah CHARLES E. GOODELL, New York ERNEST F. HOLLINGS, South Carolina. MARLOW W. COOK, Kentucky DANIEL K. INOUYE, Hawaii JOSEPH D. TYDINGS, Maryland WILLIAM B. SPONG, Ja., Virginia FRBDKRICK J. LOHDAN, Staff Director M I C H A E L PEHISCHDK, Chief Counsel LEONARD BICKWIT, Jr., Staff Counsel AiiTiimi I'ANKOPF, Jr., Minority Staff Director J. PAUL MOLLOV, Minority Staff Counsel 32 38 74 87 18 468 6 468 76 APRIL 15, 1970 Epstein, Dr. Samuel S., Children's Cancer Research Foundation, Inc., and Harvard Medical School, Boston, Mass Appendix I Appendix II Johnson, Dr. Julius E., vice president and director of research, the Dow Chemical Co.; accompanied by Etcyl Blair, director Dow Agricultural Chemical Research, V. K. Rowe, director, Dow Toxicological Laboratory, arid George Lynn, director, Government Regulatory Relations, tlie Dow Chemical Co Steinfeld, Dr. Jesse, Surgeon General, Department of Health, Education, and Welfare; accompanied by Dr. David Gaylor, Dr. Diane Courtney, and Dr. Dale Lindsay _~_ Verrett, Dr. Jacqueline, Food and Drug Administration, Department of Health, Education, and Welfare SUIICOMMITTEE ON ENERGY, NATURAL RESOURCES, AND THE ENVIRONMENT PHILIP A. HART, Michigan, Chairman FRANK E. MOSS, Utah, Vice Chairman JOHN O. PASTORE, Rhode Island HOWARD H. BAKER, JR., Tennessee KURSKLL B. LONG, Louisiana CHARLES E. GOODELL, New York JOSEl'H D. TYDINGS, Maryland HUGH SCOTT, Pennsylvania WILLIAM B. SPONQ, JB., Virginia MARLOW W. COOK, Kentucky I I 1 | | i ADDITIONAL ARTICLES, LETTERS, AND STATEMENTS A reporter at large: Defoliation, article from the New York Times Byerly, Dr. T. C., assistant director, Science and Education, Department of Agriculture, letter of April 21, 1970 Chemical and Toxicological Evaluations of Isolated and Synthetic Chloro Derivatives of Dibenzo-p-dioxin, article from Nature Chick Edema Factor, article. Chick Edema Factor: Sonic Tissue Distribution Data and Toxicologic Effects in the Rat and Chick, article -. Clinical Picture and Etiology of Chloracne, article Collabotative Bioassay for Cluck Edema Factor, article Gushing, R. L., on behalf of the Hawaiian Sugar Planters' Association, Honolulu, Hawaii, statement Decontamination of Pesticides in Soils, article from Residue Reviews Defoliants, Deformities: What Risk? article Determination of 2,3,7,8-Tetrachlorodibenzo-p-dioxin in 2,4,5-Trichlorophenoxyacctic Acid by Gas-Liquid Chromatography, letter of June 22, 1965 (Hi) 405 419 431 360 167 190 107 467 326 204 308 330 227 469 384 104 367 DuBridge, Or. Lee A., Director, Office of Science and Technology, DepartIlcallh, Education, and Welfare, statement 452 l Persistence of Herbicides Applied to Soil in Puerto llican Forests, from Weed Science 47 Rlectron Microscopic Alterations in the Liver of Chicken Fed Toxic Fat, article 303 'Growth of Crops in Soils After Hcrbicidal Treatments for Brush Control in the Tropics, article from Agronomy Journal 45 Hays, Harry W., Ph. D., Director, Agricultural Research Service, Pesticides Regulation Division, U.S. Department of Agriculture, letter of November (I, 190G 12 'Herbicides in Soils, article from Agricultural Research Service 57 Identification and Crystal Structure of a llydropericardium-Produeing Factor: 1,2,3,7,8,9-Hexachlorodibenzo-p-dioxin, article from Acta Crystallngrapilica . 330 Injection of Chemicals Into the Yolk Sac of Fertile, Kggs Prior to Incubation as a Toxicity Test, article from Toxicology and Applied Pharmacology 278 Internal Preliminary Report Analysis of Commercial Chlorophenols for Trace Amounts of Their Condensation and Polymerization Products, article 328 Lawrence, J. F., Brig. Gen., U.S. Marine Corps, Deputy Assistant to the Secretary for Legislative Affairs, Office of the Secretary of Defense, letter of Aprif'21, 1970 467 Light and Electron Microscopic Observations in Macaco, mulaUa Monkeys Fed Toxic Fat, article 314 Lipsnn, Dr. Steven, chief. Division of Epidemiology and Surveillance, Montgomery County Health Department, letter of March 17, 1970 • 17 McCarthy, Hon. Richard D., U.S. Representative from New York, statement 2 Probe Into Use of Herbicide, hearings 128 Metcalf, Hon. Lee, U.S. Senator from Montana, statement 466 Note on an Improved Cleanup Method for the Detection of Chick Edema Factor in Fats and Fatty Acids by Electron Capture Gas Chromatography, article : 321 Nutritional Adjuncts, Chick Edema Factor. III. Application of Microcoiuoinetric Gas Chromatography to Detection of Chick Edema Factor in Fats or Fatty Acids, article 265 'Occupational Intoxication Occurring in the Production of Chlorophcnol Compounds, article 241 Occurrence of the Chick Pericardial Edema Factor in Some Oleic Acids and Products Derived Therefrom, article 219 Persistence of 2,4-D, 2,4,5,5-T, and Dicamba in Range Forage Grasses, article from Weeds 53 Pierovich, John M., assistant regional forester, Albuquerque, N. Mex., letter of February 2(i, 1970 153 Progress in the. Chick Edema Problem, article 235 Report on Methodology for Chlorinated Aromatics in Fats, Oils, and Fatty Acids, article 341 Response of Rabbit Skin to Compounds Reported to Have Caused Acneform Dermatitis, article 362 Role of "Toxic Fat" in the Production of Hydropericandium and Ascites in Chickens, article 294 Ryan, M. J., acting director. Office of Legislative Services, Department of 'Health, Education, and Welfare, letter of March 12, 1970-. 126 Soil Persistence of 2,4,5-T, article from Chemical Fallout 41 Stcinfcld, Dr. Jesse L., Surgeon General, Department of Health, Education, and Welfare, letter of January 21, 1970 38 Studies of the Chick Edema Disease, article from Poultry Science 255 Studies of the Chick Edema Factor. II. Isolation of a Toxic Substance, article 223 Studies of the Chicken Edema Disease Factor, article from the Journal of the Association of Official Agricultural Chemists 207 Studies on the Metabolism of Chick Edema Factor: Distribution in Chick TUsui's, nrticlo _ __ 311 Tochnic for Tolling Arnegmic Potency in 'Rabbits'~ Applied" to "the" Potent AciifKrn, '^.T.H-Tetrnchlorodibenzo-p-Dioxin, article 249 Teratogenic Evaluation of 2,4,5-T, article U.S. Shows Signs of Concern Over Effect in Vietnam of 9-year , Program, article from New York Times _" _ ' ^^ Use of the Chicken Embryo in the Assay of Adatoxi'n Toxicit^artiYle from the Journal of the Association of Official Agricultural Chemists Whiteside, Thomas, Department of Amplification, letter of March 5 1970' 98 102 290 123 EFFECTS OF 2,4,5-T ON MAN AND THE ENVIRONMENT TUESDAY, APRIL 7, 1970 U.S. SENATE, COMMITTEE ON COMMERCE, SUBCOMMITTEE ON ENERGY, NATURAL RESOURCES, AND THE ENVIRONMENT, Washington, D.C. The subcommittee met, pursuant to notice, at 10 a.m., in room 1318, New Senate Office Building, Hon. Philip A. Hart, presiding. Present: Senators Hart, Inouye, and Baker. OPENING STATEMENT BY THE CHAIRMAN Senator HART. The committee will be in order. Permit me a brief opening statement. For the Subcommittee on Energy, Natural Resources, and the Environment, I welcome those present for tho first of these 2 days of hearings we are holding to examine the effect of the herbicide known as '2,4,5-T on man and the environment. I suggest that what is at stake at these hearings is virtually impossible to evaluate at this moment, in light of the uncertainty about this frequently used pesticide. The questions which have been raised recently concerning the hazards of 2,4,5,-T and related chemicals may in the end appear to be much ado about very little indeed. On the other hand, they may ultimately be regarded as portending the most horrible tragedy ever known to mankind. What does emerge clearly from this uncertainty is that we must take steps to eliminate it. In view of the potential disaster that could befall us—or conceivably has insidiously already befallen us —absolutely no delay is tolerable in the search for answers to the questions posed. It is with the hope that Congress will be able to play an active role in that search that these hearings have been scheduled. Although the title of the hearing refers to the effects of 2,4,5,-T alone, it should be made clear early in the game that other similar chemicals may give rise to similar problems for the environmentWitnesses should feel free, therefore, to address themselves to any such chemical to the extent that it bears upon the central problems at issue. Our first witness was to have been Congressman Richard McCarthy, of New York, but unfortunately he will not be able to be Staff member assigned to this hearing: Tveonard Bickwit, Jr. '. (1) ay. We will accordingly, if there is no proxy, submit his t e s t i i r \ y for the record. (The statement follows:) STATEMENT OF HON. RICHARD D. MCCARTHY, U.S. REPRESENTATIVE FROM NEW YORK Mr. CHAIRMAN: I appreciate the opportunity to appear before this distinguished committee in order to comment on tho policies controlling the regulation mid use of the herbicide commonly known as 2,4,5-t. Your examination is most timoly, for the effects of this defoliant on plant, mid animal life suggest t h a t more stringent regulations regarding its application are called for. As YOU may know, I have spent much of the past year investigating the government's chemical and biological warfare program. I am happy to report that several significant changes have taken place in U.S. policies: changes which 1 have advocated for many months. These include a promise to resubmit the 1025 Geneva Protocol outlawing chemical and biological agents in warfare, and u bun on any further biological warfare development, including toxins. These lire small steps forward, hut the chemical warfare prograin still contains many features which are of questionable value and safety. One of these is the defoliation program in Southeast Asia. There is evidence that one of the compounds sprayed on a widespread basis is teratogenic, or birth-deforming. Without, objection, I wish to insert an article from the New York Timcn of March 15 which examines possible birth defects in babies horn in areas where compounds containing the chemical 2,4,5-trichlorophenoxyacetic acid are used. This spray, containing an agent known as 2,4,5-tricholrophenoxyacetio acid, has also been in common use as a brush killer in the United States. The commercial compound is known as Silvex, and is produced commercially by Dow Chemical Company under the trade name, Kuron. It is used by the U.S. Forest Service to kill brush as part of watershed projects in the Southwest United States. Last year, 1 learned some startling facts about 2,4,5-t. As I have, noted before, laboratory tests conducted by the Bionetics Laboratories for the National Cancer Institute indicated that the compound is teratogenir. A recent discussion of these laboratory tests appears in the Medical "IVor/rf News of February 25, 1!)70. Without objection, I wish to insert the article, "Defoliants, Defonnatii'K: What. Risk?" in the Record at this point. 1 was therefore relieved when the White House, on October 29, I960, announced that the Agriculture Department was to terminate its use around food crops after the lirst of the year. The new year came, hut instead of receiving word of compliance, I was informed that the Department of Agriculture had no intention of restricting its use. In their words: "We are awaiting advice from DIIEW as to whether or not. they intend to establish tolerances for 2,4,5-t before we decide wliether to cancel or extend uses of 2,4,5-t on food crops. Our January 1, 1970, date was based on DIIIOW's expectation that they would have reached a decision by that time. That agency believes that the public interest, would best be served by waiting for additional research data which will be available shortly. We concur in their judgment." 1 tried in vain to find out why the White House directive had not been put into force. The explanations from all agencies involved, the White House, the Department of Agriculture, and the Food and Drug Administration, simply ignored the original directive, and I was informed that the spray would be authorized until further laboratory tests were completed sometime this Spring. The best, analysis of this confusing situation appeared in the AVir Yorker Mmjnxinc of February 4, 1970. Without objection, 1 wish to insert the article entitled "A Reporter at Large: Defoliation", by Thomas Whiteside in the Kccnnl at this point. 1 have not had the opportunity to examine the effects of defoliation in Vietnam f i r s t h a n d , but I am happy to know that Dr. Matthew Messolson, a distinguished biologist and expert on chemical and biological warfare, is in the process of initiating a thorough investigation on this matter under the auspices of the American Association for the Advancement of Science. I can only say that I am shocked by the unregulated manner in which the Departinent of Defense permits its widespread application, and I urn appalled t h a t no field investigation regarding its long-range effects has been conducted by the military. 1 have, however, had the opportunity to review the policies and examined the effects of 2,4,5-t spraying operations in this country. I hope my observations on this matter will be of interest to the members of^^is committee. Last year, I learned about a controversy over one spray p^Bt carried out by the Forest Service in Eastern Arizona. Reports from GloS^Arizona indicated that the Chapparel Management Program, a part of the Salt Hiver Watershed Project had been suspended because residents of the area had complained of irregularities in the program. • Because of the difficulties I had in obtaining adequate information from Administration officials in Washington, and because of the nature of the controversy in Globe, Arizona, I made arrangements to conduct two days of public hearings in that town to learn first-hand about the spray program and its problems. The Department of Agriculture plays a unique role in a State like Arizona. It has jurisdiction over approximately 80 percent of the land area, most of which is administered by the U.S. Forest Service. Originally established to preserve forest regions in their natural state of wilderness, the Forest Service now participates in a series of commercial projects which attempt to increase the water supply for human consumption. One step is to kill unwanted flora—in this case chapparel trees, which absorb what officials believe is too much water. Traditionally, burning accomplished this mission. In the past few years, however, chemical sprays, including Silvex Kuron, have been used extensively. According to Forest Service officials, Kuron has been sprayed on four occasions since 1965. Following the latest spraying operation in June 1969, residents complained of illnesses to themselves and to their livestock, including respiratory problems and deformed offspring to their animals. My purposes in conducting hearings on February 12 and 13, 1970 were the following: (1) To learn the regulations under which the Department of Agriculture authorizes the use of the spray, and how they are enforced. In addition, I wanted to learn how new policies from Washington were transmitted and implemented, and how each regional office of the U.S. Forest Service was advised of the latest scientific information on chemicals which were in use. (2) To ascertain the degree of scientific understanding of the ecological effects of the herbicide 2,4,5-t, and (3) To learn the scope and nature of citizen complaints, and what if any, relation they have with the chapparel spraying operations. Accordingly, I requested six witnesses to appear at the public hearing. They included Dr. Arthur Galston, Department of Biology, Yale University; Dr. John rieroVich, Assistant Regional Commissioner, National Forest Service, Albuquerque, New Mexico; Dr. F. I. Skinner, Veterinarian, Globe, Arizona; and Dr. Paul Martin, Department of Geochrouology, University of Arizona, Tuscon, Arizona. In addition, two residents of Globe, Mrs. Billie Shoecraft and Mr. Robert McKusiak gave accounts of their experiences following the June I960 spraying. Mr. Pierovich gave me removing water from this area you are going to partially change the rogrtatlon. IVrhnpn you are going to denude some of the areas in order to increase HJw runoff. Thin Involved a comparative set of valves. Whose ox (is going to be ***"«! bwnc? WtK«* interests are paramount? Clearly, cities are not going to be > la ttow Indefinitely; we nre going to have to put some limit on them. We """'ill* ***t?'»1«'' Umt HM> °"y °f Las Angeles got into a 'lot of trouble with I lira*** llwm are Jl»t too many people there. In the same way, ctities in the mrm may haw to limit their size ultimately based on the number of v j*»W« tteMr «*» rl on tlm Imxlx of the amount of water resources available. .:.:;• *X«*r' that PmMifnt Nixon among others is calling for a campaign to .;• tw»l«r* Ifc* ravirotuwnt. It might b» that we would want to look at this project in the context of what we are doing to the enire Stae and to the entire countryside." 1 cannot help but conclude that such land management piAuuus involve much more than the immediate water needs of urban spra-M^The natural regions of the Southwest are a great natural resource. They must not be sacrificed so recklessly. My third interest was to determine whether any relationship existed between the maladies of certain residents and their livestock and the spraying of Silvex. The Agriculture Department readily admitted that the spray drifted out to private property. The owners have a rightful complaint regarding this fact which has not yet been resolved. The local veterinarian, Dr. F. I. Skinner informed me that he would not have recommended the use of 2,4,5-t compounds had he been familiar with the results of the Bionetics Laboratory's tests. It is also true that illnesses developed whose symptoms are similar to those which are known to he associated with herbicides. In addition, I saw malformed animals who were born after the incidents of last June. Statements were made regarding this fact by persons testifying in good faith, and should not be dismissed outright. They obviously have some bearing because the Forest Service has suspended further sprayings in the area. This decision followed the complaints, and while Federal investigation found no direct relationship between the spray operations and the illnesses, Forest Service officials await further developments before resuming. No one is being helped by the procrastination of officials in Washington. Three agencies are now involved in the 2,4,5-t controversy, yet none have assumed responsibility for regulating this herbicide. The Food and Drug Administration, which under the 1954 amendments to the Cosmetic Act of 1938, has the obligation to establish safe tolerance levels before a chemical of this kind is put on the market, has failed to enforce the Law. The Agriculture . Department continues to ignore other agencies in administering the Federal Insecticide, Fungicide and Rodenticide Act. The Pesticide Regulation Division established by this act, was sharply condemned by the House Government Operations Committee report of November 13, 1969 for not carrying out its responsibility to police the licensing of herbicides. In addition to the charge that no legal steps have ever been taken against firms which violate licensing regulations, the Committee report brought to light repeated instances of conflict of interest among various officials of the Pesticide Regulation Division and agro-chemical companies. Finally, the White House has backed down from its assertive position of last October. After reversing an earlier ban, I am now told boldly by Dr. Lee A. DuBridge in a letter of March 2, 1970, that "we anticipate, indeed we will insist upon final action of 2,4,5-t before its period of principal usage in late spring." I will not hold my breath. Mr. Chairman, there are obvious irregularities in the regulation and management of herbicide compounds containing 2,4,5-t. It is clear that the National Forest Service no longer regards preservation of lands in their natural state as a primary responsibility. There is insufficient information regarding Its risks and inadequate statistics on its effects to animal and plant life. Its use must not be continued until its safety is assured. Accordingly, I recommend an immediate five year ban on the use of herbicides containing 2,4,5-t. During this period of suspension, the Food and Drug Administration should establish, once and for all, whether the chemical has a. safe tolerance level. The latest word from FDA Is that the officials "are in no position to say that the chemical 2,4,5-t or the dioxin is without hazardous effects". A letter to me of March 12, 1970, reflects the inconclusive evidence regarding its safety. It is worth repeating in full. Without objection, I wish to insert the letter from M1. J. Ryan of the Food and Drug Administration, along with an attached fact sheet in the Record at this point. In addition, steps should be taken immediately to collect information on the number of children born with birth defects, including those which might be caused by herbicides. Accordingly, I will be writing to the Secretary of Health, Education and Welfare urging him to instruct the National Institute of Neurological Diseases and Strokes of the National Institutes of Health to begin gathering information on these phenomena. Mr. Chairman, I am confident that these hearings will help resolve a most important issue. There can be no more delay. Jj^tehairman, without objection, I wish to insert various documents ing^^ny hearings in Globe, Arizona for inclusion as an appendix to hearings.1 Senator HART. We will move to the next scheduled witnesses, James Turner and Harrison Wellford of the Center for Responsive Law. STATEMENT OF HARRISON WELLFORD, CENTER FOR STUDY OF RESPONSIVE LAW, WASHINGTON, D.C. Mr. WvH.LPcmi). Thank you, Mr. Chairman. We appreciate A'ery much the invitation to appear at these hearings this morning. I would like, if I may, to road excerpts from my testimony rather than reading the entire testimony. In this testimony we wish to bring to your attention that herbicides containing 2,4,5,-T are widely marketed for casual use by the individual consumer in residential and other populated areas. The herbicide 2,4, 5-T even in extremely small doses, causes massive and severe birth defects in tost animals, including mice, rats, and hamsters. The herbicide 2,4-1), which is often mixed with 2,4,5-T in the popular herbicides such as Ortho Weccl-Be-Gone, is also teratogenic, but at higher dose levels. We feel that these herbicides, as currently used, may pose a grave and unnecessary danger to public health. In a recent article, the biologists Arthur W. Galston, William Cooke, and William Haseltine stated that 2,4,5-T "may represent the ecological equivalent of thalidomide." (Congressional Record, Feb. 19, 1970, S. 1984). Professor John T. Edsall of Harvard University, stated before the American Association for the Advancement of Science's Committee on Chemical and Biological Warfare that "the use of these compounds is much more seriously questionable than the use of cyclamates. If one applies the same criteria, one would consider the risks quite unacceptable." (Quoted in the New York Times, Dec." 29, 1969). Tim tests performed by the Bionetics Laboratory, by the Food and Drug Administration, National. Institute of Dental Research, and the National Institute of Environmental Health Sciences clearly show that these chemicals are potentially harmful. Whether or not human beings are more or less susceptible than test animals to these chemicals, we do not yet know. But clearly on the evidence now available, the burden of proof should be on the industry to demonstrate that they are not harmful. In the meantime, all uses of these herbicides around the home and in populated areas should be immediately suspended. Wo wish to discuss this morning the nature of the clanger as we see it and the serious failures of both the Federal Government and private industry to reduce the unnecessary risks to public health of exposure to 2,4,5-T and other suspicious weedkillers. Specifically I am concerned about : (1) The attempts by Government and industry to conceal the facts about 2,4,5-T and 2,4-D from the public and from other scientists. 1 HCP p. 102. (2) The hazards to public health which may res^^from widespread use of 2,4-D and 2,4,5-T in populated areas. WP My colleague, Mr. James Turner, will discuss the profound implications which the 2,4,5-T-2,4-D case has for the effectiveness of Government efforts to protect the public from pesticide hazards. 2,4,5-T was developed from research done at the Chemical and Biological Warfare Center at Fort Detrick during the 1940's. It has. been massively applied to the human environment for 20 years, but until very recently no studies have been conducted by any Government agency 011 the possible carcinogenic, mutagenic, or teratogonic properties of this herbicide, or on the ecological consequences of its use. In 1966 the Bionetics Laboratory was commissioned by the National Cancer Institute to begin research into the birth defect properties of a variety of pesticides and herbicides, including 2,4-D and 2,4,5-T. By 1968 these tests had revealed substantial evidence that 2,4,5-T caused birth defects in test animals. In February of 1969, the preliminary results from the Bionetics testing were to be presented to the annual meeting of the American Society of Toxicology. The report included tests results Avhich showed that 2,4-D and 2,4,5-T cause gross abnormalities and birth defects in mice. 2,4-D was termed ''potentially dangerous, but needing further study," while 2,4,5-T was labeled "probably dangerous." Tins report would have provided an early warning on potential hazards from these herbicides, but for reasons still unknown, the Bionetics presentation was canceled at the last minute, although its paper was listed on the printed agenda of the meeting. The Bionetics report thereafter returned to obscurity. Only a few scientists in the Government knew of its existence. Even members of the Mrak Commission, Panel on Teratogenicity were initially rebuffed when they asked to see the report. The report, however, had been presented to selected officials in the Food and Drug Administration, the U.S. Department of Agriculture, and the Defense Department. These delays contradict the recommendation of the Mrak report that "any teratogenic pesticide to which the population is exposed should be promptly identified so that appropriate precautions can be taken to prevent risk of human exposure "(p. 657). On October 29, after the existence of the report finally became public (it has still not been officially released), Dr. Lee Dubridge, the Science Adviser to the President, announced that "a coordinated series of actions" was being undertaken by several Federal agencies to ban the use of 2,4,5-T on food crops and Government use of the herbicide in populated areas. Dr. Dubridge stated that these actions • we,re being taken to "assure the safety of the public while further evidence is sought." The ban on use of 2,4,5-T on crops was to go into effect on January 1, 1970 unless by that time the FDA had found a means to establish safe residue tolerances. Apparently because Dr. Dubridge estimated that "almost none (of 2,4,5-T) is used by home gardeners, or in residential areas," nothing was said about stopping individual consumers from using 2,4,5-T in their own backyards. The Department of Interior did follow Dr. Dubridge's recommendation and ceased the use of 2,4,5-T in its operations. The deadline of ^fcuary 1, 1970, passed without FDA setting a tolerance. By JanBwy, it was clear that the Department of Agriculture and Federal agencies (with the exception of Interior) had no intention of restricting the use o:f 2,4,5-T. On February 6, the Department of Agriculture.announced that the original 2,4,5-T used in the Bionetics test had been contaminated with a tetradioxin and that further testing with a purer batch of 2,4,5-T had shown no adverse effect. The Department relied on tests conducted by the Dow Chemical Co., a major manufacturer of 2,4,5-T. The Dow evidence was immediately contradicted by tests conducted at several other Federal agencies which clearly showed that even the purest 2,4,5-T, where the dioxin contaminant was present at less than one part per million, still produced birth defects in test animals at significant levels. Recent tests on 2,4-D conducted by the FDA have confirmed that this herbicide, which is very often mixed with 2,4,5-T, is also teratogenic. Many other questions with significance for public health remain to be answered: How persistent are 2,4,5-T and 2,4-D once applied? How persistent is the dioxin contaminant? Is it cumulative in human tissue? How much dioxin is present in herbicides already on the shelves of local hardware stores? Therefore, Mr. Chairman, 5 months after Dr. Dubridge urged that most uses of 2,4,5-T be banned to "assure the safety of the public while further evidence is sought," these herbicides continue to be teratogenic and are as widely used as ever. The most recent tests provide the "further evidence" Dr. Dubridge asked for. The burden of proof on those who wish to demonstrate the safety of these herbi. cides has greatly expanded since the first Bionetics test. When tests indicate that a pesticide is teratogenic in animals, the burden of: proving that it is safe should be placed on the manufacturers of the pesticide, not on its possible victims in the general population. If the manufacturers do not cooperate, the Federal Government has a statutory responsibility to minimize human exposure to teratogenic pesticides by appropriate regulatory preventive action. The Government must not fail in its trust, for nationwide statistics on birth defects are so inadequate that even an increase of several thousand deformities could probably go undetected. Therefore, if Government and industry act irresponsibly, there will probably be few complaints from the medical profession or the general public to call them to task. Present population monitoring techniques do not provide adequate gages of the incidence of birth defects in the population. Federal regulators charged with protecting the public from pesticide hazards are being very irresponsible if they assume, as did Dr. Lindsey of the Food anc^ Drug Administration in a recent interview that, "the National Institute of Neurological Disease and Stroke has recorded birth defects for some 15 years and would be telling us if they were on the rise,'-'1 Dr. Hincs Berendes, Chief of NiNDS' Perinatal Research Branch, has unhappily conceded that, "no nationwide data are available on the frequency or incidence of malformation." * Even in States whore birth certificates request that doctors record birth defects, the completeness and accuracy of the reporting depends on 1 Medical World News, Feb. 27, 1070. the interest and diligence of the physician and on tin onspicuousness of the abnormality. Nationally, no attempt ^^ made to collect and evaluate all the data on birth defects tha^Tre presently available on birth certificates. After careful study of this problem, the writers of the Mrak report concluded that "epidemiologic data on possible effects of pesticides on human reproduction and teratologf are grossly inadequate." 2 The Mrak Commission report states that when animal experiments , indicate that a pesticide is teratogenic, the effect should be retrospectively evaluated when possible by a study of pregnancies during which the mothers were inadvertently exposed to the pesticide, such as in farmwork and industrial exposure and through accidental ingestion. As far as we know, this has not been done for 2,4,5-T and other weedkillers. Because of the need to minimize human exposure, it is not possible to test on human populations pesticides previously shown to be teratogenic by experimental animal studies. The Mrak report states unequivocally that there is little comfort to be gained from the expectation that present epidemiological surveys of: pesticides in current use will discover in time chemical cornpounds causing birth defects. It states that no major teratogen (term for sxibstance causing birth defects) has been discovered in this way. The malformations induced by X-rays, german measles, thalidomide, and mercury were each recognized by "an alert medical practitioner who observed a cluster of cases and then traced the cause to its source."3 Tracing observed defects to a specified cause is much more difficult when the defect commonly occurs. In the case of 2,4,5-T, the most common defects produced in test animals are kidney abnormalities and cleft palates, neither of which is unusual in humans. Had thalidomide produced such ordinary malformations instead of bizarre and unusual ones, it probably would never have been discovered. Thus any birth defects produced by human exposure to 2,4,5-T are unlikely to be traced to the weedkiller because they are already common in the population. If 2,4,5-T and 2,4-D, as commonly used in populated areas, do produce birth defects in humans, the birth defects will remain a very private family tragedy. Because 2,4,5-T leaves no unique fingerprints on the fetus to indicate its specific role as the teratogen, the parents of the deformed child would probably remain silent, with no knowledge of the caiise of their distress, They would probably never know that they belong to a class of victims of a preventable tragedy. There is another reason why Government has a special responsibility here to protect the public. Because a 2,4,5-T or 2,4-D induced birth defect is not unique, the parents of deformed children will have great difficulty in using the courts to discipline the manufacturers of dangerous herbicides. The parents will gain no compensation for their loss. Moreover, there will be no lawsuits to force the chemical companies to test more thoroughly their products for teratogenic effects before they are released on the market or to maintain strict quality control standards which will keep the level of contami1 Report of the Secretary's Commission on Pesticides (the Mrak report, U.S. Department of Health, Education, and Welfare. December 1008, p. 674. , ' Mrak report, p. 661. 10 11 natiowof dangerous dioxins as ]ow as possible. In the absence of legal remedies for private citizens, protection must come from the Federal Government. As a first step, we suggest that the committee consider the recommendation of the Mrak Commission that efforts must be made to improve and use information on congenital malformations recorded . on birth certificates and that new systems of collecting birth defect data.be established. for example, Scotts Turf Builder Plus-1, which contd^Ja mercurial compound, often disguise or play down the herbicide content. Mixing herbicides with fertilizer tends to identify potentially dangerous chemicals with innocuous fertilizers and promotes unnecessary, as well as careless use. (5) The manner in which these herbicides are used magnifies their potential risk. Their labels all read: "Avoid contact with skin, eyes or clothes. Avoid inhaling dust," Yet these products are dispensed in a manner which makes contamination of the user with either dust or spray inevitable. Herbicides in dust or powder form are often applied by dumping them into a wheelbarrow, with a hole in the bottom and a propeller underneath which the user pushes across his lawn raising a cloud of dust. Liquid herbicides are applied through attachments to a garden hose or through hand applicators. In both cases, contact with the spray is unavoidable. Many of these products on their packages depict homeowners spraying with bare hands and bare arms. On no products, even the most poisonous, was there any suggestion that the user wear rubber gloves. Several of the herbicides did contain the difficult instruction that children and pets be kept off the treated areas, sometimes for an unspecified amount of time, until after the area had been watered and dried or it had rained. Whether applied in spray or dust form, the application of herbicides containing 2,4,5-T presents serious problems of drift. The report of the Subcommittee on Weeds of the National Research Council stated in 1968 that spray with "droplets of 10 microns in diameter can drift up to 1 mile when released at a height of 10 feet with a 3-mile per-hour wind." (p. 248). Even when kept in perfect condition, few nozzles used for spray application would produce uniform droplets large enough to minimize drift and yet small enough to provide even coverage. The hazards of drift, even when the herbicide is applied in dust or powder form is also great. The Department of Agriculture, in its caution suggested for use on weed-killers containing 2,4,5-T and 2,4-D, warns that "this dust may drift for miles even on quiet days." (Federal Register, May 21,1969). It is a conservative estimate that even on a relatively calm day children playing within 100 yards of an area where a yard is being sprayed or dusted with 2,4,5-T are probably going to be exposed to the chemical. Droplets and dust particles of 2,4,5-T can be carried by the wind into open windows and onto screen porches. In heavily populated residential areas, one simply cannot defoliate his backyard of chickweed and dandelions without running the risk of contaminating his neighbors or their children. The economic and horticultural .benefits of these herbicides in residential areas do not outweigh their risks to those who wish to enjoy the outdoors without being contaminated by teratogenic spray. (6) A few products did not bear even the minimum federally required warning, "Caution, Keep out of reach of children," which must be displayed prominently on the front. For example, Scotts Kansel Weed Killer, in a salt-shaker like container, had no warnings at all on the front, and only "Avoid contact with skin, eyes, clothing, etc." written very inconspicuously on the back. The same was true of an identically packaged Amchem Garden Weeder. POTENTIAL HAZARDS FROM CONSUMER USE OP TERATOGENIC HERBICIDES IN RESIDENTIAL AREAS Weed-killers containing 2,4,5-T are readily available to the home gardener. These products come both as premised, ready-to-use liquid, spray or dust, and as liquid preparations which the user dilutes at home, or uses with the garden hose atomizer. The concentrated liquids are obviously the most dangerous. Last week two of my assistants made a survey of herbicide products in 10 Washington area stores: Eight hardware stores, including Meenehans, Sears, Hechingers, Mclntyre, Community Paint and Hardware, Kresge's at 7th and Pennsylvania, Chevy Chase Hardware; one grocery store, the Giant at Western Ave. and Wisconsin; and one gardening store, Sheridan Garden Store on Old Georgetown Road. My assistants found the following: (1) Eight of the stores carried lawn and garden weedkillers containing 2,4,5-T, nine of the stores carried product lines which included 2,4,5-T products. All of the stores carried weed-killers containing 2,4-D. They found nine product lines containing 2,4-D; six with 2,4,5-T. (2) In addition to 2,4-D and 2,4,5-T, at least seven of these stores carried all but one of the other products cited in the Bionetics Eeport' as at least potentially causes of birth defects: Captan, Folpet, Sevin; as well as organo-mercurial products which have been known to produce birth defects in humans since an epidemic in Japan led to their'being banned in that country.4 (3) Some of the products, the Scotts line in particular, were very badly packaged. Most Scotts products and some others are packaged in flimsy paper bags; a number of the bags which my assistants examined were in such bad repair that they could not be handled without spilling dust on the handler. As a result, there was frequently a coating of chemical dust on products nearby. In an unintended irony, most of the packages bore warnings to the user to "avoid contact with skin, eyes or clothing." (4) A number of products and again the Scotts line in particular, were fertilizers with various herbicides added, including 2,4-D, and 2,4,5-T (in Greenfield products.) Since the contents were not very conspicuously displayed, many users might well assume that these were only "super fertilizers," and therefore not handle them with thp care that their contents warranted. These products, containing a mixture of fertilizer and herbicide are heavily promoted on radio and television, with no warning of potential danger. Their names, 1 Mrnlt report, p. 001. 45-302—70 2 12 The potential hazards of 2,4,5-T herbicides are increased 'act that consumers frequently do not read the label on the pes ticide package, or if they do, do not usually understand it. The Mrak report cites the 1969 study of home pesticide use in Charle'ston, S.C. which found that of the 83 percent white and 97 percent nomvhite families using pesticides: 13 There is no objection to statements that are aimed against damages from careless or improper handling, are not false or misleading. the seller so long as they EXAMPLES OF DISCLAIMER STATEMENTS WHICH ARE NOT OBJECTIONABLE It is unlikely that the labels of the 2,4,5-T herbicides we examined in local stores could be changed sufficiently to prevent users from contaminating vulnerable individuals. I might add while it is not included in my prepared testimony that we did come across yesterday herbicides on local hardware shelves which have disclaimers of liabilities which are clearly in violation of the notice to manufacturers, formulators, and distributors issued by the Pesticide Eegulation Division of the Department of Agriculture on November 16, 1966. (The notice follows:) (1) Seller makes no warranty, expressed or implied, concerning the use of this product other than indicated, on the label. Buyer assumes all risk of use and/or handling of this material when such use and/or handling is contrary to label instructions. (2) Follow directions carefully. Timing and method of application, weather and crop conditions, mixtures with other chemicals not specifically recommended, and other influencing factors in the use of this product are beyond the control of the seller. Buyer assumes all risks of use, storage or handling of this material not in strict accordance with directions given herewith. (3) Buyer assumes all risks of use, storage or handling of this material not in strict accordance with directions given herewith. (4) Seller's guarantee shall be limited to the terms of the label, and subject thereto the buyer assumes any risk to persons or property arising out of use or handling and accepts the product on these conditions. (5) Our recommendations for use of this product are based upon tests believed to be reliable. The use of this product being beyond the control of the manufacturer, no guarantee, expressed or implied, is made as to the effects of such or the results to be obtained if not used in accordance with directions or established safe practice. The buyer must assume all responsibility, including injury or damage, resulting from its misuse as such, or in combination with other materials. UNITED STATES DEPARTMENT OF AGRICULTURE ' . AGRICULTURAL RESEARCH SERVICE PESTICIDES REGULATION DIVISION, Washington, D.G., November 16, 1966. Mr. AVELLiroRD. This order stated that the following disclaimer would be considered both false and misleading, and a product which had this kind of disclaimer on it would be rendered misbranded : Both white and nonwhite families commonly ignored safety precautions in the use of household chemicals. Locked storage was not employed by 88 percent of all families; 66 percent stored the pesticides within easy reach of small children; 54 percent stored the chemicals near food or medicine; and 66 percent never wore protective gloves during use or washed their hands after application. 0 , . , ' NOTICE TO MANUFACTURERS, FOBMULATOHS, DISTRIBUTORS, AND REGISTRANTS OF ECONOMIC POISONS The information furnished hereon is provided gratuitously by the manufacturer who assumes no responsibility whatsoever for the effectiveness or safety of this product regardless or whether or not it is used as directed. STATUS OP DISCLAIMER STATEMENTS ON PESTICIDE LABELING Attention : Person responsible for Federal Registration of Economic Poisons. The Federal Insecticide, Fungicide, and Rodeuticide Act, and the regulations promulgated thereunder, provide that an economic poison is misbranded if its labeling hears any statement, graphic representation, or design which is false or misleading in any particular. Labeling disclaimers which negate or detract from labeling information required under the Act and regulations are not acceptable on products proposed for registration. , . . An example of a disclaimer which would render a product misbranded is as follows: "The information furnished hereon is provided gratuitously by the manufacturer who assumes no responsibility whatsoever for the effectiveness or safety of this product regardless of whether or not it is used as directed." Such disclaimer is both false and misleading, since adequate directions for use, necessary warnings and cautions, and other essential information on the safe handling and use of a product are required under the Federal Act. Labeling for registered products bearing disclaimer statements which are either false or misleading must be revised or deleted. Such revisions or deletions will not require reregistration by this Division. However, five copies of such amended labeling should be submitted for our records. HARRY W. HAYS, Ph. D., Director. Here I have two identical cans of Ortho Poison Ivy Killer, both purchased in the last 2 days. One of them has the proper disclaimer. It says "Notice, buyers assume all responsibility for safety and use not in accordance with directions." Senator HART. Would you restate that ? Mr. WKLLFOUD. Yes, sir. The buyer assumes all responsibility for safety and use not in accordance with directions. That is a proper , disclaimer. This other product was purchased at the same time. It is the same substance, but it has the illegal disclaimer on it : There are two major provisions of disclaimer statements which have been used by many registrants that are considered to be false or misleading. These are the claims "that the information is provided gratuitously," and disclaiming on any responsibility "whether used in accordance with the directions or not." ' Mnik report, p. 148. Because of critical, unforeseeable factors beyond the manufacturer's control prevent it from eliminating all risks in connection with the use of chemicals even though reasonably flt for such use, buyer and user acknowledge and assume all risks and liability resulting from handling, storage, and use of this material. These risks include, but are not limited to, damage to plants,- crops and animals to which the material is applied, failure to control pests, damage caused by drift to other plants or crops and personal injury. Buyer and user accept and use this material on these conditions whether or not such use is in accordance with directions. Now, either these products Avere being marketed in violation of the order of the Pesticide Regulation Division or they have been on the shelves since 1966. The manager of the store at which we bought the one with the illegal disclaimer stated that the spray had been purchased 1 month ago and that the distributor' got it 4 months before that, So, we would assume that these products are still being manufactured with that disclaimer. 14 percent of all the herbicides we examined in all the stores, contained either 2,4-D or 2,4-,5-T or a combination of both. By the way, if the product—Ortho Poison Ivy Killer—which you have just examined has been on the shelves since 1966, it raises questions of when the new quality control standards introduced by Dow Chemical Co. and by probably other chemical companies •which have •reduced the level of the dioxin contaminant took effect, it may be that many products that we are now able to buy off the shelves of ' local hardware stores were produced before the new quality control standards were introduced. If that label is not illegal, it was produced at least 3 years ago, maybe more, and this raises one of the basic questions we want to stress this morning, to bring to your attention this morning, and that is we really do not know at all how much of the dioxin contaminant is in the herbicides which are so widely available at the local hardware stores. Some may be. very old. We do not know whether til] the chemical companies that produce 2,4,5-T are adhering to the same quality standards that Dow uses. This is a major area that needs investigation. In short, the general population is being exposed to 2,4,5-T herbicides in use in residential areas. Over 60 percent of all herbicides we examined in local stores contained either 2,4-D, 2,4,5-T or a combination of both. Estimating the precise hazard posed by weedkillers is difficult due to the scantiness of scientific information and the difficulty of extrapolating whatever information there is from animals to humans. For instance, the level of dioxins in these products is totally unknown. In many of the products 2,4,5-T is slightly modified chemically, although this modification would probably not effect the dioxin. Therefore, without further data there is no good way to extrapolate the effects of high doses on small numbers of animals to krgo populations exposed to low doses. Moreover, human sensitivity to 2,4,5-T probably varies greatly from person to person. Finally, it is worth recalling that thalidomide proved 50 to 200 times more teratogenic in humans than in the test rats and animals which had been used to demonstrate its safety. The potential harmfulness of the 2,4,5-T products marketed for individual consumer use depends in part on the amount of dioxin contaminant they contain. I have just made my point on that, the fact that we do not know how much they do contain. ~r x f~-t i • i /~* i knows how much dioxin is in Dow's older products still on the market, which may have been made before it instituted its present quality controls. Moreover, no one knows how much dioxin is in '2,4,5-T products produced by other chemical companies which may not have the standards of quality control employed by Dow Chemical. Moreover,'even Dow's current production of 2,4,5-T contains oyoi'^5 percent of other impurities, including some other kinds of dioxins. The effects of these impurities are totally unkown. Dr. Meselson, appointed last year by the American Association for the Advancement of Science to head a 2,4,5-T evaluatioi^|oject, says: Tlie tetrachlore dioxin represents just one of 12 or 13 wlW the chlorine atoms can arrange themselves on a benzene ring to form dioxin molecules. How do we know about the hexa, hepta, and octyclors, or about how persistent the tetraehlor itself is? Moreover, I'm very concerned about the dioxins that might be formed by unreacted trichlorphenol (2,4,5-T precursor) wht>n the product is exposed to heat. If it were taken up by plants or wood and these were burned, you'd get more dioxin. Finally, I'm bothered by the bizarre mental effects suffered by German workers making 2,4,5-T. I say when in doubt.stop it" A mystery with disturbing implications for public ^health is whether the' dioxin contaminant in 2,4,5-T accumulates in human tissue. There is as yet very little scientific evidence on this point. If the dioxins do accumulate, they pose a danger, not only to the children of women who were pregnant when exposed, but also to women who become pregnant subsequent to exposure. If the dioxin or other teratogenic substances in 2,4,5-T are cumulative, it becomes academic whether a single or even several, exposures to herbicides used on residential lawns and gardens are of sufficient magnitude to cause birth defects. CLTNlCAIj EVIDENCE OF 2,4,5-T POISONING The clinical evidence of injury to-persons coming into contact with herbicides containing 2,4,5-T is difficult to compile because techniques for monitoring 'pesticide poisonings are nearly as inadequate as those for measuring the incidence of birth defects. Poison control centers in many cities regularly treat victims of pesticide poisoning, but only rarely are their records of poisoning broken down as to particular pesticides. Nevertheless, there are enough case histories on record to substantiate the laboratory findings that the herbicide 2,4,5-T, in addition to being a possible teratogen, can be severely toxic to human beings, causing nausea, diarrhea, chloracne (a serious skin disease), mental distress, kidney damage, and other maladies. (1) Thomas Whiteside, in an article in the New York magazine, reported that chloracne has been found among workers in plants manufacturing 2,4,5-T. In the mid-1960's the Dow Chemical Co. closed down part of a 2,4,5-T plant in Midland, Mich., because workers there contracted chloracne apparently as a result of contact Math the dioxin contaminant in 2,4,5-T. The symptoms of this disease included extensive skin erruption, central nervous system disorders, fatigue, lassitude, and depression. Similar symptoms were reported in German workers in plants producing 2,4,5-T as early as 1955. (2) Professor Arthur Galston, at a hearing on herbicides conducted by Congressman Richard McCarthy, in Globe, Ariz., discussed a 'scientific report entitled "Dermatitis and Kidney Damage Ascribed to Weedkiller 2,4,5-T." This report told of < two girls, aged 4 and 6 years old, \vho had played for several hours in a yard which had been sprayed heavily a short time before with Ortho Brush Killer. The girls developed general reddening of the skin and swell' Medical World News, Feb. 27,1970, p. 17. ing oL the oval and vaginal mucous membranes. The limbs and eyclids^lro also slightly swollen. On the third day after exposiiro, kidney damage was indicated by the discovery of albumen in the urine which persisted for about 2 weeks. (3) • Several"-suspected cases of 2,4,5-T and 2,4-D poisoning are being studied in Globe, Ariz. (4) The Montgomery County Health Department has received at least one report from a Bethesda woman whose child suffered nausea, diarrhea, swelling of: the lymph glands, and prolonged mental distress, as a result of exposure to a spray containing 2,4-D, 2,4,5-T and petroleum distillates, which had drifted from a neighbor's lawn being treated with the herbicide. More clinical evidence might easily be compiled if an cpidemiological study were made of particularly vulnerable groups. For example, it would be interesting to know if Dow and other companies producing 2,4,5-T have surveyed their female workers to see if they < have a higher incidence of birth abnormalities. Similar studies should be made of farmworkers', especially those associated with rice and sugar production where 2,4,5-T is used. A special concern should be females who work in the timber industry, where 2,4,5-T spray is used to kill trees. I worked for several summers spraying trees myself, and my colleagues among the sprayers were very frequently young women who carried spray cans on their backs and sprayed the spray into cuts on the trees made by men. There is simply no wav of, after using that spray for more than 30 minutes that you would not be literally drenched with it. It would seem very likely that many of these women, being young, were in the early stages of pregnancy at some point in their' work, and that they may have received very heavy doses of herbicide from vapors and wotting of the skin. In this work, herbicides containing 2,4,5-T or 2,4-D plus 2,4,5-T in concentrations ranging from 8 to 16 pounds of acid equivalent per 100 gallons of fuel oil are commonly used.7 These herbicides are sprayed into cuts made from girdling trees. It is common practice in Virginia, for example, for women to be hired to carry spray cans and apply herbicides 011 the trees after they have been cut by men. The sprayer is invariably heavily exposed to the herbicide, through inhilation of the vapors and wetting of the skin. Scores of women, many of them in the early stages of pregnancy, have probably been exposed to heavy concentrations of 2,4,5-T and 2,4-D in this manner. The Government should take immediate steps to warn women engaged in this work of the risks they are running, especially since many of them are working on projects sponsored or'advised'by the Forest Service of the U.S. Department of Agriculture. In conclusion, Mr. Chairman, it seems an unnecessary risk to ' Wood Control, National Acndemy of Sciences, 1968, p. 330. public health that a herbicide with the dangerous potential of 2,4,5T should be widely available for casual use 'by the^Biividual consumer. In a recent letter, Dr. Steven Lipson of tr^^'Iontgomery County Health Department, stated this view very well. (The letter follows:) MAUCII 17, 1970. Mn. HARRISON WELLFORD, Center -for Study of Responsive Law, Washington, D.G. DEAR MB. WELLFOIUI : In response to your request, I nm writing you wltli regard to my personal and professional feelings about the use of 2,4-D, 2,4,5-T and related herbicides. I should like to preface my remarks with the note that these comments are made as an individual and not as an official of the Montgomery County Health Department Over the past years this Department has received a number of inquiries as to the possible toxicity of these chemicals. Our information relating to this is drawn from standard sources and it appear,? that there is no question but that these are dangerous substances when used improperly. As you know, exposure to these chemicals may cause central nervous system depression, weakness, loss of appetite, diarrhea, coma and sudden death. There Is also a risk of skin irritation and damage to peripheral nerves. Extensive discussion of this problem within the Health Department leads us to the conclusion that local control is impractical. One may pass any number of regulations regarding the use of herbicides in suburban and residential areas but enforcement is completely impractical. Public education appears to be the only feasible local alternative but as the continuing smoking problem demonstrates daily, this is ,a long and slow process. Our conclusion then was that the only reasonable means of control is at the point of sale. While again one could limit this locally, the movement of the consumer to different marketing areas is great, particularly in the Washington Metropolitan area, and limitation of sale of a substance by any single jurisdiction would have little effect. Accordingly, as a parent, physician and public health worker, I would strongly support the limitation of use of herbicides to those situations in which it is economically and scientifically indicated. To my mind, this would not include suburban residential use. If the question is taken even more broadly, I would agree fully that no foreign substance, irrespective of whether toxicity has been demonstrated, should be introduced into the environment unless there is a clear and evident need for its use. This argument becomes even stronger when we talk about the exposure of children to potential hazards. I strongly support and concur in your attempt to remove 2,4-D and 2,4,5-T from the individual consumer market. Sincerely yours, STEVEN LIPSON, If./)., M.P.If. Chief, Division of Epidemiology anil Surveillance, Montgomery County Health Department. Mr. WELLFOBD. The economic value of using these herbicides to defoliate residential backyards of dandelions and chickweed is manifestly outweighed by their potential hazards. Until the manufacturers establish that exposure of the population to 2,4,5-T and 2,4-D is not dangerous, they should be banned from the individual consumer market. We would also support efforts to ban the use of these herbicides on food crops. Thank you very much for inviting us to make this statement. My colleague, Mr. James Turner, now has a statement to make. 18 19 EATEMENT OF JAMES TUENEE, CENTEE FOE STUDY OP EESPONSIVE LAW, WASHINGTON, D.C. Mr. TTJRNEH. I too would like to thank you for your kind invitation to be present hero today to discuss the way in which pesticide safety is regulated by the Departments of Agriculture, and Health, Education, and Welfare. Mr colleague, Mr. Well ford, has outlined to you the details of the very serious potential health problem presented by the widespread use—particularly around the homes of many American families—of the pesticides 2,4,5-T and 2,4-D. There are possibly many other widely used pesticides which pose health hn/imls to the public, because under the current pesticide control system effectiveness is considered more important than safety. Tt is also important to note that pesticides are only part of the chemical problem. An increasing number of scientists believe that many of the thousands of chemicals to which the average American is exposed—food additives, drugs, industrial wastes, cosmetics and others, as well as pesticides—present a massive threat to future health and well-being. The potential health hazard presented by chemicals in the envi- . ronmerit is difficult to exaggerate. Americans have been much too secure in the belief that their health is the best in the world. In fact, the health of Americans is not good. The life expectancy of an American female ranks 21st in the world; that of an American male, 87th. That is for people who have reached the age of 20. Infant mortality figures, called by many statisticians the best measure of a nation's overall medical ability place the United States approximately 15th in the world, a drop from fifth in 1950. Since 1900 .the average American reaching the age of 4-0 has had only 4 years added to his life expectancy. The National Foundation of the March of Dimes reports that one in seven births ends in death or deformity of the infant. An increasing number of scientists believe that much of this deterioration of American Health can at least in part be traced to the strain placed on individuals by chemicals in the environment. Heart disease, the major killer, may be made more likely by chemicals such us caffeine. Cancer accounting for 20 percent of all deaths is being increasingly related to chemical causes. But more important is the potential for genetic damage that many chemicals present. At present, the knowledge gained, about potential mutagenic perils from research efforts by scientists in the field of genetics has far outreached the efforts of the Federal Government to establish procedures to insure that the general population is not exposed to mutagenic chemicals. The regulatory history of 2,4,5-T and 2,4-D serves as an example of the governmental failure to protect the public from potentially dangerous chemicals. There are some general antidotes that might possibly begin to reverse the Government failure to deal with what is potentially a very serious health hazard. It might be of interest to this committee to examine the current relationship and overlap between the Food and Drug Administra- tion 'and the Department of Agriculture in the pest^^j field. Currently the law requires all pesticides to be certified by the Pesticide Regulation Division of the Department of Agriculture, The main criteria for registration is the effectiveness of the pesticide for its intended purpose. At one stage in the process of register- . ing a new pesticide, the Agriculture Department is required to seek an advisory opinion on safety of the pesticide product from the Food and Drug Administration. As the situation now stands the recommendations of the FDA that certain products are unsafe and should not be marketed as intended have been rejected nearly 100 percent of the time by the Agriculture Department. In addition, thousands of FDA recommendations concerning the proper labeling of pesticide products have been rejected. As the law now stands this procedure is perfectly legal. Agriculture is merely required to seek advice. It is not required to act upon it. In the present time when the safety of pesticides has become a more important concern than their economic usefulness, it might be wise to readjust, the relationship between these two agencies. Perhaps pesticide registration should be officially the responsibility of the Food and Drug Administration, with the Department of Agriculture providing only an advisory opinion as to the potential usefulness and effectiveness-of the chemical. Stich a readjustment would direct Government attention toward the problem of safety and environmental contamination which have become the major concerns in the pesticide field and away from the much simpler task of determining the potential usefulness of pesticides in killing particular bugs and weeds. It might aJso be of interest to this committee to explore the relationship between the producers of chemical pesticides and the agencies that must certify and offer opinions on them. Currently the overwhelming majority of scientific information relied upon to establish the effects of pesticide use is provided by industry sources. This, by the way, is true in all chemical fields—foot additives, cosmetics, drugs, as well as pesticides. Often the manufacturers of these chemicals work out relationships with independent testing facilities which tend to seek the answer most advantageous to their clients. An illustration of the situation is what recently happened in the Food and Drug Administration itself, to a scientist who sought some testing results from an independent laboratory. He waited for several weeks for the results of what should have been a relatively easy experiment.-. Finally he called the researcher and was told that there was trouble with the experiment. The Food and Drug Administration official asked what the problem was, and was told that as hard as he tried, the scientist conducting the experiment could not find anything wrong with the chemical he was testing. He was under the impression that he had to find something wrong, or the Food and Drug Administration would be dissatisfied with the work. This kind of bias exists in both directions throughout-much of the community that is currently testing the effects of chemicals. To solve this problem in the drug field, Senator Nelson has proposed a'large^ Government-run testing facility that would establish the safety 6V 20 21 dtu^B of all drugs submitted to the Food and Drug Administration^ for approval. Such a program', if feasible in the drug area, could easily be extended to pesticides, as well as the food additives and cosmetics. Another approach to the problem of how to separate the manufacturer from the researcher would be the establishment of a Govern- • mont referral board. Such a board would receive requests from the manufacturer that wishes to have a certain pesticide or food additive certified. This board would refer these requests—by a prearranged lottery system—to one of a series of independent laboratories previously certified by the board for such testing. The rules for such referrals could include a prohibition against any laboratory testing two or more chemicals from the same manufacturer in succession, thereby breaking up the economic dependence that some laboratories have on one manufacturer. The rules could also outline in some detail the methods that must be followed. And the laboratories could be inspected to insure that they do in fact have the facilities and the expertise to conduct the experiments necessary. Such a board could be useful in maintaining the economic independence of scientific research facilities as well as in upgrading the caliber of research done on environmental chemicals. It might also be of interest to this committee to examine the interlocking relationships of the various groups that make up the scientific establishment in the pesticide field. Currently the FDA runs three separate programs in the pesticide"field. It sets tolerances and then conducts investigations to insure that tolerances once set are not exceeded. Second, it conducts a total diet survey which is the major program to determine whether or not too much pesticide is finding its way into food. Third, it advises the Agriculture Department on pesticide safety. I have already indicated that the FDA program of advice to Agriculture is not particularly effective. Unfortunately, the other programs pursued by FDA are not much more effective. The total diet program has been seriously criticized by one former FDA science advisor—"former," apparently as a direct result of the criticism leveled at this program—because of its failures to be chemically relevant. Apparently claims were made for scientific test methods that were' scientifically untrue, operators were sloppy in the use of scientific procedures and equipment, and officials did not wish to alarm the public. As a result, it appears that the reports of the total diet study which strongly suggest that pesticides in food present little danger seem to be over optimistic. Presently the FDA is conducting a review of the program based on these criticisms. The tolerance setting program has been subject to the twin problems of limited scientific ability and information and a desire not to alarm. What is most important about the FDA programs in these fields, however, is that they tend to seek support for their effectiveness from the Association of Official Agricultural Chemists and the National Academy of Science's National Research C^fcil committee on pesticides. ^F Both of these groups are presented as independent evaluatprs of FDA methods and programs. In fact, however, there is a significant overlap between the groups. FDA officials are also officials of the AOAC, and sit on the NAS-NRC committee. In fact, FDA itself is • proud of their claim that it would be difficult to find any experts in pesticide tolerance and residue monitoring techniques outside of the agency. Interlocking relationships of this kind, however, are notorious for their ability to xmdermine scientific excellence. Without vigorous and challenging debate, little scientific advance takes place. The empirical evidence on pesticides suggests that a problem of major magnitude threatening the health and well-being of thousands of Americans may exist from exposure to pesticides. The FDA, supported by the AOAC and NAS-NRC tend to discount this evidence. It would seem that information about these groups and how they relate to each other would be useful in evaluating the meaning of their pronouncements. More critical examination of official reassurances about the effect of pesticides on the environment, an evaluation of the usefulness of either greater Federal testing for safety of pesticides or the establishment of a Government referral board and the shifting of the Government focus away from pesticide effectiveness and toward pesticide safety could begin to place the serious potential dangers from pesticides into the proper focus. Thank you for your consideration of these thoughts and the opportunity to appear before you. Senator HART. Gentlemen, thank you for expressing it so effectively. Shortly after you began your testimony, Mr. Wellforcl, we were joined by the Senator from Hawaii, Mr. Inouye, and the Senator from Tennessee, Mr. Baker. I have just a few questions. First, you talk about two potential disastoi-s in your testimony. The first is< one that may have already occurred, relating to past birth deformities. You suggest that these perhaps are attributable to the use of 2,4,5-T or related chemicals. The second I think is implicit in your suggestion that 2,4,5-T and 2,4-D and the dioxin contaminant of these and other chemicals may accumulate in the human tissue and so build up in each of us on sort of a regular basis. The end result of an accumulation of such highly toxic or teratogenic material is depressingly clear. Now, there are many—if past experience is our guide—who will criticize you, and perhaps me, for talking about these possibilities. They will suggest that it is scare tactics, or some other label. Anticipating that kind of reaction, what comment do you make? I'm sure you have been exposed to it before. Mr. WE^LFOIW. If people think we sound alarmist, they should talk to the scientists we talk to, because they are very alarmed. I think our main point is, there is just too much that we do not know about these herbicides, and, secondly, we're saying that the burden of proof should be placed on the mamifacturers to establish their safety. 22 TiffTm meantime, the public should not continue to bo the unwilling guinea pigs of their casual, and I think frequently unnecessary : use, • • •• There is substantial evidence that these herbicides are potentially dangerous. There will be many more tests to find but exactly hoty dangerous they are, and maybe in the end there will be a determiria'tion that the amount of exposure that the average American receives is nothing to worry about. ' •' But at this point, there is no evidence that we shouldn't be concerned, and in light of what^we feel that the use of these herbicides should bo restricted. Senator HART, In your prepared statement'—and I think as you summarized it you repeated it—you make reference to the Whiteside * articles in the New Yorker. If there is no objection, I would ask that thov be made apart of the record at this time. . ' _ Mr. WELI/FORD. I would also say, in further response to your question, that there has been a serious credibility gap on the part, of the Government in the whole herbicide case. The Bionetics report was withheld for a long time, even from scientists. Dr. DuBridge made a statement in October. The public was reassured that something was being done. And yet the statement was ignored. I think in this situation there'is a particular necessity for people to speak out. And that is one of the reasons we have appeared this morning. Senator HART. One specific reason I asked that the Whiteside articles go in is to acknowledge that they are responsible in major part for our hearings, The first of those two articles mentions the important part played in the release of the Bionetics report by Nader's Raiders. Though you were not introduced as such, can you respond by telling us the story of the involvement of your organization in the release of the information ? Mr. TURNER. Yes, Senator. That activity regarding the Bionetics paper revolved around the group that was investigating the activities of the Food and Drug Administration, of which I was the project director. Before going into the specific story, I would like to add one comment to the statement in answer to your first question. If anything, we have understated the scientific concern about the problems of chemicals in the environment, of which pesticides is one part. But what we have tried to talk about here is how to bring the legal system into some kind of meaningful response to what We admit to be a very unclear field of scientific endeavor. We don't know, nor do many other people, what, exactly, is happening in the chemical environment. How, then, are we as'a society, as the Congress and as people—how are we going to respond to the legal challenge that scientific uncertainty presents ? And the kinds of things we have been talking about today are designed to suggest answers to these questions. Concerning the Bionetics paper, to me it has represented since we first came across it, oh, sometime in August of last summer, one of the real tragedies and one of the sad parts of the kind of a job we 1 Sec p. 107. 23 try to do on a day to day basis. And that is, in our j^nties we constantly find scientists and regulatory officials in the Grovernment who have very important concerns which they would like to bring to the attention of someone, to have some kind of resolution made about the issue that they are concerned with. This was the case in the Bionetics paper. This, by the way, is not the only paper of that nature that we have run across in the course of the last year. We received the paper from two sources, basically, that were unhappy scientists who saw a very serious potential problem and said that no one would really listen to what was being said—internally, this is. We talked to the MEAK Commission and asked them if they were aware of the report, and they had been aware of the announcement and aware of the withdrawal, but had not seen the report. So we did then try to make it known to certain officials gradually, not knowing the full impact or not knowing the full meaning of it. It is not all that clear, although it does raise some serious problems. What was clear to us, though, were a number of scientists in Government who were concerned about a specific, problem that they wanted an answer to, or wanted attention focused on, and could not find any way to bring such attention to focus. Then we began to talk to people who we thought would raise the issue in the proper circles. That is essentially the way the paper began to emerge. I would like to emphasize this is not the only one of those situations which we discovered. Senator HART. You discovered other situations that bore on pesticides as potential dangers ? Mr. TURNER. There are some other problems on pesticides which we have alluded to which, are disturbing. The question of trying to bring to the attention of regulatory officials the problems of Folpet and Captan has been one that I have been concerned with for over 18 months, and have tried to alert various people in the regulatory agency, the Food and Drug Administration, to. The scientists in that agency are concerned about these two particular pesticides, and they are asking why are they on. the market? The first time that question was asked of me was in the summer of 1968, and wo are not approximately—well, almost 2 years later, still selling these same pesticides. Senator HART. In your testimony you have talked about the tests that several federal agencies have undertaken which clearly show in Mr. Wellford's prepared statement that even the purest 2,4,5-T still produces birth defects in test animals at significant levels. You go on to say that a recent test on 2,4-D confirmed that this herbicide which was very often mixed with 2,4,5-T, is teratogenic. I have been told, however, that the tests which have been conducted are merely preliminary and that although they suggest certain conclusions they cannot confirm them. Is that correct? In other words, as of now, we can't say that we know that the currently produced 2,4,5-T is teratogenic, can we ? Mr. WELIJTORD. Certainly, as far as the effect on .human beings, you are entirely right. There is no clear evidence—we haven't been able to find evidence through epidemiological surveys for reasons 24 described, that these herbicides are definitely a danger to/ hur beings that come in contact with them. " It is also true that there has not been time for the tests, which raise the suspicion to be checked and rechecked by many other scientists. But I think one thing that is certain is the tests do raise very serious questions, and we can take no comfort whatsoever from them, that these herbicides are not dangerous as they are not being used. Again, it is a question of burden of proof. Senator HART. You would turn it around and say—well, yes, you can't say they are harmful, but you can't say they aren't? Mr. WELLFORD. I would say the more recent tests have expended the burden of proof upon those who wish to prove that they are not harmful. Senator HART. You suggest, or mention, that the advice on registration that Food and Drug has given the Department of Agriculture frequently, or sometimes, or however you phrase it, has been ignored. Mr. TURNER. What was said was 100 percent of the time, Senator. Senator HART. Almost. Mr. TURNER. Almost. Senator HART. Almost 100 percent of the time. Isn't it the fact, however, that Food and Drug could make its advice stick by setting residual tolerances which a pesticide, if registered, would exceed ? Of course, that is a shorthand way, and perhaps arbitrary. Mr. TURNER. It is somewhat arbitrary. The problem is, the kind of recommendations that are made are directed to products that have some usefulness, and to arbitrarily say there should be no residue could seriously affect the use of these pesticides which—in themselves—may not be particularly dangerous. The kinds of things we have been talking about, on a number of occasions FDA recommended that certain marketed home use products that are used to kill rats should not be directed for use on bread crumbs or bread particles to be left where rats can get them, because then children can get them. On a number of occasions this particular recommendation has been ignored by Agriculture, and they have gone ahead with that kind of a direction. There, are others. For example, the Food and Drug Administration has recommended on a number of occasions that seeds which will find their way back into the food supply, after they have been treated with pesticide, be dyed with a color, so that you can see them—blue or red. This is the general procedure in this kind of pesticide program. But there are some products to which this is not done. When FDA on several occasions has said this should be done before there is any more marketing of that product, they have been rebuffed by" Agriculture on those particular products. Senator HART. In those situations where FDA has fixed a tolerance, do they have the facility to enforce adequately the tolerance that is established ? Mr. TURNER. It is my judgment that they do not. There are two ways that the residue problem is dealt with by FDA. One is by r by the gentrying to enforce established tolerances, and the otherjs eral survey called the total diet study, which I Approximately In the former program, the FDA conducts i^P 7/100ths of 1 percent of an investigation process. That is how many interstate shipments they examine for pesticides— seven-one hundredths of a percent. _ . The way that they do many of these investigations is by being alerted beforehand that there is some kind of problem with the particular shipment. The situation, as the statistics emerge from FDA —by the way, that is one-half of their announced program for the years of 1963 to 1966; they did one-half of what they thought they were going to do — shows that they find a 3 percent violation of tolerance in the seven hundredths of a percent investigation that they conduct, and this would indicate— if the samples are statistically sound— a minimum of 70,000 shipments going in interstate commerce with pesticide residues that are not detected each year. My judgment is that that is not enough to provide the kind of incentive to a person using pesticides, who must then ship his products, to be deterred from using too much. I also would like to point out that the second program, the total diet program, which is the one that is done to survey the entire food supply, also has varieous weaknesses. The way the program works is that the total diet of a 19-year-old boy— he is considered to be the major eater in the country — is bought at a supermarket at various areas in the country. Then it is in various ways broken down and tested to see how much pesticide exists in the 2 weeks diet. On the basis of this particular program, the Food and Drug Administration has assured the American people for several years tlmt there is no real problem of pesticide residue in food. When the science advisor began to look closely at this particular program, he came up with a series of very disturbing findings. There were no verified test methods for evaluating what, in fact, was being found by FDA when they conducted these tests. FDA had reported residue amounts that were lower than the error that was granted to the system. The system could find down to perhaps 10 parts per million without error, and they were finding residues as low as five parts, or below, per million. The science adviser in Baltimore made a detailed presentation to the agency and he shortly thereafter found that his contract for that year was not renewed. It was at that point that I found another one of these tragic cases in Government, and began talking to the Food and Drug Administration about this particular problem. And at our insistence and oitr negotiation, a hearing within the agency was impaneled and a discussion was held, and a review of the program was decided upon. As yet, I don't know what the results of their review are, but my ' feeling is we do not have good pesticide residue monitoring from that program either, at this point. Senator HART. I have indicated, and you have agreed, that for FDA simply to fix a tolerance level not to be exceeded as a device to keep an item off the market, or a particular use, would' be arbitrary. And you indicate that FDA's ability to ride herd on tolerances that are established is inadequate. 2u • Avind up, I tales it, by. making the case that the key to that we are seeking to insure some protection involves business of registration. You don't quite put it this way, but what you are telling us is that registration is presently under the control of the inappropriate agency. Mr. TURNER. That is my feeling, Senator. Senator HART. This is not necessarily to be critical of Agriculture, because, as you point out, they have always been pretty occupied, understandably, determining the potential usefulness of pesticides on bugs, weeds and what have you, and FDA more likely would be concerned with the problems of environmental contamination and safety. My last question. You have told us how the lable content is regulated. What agency would have jurisdiction to require more adequate packaging to avoid breakage? Maybe I should say what agency, if any ? Mr. WELLFORIX It is cleai'ly a public health problem, and to be perfectly frank, I do not know precisely who has direct jurisdiction over packaging. I would presume, though, that' the first responsibility is the pesticide regulation division of the Department of Agriculture, but Mr. Bayley will be testifying and you can ask him that question. Senator HAKT. That does not require a great deal of scientific interchange to figure that out. Mr. WELIYFORD. No, it does not. Senator HART. If we have given anybody the authority to fix the package so it docs not bust, they ought to be able to figure it out, and if we have not, we ought to fix the law. Senator Inouye? Senator INOUYE. Thank you very much, Mr. Chairman. Gentlemen, are you convinced that the presently available data on the potential dangers of 2,4,5-T and 2,4-D are sufficient to outlaw the iise and sale of these herbicides and pesticides? Mr. WELT/FOUD. I think that the data is sufficient to at the very least suspend their use until the tests that need to be undertaken to answer the questions we have raised have been performed. I think it is^ au unnecessary risk for the public to have these substances so widely used when we know so little about them. Senator INOUYE. In other words, you are suggesting that further and more intensive studies be made before a final decision be issued by the government? Mr. WELLFORD, No, I think that one decision can be taken by the Government right away, and that is to suspend the use of these products until more tests are done. I think to delay that, to wait until tests and retests are performed might unnecessarily expose people to hazards which you will eventually regret. That is the point. Senator INOUYE. I am convinced of the potential dangers of these pesticides and herbicides, but I cannot help recalling the recent cyclinnate scare that we had here in which our shelves were rid of all these canned fruit cocktails with cyclamate. Now recently I recall reading an article in which scientists indicated that in order to sull'er the dangers involved in the use of cyclamate, a child would . , ,. 27 have to consume about 120 servings of fruit cockta^pr day for 10 years. As a result, I believe cyclamate fruits can be sold on the shelf with a warning of some sort. That is why I asked this question. Are you convinced that the data are sufficient to suspend present use ? Mr. TURNER. I would like to comment on the analogy to the cyclamate situation. There has been a great deal of misinformation circulated about cyclamates, and it should be brought back into perspective. First of all, it is my belief that the present marketing of cyclamates as it is being conducted is a violation of the law, and we will proceed to undertake some kind of action to either bring that to the attention of Congress or to try to do something in the courts about it. In fact, when the Food and Drug Administration gave its warning about cyclamates in April of 1969, before they knew that there was any involvement with cancer, they announced that people, in order to be safe, should consume only a certain amount of cyclamate. That amount for children was two-thirds of a package of presweetened Kool-Aid. When the final cancer tests were done by Abbott Laboratories, the ones that Secretary Finch relied on to take the substance off the market, the National Academy of Sciences reported that applying the hundredfold safety measures that is used generally in food additive areas and applying the general methods that are used scientifically to deal with food additives would show certain levels of cyclamate to be safe. The amount that they were talking about as safe was the amount that would go into three cups of coffee in a day. Subsequent to that time, the Food and Drug Administration conducted experiments which showed that the same kind of cancer problem that was found by Abbott Laboratories was also found at FDA at one-sixth of the level of intake over an 18-week period rather than a 2-year period, thereby greatly increasing the amount of problem that a person is perhaps subjected to when he takes cyclamate. In addition to that, there is on file at the Food and Drug Administration or in the Health, Education, and Welfare Department a memorandum which specifically points out that there is a serious question that diabetics may be more susceptible to the dangers of cyclamate than other people. The question that you raise is very apropos to both of these chemicals. We cannot really say at this point that there is anything that we can prove that will definitely say people are going to get these results. The problem we have is how do we structure the legal system to deal with the problems of uncertain science. What we have done in the food additive area is to say that a food additive must be proven to bo safe before it goes into the food except for what was to be a very small loophole in the law. Unfortunately, as that law has been administered, that list has nearly become as large as the additives proven safe. In dealing with 2,4,5-T, we would like to provide, that is, those of us testifying today, would like to provide the same kind of standard • in the pesticide area that should be prevalent in the food additive 45-362 0—70 28 29 area, and that is until the substance is proven to be not harwRil it should not come on the market. Once it is on the market, if there are questions raised and they cannot be disposed of, then it should be removed from the market until those questions are disposed of. Senator INOTJYE. Your testimony indicates that you have done a lot of study in this area, and I commend you for this. I am certain you are aware that the State of Hawaii uses a lot of this herbicide. In the year 1968 to cover an area of 120,000 acres we used 197,000 pounds of 2,4-D and 6,000 pounds of 2,4,5-T. Obviously, it is an important part of our economy, because this constitutes'the major portion of our income. In your studies, have you come up with any substitute for 2,4,5-T or 2,4-D ? Mr. WELLFORD. There are other herbicides on the market. I am not really qualified to explain the virtues of one against the other as far as the uses that 2,4-D and 2,4,5-T now have. Again, I think that if there are not adequate substitutes that there probably could be with more research and investigation. I think that at the very least having these herbicides suspended for use in populated and residential areas could help bring that search about. Mr. TURNER. I think there is another point to make in regard to your question, and that is in much of this area of chemical environment we are presented with some minds set or misconceptions that lead us down wrong tracks. One of the problems with, using pesticides such as 2,4,5-T and 2,4-D and many others is that they are what we might call broad spectrum pesticides. They can be used on many different pests. If we are going to find alternatives for them we cannot think in terms of finding a pesticide that can do as much as these will. We have to think of what sort of pest is sought to be controlled and find a method for it. The Agriculture Department has found many methods of controlling problems, for example, alfalfa and cotton. These are nonchemical methods. Sterilization of pests is one of the approaches. It depends on what kind of particular thing you happen to be directing 'the pesticide at. There can be alternatives. I think we have not found them largely because we thought in terms of these massively effective chemicals rather than thinking in terms of how do we control this pest with the least possible strain on the population around it. Senator INOTJYE. Last week when I did my shopping to prepare myself for the spring, I bought some of the items on your desk there. Do you suggest that I get rid of them ? Mr. WELLFORO. I suggest you be careful how you get rid of _ them. I think one of the real problems with these substances is the difficulty of knowing how to dispose of them if you decide you do not want to use them. .You are not supposed to put them in a trash can; you are not supposed to bury them; you cannot put them down the sink. I think if you call the various public health authorities to try to find out the best way to dispose of them, they eventually tell you, take them out to an incinerator on the outskirts of Washington, but they do not really have any other answer. I think that problem indicates the toxicity olWe substances that we are dealing with, and in direct answer to your question, I would not apply them to my lawn if there was any chance that pregnant women or small children could come into contact with them. Senator INOUYE. Thank you very much. Thank you, Mr. Chairman. Senator HART. Senator Baker? Senator BAKER. Thank you, Mr. Chairman. I must say I, too, commend you for a rather extensive and well prepared presentation this morning on Mr. Wellford's part and Mr. Turner's part. There are two or three things that occur to me about which I would like to inquire that relate to Senator Inouye's concern, and that is what do we do if we discard these pesticides and herbicides? I wonder, for instance, if there is a study or if you have any statistical evidence on what the economic loss would be, say, to the State of Hawaii in the case of a suspension of 2,4,5-T and 2,4-D? Before you answer, do not assume I am arguing against such suspension. I am rather concerned about a complex life, and to be facetious for a moment, none of us really will live through it. Is there some way to judge the relative good or the relative bad that flows from the use or nonuse of any of these pesticides? Mr. WELLFORD. One quick point on that. There is a report commissioned by the Defense Department which is on the ecological effects of continued herbicide use, and in that report they, state that in many cases the chemical weed clearance methods are often no more expensive and just as good as the use of herbicides. • It has been my experience that herbicides or their massive and popular use reflects the American penchant for technological gadgetry and marvels and perhaps we could find many more ordinary ways to get the same job clone. Senator BAKER. I agree with that. There are other ways. Take the extreme case of the mosquito and malaria. Clearly there must be some way to control the breeding grounds of the mosquitoes. It is done in some areas by raising and lowering the lake levels. It is done in other areas only by the use of pesticides. . Once again there is a trade-off. In those cases and in remote areas of Vietnam or the United States, it might be demonstrably better to run whatever risk _there is of mutagenic danger in order to dispose of the more immediate and more imperiling danger of malaria. Is there any sort of study ? Is there any sort of weighing of these equities by your group ? Mr. TURNER. There is not anything done by our group and I do not know if there has been anything done by any other group. Our approach to this problem is this, and I think it bears directly on this question: I am concerned about the cyclamate case, for example, because of the tremendous economic impact that it had, but that economic impact came largely from the delay and the lack of effective action over the period of 4 or 5 years when cyclamate was developing. What concerns us in the area of 2,4,5-T, is that it was announced its use would be curtailed. We in the public were under the impres- , 30 sion it was being curtailed. We began to look at what was happening, and it was not being curtailed. We have urged specifically that the use in the home, in and around the home be the first one to be specifically curtailed immediately. My feeling is that since we have known for approximately 18 months that as a sprayed pesticide or herbicide it (2,4,5-T) has been a problem,—that since someone has known for probably the last 15 years that it did present a problem, action should have already been taken. The workers at Dow Avere having serious occupational problems from the manufacture of the substance. Now, admittedly when a substance appears in the public eye for the first time it does create the kind of crisis situation where you perhaps must balance economics versus the potential harm from the substance. However, what I am concerned about is how we prevent that kind of crisis situation from developing on other substances and on this particular one. In my area, which is more directed at the food chemicals, I would be more than willing to say that we should have an interim period to solve the kinds of problems you are suggesting if there was any kind of guarantee that that interim period would be lived up to by the producers of these chemicals. The problem is in any area where the interim period has been allowed in the food area it has been used to erode the provisions of the law, so at the end of the interim period there is not any kind of authority or force to keep the product in control. That is what concerns me about trying to accommodate the economic situations which you are raising, although I consider them to be rather serious. Senator BAKER. They are not only economic conditions. What I am I referring to is health. I am asking if there has been a study by anyone in your group or any other group on the relative merit of the discontinuance of the use of pesticides on the one hand and the prevention of mosquitoes and malaria on the other. Mr. TURNER. For example, you mentioned the mosquito problem. I notice that one major company has just developed a nontoxic. method of controlling mosquitoes. This would be an example. I think with the proper kind of direction and the proper kind of focus, these alternatives can be developed. The reason I was talking about economic importance, is the real impact of these particular pesticides we are talking about are economic. I do not believe they have use as a health control method. Senator BAKER. Can you tell me whether FDA or anyone else is conducting any extensive research on substitute agents for pest controls? Mr. TURNER. As far as I know they are not, they are not conducting extensive research, but Agriculture does have a research program on this subject, but I do not believe it is extensive. Senator BAKER. Are you familiar with the plan by the Forest Service and, of a]] people, by the Atomic'Energy'Commission to,, develop sterilization techniques for certain pests that attack forests? Mr. TURNER. Presently, as I understand the Agriculture Department, it is dealing with this area and it believes at present they are 31 able to control about ten percent of the pest problelWvith those kinds of methods. Senator BAKER. To control completely ten percent of the pests rather than ten percent control ? Mr. TURNER. Eight. Senator BAKER. Would it seem to you that further research in these fields might very well produce acceptable substitutes, acceptable as we know them by these standards? I freely predict that 10 years from now they will not be the standards? Mr. TURNER. That is right. Agriculture is working on research methods whereby they can use much smaller amounts of pesticides to achieve the same results that are achieved by the very large use of chemical pesticides. In some cases more than a million or so pounds of 2,4-D is used on certain submerged weed areas to control the weeds per acre. This is an incredible amount of pesticide use. Now they are down in some cases to using chemicals to one one hundredth of an ounce on an acre. This kind of a balance is going to go a long way toward controlling some of the environmental hazards that we have. _ There are experiments being conducted at the University of Georgia which indicate that perhaps as much as 99 percent of all sprayed pesticides come out of nozzles in droplets which are so large that they are useless for this purpose and they are absorbed by the environment. Senator BAKER. Are you familiar with the use of underground watering for microporous piping? Mr. TURNER. No, I am not. Senator BAKER. That, of course, would stop air contamination by pesticides and herbicides and it would increase the concentrations and the effectiveness and eliminate the resin problem. Mr. TURNER. Right. Senator BAKER. I really commend that to further study. Senator HART. Thank you, Mr. Baker. I think the committee is fortunate to have Senator Baker on it. He either has done homework or somehow or other is involved in the field. Mr. Bickwit? Mr. BICKWIT. I would just like to clear up one point on my own mind. On several occasions in your testimony you have complained that Dr. DuBridge's statement made in October of 1969 was not complied with. However, in other parts of your testimony you have suggested that mere compliance with that statement would not be ' sufficient in your view. Am I right in understanding that you feel that Dr. DuBridge's statement does not go far enough? Mr. WELLFORD. Absolutely. I think its biggest gap is the fact it does not discuss pesticides which are used in residential lawns and gardens. I think there he was under the misapprehension that 2,4,5-T was not widely used for this purpose. In fact it is. I suspect if ho had known it, he would have included that in his1 statement. Senator HART, Gentlemen, thank you very much, for a .very helpful presentation. You have raised some questions that clearly will have to be resolved. _ Next, we welcome Ned Bayley, the Director of Science and Education of the Department of Agriculture. 32 33 'EMENT OF DR. NED D. BAYLEY, DIRECTOR OF SCIENCE A EDUCATION, DEPARTMENT OF AGRICULTURE; ACCOMPANIED BY DR. T. C. BYERLY, ASSISTANT DIRECTOR OF SCIENCE AND ' EDUCATION Dr. BAYLEY. I will very briefly refer to that. liRrux of the problem was that there was an interagency agreement for resolving differences in regard to registration. There was a procedure within the agreement of bringing differences to the attention of the three Secretaries involved. Unfortunately, however, over the years this agreement was in existence, not one of the departments ever used this procedure to resolve their differences. We in the Department of Agriculture must share the major responsibility for not getting the differences resolved primarily because the enforcement of FIFBA was primarily our responsibility. But I am glad to say that since that time, with the initiative of Secretary Hardm and Secretary Finch and Secretary Hickel, there has been a new agreement worked out between the departments. This agreement specifically provides the basis whereby differences in judgments regarding pesticide regulation can be brought up through the decisionmaking procedure and the three Secretaries can share in this as needed. We believe this is a sound basis for increasing the interdepartmental relationships and providing a basis for all three departments to have a rightful input into this. I think you are also acquainted Avith the fact Senator HART. Doctor, if you are going to leave that new agreement, I would like to ask one question. In the event of disagreement, when the three Secretaries' attention is invited to the competing claims, is the decision made by majority vote, or does the Secretary of Agriculture retain the final voice ? Dr. BAYLEY. It is my understanding that they will pursue the disagreement until they agree. The Secretary of Agriculture does retain the final voice according to the law, however. We believe that based on the way we are operating today this procedure can be effective. Senator HART. A meeting in Paris ? Dr. BAYLEY. Well, we are not dealing with that. I think Aye recognize that three Cabinet officers have the public interest in mind when they get together and can make a decision along these lines. Senator HART. Just to push you a little harder on it, and I suppose this is academic at the moment since no such dispute has yet reached the three Secretaries ? Dr. BAYLEY. This is correct. Senator HART. If one does get there, it Avill involve the tricky balance that Senator Baker was talking about, the economic claims, the public health claims, and the environmental concerns. HEW Avill tend, I assume, to emphasize the health factor. Is Interior the third department ? Dr. BAYLEY. Yes. Senator HART. They would I suppose, be concerned principally with effects on fish and wildlife, and Agriculture would think primarily of the utility to the agricultural economy. To put it harshly, why shouldn't the fellow who says it lias not yet been established as safe for humans have the ultimate vote and voice ? Dr. BAYLEY. Mr. Chairman, I am Ned Bayley, Director of Science and Education, Office of the Secretary, Department of Agriculture. I have with me Dr. T. C. Byerly, Assistant Director of Science and Education. Before I proceed with the formal statement, I Avould like to respond to some extent to the information Avhich has .been presented already this morning. Senator HART. Let me make it easier. Let me encourage you to do it, and any succeeding Avitnesses, too. To make the record as useful as possible, we would welcome exchanges in the nature of reply. Dr. BAYLEY. I appreciate in doing this I am putting myself in an impromptu position and, therefore, would appreciate the privilege to provide to the committee, for the record if they desire, fuller statements regarding the activities of the Department of Agriculture in regard to pesticides. Senator HART. Very well. Dr. BAYLEY. I testified before this committee previously regarding the broad policies and positions of the Department in the pesticide area, and stated that we recognize that all pesticides are economic poisons. They are only one group of the tremendous number of economic poisons which we use for a large number of useful reasons, not only for economic purposes but also to take care of public health. We recognize also, as part of our civilization and as part of the standard of living and the food supply that we already have, that Avithout these economic poisons and their judicious use, AVC Avould be in a very serious situation from the standpoint of our ability to produce food and fiber for this country. Now, I also want to point out very briefly the references to the activities of the Department of Agriculture regarding the registration of pesticides. I will be the first to agree that there have been some problems in regard to these registration procedures and I will be the first to agree that we haven't resolved all of them. We have, hoAvever, particularly during the past year, taken a number of steps toAvards eliminating some of the complexities and bureaucratic difficulties which have existed in the area of registration. The references which have been made here this morning primarily reflect the relationship among the departments that did exist, but, I think I am safe in saying, do not exist now. Senator HART. I remember in those earlier hearings we discussed this problem. Dr. BAYLEY, Yes, AVB did. Senator HART. And I had the impression that there was a transition period. 34 35 Jr. BAYLEY. ^ May I say this, that from the standpoint of the Department of Agriculture, we recognize that issues involving human health should have priority over all other issues. The reference which was made earlier that the emphasis in USDA had been primarily on effectiveness is not only incorrect regarding the past but it is utterly incorrect regarding our position now. Senator HAUT. Whatever the past, I would hope that human health does have the overriding concern of three or any other nnmbers_of men that meet on this kind of claim. I interrupted you. Dr. BAYLEY. Surely. With those preliminary comments, I will be glad to turn to the issue of 2,4,5-T and the facts as we see them at the present time. The _ herbicide 2,4,5-T has been recognized as the most effective herbicide registered for use for control of certain weeds and brush species for^more than 20 years. About four-fifths of the domestic use .of 2,4,5-T is.for nonfarm use, the largest such use being for control of brush on rights-of-way. It is also used extensively to control brush on forest lands and certain weeds in turf. 2,4,5-T has been used in the production of fruit crops, cereal grains, and sugarcane. It is the most effective herbicide for control of brush on several million acres of rangeland in the Southwestern United States. 2,4,5-T is degraded in the environment within a few months after application so that residues do not persist from one season to the next. Residues on foods are unusual. Among 5,300 food samples analyzed by FDA for 2,4,5-T during the past 4 years, 25 were reported to contain trace amounts; i.e., amounts less than the 0.1 p.p.m. limit of accuracy of present analytical procedures for foods. Two samples , showed residues of 0.19 and 0.29 p.p.m., respectively. No finite tolerance has been established for 2,4,5-T in food. In the absence of such tolerances, any detectable amount-of 2,4,5-T in food would make such food subject to seizure if found in the channels of interstate commerce, From the data cited above Senator PERCY. Do I understand your statement to mean, Doctor, that in the absence of the establishment of human tolerances for 2,4,5-T, it is the present policy of the Department of Agriculture to seize any shipments that show any measurable trace of 2,4,5-T on food shelves ? Dr. BAYLEY. It is the responsibility of the Food and Drug Administration to enforce the procedures and make the seizures. Senator PERCY, I understood you to say there are no tolerances established; therefore in the absence of any established human tolerances for 2,4,5-T, it is the present operation of the U.S. Department of Agriculture to seize any food stuffs that contain any measurable trace amounts of 2,4,5-T. Dr. BAYLEY. It is the present obligation of the Food and Drug Administration to do so. Senator PERCY. Thank you very much. Dr. BAYLEY. From the data cited above, it is apparent that contamination of food with 2,4,5-T is very infrequent and then only at very low levels. There is current concern over the continued use of 2,4,5-T arising from the report of a research study completed under contract by the National Cancer Institute by Bionetics, Inc. This sma\ sWray was based on a commercial lot of 2,4,5-T acquired for the study in 1965. It was fed to pregnant mice and rats. Many of their developing young had birth defects. After review of this information and after consultation with Federal agencies concerned, Dr. Lee A. DuBridge, the President's Science Adviser, announced on October 29, 1969, a coordinated series of actions being taken by those agencies with respect to the use of 2,4,5-T. Among them was the announcement that: "The Department of Agriculture will cancel registrations of 2,4,5-T for use on food crops effective January 1, 1970, unless by that time the Food and Drug Administration has found a basis for establishing a safe legal tolcranco in and on foods." USDA was informed in January that the lot of 2,4,5-T used in the Bionetics study contained significant amounts of a highly toxic contaminant^ tetrachlorodibenzo paradioxin. The Department was further informed that lots of 2,4,5-T of current and recent manufacture were reported to contain less than 1 p.p.m. of this contaminant in contrast to the 27 p.p.m. reported for the lot used in the Bionetic study. Extensive studies are underway to determine whether 2,4,5-T is itself teratogenic. Preliminary reports are consistent with the hypothesis that the teratogenic results reported in the Bionetics study were due to the contaminant dioxins or to interactions of such contaminants with the 2,4,5-T rather than to 2,4,5-T per se. The Department announced on February 6 that it would undertake examination of 2,4,5-T and 17 related compounds registered for pesticidal use to determine whether or not they are contaminated with dioxins. Preliminary results on 2,4,5-T show that those lots examined of current manufacture and those now in channels of trade gave the following results—I can summarize these quickly— the amounts ranged from a trace to 2.9 parts per million, and they were conducted both by the Department of Agriculture and the Food and Drug Administration. (The table follows:) TABLE 1.—AMOUNTS OFTCDD FOUND IN COMMERCIAL 2,4,5-T BY TWO METHODS TCDD content p.p.m.' Sample 2.4, 5-T 2 4 S-T 2,4,5-T 2,4,5-T« Manufacturer -. Dow .. -- Dow.. .. Lot Grade i 120110 07-020 X-17394-21-5 MM-1 20449 TO TG TG TG Collected 2/70 2/70 2/70 2/70 FDA USDA trace 1.1 . . NO.' .48, . 0.07 2.9 N.D .47-. 52 i TG>=>Technical grade. »TCDD Relers to the 2,3,7,8-tetrachloro-dibeiuo-p-dioxin (TCDD), 'N.D.^Levels of TCDD are below the limits of detection or below 0.05 p.p.m. 1 Sample supplied by Dow as a reference check and reported to contain about 0.5 p.p.m. TDCD. These data are preliminary and are obtained from first drafts of methods developed by chemists in the Crops Research Division of the USDA and in the Pesticide Chemistry and Toxicology Division^ of the FDA. The dioxin values refer only to the 2,3,7,8-tetrachloro- 37 nzo-p-dioxins (TCDD) and do not indicate levels of other d dioxins (containing 5, 6, 7, or 8 chlorines) in the 2, samples. In view of all the information now available, we have not found that registered use of 2,4,5-T without a finite tolerance on food crops constitutes a hazard requiring cancellation or suspension of such registered uses. There has been and is concern over the ecological effects of 2,4,5-T used as a defoliant in Vietnam. Dr. Fred Tschirley, Assistant Chief of our Crops Protection Research Branch, has reported the results of his examination of areas treated in Vietnam. He has reported no evidence of irreversible ecological damage. Allegations that defoliation will lead to extensive laterization of Vietnamese soils, that Mangrove areas will not recover, that fish production in wetland areas will be reduced were not verified. Dr. Tschirley also headed a team of scientists who investigated allegations of injury to humans and animals due to herbicide treatment for control of Chapparal by the Forest Service on the Tonto National Forest near Globe, Ariz. They found that apparent damage consisted of damage to susceptible plants near the treated area from drift of the herbicides used. The alleged injuries to a duck and a goat were found to be groundless. Human illnesses were those expected in a normal population with the possible exception of one man with skin irritation on his eyelids. Clinical chemistry on specimens obtained during the investigation is in process. Mr. Chairman, that is the completion of my formal statement. We primarily presented it to provide you with the latest findings that we have in the particular area. Senator HART. Thank you very much, Doctor. To summarize with respect to the sequence of events on the DuBridge announcement of October 29,1969, the Department did not in fact cleregister 2,4,5-T as DuBridge indicated would occur unless these affirmed findings came along. But you tell ns your action was based upon information that the tests by Bionetics used samples that contained the contaminant dioxin, and that the current production of that product was free of the dioxin; is that right? Dr. BAYLEY. Not completely free. The dioxin was at a sufficiently low level that Ave believed that Senator HART. That the test material had sufficiently more dioxin than the normal production amount ? Dr. BAYLEY. Yes. Senator HART. Are you aware that the preliminary results of tests conducted by Food and Drug, Dow, and by the National Instititute of Dental Research and by the National Institute of Environmental Health Scientists all indicate that 2,4,5-T contaminated with no more dioxin than is found in the currently produced 2,4,5-T is terato genie? Dr. Bayley. We arc fully aware of this. The critical facts in regard to those experiments is that those low level dioxin contaminated 2,4,5-T samples were fed at sufficiently high dosages that they would be comparable to the dosages used for the 27 parts per million or nearly so. _ Therefore, 'we do not believe this in any way changes the hypothesis that the IOAV level of dioxin is safe. Senator HART. You say in your statement "Shouldi^ teratogenic nature of 2,4,5-T be confirmed, registration for usd^P food crops will be canceled." I am attempting to establish what will confirm it. What events do you look to to determine whether these preliminary indications which you say resulted from the contamination in fact did? Is • there something in particular that you look to ? Dr. BAYLEY. Yes, the important considerations here are the usages for which the 2,4,5-T are permitted or which in actual practice are carried out. The difference between the possibility of teratogenicity of the contaminate and the teratogenicity of the material that is used in the field is based on the rates of application, the losses which occur. All these effect the possibility of contamination of human beings. Incidentally, my advice is that the one part per million level is at least tenfold below what they would consider a safe level in terms of allowances. In other words, that is the safety margin in this estimate. Senator HART. In other words, there would have to be a finding of 10 times mo re? Dr. BAYLEY. That is what I am told, yes. This is a statement based on scientific information provided to me. Senator HART. Do you have any opinion as to whether Food and Drug might set a safe tolerance level in food ? Dr. Bayley. The action we have taken is to extend the time in which information can be provided or application made with Food and Drug Administration regarding the establishment of tolerances on food. I would not in any way want to prejudge .what their actions should be, because it should be based on the data provided. If I may elaborate on that, a petition was filed with the Food and Drug Administration in December of 1967 requesting the establishment of tolerances of 0.2 parts per million for residues of 2,4,5-T on apples, barley, blueberries, corn, oats, rye, sugarcane, and wheat. Those were the only crops to which that petition would apply. The petitioner withdrew his petition on December 29, 1969, as provided under the pesticide regulations. We, have extended to December 1970 the opportunity for him to provide the data needed to reach a decision on this. There is one thing I think is important here, and that is the earlier reference to the concept of first, the burden of proof, and secondly, that we should not believe their data. We have to watch out for this paradox. Wo in the Department of Agriculture, as you know, with the cooperation of the Food and Drug Administration, are not simply accepting the proof from industry in these cases. We are going out to obtain samples and testing them ourselves in order to verify the kind of information that is coming in. Senator HART. I am not sure it is a paradox to say that the burden of proof is on the fellow that wants to expose the public to a product and some saying you cannot trust his data. They are two separate problems. Dr. BAYLEY. I recognize that. Senator HART. Let us be assured that the data is reliable and octive, and let us insist that the burden of proof may be on, low who may or may not be proposing the introduction of' imical. Dr. BAYLEY. I suspect I am sensitive to this because some people challenge why we test products. We think it should be done in the public interest when it is needed. Senator HART. On the matter of the dioxin that was found to exist in the samples in a much higher percentage than the normal production thereof i Dr. BAYLEY. You mean the samples that the Bionectics group had ? Senator HART. Yes, that the Bionetics group used. That was the reason, was it not, that led you not to follow through on the DuBridge pronouncement of October? Is that the meat and potatoes of it? Dr. BAYLEY. This is an often misunderstood situation. I think it is important to realize that the date we chose in regard to the possibility of taking such action was chosen because the Food and Drug Administration had agreed to complete their action on the petition by that time. When they had not completed their action, I wrote Dr. lioger Egeberg a letter on January 7, asking for a statement regarding the status of their considerations on the petition. On January 21, I received a response indicating that they had further data which changed the position in regard to the need for immediate cancellation. We will be glad to provide these letters for the record. Senator PlAirr. I think it may be helpful. (Theletters follow:) f JANUARY 7, 1970. DR. ROOER O. KOEDEKO, Assistant Secretary for Health and Scientific Affairs, Office of the Secretary, Department of Health, Education, and Welfare, Washington, D.G. DEAR DR. EGEBEUQ : On December 13, 19C7, a petition was filed with the Food and Drug Administration to establish tolerances for 2,4,5,T on specific food crops. In accordance with the interdepartmental agreement reached in Dr. DuJiridge's office on October 29, 1909, we announced that we would issue notice of cancellation of the registered uses of 2,4,5,T on these crops unless the Food and Drug Administration found a basis for establishing tolerances by January 1, 1970. This date was chosen because the Food and Drug Administration agreed to complete action on the petition by that time. We would appreciate receiving without delay a statement from the Food and Drug Administration regarding the status of their considerations on the petition in order that we may take appropriate action. This request is made in accordance with our mutual interest to take responsible action on this matter and also in full cognizance of the exchange of letters between Secretary Hardin and Secretary Finch regarding public health responsibilities in pesticide registration. Sincerely, NED D. BAYLEY, Director, Science and Education. SUBOEON GENEBAL OF THE PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE, Washington, D.O., January 21, 1910. Du. NED N. BAYLEY, Director of Science and Education, Office of the Secretary, U.S. Department of Agriculture, Washington, D.G. DEAR Dn. BAYLEY : In reply to your letter of January 7, 1970, inquiring as to the status of Pesticides Petition 8F06G9 (2,4,5-T), submitted by the National Agricultural Chemicals Association, the following is the current status. Wo liuve been advised by Dow Chemical Company: (1) That a sample of the 2,4,5,-T herbicide used in the Bion4k study contnlned 27±8 ppm of a highly active (biologically) contaminalHtetrachlordiiH'iizo-pftni-dioxiii. (This has been confirmed by the National~Institute for Knvlronmentnl Health Sciences.) (2) That tills material produced serious inflammation in rabbit ear tests and Mint tho presence of the contaminant had been confirmed by chromatography. (8) That standard production 2,4,5-T contains less than 1 ppm of this contninlmukt and does not produce inflammation in the rabbit ear test. (4) That Sprague-Dawley female rats as clams have been under test in their Inltonifory at Zionsville, Indiana, at five levels (25 rats each) of the standard production line material. They came to term January 7, 8 and 9, 1970, at wlilcli time they were killed by carbon dioxide inhalation and fetuses were removed by cosarean section and subjected to standard examinations for malformations and anatomical anomalies of various kinds; further examination will Include clearing, staining and histopathologie procedures. Hr. Howard L. Richardson, Chief, Pathology Branch, FDA, participated in tlio evaluation which related to full-term rat embryos subjected to 2,4,5-T during gestation, as well as a number of full-term rabbit embryos. He reports Unit no signs of malformations were found in gross and microscopic dissection «f MICKC embryos, but that histologic examinations are yet to be made. PersonIH'I from the National Institute of Environmental Health Sciences were unable to participate, but will be involved in the examination of this and other informiitlonnl material. Further characterization of the contaminant tetrachlordibenzo-para-dioxin is currently under way and Dr. Leo Friedman will welcome participation by your rw'itrch start in this effort, We would point out the resemblance if not the "practical identity" of the tetrachlordibenzo-para-dioxin with the "chick edema fiictor," Tl>is substance is of extremely high toxicity to all species of animals Mint have been exposed, and until now, its source in contaminated fatty materials has been a mystery. Considering the- imminence of the availability of this additional information and the legitimate question as to whether or not the teratology reported by Mic Bionetics study was due to the 2,4,5-T or to the contaminant, we have elected to delay action on the petition for a few more days. As you know, the petitioner had requested, on December 5, 1969, an extension of 3 additional months. We will advise you of our decision as soon as our scientific staff assays the results of this nearly completed test and considers them together with the rtwilts of other current research on 2,4,5-T at the National Institute for Dt'iitnl Research and in the Food and Drug Administration. Thus far, no one has confirmed the Bionetics results although 2,4,5-T (with 27±8 ppm contniniunnt) has been found to be embryotoxic. Sincerely yours, JESSE L. STEINFELD, M.D., Surgeon General. Senator HAKT. At the beginning of your testimony you say residues on food are unusual. AVhen they do occur, are they the result of unauthorized use of 2,4,5-T or authorized use, or both? Dr. BAYLEY. Mr. Chairman, I am going to ask Dr. Byerly to respond to that. Dr. BYEHLY. Sir, one of the two significant values, 0.19, was on milk, and the other was on sugar beets. As far as sugar beet use, I would have to verify whether or not there is a registered use on sugar beets. There is on sugarcane. There is certainly none on milk. This would be unauthorized use in the case of milk, certainly. Senator HAHT. Is it authorized for use on grass? Dr. BAYLKY. Yes, sir. Senator HART. It is? 40 41 r. BAYLKY. It is authorized for use on grass. enator HART. And clearly not on milk? Dr. BYERLY. No. Senator HART. _ What do you say to the suggestion that if you know that there is a regular unauthorized use of a pesticide go'ing on you ought not permit it to be registered? How can you register a pesticide even for safe usage when regularly it is used in an unsafe, unauthorized manner ? Dr. BAYLEY. The law provides, and I will not pretend to quote it exactly, that if despite the registration restrictions, including use, the Department finds there is injury to people and to the environment or desirable environmental organisms, that we can consider tins as misbranded and cancel the registration. So the pattern of the enforcement of that part of the law is for a surveillance program to determine the extent to which their unauthorized. uses are providing injury and then we take action. _ Phis is very clearly shown in the action Ave took recently involving a mercurial seed treatment program compound, It was this type of action, where unauthorized use was creating an in-jury and we immediately suspended it. Senator HART. You say the use is authorized on grass, not milk. Uould not cows cat grass, thus producing residues in our milk. Dr. BAYLEY. This is based on the recommendations for use and also based on the degradation properties of 2,4,5-T itself. Good practice would require withholding the grazing of cows from these pastures until such time as we can be sure there will be no residue in the animal product. The widespread use of this as herbicide on ranges and pastures indicates that farmers are following these practices with the possible one exception that we know of at this point. Senator HART. What information can you add to this record substantiating the statement that 2,4,5-T degrades in a matter of— how did you put it? Dr. BAYIJCY. We will be glad to supply for the record the scientinc mi-ormation indicating the degradation time of 2,4,5-T as well' as the circumstances under which this will vary. Senator HART. That will be printed in the record. (The information follows:) There is a voluminous body of published literature on the degradation and persistence of herbicides. Enclosed are five reprints availaWe to u , tha deal 2 4 5 T F r a m re K ,°a^ n i t h we ' 'recommend ' - ° ° following comprehensive fess: on of 1,45-T and other pesticides, the publications of Her- of (from: Chemical Fallout-Current Research on Persistent Pesticides Ed. by MorSTW. Miller & G G Berg. Charles C. Thomas, Publisher. 1969] Soil Persistence of 2,4,5-T The persistence of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), when applied at recommended rates (0.25 to 8 Ib/A) is 4 to 5 months as compared to about 1 month for 2,4-dichlorophenoxyacetic acid (2,4-D) »i shown in the figure below. FHENOXY. TOIUIDINE ond NITRIU HERBICIDES DcRose (3) found that a 3 Ib/A field application of 2,4,5-T was no longer phytotoxic to soybeans 3 months later, However, rates of 10 and 20 Ib/A remained highly phytotoxic after 3 months. Newman, et al (4) found that 2,4,5-T lost its phytotoxicity after 4 months when •pplicd at rates up to 26 Ib/A. Burger, ct al (1) found a similar loss of phytotoxicity to alfalfa 4 months after the application of 25 ppw (50 Ib/A). However, these were both laboratory studies in which the soils were maintained moist and warm, two conditions that facilitate the microbial inactivation of phenoxyacetic acid herbicides. The 6 month persistence of 2,4,5-T in soil, DeRosc and Newman (3), appears to be one of the largest persistences reported. Microorganisms are primarily responsible for degrading phenoxyacetic acid herbicides in soils. The kinetics of 2,4-D, 2-methyl-4-chlorophcnoxyacetic acid (MCPA), and 2,4,5-T detoxification in soil-perfusion experiments were exactly what would be expected if microorganisms were tho detoxicating agents (4); in addition, detoxication was blocked by the bacterial inhibitor sodium azide. The bacteria and actinomycetes responsible for degrading phenoxyacetic acids are shown in Tables 1-2 (4). Tho metabolism of the phenoxyacetic acid herbicides has been studied extensively (4). There appear to be two major pathways of degradation, i.e., via a hydroxyphcnoxyncetic acid intermediate and degradation via the corresponding phenol. Some of the important steps in microbial BCtnbolism of representative phenoxyacetic acids arc shown in Figures »-6 (4). 43 42 TABLE 1-2 CHjCOOH Bacteria and Aclinomycclcs which Degrade Phouoxyaeclic Acids o JL.a Phcnoxyacclic uoids niclabolucd 1 1 1 '-3 •£ | i * References 1Ml.lI H UiMiBHjj i i'HIHI U ^ U 4 ^ N" ^ « «i ^ 4 M' 1 i *** ^ a a \ (17) (18) \ CHjCOOH * i r f Achrt)iii(>baftcr ip. s 5-/.W * 57 5nitnias sp. PieiKhittflHtis sp. Myeopluna sp. AcltruHiabftcter sp. HOY^^y0 A* 1 Organism |^Va ^. * g K + * 3 +' * £ g OH JL ? QH V\ ' OH COOH °3. ^ >o (- a do ' a (ID a («> a c1- (14) CHjCOOH F^'rbwn f\ rfresrintin, Cor)iirbaclcrittin sp. organism Ariltiobaeitrftltibt/ortnts iBatttriiiiitfloittforme) Arilirobdctcr sp. Spororylupltfiga • 99 P-^ +* 78,79 +' 57,95 95,96,100 + + + +• +* 0 OH y § w + + a o (15) ' (M) fff/VVCfO/fl ( /"/oi obfiftcritiin oi/ittitik) AclmORiyjclCt NocardM Sp. Streptomycin 9 5J 101 SubjiMtc in enrichment and isolation media. + +. + 4- • l;i|.. 1-6. Microbial dcyradation of phcnoxyacclic acids via the corr^ponding phenols. ( \] •> \-D (17) douradaiion via 2,-t-diclilorophonol (18), ?,5-dkl\lor.>GiUxliol (19), and .,-chloroimiconic acid (20); (11) 2,4-D dcara^lalion via 2.4-diehlorophcnol, -1-chloroc.iu-ch..l (2.1), and /3-chloronuiconic acid (2-1); (C) -1-CI'A (21) dcgradaiion via .1-cliloroplivno! (22), 4. l}:2 t i(r5-T), and a 2:2:1 mixture of (he inooctyl ester* of 2,4-n:2,4,5.T:pliloram at 6.7, 26,9, and 16.8 kg/ha (6, 24, and 15 lb/A) respectively, were applied as foliar ipravs (o control guava (Psldnttn gitajtwa 1-), Six crop i tpftltR were planted in soil collected from each plot, 1, 2, fl, GI/J, 9i/a, nnd i3iA month* alter treatment, to detect herbicide residues nnd lo determine crop tolerance. Corn, loighum, wheat, rice and cotton could be grown without mlurtion in (rcsh weight ai early as S monthi after application. Soybeans were the most susceptible crop to herbicide rcnldues. Additional index words: herbicide reilduei, plcloram, 2,4-D, 2,4,5-T, guava. of 2,4-diclilorophenoxyacetic A COMBINATION acid (2,4-D) and 2,4,5-tiichlorophenoxyacetic acid (2,4,5-T) is an established herbicide for woody plant control (2,4,5). Herbicide 4-amino-3,5,6-trjthlorupicolinic acid (picloram) and combinations of pidoram with 2,4,5-T show promise for control of woody species which exhibit resistance to phenoxy herbicides (1), Since high herbicide dosages are re<)uiicj)llcmloa to date ot iniiiplliig Mil (Dec. 6, 1007), 679 iwiimcd (mm H'EKII SKIKKVH Vol. 16, Nit. I. January, l!W8 Table 2. Percentage growth of nix crops in loih from foliar applied herbicide* an RII.IVJI ], Z, 3, fiyv t>l/4 anil J.lt^, monlhl nflcr trciilmcnt (growth of control pl;tim — Kin*), •W 1 I . I B r»h. I, u* Pel. H. ll 2, AH crops showed a significant reduction in growlh . when scccletl 1 ami 2 months after application of the htrbicitlc. Rice, soybeans, and cotton were severely affected ami showed essentially no growth when seeded in soils 1 mont|i after treatment. Soybeans and corn continued to be adversely affected when seeded in soils 5 months after treatment; while sorghum, wheat, rice and cotto'n were not significantly affected. Since the disappearance of picloram is much depenilcnl upon leaching the 58.4 cm (28 inches) o£ rainfall received in the 8 months after treatment (Table 1) undoubtedly removed; lethal amounts of picloram. These data suggest that-, all crop species studied could IK safely seeded in soils G months after picloram treatment without adversely'affecting growth. 1,4-D:2,4J-T, Crop growth was retarded in soil treated with the 2,-l-D:2,4,5-T combination 1 and in some cases 2 months before seeding (Table 2). At 1 and 2 months this combination of herbicides was less inhibitory than picloram although the rate of treatment was four times as great. This suggests that the phenoxy compounds either dissipated more rapidly than picloram or that the crops were more tolerant to the herbicide combination or both. Wheat and cotton produced growth comparable to the control when iceded in soils 2 months after treatment. Soybeans were the most readily inhibited of all crops grown. Reasons for reduced growth in soils treated (or 1S>/Z months are unknown. That this may be a chance variation i.i indicated by'over 1009ip production in samples of soil taken in plots having a shorter time after these same treatments (excepting soybeans). Average (means) for all crops did not differ significantly at the 5% level of Duncan's Multiple Range Test. M-3HO. Blended rates of treatment for M-SHO were 6.72, 6.72, and 8.86 kg/ha (6, 6, and 8 Ib/A) of 2,4-D, 2,4,5-T, and picloram, respectively. No significant differences (5%) in growth occurred among averages for months but did for crop species when M-3HO was applied. Soybeans showed injury in soils treated for 9i/j months or less before seeding (Table 2). Cotton seeded S months after treatment was slightly injured. Apparently, sufficient picloram persisted '(or the described intervals to cause damage to soybeans and cotton; but other crops were unaffected, DISCUSSION These studies indicate that high rates of herbicides used to control vegetation in tropical and subtropical areas disappear rapidly, even though per1 sistciu compounds such as picloram are used. Hence, most crops could be safely grown within 6 months after application with few if any adverse effects. Soybeans were the crop most sensitive to herbicidal residues in soil. Use of an alternative crop should be considered, if seedings are made during the first i l J 4-Pj Li l t 4,ft-TllIt'll"'l jJMtj*/l'». i' 01 b Mb D< a 11 be 1Mb Mb 71 b lUtb TO 0 VH bfl ltd li > 51 bo ID lo IN li HI h Hfl 1 nt «b t»b« ua b 71 e TOb 111 (1 • 71 t 14 t i IMt i H) nh 131 H*b »I|M> 11H ElEect and Persistence of Herbicides Applied to Soil in Puerto Rican Forests' • 111 ll> » iM 71 be AVJ (Crop) M-JUIj .pplrod 1 A»I (Crep.) 111 * lib in ib 111 tSb RBb 111 1 1M » lb 117 ft 16 b (1 • 11T » 111 t i 111 I 1ST 111 CLYDE C. DOWI.EB, WILSON FORESTIER, and F. H. TSCIIIRLET' H.tkflA* in * • PI >b 11 lib 90 0 U at) 18 bo 10* 104 « let* llll 101* Mb DH 101* Dunoui'i tsulHple ruin I*'1- few months after treatment. Graminae species such as corn, sorghum or wheat may be used first in treated areas, because these are more tolerant to the residues of herbicides used for controlling brush. Rainfall (Table 1) is important in leaching herbicides from soil profiles. In areas of more abundant . precipitation one could expect more rapid disappear• ance than was encountered in this study, providing that other soil and environmental factors are equal. We have concluded that harmful residues of herbicides in the soil would not occur in tropical areas after chemical brush removal if a reasonable deferment of land was observed after application. thrttMI. M« hi'ihlciik-i wnc applied nl 3. 0. and 27 ib/A to the •U in f««m nf lime li|ic. in I'urrln Itico. The forests clllfcrcd in .iiirll»ixv-3A-dlcliloiiil>ciinile and (•hilnilul 2 iiirlhnxi -IJI.|il<(i«ipiii|)) 1 lainino)-<-trin/illi.' (iiinmelone), !*.! Vlmniin J.IIT l n i i ) l - f l • i i i e l l i ) l i n a r i l (Inomacil) were I™ cffeci»o Kkin iiMoMin iinib U.3 (i'liidilnioiihenvlaeellc acid (fenac) and *|ilirri)l).l.Uliiiiriliyluica (clluron) were incffeclivc. IkMillkin it.it gittilnr and more plant! wctc killed In Hie driest «hw mi wfirr U r a l nl, fenac wai (tic most persistent hcrbiIklr In llic .oil. Mrililcidn wei c found lo a 30 to 4S-in dcplli •llhlli 1 I,i,i,nil, afn-l a j i p l i r a l i a n . MHlunnm anil wikwh .WflliriRS wcic present at all site! willun t* pirtiilit alicr hi'ihicitle applitanon. There was no delinile n'laffcwililit l*iwrni lii'ibicidal uciitiunit and secondary succession, li<«K Iliai llu- nmnliri and fircpiency of Miccessional ipcciei wnc f»r»i*l rm plnH basing llic highest petccntaRC of defoliation, LITERATURE CITED 1, Bovcy, R. W., F. S. Davis, and H. L. Morton. 196B. Herbicide combinations for woody plant control. Weed Scl. 16: 332-335. 2. Crafts, A. S., and W, W. Robbing. 1962. Weed Control. . McGraw-Hill Book Company, Inc., New York. 8.,Goring, C. A, I., C. R. Youngson, and J. W. Hamaker. 1965, Picloram herbicide disappearance from soils. Down to Earth 20:3-5. 4. King, L, J, 1966. Weeds of the world. In Biology and Control. Interscicnce Publishers, Inc,, New York. 8, Klinginan. G. C. 1961, Weed control: Ai a science. John Wiley and Sons, Inc., New York, 6. Mcrklc, M. G., R. W. Bovcy, and R, Hall. 1966. The determination of picloram residues in soil) wing gat chroma tography, Weedi 14:161-164. 7. Mcrkle, M, G.', R, W. Bovey, nnd F. S. Davis. I9fi7. Factors affecting the pertinence of picloram in toil. Agron. J. 59:413-415. 8, Motooka, P. S., D. F. Saiki, D. L, Pluclmett, O. R. Younge, and R. E. Daehler. 1967. Control of Hawaiian jungle with aerially applied herbicide*. Down to Earth 23:18-22. , , , 9, Sheets. T. J., nnd L. L. Danielson. 1960. Herbicides In (oils, in The nature and fate of chemicals applied to ioU«, plants, and animals. ARS 209, USD A. 10. Tschirley, F. H. 1967. Problems in woody plant control evaluation in the Tropics. Weed! 15:233-237. H.,Tschirley, F. H., Rene T, Hcrnander, and C, C. Dowler. 1%7, Seasonal susceptibility of guava to selected herbicides. Wcedi 19:217-219, 12. Young&on, C. R., C. A. I. Goring, R. W. Meiklc, H. H, Scott, and J. D. Griffith, 1967, Factors influencing the dei composition of picloram herbicide in toll). Down to Earth 23:3.n, ' "<• IN'IROUUCTION lir rflnl of herbicides applied to the soil for control nf wpnilr plants in tropical forests lias not been Uliilird imi'ii'ivrly or extensively. A short review by M»)o Mcnrmlf* (7) indicaicd tbat frill treatment to Urn ».n llic most ellective method of application. ttnriiiliii! (2), SIMMS ( I I ) , and Wyatt-Smith (17, 18) nvnl f i i i l iiraiiticnis surccssfiilly on a large number of irit|iicil >|"'ri" but nuled differential susceptibility «mMiK «!«•( io. llnwkini ('I) reported that a basal spraying Of |Kimling was ;n eltoclivc as frill treatment. Man) hniclwoodi can be controlled effectively with jillrrinxiiui'iic or picolinic acids applied in basal frills m .11 HIT injrnioni (li, 15). Many compounds arc effective j. loliar ( i c n t m r n l i for controlling woody plants (3, 16). Njtinil (')) irpnilai that 4-;imino-3,5,6-trichloropicolinic »riil fpirlorani), when applied as a soil treatment, was • in tffrciivc licrblridc for controlling woody plants. He «Ui nolfd dilk'irntial susceptibility among species. Inu-iurnhle from the biological effect of herbicides >|i|ilinl lo the soil are their movement and persistence. T ""•«.',7licil t..r iiiibllrallon February 15, 1907, . •Hivitcli ARtonoinlit, AgiIcullnral Research Technician, Re. « i M b RJIIBI? Vlemisl. reiprclholy. Crops Research Division. Vintilmul Kriraiih Seivicc. U.S. nepaitment of Agriculture, Ittktll K\|»criinent Station. Mayaguez, Puerto Rico. Factors such as rainfall, physical and dicmical iharacter. istics of the soil, microorganisms, chcmiral chniaclcristio of the herbicides, and method of application may influence herbicidal movement and persistence- (5, 8. 10, M). Our objectives were (a) to determine the toxicity of selected herbicides on tropical arboreal vegetation, (b) to determine the movement and persistence of these chemicals in the soil, and (c) (o observe the short-term effects of these chemicnls on secondary plant succession. MATERIAI-S AND METHODS Our studies were located in Guanica Conimonwcallh Forest, Maricao Commonwealth Forest, and I.uqudlo National Forest. The soil type at the Oiunica Commonwealth Forest is |acana clay. It is an alluvial soil normally less than SCi in deep, with very low permeability. The vegetation is xerophytic. There were 35 woody species within the test area, but Lciicarna lr\iracrtihah (Lam.) DeWit and Harmr,5 was M.IM and 100.88 in, respectively. The soil type at the Luqtiillo National Forest site is I.os Guineos clay loam, a plastic clay with poor internal drainage. The vegetation is a tropical rain forest (1) with a mean canopy level ot about 60 ft. There were R8 tree species on the test site. Mean annual rainfall is estimated to be over 100 in. The highest.rninfall normally occurs from July lo October, but droughts arc unknown. The recorded annual rainfall near the site was 85.78 in for 49 I9(i'l and 12fi.l2 in for I9fi5. Tschirley et at. (14) characterized the Maricno and Luquillo sites in greater detail. The herbicides used in this study were 5-bromo-3-JCCbutyl-fi-niethyluracil (bromacil), dimethylamine salt of 2-metlioxy-S,G-dichlorobenzoic acid (dicamba), S-(3,4dichlorophenyl)-1,1 -dimethylurca (diuron), Na salt of 2,3,6-irichloropiienylacetic acid (fcnac), K salt of piclonuu, and 2-methoxy-4,f)-bis(isopropylarnino)-s-triazine (promctone). Each herbicide was applied at 3, 9, and 27 lb/A. A randomized complete block design was used at each location. The treatments were replicated three times at Guanica and Luquilio, but only twice at Maricao because of topographic limitations. Eacli plot was 60 by 80 ft. with A 20-ft buffer separating adjacent plots. Data were collected from a centrally located 40 by fiO-ft subplot, Narrow walkways were cut through the long axis of each plot to facilitate treatment and data collection. All woody plants having a diameter breast high (hereinafter referred to as dhh) mciisurcment of 0,75 in or greater were marked, mapped, and identified (at least to genus) at the Guanica site. At Maricao and Lucjuillo, only plants having a dbli greater than 1 in were marked, mapped, and identified. All defoliation data were collectrd only from the marked plants. The herbicides were applied with a cyclone hand spreader as granules, pellets, wci table powder, or liquid adsorbed on vcrmicnlitc, Vermiculite was used as a carrier to provide additional bulk for all treatments. The application dates were: Gunnica—October, 1963; Maricao—December, 1963; and I.uquillo—January, 1964. Percentiigc of defoliation of each marked plant was estimated periodically after treatment. Plot defoliation, calculated from i n d i v i d u a l plant defoliation, represents a mean percentage weighted according to the relative prevalence of various species. The downward movement of herbicides and their persistence in the soil were studied by sampling 3, 6, and 12 months after treatment. Duplicate soil samples were collected, at random from centrally located 20 by '10-fi subplots. Soil from depths of 0 to 6, 6 to 12, 12 to 24, 24 to 3fi, and 36 to 48 in was analyzed separately, If the soil was not 48 in deep, samples were collected to the maximum possible depth, The samples were placed, into 1-qt styrenc cups, then sealed and transferred to the greenhouse where a bioassay was immediately initiated. Cucumber (Cucumis salivits L,, vsir, Puerto Rico 59) was u'rtl as an indicator plant. Approximately six cucumber seeds were planted in each sample. After emergence, the cucumbers were thinned to two seedlings per sample. A mean injury raling of each sample was m.-idc lifter 28 days of growth and compared to an established standard curve, Abnormal growth characteristic* were expressed on a 10-point scale, where 0 = no cffcci and 10 K plants killed. Succession data were collected from one replication at each site. Observations on the species present and their relative importance were made at Maricao, Guanica, and Luquilio in April, May, and September, 19G5, respectively. The successional species on each plot were identified to family, genus, or .species and grouped according to tree seedlings, grasses and sedges, or herbaceous plants and vines. RESULTS , ' Hcrbicidal effect on woody plants, The correlation co> efficient of percentage defoliation and plants killed was significant at the 1% level of probability; consequently, results are based on defoliation only. Guanica. Picloram was clearly the most effective herbicide (Table 1). Bromacil and prometone caused high P I C L O R A M 37 LB/A Table ). Percentage of defoliation of the nine principal specie* in Maricao test site after herbicidal treatments to (he soil. Treated December, 196S and rated October, 1065. Hcrbtclde am) DefolUtl rale, lb/A ling ordct ortmpoTlancc* Picto ram 2 Table I. Percentage of'defoliation and plants killed of all arboreal species ill tlie Gtianlca, Maricao, and Luqtiillo forests approximately 2 years alter her Wci da I treatments, 96 12 r»n hl) S£ra„ <,,r. A •«.,(.«,> (Urn.) (Sw ) MCI. 7, Mittnio linltn, K Cogn. 8. C»ffW«(* ip. 9. Cfl»r,»VW"'""'<'**Jacq. ^:r Kn bl. •Def - percentage defoliation] PK - percentage planti killed. defoliation at the higher rates but were less effective when only S lb/A was used, Dicamba, diuron, and fenac caused only slight defoliation even at the highest rate, Differential susceptibility of die five principal species at Guanica is shown in Table 2. Picloram, considering all Table 2. Percentage of defoliation of the five .principal species In Giianka test site Z yeais after lierbicklal trc-iumcnis to tlic soil. Herbicide and rate, lb/A liation of major ipcclti order nf importan and density of the seedling complex could not be definitely associated with herbicidal treatment. The most common succession species in the Maricao site were Tcrebraria resinosa (Vahl) Sprague, CiutiVi spp., Ichnanthus pallens (Sw.) Munro, PaniVtim g/ufmcuum Sw., MONTHS FOL'.OWING APPLICATION and Poly podium spp. Lnquillo. Picloram caused the highest percentage of figure I. Percentage of defoliation at specified intervals after picloram was applied to the soil in three forested defoliation at the Luquilio site (Table I). The highest areas in Puerto Rico. rates of bromacil and dicamba caused appreciable defoliation, but both herbicides were much less effective when remained essentially constant for the rest of the 24-month only 3 or 9 Ib/A were used. Prometone, diuron, and fenac period. caused only slight defoliation at all rates. 1'icloram wai Total vegetation control was short-lived, even on plots as effective at 9 lb/A as dicamba and bromacil at 27 lb/A. that had been treated with 27 lb/A. No treatment preDifferential susceptibility was apparent among the vented plant succession for more than 18 months. Two eight most numerous species (Table 4). Tabfbuia heteroyears after treatment, Ifromoea spp., Indigofera sufphylia (DC.) Britton was the most resistant species in that frulicosa Mill., and cacti (Oftuntia spp.) were abundant, no treatment caused more than Sl% defoliation. PsychoGrass and tree seedlings were less numerous. Leucaena tria berteriana DC. on the other hand, was completely deIcucocephala (Lam.) DeWit was the most abundant tree seedling. Seedling density was directly related to percentage o£ defoliation. Table 4. Percentage of defoliation of the eight principal spedei In Luquilio t«t lite 21 months after heiblcldal treatments to Marieao. Picloram caused the highest percentage plot the soil. defoliation (Table 1). Dicamba at 27 lb/A caused 50% defoliation, but this was considerably lower than that Defoliation of major iprcki In otdrr of Herbicide and rate, lb/A „ (^creating Impm-t.nce' caused by picloram at 9 lb/A. Bromacil, prometone, diuron, and fenac were considerably less effective. Pick ram Picloram affected the broadest spectrum of tree species 100 SI IT i 100 ! 100 100 100 (Table S). Miconia sintenisii Cogn. was more susceptible 1 40 to picloram than were the other eight species. Rapanea 3 1 SO Jermginca (R. & P.) Mez appeared to be most susceptible 2 i i 1 0 Drd melt 13 0 1) 5 too to hromadl. Ocotea leucoxylon (Sw.) Mez tolerated all 7 100 ) too : .. . the herbicides except picloram at 9 and 27 lb/A. Prometonc j a : SS Maximum defoliation resulting from treatment with 0 0 0 c 0 I on picloram was obtained at Maricao about 1 year after ; 11 a 3 5 0 treatment (Figure 1). There was essentially no change 43 3 0 during the second year of observation. : ... No herbicidal treatment prevented the succession of D Cheelt.,.. tree seedlings, grasses and sedges, or vines and herbaceous 1 . TtMuia htirtpWlt (DC.} BrUton. 2. Cjatfc. „»,„• 1L.)1. T_ Smith. S:Cwi plants at Maricao. Tree seedlings were sparse on plots tori fitmu Utbati. 41 A.jlwj *I*»MII* (Gr*ham) Nicholwn. S Minttit *r«iiiM (5t DC 6. C*nm»-D,fp to 48-in depth. The bioassay data for all sampling depths indicated persistence of the herbicides in the soil 1 year after treatment for all locations was in the order of fenac > promctone > piclornm > diiiron > bromacil > dicambn, An example of. the residue d:\ta is shown in "Figure 3, Dicambn had almost completely disappeared 1 year a f t e r treatment, Two years after treatment, fcnnc was still the most persistent herbicide, followed by promctone and picloram. The persistence of the herbicides generally was greatest in the driest area (Guanicn) and least in the wettest area (Uiqulllo) (Figure 4). One year after application, the residue of picloram in plots treated at 27 lb/A remained in relatively high concentrations at all test sites, as determined by the cucumber bioassay test. The presence of picloram in plots treated at 9 lb/A could be easily detected 1 year after treatment, but the concentrations were about 10 times less than in plots treated with 27 lb/A. The residue data for all locations indicated a trend for all the herbicides to dissipate more rapidly in the top 12 in of soil. 51 AGIO***1 ffCS'DUE >E MONTHS and 5.94 in at Luquillo. Sufficient rain to leach the herbicides into the soil fell at all three locations within a few days after application. The rapid increase in retaliation shown for dicamba and check plots in the Guanica site is the result of refoliation occurring during the rainy season (Figure 5). "YftOUETONL \ 87 i I C - M B A VI i5 6 9 12 15 16 21 24 MONTHS FOLLOWING APPLICATION GUANICA figure 5. Percentage of woody defoliation in the dry, On an lea Forest site of Puerto Rico alter treatment with three hcrbicidci applied to the toil. fkfMt 4. I'lilorain residue at various depths In i h t t c (on'Kl aieas of Puerto Rico 12 m»nihi after application, '[;"- Figure 2. Fores! lloni nf pint treated w i t h nlcloifim nl 27 Hi/A * J.iiqiiillo. Top: Kiglit months a CUT treatment; bottom: T«* Jem's after treatment. Noio saonilary succession, SOIL DEPTH liKCHril ''gnre 3, Concentration of six hnbiddrs I year after to Jacana clay (Gtianica site) at 9 lb/A. DISCUSSION 3^ '• Om 2«l tKtfxly *|irdc* were represented on the three l^if'.iWw riirt, hut M'vcnil species were represented by only a ,;iX~ *kr* terfmdital*. Tschirlcy (13) enumerated some of the sfey (MiMfiBi Imolvrd in evaluating herbicides in tropical ", ftwinafc Other workers (2, {I, 11, Ifi) have shown distinct | i&frfffntul *iiMC|iiibility of woody plants to various ? IwrWsfclri. D t l f n c u t i a l species susceptibility also was ! nttJwtl »i our lot utcv When all the treatments at all n ate tnmideretl, Tnbebtilt , Cor din (tor nfiitmsis Urban, 7»g/i (tigifoJia (L.) *(iit Ofntra Icucoxylon (Sw.) Me?, were most and I'syclialrta bertcriann DC,, Miconia sinSrnr%alii\ wcstiana (DC.) Britton &: Rose, letintrrfihnla (Lam.) DeWit were most innt>nt-I)ryfietes, fdironin sinle.nixii Cogn. /Hflim/t (Sw.) DC. were represented at both nd LtK|tiillo. The reaction of these species to [ollowcd the same general trend at both , drfntiiilion was greater at Maricao. ^^f '!fcr*ff«t of climatic and cdaphic factors on herbicidal 1;^*****&'I «antifit l« clearly elucidated in this study be.-'}!'***•» il ililfcrrnin in specie* composition. At all three 1 *w< i^ift, rainfall l>efort' application was sufficient to in adcqunic plnnt growth, Rainfall for 2 months aintfitt was 2.41 in at Guanica, 5.16 in at Maricao, The rainy season \mtally occurs during July lo October, Most o£ the woody plants grow vigorously during the rainy season and are deciduous during the long dry season. Retaliation on plots treated with 27 lb/A of dicamba attests to its herbicidal ineffectiveness at the Guanica site. The lack of retaliation of woody plants on plots treated with picloram and prometone is indicative of their effectiveness. In this study, sufficient rainfall occurred after treatment to leach the herbicides into the soil and prevent large tosses from volatilization and photodecom position. The highest concentration of a herbicide in the soil profile was consistently found at the low r a i n f a l l Guanica site. On the other hand, the lowest concentrations of herbicides occurred at the continually moist Luquillo site. Persistence was related to the amount of herbicide applied, but the effectiveness of a herbicide on woody plants.was not related to its persistence. Fcnac, the most persistent herbicide, was ineffective for defoliating woody plants at all test sites. Prometone was more jwrsUtetu than was picloram but effectively defoliated plants only at the Guanica site, Picloram effectively defoliated woody plants at all three test sites. Although a high degree of woody plant defoliation was obtained from several treatments, total vegetation control was short-lived. Secondary succession occurred within 18 months on all defoliated plots at nil test sites. Grasses, herbaceous plants, and vines generally were more numerous than were woody tree seedlings. The amount of rainfall and increased light penetration appeared to influence secondary succession more than did the herbicidal treatment. The number and density of successional species were greatest on the wet Luquillo site and smallest at the dry Guanica-site. In general, the number and density of successional species were greater on plots that had been defoliated. This suggested that 53 52 increased light penetration was one of the major factors influencing secondary succession. There did not appear to he any relation between herbicidal residue and invading species. For example, several species such as Ptyrlwtria berteriana DC. were extremely susceptible to initial application of herbicides, but they were found on nil treated plots 18 months after application, ACKNOWLEDGMENTS This study was supported by the Advanced Research Projects Agency, Department of Defense. The herbicides were donnted by Amdicm Products, Inc., Ambler, Pa.; Dow Chemical Co., Midland, Michigan; E. I. DuPont de Nemours R: Co,, Wilmington, Delaware; Geigy Chemical Corp., Ardsley, New York; and Velsicol Chemical Corporation, Chicago, 111, LITERATURE CITED !, llKAHn, J. S. J948. The natural vegetation in (he windward and leeward islands. Oxford Forestry Mem, 21. 192 p, 2, 11 KVF.nl no E, A. E. 1057. Arboricide trials In lowland dipteroraip 'ail forest of Malaya. The Malayan Forester 20:211225. 3, llovEV, R. W,, F, S. DAVIS, M. 0. MUKLR. R. E. MEYER, H. L. MOHTON, and L. F. HOUSK. l%5. Defoliation and control of huifh. IVoc. SWC 18:288-202. 4, DAWKINS, R, C, 1957. Contact arboricides for rapid tree weeding in tropical forests. Trop. Silvicult. FAO Collcclion No. 13(2}:109~112. 5, FRISSFL. M. J, and C, H. HOLT. 1952, Interaction between certain lonlzable organic compounds (herbicides) and clay minerals. Soil Sci, 94:2B4-29J. 6. Ginns, CARTER B, 1959. Amines of 2.4-D hold promise for hardwood control. Down in Earth 15(3):6. 7. MAVO-MENENDEZ, ENRIQUE. 1964. Klltninadon de arbolcs indcscabtes incdiantc agcntcs quimicos. Rev, Interanicr, Cicnciai Agr, Turrialba MM); 199-202. 8. MKRKI.K, M. G., R. W. BOVBY, and K. HALL. 19(56. The determination of picloram residues In soil gas chromalography. Weeds 14:161-164, 9. NATION, HOVT A. 1965. Woody plant control on utility lichts-of-way with "Tordon" herbicide pellets. Proc. SWC I ft: 387-391. 10. SciiwmEk, E. E. and J. T. HOUTUH, JR. 1966. Persistence of live cotton herbicides in four southern soils, Weeds 14:22-26. H, SPOSTA, J. W. i960. Elimination de espccics tropieales infcriores por tncdio de suhstanclas quimicas. Apuntes Forest. No. 4. Trop, Forest Ren. Center, Rio Piedras, Rncrto Rico. t p. 12. THIEGS, B, J. 1962. Microbial decomposition of herbicides. Down to Earth 18(2);7-10. 13. TSCIIIRLKV, F. H. 1967, Problems in woody plant control evaluation in the tropics. Weeds 15:283-237. 14. TSCIIIRLEV, F, II., CLYPE C. DOWI,ER, and J. A. DUKE. Species diversity in two plant communities of Puerto Ulco. A Tropical Rain Forest (In Publication). 15. WATSON, A. J. and II. J, MESLF.R, JR. I96'l. Effect of tordon herbicide as basal mil and tree injection treatments on certain hardwood trees. Down to Earth 19(4):20-23. 16. WATSON, A. J. and M. G. WILTSE. 1963. Tordon . . . for brush control on utility rlghts-ot-way in the eastern United States, Down to Earth IO(1):11~M, 17. WYATT-SMITH, J. I960. Further arboricide trials in lowland Dipterocarp rain forest of Malaya. The Malayan Forester 23:814-33]. 18. WYATT-SMITH, J. 1961. Arboricide trials usin K animate X, 2,3-D, 2,4,5-T, and sodium arscnitc. The Malayan Forester 24:81-84. Reprinted from WEEDS Vol. 15, No. 3, July, 1967 Persistence of 2,4-D, 2,4,5-T, and Dicamba in Range Forage Grasses1 HOWARD L. MORTON, E. D. ROIHSON, and ROBERT E. MEYER* Abstract, The herbicides 2,4,-dlchlrjiophcnoxyacetic acid (2,4-D), 2.-tr(cl]Ioropliciioxyacetic acid • (2,4,5-T), and 2-mctlioxy-3,6-diUilorobcnzoic acid (dicamba) each labeled in the caiboxyl posilioit wore spiaycd on a pastme consisting of a mixture of silver bcardgiass (Atidrofrogan saccliaroides Swart?.). Hllic blucstcm f/J. scoftariiii Michx.), and dalli.igrass (Pasl/atum dilatalum Poir.) and a sidcoais giama (Houtrlaita citrtifietiuula [Michx.] Torr.) pastuic over a 3-ycnr ])criod. riant samples were harvested at Intervals between 1 hr and Ifi weeks after tieatment and residues determined hy raclionssny, No important differences were found in the persistence of herbicides or of different foundations of the same herbicide. Rainfall was the moat important factor influencing ihe peisistencc of ilie herbicides. The little blucstem-sllver beardKiass-diilliagiass samples haivcsted 1 hr after treatment with the buioxyclhyl ester of 2,4,5-T contained holK this ester and the acid of 2,4,5-T. One week after tiralmcnt, the acid of 2,4,5-T and unknown metabolites were found but no ester, INTRODUCTION VARIF.TY of herbaceous and woody plants are con\ trolled by 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), and 2-methoxy-3,6-dichlorobcnzoic acid (dicamba). Although the phenoxy acids have been registered and are used for weed control on lands devoted to forage production, [he substituted benzole acids have restricted usage on these areas. Little direct evidence of the persistence of these compounds in forage grasses has been published. Glastonbury ct at. (3) spiaycd peas (Pisum sativum L. vnr. Onward) with the sodium salt of 4-(2-mcthyl-4-chloro. phcnoxy)butyrir acid (MCPli) and found that the halflife o£ the retained chemical was 3 days. Gutenmann and Lisk (4) sprayed the diethylamine salt of 4-(2,4dichlorophenoxy)butyric acid (2,4-DIl) on a pasture containing birdsfoot trefoil (Lotus cornicitlatus L.) and timothy (Phleum f>ratense L.) and found a rapid decrease in herbicide concentration in the forage after rainfall. Concentration! of 2,4-DB in the forage immediately after application of 1.5 and 3.0 Ib/A rates were about 70 and 160 ppm, respectively, but were O.S2 and 0.80 ppm, respectively, after 48 days. Klingman et al. (5) sprayed a Kentucky bluegrass (Pan pratensis L.) pasture with either the butyl ester or the 2-elhylhexy! ester of 2,4-D and found that most of the butyl and about 75% of the 2-ethylhexyl ester were hydrolyzed to the 2,4-D acid within i/2 hr after spraying. Total concentrations of 2,4-D residues from the butyl and 2-cthylhcxyl esters dropped from 58.4 and A 'Received for publication November 7, 1906. Cooperative In. vcsllgalfons of the Crops Research Division, Agricultural Research •Service and Texas A&M University, 'Research Agronomist, Crops Research Division, ARS, U, S, Department of Agriculture, College Station, Texas; Assistant Range Scientist, Rolling I'lains Livestock Research Station, Texas A&M University, Spur, Texas; and Plant Physiologist, Crops Research Division^ ARS, U. S. Department of Agriculture, College Station, Texas, respectively, ' 48.4 ppm i/2 hr after treatment to 5.0 and 15.1 ppm, respectively, 7 days after treatment. The investigation reported herein was conducted to determine the persistence of 2,4-D, 2,4,5-T, and dicamba in range forage grasses, to compare the persistence of attune and acid formulations of 2,4,5-T, ami to determine the influence of rate of application on the persistence of 2,4-D and 2,4,5-T. MATERIALS AND METHODS Two field sites were fenced for the study. One was at College Station, Texas, in a pasture in which silver beardgrass (Andropoeon saccharoides Swartz.), little bluestem (A, scoparius Michx.), and clallisgrass (Paspalum dilalatnm Poir.) were the dominant species. The other was at Spur, Texas, in a pasture in which sideoats grama (Roitleloua curtipendula [Michx.] Torr.J was the dominant species, Different areas were treated at each site each year. Herbicides labeled in the carboxyl position with carbon-14 were mixed with technical grade herbicides in the proportions necessary to give the specified radioactive levels as well as the specified rate of herbicide per acre. In all experiments, sprays were applied at volumes equivalent to 20 gpa with a compressed air sprayer. Two replications of each treatment were used. In 1962, the plots were 2 by 10 ft and they were 2 by 12 ft in 1963 and 1964. Jn 1962, we applied butoxyethyl ester of 2,4,5-T al rates equivalent to yz and 2 Ib/A. Sprays were applied June I I and _|une 19 at Spur and College Station, respectively, which contained 5 u,c of radioactivity per plot. The carrier consisted of 7 parts water and 1 part diesel fuel (v/v). In 19G3, we applied 2,4-D and 2,4,5-T acids to the silver beardgrass-little bluestem-dallisgrass pasture June 14. Each solution contained 50 |»c of radioactivity and sufficient herbicide to provide i/j, or 2 Ib/A rate. The carrier was acetone-water (1:1) containing 0.5% (v/v) surfactant 3 . In 19G4, we applied dimcthylamine salt of 2,4-D, dime thyla mine salt of dicamba, and triethylaminc salt of 2,4,5-T to the silver beardgrass-little bluestcm-d.illisgrass pasture July I, We applied both the ainme and acid of 2,4,5-T and dicamba to the sideoats grama pasture July 7. The carrier was water containing 0.5% (v/v) surfaciant* for amine salt formulations and acetone-water (1:1 v/v) for the acid of 2,4,5-T. Each solution contained GO uc of radioactivity and sufficient herbicide to x provide a rate of 1 Ib/A. •.Surfactant contained alkylarylpolyox veiny ten e glycols, free fatty acids and isopropanol. \ \ 54 In all years, we sampled the treated plots 1 hr (0 week), ], 2, 4, and 8 weeks after treatment. An additional sampling was obtained on the fourteenth and sixteenth weeks in 1003 and 1964, respectively. We harvested 2-sej-ft subplots from each main plot by clipping the priiss plants at ground level, The clipped plants were separated into those tissues produced during the current year, designated green tissues, and those tissues produced during previous seasons, designated litter tissues. P a r t i a l l y decomposed plant tissues were gathered from the soil surface of the subplots and were added to the Utter tissues. After separation, the samples were placed in polyethylene bags, sealed with rubber bands, weighed, and stored at —10 C. .Samples harvested as Spur were transported to College Station in an ice chest for analysis. The harvested samples were shredded, and a 20-g portion was homogeni/.ed in a blender with 80% ethanol and filtered, The liomogenization \vas repeated until the radioactivity of the residue was less than two times background. The filtrates were combined, reduced in volume under vacuum in a rotary evaporator, and brought to volume in a 25-nil volumetric flask. Duplirate I-ml samples of each concentrated filtrate were dried in I-in planchets, weighed, and the radioactivity assayed with a Geiger-Miillcr tube. Counts were converted to weight of herbicide from standard curves with appropriate corrections for background and self-absorption, file q u a n t i t y of herbicide recovered on and in the forage was calculated for each subplot and converted to parts per million equivalents of fresh weight. In 1962 and I9f>8, identification and characterization of the radioactive compounds in the concentrated ethanolic extracts were made by descending chromatography on W h a t m a n No, 1 filter paper. An isopropanoliammnnium hydroxide:water (10:1:1 v/v/v) developer was used. After development and drying, each chromatogram was scanned with an autoscanner to determine the location of radioactive substance or substances on the chromatogram, Chromatograms of the ester of 2,4,5-T treating solulions contained radioactive butoxyethyl ester o[ 2/1,5-T and small amounts of arid. Identifications of the butoxyethyl ester of 2,'1,5-T, and acids of 2,4,5-T and 2,4-1) were made by co-chromatography of the ethanolic extracts and standard solutions of these compounds. RF.SIU.TS AND DISCUSSION Recovery of herbicidrs from sprayed plots. The amount of heibicidc recovered from grass tissues harvested 1 hr a f i c r treatment, calculated as a percentage of the amount applied, varied from 28% (20% green tissue and 8% litter tissue) to 102% (42% green tissue and 60% litter tissue) (Table I). In all but two plots, greater quantities of the herbicides were recovered from the green tissues t h a n frnni the litter tissues. Although the silver beardgrassl i t t l e bluestem-dallisgrass stands were relatively uniform, the plants and litter did not cover all of the plot areas. The low recovery percentages were due to sparse stands and the higher recoveries to dense plant and litter rover. Exprriments in 1962, Figure IA presents a semilogarithmic graph of the concentrations of the ester of 2,4,5-T in green tissues of silver beardgrass, little hint-stem and 55 Table I. Percentage of herbicides recoveied fn green and lid« 1 Issues of silver beardgrais, little uhieslem, and dallisgraw ind sldeoats grama harvested I hr after treatment.* Acirt or I,*-])..', Aci.i of 2,4-n Acid of 2,4,5-T Add or2,4,5-T Amine t«l( of 2,4-D, ... A mini! milt of 2,4,5-T.... Acid of2,4,5.T Diramba »», liitlr liIiiMiem, and dallisgtass. Disappearance *•« (twxi T»tild during the second week after treatment. 'IV HI* of [1ki|i]icarance in litter tissues was slower "Hivwl tn ihtr KTCCII tissues. Tlic apparent half-life of ester «l £,t.*T In ilie liner tissues was about 4 weeks under *fl# ffwwfifiont of this experiment. Two factors were imjr*'**.!'** in OH" slower jalc of disappearance, first, growth •i it* K»«II tinues would have diluted the herbicide, • ftirt »h» lilltr umplrs were com|)oscd of non-living tis;','«!« tnd Riowih was not a factor in lowering the conceni In these samples. Second, conditions for micro- lideoats grama. The ester oE 2,4,5-T disrapidly from sidcoats grama than from dallisgrass harvested at five dates after treatment. The lines for the two rates are essentially parallel, indicating that the rate of disappearance was not affected by rate of application. Concentrations of the ester of 2,4,5-T residues decreased rapidly during the second week after treatment when 2.18 in of rainfall occurred. The ap2,0 Lfl/A ,. 2.0 LB/A ..0.5 LB/A O.9 LB/A WKS.AFTER TREAT. I. ONMrmralUini of ester of 2,4,5-T residues found in sideaim puna llwuri hancslcd at live dates after treatment June 11, ' WHIT., M OS mil J.I) Ili/A at Spur, (A) Green tissues, (B) litter «*•**«. fcaU tedical lines indicate rainfall which occurred durb*H Ilk MwlkltH Interval nftcr treatment, 0 1 2 4 0 1 2 4 a WKS. AFTER TREAT. WKS. AFTER TREAT. Figuie I. Concciilintioiis of t-slcr S/J.S-T residues found in lilver iK'nidgi.iss-litllr hhiesIcm-d;Ulisgms!i (issues haivested a( five rlatrt aficr treatmrnt June 19, 1962 nt 0.5 and 2,0 Ib/A at Co\\tyf .Station. (A) Green tissues, (H) titter .issues. Solid vertical lino indicate rainfall which occurred during (lie indicated interval after h'catmcnt, parent half-life of the ester of 2,4,5-T (half-life equal* average length of time necessary for one-half of herbicidal residue to disappear) under the conditions of tliii experiment averaged 2.6 weeks. Concentrations of csler of 2,4,5-T in the green tissues 8 weeks after treatment were 25 and 7 ppm, respectively, at the 2.0 and 0.5 Ib/A rates. Figure lli is a semilogarithmic plot of the ester of 2,4,5-T concentrations in the litter tissues of silver iin, liitlc bluestcm, and diillisjjrass. More Tft&H foil mt thr sidcoats grama than on the silver beardwMt liitle liliifMctn, and dullisgrass, The apparent half, Mr <4 ilw hcrl>iciilc aveiitged 1.6 weeks in the green • »mt 1.7 weeks in the litter tissues. The amount liwiurnty ol the r a i n f a l l were conducive to leachCTlrmbi.il decomposition of the herbicide, and i al liilnuit grama plants. All of these factors con> * rapid reduction in herbicide concentrations. f»ff>imtnt in 1961, The concentrations of 2,4-D and • t.f,*-T roliliics found in green and litter tissues of ,!,"«***» IvjitlgraM, l i t t l e blucstcm and dallisgrass har,,-i»w*n) «t u\ djtci alter treatment June 18 are shown in „ Itfw* J. A (MiO-in rain occurred during the first week •fwr liMlmrnl. and the concentrations of both herbipear- O.SLI/A t.«.S-T «"I.OLB« M.S-T — aate/A M-O f.Ot»/A M-D figure 3. Concentration! of 2,4-1) ami 2.4.5-T Kilduei found in silver tcantarass-Httlc blucjtem-ilallisgrajl tissues harvcsltil al lix dat» aficr irealmenl June 14. I%J al OS and 2,0 Ib/A at College Station. (A) Green tissues, (B) litter tissues. Solid \emcal Imel indicate rainfall which occurred during the Indicated interval after treatment. ance was more rapid in roost of the plots than during the second week. During the fourth through the eighth weeks after treatment, only 0.08 in of rainfall occurred and relatively small decreases in herbicide concentrations were found. The 2.78 in of rainfall which occurred during the eighth through the fourteenth weeks after treatment probably was the primary factor responsible for the rapid rate of herbicide disappearance during this interval. The average half-life for 2,4-D in green and litter tissues was 2.3 and 2.8 weeks, respectively. ' The average half-life of 2,4,5-T in green and litter tissues was 2.9 and 3.4 weeks, respectively. Experiments in 1961, Residues of amine salts of 2,4-D, 2,4,5-T, and dicamba disappeared from silver beardgrass, little bluestem, and dallisgrass tissues at about the same rate (Figure 4). The apparent average half-life for each Figure 4. Concentrations of amine of 2,4-D, amine of 2,4,5-T, and dicamba residues found In silver beardgrass-little blueslcm-dallisgrass tissues harvested at six dates after treatment July I. 11164 at 1 Ib/A at College Station. (A) Green tissues, (B) litter tissues. Solid vertical lines indicate rainfall which occurred during the indicated interval after treatment. of the three compounds in green tissues was 2.0 weeks under the conditions which existed during the experiment. Because of heavy rainfall during the fifteenth week after treatment, the concentrations of the three compounds were reduced to 1 or 2 ppm in the green 56 57 HERBICIDES IN SOILS * T. J. SHEETS AND L. L. DANIELSON '• [issues. Concentrations o£ the three herbicides decreased rapidly in the Iitier tissues during the first 8 weeks after treatment (Figure 4B) when frequent rainfall kept the soil and litter tissues moist. The average half-lives of 2,4-D, 2,'i,5-T, and dicamba in the litter tissues were 2.8, 2.7, and 2.6 weeks, respectively. A relatively slow disappearance rate was found for all three herbicides in the green and litter tissues of sideoats grama (Figure 5). This slow disappearance oc- Figure S. ConccnMittionj of ncid of 2,4,5-T, nminc of 2,4,5-T, nnd dicamba residue* found in sidcoats grama tissues harvested nt six dates after treatment July 7, 1064, nt 1 Ib/A at Spur. (A) Green tissues, (B) litter tissues. Solid vertical lines Indicate rainfall which occurred during the indicated interval after treatment, rui-rcd during a period of low rainfall. Although considerable variability occurred in the concentrations of the three herbicides nt each sampling date, all herbicides had essentially the same rate of disappearance during the Ifi-week duration of the experiment. Concentrations of the acid of 2,4,5-1', amine of 2,4,5-T, and dicamba were 6, 5, and 4 ppm, respectively, in the green tissues at the time of final sampling 16 weeks after treatment. The concentrations of the three herbicides in litter tissues of sideoats grama are shown in Figure 5B, The acid of 2,4,5-T disappeared more slowly than the amine of 2,4,5-T and dicamba in litter tissues during weeks 2 to 4, but all three compounds were present after 8 weeks in approximately equal concentrations. Data indicate that formulation had no significant effect upon the persistence of 2,4,5-T in the tissues of silver bcnrdgrass. little bluestem, dallisgrass, and sideoats grama. While there were minor differences in the rates of disappearance of the three herbicides applied at College Station and Spur, their persistence in forage tissues appears to be essentially the same after several weeks. "1 he most important factor influencing the persistence of these herbicides was rainfall. Both amount and frequency oE rainfall were important. Even when rainfall did not occur, there was a gradual reduction in the herbicide concentrations in the green tissues, particularly if rainfall had occurred prior to the interval when herbicide concentration was being measured. Dilution of the herbicides by plant growth was an important factor during the intervals after rainfall had occurred and soil moisture was adequate for growth of the plants. Important reductions in the concentra- (Reproduced from ARS 20-9 :170-181, Sept. 1960) tions of the herbicides were not found in the litter tissues when no rainfall occurred. This is evident in Figures 111, 31i, and 5H, The exception to this statement is found in Figure 411 when a reduction in the concentration of the amine of 2,4,5-T from 146 ppm to 78 ppm occurred during the first week after treatment. It is not surprising that formulations had no influence on the persistence of 2,4,5-T. Phenoxy herbicides deposited on the surfaces of plant leaves as ester formulations are hydroly/ed to the acid in a relatively short period of time (1, 2, 5). Identification of herbicide residues. Attempts to identify the radioactive components in the ethanolic extracts by paper chromatography were only partly successful. All extracts from silver beardgrass, little bluestem, and dallisgrass green tissues harvested 1 hr after treatment witli ester of 2,4,5-T contained the applied herbicide find the acid of 2,4,5-T. The Rf values ranged from 0.59 to 0.75 and 0.85 to 0.89, respectively, for the acid and ester of 2,4,5-T. Approximately 10% of the radioactivity was attributed to the ester and 90% to the acid of 2,4,5-T. These data confirm the results of Klingman et al (5) who found rapid hydrolysis of the ester of. 2,4-D by Kentucky blucgrass. Extracts of green tissues of silver beardgrass, little bluestem, and dallisgrass harvested 1 week after treatment contained the acid of 2,4,5-T and unidentified metabolites which had Rf values ranging from 0.10 to 0.30 but no ester of 2,4,5-T. Approximately 50% of the radioactivity was attributed to the acid of 2,4,5-T and 50% to the unknown metabolites. The extracts 6f green'tissues of silver bdardgrass,' little bluestem, and dallisgrass harvested I hr after treatment with acid of 2,4-D or acid of 2,4,5-T yielded only the acids of 2,4-D or 2,4,5-T. Tissues harvested 1 week after treatment contained both the acid and unknown metabolites. The metabolites of 2,4,5-T had Rf values ranging from 0.10 to 0.30, and those of 2,4-D had Rf values ranging from 0.07 to 0.25. ,> ACKNOWLEDGMENT The butoxyethyl ester of 2,4,5-T used in this study was provided by Amchem Products, Inc. and the dicamba was provided by Velsicol Chemical Corp. The authors are grateful for the technical assistance of Gloria C. Taylor and T. O. Flynt. LITF.RATIIPE CITRD 1. CRAFTS, A. S. 1PGO. Evidence for hydrolysis of esters of 2.4-D during absorption by plan is. Weeds 8:19-25. 2. ERICKSON, LOUIS C., B. L, H R A N N A M A N , and CiMRi.rs W. COCGINS, In. 1003, Residues In stored lemons treated willi various formulations of 2,4-0. J. Agr. Food Chem, 11:437-440. 3. Gi,AsroNnuRV, H, A., MARIJARCT D. SIFIVKNSON, and R. W, E, DAM,. 1959. The persistence of 4-(2-mcthyl-4-chloropIienoxy) butyric add in peas. Weeds 7:3(12-36,1. 4. GDTF.NMANN, WALTIR H. and DONALD J. LISK. 1963. Rapid determination of 4(2,4-DB) and a metabolite, 2,4-D, In treated forage by electron affinity spectroseopy. J, Agr, Food Chem. 11:304-300.. 5. KLINGMAN, DAYTON L., CHiatFR II. GORDON, GEORCK YIP, and H. P. BURCI(FIELD. IPG6. Residues in the forage and in m i l k from cows grazing forage treated with esters of 2,4-D. Weeds 14:164-167, INTRODUCTION Many herbicides are applied directly to the soil surface as selective preemergence sprays and as nonselective soil sterilants. Other chemicals are applied subsurface or are thoroughly mixed with the soil after surface application. Residues remaining on leaves after foliar applications are carried to the soil In rainwater or fall to the soil when injured leaves abscise and fall. Therefore at least part of all herbicidal sprays eventually reach the soil. Soils vary greatly in composition and reactivity. Many complex nnd everchanging processes occur continuously in most soils. Soils are composed of mineral matter, organic matter, water, and air. The mineral fraction varies in amounts of sand, silt, and clay, and in types and amounts of clay minerals. The hydration and base saturation of the clay minerals also vary. The organicmatter fraction consists of decaying plant and animal -residues and active soil flora and fauna. The organic and mineral colloids present in the soil contribute directly and indirectly to the extremely active nature of soil systems. This mixture of mineral and organic matter is permeated by pore spaces of various sixes. These spaces are filled with water and air in a more or less reciprocal relation. The soil water contains many soluble compounds and serves as an essential medium for many physical and chemical processes. The soil atmosphere is composed of oxygen, carbon dioxide, nitrogen, and several minor gases. The composition of the soil atmosphere varies, particularly the oxygen and carbon dioxide contents. The complexity and variation of soil systems ' make the study of the fate of herbicides therein complicated and time consuming. METHODS OF ASSAYING HERBICIDE RESIDUES IN SOILS In most investigations on the persistence of herbicide in soils, researchers determine the presence of the active entity of the herbicide by growing sensitive plants. The influence of time on herbicidal residues has been measured by the growth of successive crops of test plants. This method, which has been used in both field and greenhouse experiments, is qualitative only. Quantitative bioassays have been developed for some herbicides. Holstun and Loomis (85) measured the elongation of young shoots of germinated millet seeds to determine the concentration of the sodium salt of 2,2-dichloropropionic acid [dalapon] in soils. Burschei and Freed (11) used heights and weights of seedling oats to determine the concentrations of isopropyl N-phenylcarbamate [IPO], isopropyl N-(3-chlorophenyl) carbamate [GIPC], and 3-amino-l,2,4~ triazolo [amitrole] in soils. Rahn and Baynard (45) used weight of oat seedlings to assay quantitatively 3-(p-chlorophenyl)-l,l-dimethylurea [monuron] in soils. Biological assays developed for solutions and vapors of herbicides could be adapted for use with soils 11, 46, 55). Some herbicides have been extracted from soils and their concentrations determined by physical or chemical methods. Methods are available for monuron, amitrole, CIFC, and pentachlorophenol [PGP] (10, 11, 30, 62). Whiteside and Alexander (61) followed the breakdown of several chlorinated phenoxy aliphatic acid herbicides in solutions inoculated with soil by the disappearance of the specific ultraviolet absorption. A physical or chemical assay may be most suitable in one situation and a biological assay in another. Both types of analyses are useful in some cases. Rahn and Baynard (45) reported that the chemical method for the determination of monuron in soils was accurate if the assay was made within a few weeks after application. If soils were chemically assayed more than 1 month after treatment, values for monurou concentration were greater than those obtained by bioassay. Rahn and Baynard (45) suggested that this apparent disagreement could be explained since the chemical assay for monuron was based on p-chloroanaline, a nonphytotoxic, hydrolytic product of monuron. 1 A contribution from the Crops Rosenrch Division, AgrlcnUnrnl Resonrnh Service, U.S. Department of Agriculture, nnd the Mississippi Agricultural Experiment Station. 3 Plant PliyRlologlntfl, Crops Research Division, Agricultural Repenrch Service, U.S. Department of Agriculture, Stonevllle, Miss., and Beltsville, Md.( respectively. 58 *•:,.•••• fey ? ' 59 | | | bwtilcldes appeared to follow afirst-orderreaction ^F$. The rate of dis6j|!*pf»ttirnnca of monuron from soil was proportional to the concentration (53). Factors affecting the movement and persistence of herbicides in soils hnve ppllil, Cl al. (33) concluded that although soil moisture and temperature often been reviewed by several workers (1, 8, 25, 20, 32, 41, 43). Leaching, fixation |I|:*ll*rwl the rates of inactivation of monuron and 3-(3,4-dichlorophenyl)-l,lby soil colloids, chemical and microbial decomposition, and volatilization were rilBMrtliyluren [Uluron], the first-order equation was probably applicable under stressed in one or more of these papers. In this discussion microbial action, *wu»l field conditions. When monuron and diuron were applied at rates of 1 volatilization, adsorption, leaching chemical reaction, photodecompositiou, nnd :**>A a wounds per acre in more humid regions of the United States, major absorption by plants will be considered for their significance in the performpmrU ot tho herbicides were inactivated each year. Accumulation from appliance and fate of soil-applied herbicides. cation* on the same soil 2 years in succession was negligible. Hahn and BayMicrobial action.—Most organic herbicides subjected to appropriate tost* i *»«! (48) found that monuron applied at 3.6 pounds per acre in two applicahave been inactivated more rapidly in soil under conditions favoring growtli IfeMW for 3 years in succession did not persist from one year to the next. Wheu and proliferation of soil microorganisms. Absorption by microorganisms is one •iMIwl lit 0.4 pounds per acre, monuron toxicity persisted from one year to of the major pathways by which organic herbicides are detoxified. Perhaps ; th* nwxt, but no accumulation occurred. Research conducted in the arid South2-(2,4-dichlorophenoxy) propionic acid [2-(2,4-DP) ], 2,4,5-trichlorophenoxyacetlu |r;;'i***t; (luring the last 7 years indicated that monuron and diuron did not acid [2,4,5-T], 2-(2,4,5-trichlorophenoxy) propionic acid [silvex], and 4-(2,4,5: ,«mtmtilnti> significantly from successive annual applications at rates used for trichlorophenoxy) butyric acid [4-(2,4,5-TB) ] are exceptions (7, 61). Optimum wH*rtl«ro weed control in cotton. Some carryover often occurred, and the oxygen, moisture, temperature, and nutrients favor microbial activity and nlsn ., amount of carryover appeared to be related to weather conditions. herbicidal detoxication. Numbers of soil microorganisms capable of inactivating ,; Can-out research indicates that the solvent used in the application of an 2,4-dichlorophenoxy acid [2,4-D] apparently increase when 2,4-D is present In * Iforrlvfrlde may have a profound influence on the persistence of herbicidal the soil (8, 42, 48, 61}. Thus repeat applications of 2,4-D were less persistent •j MllrKjr ( 1 8 ) . Ethel N,N-di-n-propylthiolcarbamate [EPTC] was applied in : in soil and therefore may be less effective herbicidally than the initial applica.*if*ral Holvents and incorporated into the soil. At weekly intervals up to 6 tion. With the phenylureas and s-triazines such an increase in microbinl !,y,***k* nfter treatment, the soils were assayed by the used of oat plants as Indlactivity apparently does not occur, because soils appear to exhibit about tlie :-:.''!tHl in water. However the rate of inactivation of technical EPTO applied occurring in the soil as products of microbial activity and that the herbicides 4 ftt tK>rvMw>np was much more rapid than that of the commercial formulation are not utilized directly by microorganisms as energy sources. ; ;•:• iHT'lwl In water. Four weeks after treatment the 2 Ib./A rate of technical Bacteria, Bacterium globiforme and Flaviobacteriiim aquatilc, which were . :.; SITO Applied in kerosene did not inhibit growth of oats. :!-•- IVrKlwtom.'o of several groups of herbicides in the soil is related to halogenacapable of inactivating 2,4-D were isolated from soil and grown in pure cul.; Uwi of (he benzene ring. This relation was demonstrated for certain chlorinated ture (G, 8, 86). Evans and Smith (27) isolated a small, Gram-negative, motile ''p&aMjoxyncPtlc acids, carbamates, and phenylureas (21, 22, 23, 50). The results soil organism which grew freely in a mineral-salt medium containing p~. 4-D exhibit various rates of vaporization. previously. • " "Hill, et al. (33) reported that a soil bacterium of the Pseudomonas group' S,.» Vajwrs^of soil-applied herbicides have caused severe injury to treated crop S**nt* tn Home instances. Vapors of 4,6-dinitro-o-seo-butylphenol [DNBP] after was capable of oxidizing monuron particularly in the presence of yeast extract. |MM>AOTi!«mcQ applications caused extensive injury to cotton in the Mississippi One group of herbicides, the esters of clilorophenoxy alcohols, becomes herbll*»tw In 1952 (25, 26, 34). DNBP injury was associated with high temperacidally active only on contact .with the soil. In warm, moist soil sodium, 2,4,t*m*. lIolllnRsworth and Ennls (84) found that vapor injury to young cotton dichlorophenoxyethyl sulfate [sesone] is hydrolyzed to 2,4-dichlorophenoxyftoiil* Increased ns soil moisture increased. ethanol in the presence of either microorganisms or acids (12, 13). Tlie -";; IWBl* Injury to cotton was reduced by application of lime and other basic hydrolysis by microorganisms was attributed to acids secreted during their Hi ***<('HfllH to the treated soil surface (9, 19). Upon addition of a base, the metabolism. The ethanol product is oxidized in the soil to 2,4-D, the active IJf.fcSwttfll-plipnnto equilibrium was probably shifted to the phenate, which Is less entity. ||:*o*«illi» than the phenol ( 0 ) . In experiments conducted by the senior author, 2-ehloro-4,6-bis(diethylamino)P:.;,-•'TatatHUnHon of the carbamates has been related to their effectiveness as s-triazine [chlorazine] mixed in the soil became more toxic to seedling onts j*|i; fr*wro»nrimce herbicides. IPO and CIPO volatilized rapidly from tinfoil and with time. This trend reversed after several months, the time depending on ||pj(tftMHf mirfnces at high temperatures (4). IPO volatilized more rapidly than the soil type and concentration, and thereafter the herbicidal activity of cull/l.CtPO, particularly at temperatures of 60° to 85° F. The most volatile cartures containing chlorazine decreased with time. The increase in toxicity could Jit: MttmtM were found to be most phytotoxic (38) ; however, loss by volatility not be accounted for completely as a response to growing conditions iu the |;; TBlte-wliiR prceuiergence application reduced the concentration of the more greenhouse. Autoclaving the soil prior to treatment retarded the rate of onset Jit : *** Chemical reaction. — The formation of salts of 2,4-D and DNBP and a possible reaction scheme for inactivation of 2,4-D in soils have been discussed. 63 Relatively little is known of the chemical reactions that &;/ plants.—Herbicides are absorbed ^Aplant roots and areunderdo in soils. Hydrolysis, oxidation, and formation of comple:Bire known HWimtly translocated to the aerial parts. Within thewmt the herbicide molereactions for certain herbicides. WiraJwi nro subjected to various physical and chemical processes. Crop plants may :"rfc» removed from the land or they may be returned to the soil along with weed Amitrole forms stable complexes with cobalt, copper, nickel, iron, and magnesium, Sund (54) suggested that complexing with metal ions in the soil :,*twwUi. Most plant roots remain in the soil, Therefore a portion of herbicides solution was one mechanism by which amitrole was detoxified. '.,'vinMwrbed by plants and the metabolic products of herbicides in plant tissues The 2-chloroacetamides, for example 2-chloro-N,N-diallylacetamide [ODAA], !:;!tt*jr«>vi»ntwilly be returned to the soil. can be hydrolyzed in the soil (31). The chlorine atom and the amide linkage II IUIB been stressed previously that soils reduce the initial effectiveness of are sites on the molecule where hydrolysis may occur. Begardless of the site whlcldra nrnl that the degree of effect varied among soil types and herbicides. where hydrolysis begins, the end products are glycolic acid and secondary Uii|iijl-w»t tf»(w following simazine treatment through the roots was reduced 50 per[CDEG], can be broken down in the soil by oxidation and hydrolysis (31). ; ft-nt by 7.2 ing. of the herbicide (C1* expressed as simazine) per gram of dry If hydrolysis of CDEO precedes oxidation, allyl alcohol is an'intermediate; ItaoiKt lit Imrvest 0 days after initial exposure (49). The amount of simazine •vwhereas if oxidation precedes hydrolysis, 2-(diethyldithiocarbamyl)acetice acid '•••.'1C*' expressed as simazine) required in seedling oat plants to reduce plant "is an intermediate. The two reactions can occur separately or concurrently '•'" wwiBlit wns less than 2 percent of that present in 400 ml. of the 0.05 p.p.m. by -In the soil. End products of breakdown are formic acid, glycolic acid, carbon nMKlit culture solution initially. Although the conditions of this experiment ••tlisulfide, and secondary amines for both reaction pathways. The breakdown ]•-'• «»«!•*• markedly different from those which occur in the field, plants probably x products may undergo further reaction in the soil. x'. •Afawb only a small fraction of the total amount of an herbicide applied to the Freed, et al. (89) suggested that EPTO was hydrolyzed in water; and HOll. ; according' to the reaction scheme which they proposed, a secondary amine, ',,-" CONCLUSIONS • carbon dioxide, and ethylmercaptan were end products. Soil treated with 3,5-dimethyltetrahydro-l,3,5,2 .ff-thiadiazine-2-thione [DMTT] : S '" Alftiiiiiijti considerable progress has been made, much additional information • evolves formaldehyde, which is thought to be the first product of DMTT breakon (he fate of herbicides in soil Weed research scientists need to •down in the soil. (40). Methylaminomethyldithiocarbamate forms next and • About the persistence of herbicides in soils under varying environactivation continues by forming monoethylamine, methyl isothiocyanate, and Mjcriinl conditions so that they can establish safe rotational practices. Mamhydrogen sulfide. Monoethylamine and hydrogen sulflde react with formal(rtnllnn toxlclty of some soil degradation products should be determined, bedehyde and form methylamirioethanol, dimethylarninorneUinne, and 1,3,5fl*uj*< fliww products can also be absorbed by plants. Information on adsorptiontrithiocyclonexane. Eventually this reaction proceeds to carbon dioxide, amtfwwirplUm relationships: on the interrelationships of adsorption, volatility, monia, sulfur dioxide, and water. The methyl isothiocyanate and water react M'llillll.v, mil leaching of herbicides; on the nature and extent of microbial to give carbon dioxide, hydrogen sulflde, and methylarnine; and the methylflipinicnl innctlvntion ; on the importance of photodecomposition; and on amine degrades into carbon dioxide and ammonia. Inllwm-e of various environmental factors on these processes is essential Monuron is thought to be hydrolyzed slowly in the soil to p-chloroamiline (4$)- '"' understanding of the behavior of herbicides in soils. In the presence of moisture tris-(2,4-dichlorophenoxyethyl)phosphite [2,4Ml scientists should determine the component or components of the soil DEP] is slowly hydrolyzed step-wise to form one mole of phosphorous acid Jff'sj:nwrt whleli dosage requirements of soli-applied herbicides can be predicted. and three moles of 2,4-dichlorophenoxyethanol (28). |§y'!J*ww«r of this information is available (48, 56), but more is necessary. EvenThe reactions which herbicides and agricultural pesticides in general undergo 0fSi!"*"' H|MwUlc recommendations of rates, times, and methods of application of in soils and the products formed are important with respect to residues in soils. 4 r£':K"^'''>Ip'<'''« nmy be based on weather forecasts and analyses of soil samples Weed research scientists should- emphasize this phase of herbicide research. pi^Jfmin fanners' fields (25). PhotodeeomposiUon.—Less is known about the direct effect of light on the fei^'s"* *)"1* "f "I0 "1(>st ur ^ent needs for research on the fate of herbicides in soils breakdown of herbicides than other factors suspected of being involved. How- i||4;M* MiofhwlH of Isolation and identification of herbicides and breakdown products. ever, photodecomposition of monuron was demonstrated by Hill, et al. (SS). S!fJ'"'°'°'{'f'n' an< ^ chemical assays must be improved and new ones derived. RadioWhen a solution containing 88.3 p.p.m. of monuron sealed in quartz tubes was |i;i|.pf««illv(» lHnfo])es bnve lieen used very little to study the decomposition of soilexposed for 48.days to sunlight, an 83-percent loss of monuron occurred. Hill, s'iif':*;tff*pl!wl herbicides. Soil samples could be treated with different lots of an et al. .(S3) concluded that in dry areas of the Western United States monuron *V;.! IwrWoUle with each lot tagged with C" at different positions in the molecule. may be inactivated by ultraviolet irradiation. They suggested that this factor •5 HJT known analytical techniques the unchanged herbicide and many reaction would account for disappearance of only a small part of the herbicide in •;,.:." fwxlurfu could be separated and identified. Radioisotopes should be most useful humid regions where frequent rains move it into soils. j,'.-ilO«l* In future research of this nature. Neburon, diuron, monuron, fenuron, and DMU were applied as alcohol solui" An new herbicides are developed, their behavior in soils in response to varitions to filter paper ( H ) . After the paper dried, it was exposed to ultraviolet *Wi« soil characteristics, weather conditions, and cultural practices must be light for several hours. The herbicides were not visible prior to exposure, but t the awns in white light tiejr were readUj- T'isiMe after erpwGrrw as light fan spots. The compounds were apparently changed during1 exposure. The effects of shade, moisture, and position in the soil on the residual activT.ITEKATUKE til til ity of monuron, diuron, and simazine were investigated in cooperation with the California Agricultural Experiment Station. The activity of monuron and (1) Aldrich, R. .7. Herbicides, residues in soils. Agric. and Food Chem. 1: diuron disappeared more rapidly from soil exposed to the sun from shaded 257-260. 1953. soil. The activity of monuron and simazine disappeared more rapidly from (g) a nd Willard, C. J. Factors affecting the pre-emergence use of moist soil than from dry soil. Monuron, diuron, and simazine were not affected 2,4-D in corn. Weeds 1: 338-345. 1952. to the same degree by these variables. Soil temperature was measured but not (S) Alexander, M. and Aleem, M.I.H. Effect of chemical structure on microcontrolled in this experiment, and soil temperatures varied considerably among bial decomposition of aromatic herbicides. (Manuscript submitted to the treatments during the day. Temperature markedly Influences vapor presAgric. and Food Chem,) 1960. sure and chemical reactions. Therefore the difference in the rate of disappear(//) Anderson, W. P., Linder, P. J., and Mitchell, J. W. Evaporation of some ance in shaded soil and soil exposed to the sun cannot be attributed unquesplant growth regulators and its possible effect on their activity. Science tionably to light inactivation, 116: 502-503.1952. 64 65 per presented (38) Hill, G. D., Soil factors as related to herbicides actio 5) Ashton, If. M. and Sheets, T. J. The relationship of soil before The Weed Society of America. New York. 1956. of EPTC to oats injury in various soil types. Weeds 7: 88-90. Bingeman, (S3) .- , McGahen, J. W., Baker, H. M,, Finnerty, D. («) Andus, Ij. J. Biological detoxication of 2,4-dichlorophenoxyacetic acid in C. W. The fate of substituted urea herbicides in agricultural soils. soils: isolation of an effective organism, Nature 166: 356. 1050. Agron. Journ. 47 : 93-103. 1955. (7) -. The biological detoxication of hormone herbicides in soil. Plant (34) Hollingsworth, E. B. and Ennis, W. B., Jr. Some studies on vapor action and Soil 3 : 170-102. 1051. of certain dinitro compounds upon young cotton plants. Proc. SWC 6: Plant Growth Substances. Leonard Hill Limited, London. 1959. (S) 23-31. 1953. (0) Barrens, K. 0., Lynn, G.' E., and Eastman, J. D. Experiments on the (35) Holstun, J. T., and Loomis, W. E. Leaching and decomposition of 2,2reduction of high temperature injury to cotton from DNOSBP. Proc. dichloropropionic acid in several Iowa soils. Weeds 4: 205-217. 1956. SWC 6 : 33-37. 1053. (36) Jensen, H. L., and Petersen, H. I. Detoxication of hormone herbicides by Bleidner, W. 0., Baker, H. M., Levitsky, M., and Lowen, W. K. Detersoil bacteria. Nature 170 : 39-40. 1952. mination of 3-(ip-chlorophenyl)-l,l-dimethylurea in soils and plant (37) Leopold, A. C., Van Schaik, P., and Neal, Mary. Molecular structure and tissue. Agric. and Food Chem. 2 : 476-479. 1954. herbicide adsorption. Weeds 8: 48-54. 1960. (11) Burschel, P. and Freed, V. H. The decomposition of herbicides in soils. (38) Linder, P. J., Shaw, W. C., and Marth, P. C.. A comparison of the relative Weeds 7 : 157-161. 1959. vapor activity and the relative rates of evaporation of several carW) Carroll, R, B. Factors influencing the activation of 2,4-dichlorophenoxybamates. Proc. SWC 8: 306-308. 1955. ethyl sulfnte. Proc. SWO 4 : 13. 1951. (39) Marth,: P. C. and Mitchell, J. W. Comparative volatility forms of 2,4-D, (13) -. Activation of sodium 2-(2,4-dichlorophenoxy)ethyl sulfate. ConBot. Gaz. 110: 032-637. 1949. trib. Boyce Thomp. Inst. 16 : 409-417. 1952. (!>(>) McKenzie, R. E. Activation of mylone, a temporary soil sterilant. Res. Coggins, 0. W. Department of Horticulture, University of California, Prog. Rpt., WWCO, p. 105. 1957. Riverside, California, personal communication. Nov. 0, 1957. (1,1) Minarik, C. E. Pre-emergence herbicides and their behavior. Proc. (15) - and Crafts, A. S. Substituted urea herbicides : Their electroNEWCC (sup.) 5: 29-39. 1951.' phoretic behavior and the influence of clay colloid in nutrient solution (42) Newman, A. S., and Thomas. R. J. Decomposition of 2,4-dichlorophenoxyon their phytotoxicity, Weeds 7 : 349-358. 1959. acetic acid in soil and liquid media. Soil Sci. Soc. Am. Proc. 14: 160(1G) Crafts, A. S. Toxicity of 2,4-D in California soils. Hilgardia 19: 141-158. 164. 1949. 1049. (43) Norman, A. G., and Newman, A. S. The persistence of herbicides in soils. (-17) Danielson, L. L. Mode and rate of release of isopropyl N-(S-chlorophenyl)Proc. NEWCC 4: 7-12. 1950. carbaumte from several granular carriers. Weeds 7 : 418-420. 1959. (18) -, Genrner, W. A., and Jansen, L. L, Besearch in progress, Agri- (44) Ogle, R. E., and Warren, G. F. Fate and activity of herbicides in soils. Weeds 3: 257-273. 3954. • cultural Research Service, U. S. Department of Agriculture, Beltsville, (45) Rahn, E. M., and Baynard, R. E., Jr. Persistence and penetration of Maryland. I960. monuron in asparagus soils. Weeds 6: 432-440. 1958. (19) Davis, D. JO. Some factors that, affect the phytotoxicity of water soluble (46) Ready, D., and Grant, V. Q. A rapid sensitive method for determination DNBP. Weeds 4 : 227-234. 1956. of low concentrations of 2,4-dichlorophenoxyacetic acid in aqueous solu(20) Davis, F, TJ. and Selman, F.' L. Effects of water upon the movement of tions. Bot. Gaz. 109: 39-44. 1947. dinitro wood killers in soils. Weeds 3 : 11-20. 1954. Sheets, T. J. The comparative toxicities of four phenylurea herbicides in DeRose, H. H. Persistence of some plant growth regulatoi-s when applied several soil types. Weeds 6: 413-424. 1958. to the soil in herbicidal treatments. Bot. Gaz. 107: 583-589. 1946. •. The uptake, distribution, and phytotoxicity of 2-chloro-4,6-bis (ethyl(48) -. Crabgrass inhibition with 0-isopropyl N-(3-chloropheny])-carbaamino)-s-triazine. Doctoral thesis, Univ. of Calif., Davis, California. mate. Agron. Journ. 43: 139-143. 1951. 1959. , (23) and Newman, A. S. The comparison of the persistence of certain The toxiclty of simazane to seedling oat plants (Manuscript sub(40) plant growth regulators when applied to soil. Soil Sci. Soc. Am. Proc. mitted to Weeds). 1960. 12:222-220.1948. and Crafts, A. S. The phytotoxicity of four phenylurea herbicides (50) (24) Dowler, 0., Baughman, N. M., and Veatch, C. The effect of soil type, in soil. Weeds 5 : 93-101. 1957. soil pH, and simulated rainfall on the distribution of DNBP in the (51) Sherburne, H. R., and Freed, V. H. Adsorption of, 3(p-chlorophenyl)-l,lsoil. Weeds 6 : 281-288. 1958. dimethylurea as a function of soil constituents. Agric. and Food Chem. (25) Ennis, W. B., Jr. Some soil and weather factors influencing usage of pre2: 937-939. 1954. emergence herbicides. Proc. Soil Sci. Soc. of Florida 14: 130-139. 1954. (58) _J——, Freed, V. H.; and Fang, S. C. The use of C1' carbonyl labeled (2C>) . Weed control in principal crops of the southern United States. 3(p-chlorophenyl)-l,l-dimethylurea in a leaching study. Weeds 4: 50-54. Adv. in Agron. 7: 251-297. 1955. 1950. (27) Evans, W. C., and Smith, B. S. W. The photochemical inactivation and '(53) Smith, R. J., Jr., and Ennis, W. B., Jr. Studies on the downward movemicrobial metabolism of the chlorophenoxyacetic acid herbicides. Bioment of 2,4-D and 3-chloro-IPC in soils. Proc. SWG 6: 63-71. 1953. cliem. Jour. Vol. 57. 1954. (54) Sund, K. A. Residual activity of 3-amino-l,2,4-triazole in soils. Agric. and (£8) Feldman, A. W., Technical summary on Falone. Naugatuck Chemical Food Chem. 4: 57-00. 1956. Division, United States Rubber Company, Naugatuck, Connecticut. (55) Swanson, C. R., Shaw, W. C., and Hughes, J. H. Some effects of isopropyl Undated. N-(3-chloropheuyl) carbamate and an alkanolamine salt on germinating (29) Freed, V. H., Montgomery, M., and Traegde, S. C. Physical properties of cotton seeds. Weeds 2: 178-189. 1953. S-ethyl-di-»-propylthiolcarbamate. Res. Prog. Rpt., WWCC, pp. 89-90. (56) Upchurch, R. P. The influence of soil factors on the phytotoxicity and 1058. plant selectivity of dim-on. Weeds 6: 161-171. 1958. (30) Gnrd, L. N., and Rudd, N. G. Herbicides determination: isopropyl Nand Pierce, W. C. The leaching of monuron from Lakeland sand (57) (3-chlorophenyl) carbamate (CIPC) in soil and crops. Agric. and Food soil. Part I. The effect of amount, intensity, and frequency of simulated Chem. 1: 630-632. 1953. ) rainfall. Weeds 5: 321-330. 1957. (3t) Hannah, L. H., Field studies with two new classes of herbicidaL'chemi(58) —'— and Pierce, W. C. The leaching of monuron from Lakeland sand cals; Proc. SWC 8: 316-321. 1955. soil. Part II. The effect of soil temperature, organic matter, soil moisture, and amount of herbicide. Weeds 6: 24-33. 1958. 67 Weaver, E. J. Reaction of certain plant growth regulators with iol^Kchangers. Bot, Gaz. 109 : 72-84. 1047. (60) - . Some uses of activated carbon in contratoxiflcation of plant growth regulators. Bot. Gaz. 110 : 300-312. 1948. (61) Whiteside, Jean S., and Alexander, M. Measurement of microbiological effects of herbicides. Weeds 8: 204-213. 3960. (62) Young, H. 0., and Carroll, J. 0. The decomposition of pentachlorophenol when applied as a residual pre-emergence herbicide. Agron. Jour. 43 i 604-507i 1951. Senator HART. Do you have any comparable information about dioxin ? Dr. BAYLBY. The answer at this point is no. Senator HART. Are any tests being run? Dr. BAYLBY. Not yet. One of the problems we had here is developing the methodology for testing these in products. As soon as this is settled, we will be able to expand our efforts and find this information. Senator HART. And you hope you will find that it has not been building up in our bodies every day ? Dr. BAYLEY. We sincerely do yes, sir. But that will not bias our results, I assure you. Senator HART. Perhaps Dr. Byerly might help us with this. I am told by my notes here that dioxins are chlorinated hydrocarbons and that these tend to be stable, and more significantly, in view of the fact that other dioxins are known to be absorbed and retained in the tissue of animals, isn't it likely that there is a build-up in the human of the dioxins found in 2,4,5 ? Dr. BYERLY. I will give you an opinion first. You stated since they are chlorinated hydrocarbons, they therefore would be persistent. Tli is does not necessarily follow. There is a very wide range from almost complete persistence, if you like, long-time persistence, to a very short-time persistence on the part of some other chlorinated hydrocarbons. With respect to these which are in the family of chlorinated hydrocarbons, of which 2,4,5-T is a member, the time of disappearance substantiated by empirical evidence, it is a matter of a few months; With respect to the dioxins themselves, Dr. Bayley answered you quite frankly, we do not have the information. We will seek it when the methods are complete. ' Senator HART. In your report you state that you agree that more rigorous tests on teratogenicity should be imposed before registration. If you favor such test as a requirement for registration, isn't there an inconsistency in allowing 2,4,5-T, which is a pesticide with suspected teratogenicity to be allowed to continue in a registered status pending the outcome of the tests? In other words, should you not deregister it now and then if the tests prove negative, register it? Dr. BAYLBY. The basis on which we would take such action would have to be on the consideration that we believe a hazard now exists. Based on the information which has been provided to us from the Department of Health, Education, and Welfare, and on our own analyses of the levels of the containment we do not believe such a hazard exists at this time. in terms Senator HART. You cite the content of four lots of producers of the content of, as you put it, TCDD. How many • of that product are there ? Dr. BYERLY. There are only three primary producers, sir. There are many f ormulators. Senator HART. Is a formulator engaged in a process which changes the generic business ? Dr. BYERLY. No. Senator HART. Almost the total of production is from three sources, right? Dr. BYERLY. Primary production. I understand the check has been made and the importation is very small. Senator HART. After you have registered the product, how frequently do you check up on the amounts of dioxins in the products that are being produced ? Dr. BAYLEY. Mr. Chairman, one of the requests which we are making of the Congress this year is to strengthen our law so that we can have plant inspection and insist on quality control within these particular plants. We have asked the Congress to help strengthen our activity in this area. It is not adequate, at the present time. We very definitely need legislation to improve it. Senator HART. Is that sort of a way to say that you do not check the dioxins ? Dr. BAYLEY. This is the first time. One of the reasons, of course, is that the methodology has only been newly developed. The results presented here were developed through check procedures with our laboratories, the Food and Drug Administration, and the industry group to see that our methodology was technically correct. So we are just getting started. Senator HART. You will include in your budget request moneys to provide what ? Dr. BAYLEY. The President's budget includes an increase of approximately $2.4 million for the pesticide regulatory division. This is between 50-and 100-percent increase in the funding for that organization. Senator HART. Do you believe that with that sum you would be in a position to have plant inspections on a regular basis ? Would you be able to have an enforcement staff which would be able to move in the event a violation was discovered? Is this the sort of thing that you visualize ? Dr. BAYLEY. Yes. We based that estimate in our budget request OB what we currently thought was necessary to do this. Now, we all recognize that the problems of concern about chemicals are expanding, and I would not want to suggest that is the final request that we would make in order to improve our operations. We put those in believing they currently were adequate from the standpoint of the enforcement and registration procedures. Senator HART. Is the table which shows the amount of dioxin of this particular type—is there a test and do you have, facility and personnel to attempt to identify the existence of any of the other seven possible dioxins ? , Dr. BYERLY. This, sir, is in the process of development in coopera-. tion with the Department of Health, Education, and Welfare and 69 dustry. These methods are in the process of development. Tor some of them the methods are quite adequate; for others, the methods have to be developed. Senator HART. For some you feel you can ? Dr. UTTERLY. Yes, sir. ; Senator HART. In that case you do. Dr. BYBRLY. We will. Senator HART. Hinged on the money problem ? Dr. BYERLY. No, it depends primarily on the development of the competence of personnel. This is the thing that has to grow. We have to have a cadre of people who are highly competent. These are most sensitive methods and we cannot just create people who can handle them overnight. Senator HART. In the testimony of the first two witnesses, and I do not recall whether it was that of Mr. Wellford or Mr. Turner, it is my impression there was a reference to a chick embryo study. Do • you recall the comment they made? Dr. UYERLY. Mr. Chairman, I do not recall the specific comment. There is a chicle embryo test. It is highly sensitive. I would point out that it lias, as a screening test, possibilities. Again, its sensitivity ' would make me want to suggest that we be very careful with respect into whose hands the tests were put. With respect to the teratogenicity, with respect to the chick embryo, the application in this test' is hardly like the applicationj either to the skin or in the food of a mammal. So a direct compari son of the effects in the chick embryo with rats and mice, the tradi-r tional ones, would require a great deal of review. Dr. BAYLEY. It would really require a correlated study to ascertain the relationship. Dr. BYERLY. It would indeed. Senator HART. I think just from what we have heard this morning, everyone on this subcommittee will be'eager to assist you ' in obtaining the additional moneys, whether it is 2.4 million or more to insure the development just as rapidly as possible of the technical data on which to base tests and the human hands to administer them. Let me get into this burden of proof again—we sort of dismissed it—very briefly. The basic conclusion of your testimony is you have not found that registered uses of 2,4,5-T without a finite tolerance on food crops constitutes a hazard requiring cancellation or suspension of such registered use. Dr. BAYIYEY. That is correct. Senator HART. And yet this morning we have heard testimony that preliminary tests suggest that 2,4,5-T when contaminated by dioxin comparable to that found in currently produced 2,4,5-T is tcratogenic in three species; that the Mrak Commission or a panel advisory to it said that the teratogenic effects in one or more such species should be grounds for immediate restriction of pesticide use; that residues of 2,4,5-T arc now found on approximately one out of every 200 food samples analyzed by FDA; that we can't be sure of (lie amounts of totradioxin 'in 2,4^5-T now being sold, nor do we have as yet clear ideas on the amount of other dioxiraWn the pesticide, some of which may be more potent than tetra; that no evidence suggests that these dioxins are not persistent or cumulative in human tissue, and that some evidence which would indicate perhaps they are. If you accept that as a premise, in view of all of this, would you say that you are sure that registration of 2,4,5-T for use directly on food crops does not constitute a hazard to man ? Dr. BAYLEY. I would say that the information we have does not give us indication that it is a hazard to man in accordance with the registered uses. I think we have to recognize that—and I am sure the committee and we are in agreement—these are all economic poisons, and the purpose of registration is to provide for their use in such a way that they are not a hazard. That is the basis on which we make our judgment. Senator HART. Your position is that they do not constitute a hazard ? Dr. BAYLEY. Yes, sir. And our position is based not only on our own data but, that provided to us from the medical authorities of the Department of Health, Education, and Welfare, and to add, 20 years of safe use. Senator HART. The first two witnesses described the difficulty of finding the brand name on a deformed infant. There are lots of birth defects. How can anyone say over 20 years that this has not been a factor in some of these private tragedies. Dr. BAYLEY. I do not in any way want to be facetious, but I think we have to recognize that one of the compounds closely related to this contaminant is lysol, a rather common household disinfectant, and I hear no suggestion that we take this off the market. We in the Department of Agriculture recognize that there is a large group of chloropheuols that we are going to have to examine to find out whether there is a real hazard or not. I am not here to raise a scare, but I think we recognize that in. dealing with these compounds we must have evidence that they are a hazard or .we will bo dealing : with emotional conjecture based on inference from various scattered data. Senator HART. To make clear what is meant, what is your position with respect to lysol ? Dr. BAYLEY. Wo have no reason to take action at this time. Senator HART. Do you have in process studies or evaluations to see whether you modify that ? Dr. BAYLEY. Within our capability, for example, we have already moved out to ascertain the dioxin content of 17 other pesticides in this area, and we recognize that this is a field in which we want to make intensive study. I do not single out this particular product as one which I would consider as more hazardous than any of the rest, but merely as an example of the total problem that we have in meeting these pesticide issues. It seems to me that from the standpoint of protecting the public health, the important thing for us to do is to take those which, based 70 ^Bn scientific data appear to have the greatest potential hazaro^md put our resources on evaluating these as we go ahead. This is true of the mercurials. We are looking very closely at all •of these compounds. I think we must recognize 5iat we are going to have to do this on a priority basis as we go. We are giving attention to 2,4,5-T, but wo need to have the appropriate facts as we proceed. Mr. BIOKWI.T. I think what has come out here is that really it all boils down to a question of burden of proof. While you say there is no evidence that 2,4,5-T is hazardous, I would have to dispute that. Assuming that you are right, that there is no evidence that it is hazardous, and yet it cannot be shown that it is not hazardous; on what do you base your inaction ? Dr. BAYLEY. Let's recognize first of all that these are economic poisons. We should all agree to that to start with. And when I USB the word "hazardous" I use it in terms of sufficiently hazardous to take action. This is bound to be a judgment based on scientific— including medical scientific data. If there is a disagreement between us, then it is in this judgment, not in anything else. Mr. BICKWIT. I recognize my inability to make adequate scientific judgments and, as a result, defer to those who I regard are .capable of making such judgments. The panel which reported to the Mrak Commission has recommended whenever teratogenic effects of a given pesticide are shown in one or more mammalian species, that immediate steps should be taken to restrict the use of that pesticide. Are you rejecting that advice ? Dr. BAYLEY. No. The use of this pesticide is already restricted because of the registered uses. Mr. BTCKWTT. I suspect that the thrust of their statement would require that it be further restricted but perhaps we would have somo difficulty pursuing what the exact intention of their statement was. You say that you must believe that a hazard exists before you can take a pesticide off the market and that in the case of 2,4,5-T you do not believe that a hazard exists. Dr. BAYLEY. We do not believe that a hazard exists which would authorize us to take it off the market, yes. Mr. BICKWIT. Are you sure that a hazard does not exist? Dr. BAYLEY. One can never be absolutely sure that a hazard does not exist, even if we are talking about table salt. , In fact, we know that table salt is hazardous if taken improperly, and we don't even register it. Mr. BICKWIT. There is a distinction from table salt in this case, and that is that there has been evidence that suggests, and to my mind rather strongly suggests, that there is a hazard here. Dr. BAYLEY. I do not see the difference that you are trying to point out. Mr.- BICKWIT. You do not think that the studies that have been done by FDA, by NIEHS, although preliminary, establish that there is any greater hazard than the hazard of table salt? Dr. BAYLEY. No; I did not say that. Mr. BICK.WIT. I misunderstood you. Dr. BAYLEY. I did not say that. But again I come back to the point that we are dealing with economic poisons. There are hazards • " A use of all of them. The decision that has to be WGe is are the ^.ww^ls sufficiently great to take action at this particular time. That pii!$a, * hn di (Terence. F&-*w\t4t ]jiOKwm you do say issues involving human health should priority over all other issues. .*•!* 1 » -A,FfV A. l l l J It J. UO* OJ.J. • ?«.; Mr. BICKWIT. What I am not clear on is whether you have to ||t*«tively believe there is a hazard before you take a pesticide off the Jjfgiwarket, J,: ;1» Uiat a legal requirement? ;* Dr. BAYLEY. I don't know what you mean by actively believe. £::.Mr. BiCKwtT. I conceive of relative states of mind as being belief, «t*loof suspension, and state of disbelief. ij1". Do you think you have to be on the belief side of state of suspenjiion in* order to take a pesticide off the market? •;''•,3f you are in a state of suspension, would that authorize you J|epilly to take it of! the market? |r Dr. BAYI,KY. The information provided to us has not shown that ilhere "m sufficient hazard for us to take action, and the information ,,-!f»!widcd to us from the Department of Health, Education, and ||;W«lfare is the information primarily that we have used. fel'Mv. BICKWIT. And you do not feel that if you are not sure one „,-.-,- or the other that that would authorize you to take it off the *;Wrket? ,*iy, Dr. BAYLEY. The data that we have at this time are not adequate P$tO show us that there is a hazard, and the data to the contrary are p|!»wflki' The lh»imrtmont announced on February 6 that it would undertake examina'**•*• Of 2,4,5-T nnd 17 related compounds registered for pesticidal use to deter,; M!IN> tthi'lhei- or not they are contaminated with dioxins. Preliminary results . «•* 3M.&-T dhow that those lots examined of current manufacture and those ; V&* In clmnnelB of trade gave the following results: i TABLE 1.—AMOUNTS OF TODD FOUND IN COMMERCIAL 2,4,5-T BY TWO METHODS TCDD Content p.p.m.' Manufacturer Lot Grade 1 Collected USDA FDA 120110 07-020 X-17394 21-5 MM-120449 TG TG TG TG 2/70 2/70 2/70 2/70 trace 1.1 W D» . 48 0.07 2.9 N.D. . 47- 52 ?•; SKJIUM ...... l**~— - ... Dow 1S"*t«hnlt«l grade. ttaOlMm lo the 2,3,7,8-tetraohloro-dibenzo-p-dioxin (TCDD). """• •- - 1 - I TCDO are below the limits of detection or below 0.05 p.p.m. I by Dow as a reference check and reported to contain about 0.5 p.p.m. TCDD. data are preliminary and are obtained from first drafts of methods r, Ttachlrley also headed a team of scientists who investigated allegations' |tf'toinry to humans and animals due to herbicide treatment for control of by the Forest Service on the Tonto National Forest near Globe, ArlTlioy found that apparent damage consisted of damage to susceptible W nour the treated area from drift of the herbicides used. The alleged rlwi lo a cluck and a goat were found to be groundless. Human illnesses »thone expected in a normal population with the possible exception of one :wlth skin irritation on his eyelids. Clinical chemistry on specimens to«l during the investigation is in process, HART. I think in fairness to all we should recess to resume |ff'^(Whereupon, at 12:45 p.m., the subcommittee was recessed, to. rei nt 2:15 p.m., this same day.) 4ft-362—70 0 76 77 AFTERNOON SESSION lar scrutiny because of their potential ability to dSBlate certain classes- of living organisms, and thereby to upset the balance of nature—to disrupt what the ecologist refers to as the "ecosystem." I shall here limit my remarks to the potential dangers to the environment that might be expected from the excessive or otherwise incorrect use of one such substance: 2,4,5-T. As we have hoard this' morning, this compound has recently gained a degree of notoriety owing to its massive military use in Vietnam despite the suspected ability of it, or an associated impurity, to cause birth defects. I need not refer you to Thomas Whiteside's article on this :subject •- •>.•> , 1 - 1 _ ji _:_* Senator HART. The committee will be in order. Resuming this afternoon, our first witness is Dr. Arthur H. Westing. Dr. Westing3 is chairman of the Biology Department of Windham College in I utney, Vt. STATEMENT OF DR. ARTHUR H. WESTING, CHAIRMAN, BIOLOGY DEPARTMENT, WINDHAM COLLEGE, PUTNEY, VT. Dr. WESTING. Senator Hart, I consider it a privilege to be able to testify before your committee. Actually, I am very pleasantly surprised that you and your staff show such tolerance toward me despite a fairly questionable record with respect to your state, First of all, most of the year I lived in Michigan, I devoted to spraying your forests with 2,4,5-T, and perhaps even worse, when I left Michigan I took with me one of your most desirable natives as my wife. Senator HART. I don't know whether that makes an even trade or not. Dr. WESTINO. AlLthe time I was listening to this morning's testimony and realizing*^how muddled the situation was with respect to the medical and public health aspects and the legal and administrative aspects, I kept thinking that those aspects were crystal-clear in relation to the aspects that I am going to try to talk about, and that is the impact of 2,4,5-T on the environment. It is such a difficult field to cope with because ecology is still really in its infancy, particularly when it comes to the study of the full impact of a toxic introduction to the environment. To judge from the popular press, our nation is on the brink of environmental disaster. Ecology has achieved some sort of a mystical significance to many people, and a whole new vocabulary has emerged overnight utilizing that wonderful avant-garde prefix "eco." Over and over again we are being reminded of our collision course with "ecocatastrophe" leading to "ecocleath."'Wc are told that we have to really use drastic "ecotactics;" a Senator like you should be using aggressive "ecopolitics." The whole world is being challenged to accept a protocol on "ecocide." And I suspect that psychiatrists are very soon going to be diagnosing "eeoneuroses." Actually, the unhappy truth of the matter is that there may well be good cause for'Concern over the future of man's environment. It is being assaulted from all quarters with a gusto that is hard to grasp. Man has habitually ignored the impact that lie has had on the environment, the environment that all of us depend upon for our well-being and survival. Western man has always considered himself master of his surroundings. Until the present, with far smaller numbers and very ineffectual technology, this self-delusion made very little difference. P>iit today we are introducing a great diversity of chemicals into our environment in vast, continuing, and exponentially increasing quantities. Among these chemicals, pesticides are worthy of particu- it is one of a class of potent herbicides or plant killers, the one preferred by utility employees, foresters, range managers, and farmers, and by our armed forces in Vietnam for the destruction of unwanted woody vegetation. It is one of a class of growth-hormonemimicking herbicides whose close chemical relatives include 2,4-D, MCPA, and Silvex. 2,4,5-T was developed during the early 1940's (as a possible chemical warfare agent) and came into widespread domestic use during the mld-1950's. In 1964, some 13 million pounds of 2,4,5-T were manufactured in the United States. About a million of these pounds were applied to about 3 million acres of U.S. croplands, another million pounds were sprayed on perhaps 80,000 acres of forest lands in Vietnam, and most of the remaining 11 million pounds wore presumably used domestically on an undetermined number of acres of noncroplands. This morning we were vividly reminded that a small portion of this is also used by home gardeners. ». Now, these are the 1964 figures. Although I am not sure of the current ones, I understand that the domestic use of herbicides in general has been increasing at a compounded growth rate of 10 percent per annum. 2,4,5-T is commercially available in a number-of formulations of which the most important are the oil-soluble esters and the slightly less effective water-soluble amines. Whereas the amine formulations are very low in volatility, some of the ester formulations are relatively high and others are relatively low. The low volatility esters are actually somewhat more effective than the high volatility ones, but they arc also slightly more expensive. 2,4,5-T is also available in combination with 2,4-D, a mixture which is known domestically as "brush killer" and by the military as "agent orange." The 2,4,5-T is effectively applied either to the foliage of unwanted woody vegetation from ground- or aircraft-mounted spray rigs, or to their stems by a variety of techniques. Domestically, it is very often applied highly diluted by oil or water, although some domestic teclmiques of individual application call for strong concentrations. In Vietnam, it is aerially applied in totaly undiluted form. Recommended broadcast dosages—these are domestic recommenclations-^-rango* from one-half to three pounds of active ingredient per 78 79 re. At these levels of one-half to three pounds per acre, the quite 'selective, killing many species of broad-leafed woody plants •and sparing most grasses and conifers. At the high rates the military use in Vietnam—which is about 13 pounds per acre, together with as much again of 2,4-D—it becomes far less selective and kills - a high proportion of the vegetation. In their silvicultural applications, foresters do some aerial spraying, but often resort to individual application to unwanted trees. However, in range improvement and in the control of vegetation on rights-of-way, and in Vietnam, application is mainly or entirely from the, air. Overall, the domestic applications average out to about one-third to one-half pound per acre treated. That is a very brief summary of the use of 2,4,5-T. Now I would like to spend a few minutes on the potential dangers from the use of 2,4,5-T. I am limiting my remarks, by and large, to the dangers to the environment since the medical and public health aspects were covered previously, and I understand will be covered by subsequent speakers. Senator HART. Doctor, as you leave the use section and before you get into these potential dangers, can you describe for the record—I think it has not yet been stated in layman's language for the record— what the bush or tree or grass or area of earth surface looks like when this is applied to it, you say 1 to 3 pounds an acre. Dr. WESTING. That is right. Senator HART. If you can in language describe for the reader and me what it looks like. I frankly have not seen it. Dr. WESTING. Stretching my memory back to the Upper Peninsula. Senator HART. The beautiful Upper Peninsula. Dr. WESTING. I might interject here that a lot of pioneering work in aerial forest spraying was actually done in Michigan. The leaves on unwanted oaks or maples very rapidly turn brown, within a matter of 3 or 4 days. In 5 days they start showing signs of shriveling up. They usually hang on that way for 6 to 8 weeks, and perhaps longer; so, one sees a lot of trees that have brown, shriveled up leaves. If conifers are intermingled, they show no damage so they stand out like green thumbs, and a good bit of the forest floor stays green; grasses and so on stay green, ferns and so on will turn brown; some plants stay green and others do not, depending upon the type. What it looks like really in this country is as if fall had just decided to come a few months early. Senator HART, flow would you describe the same scene if there was applied to it the 13 pounds per acre which you say is the current application on the average in Vietnam? Dr. WESTING. It is actually about 25 or 26 pounds. It is 13 of 2,4,5-T plus another 13 of 2,4-D. Senator HART. The picture you described Dr. WESTING. Was for one to two pounds. Senator HART. Of 2,4,5-T only ? Dr. WESTING. Right. I have not seen an area myself that has been, hit this heavily, but I have seen pictures. Within a very short period of time, all the. leaves look brown and shriveled up and within a matd^P>f perhaps two to three weeks most of the leaves drop off the trees, vines, and shrubs. _ This, of course, is the reason why the military spray these herbicides and sprays them in such heavy dosages, in order to get as rapid a leaf defoliation as possible. But in the process of getting rapid defoliation, there is a high degree of kill, which is an unhappy corollary. I am not sure if this is really intended: it happens, particularly in certain types of vegetation. Subsequently, grasses, bamboos, and a variety of other weeds grow hack fairly rapidly. So, after several months you see lots of large •dead trees and then a very heavy new undergrowth. Senator HART. You say the tree does die ? Dr. WESTING. Well, it depends upon the species, Senator Hart. Mangroves would be killed by one application in Vietnam whereas some other trees might not be killed unless they were sprayed a second time. A single spraying seems to kill about 10 percent of the trees. There is a great diversity of tree species there. I have flown over areas in southeast Asia that have been sprayed •once and it seems that roughly one tree in eight or 10 is dead. If these were sprayed a second time 6 months later, perhaps two out •of three trees would be dead, or maybe even more. Senator HART. Thank you. . Dr. WESTING. I wish now to touch upon some of the potential dangers to the environment from the use of 2,4,5-T, and I am speaking again primarily domestically. The dangers can arise not only from the 2,4,5-T itself, but also from its contaminants, (such as were discussed at great length this morning), from its additives, (and 'there are endless kinds of additives: wetting agents, emulsifiers, •stickers, penetrants, thickeners, humectants, spreaders, etc.), from its •carriers or diluents, (such as fuel oil, kerosene, seal oil), and from its degradation products (or perhaps degradation products arising from subsequent burning). All of these various possibilities I shall lump together for purposes of my comments here, just calling them :2,4,5-T. _ The dangers from the use of 2,4,5-T need not be confined to the site of application, but can be carried elsewhere by wind, either as liquid or as vapor, or carried elsewhere by water, either surface 'water or ground water. Moreover, the potential dangers are not confined to the time of application, but last, of course, until the 2,4,5-T 'degrades to the level of insignificance. Under wet and warm field, •conditions, one of the advantages of 2,4,5-T is that it breaks down within a matter of several weeks, 6 or 8 weeks perhaps. But under dry and cool conditions, this may take well over a year. Furthermore, the rate of degradation in the groundwater may also be very slow. The dangers from the use of 2,4,5-T can result from damage to plants, damage to animals, both higher and lower, possibly from damage to microorganisms, and from direct and indirect combina'tions of these effects. I shall elaborate very briefly on some of these possibilities. The most spectacular effect of 2,4,5-T—when used as recommended •domestically—is, of course, on certain classes of plants, particularly 80 81 but not exclusively the broadleafed woody vegettat,ion. In selectively destroying such plants and sparing others, the species composition of the treated area is altered, the overall diversity of species is reduced, and the total mass of living things is probably diminished. And such changes are considered by ecologists to be an unstabilizing and therefore detrimental influence on an ecosystem. In other words, they make the balance of nature more precarious. A properly functioning, relatively undisturbed ecosystem owes its stability—indeed, its very integrity—to a highly complex set of interactions amongst all of its many living and nonliving components. Nutrients cycle and recycle from the soil up through the interlocking food chains and back again to the soil. Population levels of the many component plants, animals, and microorganisms are kept in balance by a staggering multitude of predator/prey, host/parasite, and other long-established interactions of mutual dependcncjr. As soon as a toxic factor such as 2,4,5-T intrudes upon this highly complex, totally interacting system, a certain amount of the so-called ecological buffering action (of the many inherent checks and balances) is lost, and things start going wrong. Erosion may bo accelerated, particularly in hilly terrain and even more particularly when streamside vegetation is killed. This effect, together with a reduction in the total mass of .the living component of an ecosystem inevitably leads to a loss to the area of vital nutrient materials. Especially following heavy or repeated applications, the result is a steady decline in the productivity of the treated ecosystem—something that may take it centuries from which to recover. Ou top of this there are all sorts of subtle things that can go wrong. For example, a continuing supply of available nitrogen—one of the elements.essential to all life, and often in short supply—depends to a large extent on. the presence of certain 2,4,5-T sensitive plants, whose roofs play host to various microorganisms crucial to this process. Actually, there has been some evidence of this occurring in the Pacific Northwest, where ponderosa pines are the crop tree and alders are being removed by 2,4,5-T as weeds, with a resulting loss to the area of available nitrogen. Additionally, the birds and other animals that depend upon the 2,4,5-T decimated plants for food or cover are placed at a groat disadvantage and may be partially or even completely eliminated from a treated area. • The direct toxicity of 2,4,5-T to most higher animals is known not to be very severe, particularly at the recommended rates of application. However, that there is also potential danger in this regard is suggested by its known effects on humans. The U.S. Department of Agriculture categorize 2,4,5-T as "mildly" irritating to the skin in a standard dermal response rating, and as "moderately" toxic when ingested. In fact, one can onote the following precaution from the product label: "Do not contaminate irrigation ditches or water used for domestic purposes;" and also the following warning: "Causes irritation of skin nnd eyes." 'Moreover, in aquatic habitats, the death of trout find some other fish has been reported when 2,4,5-T is applied at recommended rates for weed killing. Certain crabs, shrimps, and mollusks are also harmed by low concentrations of 2,4,5-T. The adverse effects on wildlife are not limited to the ones already alluded to. Some plants exposed to sublethal doses of 2,4,5-T (or 2,4-D) start producing abnormally high levels of nitrates (and in some cases there has been a suggestion of even cyanide). It has been noted with livestock that when such plants are ingested, the excess nitrates are converted ,to nitrites, toxic or even lethal to the animals. Another occasional result of 2,4,5-T application is that naturally poisonous, and usually avoided, plants are made attractive to animals as a result of 2,4,5-T spraying; and then the animals feed on these newly attractive plants and are poisoned. The known ability of 2,4,5-T to cause chromosomal damage in some plants and the fact that in some animals it, or an associated impurity, results in deformed offspring when ingested during pregnancy, suggest that the plant and animal populations thus affected will be less able to cope with their environment. All of these debilitations that I have been cataloging, and additional ones that I have not, do harm not only to the affected species, but, of course, thereby also to the ecosystems of which they are a partSince man is also a part of nature, I can bring out here once again for emphasis that there is strong reason to suspect that 2.4.5-T or an unavoidably associated impurity, the dioxin we have been hearing about this morning, 2,3,7,8-tetrachlorodibenzo-p-dioxin (or, by the way, a dozen or so closely related compounds all coming under the name of dioxin), are now known to be highly teratogcnic. In other words, they result in malformed offspring when ingested during pregnancy. Until this issue is clarified, I think it should go without saying that the use of 2,4,5-T both domestically and in Vietnam be restricted to locations and amounts that would preclude its possible human ingestion. Well, let me now make a few concluding remarks. Senator HART. Doctor, I think it would be wise if we interrupt briefly for a recess. That was a signal that sounded for a vote. I thin]?: this is the time to suspend. (Short recess.) Senator HART. Doctor, with luck we will finish before there is another vote. ' You were just about to begin with your conclusions. Dr. WESTING. It is possible that I have been painting somewhattoo grim a picture of the domestic use of 2,4,5-T. But I have no particular fears that detailed exposition of its safety and benefits can be left to the herbicide manufacturers and others. So, I figure that what I am describing here from the environmental standpoint is one side of the picture, and let the manufacturers tell us the other side. Senator HART. Let me react to that, but very briefly. It is not inappropriate or a matter of surprise, nor in my book, should it be the basis of criticism, if the manufacturer of the product describes it in glowing terms if society and its government permits him to market it. If those responsible for the protection of the health of the society conclude that he can market the product with those claims, then why get mad at him? Why don't we get mad at the society's institutions? • • F 82 83 Dr. WESTING. I agree. > -. ' The low volatile formulations are not only more effelWe as herbicides, but they are also much safer with respect to the problem of Senator HART. You can't have it both ways, if I make myself clear. drift and volatilization. Aerial spraying should be confined to relatively windless periods, • Dr. WESTING. Yes, I certainly am in full sympathy with this. I (wind speeds of less than 5 mph) and to air temperatures of less think the burden falls upon our regulatory agencies. Senator HART. Clearly. | than 85 degrees. Only nozzles equipped with course sprays should be Dr. WESTING. I don't think Dow is the culprit here at all. It is [' used. The cleaning of spray equipment or the dumping of excesses FDAandUSDA, and soon.', ' • near lakes or streams must be avoided; and getting rid of the empty cans and so on should be limited to sanitary land-fill dumps or simiSenator HART. This goes beyond the immediate product line we are talking about. This goes to the marketplace and the role of larly safe locations. To insure all of the above, State and Federal regulations should society in protecting itself, establishing regulations where needed, and enforcing them as established. be tightened both for manufacturers and users, and educational efforts increased with the aim of minimizing unnecessary or excess, Dr. WESTING. I would certainly have to admit that the vast sucapplication. Our flagrant misuse in Vietnam should be halted immecesses of productivity upon which our nation's current affluence diately (see, e.g., my article in the Friends Journal of 1 April 1970). hinges, depend to a large extent' upon the use of pesticides such as Finally, I wish to stress once again the complex and as yet little 2,4,5-T. And it seems clear that the use of pesticides will continue, understood nature of our environment. The study of ecosystems asperhaps even unabated, without a highly unlikely downward trend in such is still in its infancy. And since hormonal herbicides have been our population, and, even more particularly, in our collective desires and demands. in general use now for only two decades or less, we simply are not yet able to predict the full range of potential disasters that their Plowever, the time seems to be fast arriving when certain precauunrestricted use may inflict upon us and all other living creaturestions must be taken so as not to overload our environment with such potent pollutants. A number of suggestions are thus in order to with which we share this small world. Senator HART. Doctor, for all its brevity, this is a very helpful forestall the need for a basic change in our way of life. •' , First of all, research efforts' should be expanded on several fronts^ | statement. I have a couple of questions that I would like for you to react to. Effective cultural and biological controls of pest species should be You tell us in dry and cool conditions it may take well over a sought and developed with renewed vigor. With respect to the pestiyear for 2,4,5-T to degrade. I think you were here this morning. The cides themselves, highly selective and rapidly degrading ones should be aimed for. Department of Agriculture is not in agreement with that statement. Can you give us some evidence for your statement, or refer us to In the light of the current 2,4,5-T affair, I must add here that all pesticides, existing and potential, must be rigorously tested prior to sources that are in agreement with your statement ? Dr. WESTING. To my knowledge, there has been precious little their general release for possible toxicity, carcinogenicity, teratogenresearch done on the life of 2,4,5-T in the environment. I am aware icity, and mutagcnicity to humans; and additionally, for possible of one study that was done in a forest environment in which it was adverse effects on livestock, on wildlife, on game, on fish, and on other components of the ecosystem.shown that 2,4,5-T degraded to insignificance in a matter of several With respect to 2,4,5-T, its use—in my considered opinion—must months, as I recall. On the other hand, it has been well established, and it is clearly be limited to areas remote from human habitation. Control of vegeknown, that 2,4-1)—a compound similar to 2,4,5-T—degrades much tation on rights-of-way must be regulated with particular care since more rapidly than 2,4,5-T. It has been demonstrated a number of utility, transportation, and other rights-of-way are by their very times that under dry conditions, 2,4-D can persist in the environnature frequently close to civilization. I want to emphasize here, ment and have detrimental effects for as long as a year or a year Senator Hart, that one of the major uses of 2,4,5-T—one of its preand a half after application. From this I infer that 2,4,5-T?>which is ferred uses—is in woody vegetation control along rights-of-way. more persistent than 2,4,5-D, would have at least a similar life under This is a major place where 2,4,5-T is likely to impinge upon human habitation, to come in contact with civilization. dry conditions. Senator HART. Then, adopting your reasoning, it would mean that Broadcast applications, where safely remote from human habitaunder those conditions, 2,4,5-T might be found on food that istion, should not exceed 3 pounds per acre; and where spraying covers served months after the spraying of the crop; is that correct? •extensive areas, unsprayed zones should be left as oases for wildlife, .and so forth. Dr. WESTING. I have no direct information, but one could surmise that this could happen. This is a possibility. Repeat applications should be controlled, perhaps to intervals of Senator HART. What would you think the possibility of 2,4,5-T's3 years or more. Aerial broadcast spraying should be avoided where capacity is to persist within the organism, plant or animal, which possible, and always avoided near bodies of water, in favor of spot applications, or individual applications. had ingested it, including the humans Dr. WESTING. I have no first-hand knowledge on this whatsoever f In those areas where aerial spraying is permissible, the highly volso I prefer not to try to answer it. , atile (though cheaper) formulations should be banned completely. Senator HART. You would agree that it is impossible to t possible? Dr. WESTING. The likelihood is there. As far as I know, it may persist, or even build up in the human body. Some other chemicals that are fat soluble (as are the ester formulations of 2,4,5-T) are known to deposit and be stored in the fatty tissue of humans; so, it is highly possible that 2,4,5-T does this, but I simply do not know whether it does or not. Senator HAIIT. You suggest that nso of 2,4,5-T be limited to areas remote from human habitation, and that it should be restricted in .other respects. -Does that mean that you would feel that Dr. DuBridge's suggestion that pesticides be deregistered for food use, assuming there can be lio tolerance level set by FDA doesn't go far enough? Dr. WESTING. '2,4,5-T as it is commercially available with its impurities, is a substance that should not have any food tolerance at all. It should haA^e zero tolerance, 'at least given the current state of knowledge. ' • . . . - , > Certainly, the suggestion made this morning by—I think it was Mr. Well ford—that its use should be curtailed severely, or suspended until Ave clarify this whole issue is one that I fully support. I think that 2,4,5-T is probably a safe chemical to. use at, the Iqw^ t recommended doses in areas remote from human habitation. I don't think it need be banned under such conditions in the forest environment, or on range lands. On the' other hand, along power line rights-of-way, railroad rights-of-way, and so on, that get near houses, I think there should be severe restrictions. ' Senator HART. What about proximity to crops? Dr. WESTING. Food crops? Senator HART. Yes. Dr. WESTING. I think that certainly for the time being, it should not be registered for use on food crops and not be used near them. Senator HART. In these areas that you have described where 2,4,5-T has been applied you have said that some of the birds and animals that depend on the plants that have been destroyed may be eliminated. Which birds and which animals are likely to be affected ? ' One way to answer that I suppose is any that are in that area, but I am trying to find out if some are and others are not affected. Dr. WESTING. I wish I could give you some spectacular answer about bird X or T having become extinct as a result of the use of 2,4,5-T, but I cannot. I can quote a recent statement made by a British authority on pesticides, Dr. N. W. Moore, director of tlie Monks Wood Experimental Station in England: ' The vi.se of 2,4-D and 2,4,5-T to control scrub by roads and in woods reduces the essential habitnt of almost; all British land birds, which, because they are mirvivnvs of the original forest fauna, are still dependent on trees and bushes. (Advances in Ecological Research 4:108; 1907) To judge from this statement Dr. Moore is concerned over the fate of the native British birds as a result of the routine use of these herbicides. In this country there is an extensive program over many tens of thousands of acres in the West of sagebrush control in which herbi- cides of this nature are used, primarily 2,4-D. There is some evi•dence that the sage grouse population has been deplelj^fc at least the hunters are not as happy as they used to be. ^^ I have to warn you, Senator Hart, this is one area where the herbicide proponents will jump up and say that there are a number of clear cases where the use of herbicides has actually benefittecl wildlife populations. Senator HART. I made an interjection earlier to say that if we are going to get mad at somebody let's get mad'at ourselves first of all as a people for not recognizing dangers and setting down the laws that will prohibit the marketing of certain things, but equally true, •of course, is that the producer is obligated, absent any explicit regulation, to make truthful representation about its product—again, I am thinking not of chemicals alone but anything—and report factually the experience that has come to his attention to whatever public agency there is that is expected to make the judgment for all of us as to whether that product in fact should be marketed. So, if they jump up and explain it is good for us, I hope, they will not do so unless they can explain why/ . •• You noted, among other things, in your conclusion that we should expand research, attempting to develop other controls of pests. What development do you imagine would be fruitful? Dr. WESTING. Well, the main thrust of alternatives to the use of insecticides has been to introduce predators or diseases of the insect pests. This same approach can also be used with herbicides such as 2,4,5-T. Plant pests are a little less amenable to this sort of an approach, but one could push ahead on research on. possible virus diseases or fungus diseases or insect enemies of weed species. I am familiar with one success story in this regard. A serious weed in the Northwest is St. Johnswort, and a beetle (chrysolina) has been introduced from Australia that feeds on the St. Johnswort, in a highly successful alternative to. chemical herbicides. This general type of approach should be exploited to the greatest extent possible. There are all kinds of other possibilities. Just in forestry, for example, closer spacing of crop trees shades out certain weeds. You can go back to a greater emphasis on some of the mechanical methods that are now avoided because of the high cost of labor: mowing, weeding (pulling out the weeds or cutting them doAvn), burning. Flamethrowers are used in certain instances and even controlled fires. These methods have a much more selective effect on the actual weed and a minimum of lasting untoward side effects. If chemicals are to be used, the forester's approach of individual application is far preferable to the utility and range manager's approach of broadcast spraying from the air. So, there are a variety of alternatives available. With just the slightest amount of urging,' the slightest realization that there is a necessity to worry, these alternatives would at least be explored. In the past it had never even been realized that there were possible ill side effects to the use of herbicides. Senator HART. Now, you have lectured us quite thoroughly on the dangers inherent in changing the ecological pattern. Yet every one 86 87 Bf these alternatives that you talk about suggests similar ' and some additional ones. The Australian beetle is not native to the Northwest, I take it, but you are going to bring Australian beetles in. The flamethrower is not really an altogether acceptable Dr. WESTING. There is a history of introducing something to< combat a pest and thereby introducing a worse pest, so there has tobe some very careful preliminary testing and evaluating before this approach is iise_d. With this in mind, it is safer to use something like' a virus than it is to use something like an insect or a fungus because the virus one will be far more host-specific and therefore will not switch to an alternate host after it does its job and then become a pest in its own right. This is a danger that has to be kept in mind. With regard to the pesticides, I suggested that we keep searching for much more highly selective ones. The problem with 2,4,5-T is. that although selective in a certain sense it still is relatively unselective and kills lots of things that you do not want it to kill. This isthe sort of thing that has to be watched out for. Senator HART. I must admit that I got the impression clearly this. morning that the existence of a realistic alternative to some of these things might help to convince the Department of Agriculture totake action; that is if they knew they had a realistic alternative,. maybe the evidence which the Department now says is not sufficient to alarm them might have higher credibility. I don't know whether I make myself clear. Dr. WESTING. Yes; that:is why it is important to mention that, there are possible alternatives or at least that a goodly researcheffort should be aimed in that direction, to provide possible alternatives. We have come to depend upon the chemicals to such an extent that I think other possible control methods have become less interesting. Senator HART. I think it should be said, and not necessarily as a direct criticism of anybody, but humans are humans and if there is; some acceptable alternative for what would otherwise be a decision that would put a lot of heat on the fellow making the decision, it would be much easier to make and somewhat unconsciously perhaps the existence of an alternative might change the attitude of some of these individuals. Mr. Bickwit. Mr. BICKWIT. Part of your evidence for the persistence of 2,4,5-T under certain conditions for over a year stems from experiments; establishing the persistence of 2,4-D. I think for the record we ought to have some reason why you can jump from evidence of the persistonce of 2,4-D to conclusions about the persistence of 2,4,5-T. Can you meet the argument that the 2,4-D evidence might show that 2,4D is just more persistent than 2,4,5-T? Dr. WESTING. No, I think one could be on completely safe ground' in saying that 2,4-D is considerably less persistent than 2,4,5-T. 2,4D will degrade under normal, moist environmental conditions in a-, matter of weeks. 2,4,5-T is perhaps twice as persistent. There are a lot of studies to show that 2,4-D degrades more readily than 2,4,5-T; lots of short-term experiments have shown this. I am not familiar •with any definitive long-term 2,4,5-T studies. It is yerj^pisonable to assume that as long as 2,4-D will, under dry conditions such as you find in Idaho, have harmful effects on crops a year or more after use that 2,4,5-T would also. Senator HART. Doctor, thank you very much. It was a helpful • paper. The signal a few moments ago indicated another roll being called in the Senate. I apologize to Dr. Kotin, but we will have to take another recess, and I will be back just as soon as I can get on the roll. (Recess.) Senator HART. The Committee will be in order. Our concluding witness on this first day of hearing is the Director •of the National Institute of Environmental Health Sciences, Dr. Paul Kotin. -STATEMENT OF DR. PAUL KOTIN, DIRECTOR, NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES Dr. KOTIN. Mr. Chairman, I am privileged to be here today •engaging in the practice of one of my most pleasant responsibilities—that of discussing the programs and plans of the National Institute of Environmental Health Sciences of which I am Director. Our Institute is a newcomer in the executive branch; we have been in existence since 1966, achieving the status of National Institute of Environmental Health Sciences only in January 1969. This activity started as a small segment of the Department of Health, Education, and Welfare's effort in environmental health in response to recommendations made by several public advisory committees during the late 1950's and early 1960's. These committees— starting with one chaired in 1958 by Dr. Stanhope Bayne-Jones and concluding with one headed in 1965 by Dr. Detlev W. Bronk—repeatedly emphasized the necessity of establishing within the Public Health Service an organization dedicated to performing fundamental research into the real and potential effects, of human health wrought by a rapidly changing environment. The decision of the Surgon General in 1966 that this research program be located within the National Institutes of Health—that Federal agency responsible for building the Nation's base of fundamental biomedical, health-related research—made clear the mission envisioned for our program. That mission was and is: First, to determine the magnitude and significance of-the hazard to man's health from long-term exposures to low-level concentrations of chemical, physical, and biological agents in the environment; and second, to elucidate the underlying mechanisms of adverse response with the hope that principles and generalizations would be identified to provide a scientific base for criteria upon which control agencies could set standards for protective and preventive measures. During the present (1970) fiscal year, Congress and the President have authorized $17,730,000 to be expended in the conduct of this program. Since you may be familiar with other programs of the National Institutes of Health, I would like to take just a moment to point out • to you some ways in which we are similar to other part^^f NIH I uiul critical review of other researchers who have an opportunity to and some ways in which we differ. I might preface this l^fcsaying question our methods and conclusions by usual strujpit^ standards. I hope that the preceding discussion has placed tl^pational Instithat we are similar to other NIH research components in inore ways i that we are different. [ tvito of Environmental Health Sciences in perspective for you. I would now like briefly to tell you in somewhat greater detail Like the other research institutes of NIH, our mission reflects twovery important principles of operation: (l)We are in business prisome of the things we are doing, why we are doing them, and how marily to add to the fundamental knowledge and understanding of we come to be involved in the resolution of the problem which is the environmental agents which as biomedical hazards immediately or subject of these hearings. ultimately affect human health. Speaking quite broadly, the NI.EHS program attempts to employ In other words, we are concerned about the what and how of u wide spectrum of scientific disciplines and bring them to bear on health effects first and foremost in human beings. That we must also real and potential human health problems resulting from: 1. Changes in the makeup of the environment in consequence of understand that what and how of the complex constituents of our environment in order to perform the primary task is obvious. technological progress and industrialization; 2. Changes in the size and characteristics of the population; and Nevertheless, it is the results in humans which is of overriding concern to us. (2) The responsibility for taking direct action to con3. Cha.nges in the character of interactions between those two. In order to best understand the significance of changes in the trol or eliminate the hazards which we must identify resides in other components of HEW. makeup of the environment, we employ the disciplines of analytical I hasten to emphasize that we do not consider our job done until and synthetic chemistry, pharmacology, and of biophysics. our findings are made available to the appropriate components of In order to better understand our changing population and the Government. To accomplish this, we maintain effective, close, and subtle interactions of new and changing environments on people, we continuing relationships with the Environmental Health Service, the employ the sciences of epidemiology, biometry, pathology, and toxiFood and Drug Administration, the Department of Interior, the cology. Department of Agriculture, the Federal Trade Commission, .and In order to establish the mode and mechanisms of interactions, we other agencies with control responsibilities. employ all categorical divisions of scientific inquiry with special The reasons for the distinction between fundamental research and emphasis on comparative-biology to assure maximum relevance of control powers are, I think, important. First, the urgency in the research data to man/ need for control measures requires research directed to answering These varied resources and methods have so far been brought to today's questions witli today's techniques. bear in programs studying the potential health hazards of: There is, however, an equally, or perhaps more, important need Natural products including fungal contaminants of food; fibers for research directed to questions having long-range implications and polymer dusts, asbestos and fiberglas; alpha radiation; trace extending for decades and perhaps even generations into the future. metals (such as lead) and their compounds; hydrocarbons and their It is in response to this need that our Institute's program, is reaction products; tobacco smoke; and pesticides and pesticide designed. flyncrgists (includingherbicides). : While techniques frequently used in attacking these two sets of In all of these studies we are concerned with the effects of longquestions are similar, 'the orientation and end points stand in sharp term exposures to low levels of concentration-because these are the contrast. usual characteristics of exposure during life in the environment we Second, freedom from control activities permits us to devote our have created for ourselves. total effort to research. Effects are likely to be gradual in appearance, and most commonly Third, control activities are performed by experts in an environthe result of interactions of numberous agents combining in addiment in which the guidelines for operation are completely dedicated tive, synergistic, or antagonistic manners. • to this responsibility. To dissect these complexities we must identify interactions at all _ Fourth, our relationship with industries, communities, and indilevels from the intracellular organelle to the whole organism. viduals is one based exclusively on scientific grounds rather than one' Our goals include determinations of threshold for response, effects of regulation, monitoring, and enforcing. of repetitive exposures, effects of storage of the agents in living Finally^ our inputs to control agencies are objective and provide organisms, and the roles of such host factors as age, sex, antecedent an impartial basis for the very real practical considerations which or concurrent illness, nutrition, behavioral characteristics, and genemust be faced in formulating and inaugurating control measures. He make-up. As noted, the fruits of our work are promptly forwarded,. toIt may seem that our approach is somewhat complex, but it must appropriate Government agencies for use in the pursuit of their misbo so in order to resolve the complex problems wrought by the sion with virtual simultaneous publication in professional journals, changes in our environment intrinsic to technological progress. rather than in the popular press. We have attempted, in. the process of establishing, the program of This practice assures that our findings are subject to the scrutiny (lie Institute during the past 3 years, to maintain a measure of flexibility amid this essential complexity to provide for response to 90 91 nanticipated problems. Our current efforts in response to over the widening use of herbicides is in a way a case in point. You are aware, I am now certain, that the recently completed study which revealed information about the toxicity of the herbicide 2,4,5-T, in fact, was initiated by the National Cancer Institute in 19G3. _ As indicated earlier, our Institute was not in existence at that time. However, I was the scientific director for etiology in the 'Cancer Institute at that time, and along with members then and now .on my staff played a leading role in the initiation of the research .contract with Bionetics Research Laboratories, Inc., which yielded the information under discussion. Very briefly, that study was undertaken primarily to identify any •potential carcinogenic (cancer causing) or teratogenic (birth defect •causing) agents in a wide variety of pesticides and allied compounds in commercial use. We also anticipated that the study would provide data on which •to develop improvements in pur methods for identifying carcinogenic agents and hopefully identify any correlations that might exist between the carcinogenic and teratogenic capabilities of single •specific compounds. Pesticides were selected for inclusion in the study on either of two bases; First, a projection of the potential extent of their use in terms of their utility in the community; and; second, a judgment as to potential carcinogenicity by virtue of chemical structure or.meta-, bolic fate. _ In consequence, some 86 pesticidal products—including insecti.cides, fungicides, and-herbicides—were subjected to controlled, longterm studies on mice. As had been intended from the start, the study •continued through the 1960's. In the interim, the then Division of Environmental Health Sciences was^ established, and I was asked to become its first dir'ec•tor. In agreeing, I was granted approval to take with me one or two key staff members—scientists, as it happened—who had also been associated with the Bionetics contract. Since intensive programing and developmental responsibilities faced my staff and me during the first years of our Institute, we ' were quite satisfied to leave the. management of the Bionetics pesticide study in the able hands of our successors in the Cancer Institute. Furthermore, it should be recalled that the one major basis for •the study was quite clearly related to the mission of the Cancer Institute, the identification of cancer-causing agents in the environment. Upon completion of the study in early 1969, the Cancer Institute released the results of the study. The results of the teratogenic studies were released to the Mrak Commission immediately as they became available. The popular press took intense interest in the findings reported, and pressures developed for more complete information on several of the pesticides included in the study. The herbicide 2,4,5-T came under special scrutiny because its use is especially widespread, particularly in military operations in Viet:nam. Word that the Bionetics study had shown this chemical com- pound as "causing significantly more deformities thai^cpected" was especially alarming in some quarters. ^^ Dr. Endicott, then director of National Cancer Institute, requested that NIEHS staff familiar with the study in question, and also familiar with teratogenicity and pesticide chemistry generally, be assigned to data analysis and interpretation. NIEHS assumed sole ' responsibility for the statistical analysis of the very large volume of data. During the early stages of .the now public discussion, it was pointed out by the Dow Chemical Co., a major supplier of 2,4,5-T, that the materials used in the Bionetics study were significantly different than those which had been supplied by DOAV since 1965. It is certainly true that the 2,4,5-T used in the study contained significantly Jarger amounts of an impurity, dioxin. This impurity is highly toxic" and its presence occurs incidental to minor alterations in the reaction conditions during the manufacture of'2,4,5-T. DOAV Chemical Co. scientists contended that it was the dioxin derivative rather than the 2,4,5-T which had caused the deformities in test animals. A sample of the original 2,4,5-T used in the Bionetics study was analyzed and was found to contain 30 parts per million of this dioxin compound. In consequence, it became necessary to restudy the sitxiation to see whether the virtually no-longer-existing impurity in 2,4,5-T could be -held responsible for the adverse effects. In order to verify the possible role of dioxin, NIEHS brought its available resources to bear and undertook an accelerated program of research. Pure 2,4,5-T—and by pure, I mean that which is now in the marketplace with a dioxin concentration of less than one tenth of a part per million—has been made available to us and recently we received the dioxin in pure state so that experiments can be repeated with the pure material, as well as with a combination of the two ingredients. These studies are now underway. As indicated in prior discussions with the subcommittee staff, the results of this research are not yet complete. At such time as they are, in the very, near future, we will be pleased to supply them to this committee. I would be happy at this time to answer any questions of the committee regarding the mission of NIEHS or the circumstances leading to our current study of 2,4,5-T. Senator HART. Thank you, Doctor. It was thoughtless of me—I should have suggested, since you commented on having a sore throat before, that you not read the statement, but merely put it into the record. But I think as long as you were able to get through it, it helps all of us to hear it, rather than waiting for the printed record. On this business of the study, do you know when the National Cancer Institute received its first data from Bionetics suggesting that 2,4,5-T was teratogenic? Dr. KOTIN. I can't tell you offhand, but I would be. very happy to get it for the record, sir. (The information was subsequently received for the record:) 45-362-JTO 7 92 93 p .Tune of 10GC, we received the first data indicating that 2,4,5-T adij tored by injection at a dose of 113 mg/kg of body weight produced terato|BIc effects, In May of 1968, data indicated teratogenic results from oral administration of 2,4,5-T at a dose of 113 mg/kg of body weight." sis of data from a series of experiments in which mij^fcple species were used, multiple doses were used, were terribly time-Wlsuming. _ So that all I can do is vouch for the commitment of resources it took from the National Institute of Environmental Health Sciences to do its little share, provide its little share of the total. Senator HART. Doctor, I am going to ask Mr. Bickwit to continue with these questions. We have reviewed them prior to the hearing, and I will remain, using the time to road a memorandum that explains what this vote that was just signaled is all about. I hope by the time he finishes, and I finish this, we will have the answers. Dr. KOTIN. I hope I don't disturb you. Mr. BICKWIT. I'm frankly not clear on the major dates that arc involved here, the dates that you received the Bionetics information, the date that you came out with your first report on it, and the date that you came out with your final report on it. Now, if I'm right in thinking that those are relevant dates, could you tell mo what those dates are? Dr. KOTIN. Right. Well, the dates are relevant. I think it was, again, the date I offered for the record, which I don't remember offhand, is the date the Cancer Institute received the Bionetics report. You will recall Dr. Falk and our associates instigated the Bionetics study, and it wasn't a personal contract with vis. It was with the Cancer Institute. So the report went to the Cancer Institute and I don't know when they received that. Fundamentally, the only reason I suspect that we would have gotten involved at all in terms of the Bionetics report, as distinct from our own commitment by virtue of our mission in this, was the fact that Dr. Endicott did have a need for a tremendous amount of statistical and chemical analytical competency, and it was more than he had available in the Cancer Institute. So I can give the date at which the material was forwarded to us. This was in 1968, and again, I will get the date for the record. But it was—actually, the material was forwarded to us coincidental with the request to get involved with some of the analyses. Mr. BICKWIT. About when in 1968 ? Dr. KOTIN. I will be happy to give you the exact date for the record, sir. (The information was subsequently received for the record:) NIEHS performed analyses of the raw data between January and June 1969. Senator HART. We would appreciate that, and it will be made a part of the record. I am under the impression that it was sometime in 1966. In a sense I guess that's about the time you departed the promises ? Dr. KOTIN. Exactly. Senator HART. Let's assume that the date is June 1.966, that being the time the first data was received from Bionetics by the Institute. Do you recall \vhen the final report came out? Dr. KOTIN. Yes, the final report, in 1969—late 1968 and early 1969, as I recall. A little over a year ago, as I recall. Again, I can't be sm-e of that, but I would be pleased to get the exact date. I had left the Institute. (The information was subsequently received for the record:) "Bionotics supplied a draft "final" report in September of 1908. Questions raised by NIH required additional work by Bionetics and subsequent revisions of the report. Bionetics completed this work and submitted a truly "final" report in September of 1969." Senator HART. The NIEHS report—when did that come out? Dr. KOTIN. The final report was last fall, when we were providing the results of our statistical analysis, and the data on the teratogenicity to the Mrak Commission. Senator HART. If it develops that the June 1966 date is the time that the National Cancer Institute got its first data from Bionetics, and the final report by NIEHS came out in the fall of 1969, why in the world did it take so long to come up with the information for that final report ? Dr. KOTIN. I really can't answer that, other than to say that at the time the National Institute of Environmental Health Sciences was asked by Dr. Endicott to provide the statistical and analytical competency for the review of the data, the work Avas done very promptly. In fact, Aye didn't even wait until the end of the report to make the information available to the Mrac Commission. As each little increment of information that represented a part of the total became available, this was made immediately available, to the Mrak Commission, and the Food and Drug Administration. Senator HART. I am trying to get these dates clearly fixed, if I - can. You state that NCI released the results of the study in early 1969. Was this the preliminary report of Bionetic's findings? Dr. KOTIN. No, sir; this, I think, represented the first report in which conclusions were published, both in the scientific literature and in the Journal of the National Cancer Institute, as well as made available to the various responsible government agencies. The really important aspects of the conclusions, the necessity for voluminous work—there were some 86 compounds—the National Cancer Institute justifiably felt that in-house staff should at least on a random basis review the data. There was much, much new information that heretofore had been unknown. And just the histological review of the slides from the autopsied animals, the statistical analy- Mr. BICKWIT. Then you released reports periodically ? Dr. KOTIN. To the Mrak Commission only, and to the relevant Government agencies. Mr. BICKWIT. About how many reports were there ? Dr. KOTIN. These were not formal reports, but they were presented quite informally—we finished the analysis of the White Swiss Mouse data, the C-57 black data, the DBA data. We checked the statistical significance of the differences between test and controls, between the various dose levls, between the various modes of admission. So that, rather than adorn the data with 94 95 sc, we just gave them the statistical material with the listing^B conclusions. ^^ Mr. BICKAVIT. So, whenever you had anything of any importance, it went to Mrak. Dr. KOTIN. Promptly. • > , , Mr. BicicAvrr. Your final report came out in the fall of 1969, is' that right? Dr. KOTIN. Yes, we have submitted a paper for publication in the journal Science which relates pur analysis on the teratogenicity of 2,4,5-T, and it should be appearing shortly. Again I would be happy to make a preprint copy of the manuscript available for the Committee if you desire.1 Mr. BICKAVIT. Thank you. That would bo fine. Now, if you got your information sometime in 1968, and we don't know when, let's assume it was late 1968, and it took until the fall of 1969 to como up with a final report, why did it take that length of time ? Dr. KOTIN. Just the difference.between the magnitude of the job and the availability of professional resources within our institute. At that time, our Biometry branch consisted of two professional biometricians at the doctorate level. This staff was involved in a series of studies including one on the relationship of asbestos to lung cancer, and another on a quantification of the hazard to uranium miners. This limited staff had to be literally redeployed in order to perform the necessary analyses of the Bionetics data. Mr. BICKWIT. On the carcinogenicity studies, when did you get the information from Bionetics? Dr. KOTIN. We really didn't, other than as information. It came as part of the same report. But the analysis of the carcinogenicity study remained entirely within the Cancer Institute, since it was clearly relevant to their mission and responsibility as the National Cancer Institute. Mr. BICKWIT, You were not responsible for analyzing that? Dr. KOTIN. No, sir. Mr. BICKWIT. You have stated the results of' the toratogenicity. studios were released to the Mrak Commission immediately when they became available. I am sure you are familiar with Mr. Whiteside's allegation that Dr. Samuel Epstein of the Mrak Commission had a great deal of difficulty acquiring information on the studies. I Avonder if you could reply to, this allegation? If you are not familiar Avith it Dr. KOTIN. I am familiar with the allegation. I read it in the story in the New Yorker, of course. No, I think that we are probably speaking of two different things. There was, at no time, the necessity for the requesting of any information from us. There was a mechanism for the fonvarding of the information to the Mrak Commission; the best evidence that this allegation is not so in another sense is that the head of our Biometry Branch, Dr. David Gaylor, was on the Mrak Teratogonicity Committee, the very committee to which the data were being supplied. Avn cornSo, essentially it would be denying his own data to mittee if this were so. Do you follow me. Mr. BICKAVIT. I am sorry, I don't. Senator HART. I am going to have to interrupt again, I am sorry. I hoped Ave could avoid the necessity of holding you, but I will miss the vote. I will not be able to return as promptly as I like, because I must remain on the floor to get something done, a matter that will be voted on tomorroAv. So, Ave will have to recess in the very unhappy condition of not knowing exactly when I will get back, but as quickly as I can. (Recess.) Senator HART. We Avill resume, and Avith better luck than AVG have been having in the last hour or so, maybe we can conclude before the next vote is signaled. Mr. BICKAVIT. I believe the last statement which you made I had some difficulty with. Dr. KOTIN. What I Avas saying Avas that Dr. Epstein and Dr. Gaylor Avere on the same teratogenicity panel of the Mrak Commission, and each meeting they held Dr. Gaylor brought the data "P_So the only information Dr. Epstein might have asked for that he did not got Avere data that just were not complete. But certainly in relation to the teratogenicity, I cannot conceive of any aAvailable data that would not have been made available. Mr. BICKAVIT. Was the final Bionetics report made available to the Mrak Commission when they asked for it? Dr. KOTIN. It is my impression that it Avas. And again they would not have come to us, because the final report Avas the property of the National Cancer Institute, as the contracting institution. Mr. BICKAVIT. If they did come to' you, would you haA'e had authority to give it to them ? Dr. KOTIN. Actually, I suspect I Avould have picked up the phone and asked Dr. Endicott Avho was responsible, and I would have gotten authority for it because the information contained in it was germane to itho Mrak Commission. But again I would emphasize that the final report of any contractor Avould not include the interpretation and the analysis of the data. This was not part of the purchase. Mr. BICKAVIT. I realize that, but if Dr. Epstein of the Mrak Commission had asked you for the final Bionetics report, Avithoutx an analysis from NIEHS, you would have furnished i t to him immediately ? Dr. KOTIN. I would have furnished it to the Mrak Commission. Mr. BICKAVIT. Would you not have furnished it to Dr. Epstein ? Dr. KOTIN. The data itself ? Mr. BICKWIT. Yes. Dr. KOTIN. Uninterpreted? Mr. BICKWIT. Yes. Dr. KOTIN. Oh, I probably would not have, no. Mr. BICKWIT. Why not ? t 96 97 'Dr. KOTIN. Essentially the data are crude data that require imKprotation, and essentially the implications, the results of the report, arc the conclusions, and the responsibility for those conclusions would have been ours,—that is, the responsibility of the NIH. Mr. BicitAvrr. These data, I understand, did raise doubts, about the toratogenicity of 2,4,5-ff. { Dr. KOTIN. You mean, rather than raise doubts, established the J experimental teratogenicity of this. After the data were analyzed, jj yes. | Mr. BIOKAVET. You are saying that you do not believe that a J member of the teratology panel of the Mrak Commission should have the right to examine those data unalyzed? Dr. KOTIN, Oh, not at all. All I am trying to say is the data themselves, short of total package, once the data were analyzed, and conclusions made, then by no stretch of the imagination would the date bo withheld from anybody. Mr. BIGKWIT. But unanalyzcd, he should not be entitled to look at them ? . Dr. KQTIN. I do not think so, no, sir. Mr. BICKAVIT. Should anybody other than the organization entrusted with the analysis of the data be entitled to look at them? Dr. KQTTN. Oh, surely. Mr. Hart's Committee, or there are a whole spectrum of responsible agencies. Mr. BICKAVIT. Could you list those agencies that would be entitled to look at this data? Senator HAKT. You are inquiring about before analysis? Mr. BICKWIT. Yes. Dr. KOTIN, The hierarchy above me, as a lowly director of an institute, the director of NIH, The Surgeon General, the Secretary of HEW, all of the way up, any member of the legislature, any member of the executive branch, with the authority, surely. ' Mr. BICKWIT. But you would not want to allow a nongovernmental scientist with, some expertise in the field to look at this data? Dr. KOTIN. Again, there is no flat yes and no. There are many instances when we call people in nongovernmentally to look. Mr. BICKWIT. What I am asking you to do is draw the line. I know it is hard, but you have excluded one nongovernmental scientist. I would like to know how you formulate your opinion in deciding who should be excluded and who should be included. Dr. KOTIN. That is a matter of judgment. How much help I think we can get from them, how much help we can provide them. Mr. BICKWIT. Is that the only basis for your decision? Dr. KOTIN. I would have to think. I suspect that is the major one. We have crude data a.nd what we try to do is get the best expertise. We have everything from advisory committees to councils to study sections to consultants to the institutes, who are not Government employees, who are on call at all times and who are used rather consistently, particularly by a young institute like our own, (we are 3 years old; our $17 million budget, when contrasted with the $150plus million budget of the larger well established institutes is probably as good an indication of our size as anything.) I think a corollary of our small size is the great consistency with which we get outside help in terms of consultation. We just had a task force that spent 8 weeks preparing a consultatiA^puide, as it were, for the Institute. So there is no tendency on pur part at all to treat anything that we got as either clandestine or in any way not open to scrutiny. In fact, as I said in my testimony, I made a special point that scientific scrutiny is something that we insist on in all of our data before wo accept it as fact. As our critical mass at NIEHS enlarges, we will probably be more certain. But we are a small outfit and we use outside consultants a lot. So in answer to your question specifically do I feel categorically that data should not be seen by outside scientists, not at all. There are instances where you call them in and they see it initially with us, as it were, around the table for the first time. Mr. BICKAVTT. On the pro side yon are weighing the potential helpfulness of the scientist who would be asking to see the data. Dr. KOTIN. Oh, no. Also what he can contribute to the maximum utilization of the data. In the years I have been in NIH when there are implications of the data that aflect other executive branches, or have socioeconomic implications, the people who yon try to get help from and proxide help to are judged on an individual basis. This is really so. Mr. BICKWIT. What is on the other side? In formulating your opinion what is it that would keep you from giving the information out? Dr. KOTIN. Number one, concern over data where the interpretation would be such that we would want our interpretation to be on the record at the time the data were made available. That would be one example. Another example, where there is some question we have about the data ourselves, so we want to go back and verify techniques, verify the workbooks from which the reports were made. And in fact this was done in this case. So there are lots of reasons. Not as many as on the other side, but you just have to do it on an individual basis, decide what is the best way to get maximum returns from the data. Mr. BICKAVIT. With respect to Dr. Epstein, a member of the teratology panel of the Mrak Commission, would you rule out the possibility of his being able to contribute to the utilization of this material? Dr. KOTIN. Yes. Mr. BICKAVIT. Could you elaborate on that ? Dr. KOTTN. Fundamentally it is a matter of judgment. I felt at that time that the data themselves needed analysis for the reasons I mentioned, that the conclusions Avere integral to the data because again the mere fact that you had chi square indicated there that the significance Avas in large measure determined by statistical methods. It wasn't a situation where, as the data amply attest, an all or none response occurred, Avhere all of the controls did one thing, all of the test animals did the other. There Avere statistical differences. There were differences in degree and intensity and in time. These had to be determined by statistical techniques. Senator HART. Doctor, I will be brief in my thanks,' since I am under the compulsion of another vote signal. (The information referred to on p. 94 folloAvs:) aa TEKATOGENIO EVALUATION OF 2,4,5-T ABSTRACT The herbicide 2,4,5-T (2,4,5-trichlorophenoxyacetic acid) has been shown to be teviitogenic and fetocidal in two strains of mice using either subcutaneous or oral routes of administration, and in one strain of rats by oral administration. The incidences of both cystic kidney and cleft palate were increased in the G57BL/6 mice as well as the incidence of cleft palate in the AKR mice. The incidence of cystic kidney was also increased in the rats. In addition, an increase in liver to body weight ratio in the mouse fetus and the occurrence of hcmorrhaglc gastrointestinal tract in the rat fetus suggest that this compound also has fetotoxic properties. The chlorinated.herbicide 2,4,5-T (2,4,5-trichlorophenoxyacetic acid) is used extensively for weed control (1). However, there have been relatively few reports concerning its pharmacologic and toxicologic properties in animals (2,3). Indeed, there are no data available concerning the effects of this compound on the developing embryo and fetus1. Therefore, this report evaluates the teratogenic and fetotoxic potential of 2,4,5-T in mice and rats (4). Breeding colonies of O57BIJ/6 and AKH strains of mice were established at Bionetics Research Laboratories, Inc., to supply the mice. For the study, breeding was by random mating. Detection of a vaginal plug indicated day zero of pregnancy. Rats were procured from the Holtzman Co., with knowu insemination dates. • Detection of sperm indicated day zero of pregnancy. All animals received chow and water ad libitum. 2,4,5-T (5) was administered by one of two routes, subcutaneously or orally. A solution of 2,4,6-T in 100% dimethylsulfoxide (DMSO) in a volume of 100 /il/inouse was used for each subcutaneous administration. For oral administration by gastric intubation, 2,4,5-T was suspended in a honey solution (honey: water, 1:1) and volumes of 100 /jl/mouse and 200 /d/rat were used. Jn the studies with the C57BL/6 strain, 2,4,6-T was administered daily beginning on the sixth day of pregnancy and continuing through the 14th day or from the Oth through the 17th day. The mice were sacrificed on the 18th day of gestation for examination. In the studies with the AKR strain, 2,4,5-T was administered daily beginning on the 6th day of pregnancy and continuing through the 15th. These mice were sacrificed on the 19th day of gestation. The rats were treated on the 10th through the 15th and sacrificed on the 20th day of gestation. Upon sacrifice both mothers and fetuses were examined carefully. In addition, about one-third of the mouse fetuses were stained with alizarin red S in order to detect skeletal anomalies. Tables 1 through 3 contain data on fetal mortality, abnormal litters, abnormal fetuses per litter, fetuses with cleft palate, fetuses with cystic kidney, maternal weight gain, and maternal and fetal liver to body weight ratios. The following conventions were observed in compiling these data. If a fetus was either dead or resorbed, it was regarded as a dead fetus. A fetus was classified abnormal if it was alive and had at least one anomaly (regardless of type). Similarly, a litter was classified ns abnormal if it contained one or more abnormal retuses. A fetus was said to have a cystic kidney if at least one of its kidneys was affected. In calculating the maternal liver/body weight ratio, maternal body weight was defined as the difference between the weight of the animal on the day it was sacrificed and the gravid uterus weight. Finally, the maternal .weight gain was defined as the difference in the corrected maternal body weight on the day it was sacrificed and its weight on day zero of pregnancy. The percentages for fetal mortality, abnormal fetuses, fetuses with cleft palate and fetuses with cystic kidney were computed by first, obtaining the percent for each litter and then calculating the average of these percentages. ' The percentage of abnormal litters provides a measure of the prevalence of abnormal fetuses across litters, while the percentage of abnormal fetuses per litter gives an indication of the prevalence of abnormal fetuses within litters. In this report, the control animals are those that were on a large study during the 3-year time period in which 2,4,5-T was evaluated. The data from ( h o DMSO and honey treated control groups were compared with the data for the non-treated control group. Then the results from animals treated with 2,4,5-T in either DMSO or honey were compared to the appropriate control data. Standard corrected 2x2 chi-square tests (6) were used to compare the results of 2,4,5-T treated animals with the appropriate cont^^data for the proportion of litters containing abnormal fetuses. VP The distribution of the percent of abnormal fetuses per litter for 2,4,5-T treated litters was compared with the appropriate control distribution by use of the non-parametric Mann- Whitney U-test (6). Also, this test was used for comparing the percent fetal mortality, cleft palate, cystic kidney, and enlarged ronal pelvis per litter. This test requires that the proportion of dead or abnormal fetuses per litter is independent from litter to litter, but requires no assumption about the frequency distributions of these proportions. Initial analyses of the data indicated that occurrences of anomalies among fetuses within litters were correlated. That is, anomalies were not randomly distributed across all litters but tended to cluster within litters. Many litters possessed no anomalies whereas all of the fetuses in some litters were abnormal. Since fetuses within the same litter tend to be more alike, pooling the data across Utters before performing statistical tests is not appropriate. The experimental unit (7) is that entity to which treatments are applied, in this case the pregnant animal. Hence, all calculations of averages and all statistical tests were performed on the independent responses of the experimental units (litters). The administration of DMSO or honey to mice or rats did not adversely affect the development of the fetuses. The incidence and type of naturally occurring anomalies observed in the DMSO and honey treated animals did not show an increase compared to the non-treated group. The alizarin stained fetuses of the control mice showed very few skeletal anomolies. No skeletal anomalies were detected by staining in the treated mice. For both mice and rats, there were no differences in the average number of implantations in the control and experimental litters. A few values for treated animals were less than those of their appropriate controls. None of these differences were statistically significant including the 8% fetal mortality observed in the C57BL/0 mice receiving a 21.5 mg/kg dose of 2,4,5-T reported in Table 1. This value of 3% reflects a period of low fetal mortality (9%) observed in the control mice during the initial few mouths of the study. This difference in mortality is not statistically significant. There were no other significant changes in these control data during the 3-year period. As shown in Table 1, the administration of 2,4,5-T to C57BL/6 mice on days 0-14 at a dosage level of 113 mg/kg produced significant increases in percent of abnormal litters and percent of abnormal fetuses per litter. The anomalies produced by 2,4,5-T were almost exclusively cystic kidney and cleft palate. Similar results were obtained regardless of whether the compound was administered subcutaneously or orally. A dosage level of 46.4 mg/kg administered orally did not produce a significant increase in fetal mortality or an effect on palatal development, but did cause a significant increase in the percentages of fetuses with cystic kidney. Administration of 2,4,5-T subcutaneously at a dosage level of 21.5 mg/kg did not affect the visability or development of the fetuses. Thus, a dose-response relationship for the fetocidal and teratogenic properties of 2,4,5-T in mice is suggested for both routes of administration. It was also observed that in mice treated with 2,4,6-T on days 6 through 14, there was a significant decrease in the incidence of naturally occurring anomalies. These consist of microphthaliuia followed by anophthalmia and are in accord with other O57BI>/6 colonies (8). Although the fetuses from mice treated on the C-14th days had fewer naturally occurring anomalies, the fetuses from mice treated on the 9th to 17th days did exhibit these anomalies. Thus, it appears that the interval of days 6 to 9 of gestation is one of the sensitive periods of development with respect to 2,4,5-T. Two other sensitive periods are during development of the palate and kidney since they are so highly affected. The occurrence of these two anomalies are statistically unrelated. In the study where 2,4,5-T was administered on the 9th to the 17th day of gestation with the C57BL/6 mice, maternal and fetal liver weights were determined. As seen in Table 2, this treatment produced a significant increase in maternal and fetal liver to body weight ratios. The significant increase in fetal liver to body weight ratio reflects both an increase in fetal liver, weight and a decrease in fetal body weight ,The significant increase in the liver to body weight ratio suggests a change in activity of drug metabolizing enzymes of the 101 100 |mdoplasmic retlculum which has been studied (9). Again, the Mann-'fl^Bey J-test was used to compare the animals administered 2,4,5-T with the appropriate DMSO control mice. Thus, in the C57BL/6 mice, 2,4,5-T is fetocidal, teratogenic and capable of producing an increase in the liver to body weight ratios. Treatment of mice of the AKR strain with 2,4,5-T in honey produced a significant increase in fetal mortality. The incidence of cleft palate was increased with both routes of administration. However, 2,4,5-T did not produce an increased incidence of cystic kidney in this strain. There was no effect of 2,4,5-T administration in this strain on the maternal weight gain with either route of administration. However, the maternal liver to body weight ratio was increased using either route of administration. In addition, hybrid litters resulting from mating C57BL/6 females with AKR males were evaluated. The administration of 113 mg./kg in DMSO from days 6 through 14 of gestation produced a high incidence of both cystic kidney and cleft palate. There was no effect on maternal weight gain. The oral administration to rats of 2,4,5-T at a dosage level of 10.0 or 46.4 mg/kg on the 10th through the 15th day of gestation produced a significant increase in fetal mortality (Table 3). The two lower dosage levels, 4.6 and 10.0 mg/kg produced a significant increase in the percentage of abnormal fetuses. These fetuses displayed a high incidence of cystic kidney. At the highest dose level, 46.4 mg/kg, the marked increase in fetal mortality reduced the population of live fetuses to a small sample. However, cystic kidneys were observed. In a limited study, the administration of 2,4,5-T at dosage levels of 21.5 or 40.4 mg/kg from the 6th through the 15th day of gestation was highly fetocidal. At all dosage levels studied in the rat, hemorrhagic gastrointestinal tracts were observed in the fetuses. The percentages of fetuses per litter with hemorrhagic gastrointestinal tracts showed a dose-reponse relationship; i.e., 3%, 50%, and 83% at,doses of 4.6, 10.0 and 46.4 mg/kg, respectively. None were observed in the fetuses from the control animals. Drill and Hiratzka (2) have reported that dogs which received 2,4,5-T in the diet showed some necrosis and inflammation of the intestinal mucosa. The hemorrhagic gastrointestinal tracts observed in the rat fetuses is probably a toxic effect of 2,4,6-T on the fetal organ as opposed to a developmental defect. In conclusion, these studies show that 2,4,5-T adversely affects the development and viability of the mouse and rat fetus. TABLE l.-TERATOGENIC EVALUATION OF 2,4,5-T IN MICE Average number Compound Vehicle Dose of (mg/kg) litters Percent fetuses/ mortality/ abnormal litter litters litter Percent of fetuses per Percent litter with— i~i fetuses/ Cleft palate Cystic kidney litter C57VL/6 STRAIN TREATED DAYS 6-14 Nontreated.. Control Control 2,4,5-T 2,4,5-T 2,4,5-T 2,4,5-T , . None... . DMSO.. . Honey.. DMSO.., DMSO., Honey... ...do.... Nontreated.. Control 2,4,5-T . None.. . DMSO.. DMSO.. None None 113.0 Nontreated Control Control 2,4,5-T 2,4,5-T None DMSO Honey DMSO Honey None None . None 113.0 113.0 None None None 72 21.5 106 32 6 46.4 18 6 1)3,0 113.0 12 5.8 5.5 ?:i 7.7 4,4 8.5 4.8 26 29 15 3 42 41 50 42 8 147 i 100 > 100 38 »8S 11 12 14 12 " 57 237 "70 1 2 1 0 0 0 «22 2 "23 »4J i 33 I 48 C57BL/6 STRAIN TREATED DAYS 9-17 10 10 5.1 6.1 7.7 36 23 11 ! 100 !77 0 0 "29 7 0 >60 19 24 0 5 4 0 329 3 55 <1 <1 0 »28 ' 55 <1 <\ (\ 1 0 71 30 31 8 AKR STRAIN TREATED DAYS 6-15 > Statistical Significance Levol=0.10; 58 72 12 14 7 7.1 6.9 8.8 6.9 5.3 16 15 9 23 M2 i Statistical Significance Level°»0.05; 3 71 » 100 > Statistical Significance Level=O.OI. iOUSLY IN DMSO W8U Z.-LIVER WEIGHT STUDY: 2,4,5-T ADMINISTERED DAILY AT 113 MG/KG FROM THE 9TH THROUGH THE 17TH DAY OF GESTATION IN Maternal Fetal Treatment Liver wt. .047 . 046 J.057 . ' Slatlslical significance level = 0.10. Wt. gain (6ms) Liver wt./ Body wt. .810 .818 .058 .056 1.738 ".076 6.00 5.99 4.65 «.120 (8ms) (8ms) Nantiealed D«SO M.S-T... Liver wt./ Body wt. Body wt. »Statistical significance level = 0.05.' .069 .068 »Statistical significance level = 0.01. TABLE 3.-TERATOGENIC EVALUATION OF 2,4,5-T IN RATS Compound Nontreated Control 2,4,0-T Z.45-T M.5-T 1 Vehicle None . Honey do do. do Number of Dose (mg/kg) litters None None 4.6 10.0 46.4 Statistical Significance Level = 0.10. 7 14 8 7 6 Average number Percent fetal Percent abnormal live fetuses/ mortality/ abnormal fetuses/ litter litters litter litter 9.9 8.7 8.2 7.1 2.7 11 1 12 128 '59 s Statistical Significance Level = 0.05. 43 57 88 86 67 9 12 »36 '46 60 Percent of feluses per litter with— Enlarged renal pelvis Cystic kidney 9 12 18 17 27 0 <1 »21 »30 33 a Statistical Significance Level=0.01. HEFERENCES AND NOTES (1) Audus, Ii. ,T., The Physiology and Biochemistry of Herbicides, Academic Tress, New York, 1964. (2) Drill, V. A. and Hiratzka, T., Arch. Industrial Hygiene Occupational Mca. 7, 61, 1953. (3) Howe, V. 1C. and Hymas, T. A., Am, J. Vet. Res. 15, 622, 1954. (4) These results are from a large study designed to screen selected compounds for teratogenic effects in mice which was performed at the Bionetics Research Laboratories, Division of Litton Industries, under contract numbers I'll 43-64-57 and PH 43-67-735 from the National Institutes of Health. During the performance of this study, Dr. Courtney was a staff member of the Bionetics Research Labs., Inc., and Dr. Falk was a member of the National Cancer Institute. (5) 2,4,5-T was produced by the Diamond Alkali Co., 98%, Tech., m.p. 149151°. (6) Snedecor, G. W. and Cochrau, \V. G. Statistical Methods, 6th ed., Iowa 'State Univ. Press, Ames, Iowa, 1967. (7) Kempthorne, O., The Design and Analysis of EsDperime^lts, Wiley, N.Y., 1052. (8) Kalter, H., Teratology 1, 193, 1968. (9) Courtney, K. D. (In preparation). Note added in proof: The sample of 2,4,5-T used in this study contained approximately 30 ppm of 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) (10). Dioxin as well as purified 2,4,5-T are currently being evaluated for their teratogenic and fetotoxic potential. (10) We thank Dow Chemical Co., for the analysis of 2,4,5-T. K. Diane Courtney D. W. Gaylor M. D. Hogan H. L. Falk National Institute of Environmental Health Sciences, National Institutes of Health, Post Office Box 12233, Research Triangle Park, N.C..27709 R. R. Bates I. Mitchell National Cancer Institute National Institutes of Health Rethesda, Md. 20014 102 103 Senator HAIIT. Are there any additional questions? Mr. BICKWIT. No, Mr. Chairman. Senator HAHT. If any arise we will submit them in writing and receive the replies in the record. I appreciate the cooperation of everyone through the day, and apologize for the erratic scheduling this afternoon. (The following was subsequently received for the record:) Saigon's leading maternity hospital, Tudu, from which rum^Af an increase of abnormal births emanate periodically, has not even compilerKnnual reports of statistics for the last three years. Recent monthly figures show an average oC about 140 miscarriages and 150 premature births among approximately 2,800 pregnancies, but the hospital is not prepared to say whether this represents nn Increase and, if so, what the cause might be. A high Agriculture Ministry official said: "I don't think the Aznericnns would use the chemicals if they were harmful." He conceded that his ministry had made no tests and asserted that his experts had been unable to get any information about the defoliants from the Defense Ministry, which considers such data secret The main defoliant compounds and some information about them are available in the United States. Last Oct. 29, President Nixon's science adviser, Dr. Lee A. Du Bridge, announced that as a result of a study showing that one of the defoliants used, 2,4,5-T, had caused an unexpectedly high incidence of fetal deformities in mice and rats, the compound would henceforth be restricted to areas remote from population. That directive appears to be ambiguous in South Vietnam for military spokesmen assert that 2,4,5-T continues to be used only in "enemy staging areas"—by definition populated regions. Appendix 1 U.S. SHOWS SIGNS OF CONCERN OVER EFFECT IN VIETNAM or 9-YEAR DEFOLIATION PROGRAM (By Ralph Blumenthal, special to the New York Times) SAIGON, South Vietnam, March 14—Many South Vietnamese who live adjacent to areas that are being defoliated by spray from United States planes are convinced that any ailments or misfortunes that they suffer are related to the sprayings. There is no proof that they are right about the effect of the chemical sprays on the human body, but neither is there any assurance that they are wrong. Although the defoliation program, organized and run by the United States, has been in operation for nearly nine years the full effect of the chemicals on . animal and human life remains largely undetermined. The United States military command says the program, which is designed to strip plant cover from areas occupied by the enemy and to destroy crops that might yield him food, has covered about 5,000 of South Vietnam's 60,350 square miles. U.S. TERMS IT VALUABLE The United States command says the program has proved its military worth. "It has contributed materially to the security of units operating in the field by increasing their visibility from the ground as well as the air," the command said. About 13 per cent of the program has been directed against crops, presumably food grown by and for the enemy. Because of the drifting of defoliants and the difficulty of assessing the results on the ground, it is virtually impossible to say how much of the crop has been destroyed by the chemicals, but it would not appear to be a significant part of the country's capacity. It has brought hardships, however, to individual farmers. After yeiirs of assuring the South Vietnamese that this extensive spraying was harmless to animals and humans, United States officials are showing signs of concern over recent reports the the chemical sprays may have some littleunderstood and alarming effects. , , , PANEL STUDYING EFFECTS In the last several months, reportedly on instruction from Washington, the United States military command and the United States Embassy have formed a special committee to review the effects of the defoliation program, especially on humans. The sensitivity of the issue has foreclosed official comment, but according to informed sources the science advisory office of the command is responsible for gathering data in interviews and tests that embassy officials will then evaluate, The South A'ietnamese Government regards the entire subject as taboo. Vietnamese newspapers have been suspended for publishing articles about birth defects allegedly attributed to the defoliants, and the public Health Ministry declines to provide any statistics on normal and abnormal births. However, the concern felt among the Americans is shared by many South Vietnamese scientists, physicians, health officials and villagers interviewed in a throe-week survey of the effects of the program. Officers of the United States command are aware of the allegations of birth defects but they generally discount the reports. KoHponsible South Vietnamese scientists and officials say they know virtually nothing about the effects of the chemical sprays. DEFOLIANTS WERE CONCEALED Don That Trinh, Minister of Agriculture from November, 1967 to May, 1008, nnd for 10 years professor of agronomy at Saigon University, said that while he was minister, the Defense Ministry "would try to conceal the defoliant products from me." "I did not believe in defoliation," he added. According to one of the Vietnamese directors of a Government research laboratory in Saigon: "We didn't know anything before the United States started spraying. It wsts only when we received complaints from the livestock people that we started getting interested." But, he added, there are still no Vietnamese studies. Even the village of Tanhiep, 20 miles north of Saigon, on which 1,000 gallons of defoliants were jettisoned on Dec. 1, 1968, has not been the object of attention or study. An American O-123 flying out of Bienhoa air base, Northeast of Saigon, developed engine trouble shortly after takeoff. To lighten the craft, the pilot sprayed the full load of chemicals over Tanhiep and nearby Binhtri in 30 seconds instead of the usual 4 minutes 30 seconds, which spreads the defoliant nt the rate of three gallons an acre in unpopulated areas. The defoliant involved, according to the United States command, was a 50-60 mixture of 2,4-Dichlorophenoxyacetate, or 2,4-D, and 2,4,5-Trichlorophenoxyacetate, or 2,4,5-T, in an oil base. It is one of three compounds the ' military says it uses here, the others being a Dow chemical product called Tordon 101, a mixture of arnine salts of 2,4-D and Picloram, and an arsenic compound of cacodylic acid. No physicians visited Tanhiep to examine the people after their exposure, which, like eight similar emergency dumpings since 1968—some over unpopulated forests—was not made public by the United States command. A United States Air Force medical team visited Binhtri shortly after the spraying and, according to American district officials, found the villagers had suffered no ill effects. There was no later inquiry. Mrs. Trail Thi Tien of Tanhiep, hwo says she has four normal children, is convinced that the malfunction of her son, who still looks like a newborn at 14 months of age, "must be due to I ho, chemicals I breathed." Her neighbors, Mrs. Nguyen Thi Hai and Mrs. Tong Thi An, blame the spraying for the fact that their children, one year and 20 months old respectively, still crawl instead of walk. Nguyen Van Nhap, a farmer, complains of suffering bouts of fever, sneezing and weakness. "I was working in the field when the spray came down," Mrs. Tien said through an interpreter. "I felt dizzy, like vomiting and had to' stay in bed three or four days." i Many other villagers reported feeling the same sensations as Mrs. Tien,1 but, except for the two children described as retarded in learning to walk, no other abnormal children were described to visitors at the village of 1,200 residents. 104 Tran Van Dang, a farmer in neighboring Binhtri, recalled that .,., after the spraying two villagers, Tarn Ten and Mrs. Hal Mua, died aOTr suffering respiratory difficulties and trembling. The next day, he said, a third villager, Mrs. Hai Nuc, died after showing similar symptoms. Mr. Ten was an old man and could have been expected to die soon anyway but the two others, Mr. Dang said, were middle-aged and seemed healthy. Such complaints are not limited to Tanhiep and Binhtri, where villagers were admittedly exposed to concentrated doses of defoliant—though just how concentrated has not been established. In Bienhoa city, 10 miles from Tanhiep, any defoliant in the air drifts down from the heavily sprayed battle areas to the north. Dr. Nguyen Son Oao says he finds a clear correlation between the days when there is spraying and the number of patients who come in with respiratory ailments, mostly sneezing and coughing. Dr. Cao, who has been practicing in Bienhoa for 21 years, said he had also noticed that in the last two to three years the number of miscarriages among his patients had doubled. "These women are convinced they are the victims of the chemicals," he said. "I only suspect there could be a relationship. This suspicion is very well known. The increase in miscarriages is very obvious, very significant." However, the manager of another clinic reported no increase in miscarriages over the last several years. Any increase in miscarriages has many possible explanations: perhaps tlie deterioration of the daily diet, the cumulative effect of the hardships of war, population and economic movements that register statistics of only certain groups, or air pollution, of which the defoliant chemicals are a part. Appendix 2 DEFOLIANTS, DEFORMITIES: WHAT RISK? Dr. Jackie- Vorrctt is fascinated and horrified by what has now become an everyday sight at her FDA toxicology lab in Washington, D.O.: several white leghorn chicks struggling to get to their feet and then finally walking—on then knees. Besides slipped tendons in their legs, some of the chicks have cleft palates and beak deformities. All this has been wrought by injecting fertilized eggs with an etlinnol solution containing just 2.5 micromicrograms (or 50 parts per trillion) of 2,3,6,7-totrachlorodibenzo-p-dioxin, a contaminant in 2,4,5-trichlorophenoxyaceticacid (2,4,5-T). Over the past nine years, 40 million pounds of this defoliating herbicide have been sprayed in very heavy concentrations across at least five million acres of Vietnam to destroy crops and expose the enemy. By MWN'S reckoning, some 30 million pounds have been spewed out in lesser concentrations during just the past five years across perhaps 30 million acres of range, forest, and farmland (not to mention home gardens) in the TJ.S—an area three times the size of Texas. Thus, Dr. Verrctt's preliminary findings are not just of interest to poultrymen. The 11 crippled chicks in her study were among 15 survivors of a clutch of 25 eggs. In the unhatched chicks, Dr. Verrett found pronounced evidence of chick edema—swollen tissues, cysts on the back, necrotic livers, and the same deformities the live birds have. The FDA researcher is diluting the dioxin content to try to find a "no effects" level. In another brood, she has produced a similar pattern of birth defects with just 2% parts per trillion of dioxin, 1/400,000 the 1 ppm found in currently marketed products. Now she's experimenting with .25 parts per trillion. (The work is so politically sensitive that she doesn't even know the origin of the 2,4,5-T involved and feels "like I'm in the CIA.") When told that HEW Secretary Robert Finch is doubtful about the applicability of the chick embryo work to human risk, Dr. Verrett snapped, "I know, I know, but the only time Bob Finch sees eggs is when he eats them for breakfast" While Dr. Verrett labored in the lab early this month, Dr. Samuel Epstein, • chief of toxicology at Children's Hospital Medical Center in Boston, was out in Globe, a foothill town in southeastern Arizona, to evaluate reports of toxic and & sK 105 ^ ' tnTYitr>K-Trichloroplienoxy) proprionic Acid (Silvea;) in adjacenWonto National These reports have disturbed the nation and drawn experts to the ODD EFFECTS AROUND GLOBE la Globe, Dr. Epstein saw two goats and a duck with leg deformities similar • ,ln those in Dr. Verrett's chickens, and studied the histories of sick people. "H's Impossible to say for certain whether the claimed symptoms and effects art1 attributable to the spray," he said. But at the same time he lashed out at the U.S. Forest Service for risking the contamination of water sources against tl* own policy, for contributing to drift by using water as a 2,4,5-T solvent, i mid for failing to post the area before spraying. MWN found that the Department of Agriculture keeps such casual tabs of fJM.fi-T spraying that it would take officials a week just to find which of the 33 national forests besides Tonto have been bombed with the two million pounds Forest Service,has jetted out over the past six years. "But Interior uses more tlum we do," said one official. Replies an Interior spokesman, "We used only 44,232 pounds last year." In the Globe area, the Forest Service has sprayed 2,4,5-T and Silvea; four of Hie past five years to promote growth of grass in a burned-over section and to eliminate chaparral. But most 2,4,5-T use is unmonitored. The defoliant is bought by ranchers and private foresters and it's pretty much up to them what happens to it. Human teratogenicity is the chief worry; it is fairly well known by now that Dr. Verrett's work is not the first study to dramatize the risk. Yet MWN leained that the U.S. doesn't keep nationwide birth-defect figures. Dr. Edward Burger of the government's Office of Science and Technology dons not seem worried by this absence of monitoring and supervision, nor, Indeed, about the risk of 2,4,5-T teratogenicity. Dr. Burger, technical assistant to Presidential science advisor Lee A. DuBridge, acknowledges that a study done by Bionetics Research Laboratories for the National Cancer Institute showed last March (it was suppressed for six months) that nearly all offspring of mice and rats given 2,4,5-T early in gestation at the relatively high levels of 21.5 mg/kg or 46.4 mg/kg were born dead or deformed—in some cases with no eyes, with cleft palates, and cystic kidneys and enlarged livers. Kven at 4.6 rng/kg dosage, 39% of the animals were born malformed. The OST expert is more familiar than most with the high-level decisionmaking that went into Dr. DuBridge's declaration October 29 that on the basis of the Bionetics study, the use of 2,4,5-T in populated areas would be restricted. Dr. DuBridge said Agriculture would, by Jan. 1, 1970, withdraw licenses for its use on crops (corn, bluberries, peaches, pears, and several leafy vegetables) unless the FDA found that the residue was negligible and humans were tolerant of it. Dr. Burger explains that the FDA missed this deadline for a number of reasons. First, Dow Chemical Co., a major maker of 2,4,5-T, discovered last December that the sample used by Bionetics contained 27 ppm of the tetrachloro dioxin instead of the "less than 1 ppm" Dow says is in its product. So the study is now being re-run with a Dow sample at Dow labs in Zionsville, Ind., and Midland, Mich., and at the National Institute Environmental Health Sciences. Next, says Dr. Burger, even after the teratogenic potential is re-evaluated in a rodent model, the disappearance rate of the contaminant in the animal blood stream must be determined and calibrated with that in human volunteers. He concludes: "The possibility of exposure to 2,4,5-T, vis-a-vis the small teratogenic risk, is, certainly not sufficient at this time to justify wiping the chemical off the market." Comments Associate FDA Commissioner for Science Dale Lindsay: "Dr. DuBridge had no damned business setting a tolerance deadline. Our marketbasket surveys for 1968 and 1969—thousands of samples of 120 foods and vegetables are constantly being assessed—show only five recoveries of 2,4,5-T— three from leafy vegetables at negligible levels, plus one from, vmilk, and one from meat at the .01-mg level. "Yet if we Had to set a tolerance today it would be zero. The trouble with this very active dioxin contaminant is that while it may be a known quantity in a product, you can't extract it in the same quantity." 107 106 w Harvard microbiologist Matthew Meselson is worried for the same reasonand many others. Dr. Meselson—appointed last year by the American Associa tion for the Advancement of Science to head a 2,4,5-T evaluation project— says: "The tetrachloro dioxin represents just one of 12' or 13 ways the chlorine atoms can arrange themselves on a benzene ring to form dioxin molecules. How do we know about the hexa, hepta, and octychlors, or about how persistent the tetrachlor itself is? Moreover, I'm very concerned about the dioxins that might be formed by vmreacted trichlorphenol [2,4,5-T precursor] when the product is exposed to heat If it were taken up by plants or wood and these were burned, you'd get more dioxin. Finally, I'm bothered by the bizarre mental effects suffered by German workers making 2,4,5-T. I say when in Dr. doubt, stopJohnson, it." Julius vice president and director of research for Dow, regards these concerns as speculative. "If we thought 2,4,5-T was harming anybody we'd take it off the market tomorrow," he says. "We've been dedicated to cleaning it up ever since 1964 [when the contaminant was linked to an outbreak of chlor-acne in Dow workers at Midland]." Dr. Johnson says it would take a 200-degree jolt to produce reaction- of dioxin, and the contaminant disappears within hours under ultraviolet light. So far, he adds, Dow tests show that its, 2,4,5-T has no teratogenic effect on rats at a dosage of 24 mg/kg and on rabbits at 40 mg/kg. But how about Dr. Verrett's new findings in the chick embryo test? The Dow executive confesses surprise. "But I'm confident," he says, '"that we'll be safe when we propose a new specification for all 2,4,5-T products .1 ppm of dioxin." Safety ofalso assumes gauges of teratogenicity in the population, however. FDA's Dr. Lindsay spoke with certitude when he told MWN that "the National Institute of Neurological Diseases and Stroke has recorded birth defects for some 15 years and would be telling us if they were on the rise." He's wrong. Dr. Heinz Berendes, chief of KINDS' perinatal research branch, admits dolefully that "no nationwide data are available on frequency or incidence of malformation." Adds Yale biologist Arthur Galston: "It's shocking, but absolutely no studies have been made in Vietnam either. There have been reports of birth defects in Saigon papers since last June but hospital records haven't been made available." State Department' officials say they know of no policy whereby such data would be classified or withheld. Significantly, however, Dr. Malcolm Phelps, chief of the Vietnam medical section of the Agency for International Development, says he is acting on a recent White House request to collect figures on tcratological occurrences in Vietnam civilian hospitals. As for all the toxic effects reported by Globe residents after the June 8-11 spraying—a helicopter released 935 gallons of Silvem, 30 of 2,4,5-T, and 20 gallons of a combination called "Orange" over 1,900 acres of forest;—an MWN reporter inquired into the histories of 18 patients with four of the five doctors who treated them, and checked on the two crippled goats, the crippled duck, a bleeding bull terrier, and two other dogs with pneumonia. Net result: two strongly suspected herbicide poisoning cases linked to the spraying, and one "definite." There's one-year-old Paul McOray, who lives on the edge of Tonto National Forest and whose father drove the family right up to the 'copter landing spot during spraying. The boy has had respiratory attacks and convulsions. Phoenix pediatrician W. Scott Ohisholm finds Paul has lymphositosis, with a white coll count twice normal. The second suspected case, a smeltery worker named James Andrews who has complained of a number of symptoms associated with herbicide poisoning —nausea, muscle weakness, vertigo, numbness, and stabbing pain—ia vouchsafed by Dr. Granville Knight of Santa Monica, Calif. In the third case, that of Mrs, Billee Shoecraft, Dr. Knight says he has found 2-4-D in tissue. Dr. Bernard Collopy would not label the muscle spasms and stabbing pain suffered by potter Hobert McKusick, owner of the defective goats and ducks, ns herbicide-related. Dr. William Bishop would not credit the chest pains of ^<* McCray, father of little Paul, or bis wife's tingling fingers and toes, as 1 i,' ' %C;^ ov SitDcoj poisoning. And veterinarian EVI. Skinner hadn't seen any of *,*^'v\ cases. *«*>' wn»vXi'lJl> ^ lR bop: "There's a good possibility some of the human cases "raying, but symptomatic connections aren't connections and I'm no toxicologist. People here are emotional and each mornlMlrake up with new nails pounded into their palms. What's needed is solid scientific investigation. All I hope is they don't leave us hanging in the air for the next 20 years." Appendix 3 [From the New Yorker, Feb. 7, 1070] A REPOBTEB AT LARGE: DEFOLIATION By Thomas Whiteside Late in 1961, the United States Military Advisory Group in Vietnam began, as a minor test operation, the defoliation, by aerial spraying, of trees along the sides of roads and canals east of Saigon. The purpose of the operation was to increase visibility and thus safeguard against ambushes of allied troops and make more vulnerable any Vietcong who might be concealed under cover of the dense foliage. The number of acres sprayed does not appear to have been publicly recorded, but the test was adjudged a success militarily. In January, 1962 following a formal announcement by South Vietnamese and American officials that a program of such spraying was to be put into effect, and that it was intended "to improve the country's economy by permitting freer communication as well as to facilitate the Vietnamese Army's task of keeping these avenues free of Vietcong harassments," military defoliation operations really got under way. According to an article that month in the New York Times, "a high South Vietnamese official" announced that a seventy-mile stretch of road between Saigon and the coast was sprayed "to remove foliage hiding Communist guerrillas." The South Vietnamese spokesman also announced that defoliant chemicals would be sprayed on Vietcong plantations of manioc and sweet potatoes in the Highlands. The program was gathering momentum. It was doing so in spite of certain private misgivings among American officials, particularly in the State Department, who feared, first, that the operations might open the United States to charges of engaging in chemical and biological warfare, and second, that they were not all that militarily effective; Roger Hilsman, now a professor of government at Columbia University, and then Director of Intelligence and Research for the State Department, reported, after a trip to Vietnam, that defoliation operations "had political disadvantages" and, furthermore, that they were of questionable military value, particularly in accomplishing their supposed purpose of reducing cover for ambushes. Hilsman later recalled in his book, "To Move a Nation," his visit to Vietnam, in March, 1062; "I had flown down a stretch of road that had been used for a tost and found that the results were not very impressive. , . . Later, the senior Australian military representative in Saigon, Colonel Serong, also pointed out that defoliation actually aided the ambushers—if the vegetation was close to the road those who were ambushed could take cover quickly; when it was removed the guerrillas had a better field of fire." According to Hilsman, "The National Security Council spent tense sessions debating the matter." Nonetheless, the Joint Chiefs of Staff and their Chairman, General Maxwell Taylor, agreed that chemical defoliation was a useful military weapon. In 1962, the American military "treated" 4,940 acres of the Vietnamese countryside with herbicides, In 1963, the area sprayed increased five-fold to a total of 24,700 acres. In 1964, the defoliated area was more than tripled, In 1965, the 1964 figure was doubled, increasing to 155,610 acres. In 1966, the sprayed area was again increased fivefold, to 741,247 acres, and in 1967 it was doubled once again over the previous year, to 1,486,446 acres. Thus, the areas defoliated in Vietnam had increased approximately three' hundredfold in five years, but now adverse opinion among scientists and other people who were concerned about the effects of defoliation on the Vietnamese ecology at last began to have a braking effect on the program. In 1968, 1,267,110 acres were sprayed, and In 1969 perhaps a million acres. Since 1962, the defoliation operations have covered almost five million acres, an area equivalent to about twelve per cent of the entire territory of South Vietnam, and about the size of the'state of Massachusetts. Between 1902 and 1967, the deliberate destruction of plots of1 rice, manioc, beans, and other foodstuffs through herbicidal spraying—the word "deliberate" is used here to exclude the many reported instances of accidental 45-362—70 8 108 109 'spraying of Vietnamese plots—increased three hundredfold, from an 741 acres to 221,312 acres, and by the end of 1909 the Vietnamese cropgrowing area that since 1902 had been sprayed with herbicides totalled at least half a million acres. By then, in many areas the original purpose of the defoliation had been all but forgotten. The military had discovered that a more effective way of keeping roadsides clear was to bulldoze them. But by the time of that discovery defoliation had settled in as a general policy and taken on a life of its own—mainly justified on the ground that it made enemy infiltration from the North much more difficult by removing vegetation that concealed jungle roads and trails. During all the time since the program began in 1901, no American military or civilian official has ever publicly characterized it as an operation of either chemical or biological warfare, although there can be no doubt that it is an operation of chemical warfare in that it involves the aerial spraying of chemical substances with the aim of gaining a military advantage, and that it is an operation of biological warfare in that it is aimed at a deliberate disruption of the biological conditions prevailing in a given area. Such distinctions simply do not appear in official United States statements or documents; they were long ago shrouded under heavy verbal cover. Thus, a State Department report, made public in March, 1900, saying that about twenty thousand acres of crops in South Vietnam had been destroyed by defoliation to deny food to guerrillas, described the areas involved as "remote and thinly populated," and gave a firm assurance that the materials sprayed on the crops were of a mild and transient potency: "The herbicides used are nontoxic and not dangerous to man or animal life. The land is not affected for future use." However comforting the statements issued by our government during seven years of herbicidal operations in Vietnam, the fact is that the major development of defoliant chemicals (whose existence had been known in the thirties) and other herbicidal agents came about in military programs for biological warfare. The direction of this work was set during the Second World War, when Professor K. 3. Kraus, who then headed'the Botany Department of the University of Chicago, brought certain scientific possibilities to the attention of a committee that had been set up by Henry L. Stimson, the Secretary of War, under the National Research Council, to provide the military with advice on various aspects of biological warfare. Kraus, referring to the existence of hormone-like substances that experimentation had shown would kill certain plants or-disrupt their growth, suggested to the committee in 1941 that it might be interested in "the toxic properties of growth-regulating substances for the destruction of crops or the limitation of crop production." Military research on herbicides thereupon got under way, principally at Camp (later Fort) Detrick, Maryland, the Army center for biological-warfare research. According to George Merck, a chemist, who headed Stimson's biological-warfare advisory committee, "Only the rapid ending of the war prevented field trials in an active theatre of synthetic agents that would, without injury to human or animal life, affect the growing crops and make them useless." After the war, many of the herbicidal materials that had been developed and tested for biological-warfare use were marketed for civilian purposes and used by farmers and homeowners for killing weeds and controlling brush. The most powerful of the herbicides were the two chemicals 2,4-dichlorophenoxyacetic acid, generally known as 2,4-D, and 2,4,5-trichlorpphenoxyacetic acid, known as 2,4,5-T. The direct toxicity levels of these chemicals as they'affected experimental animals, and, by scientific estimates, men, appeared then to be low (although these estimates have later been challenged), and the United States Department of Agriculture, the Food and Drug Administration, and the Fish and Wildlife Service all sanctioned the widespread sale and use of both. The chemicals were also reported to be Shortlived in soil after their application. 2,4-D was the bigger seller of the two, partly because it was cheaper, and suburbanites commonly used mixtures containing 2,4-D on their lawns to control dandelions and other weeds. Commercially, 2,4-D and 2,4,5-T were used to clear railroad rights-of-way and power-line routes, and, in cattle country, to get rid of woody brush, 2,4,5-T being favored for the last, because it was considered to have a more effective herbicidal action on woody plants. Very often, however, the two chemicals were used in combination. Between 1945 and 1903, (lie production of herbicides jumped from nine hundred and seventeen thousand pounds to about a hundred and flfty million pounds in this country; bercent—more since 1903, their use had risen two hundred and seventy-c^^perc than of increase^ in the use thouiHpesl i.imu double wumj.c the •,«« rate *..*..~ ~~ - -- of pesticides, . pesticides are Htlll far more extensively used. By 1900, an area equivalent to more than three per cent of the entire United States was being sprayed each year with herbicides. Considering the rapidly growing civilian use of these products, it is perhaps , not surprising that the defoliation operations in Vietnam escaped any significant comment in the press, and that the American public remained unaware of the extent to which these uses had their origin in planning for chemical and biological warfare. Nevertheless, between 1941 and the present, testing and experimentation in the use of 2,4-D, 2,4,5-T, and other herbicides as military weapons were going forward very actively at Fort Detrick. While homeowners were using herbicidal mixtures to keep their lawns free of weeds, the military were screening some twelve hundred compounds for their usefulness in biological-warfare operations. The most promising of these compounds were testsprayed on tropical vegetation in Puerto Rico and Thailand, and by the time fullscale defoliation operations got under way in Vietnam the U.S. military had settled on the use of four herbicidal spray materials there. These went under the names Agent Orange, Agent Purple, Agent White; and Agent Blue— designations derived from color-coded stripes girdling the shipping drums of each type of material. Of these materials, Agent Orange, the most widely used as a general defoliant, consists of a fifty-fifty mixture of n butyl esters and of 2,4-D and 2,4,5-T. Agent Purple, which is interchangeable with Agent Orange, consists of the same substances with slight molecular variations. Agent White, which is used mostly for forest defoliation, is a combination of 2,4-D and Picloram, produced by the Dow Chemical Company. Unlike 2,4-D or 2,4,5-T, which, after application, is said to be decomposable by micro-organisms in soil over a period of weeks or months (one field test of 2,4,5-T in this country showed that significant quantities persisted in soil for ninety-three days after application), Picloram—whose use the Department of Agriculture has not authorized in the cultivation of any American crop—is one of the most persistent herbicides known. Dr. Arthur W. Galston, professor of biology at Yale, has described Picloram as "a herbicidal analog of DDT," and an article in a Dow Chemical Company publication called "Down to Earth" reported, that in field trials of Picloram in various California soils between eighty and ninety-six and a half per cent of the substance remained in the soils four hundred and sixty-seven days after application. (The rate at which Picloram decomposes in tropical soils may, however, be higher.) Agent Blue consists of a solution of cacodylic acid, a substance that contains fifty-four per cent arsenic, and it is used in Vietnam to destroy rice crops. According to the authoritative "Merck Index," a source book on chemicals, this material is "poisonous." It can be used on agricultural crops in this country only under certain restrictions imposed by the Department of Agriculture. It is being used herbicidally on . Vietnamese rice fields at seven and a half times the concentration permitted for weed-killing purposes in this country, and so far in Vietnam something like five thousand tons is estimated to have been sprayed on paddies and vegetable fields. Defoliation operations in Vietnam are carried out by a special flight of the 12th Air Commando Squadron of the United States Air Force, from a base at Bien Hoa, just outside Saigon, with specially equipped C-123 cargo planes. Bach of .these aircraft has been fitted out with tanks capable of holding a thousand gallons. On defoliation missions, the herbicide carried in these tanks is sprayed from an altitude of around a hundred and fifty feet, under pressure, from thirty-six nozzles on the wings and tail of the plane, and usually several spray planes work in formation, laying down broad blankets of spray. The normal crew of a military herbicidal-spray plane consists of a pilot, a copilot, and a technician, who sits in the tail area and operates a console regulating the spray. The equipment is calibrated to spray a thousand gallons of herbicidal mixture at a rate that works out, when all goes well, to about three gallons per acre. Spraying a thousand-gallon tankload takes five minutes. In an emergency, the tank can he emptied in thirty seconds—a fact that has particular significance because of what has recently been learned about the nature of at least one of the herbicidal substances. The official code name for the program is Operation Hades, but a, more friendly code name, Operation Ranch Hand, is commonly used. In similar fash-. ion, military public-relations men refer to the herbicidal spraying of crops sup- 110 111 dly grown for Vietcong use in Vietnam, when they refer to it at all rt-denial program." By contrast, an American biologist who is less enthusiastic about the effort has called it, in its current phase, "escalation to a program of starvation of the population in the affected area." Dr. Jean Mayer, the Harvard professor who now is President Nixon's special adviser on nutrition, contended in an article in Science and, Citizen in 1967 that the ultimate target of herbicidal operations against rice and other crops in Vietnam was "the weakest clement of the civilian population"—that is, women, children, and tlm elderly—because in the sprayed area "Vietcong soldiers may . . . be expected to get the fighter's share of whatever food there is." He pointed out that malnutrition is endemic in many parts of Southeast Asia but that in wartime South Vietnam, where diseases associated with malnutrition, such as beri-beri, anemia, kwashiorkor (the disease that has decimated the Biafran population), and tuberculosis, are particularly widespread, "there can be no doubt that if the (crop-destruction) program is continued, (the) problems will grow." AVhether a particular mission involves defoliation or crop destruction, American military spokesmen insist that a mission never takes place without careful consideration of all the factors involved, including the welfare of friendly inhabitants and the safety of American 'personnel. (There can be little doubt that defoliation missions are extremely hazardous to the members oC the planes' crews, for the planes are required to fly very low and only slightly above stalling speed, and they are often targets of automatic-weapons flre from the ground.) The process of setting up targets and approving specific herbicidal operations is theoretically subject to elaborate review through two parallel chains of command; one chain consisting of South Vietnamese district and > province chiefs—who can themselves initiate such missions—and South Vietnamese Army commanders at various levels; the other a United States chain, consisting of a district adviser, a sector adviser, a divisional senior adviser, a corps senior adviser, the United States Military Assistance Command in South Vietnam, and the American Embassy in Saigon, ending up with the American ambassador himself. Positive justification of the military advantage likely to be gained from each operation is theoretically required, and applications with such positive justification are theoretically disapproved. However, according to one of a series of articles by Elizabeth Pond that appeared toward the end of 1907 in the Christian Science Monitor: "In practice, [American] corps advisers find it very difficult to turn down defoliation requests from province level because they simply do not have sufficient specific knowledge to call a proposed operation into question. And with the momentum of six years' use of defoliants, the practice, in the words of one source, has long since been "set in cement." "The real burden of proof has long since shifted from the positive one of justifying an operation by its [military] gains to the negative one of denying an operation because of [specific] drawbacks. There is thus a great deal of pressure, especially above province level, to approve recommendations sent up from below as a matter of course." Miss Pond reported that American military sources in Saigon were "enthusiastic" about the defoliation program, and that American commanders and spotter-plane pilots were "clamoring for more of the same." She was given firm assurances as to the mild nature of the chemicals used in the spray operations : "The defoliants used, according to the military spokesman contacted, are the same herbicides . . . as those used commercially over some four million acres in the United States. In the strengths used in Vietnam they are not at all harmful to humans or animals, the spokesman pointed out, and in illustration of this he dabbed onto his tongue a bit of liquid from one of ... three bottles sitting on his desk." As the apparently inexorable advance of defoliation operations in South Vietnam continued, a number of scientists in the United States began to protest the military use of herbicides, contending that Vietnam was being used, in effect, as a proving ground for chemical and biological warfare. Early in 1966, a group of twenty-nine scientists, under the leadership of Dr. John Edsall, a professor of biochemistry at Harvard, appealed to President Johnson to prohibit the use of defoliants and crop-destroying herbicides, and called the use of these substances in Vietnam "barbarous because they are indiscriminate." In the late summer of 1006, this protest was followed by a letter of petition to President Johnson from twenty-two scientists, including seven Ncbel laureates. The petition pointed out that the "large-scale use of auticroij^M 'nonlethal' antipersonnel chemical weapons in Vietnam" constituted "danglBHs precedent" in chemical and biological warfare, and it asked the President to order it stopped. Before the end of that year, Dr. Edsall and Dr. Matthew S. Meselson, a Harvard professor of biology, obtained the signatures of five thousand scientists to co-sponsor the petition. Despite these protests, the area covered by defoliation operations in Vietnam in 1967 was double that covered in 1900, and the acreage of crops destroyed was nearly doubled. These figures relate only to areas that were sprayed intentionally. There is no known way of spraying an area with herbicides from the air in a really accurate manner, because the material used is so highly volatile, especially under tropical conditions, that even light wind drift can cause extensive damage to foliage find crops outside the deliberately sprayed area. Crops are so sensitive to the herbicidal spray that it can cause damage to fields and gardens as much as fifteen miles away from the target zone, Particularly severe accidental damage is reported, from time to time, to so-called "friendly" crops in the III Corps area, which all but surrounds Saigon and extends in a rough square from the coastline to the Cambodian border. Most of the spraying in til Corps is now done in War Zones 0 and D, which are classified as free fire zones, where, as one American official has put it, "everything that moves in Zones C and D is considered Charlie." A press dispatch from Saigon in 1967 quoted another American official as saying that every Vietnamese farmer in tint*- corps area knew of the defoliation program and disapproved of it. Dr. (Jalston, the Yale biologist, who is one of the most persistent critics of American policy concerning herbicidal operations in Vietnam, recently said in an interview, "We know that most of the truck crops grown along roads, canals, and trails and formerly brought into Saigon have been essentially abandoned because of the deliberate or inadvertent falling of these defoliant sprays; many crops in the Saigon area are simply not being harvested." He also cited reports that in some instances in which the inhabitants of Vietnamese villages have been suspected of being Vietcong sympathizers the destruction of food crops has brought about complete abandonment of the villages. In 1966, herbicidal operations caused extensive inadvertent damage, through wind drift, to a very large rubber plantation northwest of Saigon owned by the Michelin rubber interests. As the result of claims made for this damage, the South Vietnamese authorities paid the corporate owners, through the American military, nearly a million dollars. The extent of the known inadvertent damage to crops in Vietnam can be inferred from the South Vietnamese budget—in reality, the American military budget—for settling such claims. In 1967, the budget for this compensation was three million six hundred thousand dollars. This sum, however, probably reflects only the barest emergency claims of the people affected. According to Representative Eichard D. McCarthy, a Democrat from upstate Now York who has been a strong critic of the program, the policy of allowing applications for defoliation operations to flow, usually without question, from the level of the South Vietnamese provincial or district chiefs has meant that these local functionaries would order repeated sprayings of areas that they had not visited in months, or even years. The thought that a Vietnamese district chief can initiate such wholesale spraying, in effect without much likelihood of serious hindrance by American military advisers, is a disquieting one to a number of biologists. Something that disquiets many of them even more is what they believe the long-range effects of nine years of defoliation operations will be on the ecology of South Vietnam. Dr. Galston, testifying recently before a congressional subcommittee on chemical and biological warfare, made these observations: "It has already been well documented that some kinds of plant associations subject to spray, especially by Agent Orange, containing 2,4-D and 2,4,5-T, have been irreversibly damaged. I refer specifically to the mangrove associations that line the estuaries, especially around the Saigon River. Up to a hundred thousand acres of these mangroves have been sprayed. . . . Some (mangrove areas) had been sprayed as early as 1961 and have shown no substantial signs of recovery. . . . Ecologists have known for a long time that the mangroves lining estuaries furnish one of the most important ecological niches for the completion of the life cycle of certain shellfish and migratory fish. If these plant communities are not in a healthy state, secondary effects on tbo whole « 112 113 interlocked web of organisms are bound to occur. . . . In the years^fcead the Vietnamese, who do not have overabundant sources of proteins a^P&w, are probably going to suffer dietarily because of the deprivation Of food in the form of fish and shellfish. "Damage to the soil is another possible consequence of extensive defoliation. . . . We know that the soil is not a dead, inert mass but, rather, that it is a vibrant, living community. . . . If you knock the leaves off of trees once, twice, or three times . . . you change the quality of the soil. . . . Certain tropical soils—and it lias been estimated that in Vietnam up to fifty per cent of all the soils fall into this category—are laterizable; that is, they may be irreversibly converted to rock as a result of the deprivation of organic matter. . . . If ... you deprive trees of leaves and photosynthesis stops, organic matter in the soil declines and laterization, the making of brick, may occur on a very extensive scale. I would emphasize that this brick is irreversibly hardened; it can't be made back into soil. . . . "Another ecological consequence is the invasion of an area by undesirable plants. One of the main plants that invade an area that has been defoliated is bamboo. Bamboo is one of the most difficult of all plants to destroy once it becomes established where you don't want it. It is not amenable to killing by herbicides. Frequently it has to be burned over, and this causes tremendous dislocations to agriculture." Dr. Fred H. Tschirley, assistant chief of the Crops Protection Research Branch of the Department of Agriculture, who made a month's visit to Vietnam in the spring of 1968 in behalf of the State Department to report on the ecological effects of herbicidal operations there, does not agree with Dr. Galston's view that laterization of the soil is a serioxis probability. However, he reported to the State Department that in the Bung Sat area, southeast of Saigon, where about- a hundred thousand acres of mangrove trees had been sprayed with defoliant, each single application of Agent Orange had killed ninety to a hundred per cent of the mangroves touched by the spray, and he estimated that the regeneration of the mangroves in this area would take another twenty years, at least. Dr. Tschirley agrees with Dr. Galston that a' biological danger attending the defoliation of mangroves is an invasion of virtually ineradicable bamboo. A fairly well-documented example not only of the ecological consequences of defoliation operations but also of their disruptive effects on human life was provided last year by a rubber-plantation area in Kompong Cham Province, Cambodia, which lies just across the border .from Vietnam's Tay Ninh Province. On June 2, 1969, the Cambodian government, in an angry diplomatic note to the United States government, charged the United States with major defoliation damage to rubber plantations, and also to farm and garden crops in the province, through herbicidal operations deliberately conducted on Cambodian soil, It demanded compensation of eight and a half million dollars for destruction or serious damage to twenty-four thousand acres of trees and crops. After some delay, the'State Department conceded that the alleged damage might be connected with' "accidental drift" of spray over the border from herbicidal operations in Tay Ninh Province. The Defense Department flatly denied that the Cambodian areas had been deliberately sprayed. Late in June, the State Department sent a team of four American scientists to Cambodia, and they confirmed the extent of the area of damage that the Cambodians had claimed. They found that although some evidence of spray drift across the Vietnamese border existed, the extent and severity of damage in the tiren. worst affected were such that "it is highly unlikely that this quantity could have drifted over the border from the Tay Ninh defoliation operations." Their report added, "The evidence we have seen, though circumstantial, suggests strongly that damage was caused by direct overflight." A second report on herbicidal damage to the area was made after an unofficial party of American biologists, including Professor E: W. Pfeifl'er, of the University of Montana, and Professor Arthur H. AVesting, or Windham College, Vermont, visited Cambodia last December at the invitation of the Cambodian government. They found that about a third of all the rubber trees currently in production in Cambodia had been damaged, and this had happened in an area that normally had the highest latex yield per acre of any in the world. A high proportion of two varieties of rubber trees in the area had died as a result of the damage, and Dr. Westing estimated that the damage to the latex-producing capacity of some varieties might persist for twenty years. Between May and November of last year, latex production in the affected plantations fell off by^^iverage of between thirty-five and forty per cent. According to a report b^^e two scientists, "A large variety of garden crops were devastated in the seemingly endless number of small villages scattered throughout tiie affected area. Virtually all of the . . . local inhabitants . . . depend for their wellbeing upon their own local produce. These people saw their crops . . . literally wither before their eyes." The Cambodian claim is still pending. Until the end of last year, the criticism by biologists of the dangers involved in the use of herbicides centered on their use in what were increasingly construed as biological-warfare operations, and on the disruptive effects of these chemicals upon civilian populations and upon the ecology of the regions in whicli they were used. Last year, however, certain biologists began to raise serious questions on another score—possible direct hazards to life from 2,4,C-T. On October 29th, as a result of these questions, a statement was publicly issued by Dr. Lee DuBridge, President Nixon's science adviser. In summary, the statement said that because a laboratory study of mice and rats that had been given relatively high oral doses of 2,4,6-T in early stages of pregnancy "showed a higher than expected number of deformities" in the offspring, the government would, as a precautionary measure, undertake a series of coordinated actions to restrict the use of 2,4,5-T in both domestic civilian applications and military herbicidal operations. The DuBridge statement identified the laboratory study as having been made by an organization called the Bionetics Research Laboratories, in Bethesda, Maryland, but gave no details of either the findings or the data on which they were based. This absence of specific information turned out to be characteristic of what has been made available to the public concerning this particular.research project. From the beginning, it seems, there was an extraordinary reluctance to discuss details of the purported ill effects of 2,4,5-T on animals. Six weeks after the publication of the DuBridge statement, a journalist who was attempting to obtain a copy of the full report made by Bionetics and to discuss its details with some of the government officials concerned encountered hard going. At the Bionetics Laboratories, an official said that he couldn't talk about the study, because "we're under wraps to the National Institutes of Health"—the government agency that commissioned the study. Then, having been asked what the specific doses of 2,4,5-T were that were said to have increased birth defects in the fetuses of experimental animals, the Bionetics official cut off discussion by saying, "You're asking sophisticated questions that as a layman you don't have the equipment to understand the answers to." At the National Institutes of Health, an official who was asked for details of or a copy of the study on 2,4,5-T replied, "The position I'm in is that I have been requested not to distribute this information." He did say, however, that a continuing evaluation of the study was under way at the National Institute of Environmental Health Sciences, at Research Triangle Park, North Carolina. A telephone call to an officer of this organization brought a response whose tone varied from wariness of downright hostility and made it clear that the official had no intention of discussing details or results of the study with the press.. Tlie Bionetics study on 2,4,5-T was part of a series carried out under contract to the National Cancer Institute, which is an arm of the National Institutes of Health, to investigate more than two hundred compounds, most of them pesticides, in order to determine whether they induced cancer-causing changes, fetus-deforming changes, or mutation-causing changes in experimental animals. The contract was a large one, involving more than two and a half million dollars' worth of research, and its primary purpose was to screen out suspicious-looking substances for further study. The first visible fruits of the Bionetics research were presented in March of last year before a convention of the American Association for the Advancement of Science, in the form of a study of possible carcinogenic properties of the fifty-three compounds; the findings on 2,4,5-T were that it did not appear to cause carcinogenic changes in the animals studied.' By the time the report on the carcinogenic properties of the substances was presented, the results of another part of the Bionetics studies, concerning the teratogenic, or fetus-deforming, properties of the substances, were being compiled, but these results were not immediately made available to biologists outside the government. The data remained—somewhat frustratingly, in the view of some scientists who had been most curious about the effects of herbicides— out of sight, and a number of attempts by biologists who had heard about tlie JU<± « ,tologieal study of 2,4,5-T to get at its findings appear to have been arted by the authorities involved. Upon being asked to account fouflfcR nrent delay in making this information available to biologists, an offlc^^E the National Institute of Environmental Health Sciences (another branch of the National Institutes of Health) has declared, with some heat, that the results of the study itself and of a statistical summary of the findings prepared by the Institute were in fact passed on as they were completed to the Commission on Pesticides and Their Relationship to Environmental Health, a scientific group appointed by Secretary of Health, Education, and Welfare Robert Finch and known—after its chairman, Dr. E. M. Mrak, of the University of California—as the Mrak Commission. Dr. Samuel S. Epstein, chief of the Laboratories of Environmental Toxicology and Carcinogenesis at the Children's Cancer Research Foundation in Boston, who was co-chairman of the Mrak Commission panel considering the teratogenic potential of pesticides, tells a different story on the availability of the Bionetics study. He says that he first heard about it in February. At a meeting of his panel in August, he asked for a copy of the report. Ten days later, the panel was told that the National Institute of Environmental Health Sciences would be willing to provide a statistical summary but that the group could not have access to the full report on which the summary was based. Dr. Epstein says that the panel eventually got the full report on September 24th "by pulling teeth." Actually, as far back as February, officials at the National Cancer Institute had known, on the basis of a preliminary written outline from Bionetics, the findings of the Bionetics scientists on the fetus-deforming role of 2,4,5-T. Dr. Richard Bates, the officer of the National Institutes of Health who was in charge of coordinating the Bionetics project, has said that during the same month this information was put into the hands of officials of the Food and Drug Administration, the Department of Agriculture, and the Department of Defense. "We had a meeting with a couple of scientists from Fort Detrick, find we informed them of what we had learned," Dr. Bates said recently. "I don't know whether they were the right people for us to see. We didn't hear from them again until after the DuBridge announcement at the White House. Then they called up and asked for a copy of the Bionetics report." At the Department of Agriculture, which Dr. Bates said had been informed in February of the preliminary Bionetics findings, Dr. Tschirley, one of the officials most intimately concerned with the permissible uses of herbicidal compounds, says that he first heard about the report on 2,4,5-T through the DuBridge announcement. At the Food and Drug Administration, where appropriate officials had been informed in February of the teratogenic potential of 2,4,5-T, no new action was taken to safeguard the public against 2,4,5-T in foodstuffs. I]] fact, it appears that no action at all was taken by the Food and Drug Administration on the matter during the whole of last year. The explanation that F.D.A. officials have offered for this inaction is that they were under instructions to leave the whole question alone at least until December, because the matter was under definitive study by the Mrak Commission—the very group whose members, as it turns out, had such extraordinary difficulty in obtaining the Bionetics data. The Food Toxicology Branch of the1 F.D.A. did not have access to the full Bionetics report on 2,4,5-T until after Dr . DuBridge issued his statement, at the end of October. Thus, after the first word went to various agencies about the fetus-deforming potential of 2,4,5-T, and warning lights could have flashed on in every branch of the government and in the headquarters of every company manufacturing or handling it, literally almost nothing was done by the officials charged with protecting the public from exposure to dangerous or potentially dangerous materials—by the officials in the F.D.A., in the Department of Agriculture, and in the Department of Defense. It is conceivable that the Bionetics findings might still be hidden from the public if they had not been pried loose in midsummer through the activities of a group of young law students. The students were members of a team put together by the consumer-protection activist Ralph Nader—and often referred to as Nader's Raiders—to explore the labyrinthine workings of the Food and Drug Administration. In the course of their investigations, one of the law students, a young woman named Anita Johnson, happened to see a copy of the preliminary report on the Bionetics findings that had been passed on to the F.D.A. in February, and its observations seemed quite disturbing to her. Miss Johnson wrote a report to Nader, and in September she showed a copy of the report to a friend who was a biology student at Harvard. In early October, Miss Johnson's friend, in a conversation with Professor Matthew Meselson, mentioned Miss Johnson's report onJjje preliminary Bionetics findings. This was the first that Dr. Meselson had hj^B of the existence of the Bionetics study. A few days previously, he had^wceived a call from a scientist friend of his asking whether Dr. Meselson had heard of certain stories, originating with South Vietnamese journalists and other South Vietnamese, of an unusual incidence of birth defects in South Vietnam, which were alleged to be connected with defoliation operations there. A few days later, after his friend sent him further information, Mr. Meselson decided to obtain a copy of the Bionetics report, and he called up an acquaintance in a government agency and asked for it He was told that the report was "confidential and classified," and inaccessible to outsiders. Actually, in addition to the preliminary report there were now in existence the full Bionetics report and a statistical summary prepared by the National Institute of Environmental Health Sciences, and, by nagging various Washington friends, Dr. Meselson obtained bootlegged copies of the two latest reports. What he read seemed to him to have such serious implications that he got in touch with acquaintances in the White House and also with someone in the Army to alert them to the problems of 2,4,5-T, in the hope that some new restriction would be placed on its use. According to Dr. Meselson, the White House people apparently didn't know until that moment that the reports on the adverse effects of 2,4,5-T even existed. (Around that time, according to a member of Nader's Raiders, "a tremendous lid was put on this thing" within government agencies, and on the subject of the Bionetics work and 2,4,5-T "people in government whom we'd been talking to freely for years just shut up and wouldn't say a word.") While Dr. Meselson awaited word on the matter, a colleague of his informed the press about the findings of the Bionetics report. Very shortly thereafter, Dr. DuBridge made his public announcement of the proposed restrictions on the use of 2,4,5-T. In certain respects, the DuBridge announcement is a curious document. In its approach to the facts about 2,4,5-T that were set forth in the Bionetics report, it reflects considerable sensitivity to the political and international issues that lie behind the widespread use of this powerful herbicide for civilian and military purposes, and the words in which it describes the reasons for restricting its use appear to have been very carefully chosen : "The actions to control the use of the chemical were taken as a result of findings from a laboratory study conducted by Bionetics Research Laboratories which indicated that offspring of mice and rats given relatively large oral doses of the herbicide during early stages of pregnancy showed a higher than expected number of deformities. "Although it seems improbable that any person could receive harmful amounts of this chemical from any of the existing uses of 2,4,5-T, and while the relationships of these effects in laboratory animals to effects in man are not entirely clear at this time, the actions taken will assure safety of the public while further evidence is being sought." These actions, according to the statement, included decisions that the Department of Agriculture would cancel manufacturers' registrations of 2,4,5-T for use on food crops, effective at the beginning of 1970, "unless by that time the Food and Drug Administration has found a basis for establishing a safe legal tolerance in and on foods," and that the Departments of Agriculture and the Interior, in their own programs, would stop the use of 2,4,5-T in populated areas and in all other areas where residues of the substance could reach man. As for military uses of 2,4,5-T, the statement said, "The chemical is effective in defoliating trees and shrubs and its use in South Vietnam has resulted in reducing greatly the number of ambushes, thus saving lives." However, the statement continued, "the Department of Defense will [henceforth] restrict the use of 2,4,5-T to areas remote from the population." All this sounds eminently fair and sensible, but whether it represents a candid exposition of the facts about 2,4,5-T and the Bionetics report is debatable. The White House statement that the Bionetics findings "indicated that offspring of mice and rats given relatively large oral doses of the herbicide during early stages of pregnancy showed a higher than expected number of deformities" is, in the words of one eminent biologist who has studied the Bionetics data, "an understatement." He went on to say that "if .the effects on experimental animals are applicable to people it's a very sad and serious situa- 116 117 ." The actual Bionetics report described 2,4,5-T as producing "sufflciq ninent effects of seriously hazardous nature" in controlled expei'ii with pregnant mice to lead the authors "to categorize [it] as probably Aanfifirous." The report also found 2,4-D "potentially dangerous hut needing further study." As for 2,4,5-T, the report noted that, with the exception of very small subcutaneous dosnges, "all dosages, routes, and strains resulted in increased incidence of abnormal fetuses" after its administration. The abnormalities in the fetuses included lack of eyes, faulty eyes, cystic kidneys, cleft palates, and enlarged livers. The Bionetics report went on to report on further experimental applications of 2,4,5-T to another species: "Because of the potential importance of the findings in mice, an additional study was carried out in rats of the Sprague-Dawley strain. Using dosages of 21.5 and 46.4 mg/kg [that is, dosages scaled to represent 21.5' and 46.4 .milligrams of 2,4,5-T per kilogram of the experimental animal's body weight] suspended in 50 per cent honey and given by the oral route on the 6th through 15th dnys of gestation, we observed excessive fetal mortality almost 80 per cent) and a high incidence of abnormalities in the survivors. AVheu the beginning of administration was delayed until the 10th day, fetal mortality was somewhat less but still quite high even when dosage was reduced to 4.6 mg/kg. The incidence of abnormal fetuses was threefold that in controls even with the smallest dosage and shortest period used. . . . .It seems inescapable the 2,4,5-T is teratogenic in this strain of rats when given orally at the dosage schedules used here." Considering the fetus-deforming effects of the lowest oral dosage of 2,4,5-T used in Bionetics work on rats—to say nothing of the excessive fetal mortality —the White House statement that "relatively large oral doses of the herbicide . . . showed a higher than expected number of deformities" is hardly an accurate description of the results of the study. In fact, the statistical tables presented as part of the Bionetics report showed that at the lowest oral dosage of 2,4,5-T given to pregnant rats between the tenth and fifteenth days of gestal.ion thirty-nine per cent of the fetuses produced were abnormal, or three, .times,, the figure for control animals. At what could without much question be described as "relatively large oral doses" of the herbicide—dosages of 21.5 and 46.4 milligrams per kilogram of body weight of rats, for example—the percentage of abnormal fetuses was ninety and a hundred per cent, respectively, or a good bit higher than one would be likely to deduce from the phrase "a higher than expected number of deformities." The assertion that "it seems improbable that any person could receive harmful amounts of this chemical from any of the existing uses of 2,4,5-T" also appears to be worth examining for this is precisely what many biologists are most worried about in relation to 2,4,5-T and allied substances. It seems fair, before going further, to quote a cautionary note in the DuBridge statement: "The study involved relatively small numbers of laboratory rats and mice. More extensive studies are needed and will be undertaken. At best it is difficult to extrapolate results obtained with laboratory animals to man—'Sensitivity to a given compound may be different in man than in animal species. . ; ," It would be difficult to get a biologist to disagree with these seemingly sound generalities. However, the first part of the statement does imply, at least to a layman, that the number of experimental animals used in the Bionetics study had been considerably smaller than the numbers used to test commercial compounds other than 2,4,5-T before they are approved by agencies such as the Food and Drug Administration and the Department of Agriculture. In this connection, the curious layman could reasonably begin with the recommendations, in 1963, of the President's Science Advisory Committee on the use of pesticides, which proposed that companies putting out pesticides should be required from then on to demonstrate the safety of their products by means of toxicity studies on two generations of at least two warm-blooded mammalian species. Subsequently, the F.D.A. set up new testing requirements, based on these recommendations, for companies producing pesticides. However, according to Dr. Joseph McLaughlin, of the Food Toxicology Branch of the F.D.A., the organization actually requires applicants for permission to sell pesticides to present the results of tests on only one species (usually, in practice, the rat). According to Dr. Mclaughlin, the average number of experimental animals used in studies of pesticides is between eighty and a hundred and sixty, including animals used as controls but excluding litl-ci-H produced. The Bionetics studies of 2,4,5-T used both mice and rats, and their total number was, in fact, greater, not less, than this average. Including controls but excluding litters, the total number of animals u^Kn the 2,4,5-T studies was two hundred and twenty-five. Analysis of th^^sults by the National Institute of Environmental Health Sciences found them statistically "significant," and this is the real purpose of such a study: it is meant to act n s a coarse screen to shake out of the data the larger lumps of bad news. Such a study is usually incapable of shaking out anything smaller; another kind of study is needed to do that. Thus, the DuBridge statement seems to give rise to this question: If the Bionetics study, based on the effects of 2,4,5-T on two hundred and twenty-five experimental animals of two species, appears to be less than conclusive, on the ground that "the study involved relatively small numbers of laboratory rats and mice," what is one to think of the adequacy of the tests that the manufacturers of pesticides make? If, as the DuBridge statement says, "at best it is difficult to extrapolate results obtained with laboratory animals to man," what is one to say of the protection that the government affords the consumer when the results of tests of pesticidal substances on perhaps a hundred and twenty rats are officially extrapolated to justify the use of the substances by a population of two hundred million people—not to mention one to two million unborn babies being carried in their mothers' wombs? The very coarseness of the screen used in all these tests—that is, the relatively small number of animals involved—means that the bad news that shows up in the data has to be taken with particular seriousness, because lesser effects tend not to be demonstrable at all. The inadequacy of the scale on which animal tests with, for instance, pesticides are currently being made in this country to gain F.D.A. approval is further indicated by the fact that a fetus-deforming effect that might show up if a thousand test animals were used is almost never picked up, since the studies are not conducted on that scale; yet if the material being tested turned out to have the same effect, quantitatively, on human beings, .this would mean that it would cause between three and four thousand malformed babies to be produced each year. The teratogenic effects of 2,4,5-T on experimental animals used by the Bionetics people, however, were not on the order of one in a thousand. Even in the case of the lowest oral dose given rats, they were on the order of one in three. Again, it is fair to say that what is applicable to rats in such tests may not be applicable to human beings. But it is also fair to say that studies involving rats are conducted not for the welfare of the rat kingdom but for the ultimate protection of human beings. In the opinion of Dr. Epstein, the fact that the 2,4,5-T used in the Bionetics study produced teratogenic effects in botli mice and rats underlines the seriousness of the study's implications. In the opinion of Dr. Mclaughlin, this is even further underlined by another circumstance— that the rat, as a test animal, tends to be relatively resistant to teratogenic effects of chemicals. For example, in the late nineteen-fifties, when thalidomide, that disastrously teratogenic compound, was being tested on rats in oral dosages ranging from low to very high, no discernible fetus-deforming effects were produced. And Dr. McLaughlin says that as far as thalidomide tests on rabbits were concerned, "You could give thalidomide to rabbits in oral doses fit between fifty and two hundred times the comparable human level to show any comparable teratogenic effects." In babies born to women who took thalidomide, whether in small or large dosages and whether in single or multiple dosages, between the sixth and seventh weeks of pregnancy, the rate of deformation was estimated to be one in ten. Because of the relatively coarse testing screen through which compounds like pesticides—and food additives as well—are sifted before they are approved for general or specialized use in this country, the Food and Drug Administration theoretically maintains a policy of stipulating, as a safety factor, that the maximum amount of such a substance allowable in the human diet range from one two-thousandth to one one-hundredth of the highest dosage level of the substance that produces no harmful effects in experimental animals. (In the case of pesticides, the World Health Organization takes a more conservative view, considering one two-thousandth1 of the "no-effect" level in animal studies to ,be a resaonsable safety level for human exposure.) According to the standards of safety established by F.D.A. policy, then, no human being anywhere should ever have been exposed to 2,4,5-T, because in the Bionetics study of rats every dosage level produced deformed fetuses. A "no-effect" level was never achieved. t To make a reasonable guess about the general safety of 2,4,5-T for human ;s, as the material has been used up to now, the most appropriate p<^H« area to observe is probably not the relatively healthy and well-fed UlMerl States, where human beings are perhaps better equipped to withstand the assault of toxic substances, but South Vietnam, where great numbers of civilians are half-starved, ravaged by disease, and racked by the innumerable horrors of war. In considering any potentially harmful effects of 2,4,5-T on human beings in Vietnam, some attempt has to be made to estimate the amount of 2,4,5-T to which people, and particularly pregnant women, may have been exposed as a result of the repeated defoliation operations. To do so, a comparison of known rates of application of 2,4,5-T in the United States and in Vietnam is in order. In this country, according to Dr. Tschirley, the average recommended application of 2,4,5-T in aerial spraying for woody-plant control is between three-quarters of a pound and a pound per acre. Therre are about five manufacturers of 2,4,5-T in this country, of which the' Dow Chemical Company is one of the biggest. One of Dow Chemical's best-sellers in the 2,4,5-T line is Esteron 245 Concentrate, and the cautionary notes that a drum of Bsteron bears on its label are hardly reassuring to someone lulled by prior allgeations that 2,4,5-T is a substance of low toxicity: "Caution—may cause skin irriation, avoid contact with eyes, skin, and clothing keep out of the reach of children." • Under the word "warning" are a number of instructions concerning safe use of the material, and these include, presumably for good reason, the following admonition: "Do not contaminate irrigation ditches or water used for domestic purposes Then comes a "notice" : "Seller makes no warranty of any kind, express or implied, concerning the use of this product. Buyer assumes all risk of use or handling, whether in accordance with directions or not," The concentration of Bsteron recommended—subject to all these warnings, cautions, and disclaimers—for aerial spraying in, the United States varies with the type of vegetation to be sprayed, but probably a fair average would be three-quarters to one pound acid equivalent of the raw 2,4,5-T per acre. In Vietnam, however, the concentration of 2,4,5-T for each acre sprayed has been far higher. In Agent Orange, the concentrations of 2,4,5-T have averaged thirteen times the recommended concentrations used in the United States. The principal route through which quantities of 2,4,5-T might be expected to enter the human system in Vietnam is through drinking water, and in the areas sprayed most drinking water comes either from rainwater cisterns fed from house roofs or from very shallow wells. It has been calculated that, taking into account the average amount of 2,4,5-T in Agent Orange sprayed per acre in Vietnam by the military, and assuming a one-inch rainfall (which is quite common in South Vietnam) after a spraying, a forty-kilo (about eighty-eightpound) Vietnamese woman drinking two litres (about 1.8 quarts) of contaminated water a day could very well be absorbing into her system a hundred and twenty milligrams, or about one two-hundred-and-fiftieth of an ounce, of 2,4,5-T a clay; that is, a daily oral dosage of three milligrams of 2,4,5-T per kilo of body weight. Thus, if a Vietnamese woman who was exposed to Agent Orange was pregnant, she might very well be absorbing into her system a percentage of 2,4,5-T-only slightly less than the percentage that deformed one out of every three fetuses of the pregnant experimental rats. To pursue further the question of exposure of Vietnamese to 2,4,5-T concentrations in relation to concentrations officially considered safe for Americans, an advisory subcommittee to the Secretary of the Interior, in setting up guide-lines for maximum safe contamination of surface water by pesticides and allied substances some time ago, recommended a concentration of one-tenth of a milligram of 2,4,5-T in one litre of drinking water as the maximum safe concentration. Thus, a pregnant Vietnamese woman who ingested a hundred and twenty milligrams of 2,4,5-T in two litres of water a day would be exposed to 2,4,5-T at six hundred times the concentration officially considered safe for Americans. Moreover, the level of exposure of Vietnamese people in sprayed areas is not necessarily limited to the concentrations shown in Drt Meselson's calculations. Sometimes the level may be far higher. Dr. Pfeiffer, the University of Mon- 1 tana biologist, says that when difficulties arise with the spray planes or the spray apparatus, or when other accidents occur, an entire thousand-gallon load of herbicidal agent containing 2,4,5-T may be dumped in one area by means of the thirty-second emergency-dumping procedure. Dr. Pfeiffer has recalled going along as an observer on a United States defoliation mission last March, over the Plain of Eeeds area of Vietnam, near the Cambodian boi^B during which the technician at the spray controls was unable to get the a^Ptatus to work, and thereupon dumped his whole load. "This rained down a dose of 2,4,G-T that must have been fantastically concentrated," Dr. Pfeiffer has said. "It was released on a very watery spot that looked like headwaters draining into the Mekong Elver, which hundreds of thousands of people use? In another instance, he has recalled, a pilot going over the area of the supposedly "friendly" Catholic refugee villages of Ho Nai, near Bien Hoa, had serious engine trouble and dumped his whole spray load of herbicide on or near the village. In such instance, the concentration of 2,4,5-T dumped upon an inhabited area in Vietnam probably averaged about a hundred and thirty times the concentration reccommended by 2,4,5-T manufacturers as both effective and safe for use in the United States. Theoretically, the dangers inherent in the use of 2,4,5-T should have been removed by means of the steps promised in the White House announcement last October. A quick reading of the statement by Dr. DuBridge (who is also the executive secretary of the President's Environmental Quality Council) certainly seemed to convey the impression that from that day onward there would be a change in Department of Defense policy on the use of 2,4,5-T in Vietnam, just as there would be a change in the policies of the Departments of Agriculture and the Interior on the domestic use of 2,4,5-T. But did the White House mean what it certainly seemed to be saying about the future military use of 2,4,5JT in Vietnam? The White House statement was issued on October 29th. On October 30th, the Pentagon announced that no change would be made in the policy governing the military use of 2,4,6-T in South Vietnam, because—so the Washington. Post reported on October 31st—"the Defense Department feels its present policy conforms to the new Presidential directive." The Post article went on: "A Pentagon spokesman's explanation of the policy, read at a morning press briefing, differed markedly from the written version given reporters later. "When the written statement was distributed, reporters were told not to use the spokesman's [previous] comment that the defoliant . . . is used against enemy 'training and regroupment centers.' "The statement was expunged after a reporter asked how use against such centers conformed to the Defense Department's stated policy of prohibiting its use in 'populated areas.' " But the statement wasn't so easily expunged. A short time later, it was made again, in essence, by Bear Admiral William E. Lemos, of the Policy Plans and National Security Council Affairs Office of the Department of Defense, in testimony before a subcommittee of the House Foreign Affairs Committee, the only difference being that the phrase "training and regroupment centers" became "enemy base camps." And in testifying that the military was mounting herbicidal operations on alleged enemy base camps Rear Admiral Lemos said: "We know . . . that the enemy will move from areas that have been sprayed, therefore, enemy base camps or unit headquarters are sprayed in order to make him move to avoid exposing himself to aerial observation." If one addsj;o the words "enemy base camps" the expunged words "training .and regroupment centers"—centers that are unlikely to operate without an accompanying civilian population—what the Defense Department seema actually to be indicating is that the "areas remote from the population" .against which the United States is conducting military herbicidal operations are "remote from the population" at least in part because of these operations. As for the Bionetics findings on the teratogenic effects of 2,4,5-T on experimental animals, the Department of Defense indicated that it put little stock in the dangers suggested by the report. A reporter for the Yale Daily News who telephoned the Pentagon during the first week in December to inquire about the Defense Department's attitude toward its use of 2,4,5-T in the light of the Bionetics report was assured that "there is no cause for alarm about defoliants." A week or so later, he received a letter from the Directorate for Defense Information at the Pentagon which, described the Bionetics results as liased on "evidence that 2,4,5-T, when fed in large amounts to highly imbred and susceptible mice and rats, gave a higher incidence of birth defects than was normal for these animals." After reading this letter, the Yale Daily News 120 rter again telephoned the Pentagon, and asked, "Does [the Department business of manufacturing 2,4,5-T. It appears that the presence of a dioxin conense] think defoliants could be affecting embryo growth in any way! taminant in the process of manufacturing 2,4,5-T is a constant^ijpblem among Vietnam?" The Pentagon spokesman said, "No." And that was that. The exf all manufacturers. Three years ago, Dow was obliged to close^Hrn its 2,4,5-T iiuental animals were highly susceptible; the civilian Vietnamese population, plant in Midland, Michigan, for several months and partly rewmd it because which even under "normal" circumstances is the victim of a statistically incalof what Dow people variously described as "a problem" and "an accident." culable but clearly very high abortion and infant-mortality rate, was not. The problem—or accident—was that workers exposed to the dioxin contamiNearly a month after Dr. DuBridge's statement, another was issued, this one nant during the process of manufacture came down with an acute skin irritaby the President himself, on United States policy on chemical and biological tion known as culor-aene. The Dow people, who speak with considerable pride warfare. The President, noting that "biological weapons have massive, unpreof their toxicological work ("We established our toxicology lab the year Ralph dictable, and potentially uncontrollable consequences" that might "impair the Nader was born," a Dow public-relations man said recently, showing, at any health of future generations," announcced it as his decision that thenceforrate, that Dow is keenly aware of Nader and his career), say that the chlorward "the United States shall renounce the use of lethal biological agents acne problem has long since been cleared up, and that?' the current level of the and weapons, and all other methods of biological warfare." Later, a White dioxin contaminant in Dow's 2,4,5-T is less than one part per million, as House spokesman, in answer to questions by reporters whether this included opposed to the dioxin level in the 2,4,5-T used in the Biouetics study, which is the use of herbicidal, defoliant, or crop-killing chemicals in Vietnam, made it alleged to have been between fifteen and thirty parts per million. A scientist clear that the new policy did not encompass herbicides. at the DuBridge office, which has become a coordinating agency for informaSince the President's statement did specifically renounce "all other methods tion having to do with the 2,4,5-T question, says that the 2,4,5-T used by Bioof biological warfare," the reasonable assumption is that the United States netics was "probably representative" of 2,4,5-T being used in this country—aud government does not consider herbicidal, defoliant, and crop-killing operations presumably in Vietnam—at the time it was obtained but that considerably loss against military and civilian populations, to be part of biological warfare. The of the contaminant is present in the 2,4,5-T now being produced. Evidently, the question therefore remains: What does, the United States government consider degree of dioxin contamination present in 2,4,5-T varies from manufacturer to biological warfare to consist of? The best place to look for an authoritative manufacturer. What degree of contamination high or low, was present in the definition is a work known as the Joint Chiefs of Staff Dictionary, an official quantities of 2,4,5-T shipped to South Vietnam at various times this spokesman publication that governs proper word usage within the military establishment, didn't seem to know. in the current edition of the Joint Chiefs of Staff Dictionary, "biological warThe point about the dioxin contamination of 2,4,5-T is an extremely imporfare" is defined as the "employment of living organisms, toxic biological prodtant one, because if the suspicions of the Dow people are correct and the cause ucts, and plant-growth regulators to produce death or casulaties in man, aniof the fetus deformities cited in the Bionetics study is not the 2,4,5-T but the mals, or plants or defense against such action." But the term "plant-growth dioxin contaminant, then this contaminant may be among the most teratogeniregulators" is nowhere defined in the Joint Chiefs of Staff Dictionary, and cally powerful agents ever known. Dr. McLaughlin has calculated that if the since a certain technical distinction might be made (by weed-control scientists, dioxin present in the Bionetics 2,4,5-T was indeed responsible for the teratofor example) between plant-growth regulators and defoliants, the question of genic effects on the experimental -animals, it looks as though the contaminant whether the Joint Chiefs consider military defoliation operations part of biowould have to be at least ten thousand times more teratogenically active in logical warfare is left unclear, As for "defoliant agents," the Dictionary rats than thalidomide was found to be in rabbits. Furthermore, it raises defines such an agent only as "a chemical which causes trees, shrubs, and the alarming questions about the prevalence of the dioxin material in our environother plants to shed their leaves prematurely." All this is hardly a surprise to ment. It appears that under high heat the dioxin material can be produced in anyone familiar with the fast semantic legerdemain involved in all official statea whole class of chemical substances known as trichlorophenols and p'entnchlorments on biological warfare, in which defoliation has the baffingly evanescent ophenols. These substances include components of certain fatty acids used in half-existence of a pea under a shell. detergents and in animal feed. To find that pea in the official literature is not easy. But it is resonable toAs a consequence of studies that have been made of the deaths of millions assume that if the Department of Defense were to concede officially that, of young chicks in this country after the chicks had eaten certain kinds of "defoliant agents" were in the same category as "plant-growth regulators" that chicken feed, government scientists are now seriously speculating on the possi"produce death . . . in plants," it would thereby also be conceding that it is in. bility that the deaths were at the end of a chain that began with the spraying fact engaging in the biological warfare that President Nixon has renounced. of corn crops with 2,4,5-T. The hypothesis is that residues of dioxiu present in And such a concession seems to have been run to earth in the current edition. the 2,4,5-T remained in the harvested corn and wei'e concentrated into certain oC a Department of the Army publication entitled "Manvial on Use of Herbibyproducts that were then sold to manufacturers of chicken feed, and that the cides for Military Purposes," in which "antiplant agents" are defined as. | dioxin became absorbed into the system of the young chicks. One particularly "chemical agents which possess a high offensive potential for destroying or f; disquieting sign of the potential of the dioxin material is the fact that bioseriously limiting the production of food and defoliating vegetation," and goes assays made on chick embryos in another study revealed that- all the embryos on "These compounds include herbicides that kill or inhibit the growth of were killed by one twenty-millionth of a gram of dioxin per egg. plants; plant-growth regulators that either regulate or inhibit plant growth,, Perhaps an even more disquieting speculation about the dioxin is that 2.4,5-T sometimes causing plant death. . . ." The admission that the Department of may not be the only material in which it appears. Among the compounds Defense is indeed engaging, through its defoliation and herbicidal operations that several experienced biologists and toxicologists suspect might contain or in Vietnam, in biological warfare, as this is defined by the Joint Chiefs and as; produce dioxin are the trichlorophenols and pentachlorophenols, which are it hns been formally renounced by the President, seems inescapable. rather widely present in the environment in various forms. For example, A Since the DuBridge statement, allegations, apparently originating in part1 number of the trichlorophenols and. pentachlorophenols are used 'as slime-killwith the Dow Chemical Company, have been made to the effect that the 2,4,5-T ing agents in paper-pulp manufacture, and are present in a wide range of conused in the Bionetics study was unrepresentative of the 2,4,5-T generally prosumer products, including adhesives, water-based and oil-based paints, varduced in this country, in that it contained comparatively large amounts of a nishes and lacquers, and paper and paper coatings. They are used to prevent certain contaminant, which, according to the Dow people, is ordinarily present slime in pasteurizers and fungus on vats in breweries and are also used in in 2,4,5-T only in trace quantities. Accordingly, it has been suggested that the Hair shnmpoo. Along with'the 2,4,5-T used in the Bionetics study, one trichlorreal cause oC the teratogenic effects of the 2,4,5-T used in Uie Bionntics study ophenol and one pentachlorophenol were tested without teratogenic results. may not have been the 2,4,5-T itself but, rather, the contaminant in the sample But Dr. McLaughlin points out that since there are many such compounds put used. The chemical name of the contaminant thus suspected by the Dow people out by various companies, these particular samples might turn out to be —by is 2,3,0,7-tetrachlorodibenzo-p-dioxin, often referred to simply as dioxin. The the reasoning of the allegation that the 2,4,5-T used by Bionetics was unu2,4,5-T used by Bionetics was obtained in 1905 from the Diamond Alkali Comsually dirty—unusually clean. pany, now known as the Diamond-Shamrock Company and no longer in the- Dr. McLaughlin tends to consider significant, in view of the now known kxtreine toxicity and possible extreme teratogenicity of dioxin, the existej^^of Iveu very small amounts of the trichloroplienols and pentachlorophenl^in food wrappings and other consumers products. Since the production of dioxin appears to be associated with high-temperature conditions, a question arises whether these thermal conditions are met at any stage of production or subsequent use or disposal of such materials, even in minute amounts, One of the problems here seems to be, as Dr. Epstein has put it, "The moment you introduce something into the environment it's likely to be burned sooner or later— that's the way we get rid of nearly everything." And most of these consumer products may wind up in municipal incinerators, and when they are burned, the thermal and other conditions for creating dioxin materials may quite possibly be met If so, this could mean a release of dioxin material into the entire environment through the atmosphere. Yet so far the dioxin material now suspected of causing the fetus-deforming effects in experimental animals has never been put through any formal teratological tests by any company or any government agency. If the speculation over the connection between dioxin in 2,4,5-T and the deaths of millions of baby chicks is borne out, it might mean that, quite contrary to the assumptions made up to now that 2,4,5-T is rapidly decomposable in soil, the dioxin material may be extremely persistent as well as extremely deadly. So far, nobody knows—and it is probable that nobody will know for some time—whether the fetus deformities in the Bionetics study were caused by the •2,4,5-T itself, by the dioxin contaminant, or by some other substance or substances present in the 2,4,5-T, or whether human fetuses react to 2,4,5-T in the same way as the fetuses of the experimental animals in the Bionetics study. However, the experience so far with the employment of 2,4,5-T and substances chemically allied to it ought to be instructive. The history of 2,4,5-T is related to preparations for biological warfare, although nobody in the United States government seems to want to admit this, and it has wound up being used for purposes of biological warfare, although nobody in the United States government seems to want to admit this, either. Since 2,4,5-T was developed, the United States government has allowed it to be used on a very large scale on our own fields and countryside without adequate tests of its effects. In South Vietnam—a nation we are attempting to save—for seven full years the American military has sprayed or dumped this biological-warfare material on the countryside, on villages, and on South Vietnamese mena and women in staggering amounts. In that time, the military has sprayed or dumped 'on Vietnam < fifty thousand tons of heribicide, of which twenty thousand tons have apparently been straight 2,4,5-T. In addition, the American military has apparently made incursions into a neutral country, Cambodia, and rained down on an area inhabited by thirty thousand civilians a vast quantity of 2,4,5-T. Yet in the quarter of a century since the Department of Defense first developed the biological-warfare uses of this material it has not completed a single series of formal teratological tests on pregnant animals to determine whether it has an effect on their unborn offspring. Similarly, officials of the Dow Chemical Company, one of the largest producers of 2,4,5-T, although they refuse to divulge how much 2,4,5-T they are and have been producing, admit that in all the years that they had produced the chemical before the DuBridge statement they had never made formal teratological tests on their 2,4,5-T, which they are now doing. The Monsanto Chemical Company, another big producer, had, as far as is known, never made such tests, either, nor, according to an official in the White House, had any other manufacturer. The Department of Agriculture has never required any such tests from manufacturers. The Food and Drug Administration has never required any such tests from manufacturers. The first tests to determine the teratogenic efforts of 2,4,5-T were not made until the National Institutes of Health contracted for them with Bionetics Laboratories. And even then, when the adverse results of the tests became apparent, it was, as Dr. Epstein said, like "pulling teeth" to get the data out of the institutions involved. And when the data were obtained and the White House was obliged, partly by outside pressure and publicity, to act, the President's science adviser publicly presented the facts in a less than candid manner, while the Department of Defense, for all practical purposes, ignored the whole business and announced its intention of going on doing what it had been doing all along. There have been a number of reports from. Vietnam both of animal abortions nnd of malformed human babies that are thought to have resuttgl from spraying operations in which 2,4,5-T was used. But such seattered^^rts, however well founded, cannot really shed much more light on the situi^Pn. The fact is that even in this country, the best-fed, richest, and certainly most statisticsminded of all countries on earth, the standards for testing materials that are put into the environment, into drugs, and into the human diet are grossly inadequate. The screening system is so coarse that, as a teratology panel of the Mrak Commission warned recently, in connection with thalidomide, "the teratogenicity of thalidomide might have been missed had it not produced malformations rarely encountered." In other words, had it not been for the fact that very unusual and particularly terrible malformations appeared in an obvious pattern—for example, similarly malformed babies in the same hospital at abo\it the same time—pregnant women might still be using thalidomide, and lesser deformations would, so to speak, disappear into the general statistical background. As for more subtle effects, such as brain damage and damage to the central-nervous system, they would probably never show up as such at all. If such risks existed under orderly, normal medical conditions in a highly developed country, how is one ever to measure the harm that might be done to unborn children in rural Vietnam, in the midst of the malnutrition, the disease, the trauma, the .poverty, and the general shambles of war? DEPARTMENT OF AMPLIFICATION, New York, March 5, 19W. ' The EDITORS, The New Yorker DEAE Sins: In an article that appeared in The New Yorker on February 7th, I wrote that Dr. Lee DuBridge, the President's science adviser, issued a statement last October at the White House saying that because a laboratory study had shown a "higher than expected number of deformities" in the fetuses of mice and rats exposed to the herbicide 2,4,5-T, agencies of the United States government would take action to restrict the use of that substance in this country and in Vietnam, where it was being used in extensive military defoliation operations. This action, Dr. DuBridge announced, would include the cancellation, by January 1st of this year, of Department of Agriculture permits for the use of 2,4,5-T on some American food crops unless the Food and Drug Administration had by then been able to determine a safe concentration o£ the herbicide in foods. Dr. DuBridge further announced that the Department of Defense would thenceforth "restrict the use of 2,4,5-T to areas remote from the population" in Vietnam. His statement added that these actions and others "will assure the safety of the public while further evidence [of the alleged harmful effects of 2,4,5-T] is being sought" Four months have passed, and 2,4,5-T is still being used as widely as ever. The Department of Agriculture has yet to cancel its permits for the use of the herbicide on food crops in this country, and the Department of Defense is continuing to use it in populated areas of Vietnam. In the meantime, officials o£ the Dow Chemical Company, which is one of the largest producers of 2,4,8-T, have been maintaining that the samples of 2,4,5-T used in the study cited by Dr. DuBridge, which was done by the Biouetics Research Laboratories, of, Bethesda, Maryland, were uncharacteristic of the 2,4,5-T currently being produced, because the material tested by Bionetics—which did not come from Dow—was contaminated to an unusual extent by a toxic substance identified as symmetrical 2,3,6,7-tetrachlorodibenzo-p-dioxin. This contaminant, usually called dioxin, was alleged by the Dow people to be present in the Bionetica samples at a concentration of approximately twenty-seven parts per million) and they claim that the 2,4,5-T that Dow is currently producing contains the dioxin contaminant in concentrations of less than one part per million. The Dow people maintain that their currently produced 2,4,5-T does not appear to have the effect of deforming rat fetuses. In January, a Dow official told the Department of Health, Education, and Welfare, "We strongly urge that actlou concerning the status of 2,4,5-T be held in abeyance until [Dow's] testing program is completed [in] April." The United States government's .failure so fur to place the promised restrictions on the use of 2,4,5-T in this country nmy in part be attributed to this plea. 45-382—TO 8 Because of the seriousness of the issues involved, it seems to m^hat the' government's .failure to act on tlie use of 2,4,6-T here and in Vietn^^>alls for much fuller public discussion. Even though the dioxin contaminarfBKay now be present in 2,4,5-T in what the Dow Chemical Company apparently considers to be no more than tolerable amounts, the substance is of such potency that its release even in small concentrations must prompt deep concern. In the presumably more heavily dioxin-contazninated samples of 2,4,5-T that were used in the Bionetics work, the smallest dosages of 2,4,5-T that the test animals were given caused extensive deformities in fetuses. In more recent studies of the dioxin contaminant, conducted by Dr. Jacqueline Verrett, of the Food and Drug Administration (who earlier was responsible for revealing the carcinogenicity of cyclamates), extensive teratogcnic, or fetus-deforming, effects were discovered iu chick embryos when the dioxin, or a distillate predominantly consisting of it, was present at concentrations of little more than a trillionth of a gram per gram of the egg. The magnitude of this effect on chick embryos may be gathered from the fact that, according to Dr. Verrett's studies, the dioxin appears to be a million times as potent a fetus-deforming agent as the notorious teratogen thalidomide was found to be in tests on chicks. Of course, chick embryos are far down the biological ladder from human fetuses, and they a.re also extremely sensitive to many substances. But even if, for theoretical purposes, we reduced the teratogenie power of the dioxin, as shown in Dr. Verrett's chick-embryo studies, approximately a million times, we would still have to Consider that we were dealing with a substance as teratogenieally potent as thalidomide. That the United States government permits the presence, even in minute amounts, of such a. substance in herbicide I mixtures to be sold for spraying on food crops and on suburban lawns—where some of the chemical may enter shallow wells and other drinking-water supplies—is hardly reassuring. And it is particularly disturbing when one reflects that in the quarter of a century in which 2,4,5-T was used prior to Dr. DuBridgc's announcement not a single regulatory agency of the United States government, not the Department of Defense—which has been spreading huge quantities oO 2,4,5-T on vast areas of Vietnam—and not, as far as is known, the researchers for any one of the half-dozen large American chemical companies producing the material had ever so much as opened up a pregnant mouse to determine whether 2,4,5-T or the dioxin contaminant in it did any systemic or pathogenic harm to the fetus. Several studies of the sort are now under way, but the United States government still seems to take the position that the 2,4,5-T produced by Dow and other large chemical companies should be considered innocent until it is proved to be otherwise. Meanwhile, 2,4,5-T is being sprayed on certain crops and on areas where it may come into contact with human beings, cattle, and wildlife. In Vietnam, it is still being sprayed by the military in concentrations that average thirteen times as great as those that the manufacturers themselves recommend as safe and effective for use in this country. It is true that the teratogenicity of dioxin—as distinct from dioxiu-contaminatecl 2,4,5-T—has not yet been established in tests conducted on experimental animals of mammalian species. However, the direct toxic, or body-poisoning, effects—as distinct from fetus-deforming effects—of dioxin are known to be very high both in animals and in human beings. In past studies on rats, dosages of forty-five millionths of a gram per kilo of the mother's hody weight have been found to kill fifty per cent of the offspring. When dioxin was given orally to pregnant rats in recent tests, it was found, on preliminary investigation, to kill all fetuses with dosages of eight millionths of a gram per kilo of the mother's hody weight, and to damage fetuses with dosages of a half-millionth of a gram per kilo. ' •Further, the effects of dioxin on human beings, even in small dosages, are known to be serious. In the past, in plants manufacturing 2,4,5-T an illness called chloracne seems to have been widespread among the workers. In the mid-sixties, Dow was obliged to close down part of a 2,4,5-T plant in Midland, Michigan, for some time because about sixty workers contracted chloracne as a result of contact with dioxin, which seems to be always present in varying degrees during the process of manufacturing 2,4,5-T and in the finished ,2,4,5-T itself. The symptoms of this disease include extensive skin eruptions, disorders of the central nervous system, chronic fatigue, lassitude, and depression. Workers at a 2,4,5-T plant, in New Jersey run by another company suffered similar symptoms in the mid-sixties, and six years later some of them were reported to be still suffering from the effects of the disease. In Germany, since the mid-fifties, workers in factory after factory producing 2,4,5-T ami nolychlow phenolic compounds have been afflicted with chloracn^fcer absorbing apparently only minute amounts of the dioxin contaminan^Kieir symptoms have been described in several medical papers as including liver damage, nervous and mental disorders, depression, loss of appetite and weight, and markedly reduced sexual drive. ' A few weeks ago, when a reporter approached an official in Dr. DuBridge's office for information on 2,4,5-T he was told that he would be given White House cooperation "only to a certain extent," because the official didn't want "wild speculation" stirred up. He cited as an example of "wild speculation" the recent controversy over the birth-control pill, which, he said, had "caused millions of women to get hysterical with worry." The reporter replied that he didn't think the analogy between 2,4,5-T and the Pill was a particularly good one, for the reason that a woman using the Pill could employ alternative methods of contraception, whereas a Vietnamese woman exposed to herbicidai spray put down by the American military had no choice in the matter. But perhaps the comparison between 2,4,5-T (and its dioxin contaminant i and commonly used pills is worth pursuing. Suppose that such a daugerou'substance as dioxin were found to be contained in a pill offered for human consumption in this country, and suppose that the contaminant were present iii ,such minute amounts that an adult following the prescribed dosages nn.uli'v ingest a hundredth of a millionth of a gram of the contaminant per tla. . There is no doubt whatever that, according to existing Food and Drug Administration standards, the F.D.A. would imitunUately ban production and sale of the pill on the ground that it was highly dangerous to public health; in f:ic(, the amount of such a potent contaminant that the F.D.A. would permit in a pill under the agency's present policy on toxicity would almost certainly be zero. While 2,4,5-T, with or without the dioxin contaminant, doesn't come in pill form, it may be worthwhile to try to calculate, on the basis of a hypothetical pill, how much 2,4,5-T (and dioxin) a Vietnamese woman living in an nre.i sprayed by the American military might ingest in a day. It has already been calculated by reputable biologists that, if one takes into account the average amount of 2,4,5-T sprayed per acre in Vietnam, and also takes into account n one-inch rainfall—such as is common there—after a spraying, a forty-kilo (about eighty-eight-pound) Vietnamese woman drinking two litres (about two quarts) of 2,4,5-T-contaminated water per clay could be ingesting about » hundred and twenty milligrams (about a two-hundred-and-fiftieth of an ounce) of 2,4,5-T a day. If the 2,4,5-T contained the dioxin contaminant at a level <>" one part per million—which is what the Dow people say is the maximum amount present in the 2,4,5-T they are currently producing—the Vietnamese woman would be absorbing a little over a tenth of a microgram of dioxin per (lay, or ten times the amount of dioxin entering the system of an adult from the hypothetical pill that the F.D.A. would certainly find dangerous to humnn health. .Further, if this Vietnamese woman were to conceive a child two weeks. Say, after the spraying, the weight of the dioxin that hy these same calculations w'ould have then accumulated in her system (the evidence thus far is that dioxin accumulates in mammalian tissue in the same manner as the chlorinated hydrocarbons, such as DDT) would be more than the weight of tinjust-fertilized ovum. Considering the existing evidence of the frightening degree of teratogenicity of the dioxin in chick embryos and its highly toxic effects on mammalian fetuses, the presence of this much dioxin in a mother's body at the very beginning of a human life surely has ominous implications. Now, what about the safety of 2,4,5-T itself? Admittedly, the dioxin contaminant seems to be a residue from one stage of its manufacture. But if by some future chemical miracle the very last trace of dioxin could be removed froTn the finished 2,4,5-T, would the resultant "pure" 2,4,5-T be harmless? The fact seems to be that even then 2,4,5-T, as produced in this country, would have f<> be viewed with suspicion, for the breakdown products of 2,4,5-T, when subjected to heat and other conditions, are themselves capable, according to » number of responsible biologists, of producing dioxin. Given this potential, the ultimate folly in our defoliation operations in Vietnam was possibly achieved during 1965 and 1906, when the military made large-scale efforts in two defoliated areas to create fire storms—that is, fires so huge that all Hie oxygen in those areas would be exhausted. The apparent intention was to render the coil barren. (A fire storm would also, of course, have the result of burning or snf- 126 any living beings remaining in the area.) Operation Sherwood d in 1965, was an attempt to burn a defoliated section of the Woods. In October, 1066, the military began Operation Pink Hose, a project. Neither o£ the projects, in which tons of napalm were thrown down on toil of the residue of tons of sprayed 2,4,5-T, succeeded in creating the desired effect; whether they released into the atmosphere dioxin produced by the breakdown products, of the 2,4,5-T will probably never be known. There are also less spectacular ways in which conditions suitable for the release of dioxin in Vietnam may have been created. For example, after areas accessible by road have been defoliated, woodcutters move in to chop up the cloud timber, which is then carted off to nearby towns'and 'Sold as'firewood. , Large quantities of it are said to have been entering Saigon for years. Since the fires are customarily tended by Vietnamese women, and since many of them are certainly pregnant, the hazards to health and to the lives of unborn children surely cannot be ignored. In the United States, the potential hazards from the present use of 2,4,5-T are considerably less than they are in Vietnam. In the first place, the recommended concentrations of 2,4,5-T for spraying here are, as I have pointed out, about a thirteenth of what the Vietnamese population is sometimes subjected to. And, in the second place, a great deal, if not most, of the 2,4,5-T that would otherwise have been sprayed on American crops and grazing areas has for several years been sent to Vietnam. However, the shortage of 2,4,5-T in this country does not necessarily mean that the potential hazards are at a minimum. The substances known as the trichlorophenols and compounds of pentachlorophenol, which officials of the F.D.A. believe may be chemical precursors of dioxiu under certain thermal and other conditions, are used widely in the manufacture of a large variety of consumer products, ranging from paper to laundry starch and hair shampoo. Dow Chemical puts out a whole line of polychlorophenolic chemicals known as Dowicide Products. Monsanto Chemical also puts out a line of pentachlorophenol substances, known as Penta Compounds. Since a very great many consumer products wind up being burned sooner or later, and since the polychlorophenolic compounds are suspected of being capable, under particular thermal and other conditions, of releasing dioxin, the alarming question arises whether, and to what extent, dioxin is being released into the environment through the atmosphere. Pentachlorophenol, used in certain herbicides, is readily decomposed in sunlight, and in its breakdown process a number of products, including chemical precursors of chlorodibenzo-p-dioxin compounds, are produced. Because of these factors, a whole range of pesticides, as well as of herbicides, now must come under suspicion of producing dioxin compounds'. Although the chemical companies that manufacture 2,4,5-T have long taken pride in pointing out that 2,4,B-T itself is quite readily decomposable in soil, the crucial matters of how stable the dioxin contaminant is and to what extent it is cumulative in animal tissue have apparently been neglected. Consequently, the fact that traces of compounds virtually indistinguishable from dioxin have already been detected in this country in the human food chain—in the livers of chickens and in edible oils—clearly indicates that dioxin should be considered a hazard to man. Why, under all these inauspicious circumstances, the production and the use here and in Vietnam of 2,4,5-T has not summarily been stopped by the United States government is hard to understand. Sincerely, THOMAS WHITESIDE. Appendix 4 DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE, PUBLIC HEALTH SERVICE, FOOD AND DRUO ADMINISTRATION, Roclcville, MA., March 1%, 1910. Hon. RICHARD D. MCCARTHY, House O'f Representatives, Washington, D.G. DEAR MR. MCCARTHY: The Secretary has asked us to reply to your letter of February 3, 1070, requesting whether the B'ood and Drug Administration has information indicating that 2,4,5-T is now safe to use. No tolerances have been established for residues of 2,4,5-T in food or feed crops. The whole matter of the safety of this herbicide, when its use results in a residue in or on a food crop, is currently under evaluatioi^ljhis evaluation will be completed as expeditiously as possible. We are enclasp a Fact Sheet explaining the status of 2,4,5-T at this time. We shall promptly inform you of our decision upon completion of the evaluation of 2,4,5-T. Sincerely yours, M. J. RYAN, Acting Director, Office of Legislative Services. FDA FACT SHEET U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE, PUBLIC HEALTH SERVICE, FOOD AND DHUQ ADMINISTRATION, Washington, D.C. * 2,4,5-T * 2,4,5-T (2,4,5-trichlorophenoxyacet.ic acid) has had extensive registered use as a deColiant and weed killer. It has also been registered by the U.S. Department of Agriculture as a pesticide chemical (herbicide) on a no residue basis on a few certain selected food crops for some years, primarily for weed control of pasture and rangeland. TOXICITY A research study recently completed under contract to the National Cancer Institute on a commercial lot of 2,4,5-T showed that the feeding of this material to rats and mice produced abnormal birth effects on the embryos. Further investigation of the 2,4,5-T used in the feeding studies established that the material used contained a significant amount of one of more impurities called dioxins produced during the manufacture of 2,4,6-T. Improved manufacturing processes are claimed by one manufacturer to have reduced the dioxiu impurities to insignificant amounts. The dioxins are of concern because they are known to be extremely toxic to poultry and to have produced severe skin irritation to workers in plants exposed to dioxins inadvertently during the manufacture of other chemicals. At present a number of research studies are. underway in both government and commercial laboratories to determine if the reported birth defects of the earlier study are due to 2,4,5-T itself, the dioxin impurities, or a combination of the 2,4,5-T and the dioxins. Additional investigations are underway to improve our ability to detect very small amounts of dioxins in samples of 2,4,5-T and to determine whether other commonly used pesticides chemically related to 2,4,5-T contain significant amounts of the dioxin contaminants. Drinking water supplies are being tested for the presence of 2,4,5-T and other possible environmental sources of these chemicals studied, but no results are available at this time. The USDA announced on February 6, 1970, that it is investigating 17 commonly used pesticides chemically related to 2,4,5-T to determine whether they contain hazardous amounts of these toxic contaminants. FOOD IN THE UNITED STATES The Food and Drug Administration is continually engaged in examining samples of individual foodstuffs for residues of pesticides above the safe tolerances established under the Miller Pesticide Amendment In addition, FDA purchases food in the markets of several cities, prepares the food in the quantities and combinations typical of the diet of an average 10-year-old male, and determines the amounts of the several pesticides that might be actually ingested in the typical diet of a heavy eater. Of 5300 food samples tested for 2,4,P-T residues during the last four-year period, 25 samples indicated trace amounts '(less than the 0.1 p.p.m. limit of accuracy of present analytical procedures) and 2 samples showed higher residues. 0.19 p.p.m. 2,4,5-T was detected iri one sample of milk taken in 1065 In New England, and one sample of sugarbeets from Ohio in 1900 showed 0.29 ,m. 2,4,5-T. The milk had been distributed before analysis was complete and icssing of the sugar-beets removes the chemical. If food is found to c 'te residues of 2,4,5-T, it is subject to removal from the market. STATUS OF 2,4,5-T UNDEE THE FEDERAL FOOB, DBUG, AND COSMETIC ACT No finite tolerances have been established for residues of 2,4,5-T or the dioxins in food. In the absence of established tolerances any detectable amount of either chemical in food would make the contaminated food illegal and subject to sei'/.uve if found in the channels of interstate commerce. A. petition was filed in December, 1907 requesting the establishment of tolerances of 0.2 p.p.m. for residues of 2,4,5-T on apples, barley, blueberries, corn, oats, rice, rye, sugarcane, and wheat. Neither the petition as originally submitted or as later supplemented provided data to support affirmative action and the petitioner withdrew his petition on December 29, 1969, as provided for under the pesticide regulations. Petitions to establish a safe tolerance level for residues of 2,4,5-T in food may again bo submitted to the FDA in the future. However, any such submission must include scientific research data to resolve the questions that have been raised concerning toxicity of 2,4,5-T and the dioxins. CONCLUSION The Department of Health, Education, and "Welfare is continuing investigations to determine the potential hazards from the possible presence of residues of 2,4,5-T and dioxius in foods, water, and other environmental sources to which the public may be exposed. It is to be emphasized that there is no tolerance for 2,4,5-T in food today; the testing of food over the past several years has revealed no significant problem of food contamination. Appendix 5 PROBE INTO USE OF HERBICIDES BY CONGRESSMAN RICHARD D. MCCARTHY. D-N.Y. Globe, Ariz., February 13, 1070 Ladies and gentlemen, I think we should begin. I am Congressman Richard D. McCarthy, and the hearings will coine to order. For more than a decade scientists have had serious misgivings about the widespread use of herbicides and, pesticides in the environment. The .late Rnchael Carson warned of the risk of the use of herbicides, whose effects were either harmful or unknown. • . . Tn the United States 120 million acres each year are sprayed with herbicides'' Cor the clearing of railroads, for brush control, for watershed management, nud for other purposes. One of these is known as 2,4,5-T. It was developed and perfected at Fort Detriclc, Md., the army's chief Biological Warfare Research Center. The herbicide 2,4,5-T, and 2,4-D, a related herbicide, collectively account for some 83 million pounds of production per year—that was the figure in 1908. I've long been concerned with the widespread use of these herbicides in Vietnam, Each day some 100 tons are dropped on South Vietnam, and scientists for many months have been concerned about the adverse ecological effects of this herbicidal inundation. Last summer in the course of my inquiry into the Army's germ and gas warfare policies, I learned that a study, by the Bionetics Research Laboratories for the National Cancer Institute showed that the herbicide 2,4,5-T produced birth defects in rats and mice. When the conclusions of this study were known, the President's science adviser, in October, announced a ban on the herbicide beginning January 1, 1970, unless the F.D.A. had found safe legal tolerances. I was distressed 11 dnys ago to learn that contrary to the White House's announcement, the Department of Agriculture continues to authorize the use of 2,4,5-T in the United States, It's incredible to me that someone, or some people should have succeeded in overruling the science adviser to the President of the United States. We know from the tlmlidomide experience Unit if we are going to -err, we should err on the side of caution, and not on the side of danger. It is my firm conviction that such chemicals should not be used unless fuU^fcets show that they are safe. It is also incredible to me that this herbicide, ^Vh has been in •existence since its development some 25 years ago at t h e u e r m Warfare Research Center, still has not been fully tested for its teratogenic effects on human beings—that is, its power to produce birth deformities. We know that it produces birth deformities in test animals under laboratory •conditions, and we continue to receive reports from Vietnam that civilian women living in this heavily defoliated area are bringing forth deformed •offsprings. The Saigon Press has reported on these in considerable detail. Now, we have the allegations, and complaints emanating from here, Globe, Ariz. It is my hope that my investigation into these complaints and allegations will assist me in continuing my inquiry into this whole matter. I wish to determine how the White House was overruled, and why it is that we continue to use this herbicide despite the warning signals that have arisen. As the great French scientist physiologist, Claude Banard, once said, "True science teaches us to doubt, and ignorance to refrain." I want to welcome all the local State and Federal officials who are in -attendance. I hope to have a chance to meet with you personally during our "visit. Our first witness is Prof. Arthur W. Galston, a professor of biology from Yale University. Doctor Galston. Professor Galston, I wonder if, for the record, you would identify yourself, and your background, and particular expertise in the matters under inquiry. Dr, GALSTON. Very happy to do that, Congressman. I'm currently a professor of biology at Yale University. I'm also lecturer in forestry, and director of the March Botanical Gardens at Yale. I've been n professor of plant physiology for about 27 years. I was trained at the New York State College of Agriculture at Cornell University. I did my graduate work at the University of Illinois, where I earned a Ph.D. •degree in 1943. 1 then went to work for the emergency rubber project for the U.S. Government, located at Gal-Tech. During AVorld War I was agricultural officer for U.S. Navy Military Government on the Isle of Okinawa. I then Tvorked at Gal-Tech for 10 years, and I've been at Yale for the last 15 years. I've published books in the area of plant physiology, and I have over 100 articles in the subject. Congressman MCCARTHY. For the record, Doctor Galston, I wonder if you could give us a scientific information about the herbicide under investigation. DR. GALSTON. Congressmen, what I'd like to do is to give you and the audience here some appreciation of the feeling of a large number of scientists as exemplified in this report recently delivered to the Secretary of Health, Education, and Welfare, Finch. It is culled, "The Report of the Secretary's Commission on Pesticides and Their Relationship to Environmental Health." It's dated December 5, 1969, and was prepared by the distinguished panel shared by Doctor Emil Mrak, the •chancellor emeritus of the University of California at Davis. It included many academic people, and also the vice presidents of two important companies, Dow, and Eli Lilly, both of whom manufacture herbicides and other pesticides in wide use. • The Commission takes note of the fact that there are now more than 400 •different kinds of chemicals which are being used as pesticides to combat insects, fungi, weeds, and other predators. Our modern agriculture and highly technicalized food production activities •demand that we do use chemicals in agriculture. I'd like to make it clear that I'm not alining myself with people that say, "Stop nil chemicals." That's ridiculous in this day and age. We are dependent 'upon chemicals, and we have to keep using them. Nonetheless, some of these chemicals are terribly noxious when introduced into the environment. All of us are now familiar with the fact that DDT may be more of a bane •than a boom. It has become global. Even a penguin picked up on fin ice flow in .Antarctica is full of DDT, and that was 400 miles from the'application of iOU :>DT, and we know that DDT causes oversized livers, and alteration of the (teroid metabolism in everyone's genes. |^fc This Commission agrees unanimously that DDT must be phased <^^as quickly as possible as a pesticide.' With that as a background, I think it's perfectly clear that as scientific information develops, we are going to want to examine every pesticide for its possible harmful effects on man and his domestic animals, and his environment. Here I must digress to tell you about the changes that have occurred in our concept of what constitutes adequate testing for a compound of this kind. It used to be that simple toxicology tests were conducted. A laboratory animal, such as a mouse or a rat was fed a certain amount of chemical. If that animal showed serious' symptoms, the teratogenicity yas calculated on the base of how many milligrams per kilogram of body weight of this material produced the toxic effects. We now have tables which tell us roughly how toxic given materials are. Now, based on that kind of test, 2,4,5-T, for example, is not terribly toxic, it's only a mildly toxic compound in the order of 2 to 700 kilograms milligram of body weight cause toxicity. If, however, you use more subtle tests, you find out that 2.4,5-T may be more dangerous. &mong these tests are: Does the compound cause cancer? That takes a much more serious look than simply feeding and watching the dying of animals. Secondly, do the compounds cause genetic effects, that is, does it break chromosomes, or cause mutations. Thirdly, does the compound cause birth abnormalities. The word to describe that is teratogenics; that is the formation of monsters. Now, this report which I have alluded to has as its last chapter, a chapter on teratology, and I'd like to read you just a little bit out of this chapter, and out of the summary which is written here, which gives you my concern. "All currently used pesticides should be tested for teratogenicity in the near future in two or more mammalian species chosen on the basis of the closest metabolic and pharmacologic similarity to human beings possible. Pesticides should be tested at various concentrations including levels substantially higher than those to which the human population are likely to be exposed. Test procedures should also reflect routes related to human exposures. Apart from the obvious route of ingestion, attention should be directed to other routes of exposure, including inhalation exposures from pesticide aerosols and vaporizing , pesticide strips used domestically, and exposures from skin absorption. Parenteral administration is an appropriate test route for pesticides to which humans are exposed by inhalation, or for pesticides, which are systemically absorped following ingestion. "The use of currently registered pesticides to which humans are exposed and which are found to be teratogenic by suitable test procedures in one; or more mammalian species should be immediately restricted to prevent risk of human exposures." I'd like to repeat that; "Currently registered pesticides to which humans are exposed and which are found to be teratogenic by suitable test procedures in one or more mammalian species should be immediately restricted to prevent risk of human exposure. Such pesticides, in current use, include—" I'll skip a lot. of names, 2,4-D and 2,4,5-T are listed. Here's the Government's most distinguished panel saying that, there is evidence that 2,4,5-T has produced teratogenic effects in one or more mammalian species, its use should be restricted immediately. They also said no new pesticide found to be teratogenic, should be used only in circumstances where risk of human exposure is minimal. Congressman MCCARTHY. AVhat's the date of that report, Professor? Dr. GALSTON. December 5, 1969, it's now only 2 months old, Congressman, and it says a scientific group, or commission should be charged with the responsibility for continued surveillance of the whole problem of pesticide teraNow, the problem of determining whether a problem is teratogenic, whether it's given rise to birth defects is terribly complicated. If you do a laboratory test, whore you have one group of mice getting the chemical, and one grotip not, there's no problem to determine teratogenicity. By this kind of test it has been determined that 2,4,5-T as tested is one of the most teratogenic chemicals 131 known. Even as little as 4% milligrams per kilo of body weight have trebled the rate of abnormal production in mice and in rats a 113 .•ams per kilo ;rsWR 70 percent of body weight lias produced 100 percent abnormal litters abnormal individuals in those litters. Congressman MCCARTHY. I wonder if you could translate those figures into what a human being would be likely to receive in the United States, or in Vietnam. Dr. GALBTON. Well, if you take the lowest of those figures, 4% milligrams per kilogram of body weight, and you say you have a 50-kilogram woman, that's 110 pounds which is about the average weight of a Vietnamese woman, then she needs to digest only about 200 milligrams total to have a teratogenic dose, 100 milligrams per day. Now, we are spraying agent orange, which is a 1 to 1 mixture of 2,4-D, and 2,4,6-T, in Vietnam at the rate of 270 pounds per acre. I should note that is 10 times what we used locally. Congressman MCCARTHY. What would it be in Arizona? Dr. GALSTON. I think our Forestry friends could tell us, it is in the order of two pounds per acre. Congressman MCCARTHY. We will get to that with them today or tomorrow, but that's about the range? Dr. GALSTON. At the Vietnam dose rate, if you assume a 27-pound per acre sprayed, followed by a 1-inch rainfall, which is normal for that region and you know that the rainwater is collected off the roof, or stored in cisterns, or gotten from very shallow wells, then a woman need only consume less tlmn 3 quarts of water per day in combined drinking and cooking operations to receive that teratogenic dose. I have calculated on this basis that it's possible that in Vietnam people have been given this kind of teratogenic dose. Congressman MCCARTHY. Doctor, let me ask you this. Here we have the Bionetics Research Laboratory test which showed that 2,4,5-T is teratogenic in test animals, mice and rats. Is it teratogenic in human beings—do we know? Dr. GALSTON. One doesn't know for sure whether it's teratogenic in Imman beings, one doesn't experiment with pregnant, women, feeding some of them 2,4,5-T, and not feeding others. That would be inhuman, we do not tolerate that kind of experimentation, but the paragraph I was about to read here in fact deals with this. It says there are two ways that you can determine whether a chemical is teratogenic. "First, chemicals or other agents may be administered to experimental animals to determine whether they induce prenatal damage. Secondly, and on a post hoc basis, human populations may be epidemiologically surveyed to detect geographical, or temporal clusters of unusual types of frequencies of congenital malformaties. Combinations of these approaches are likely to insure early detection and identification of teratogenic hazards." Congressman MCCARTHY. Now, to your knowledge, has that been done in Vietnam, or is it contemplated, is the American Association for the Advancement of Science going to do what you just read? Dr. GALSTON. I think it's shocking that there are absolutely no studies on the possible teratogenicity of these chemicals either in Vietnam or in this country. That is why it's so important to gather data from places like globe, and from places like the Saigon area to attempt to correlate, if it's possible to do so, the use of any particular pesticide with the appearance of any birth abnormalities, or any physiological malfunctions. Congressman MCCARTHY : Doesn't the commission's study recommend that no herbicides like this be used until we are sure that it doesn't produce effects inhuman beings? Dr. GALSTON. That's correct, the Commission recommends that given the suspicion that these materials are teratogenic, given their widespread use, but given also our wide dependency on these things in agriculture, we should immediately restrict the use so that we only use these herbicides where it is absolutely necessary to do so, and where there is no possibility of contact with human organisms. I believe that is the safe policy when you think you may be doing harm. You- stop until you find out whether you are in fact doing harm. Congressman MCCARTHY. Do you have any information that you could give for the record here, which would suggest why The White House ban never went into effect? I have a letter here which I received just prior to leaving Washington, which needs further clarification. It is from Mr. Ned D. Hnyley, director of science and education for the Department of Agriculture in 132 138 Onse to a letter I'd addressed to Secretary Hardin, asking why The Wl n ....Snse ban 'didn't go into effect. Among other things, here's what he ss_ "Now, data submitted to D.H.E.W., Department of Health, Education, and Welfare, relev'ant to this position is that the 2,4,5-0? used in the bionetics study contained about 27ptm of Dr. GALSTON. Dioxin is the way it's usually referred to. ' Congressman MCCARTHY, It's t-e-t-r-a-c-h-1-o-r-o-d-i-b-e-n-z-o p-a-r-a dioxin< Dr. GALSTON, Tetrachlorodibenzo para dioxin. Congressman MCCARTHY. A highly toxic contaminant. Dr. GALSTON. Yes. Congressman MOOABTHY. I'm going to seek further clarification that one of the reasons the ban was lifted was this discovery. Now, do you know anything about this in the course of your inquiry? Dr. GALSTON. Yes, Congressman, I became aware of this new development— 2,4,5-T is a chemical synthesized from the reactants that are put together in a vehicle. Depending on the method of synthesis, and the temperature of.syn- M thesis, you may or may not get certain impurities formed in that reaction that accompany the 2,4,5-T which is realized out of the reaction fixture. One of the impurities is tetrachlorodibenzo-p-dioxin. Now, there's previous information that this compound is a highly noxious material. There have been several factory and laboratory accidents in which people exposed to this compound have developed very severe blistering, loss of sensation, and respiratory troubles. The Germans have had a similar experience. So it's natural when you have a report of this kind about the toxicity of 2,4,5-T, to inquire whether the effect is due to the chemical itself, or to the impurity. Congressman MCCARTHY. Does it matter? Dr. GALSTON. I'll mnke this statement. I think it does matter in the long run, Congressman, because if it's theimpurity, then in the future we can learn possibly how to make the chemical without the impurity, and continue its nse. Congressman MCCAUTHY. I've read in the long article by Mr. Whiteside in* the latest issue of New Yorker Magazine, at least he made the point that you can't make 2,4,5-T without getting some dioxin. Now, is that fight? Dr. GALSTON. That's correct, I don't know if any sample that has less than a part per million of dioxin, so all of the 2,4,5-T that has been sprayed both at home and abroad has some dioxin. The question is: Can you lessen the dioxin level down to the point where it is no longer so dangerous? Congressman MCCARTHY. Is there any other way that dioxin can be produced after it's sprayed? Dr. GALSTON. Oh, yes, even if you sprayed 2,4,5-T without any dioxin it might form chemicals in this Arizona sunshine. Putting all that light energy in1 could easily imagine compounds like the dioxin being formed. If there were a little fire somewhere, that's just the condition which would form the dioxin from 2,4,5-T. The only hard data on the terntogenicity of 2,4,5-T are right in this book that I have. There are no date which tell me, or anybody else, that, it's the dioxin and not the 2,4,5-T that's responsible for those teratogenic effects. 1 've had telephone conversations with people who have alleged this, Congressman MCCARTHY. Who are they? Dr. GALSTON. Well, one of them is a member of this Commission, DoctorJulius Johnson of Dow who is an old friend of mine, and 1 think he is very terribly concerned about this development. Naturally, he would be since Dow is tho manufacturer of some of this, and he told me that there are tests going on now which are not finished. He said he would not care to quote the data as of the present moment. Congressman MCCARTHY. Mr. James Hansen of the Dow Chemical Co. visited" uiy office last week and alluded to, I assume, the same tests. Or. GALSTON. Yes. Congressman MCCARTHY. That the Dow Co. itself was carrying out the following-up on this possibility that it is the dioxin. Now, in this letter from Mr. Bayley he said new data submitted to D.H.B.W. , relevant to this position indicates that the 2,4,5-T contained the dioxin. Well, it sounds as if it's the same thing. What I don't understand is how the Dow Chemical Co. could, in effect, by intervening, countt^Bnd, or negate White House orders. ^^ Now, have you discussed this with any other people in the Government, or outside the Government? Dr. GALSTON. I have not, Congressman. I don't have any information on how this operation came about. I would only say that to me it's unthinkable that, in absence of hard data, and to protect the lives and welfare of people in the country, I don't see how this order could fail to be enforced. We must be safe before we are sorry. I would say let's get the facts before we resume spraying with this 2,4,5-T and at the present time there are no published data that I, or any other scientists have seen, that would say that 2,4,5-T is not the culpable agent. I think it's very peculiar that the orders of Doctor DuBridge are not being followed by the Department of Agriculture and the Department of Interior. The Department of Defense, said it announced immediately it would not follow this directive. Congressman MCCARTHY. That's right. The next clay on October 30th, the spokesman for the Department of Defense contradicted the DuBridge order in a verbal briefing to newsmen. He said that the 2,4,5-T would continue to be sprayed in training and regroupment areas where obviously populated areas, and of course as you know it has been sprayed in rubber plantations in Cambodia, which are also populated. AVell, Professor Galston, I appreciate very much your testimony here. Dr. GALSTON. Do you mind if I make one more brief statement? Congressman MCCARTHY. No, please do. Dr, GALSTON. As a biologist, I'm terribly concerned about this because I believe in herbicides, I want to see that they continue to be used. I'm afraid there may be overreaction on the part of the public. I would like to say that there are probably ways that we can safely use these compounds, and the first recommendation of this Commission—I would like to read you just two paragraphs, short ones, because they outline to me what would be a safe procedure. It says: "A new interagency agreement is needed to strengthen cooperative action among the Department of Health, Education and Welfare, U.S. Department of Agriculture, and the U.S. Department of Interior, to protect public health, and the quality of environment from pesticides danger provided by the Secretaries of I-I.E.W. and Interior, as well as Agriculture, should be required for all pesticides registration, pesticide use determined by any of the three Secretaries to be hazardous should be restricted, or eliminated. "The agreement should further require the continuous review of new scientific information on pesticides now in use with the formal reviews made 2 years after initial registration, and subsequent formal reviews by the (hree agencies at 5-year intervals." That seems to be loiidly, essential for the continued safe use of pesticides and it's coupled with the establishment of a national testing center for pesticides, which is also recommended, I would say that we .would be well on our way for the safe use of pesticides. Congressman MCCARTHY. Do you think it's proper to delegate to the manufacturer of such a chemical the responsibility for testing its teratogenicity and carcinogenicity ? Dr. GALSTON. Well, you can certainly accept the data that are contributed by the manufacturer as relevant to the solution of the problem. I think those people have shown necessary testing laboratories which give honest data, but I would not depend on those alone. I would want to see the FDA or some other agency independently test these same compounds aiso, under completely different conditions. That's only a scientific rule, you don't believe anything anybody tells you, it has to be confirmed once or twice before you can believe it. I would certainly hope the FDA, or some other agency, HEW would continue conducting further tests on these toxic chemicals. Congressman MCCARTHY. And not really solely on the research of Dow, or other manufacturers? Dr. GALSTON. That's correct Congressman MCCARTHY. Professor, I wonder if you would bo kind enough to sit with us here, I'd like to use you as a resource person when, we have the other witnesses. Our next witness is Mr. John Pierovich, Assistant Regional Forester, from Albuquerque. « • 134 I Is he in the room? If you would be seated and identify yourself for the record, anc responsibilities in areas under scrutiny here. Mr. PIEUOVICH. Yes, sir, I'm John Pierovich, Assistant Regional Forester in Albuquerque, N. Mex. My responsibilities related to this matter are in connection with the complaints we've received here at Globe, and the overall evaluation of our Chaparral program, and our Chaparral program guidelines. The primary reasons the Forest Service is here today is because this is a Forest Service project. I think that we need to be cognizant of such hearings as this, and we do try to keep informed through the literature of regulatory rules and concerns. In fact, we share quite deeply the concern of the people in this community with their environment, we wouldn't want to do anything that would jeopardize their safety. They're our neighbors, we also live here. At the same time, we've been asked repeatedly to announce that we would not spray again in the Globe area, and like Doctor Galston, I think that we wouldn't want to overreact at this time. So we've said that such an announcement would be premature, we have ourf own studies going forward, and that these studies must be resolved before we can reach decisions on herbicide's use, or on the Chaparral program. In addition to that we believe that it would be also unwise to base decisions on herbicides used particularly from the current allegations, or suspicions here in this area. These matters need to be studied deeply, and we hope to have them studied deeply, and frankly welcome this inquiry because it will help to daylight some of the areas of concern. That's essentially our position, Mr. Congressman. I'd be glad to answer any questions you might have. Congressman MCCARTHY. Thank you very much. In the course of my study, I have come into possession of documents that have been exchanged between the Department of Agriculture and citizens in the area. Here is one from John A. Williams for the Task Group, U.S. Department of Agriculture, Forest Service. Are you familiar with Mr. Williams? Mr. PIEROVICH. Yes, I am. Congressman MCCARTHY. Is he an associate of yours? Mr, PIEROVICH. He works in our regional office. Congressman MCCARTHY. Is he here today? 'Mr. PIEROVICH. No, he's not. Congressman MCCARTHY, I'd like, to read you some of the things that he says: "Paul Boffin (phonetic) called a Dow Chemical representative at Davis, Calif., and requested information about Silvex. This man called Supervisor Courtney later and indicated that a publicity release was being prepared for submission to the news media concerning the known toxicity of Silvex. This if accepted and used by the news media will go a long ways towards improving the situation, and dispelling the fear of Silvex as a highly toxic, or poisonous agent." He then goes on to say in his conclusions, "We are fully convinced that^ many of the people in this area honestly believe they were being subjected to' a highly toxic and extremely poisonous compound with a high degree of persistence and one which would increase in concentration in the water supplies, and in the bodies of humans, and animals. These ideas are not in any way supported by research findings." Now, that is dated ,Tuly 22, 1969, and if I just would ask Professor Galston when was the Bionetics study brought to light? Dr. GALSTON. It was handed over to the Department of Health, Education, and Welfare in December of 1968, to the best of our knowledge. Congressman MCCARTHY. So that to the best of your knowledge, the Department of Agriculture Dr. GAT,STON. Might have had access to that information. Congressman MCCARTHY. Actually, the tests were run i7i 19(57. Now, Mr. Williams obviously either did not know about the Bionetics report, and I would—• I would accept that, I don't think he did just from the tone of the letter, but I'll ask you to comment. Now, which do you think it was? Mr. PIEHOVICH. First of all, Mr. Williams was heading a group for a general survey of the effects here in the Globe area at the request of the Forest Supervisor, and after the initial complaints. We've had si^equent studies go forward, one of these coining out as a second task force >^B't which is somewhat more in depth. Mr. Williams' information was then^ra general nature for an initial report for the forest supervisors. Williams himself is not an herbicide man. Mr. Boffin is, and his reason for talking with the Dow was to get more information. The second question you've asked regarding the Bionetics study was not known to these people, and only known to a few people within the Forest Service but the word of mouth communication that took place following the review of the Bionetics study for publication. This has precipitated a lot of discussion among the science community, and v in the Congressman MCCARTHY. Are you alluding to the Whiteside article in New Yorker Magazine? Mr. PIEKOVICH. No, that's the most recent and clarifying article, at'least I found it very informative. Congressman MCCARTHY. When did you first learn about the Bionetics findings on teratogenicity? Mr. PIISROVICH. I personally learned about it in November when I was assigned to this problem area, and I learned about it through reading in the literature, seeing the discussions among others. Congressman MCCARTHY. Was the present science advisors ordered ban ever transmitted to you, or here in the area? Mr. I'IISROVICH. We were furnished a policy statement from the Secretary of Agriculture in December which referred to the DuBridge statement. Congressman MCCARTHY. Did you take that as a directive not to continue using 2,4,5-T? Mr. PIEROVIOH. We understood it to be directed towards crops, and that }t was not at that time being restricted in range-land use. However, we could infer from this, and from discussions with our Washington counterparts, we learned that there were other studies underway on this compound, and as you perhaps have noted, we did defer our chaparral program in October. The last spraying on this project was in June, and these events have unfolded since that time. It's currently our position here in this region not to use herbicides until some of these matters are researched. The studies that are underway should be most helpful to us in this regard. Congressman MCCARTHY. I think there's a little confusion about just what the DuBridge announcement banned, Doctor DuBridge said—this is October 20, 1960. That 2,4,5-T would be prohibited for use on American agricultural products after January 1, 1970, until the Food and Drug Administration could develop information showing that it could be used with safety. Dr. DuBridge also announced that the use of 2,4,5-T in Vietnam would be restricted in areas remote from population. Mr. PiioROVicn. This is where we found our references to the crop production area, and the Secretary has interpreted this way. As I said the ban on crops is in effect at this time, and as near as we can tell we are also examining the future of the 2,4,5-T as it is compounded today. Dr. GAT.STON. Congressman, could I make a comment here? Congressman MCCARTHY. Yes. Dr. GALSTON. I was unable to understand why when Dr. DuBridge issued this statement he did not also take care to specify prohibition of use in regions where 2,4,5-T might find its way into drinking water. For example, supposing you are using 2,4,5-T to clear shrubs from under a power line, and that power line is going through a town where people have wells, and they draw water from these wells. Don't we need to know if the 2,4,5-T is going to seep down in the water cable and get to these people? It seems to me applying the ban to the food crops is only a halfway measure. Mr. PIEROVICH. I think we need to be concerned by this, and this is why we monitor water from treatment areas. It's significant in this Globe area. Our reference—or the Federal water quality control criterion of one-tenth part per million, this level has never been reached in any of the water, analyzed that we've had run, or had been brought to our attention. Congressman MCCARTHY. You say yon received the directive NovemberMr. PIBBOVIOH. We received the Secretary's explanatory information in ecember as I recall. Congressman MCCARTHY. Were you ever advised that the bnn had Mr. PIEROVICH. No, sir. Congressman MCCARTHY, So the last you had was the DuBridge directive? Mr. PIBHOVICH. Yes, and a statement from our Secretary to agriculture agencies o.C which we are, telling us that 2,4, 5-T was not to be used in crops, and incidentally, the Secretary has added to his statement that we would use alternative methods whenever these are available and practical, and is stressing within the department a use of noncheniical means where these are available to us. Now, this is all developments since the last spraying here1 at Globe, I hope this is clear. Congressman MCCARTHY. Are you spraying in other parts of your region? Mr. PIEROVICI-I. No, sir, and we have no plans to spray during current, or the coming fiscal year at this time. Now, if we have some break-throughs, I'm sure we will be talking about this. Again, it would be premature to say. Congressman MCCARTHY. What's the basic rationale behind the spraying here at Globe V Mr. PiraiovicH. You meanCongressman MCCARTHY : AVhat's the purpose of it? Mr. PIEIIOVIOH (continuing) : The purpose of the project. This is the part of the region, and the Tonto National Forest chaparral management program. This program has many objectives for —if I may take a minute— fire is a very common ingredient in the life history of chaparral, and in trying to bring management to Chaparral Forest, we have excluded fire, or we are using fire by prescription, rather than have the chance of holocaust. In doing this, we attempt to bring a break to the fuels in large continuous masses by developing grassy ridge tops, or grassy openings. These have other advantages for people who want to use the forest, and for game. It happens that the project here in the area was a water-yield project. We have learned through research at the 3-Bar experimental area, and particularly that we can substantially reach the flow of streams, particularly in the winter months where the vegetation is not using the amounts of water that chaparral vegetation does. Now, herbicides were used here at Globe partially because of the known flooding potential of these streams, and that they also know that fire over a large area could cause floods. So rather than use prescribed fire as initial treatment, herbicides were .used. We have plans to use some small amount of fire to continue our work here. Congressman MCCARTHY. Doesn't it say right on the container that this should not be used over water? Mr. PIEHOVICII. That's correct, and as the project instructions were followed here, the applicator pilot was to interrupt his spray every time he passed a major stream channel. Congressman MCCARTHY. "Interrupt his spray," you mean from a helicopter? Mr. PIEIIOVIOH.' From his helicopter, yes. Congressman MCCARTHY. Do you think that is that the answer? Mr. PIEHOVICH, Well, I think it's quite practical, sir. Congressman MCCARTHY. Well, wind might carry. Aren't there restrictions under the circumstances in which you use it? Mr. PIISROVICH. First, let me explain in spraying this area the primary pattorn would be along, or parallel, or to a water course so that it isn't necessary to turn valves off as you may each time he crosses at the creek, but he was going to be crossing streams at the same time he has been spraying. So he would be than instructed to interrupt the spray before making such a crossing. Some drift did occur into the bayous, we have found some of the Sycamores in the Kellner area, the tops have been hit. We don't feel that a substantial amount of herbicide came to the water course, and the pilot was instructed not to apply this over water. Water residues again haven't indicated any great amount of the herbicide in water. I Congressman MCCARTHY. Are they instructed only to spray when the wind Is blowing at a certain mile per hour? Mr. PIEKOVIOH. Yes, that's right. ^_ Congressman MCCARTHY. What is it, eight ? Am Mr. PIEROVIOH. In some projects it's 5-miles per hour, in t^pjase it was 10. Congressman MCCARTHY. Ten ? Mr. PIEHOVICH. Yes. Congressman MCCARTHY. Is that rigidly adhered to? Mr. PIEROVICH. Well, I would hope that it is, here we are depending on other people to do our work, hut we have a project area officer, aud this project had • •u project area officer who works from the helispot where the copter is operating, using a pocket anemometer, aud as lie noticed the wind picking up he would take the pocket anemometer out and keep track of the gusts. Whenever it apnroaches 10-miles per hour, the project would be shut down. i have records here with me of the shut-down on this project, if you are interested. Congressman MCCARTHY. You are undoubtedly aware that some of the residents in the area charge that spraying went on iu much stronger wind velocities? Mr. PIEROVICH. Yes, sir, I am, and I am aware that there has been drifts, and we are attempting to identify how far tin's drift went. In the task force 2 report, we identified a visual effects drift line, we are currently working on infrared interpretation, and I would be very happy to furnish you with a map which delineates how far the dead vegetation that shows up. That's not available to see by the naked eye. Congressman MCCARTHY. That would be very good to fill out the record. I would like to have that documentation very much. Dr. GALSTON. Do you mind if I ask a question at this point? As a scientist, I'm interested in following up one line of questioning here. The benefits that one wishes to derive from this program has to do with increased water ilow? Mr. PIKHOVICH. In part. Dr. GALSTON. And the other part is, I presume, to have a more accessible and manageable terrain where the Chaparral vegetation is? Mr. PIBKOVICH. That's a good generalization among other things. We would like the esthetic qualities of the area to be an indication. Dr. GALBTON. Do you see any deleterious consequences of partial denudation of the hillsides where Chaparral is growing? Mr. PiEttovion.,It's not our intent to denude the hillside. Dr. GALSTON. I'said partial. Mr. PIEKOVICJI. In the course of making a conversion, one often has to take a compromise, and we do compromise to the extent that we will—say for example, in burning—taking out a.n area, we will burn only so long a slope here because any more we would have an overflow of plants and water, and erosion while it is bare from burning', it is an opportunity for a torrential thunderstorm, or wind to cause erosion. But this is also one of the compromises that a farmer must make when he plows his field. Dr. GALSTON. And this is something you think you can keep under pretty good control with applied herbicides? Mr. PIEROVICH. In this case we used herbicides for that reason, yes. Dr. GALSTON. Was there any measurement for the relevant erosion rates before and after herbicide use in a given area? Mr. PIEROVICH. In the 3-Bar area this is being noted at this time. The studies have been in progress for some time, I don't have those data with me, but I could find them for you. Dr. GALSTON,. I, personally, would be very interested in having those data. It's been my impression that some programs have been gone into fairly massively without the comfortable feeling that there's a lot of scientific data behind the original studies to tell us that, this is really what we ousiht to do, and in calculating returns per acre, in terms of where we've applied, I think we have to have a negative quantity in there for possibly deleterious effects, that possibly are not measured. Congressman MCCARTHY. I'd be eager to see those. Mr. PiEnoviCH. I'd be happy to furnish them for you. I think something we have going right now, you may notice in the statement we've furnished you, e are looking at -alternatives, and tolerable levels, and we are approaching very tiling using projects that have been installed as a basis for arrivi Congressman MCCARTHY. On that I wonder if I could ask yon, are you giving licenses for the use of Kuron? Mr. TiEUovicH. We give no licenses for chemical uses. The answer would be no. Congressman MCCARTHY. I see. From whom do they get these licenses? Mr. PIEROVICII, The use of chemicals is done by—in our case, the approval of a project proposal by a regional and national pesticide committee. Once the forest officer who has a project wants to apply a herbicide he prepares a , formal proposal, it's submitted to our regional committee, if they approve, to a national committee. And I'll tell you right at this point, our committee won't approve such a use, but we don't license, Congressman MCCARTHY. Well, thank you very much. Will you be available today and tomorrow? Mr. PIEROVIOII. Yes, sir, I will, as will the ranger and the 'acting supervisor here. Congressman MCCARTHY. Thank you very much. Our next witness is Dr. F. I. Skinner, veterinarian from Globe. Is Dr. Skinner here? Dr. Skinner, I'm pleased to have a veterinarian testify in light of recent Indications that the use of 2,4,5-T spray may have had harmful effects on animal fetuses, I wonder if you would, for the record, identify yourself, your background and experience. Dr. SKINNER. I am Dr. Skinner, local veterinarian, I've been in the area 14 years, graduate of Kansas State University with a degree of T.B.M.. Now, these are my people, and I've lived amongst them. Now, any questions you'd like to ask I'll try to answer. Congressman MCCARTHY. Would you recommend the use of this , Silvex Kuron spray after tests have shown that is has teratogenic effects on animals? Dr. SKINNER. No, I wouldn't recommend it without further study, further research. Congressman MCCARTHY, You think it should be stopped untilDr. SKINNER. Yes, sir. Congressman MCCARTHY. You have some question about the Bionetics findings of the effects of this oh animals? Dr. SKINNER. I'm a clinician, I'm not research. I have not seen any effects of animals in this area—definitely, clinically. Now, as I say I'm not a research, I'm a clinician. I don't set myself up to be an expert on it, but I've not seen any abortions, malformations of fetuses in this area that I can clinically say it was caused by Silvex, or 2,4-D, or pesticides. Congressman MCOABTHY. As I understand it, and we hope to hear from others, that there have been allegations made that the 2,4,5-T sprayed did cause malformation in animals. Dr. SKINNEB. \ cannot speak for those, I have not seen them myself. Congressman MCCARTHY. You did not. Were you ever asked to examine the animals in question? Dr. SKINNER. No, sir. Congressman MCCARTHY. You were notDr. SKINNER. No, sir. Congressman MCCARTHY. So that you just don't know? Dr. SKINNER. I don't know, I don't pretend to know. Congressman MCCARTHY. All right. Well, maybe they will be calling on you. Dr. SKINNKR. I hope so. Congressman MCCARTHY. Well, thank you very much, Doctor Skinner. Dr. SKINNER. Thank you, Congressman McCarthy. Congressman MCCARTHY. Our next witness we'd like to call is Mr. Eobert Mc.Kuslak. Mr. McKusiak? Mr. SICOMP. Sir, I represent Mr. McKusiak as an attorney, and he's requested that he be called later. Can you pass him at this time? He wants to pass at this immediate time. Congressman MCCARTHY. Surely. In that event we'd like to call Mrs. Billee Shoecraft. Mrs, Shoecraft, I wonder if you'd identify yourself for the record, and - i Mrs. SHOECRAFT. Billee Shoecraft, Ice House Canyon, Globe, Ariz. Congressman MCCARTHY, And if you would tell us a little bit about how long you've lived here, and your own experience with the chaparra^pray program? Mrs. SHOECRAFT. We have been in the area since 1947—Mr^H>ecraft a little longer than that, ^^ Congressman MCCARTHY, I wonder if you could tell us about your experiences with the spray program, nnd some of the correspondence you've had with the various agencies of government in this connection. Mrs. SHOECBAFT. I'd be glad to, thank you. We first became aware that they were going to spray a chemical, which they asserted was harmless Congressman MCCARTHY. You say, "they" Mrs. SHOECRAM. The Forest Service. Congressman MCCARTHY. U.S. Forest Service? Mrs. SHOEORAFT. Right, in 1965. They had published in the local paper a news item dated August the 19th, 1965, in which they said the herbicide will be 2,4-D, and 2,4,5-T mixed with diesel oil, and water. The diesel oil will serve as a weight factor to insure against wind drift. Neither 2,4-D or 2,4,5-T is harmful to birds, insects, fish, wildlife, or humans. ^ Congressman MCCARTHY. Do you have a date and name on that? What was the publication, what newspaper is it? Mrs. SHOECBAFT. From the Arizona Record. Congressman MCCARTHY: Of what date? Mrs. SHOECRAM', Of August the 19th, 1965. I also have the typed-i\p version when he initiated at that time from which he deleted the word. "1 anticipate honest inquiry from many individuals and groups concerning the project! I also anticipate adverse criticism and harassment from those who devote their lives to criticizing and harassing." I forgot to read the part where he invited the general public to come and see them spray. If you are as curious as I am, you will want to drive up and watch the operation. I hope you will. Again, I read from the report No. 16, Georgia Forest Research Counsel, Macon, Ga., 1865. On page 28 it. says, "Possible harmful effects: 2,4-D and 2,4,5-T have a. low toxicity, although spray applications leave no toxic residue, a tolerance of five parts per million has been established on or in apples, citrus fruits, asparagus, pears, and quinces. We can find nothing in the Department of Agriculture to back this up." Then, they further said, "Since some persons may be allergic to the oil in the herbicide mixture, skin contact should be avoided, and when treatments are used a respirator is also a desirable piece of safety equipment Congressman MCCARTHY. Who is saying this? Mrs. SHOEORAFT. This is from the Southwestern Forest Experiment Station, Forest Service, U.S. Department of Agriculture, Asheville, N.C. Congressman MCCARTHY. And the day on that, please? Mrs. SHOECRAFT. The date on this was 19651. It further says—after mentioning the respirator, the odor, or vapors may bring on a case of nausea. The Forest Service Health and Safety cautioned that 2,4-D' and 2,4,5-T are mildly poisonous, and flammable in an oil base. However, we were invited to come and see the spray. Congressman MCCARTHY. Do you have any more documents that cast some Mrs. SHOECBAFT. Oh, I've many. v I have here this little item that was given to us, there were a few missing pages, it only had four, so I got in touch with Dr. Holston (phonetic) at Belleville, Md., because this is the U.S. Department of Agriculture, and I wondered where the rest of the pnges were. So Dr. Holston from Belleville mailed me a package in which -was included the rest of it, it totaled 25 pages, and this concerning the toxicity of some organic herbicide to cattle, sheep, and chickens. It tells about some of the things that they found in relation to the herbicides that we've been sprayed with. We don't know exactly because the reports have varied, but they have said they used 2,4-D, 2,4,5-T, and Silvex. They further said it one form, then the tests showed different forms. I quote: "We concluded—that the enlargements were caused by the chemical reaction of the diluted herbicide formulation. The ecropsy—the liver was enlarged and viable. The kidneys were congested. A small abcess was found in the parotid 45-382—70 101 lymph node. In one year that developed a swelling in the region related to the chemical reaction. Associated other lymph nodes of the body were often rged and hemologic." Congressman MCCARTHY. Mrs. Shoecraft, I wonder if just for the iight just interrupt you briefly. I would like to ask Professor would explain the difference between Silvex Kuron, 2,4,5-T, and 2,4-D just for the record. Dr. GALBTON. These are very closely related materials, and I think from the toxicology point oE view, and from the points of view—the presence of any o£ these impurities like, the dioxin we were talking about, they would all be in the same bag. 2,4-D is 2,4-dichlorophenoxyacetic acid, 2,4,5-T has one more, that is 2,4,5tridilorophenoxyaectic acid, and Kuron is simply a trade name for a similar preparation that I believe is a Dow product. Is that correct, I don't whether the foresters here would Mr. PIEROVICH. Yes, that's correct. Congressman MCCARTHY. Is there anything significantly different between 2,4,5-T and Silvex? Or. GALSTON. I would say none whatsoever from the point of view we are talking about. The toxicity would not be due to the length of the chain, but due to the fluorinated aromatic nucleus, as a chemist would call it. Congressman MCCARTHY. Mrs. Shoecraft, I realize you have many documents, and wo would like if we could to have any of these you would care to submit for the record. Mrs. SHOECRAFT. I'd be glad to. Congressman MCCARTHY. Would you, this would help very much. Mrs. SHOECRAFT. Yes. Congressman MCCARTHY. However, now, if there are any particularly salient quotations that—without being overly lengthy, you think should go into the record at. this point, we would like to have those. Mrs. SHOECHAFT. May I submit Farmers Bulletin Number 2158, U.S. Department of Agriculture, issued April 1961, slightly revised, August 10C9, referring to what their rules are on what the wind velocity should be. Congressman MCCARTHY. What does that say? Mrs. SHOEORAFT. It says, "Apply the spray when the wind velocity is less than 6 miles per hour, and the air temperature is 90" or less. Again use a coarse spray " They did not use a coarse spray, they used a fine spray. "Use a slowly vaporizing formulation." They did not use a slowing vaporizing formulation, they substituted water for oil in a very small amount and released it at very high altitudes on a very hot and windy day, and they kept no records—weather records on the job. Congressman MCCARTHY. Can you substantiate those points ? Sirs. SHOECRAFT. Yes, I can. ' ' ' ' " Congressman MCCARTHY, How? Mrs. SnoECKAFT. I'm reading from file Noi 2520, and it states in this lefthand corner to the file, it's from William H, Moehn, district ranger. Congressman MCCARTHY. How do you spell that? Mrs. SnoiccuAFT. M-o-e-li-n, district ranger, date July 11, 1909, subject: Watershed protection, • Kellner Russell chemical maintenance, fiscal ywir' 3000. This memo is a resume of the fiscal year 1909, maintenace project. "The spraying- done on June 8, 9, 10, and 11, 1969, were started at 6:40 a.m. on Sunday, June 8, and the hilltop located on the Icehouse Canyon Trail, at (i ;51 a.m. after the third load was through, the pilot flew to the O.C.O. Camp to check his spray. Wheu he landed Mrs. Shoecraft arrived and told him some of tho spray had landed on her. The pilot returned at the hill at 7 :14 a.m. and said someone should go talk to her. "I left the spray job at that time and did not locate Mrs. Shoecraft." In fact, I cnlled Washington on the third day, but they didn't find me, but I hoy could have if they had looked. "I left the spray job and we continued to spray from the hclispot until 30:57 a.m. when •" T>—^«Ar.xv«4.«4-4,,n Qf^ifyrtT'c, victims. I was informed by the Department Office that they sent it out to the printer's. My suggestion was you either get it from the printer's, or you get a copy, I need it now. I received it in 3 days. In this it refers to the formulation which they call, Orange, and it says that it is one part 2,4,5-T, and one part 2,4-D. I have before me a letter dated October 6, 1969, from the USDA, in Phoenix. The branch of the Forest Service, the Tonto National forest Service, signed by Mr, Jenkins for Mr. R. B. Cortney, Forest Service. He says: Dear Mrs. Shoecraft, following is a list of chemicals purchased by the Tonto Forest as requested by you. The mixture was two gallons chemical with seven and one-half gallons per acre. In a few cases more water was used, and all of them are 2,4-D and 2,4,5-T. Since I was curious because there was no Silvex, I further proceeded to say who bought the Silvex, and I was finally informed by Mr. Moore at Salt ttiver Project they made the decision to purchase the Silvex. They did not purchase it as they said in the Forest Service. They have lied, it's the only word I'd like to use because it's lying when it covers things when they know better. Congressman MCCARTHY. I wonder if you could submit those documents to Mr. Riddleberger for our records? Mrs. SHOECRAFT. All right. Congressman MOCABTHY. And if you are available we hope to go out this afternoon and tour the area. Mrs. SHOICCRAFT. Be pleased to. Congressman MCCARTHY. Thank you very much. We would like to move on now and hear from Mr. McKusiak. Mrs. SHOECRAFT. I had requested analysis that were clone on our plant back in September before another task force is to arrive, which I understand is next week, I've spoken with Mr. Tschirley this morning, he called, I told him before I wanted anymore samples taken. I would like the reports of what they took in September. They seem to be still evaluating these water samples we sent in, and for your information I just learned this morning the samples taken from our own drinking water last week are still highly contaminated, and I suppose I'm the first human to go on record to be able to say that they have now found 2,4-D in my pound of flesh, and that was as of this morning from two different laboratories. Congressman MCCARTHY. That's important, could you elaborate on that? Do you have those laboratory findings? Mrs. SHOECRAFT. These were found in the G.H.T. Laboratories in California, the other laboratory I'm not even aware of the name where the samples were sent. Congressman MCCARTHY. What's that, G. H. Mrs. SHOECRAFT. That's the laboratory where the Department of Agriculture, Doctor Hemton (phonetic) had recommended that the samples be sent on the plant life originally. There will be a longer report on it this afternoon. 142 ^Congressman MCCARTHY. We will check that out. Did you mean to lat a biopsy has been applied on your tissues, and 2,4-D has been foil your Mrs. SIIOKCRAFT. As of this morning they were not complete. Congressman MCCARTHY. Thank you very much. We'd like to call Mr. McKusiak now. Mr. McKusiak, do you care to be accompanied by counsel? If you do, it's perfectly all right Mr. SKOMP. We hnvp no objection, Congressman Mc( AKTHY. All right. Mr. McKusiak, I wonder would you identify yourself for the record, please, your name and your background, and how long you've resided here. Mr. McKusiAK. I'm Robert McKusiak, and I've been an Artist in tile and mosaic for somo 22 years. I have a background prior to that time, and since that time also in science. I majored in chemistry in college, Congressman MCCARTHY. What was that? Mr. McKusiAK. University of Arizona, I do not hold a degree. Congressman MCCARTHY. How long have you resided here? Mr. McKusiAK. I've lived in this area since 1932 with the exception of the time that I attended the University of Arizona. Congressman MCCARTHY. Now, I wonder if you would verbally give us a generalization of your experience with the Forest Service spray program? Mr. McKusiAK. My experience with the Forest Service spray program really didn't come into, being fully until 1969 following the June spraying. Well, let me back up, it came into being in about May 31, 1968. I was aware prior to that time that they had been spraying, but I was not aware that the things that they were 'spraying were particularly harmful. I had seen unusual effects taking place, but. I didn't know what to attribute them to. Congressman MCCARTHY. What unusual effects, could you cite a couple? Mr. McKusiAK. Yes, one in particular which I would prefer that Mrs, McKusiak documented for you because that's her field, and not mine, but specifically in 1066, in May of 1966, the brown pewee population, these are birds Hint, live in our canyon area, suddenly started dying in great numbers in our yard. We have a waterer that birds come to, and there were birds all over during May which had matter in their eyes, and seemed to be having respiratory trouble, and were dying, and at that time we continued spraying it. Congressman MCCARTHY. You don't happen to have any photographs of that, do you? Mr. McKusiAK. No, I don't, I would prefer on a discussion of birds to have MA'S. McKusiak go into this because that.was her field. But, in 1968, on the 31st of May, I was up at my property where I get my clay, it's private land in the area that wns sprayed, it was included in the area sprayed. I had my wife and three children, and the two dogs up there, and the spraying was taking place down canyon. The helicopter came up the canyon, we have a stock pond that was between us and the edge of the property, so to speak, and the helicopter came up the canyon and made a turn southerly, in other words, it made n right-angle turn toward the moxmtains, and it approached. We were waving our arms because we didn't want to be sprayed. He made a turn and he was so close to UK, and the spray descended upon us, and upon the pond, and upon oar kids and dogs, and so forth. At that time we weren't really aware that anything was wrong with it except we both rushed home, my wife and I have both had headchcs from it. Congressman MCCARTHY. The pond, is that drinking water? Mr. McKusiAK. This is a pond which is used for livestock water, but it's on private land. Congressman MCCARTHY. Now, you heard undoubtedly the Forest Service say that they stopped spraying 'when they would get over a stream, but they didn't over a pond. I suppose that would be obviously important? - Mr. McKusiAK. It's incorrect that they stopped over streams, they sprayed directly over three different semipermanent streams that I know of, and one permanent Congressman MCCARTHY. Did you see that yourself? Mr. McKusiAK, I saw them spraying in this area over it, and the devastation continues right down to tho edge of the stream, it's quite visible. Congressman MCCARTHY. Will we be able to see that this afternoon? Mr. McKusiAK. I'm sure you will. 143 f'< Congressman MCCARTHY. I think it's very important. vaSsii; Mr. McKusiAK. One canyon in particular in 1968 when I walHsprayed with my family on our property, and we did have illnesses and have had illnesses thereafter, continued since this time. This particular little canyon, when they flew up toward us—which has a permanent stream in it, and they flew right up the canyon to the pond, it's a stream that seeps out from the pond, and has never been dry. Congressman MCCARTHY. I wonder if you would, for the record, tell us about changes in livestock, and other animal life on your farm, which you would attribute to this spraying. Mr. MoKusiAK. I really don't have a farm to correct the record, I have many different animals, my wife keeps ornamental fowl, she is an archeoornithologist, and she works with archeologlcal birds, and she keeps flies of various types for comparative work, and also for our own enjoyment We have 10 or 12 milking goats that we have had for 10, or 17 years. Wo'vo kept a small population of them, and in the last 2 years we have had a number of our milk goats bear kids, they have from two to three offspring a year, each goat, and a number of these have borne deformed offspring. When I say deformed, I'm referring generally to their heads, their heads were born ^alshaped, and malformed in some cases their bodies, but generally their heads. We have one goat which is already been covered by the news media, but we have one goat which wasn't as malformed as the others. We have kept it alive simply because people were denying such things happening. I would say most of the offspring that were born were born either dead, or deformed, or both. Most of them who were born deformed were born dead. In other words, the animal miscarried deformed offspring. Congressman MCCARTHY. Did you ever ask Dr. Skinner to come out and look at these animals? Mr. MoKusiAK. No, I don't believe I've ever discussed these animals with Dr. Skinner until just recently, but Dr. Skinner and I are good friends, and we have from, time to time called him to ask how much dosage to give an animal if we were going to give them a shot. Some of our animals from time to time have suffered from pneumonia, or things of this type. For- example, many of our fowl in birth have died. I'm referring specifically to geese, and ducks, and some chickens, and many of them have died, and we found by giving them a shot of com-biotic, it's a penecillin streptomycin, I believe, combination, by giving them a shot, usually we could save them. These fowl would come down with what seemed to be pneumonia. There are many other people in the canyon whose fowl done the same thing. We found by giving them a shot we could save them. We called Dr. Skinner to find out what the correct dosage would be, and we generally didn't call back telling him it came out. Congressman MCCARTHY. Well, Mr. McKusiak, I know we could go on for some time, but we have to adjourn shortly, but we will be with you this afternoon. Mr. McKusiAK. I would like to make one other comment, if I could, for the record. Congressman MCCARTHY. Surely. Mr. McKusiAK. I was talking about 1968 when we were sprayed on our own property, and our own dogs following this spraying, we went home and washed, but our own dogs that were with us, two of them became ill immediately with what we considered to be pneumonia, at that time we didn't associate it really with the spray, we didn't think about it, and we gave the dogs —we tried to call Dr. Skinner and he was out of town, and we gave the dogs com-biotics for this, and I believe it was the next day we called Dr. Skinner, ho was hack, and, my wife checked with him and she cheeked the dosage she had given them, and he said it was twice too much, and give them half as much again, and we did, and the dog survived. It would have died if we had not given him the medication. Congressman MCCARTHY. You still have the two dogs? Mr, McKcjsiAK. Neither are malformed or anything, one of them has never been quite well, it's never been well, It wheezes a lot Ono other thing, there are many families in the canyon and many families in Globe and Miami who have dogs that are bleeding from all body openings. 145 144 ^ ' ' ^^, Wo have clogs of this type, and people who have had dogs die from this, we1 could put you in contact with. Congressman MCCARTHY. We would like to have that information. Well, thank you, Mr. McKusiak. We'll look forward to seeing you this afternoon. This hearing will stand adjourned. Congressman MCCARTHY. The hearings will come to order. I've just received the following letter from the White House which I wish to read into the record at this point. It's from the Science Adviser to the President of the United States, Dr. Lee A. DuBridge. "The White House, February 10, 1970. "Dear Mr. McCarthy: This will acknowledge your February 3rd letter cop-^ corning 2,4,5-T, the October 20th announcement that you referred to was'a statement of the actions that were planned to be taken by the various units of the Federal Government in relation to the 2,4,5-T. It was not a directive toagencies for the simple reason that statutory responsibility for these decisions rest in the separate agencies. "I'm sure that by now you have heard from the Department of Agriculture. I appreciate your views on the desirability of an investigation of reports of birth, of malformed children in Vietnam. By copy of this letter I'm calling: your views to Secretary Laird's attention since this area is primarily his responsibility. "As to 2,4-D, this compound is being reviewed along with other compounds, being singled out as requiring additional study in the Bionetics records towhich yon referred." Signed, "Lee n DuBridge, Science Adviser to the President." I'd like to contrast this with a statement as it was issued on October 20' whore DuBridge said that the Defense Department will restrict use of 2,4,5-T to tlie areas remote from population, that the Agriculture Department will cancel registration of 2,4-D for food crops effective January 1, 1970. TheDepartment of Agriculture- and Interior will stop using 2,4,5-T in their own programs in populated areas, or where the residues from use could otherwiseroach man. That the Department of Health, Education, and Welfare will complcfc action on a tolerance for 2,4,5-T, the residues on foods prior to January 1, 1970. This is obviously a retreat from the position taken by the White House in October 29. As I road the statement at that time it was in the form of a directive that the departments will do such and such, now we find that the White House is backing off from this, and is saying that the statutory authority restswith tho agencies. It snems to me that the President of the United States has authority—Hie nlh'mato authority over these agencies, and I regret very much that the President's Science Adviser has seen fit to retreat from the decision of October 29,. which 1 believe was the wise one. The use of this particular chemical should bo banned pending tests. On the plus side I'm delighted to be informed last night,.and it's reported' today in the press, that the distinguished Senator from Michigan, Philip Hart has announced he will hold hearings on 2,4,5-T. He asked Secretary Hardin,. Secretary of Agriculture, Robert Finch, Secretary of Health, Education, and Welfare, and DuBridge to testify on March 11, This is further evidence to methat the compound's effects require additional evaluation, and I expect that I will testify myself before this Senate Subcommittee when they have hearings, I will make that request. I should also announce that a report on my investigation will bo prepared in1 consultation with Dr. Galston, and will be issued at the earliest practical point, Now, we would like to hear again from Mr. Pierovich of the Forest Service. Ts he here? Mr. PusnovicH : Yes. sir. Congressman. MCCARTHY : T would like to say for the record, which I just said on tho radio station here, that T hnve boon very favorably impressed bv Hie cooperation of tho Forest Service. I think that anybody who has any smattering of knowledge about this whole thing must realize that this is something' transcending individual agencies out in the field, that we are td^lg here about national policy, and what is done out in the field really is a result of decisions made at a much higher level,, and to try to focus responsibility on a field unit I think is really to carry this too far. I've been most impressed with your cooperation, and that of your colleagues, Mr. Pierovich, and I want you to know that we appreciate it very much, and our report will so indicate. I understand you would like to elaborate on the statements you made yesterday.Mr. PIEHOVICH. Thank you, Mr. Congressman, for your kind comments, and also for the way you've conducted this hearing. I think the Forest Service is pleased with the way the hearing has gone. There are some significant elements of Forest Service concern that I felt should be made a part of the record 'this morning, and I'll read essentially from that statement. First of all, the Forest, Service has used phenoxy herbicides, but not since the nationwide controversy broke last fall. In fact, the last use of herbicides on the Kellner Russell project was June 11, 1969, and to the best of my knowledge, the last use of any herbicide by the Southwestern National Forest was the August, 1909, on the Gila National Forest in New Mexico. Second, it's apparent there are several persons in this area who believe there are unknown, or suspected characteristics of these herbicides which may have caused them damage, and this is of concern to us. Three, it's apparent we must continue our efforts to ascertain the extent of drift levels of herbicide residues, and the definite relationships between herbicides over environmental factors and the responses of plants and animals in this area. These studies are to be made public when they're completed. Lastly, the extent of continued deferment of herbicide use in the Clmpnrrnl program is dependent upon the outcome of our studies and of the Department's investigation of these matters. Congressman MCCARTHY : Thank you very much. I wonder if you could for the record, repeat what you told me yesterday relative to the drift of tho herbicide over streams, and into adjacent private property, and what steps, should this be resumed, assuming that it can be shown to be safe, what steps would be needed to correct that? Mr. PIEROVICH. At this point, this will be my own opinion, but I first mentioned to you yesterday that our instructions to the applicator pilot were to interrupt his spray application when he crosses streams, we had definite plans for the project here to call for application away from the open water, nnrt main stream courses. I do believe there was some drift into this stream course as evidenced by some top kill on the Sycamores on the stream bottom. There has been drift from the project area onto private property which we have established so far as the visual effects are concerned, and from this I'm certain that we will be developing new guidelines to both assure that the herbicides that we might apply in the future are confined to the project area, and to assure the safety of the public. One definite indicator in this is that it would be desirable to use a much more restrictive windspeed in application. Does that answer your question, sir? Congressman MCCARTHY : Yes, but what wind velocity do you think would be safe? Mr. PIEUOVICH : I wouldn't want to speculate at this time, but we do have a general rule of 5 miles per hour, and we know that herbicides .were applied here to 10 miles per hour, and we see new development in the herbicide application field, the use of inverts has become more and more popular, niul with some corrective work recently done is this area I feel this will help us a pvont deal. Congressman MCCARTHY: Another point that I definitely sympathize with you about is difficulty you have of getting information. I think the fact that you weren't apprised of the Bionetics Kesearch Laboratory finding on teratogonicity until late last year suggests a problem in communications here, and if you have any suggestions for new legislation I'd be grateful. Do you feel you get enough information from Washington on such subjects? Mr. Pircnovicn, I fool that in all of our—the exchange of information Is a very complex thing today. We do make ourselves available to conferences, with' 146 pie in these fields. Our technicians in herbicide work attend meetings ,y on this matter. We are expected to keep ourselves informed. The ll_ • ture has been quite full of the controversies on 2,4,5-T, and we have been aware of the developing controversies. The most healthy thing that could happen in this area would be a definite summary of literature that our technicians could refer to. There are abstracts available now, but the combination of inputs from the universities and from the various departments of government in one abstract bulletin would be helpfill to us. Congressman MCCARTHY. Do you have anything to add, Mr, Pierovich? Mr. I'IEHOVICH. No, I don't, sir. Congressman MCCARTHY. Thank you very much, we appreciate it. Mr. PIEROVICH. Thank you. Congressman MCCARTHY. Our next witness is Dr. Paul Martin from the University of Arizona. Dr. Martin, I understand you are accompanied by Dr. Russell? Dr. MARTIN. That's right. Congressman MCCARTHY. Would you like him to sit with you? Dr. MARTIN. Yes. Congressman MCCARTHY. Dr. Russell, would you care to join Dr. Martin? Dr, Martin, we appreciate your being here. I wonder if you would identify yourself and Dr. Russell for the record, your background and your particular interest in this? Dr. MAUTIN. I'm Paul S. Martin, University of Arizona, Department of Geology. I had training as a professional ecologist, and with me is Dr. Stephen Russell who is a zoologist in the biology department in the University of Arizona. His special interest is in birds. Congressman MOCABTHY. Thank you. Dr. Martin, I wonder before the record if you would tell us about your involvement with the spraying project, and any conclusions that you reached, based upon your analyses. Dr. MARTIN. Well, I'm not involved in the spraying project, and I'm not a herbicide expert. I .have no research experience with herbicides. I do hcive interest in the vegetation of Arizona. I've spent years studying its fossil pollen records, but the interest I had in Globe was in first seeing if indeed there was any effect on vegetation as a result of herbicide treatment that had been called to my attention, I have come up on four separate trips to visit the area that was sprayed, and see what little I could of the community. Congressman MCCARTHY. How long did you spend on these trips? Dr. MARTIN. These were 1-day visits. Congressman MCCARTHY. How many did you make? Dr. MAHTIN. Four. As a result of seeing the area, and talking to some of the people in the area, I was curious to see if just what degree the community might have been affected by this. I wasn't prepared to believe that people, or animals could be affected by herbicide sprays because the little I heard indicated that those who work with herbicides stand underneath the spray plane and are occasionally drenched by the chemicals, and don't suffer ill effects. , , , , So it seems incredible that people in this community could be complaining of such an effect, but they were. Indeed as a result it seemed to me that it was important to listen to them and try to understand what they were saying, and try to come to terms with the only observers who witnessed an event that wasn't supposed to have happened. It also seemed to me that some of the people involved in the work with herbicides were unprepared for this sort of experience, they weren't even listening to the complaints. So I presumed to do that. Congressman MCCARTHY, And what did you find in the course of your four trips? Dr. MARTIN. There is one other person that's involved in what I'm going to say next, I don't know if she's here or not. Within the last month a student from Massachusetts by the name of Miss Adolnide Friclc and she was willing to go on a door-to-door basis, nnd interview people in the community apart from the ones that I talked to. . Congressman MCCARTHY. Excuse me, is Miss Frick present? Dr. MARTIN. I have the results, a summary of her door-to^^r investigation in the area, the purpose was to see if there complaints coini^^rom any other source other than the individuals that I talked to. The trips^Klt I'd made up here and the design was to on a door-to-door basis talk to approximately 50 people in the canyons close to the sprayed area, and to another 50 over in Crestwood, which I believe is east of Globe at a further—at a point further remote from the area that was sprayed. So what Miss Frick did was then conduct a door-to-door interview with people close to the sprayed area, and another group of 50 further away from it. Congressman MCCARTHY, What did she find, do you have the report? We would like to have that for the record. Dr. MARTIN. I'd be glad to give you a copy. Congressman MCCARTHY. Would you care to summarize it? Dr. MABTIN, I'll simply read about a paragraph from the report that summarized it, and of course, the individuals are not identified in this report, and the complete questionnaire is not represented here, simply the highlights of it. There are three key questions, two that have to do with personal health, and one that has to do with livestock. It turned out that few people do have livestock in either—neither the spray area, or in Crestwood, but quite a number have pets. This is what she found. Regarding pets, 13 cases in which animals were effected, and one must presume some relationship to spraying although in no individual case perhaps could this be directly proved. This is the experiences of people living in this community who know the nature of the community, and then feel that something has happened that's a little bit out of the ordinary. Thirteen cases in which animals acted, three kittens lost; two dogs lost; infertile eggs, one; rabbits not breeding, two; chickens not laying, one; burro lost, one; sick dogs, three reports. Now, as far as people are concerned near the spray area, 23 of 56 indicated illness over the past 2 years which may be spray associated. Some people had absolutely nothing wrong with them, or were not concerned. They thought that those that were complaining were imagining it happened, an event that had no bearing in the real world, that it was in the minds of the people reporting. Other reported, and we're quite convinced that their experiences were related to the events of last June, or earlier when herbicide spraying had Imped. Of the 23 reporting illness, 21 were reporting breathing difficulties. Many of these are attributed to the times of spraying. Some are attributed to smelter smoke, there's no avoiding the fact that this area that experiences a good deal of smelter smoke. Some of these people may be reporting an effect that is indeed caused by smoke, I don't know. There were five reports of serious diarrhea, including one entire family. Four reports of chest pains, including one false heart attack, one report of coughing of blood, one report of subnormal temperature: Two reports of numb pain in arms; two reports of hemorrhaging; two reports of irregular periods; one report of miscarriage; two others by hearsay. Fifty-six people interviewed, 42 mentioned some damage to plants, although the purpose of this questionnaire was not to consider plant damage. Now, in Crestwood at a great distance from the Congressman MCCARTHY. Was the interviewer able to determine if such complaints were prevalent before the spraying began? Dr. MARTIN. I don't know how one would do that. In fairness to the people in the Forest Service who have worked with this project, one simply can't conduct a scientific experiment at this point in time. All we can do is talk to the people who were the observers, or ones—or residents in the area, and while their memories are still hopefully fresh, recover some information, just having to take them at their word. Congressman MCCARTHY. Let me jtist clarify. Is the interviewer ascribing these conditions to the spraying based on the interviews with the people? Do , ' 148 they say that these phenomena results were the results of the spraying, or "|ou't they know? Dr. MAUTIN. Yes, some of them would rather not say. The question effect, "Have you experienced any sickness which might be related to herbicide spraying of this area," It's a leading question in part. It's not a question that denies any ignorance of the fact that herbicide spraying had taken place in the area. I am sure there are many faults of a questionnaire of this sort that a professional psychologist would recognize. Congressman MCCAHTHY, Let me say as a point of information, we will shortly have put into the record a scientific data of the results on human beings of 2,4,fi-T, which I think you will find bear a similarity to phenomena you've just described. I wonder if you would go beyond Miss Trick's survey to give us benefit of your own observations of what you saw, and if you were able to reach any conclusions about the effects of the spraying,on either humans, vegetation, or animals? Dr. MARTIN, Well, the effects on vegetation impressed me as ones that have to bo watched over a period of time. Again, this problem of who's to make the investigation, and how it's to be conducted are important. The incident is over, and iu the minds of some local people, hopefully will never occur again. The problem is, what really happened? I was up on four separate trips, or 4 separate days, I saw some things that I have not seen in Arizona vegetation before. Such as the presence on Century plants of flowering way out of season, and immature new plants going on the old stocks of old ones without normal seed being set, I understand that this particular species of Century plant is known to do that, and other botanists have seen such a feature. The area that was sprayed, not all plants are dead in it, Some species like Mnnzanita are remarkably resistant up to this point. The effectiveness of the treatment is doubtful. The areas of spray aren't (lend. The effects of spray on the outside areas on different plants have to be watched over a period of time to fully appreciate the change in phenology, the changes of flowerng time, the change of time when the leaves appear, and when they fall, the way the tradition of plants may be as far as overall growth is concerned, and if one wants to demonstrate the herbicide-caused effect on vegetation. It's also necessary to take into consideration all the other environmental variations that aren't under control either, such as rainfall and temperature. Congressman MCCARTHY. But, you did find evidences of drift outside the project urea? Dr. MARTIN. Yes. Congressman MCCARTHY. Did you find evidences of 2,4,5-T in any of the adjacent streams, or did you seek to find it? Dr. MARTIN. No, I collected samples only from within the project area, soil samples and water samples. Congressman MCCARTHY. You found evidences of 2,4,5-T in the water you've collected within the project area? Dr. MARTIN. The samples that I collected and submitted to a laboratory in California came back with a report of the presence of 2,4-D, and smaller amounts'-.of 2,4,5-T. Congressman MCCARTHY. In the water? Dr. MARTIN. Thorn was a trace in the water, there was up to one part per m i l l i o n in the soil of 2,4-D. Congressman MCCARTHY. Is there anything that you or your colleague could add which would he pertinent to our inquiry? Dr. MARTIN. I would make one recommendation, and then if Steve Russell 1ms anything he would care to add. The recommendation would simply be that hospital records, doctors' records, t.ho veterinary records of those doctors and veterinarians in the Globe area be Bone over very carefully by proper professional people. Congressman MCCARTHY. At that point I think we should pnl into the record a memo of conversation with Mr. Peter Kiddleberger of my staff, and 149 Dr. Grantville Knight, M.D., 2901 Wilsbire Boulevard, Sigto 3'I5, Santa Monica, Calif. ^P This conversation took place on February 6, 1970. ^^ Dr. Knight informed Mr. Iliddleberger that he has two patients under his TOIre from Globe, Ariz. While his examination is not complete, he is of the "(Union that their malady is associated with the recent spraying of Silvex containing 2,4,5-T by the U.S. Forest Servicei Dr. Knight is of the opinion that an Investigation is warranted, and offered to submit a statement of his findings upon completion of his examination subject to the approval of his patients. Miss Frick is here now, and I wonder if she could sit next to Dr. Martin mid Doctor, if you would be good enough to reread that portion alluded to? Dr. MARTIN. This simply summarizes the interviews that Miss Frick conducted in the canyons that is Kellner Canyon, Russell, Sixsbooter, and Ice•Uouso. Fifty-six interviews in that particular area, and some people who had serious complaints to make were not considered in this interview. What I found just in tabulating what her questionnaire revealed was that 23 of 56 individuals indicated illness over the past 2 years, which may be spray associated, 21 individuals reported breathing difficulties, many of these nre attributed to the times of spraying, but not all. Some were attributed to smelter smoke. There were five reports of serious diarrhea, including one entire family. Miss FRICK. Yes. Dr. MARTIN. Four reports of chest pain, including one false heart attack; one report of coughing of blood ; one report of subnormal temperature. Two reports of pains, or numbness in arms; three reports of uterine hemorrhaging; one report of a miscarriage. There were two others that I thought were hearsay, hut I wasn't sure had really occurred in family that you interviewed, and then finally all the questionnaires wasn't directed to plant damage, there were 42 people interviewed who mentioned at least some damage to their plants in that area. Now, the Crestwood account shows much less effect, and this is what one might expect because of the distance further away from the area of spray. Congressman MCCARTHY. Doctor Russell, is there anything that you would udd to the record here that would be helpful? Dr. RXTSSELL. I don't think I would add to the record, but I'm in agreement with Dr. Martin's statement. Congressman MCCARTHY. You are, you've studied the information he lias available? Dr. RUSSELL. I have seen much of the general information, but I've conducted no investigation of my own into it. Congressman MCCARTHY. Thank you, Gentlemen, and Miss Frick, very much, I'd like to now recall I'rof. Galstori. Doctor, as we discussed here I understand you have some scientific data on the effects on human beings of 2,4,5-T. I wonder if you would cite the source of this information, and the findings? Dr. GALSTON. Mr. Congressman, I'm very happy to present this information because in the course of my wanderings around on this day I have found that certain individuals tend instinctively to disapprove any allegations of direct damage to human beings or animals. Now, as I hoped I made clear yesterday, very small doses of 2,4,5-T can cause birth abnormalities in laboratory animals, and that is now actively under investigation, and we've discussed to see whether it might be due to this Impurity called dioxin, or whether it was due in fact to the chemical. But now, the question is, can we actually produce an effect on mature individuals, let us say male individuals, totally apart from pregnant females bearing embryos in uteri, and I should say that there is a fairly sizable respectable scientific literature on this, and if one looks in a variety of sources, including the sort of encyclopedia of clinical toxicology by Gleason and CougliHn, and can find citations to many articles, and I have reference to a few here. Now, 2,4-D can produce, if it's administered in very massive quantities, it •can produce death in the small animals, and there are even a few cases 150 151 recorded oC its having produced very severe symptoms in man. ' ^^ sidered more however, comes from 2,4,5-T, and I would like to read to you a 1W account!• Mnnznnita and Oak, and the desired persistence of what t — ^.-,1^^ of an article published in 1959 by T. Flint entitled "Dermatitis and Kidne!|;; a^airnble plants such as gerardia. .: Now, I suppose a question could be raised as some of the local residents Damage Ascribed to Weed Killer 2,4,5-T." ' huve been raising undesirable, and desirable, according to whose criteria, and Flint relates an episode involving two sisters, age 4 and 0 years, who played for several hours in a yard which had been sprayed heavily a slioi* bf what judgmental values. Manzanita and Oak do live on these hillsides, they time'-before with the Ortho brand of 2,4,5-T, brush killer. This was used fo: tlo transpire to water, and I suppose their killing is desirable m the contention ..f wanting to avoid the evaporation of water. Whether after you are all the control of poison oak. through with the operation and plant to grass, which is the stated objective or This spray contained 15.4 percent of the isopro ester of 2,4,5-T in an ol' base. (hi» clearing observation, you are going to save very much water, I m not sure, «ml whether, in fact, the esthetics of the environment will be improved Now, I should mention parenthetically, I don't have the exact data at Imni another stated objective of this operation is also I'd say open to question, I but Kuron contains much more than that, I believe in excess of 60 percent of would think it would be a very useful operation for those groups charged with this same ester. iimkttiK policy to hold some public hearings at which citizens could come with The next day both girls exhibited generalized erythema—reddening of tta their points of view. I think a lot of this fracas is due to poor interchange of skin—and edematus swelling of the oral and vaginal mucous membranes. information between official agencies, and the citizens. If there had been open The pulse rate and body temperature were not elevated, but both childrn, were described as appearing slightly toxic. The limbs and eyelids were sliglitl! • lion rings, and announcements, this is what we intend to do, this is why we are swollen as the mucous membranes of the mouth were inflamed. On the 3d dwEj' doing it, and this is how we are going to do it, and have objections recorded there were signs of kidney damage. Albumen was noticed in the urine. Tlietfj|p *t the time, a lot of the acrimony that's built up here might have been ' - , , . . i *. was no evidence of liver injury, the urinary abnormalities persisted for about^5" avoided. Now, so far as the damage of plants around homes, there is no doubt about 2 weeks, but 2 months later the urine specimens for both patients normal. It, it has occurred. I have seen it, and as a plant physiologist, I could testify Now, there are other reports in which 2,4-D, and 2,4,5-T are alleged to hnrep. Hint this is typical damage due to herbicide drift. I think that this points up caused toxic effects on the nervous system as measured by the electroen-j|; n lesson when you discharge herbicides from the nozzles of spray on a helicopter, you are getting an assortment of droplet sizes, the big drops are going to cephalogram. That is after ingestion, there was a desynchronization of the electrical activities of the nervous system, I bring these points up only to rein- _ fall quickly, the small drops are going to be carried for longer distances. I force the fact that no chemical is completely innocuous. Some individuals are pi, think until the technology is improved, the so-called invert sprays is one possimore sensitive than others, and some may require a big dose, and some a pi- bility here, and new types of booms for spraying are another, it seems to me that it's very unwise to spray in areas where homes are so intimately assocismall dose to have these abnormal effects produced, but I share with Dr. ated with the forest and woodland, that you are trying to control. You cannot Martin the view that when people appear and say that they have been pinpoint the spray, you cannot, keep it out of the water, and you cannot preadversely affected by these chemicals, immediate and adequate attention vent inadvertent spray damage to the nearby residences, and I would say that should be given to the possibility that these reports will furnish yet additional there are certainly many sprays in the country where the application of aerodata to supplement the rather large amount of scientific data already existing. sol sprays is a highly beneficial practice. Congressman MCCARTHY. Thank you, Dr. Galston. I wonder if you couldFrom my cursory look here that I would say the intervening of house and give us your observations after your inspection of the sprayed area, and tlio area where it drifted. the canyons in which spraying is desired, is so intricate that the slightest miscalculation, the slightest air movement, the slightest malfunctions of the spray Is there anything that you at this point care to have in the record ? equipment would lead to damage to the property, and I don't know how that Dr. GALSTON. Well, I'll say a few words. I want to make it perfectly clear could be' worked out technically, and I would want assurance that those probthat after 24 hours in Globe, Ariz., I don't want to pose as an expert either OD lems are looked into. . , the program, or the effects on vegetation, or on people, but as a biologist workI think the people whose plants have been damaged ought to be compensated ing in this area, there is some conclusions I think I can make which point out In some way because the damage has been considerable around some homes, the need for still further investigation, and everything I sny should be held In that, light. nnd I think it's unfair to expect these people to bear the brunt of this kind of Inadvertent drift operation. What did I see on my brief trip yesterday? Well, I would classify them in several categories. Now, I did see damaged animals, and I talked with humans who alleged that they were adversely affected. Number .1, at the helispot, overlooking the picnic area, I observed and All I can say here is the damage is there, and spray operations did occur, smelled residues, there was no doubt that you could smell residual diesel oil but I know of absolutely no scientific evidence which would link the spray which was primarily the carrier for the herbicide which had been splashed operation to the damage, and I think the people who showed me the damaged during the loading operation onto the helicopter. animals showed it to me in the spirit that this could be a consequence of Now, if you could smell it, there was a good deal aronnd, and that would spray operations, but they weren't sure, and certainly I'm not sure, but unlike indicate that there are definitely residues in certain selected areas, how niucli some people I would not immediately offhand say this is ridiculous. It could be there was I can't, say, how much there might be in the soil, or in the water, 1 us I have shown from my previous reading from this scientific compendium, cannot say, but it seems to me that I could smell evidences at various points nnd I could document further a lot of the symptoms that people are reporting in my trip. So that there probably are residues here and there, and those here have been reported for massive doses of 2,4-D. So we should not leave the could serve as a continuous supply of leaching, I suppose, into the waters of possibility that this did occur, but a much more scientific information is the area, one should not discount that possibility. required. The second category was definite plant damage, and the plant damage was My overall view after one day of looking around is one of puzzlement. I both the desired plant damage in the canyon, and undesired plant damage in wonder why it's desired to initiate this kind of an operation in this kind of an the vicinity of homes, which was due to the drifting, I assume, the herbicide. environment The stated objective is to improve water runoff, and water runoff In the canyons we could see, and these were pointed out to me by some of will benefit, I presume, the citizens of a nearby urban area, Phoenix, which is our Forestry friends who were with me, the desired killing of such plants «s growing rapidly, and which has a lot of water requirements, and their water 152 153 requirements will grow as the years go by. We know this is an arid way, not being an Arizona resident, and not being a politician, I peThaps could say some things here which a lot of people were thinking, but haven't brought forth. Truly, water is going to be wilting in this area for others. So far as I can see unless nuclear technicology makes it available on a massive scale, Which I don't foresee, if you take water from this area to give to another area, you are, in fact, robbing Peter to pay Paul. If you are robbing water from here, you are going to partially change the kind of vegetation, perhaps you are going to denude some of the areas in order to increase the runoff, this involves a comparative set of rules. Whose object is going to be gored here, whose interests are paramount? Well, clearly cities are not going to be able to grow indefinitely, we are going to have to put some limit on them, we know, for example, that the city of Los Angeles got into a lot of trouble with smoke because there are just too many people there. In the same way cities in the Southwest may have to limit their size ultimately based on the number of people they can support on the amount of water resources there are. The trying to take every amount of water out of the Country brings a possibility of a very serious question. Now that President Nixon among others is calling for a campaign to restore the environment, it might be that, we would want to look at this whole project in the context of what we are doing to the entire State, and to the entire countryside. Finally, I would like to merely renew my suggestions that the people who formulated this policy, who set up this whole spray program should identify themselves, and should request the contributions of the citizenry as an input to this whole program. I think that policy should not be made without question. This is a democratic society in which citizens have responsibijity to interest themselves in the making of policy, and—my faith in the American people, and in their desire to run (heir own country has been to a certain extent reinforced by seeing a group of aroused citizens here out to protect their rights. Thank you very much. Congressman MCCARTHY. Thank you, Doctor Galston. I think the points you make are valid. One that I would just enlarge on a bit is that I am presently working on legislation to be established to support a National Growth Policy, I think growth has to be commensurate with the resources and of course, in this •case, water is a critical resource. I would conclude these hearings now with a couple of observations. I think it's important to know that 2,4,5-T was developed fit the Army's chief Genii Warfare Research Center at Fort Detrick, Md. My experiences in investigating the Army's chemical and biological warfare programs, and policies, has not encouraged me about some of the actions' that have been taken, without taking into consideration some of the unforeseen consequences. For instance, when they wanted to dispose of waste from nerve gas production at the Rocky Mountain arsenal near Denver, they first dumped this material into ponds on the arsenal's property. They didn't expect that it would find its way out. They thought it would be just absorped in the water on the pond. It wasn't, it was carried out into adjacent streams, and the neighboring countryside, and killed among other things livestock and 6 square miles of sugar beets. They then dug a deep well and figured the best way to dispose of it was by dumping it deep into the earth. That set off 1,500 earthquakes in' the> Denver area, some of them up to six on the Richter scale, and caused great alarm in the community. They finally had to pull out this material, and of course the earthquakes stopped. Then, they thought they should ship it across the entire United States. They thought this would be safe. Scientists later said it would risk the lives of thousands of people, the plans also called for dumping this large quantity of nerve gas and other materials into the Atlantic Ocean. They thought that would be safe. Scientists later said it could destroy all marine life in 600 cubic miles of tile Atlantic Ocean, with a cataclysmic effect on ocean's production cycle. Now, I cite these instances not in reproaching the Arms^fc: the C.B.W. establishment, but I think that this particular program ha^p questionable record. We find 2,4,5-T developed by the Army's Germ and Gas Warfare establishment, 25 years ago to this date. We do not know for sure whether it will produce birth defects in human beings, I find it unwise to say the least to use such a substance 'without being sure that it is safe. For some reason the burden of proof seems to be on me and my colleagues in the sense that the uttitude is, "we'll keep using it until you can prove it unsafe." Well, I quarrel with the basic assumption, I think that it should be just the reverse, I don't think that any toxic substance whether herbicide, pesticide, drug, whatever, should be used, sold ill the United States until it can be shown that it is not harmful to human beings, that it doesn't produce cancer, or birth defects, or genetic effects. One would think that we have learned from the Thalidomide experience, but apparently we haven't. I also find it incredible that the Dow Chemical Corp. could have succeeded. In helping reverse an order from The White House. Now, I read this section from the statement of October 29 wherein the President's science adviser said that certain agencies of Government, the Department of Defense, the Department of Interior, the Department of Agriculture would do certain things, will inaugurate a new policy. Now we have the letter received today from The White House addressed to me, advising me that The White House is backing off from this directive, and is saying that the statutory responsibility resides with the individual agencies. .1 find it personally unconscionable that in light of the Bionetics findings, und the scientific data cited by Doctor Galston this morning about the proven effects of 2,4,5-T on females, that this substance would be continued to be used on wide scale in the United States, and for that matter in Vietnam where even larger quantities are used. I welcome the U.S. Senate Subcommittee On Investigation into this. I will prepare a full report which will appear in the public documents that will be developed, as a consequence of our trip will be made available to not only the Semite Commerce Committee, but appropriate other committees of the Congress, as well as to the study of the American Association for the Advancement of Science under the directorship of Professor Messelson of Harvard. We finally conclude by thanking the officials who have been most helpful, and to the residents of Globe who have been most hospitable, and I would hope that this experience here might have effects far more reaching than the small area of Globe, Ariz., and that perhaps as a result at least in part of what we have discovered here, that we will stop using 2,4,6-T around Hie world until we can run a series of tests that show that it is not harmful to tills generation, and to the next generation. Thank you very much. Appendix 6 ALBUQUERQUE, N. MBX., February 26, Hon. RICHARD D. MCCARTHY, House of Representatives, Washington, D.O. DEAR MB. MCCARTHY: Thank you for yonr letter of February 16 and for the opportunity to furnish additional documents or statements for the record of' your hearing in Globe. FOB THE RECORD REGARDING WINDS In my testimony I promised to furnish you with additional data on windspeeds during the 1969 spray project. While windspeed was measured l(y tli<> 1' reject Air Officer who used a pocket anemometer, no record of observations was made. He did, however, maintain a record of application flight times JL«J'± phich shows when the work was shut down due to winds exceeding 10 miles V hour. The following table summarizes these important times £ 'cord; Data Time June 8,1969... J u n e S , 1969... JuneS, 1969... June 9, 19G9... Remarks 1505 Shutdown (wind exceeds 10 m.p.h.). . 1703 Resume operations (wind below maximum). 1935 End operation for day. 1018 Shutdown (wind exceeds 10 m.p.h.). End operation (or day. 1115 Do. 1250 Do. June 10,1969.. June 11,1969.. Because allegations.ol! "gale winds" during application have been made, it is of interest to compare the above shut-down times with winds recorded at the Globe Fire Weather Station, The Globe Station records are for observations made only once daily at 1300 hours, but do not indicate the presence of "gale winds" on any day of the project. These 1300 hours observations are as follows: Date June 9 June 10 June 11 Direction -- - - .. J SW , SW W SW Also on your field tour, there seemed to be some misunderstanding regarding application of herbicide to the live stream in Kellner C^ki. While the stream was flowing when you were in the area, it was not a !^Pstream at the point visited at the time of application. We do not deny thaTsome herbicide may have drifted to live streams, as evidenced by some tip damage to trees in the Kellner Recreation Area where there was a live stream, but that drift actually reached the water has not been established. While the Interdepartmental Panel of Scientists headed by Dr. Fred H isehirley arrived following your hearing, their findings are of sufficient importance to the matter under consideration, that we desire to have the enclosed press release issued by them inserted in the record. It was a pleasure working with you and Mr. Riddleberger during your visit ir the lorest Service can be of any further assistance, please let us know AVe will appreciate receiving three copies of the hearing record when available Sincerely, JOHN M. PIEBOVICH, Assistant Regional Forester. Speed (m.p.h.) 5 16 14 16 As can be seen from the two tables, the only day on which applications extended beyond 1300 hours was June 8, when the 1300 hours observation was only 5 miles per hour. The June 11 shut-down time of 1250 hours would tend to infer that winds did possibly exceed 10 miles per hour when compared with the 1300 hours observation of 16 miles per hour. Banger Moehn has stated that winds did not exceed 10 miles per hour in the area of the spray application, and this is quite possible since spray work was high up in Russell Gulch, in the lee of sheltering mountains to the Southwest, on that date. OTHEB ITEMS FOB THE EECOKD Additional copies of the Forest Service Interim Position Statement and of the map showing the limit of infrared detection of dead and distressed vegetation (as of October 1009) are enclosed for the record. As I recall, Professor Galston asked for additional information on the 3-Bar research studies related to water yield. Since the Interim Position Statement digests these, I suggest that the Statement will serve for the record, but would be glad to arrange for you or for Dr. Galston to receive a copy of the rough draft of the manuscript referenced in the Statement. Since the herbicide container converted to a trash barrel, and found in Kellnor Canyon during your field tour, became a matter of importance to the press, the following additional information may serve as a useful insertion for the record: (1) The Dow Chemical Company label does not specify that the container be destroyed (copy of specimen label enclosed) ; (2) As a matter of good practice, we prefer that all pesticide containers not be reused, and when it was found that trash barrels were being made of the containers by the Globe District, the Kegional Forester directed by memorandum on January 29 that all Southwestern Region Ranger Districts discontinue such uses; (3) Ranger Moehn, in response to the Regional Forester's direction, had all such trash barrels picked up earlier in the week of your visit; (4) presence of the container in the creek at the Kellner recreation area cannot be explained by District personnel who were in the area and had not seen it prior to your field tour; (5) the container had been washed with water and detergent prior to painting for use as a trash barrel. 45-382 O - 70 - 11 167 156 FOREST SSRVICiO INTERIM POSITION: K3IMSR CANYOWRUSSSU, GULCH HERBICIDE SPRAY PROJECT AND THE SOUTHWESTERN REGION CHAPARRAL PROGRAM, February 9, 1970 INTRODUCTION . •.-..' • .-Background on Kellner Canyon-Russell Gulch Project The Kellner Canyon-Russell Gulch -Project is a part of the Chaparral Management Program of the Tonto National Forost. The primary objectiTa of this project is to improve water yield, out other program objectives . and resulting benefits are inttnied tp be. met as veil. Improved water yield and other Chaparral Program objectives are discussed bel'ow. This project was initiated in 1965 following extensive local discussions and a press release which appeared in the local paper. Rather than the usual practice of applying prescribed fire as the initial treatment, herbicides were used. This was because of the known tendency for streams in this area'to produce flash floods; herbicide treatment was considered to be unlikely to contribute to flooding, ,wher.eas M large -area.s treated by fire could. Chemicals used in this project are listed by year of use in Table 1, which is appended. These.are all Federally Registered Compounds and were applied in keeping with the laws and label instructions governing their safe use, Following the 1969 Application of Herbicide, Tonto Forest Supervisor Robert Courtney received a complaint in the form of a petition bearing 15't signatures of people in and near Globe, Arizona. Following the initial complaint, Courtney requested a team of qualified individuals to visit the area for a general assessment of alleged herbicide damage. This team reported some limited damage to vegetation on certain private properties. Chaparral Management Objectives • Objectives of managing chaparral'on the Southwestern National Forests are to: 1. Improve water quality and yield through reductions of the potential for sedimentation following wildfire and through reductions in evapo-transpiration losses where modification of existing vegetation is proper. 2. To enhance the scenic value of the' Chaparral zone through development of varied patterns resembling the natural variety sometimes found in unprotected chaparral; these patterns range from savanah-like grass and forb areas to newly regrowint; chaparral, to relic,stands of mature chaparral. 3. To improve wildlife habitat through creation of "additional aodi edge effect and through maintenance of vigor and new growtli in desirable species. H. To reduce the high .costs of protecting chaparral from wildfires ' through the establishment of breaks in heavy fuel continuity, making it more possible to avert fires of conflagration proportions. 5. To increase forar;c production for wildlife and livestock through the release of native grasses and the establishment • of new grass stands. 6. To improve access for both the observer of wildlife and the hunter through a system of.near-primitive roads to strategic fire control locations and through the openings- that will result in treated areas. It is intended that each of the above objectives will be met through Multiple Use Coordination Procedures. These require that regardless of the primary purpose of any project, proper consideration be given to other forest uses and values. Because of the intense interest in improving Southwestern, water quality and yield, both Federal watershed management arid cooperator funds have been made available • for this work as a primary purpoje. Each of .the objectives of chaparral management is fairly well understood by the interested public except for this one of improvement in water yield. Even some experts have, until recently, discounted the potential for augmenting water supplies through alteration of shrub cover in the chaparral type. Much of the research leading to improved understanding of the potential for additional water has been done on the 3-Bar Experimental Watersheds near Roosevelt Dam on the Tonto National Forest, Work there was begun in 1956. Two reports from this work are of particular interest. Pase, C.P., and P.A. Ingebo, 1965, "Burned chaparral to grass: early effects on water and sediment yields from two granitic soil watersheds in Arizona," Proceedings ninth Annual Arizona Watershed Symposium, U pp illus. Hibbert, Alden R., Unpublished 1970 Manuscript on file with Rocky Mountain Forest and Range Experinent Station: "Increases in streamflow vary with rainfall after converting brush to grass." The latter report is cited because it contains data not previously available which are regarded as more reliable (due to additional years' of streamflow measurement) and which indicate greater pro-dice of improved water yields than previously expected. Increases due to watershed treatment have varied froiu 1.5 area inches to lH.0 area inches. The two tost watersheds averaged an increase in water yield, for the period 1959 through 1969, of from h to 6 area inches. 159 Progress and Direction of Studios—The Kollner Canyon-Russell Gulch Project northeast corner of the project. This corner was selected as the best to test the hypothesis that soil residues from drift might be found, since prevailing winds are from the Southwest. Task_ Forcen Ho. 1 and No. ?. (Completed Work) The first two teams to examine the area were concerned with visually detectable effects of the 1969 herbicide application. Due to the similarity of some insect and desease symptoms to symptoms of herbicide effects, the second teatn included specialists in cntovnolo^/ and plant patholo;;y. It was on the basis of this team's findings that many plants alleged to be damaged from herbicide drift were determined to be affected by other causes. Initial laboratory analysis reports have indicated low concentrations of Silvex and 2, U-D at some locations (maximum detected concentration off the project to date is 0,16 p.p.m. Silvex). Especially at these low levels of concentration, it is possible that other sources of contamination may induce "background" which could lead to erroneous conclusions. For this reason, we are proceeding to cross-check analysis procedures while, at the same time, widespread sampling north of the project is scheduled. It should be noted that while all complainants have been advised of Forest Service claim-for-damage procedures, only one formal claim has been filed. This claim was not for properties identified as damaged in the Task Force Ho. 2 Report, and has thus been disallowed. Infrared Photography and Interpretation for Distressed Vegabation ('.-fork In Progress)" While the second Task Force reported that some visually detectable herbicide drift had occurred from the 1969 spray project, extending approximately one-fourth mile north of the project, their assessment did not include previous years' effects, nor was it concerned with delineation of the sprayed area as a whole. In order to more accurately define the limits of-herbicide effect on plants from all years of spraying, aerial infrared photography has been employed. Interpretation of these aerial photo/jraphs has made possible a preliminary delineation of the exterior boundary of destressed and dead vegetation. Both the visually detected drift line reported by Task Force No. 2 and the External Limit of Infrared-detected din-tressed and dead vegetation are shovm on the appended PRELIMINARY map, It is important to note that internal exclusions have not been delineated and that field verifications are not yet completed for the infrared interpretation. Environmental Effects (Work in Progress) Work is underway in this study to assess the total effect of the Kellner Canyon-Russell Gulch Project on the environment. Some of the key considerations included in this study are listed below. 1. Possible further evidence of drift of herbicide sprays through such herbicide residues aa arc detected in soil samples north ol'. the pro.ject area. Initial soil sampling was within the project and on two transects toward the It would be premature to reach any conclusion regarding drift at this time. 2. Herbicide levels in water samples. Water sampling and analyses have been underway for some time. Project methods • called for interruption of application at all stream channel crossings, and as far as we have been able to-determine, no herbicide was applied directly to water. Some soilleaching and runoff is to be expected. All samples we have taken, or taken by private individuals and brought to our attention, are less than the Federal water quality criterion of 0.1 p.p.m. I/ 3. Effect of Treatment on Esthetics. While it is evident the dead vegetation over this area is not pleasing, our concern here is with the next needed steps to actually provide enhancement of the scenic resource. It is sometimes necessary to tolerate temporary degradation of the appearance of an area as a cost of .ultimate improvement. This study is intended to better define tolerable limits, explore alternatives, and recommend treatments to completion. Concurrently, we are assessing the past, present, and projected fire hazard in order to build conflagration control concepts into the landscape design. It. Effects on Animals and Plants. Initial observations by wildlife experts have shown no marked effect upon wildlife. I/ Surface water criteria for public water supplies table appearing in: Water Quality Criteria issued as a report to the Secretary of Interior, April 1, 1963, and published by the Federal Water Pollution Control Administration. On the other hand, repeated claims have attributed varied maladies of humans and animals to the project's herbicide sprays. Lacking private medical histories or other solid bases for evaluation, we believe it more sound to rely on published results of laboratory tests. These are to be used in determining expected effects on animals for rates of application used. Yet to be published laboratory results are needed to complete this study as it relates to animals. ! 1 H .. CM !H rd0) 4^ M o I 1 4> H) 4-> O M r-l •H f J t3 O u' 3 •H -P M Oi Id 'I M [d • H H ,0 ,D r-l .CJ rH 5 rH H H rH w ,0 . • W * » • CO 10 CM H H CM CM • :C A further consideration of this study is that for proper perspective, all of the environmental influences on the area must be weighed. Two examples help to brine this need to focus, One is the frequent presence of smoke from nearby sweltering operations, especially when an inversion and northwest winds combine to produce a thick accumulation in the basin north of the Final Mountains. The other is household and industrial uses of herbicides which may have induced additional residues Into the affected area. It is our further position that it would be unwise to base decisions on the future use of the herbicides employed, solely upon alleged or suspected effects in the vicinity of the Kellner Canyon-Russell Gulch Project. There are many environmental influences operating in this aroa which must be better understood. Also, many of the questions raised about t'ness chemical compounds can be resolved only through carefully controlled laboratory experiments. •M O A o ill tT\ rH > M VD td H O ON •H H M t.1 ^ ^ nt 01 in O II O B « • 8 •H f!J 25! (J ^f" 1 -=!• CM IA 00 J=r H o 1 h J « 13 ib id "" <2fl IA H ri 1 £ 1 OJ VD l 1 ' CM IA rH -^ _-t 00 vo iH < VO -d- r-l rn vo IA rH -d- -3- -d1 1 1 1 •rt n! rd •d 11) IS & W i 1* a S Pi sO 3 £ W 0 U ^^ rH r-H OJ ,-t O\ G»i IA rH 00 CO •— * a o 5 & M Jt! O 5jj o h a o -'H O -.H ,O JH •H ,x rj -P O H -H > rt 3 ^ t) t3 U OH 0 H Pi 3 ri O & rl rH !~i O rl d rd rrj 11 .y B fj c; -d « +» 'd o +> VH c -o fi f-i C) t-J O O (.1 r< A rl Pi td (U (!) O IA Q IA £1 -d- .d- -d- .d- H 6 OJ CM CVj CM •rH CO 13 Pi tu £ r) •H a --P i rl fH O ,« I W J3 -|5 IA 0) C-l n , EH E-i w §! EH EH EH & o ^t rl ^ UI ,d CM -•* JU B IA 1A IA rH •H Cf] -d" j-j- ^J- ^J1 6 CM CM •H rH f-l PJ !/> !^l U OJ CVI -H 'J 'rj cJ t/i II O CO -,H O CJ r| 3 O ft! 3 O C3 I .p J-i 3 O P, S A IA VO VO vo 00 vo ON H oS VO « O O 0) H r •v <0 rl IU H C 'H 'H I *-— Q o i n to ' -P O <1J tu 1 1 w £ ti o U -rl IH A3 I-i 3 O S fn O"H O 0) w O II H i-.') !••• W g o{j •"ci tua'?-< Cd P, c\l 03 r! 'M tU « fi W 'i K •P a c u at t-i P r-1 C' U H 0 FOREST SSRVICS POSITION. It is our position that the studies we have underway, as well as the outcome of public meetings concerned with herbicides and with the overall conduct of the chaparral program, must determine the ultimate decision on deferment* For this reason, we believe it would be premature to state at this time either when the deferment may be lifted, or what new Guidelines will be followed. S O a ';1ro Pi B OJ (1) 3 'H "* fi O d) While neither the effect of possible air pollution in the area nor the possible contamination by other herbicide uses are known, their importance as suspect environmental effects cannot be discounted. V/e share deeply the concern of the people in this area with their environment. The Forest Service has no intention of persuing a course which will adversely affect the health and safety of its National Forest neighbors, nor which will permanently detract from the scenic or other qualities of the Forests. Ob.. 'H O J- -I' rH 'd a w iij •p o -3 ;c! .p o o S c >!> Pi i-i *> ••"» C •» o o o o "> z && y$ c ! H ,fl C 3 3 H; H-J Oj f* -j) ^ O Ul it) p «w 163 V. ".'-1 ,1.- "•<: ' ,M . — • . .-'-• ;/" • '"," ?5«.j . :.?.<. • •••!•••••.•• i ' .S • •' $Mr£fa:r v J---v.\Li< i.:^.^.^i. -' •... .|1 -••' y_ • -..'...•Si • ...-'--"~Jj-SM -„ .-*: ^. PRESS RELKASE - February 20, 1970 Government Interdepartmental Panel of Scientists The panel ,i» carefully examining the evidence collected during its visit. The study vill continue and will include analyses of the numerous samples of blood, soil, vater, fruit and plants for the herbicides, a possible contaminant (dioxin), as veil as various agents producing disease in man, animals and plants. However, to date, ve can summarize a few of our findings as follows: 1. The application of herbicides in the Final Mountains near. Globe, Arizona vas made by the Tonto National Forest starting in 1965i The most recent application of the herbicide was made by helicopter on June 8, 9, 10 and 11, 1969. 2. The materials used in the treatments in 196$, 1966, 1968 and 1969 included 2,1*-D, 2,U,5-T, and silvex. These chemicals came from different sources. In 1969, 30 gallons of 2,1»,5-T produced by the Hercules Chemical Company and 935 gallons of silvex produced by the Dow Chemical Company were used. The silvex is reported by Dow Chemical Company to contain less than 1 ppm of the dioxin. Analyses will be made of eilvex and the other herbicides for dioxin and the active herbicide ingredients, R.16E. 165 164 7. There vaa evidence of woody plant mortality from root rot, 3. There are reports of the aircraft flying over private propertiei and also visible damage to certain yard trees from several kinds but not spraying; and other reports of the herbicide being applied of insects and woodpeckers or sapsuckers. Other plant injuries Just outside the project area. There is clear evidence of drift of ware observed that appeared to be caused by low soil moisture, the herbicides on a number of plants on some of the nearby properties. k. Human illnesses have been reported by several residents in the air pollution and unusual soil properties. 8. Globe region. The phenoxy herbicides following normal use dd not usually Many of the residents vith complaints vere interviewed persist for more than 8 months in soil and vater. Additional by a medical member of the panel. These are complaints that commonly analyses ar« in progress to determine the presence or absence of occur in the normal population; the eye irritation in one individual may be related to the spraying. Nine doctors serving the area of Globe vere interviewed and there vas general agreement that there had been no significant increa'se in human illness related to the spraying. However, blood samples vere obtained and additional studies are planned to verify or rule out this possibility. 5. Reports from the wildlife specialists indicate no significant effects on birds, deer, and other wildlife. There are reports of reductions of birds on a fev properties but there are other reports that bird and other wildlife populations in and near the project area are normal.1 6. Information obtained from ovners of livestock and observations of animals did not indicate any illnesses that do not commonly occur in other regions. It is doubtful that the spraying of the herbicides or dioxin caused the afflictions in the goat and duck because the goat vas born before the treatment and the duck was hatched about l» niles away from the treated area. herbicides. '• Senator HART. We are adjourned to resume on the 15t3i of this month in this room. (Whereupon, at 5 :15 p.m., the Subcommittee was adjourned, to resume on April 15,1970.) EFFECTS OF 2,4,5-T ON MAN AND THE ENVIRONMENT WEDNESDAY, APRIL 15, 1970 U.S. SENATE, COMMITTEE ON COMMERCE, SUBCOMMITTEE ON ENERGY, NATURAL RESOURCES AND THE ENVIRONMENT, Washington, D.G. The Subcommittee met, pursuant to adjournment,, at 10 a.m., in room 1318, New Senate Office Building, Hon. Philip A. Hart, presiding. Present: Senators Hart and Baker. Senator HART. The Committee will be in order. Our first and distinguished witness is the Surgon General, Dr. Jesse Steinf eld. f;, STATEMENT OF DR. JESSE STEINFELfy SURGEON GENERAL, DEPARTMENT Or HEALTH, EDUCATION, AND WELFARE: ACCOMPANIED BY DR. DAVID GAYLOR, DR. DIANE COURTNEY, AND DR. DALE LINDSAY Dr. STEINJTELD, Thank you, Senator Hart. Accompanying me are Dr. Diane Courtney, on my right, of the Pharmacology and Toxicology Branch of the National Institute of Environmental Health Sciences, Dr. Dale Lindsay, associate commissioner for science (FDA) and Dr. David Gaylor, chief of the Biometry Branch of the National Institute of Environmental Health Sciences. I have a prepared statement. Senator HART. Yes. I suggest you read it and if, there is any footnoting or extension that you want to make as you go along, feel free to do it. Dr. STEINFELD. Thank you, sir. I am pleased to appear before you today to discuss the herbicide known as 2,4,5-T, our efforts to determine its hazard to health, and subsequent action to protect human health. The production of 2,4,5-T (2,4,5-trichlorophenoxyacetic acid) in the United States increased from 8 to 40 million pounds per year in the last decade. In the United States, 2,4,5-T is principally used as a weedkiller in clearing range and pasturelands, roadsides and rightsof-way, in suppressing aquatic weeds, and in eliminating weeds in croplands. It is also used to reduce weeds in turf. The use of 2,4,5-T and its salts and esters on food crops has been registered by the (167) 168 f.S, Department of Agriculture on the basis of no residues i marketed food. To insure that the foods reaching markets are free of residues, the FDA has monitored the food supply in selected cities. About 5,800 food samples were analyzed for 2,4,5-T and other pesticides in the last 4 years. Residues of 2,4,5-T, at trace levels (less than 0.1 part per million), were found in 25 of these samples. In 1965, one sample contained 0.19 parts per'million; in 1966, another sample contained 0.29 parts per million. It is my opinion that the results of the monitoring program justified the registered use of 2,4,5-T on selected food crops, in the absence of any known toxicity of 2,4,5-T. The development of a balanced public policy which considers benefits and risks associated with the use of a compound such as 2,4,5-T is an exceptionally difficult matter. Great public fear of the possible implications for man has followed reports of harm in laboratory animal tests. And yet frequently it is not known with certainty what laboratory animal tests may mean for man. We are obligated to make decisions of great health and economic importance on the basis of very limited evidence of potential hazard; prudence allows no other course. We are aware that both good and bad consequences may result from our actions. The enormous strides taken in achieving the prosperous and healthy life we now enjoy in an industrial age has created problems and uncertainties which are not easily overcome. The resolution of these uncertainties and solution of these problems will require national commitment and broad public education and understanding. At this point, I would now like to read the joint announcement of Secretaries Hardin, Finch, and Hickel, prepared in accord' with the,, Tnteragency Agreement for Protection of the Public Health and the Quality of the Environment in Relation to Pesticides. This is the first public release of this announcement. Agriculture Secretary Clifford . M. Karelin, Interior Secretary Walter ,T, Hickel, and HEW Secretary Robert H. Finch today announced the immediate suspension by Agriculture of the registrations of liquid formulations of the weed killer, 2,4,5-T for use around the home and for registered uses on lakes, ponds, and ditch banks. Those actions are being taken pursuant to the Interagency Agreement for Protection of the Public Health and the Quality of the Environment in Relation to Pesticides among the three Departments. The three Cabinet Officers also announced that the Department of Agriculture intends to cancel registered uses of non-liquid formulations of 2,4,5-T iirouwl the home and on all food crops for human consumption (apples, blueberries, barley, corn, oats, rye, rice and sugar cane) for which it is presently registered. The suspension actions were based on the opinion of the Department of Health, Education and Welfare that contamination resulting from uses ol 2,4,5-X around the home and in water areas could constitute a hazard to human health. New information reported to HEW on Monday, April 13, 1970, indicates that 2,4,5-T as well as its contaminant dioxins, may produce abnormal development in unborn animals. Nearly pure 2,4,5-T was reported to cause birth defects when injected at high doses into experimental pregnant mice, but not in rats. No data on humans are available. These actions do not eliminate registered use of 2,4,5-T for control of weeds and brush on range, pasture, forest, rights of way and other non-agricultural land. Users are cautioned that 2,4,5-T should not be used near homes or recreation areas. Registered uses are being reviewed by the three Departments to make 169 certain that they include adequate precautions against graziM^treated areas until long enough after treatment by 2,4,5-T so that no contc^Mited meat or milk results from animals grazing the treated area. ^^ Wliile residues of 2,4,5-T in meat and milk are very rare, such residues are Illegal and render contaminated products subject to seizure. There is no tolerance for 2,4,5-T on meat, milk or any other feed or food. USDA will issue guidelines for disposal of household products containing 2,4,5-T. The chemical is biologically decomposed in a moist environment. BACKGBOUND INFORMATION Secretary Finch's Commission on Pesticides, which reported its findings in November and December 1960, extressed concern that research conducted at Hionetics Research Laboratories, under the direction of the National Cancer Institute, indicated that 2,4,5-T had produced a number of birth defects when fed or injected into certain strains of mice and rats. Because the test material contained substantial concentrations of chemical impurities (dioxins), the birth abnormalities could not be attributed with certainty either to 2,4,5-T, or to the impurities known to be present. Representatives of the chemical industry pointed to evidence of extreme potency of the impurities as toxic agents. They demonstrated that 2,4,5-T now being marketed Js of a greater purity than that which had been tested in the IHonetics experiments and urged that further testing be undertaken to clarify the questions raised. Responding to this suggestion and utilizing materials supplied by one of the major producers of 2,4,5-T, scientists at the National Institute of Environmental Health Sciences promptly initiated studies to determine whether 2,4,5-T itself, its impurities or a combination of both had caused the earlier findings, and whether the 2,4,5-T now being marketed produces birth abnormalities in mice and rats. The experiments were completed last week and the statistical analyses performed over the weekend. On Monday and Tuesday of this week the analyses of the data were presented to the regulatory agencies of the Federal Government and to the members of the Cabinet. The dioxin impurities and the 2,4,5-T as it is now manufactured, separately produced birth abnormalities in the experimental mice. Because absolutely pure 2,4,5-T was not available for testing, it is possible only to infer from certain of the observations that the pure 2,4,5-T probably would be found to be teratogenic if it were tested. But, since pure 2,4,5-T is not marketed and could not be produced in commercial quantities, this is not n practical issue for consideration. In exercising its responsibility to safeguard public health and safety, the regulatory agencies of the Federal Government will move immediately to minimize human exposure to 2,4,5-T and its impurities. The measures being taken are designed to provide maximum protection to women in the childbearing years by eliminating liquid formulation of 2,4,5-T use in household, aquatic nnd recreational areas. Its use on food crops will be cancelled, and its use on range and pastureland will be controlled. Maximum surveillance of water supplies and marketed foods will be maintained as a measure of the effectiveness of these controls. These measures will be announced more specifically in the Federal Register shortly. While the restriction to be imposed upon the use of this herbicide may cause some economic hardship, we must all cooperate to protect human health from potential hazards of 2,4,5-T, other pesticides and the dioxins. The three Secretaries commended the chemical industry for its prompt and willing cooperation with the National Institute of Environmental Health Sciences in the studies to clarify questions raised by the initial studies of this herbicide and for working closely with the FDA in the other studies still underway. They urged the full support of industry, agriculture and the home gardner in insuring the safe use of 2,4,5-T and other pesticides which contribute in important ways to the welfare of the Nation. 170 171 Fhat is the end of the press release and I would add that it is _ understanding that Secretary Packard of the Department' Defense sent a memorandum to the Joint Chiefs of Staff saying the Department will suspend the use of 2,4,5-T in all operations pending evaluation of the data. I will return to the prepared testimony. At this point, we would like to provide for the record a summary description of the results of these latest studies of the National Institute of Environmental Health Sciences,1 completed this past week. I shall be pleased to respond to questions about these data but suggest that the Committee not be burdened by a detailed oral presentation of the findings which have been stated briefly in the foregoing announcement. This leads me to brief mention of the.studies which will be presented next by Dr. Verrett. Commencing in the fall of 1969, Dr. Verrett reinstituted tests of the embryotoxicity and teratogenicity of 2,4,5-T, its contaminating dioxins, and related chemicals. Dr. Verrett is to be commended for promptly attacking these problems and for going to the very considerable trouble of purifying the 2,4,5-T by repeated recrystallization. However, I must express concern about the degree of reliance which has been placed upon chick embryo studies. While the studies in chick embryos arc in general agreement with those in studies of rodents at the NIEHS, it is to be emphasized that they do not clarify the uncertainties as to significance for man. I believe that it is imperative that everyone involved in the development of a national policy for dealing with the many questions posed by 2,4,5-T and other pesticides be aware of the complexity as well as the importance of the issues, together with the limitations of our ability to estimate potential hazards to human health posed by these substances. It is essential that we strive to respond wisely to the discoveries which have been made in this field, and resist the temptation to resort to measures which may be more extreme than the evidence warrants. For example, 2,4,5-T is probably the most effective means of controling poison ivy, poison oak, and other noxious weeds to which a substantial portion of the population react badly. It has been estimated that 60 percent of the American population is sensitive to either poison ivy or poison oak, and that from 5 to 10 percent of Americans suffer a reaction to the poisons from these weeds each year. Some of these individuals become quite ill and incapaci- tated by their reaction to these poisons. By contrast, we are not aware of any reliable evidence that 2,4,5-T, indeed any of the pesticidal chemicals, has resulted in human birth abnormalities. These remarks should not be interpreted as evidence of indifference to what may be a potential hazard to health. The record clearly reveals a series of responsible actions by the Administration to the results of recent laboratory tests. Prudence has characterized these decisions and actions and will continue to guide the Department in these matters. In keeping with the pattern established with the naming of the Secretary's Commission on Pesticides, the thorough s(B^ of pesticide problems by the Commission, and the Administraum's prompt action to implement the recommendations of the Commission, we HOW commit ourselves to the following actions: We shall strive to develop better means for predicting in laboratory animal systems the potential hazard posed for man by chemical pesticides. We are aware of a great need for a centralized clearinghouse for information of all types on pesticides. We plan to have such a clearinghouse established jointly by the National Library of Medicine and the FDA in the very near future. Other agencies having similar interests and needs will be invited to participate in this undertaking. The need to continue certain closely restricted uses of 2,4,5-T will require a high level of surveillance activity to insvire protection of the human population from exposure through water sources. This .will be done. The Food and Drug Administration will continue to examine a variety of foods for the possible presence of residues of pesticides, and will take appropriate action through the interdepartmental agreement to protect the public health. This completes my prepared statement, Senator Hart. My colleagues and I will be pleased to answer any questions. Senator HART. Thank you Doctor. Just as you began, we were joined by the able Senator from Tennessee, Senator Baker. I understand that the announcement you just read us relates to both powdered and liquid forms of 2,4,5-T shipped in interstate commerce. But what about the 2,4,5-T which is now on the shelf? What do we do about that? Dr. STEINFELD. You mean on the shelves in the homes and the shelves in the stores? Senator HART. Yes, the places for retail sale. Dr. STEINFELI). I think there is a distinction between the suspension of the registration and the cancelling for registration and I would like to call on Dr. Lindsay to describe in more detail, the procedures involved. Dr. LINDSAY. The suspension is a little more drastic than the cancellation, because it is a final action until some other action is taken, whereas the cancellation permits hearings and has the statutory procedure for appeal during which time the pesticide may be used while it is being reviewed. Senator HART. Well, the suspension, the more drastic remedy, was directed at the liquid form. Do I read that correctly ? Dr. STEINFEI,D. Yes. The suspension by Agriculture of the registrations of liquid formulations of the weed killer for use around the home and for registered use on lakes, ponds, and ditch banks. We reviewed the concentration of 2,4,5-T in a number'of formulations and found the concentrated form is present in liquids and could present a hazard. 1 See p. 08. P:' , 45-382 O—70 12 « 'he amount of 2,4,5-T in some of the solid fertilizer-type mates was much less and therefore, the more drastic action was^ taken regarding those compounds. ^^ Senator HART. As you read that suspension sentence I did not hear a suspension extended to the use of 2,4,5-T on food crops. Dr. STEINFELD. The three Cabinet officers announced they, intended to cancel the registered use of nonliquid formulation around the home and on all food crops for human consumption, so that all of these registered uses will be cancelled. ' ' " Senator HART; But the use of liquid formulations on food crops, as I understand the announcement, was not. Dr. LINDSAY. As far as I know all of the use on food crops is from the liquid application. Senator HART. So there would be no application to food crops under this order, as you understand it ? Dr. LINDSAY. As I understand it. I am not aware of any dry material used on food crops. Senator HART. Well, let me get back to my point of departure. You have suspended for certain applications 2,4,5-T in liquid form. As Dr. Lindsay said, that is the more drastic sanction. Now, with respect to that 2,4,5-T in liquid form, the order today has what effect on the marketing and use on shelves or in homes ? Dr. STEINFELD. Well, I don't know exactly what the Department of Agriculture will do. This is not an FDA activity. I am certain they will move quickly and appropriately. I think a significant statement is on page 2 of the release, which says the "U.S. Department of Agriculture will issue guidelines for disposal of household products containing 2,4,5-T. The chemical is biologically decomposed in a moist environment". The intent is to get rid of all 2,4,5-T around the household. 1 assume it would not be available for use in households where pregnant women would have access to it. I don't have the details of those actions. Senator HART. I see we don't have anybody on the witness list this morning for the Department of Agriculture, but would you agree it would be very inappropriate for the Department of Agriculture to permit continued vending of liquid 2,4,5-T for any of the purposes for which you have suspended it, even though it is now in retail distribution ? Dr. STEINFELD. I think this announcement will have dramatic impact. Our meetings with the Department of Agriculture on Monday and Tuesday would lead me to believe they are going to take appropriate and vigorous action. Senator HART. Would you describe as appropriate, walking into a store and seeing the thing on the shelf and saying, take it off ? That seems appropriate to me. Dr. STEINFELD. Idon't know the mechanisms which they have to insure compliance. Senator HART. If they have it and don't do it, don't you think it would be inappropriate and if they don't have it, don't you think Congress should give it to them ? \<; I Dr. STEINFELD. Certainly they should have the authority to do what is required to protect the public health, and I itijk. they do have this. ' ^P Senator HAHT. Well, we will find out. Dr. STEINFKLD. I am sorry, I don't know. Senator HART. You are talking to another nonexpert, so don't feel bad. Mr. Bickwit has greater expertise than I, so we will let him deal further with the problem. But there is one passage in your announcement that particularly interests me. In the press statement which you read, there is a paragraph which states: "The regulatory agencies of the Federal Government will move immediately to minimize human exposure to '2,4,5-T and its impurities. The measures being taken are designed to provide maximum protection to women in childbearing years by eliminating formulation of 2,4,5-T use in household, aquatic and recreational areas. Its use on food crops will be canceled and its use on range and pasture land will be controlled," You say on food crops its use will be "canceled." But is 'it not a very technical definition only of that term that permits you to say it will be canceled on food crops, because in liquid form'l take it, 'it may still be used, or am I wrong about that? Dr. STEINFELD. When the use is canceled, such a notice is published in the Federal Eegister, I believe. And then there is a 80-day period for comments, is that not correct, Dr. Lindsay ? Dr. LINDSAY. Yes. Dr. STEINFELD. After which appropriate action is taken. Senator HART. I think what I am more concerned about is my desire to understand precisely what may or may not be done with this formulation in application to food crops. In liquid form may it continue to be used ? Dr. STEINFELD. You mean during the 30-day period while the—I am afraid I don't understand. Senator HART. It has been suggested to me that there would continue to be no restrictions with respect to the use in liquid form on food products. Now, is my information correct on that? Dr. STEINFELD. No, sir, the use on all food crops will be eliminated as promptly as the law permits through cancelation of the registration, whether in dry or liquid form or any form. There will be no vise on food crops, Senator Hart. Senator HART. All right. I think this is a desirable clarification, since there were some who had felt otherwise. You say it will be eliminated as promptly as is possible under the law. .It could be eliminated more promptly by a suspension than a cancelation? Dr. STEINFELD. Yes. Dr. LINDSAY. Yes, I am not aware of what the Department of Agriculture's intent is with regard to carrying this on. The main idea was to get it into effect at the earliest possible time where it would be likely to come in contact with women of childbearing age. 174 enator HART. I am trying to ask why the different treatml Why with respect to certain forms and use is it merely canceled ? Although that sounds very dramatic, it means if you want to use it, go ahead and use it until somebody resolves differences which may arise over the action. Why handle some uses on a cancelaticm basis and some by suspension ? Is it because those uses and forms that you suspended more intimately or directly come in contact with women of childbearing age? Dr. STEINFEUX Yes, I believe that is the reason. Right now there is a zero tolerance on foods, and any foods that had any measurable toxicity would be subject to seizure. I believe the intent was to move as quickly as possible, but we wanted to alert women who may have liquid formulations around the home, who may be spraying it, that it may present a hazard. We will take appropriate steps to try to warn the female population, particularly of childbearing age. That is the reason for the more dramatic action in the one instance, and the less dramatic but, I believe nonetheless complete, action, however, nonetheless in others. I guess I have here a legal phrase; I think for suspension one must show an imminent hazard to health, and this, perhaps, is the reason. Senator HART. I don't envy you that business of interbalancing. You describe the judgement that you seek to arrive at as a product of weighing the imminence of danger against the values that are identified as following from the use of the pesticide. As a layman, probably we would tend to oversimplify it. Now, having admitted this may be an oversimplified impression, why isn't it a more prudent balancing act to say, well, there is danger here because we can't establish that there is no danger and we are not going to get hung up on the degree of imminence of the danger. We are just going to say, to be sure there isn't any danger, we are going to suspend this. Why aren't you tempted to resolve this balancing operation in that manner ? . Dr. STEINFELD. I am not sure I am the one who makes all these decisions of balancing, Senator Hart. My role of course, is concerned with public health and safety. But we are always balancing things, Certainly in medicine, in picking drugs to use for diseases, sometimes the treatment is worse than the disease. If it should turn out that these materials can be safely used on range and pastureland, that there is a.period in which there is biodegradability during which the materials will effectively disappear, and yet permit the person who raises his cattle or dairy cows to have a better—I don't really know the name, I am a city boy, a small-toAvn boy, not a farmer—but better able to have better cows, more milk, better meat, then there are appropriate reasons for using this chemical. I think the real problem, Senator Hart, is that we do not have an effective, adequate substitute for certain uses. I think this is the key issue. The other good chemical which kills poison ivy and poison oak is a carcinogen in some animals and not proven for many, but it is a very potent chemical that will destroy poison ivy and poison oak. So there is another balance that one must weigh. A Senator HART. But into that formula you have to t» of economic possibility that if this were suspended, if it ]ust permitted to be marketed for this purpose, and if there is a neec tor a cure for the ill that this thing treats, maybe there would be a renewed effort to find a third alternative. , Dr. STEINFELD. I believe the action which has been taken today will lead to more intensive research to find an alternative to 2,4,0-1 to destroy the particular kind of herbs it is capable of destroying. Senator HART. Mr. Bickwit, . , Mr. BICKWIT. I am sorry to go over the matter of use on food crops again, but I do want to clear this up so that we know piecisely what the situation is. It says in the first paragraph^of yout press release that liquid formulations of the weed killer 2,4,5-T foi use around the home, for registered use on lakes, P™ds and d itch banks will be suspended. Do you intend to include within that list ol the wording for food crops is otherN wise. It would be canceled rather than suspended. Mr. BICKWIT. I am talking about liquid formulation. Dr. STEIN™. As I read the actions taken there will be a. cancelation of registered use of nonliquid formulations around the home r ™ is clear, but what I want to know is ^ notion is proposed with respect to the use of liquid formulations on r ™ N F E L D . My interpretation of this lawyer but I now see what you are driving at. I think this have been worded, and we will have to check into it, 1 quid and nonliquid formulations around food crops." The intent is not to use liquid and nonliquid formulations on ™ speak for the three Cabinet officers. It is my understanding that the intent is not to permit use on any loocl crop for human consumption. '. „„„!«, y Mr BICKWIT. Well, you will permit use on it pending appeals < Dr.' STEINFELD. Pending the legal activities. Dr. LINDSAY. But there is no permitted residue of 2,4,5-T on any food. It would be subject to seizure. Mr BK-KWIT. Now, I would like to deal with your statement that an imminent hazard needs to be present before suspension can take place. Is that to say that there is no imminent hazard from the use . studies which have been done flja market basket sampling and the measurement of foods for 2,4,5-1, as 1 mentioned, it is a very rare instance where these things are found, and in sugar cane the herbicide is probably destroyed in the processing by heat. We do not really know. The action we are taking is based on teratogenicity in mice and the fact that dioxms also cause teratogenicity in rats and perhaps in hamsters. It- is a possible hazard. 176 177 Mr. BICKWIT. Is that what you need to cancel as opposed iend-a possible hazard ? Dr. STEINFELD. I do not know the law that well. I really do not know the exact wording of the law, do you, Dr. Lindsay ? Dr. LINDSAY. No. I am sorry. This is Agriculture's bag, and I do not know it. Senator HART. Let us order printed in the record at the conclusion of your testimony the appropriate sections of the Federal Insecticide Fungicide and Rodenticide Act. Dr. STEINFELD. Fine. Mr. BICKWIT, liave you any information derived from your tests on the degradability of dioxin ? Dr. STEINFELD. Dr. Courtney is a pharmacologist. Dr. COURTNEY. We have no information on that. Mr. BICKWIT. In other words, then, it is possible that dioxin is both persistent and accumulative in human beings ? Dr. COURTNEY. That is possible. It is also possible that it can be metabolized. Dr. STEINFELD. I would like to volunteer something, that is, that the dioxin which produced the results that we will submit for the record is a very potent teratogen for mice in 10,000 to 30,000 times smaller a dosage than 2,4,5-T as we could obtain to pinpoint which chemicals were the villains. And I think it raises another issue, that is, where else in man's environment could these chemicals be found? We have not shown that these chemicals are teratogenic for man, but we may want to take action. The Food and Drug Administration and Agriculture are presently studying a number of other pesticides in the mamifacture of which poly-chlorinated phenols are subjected to heavy temperatures and may produce dioxin. So I think we are having an important study carried out there. Mr. BICKWIT, Are you looking outside the herbicide area as well? Dr. STEINFELD. We must look wherever polychlorinatecl phenols are subjected to high temperatures. We must look for the presence of dioxin and if we find them we shall have to take appropriate action. Mr. BICKWIT. But the appropriate action is not to find that an imminent hazard exists ? Dr. STEINFELD. I do not know what the appropriate action is. I know we are going ahead with this activity. Mr. BICKWIT. I take it you do know what the data are with respect to 2,4,5-T and you do know dioxin is present and you do know it is very potent and yet you have concluded it is not an imminent hazard. If it were you would have suspended rather than canceled use. : Dr. STEINFELD. You mean suspended all use everywhere? Is this what you mean? Mr. BICKWIT. Yes. ' Dr. STEINFELD. I think the question of imminent hazard would relate to pregnant women, but we do not know it is teratogenic for man. TJse out in rangelands and forests and so forth, I do not see as a hazard to pregnant women. Mr. BICKWIT. Clearly you have no evidence that it is not. Dr. STEINFELD. No, I have no evidence that it is i^fnor that it is, actually. It is a potential. Mr. BICKWIT. And when you have no evidence either way you conclude that it is not an imminent hazard? Dr. STEINFELD. I am tempted to make an analogy, but I probably should not. It is difficult to state that there is no evidence that a number of things are not a hazard to health. I think we are in a never-never land, and where we can, we should try to get as much good hard data as we can and act accordingly. Mr. BICKWIT. Is there any evidence either way on the accumulativoness of dioxin ? Dr. STEINFELD. I do not think there is any evidence on dioxin. This is a new area which has opened up which we will have to study intensively. Mr. BICKWIT. Thank you. Senator HART. I am not sure this will come out as an effective analogy, but think for the moment of the general attitude on pot— marijuana the prevailing view appears to be that since we cannot be sure it is not harmful, it ought not to be used. Is it not correct now that there is at least disagreement as to whether it is harmful or not? Dr. STEINFELD. I think most physicians, and I am the father of teenagers, feel that pot is harmful. Senator HART. Yon cannot be sure it is not harmful. Is not that your parental attitude ? Dr. STEINFELD. I feel it is harmful because it represents an attempt to escape from reality at a time when children must adjust to the outside world and become independent. So I find it harmful as a crutch which particularly the teenagers and those growing up must not use. Senator HART. Well, you have destroyed my analogy. I was going to pursue it on the assumption that you would agree you cannot be sure it is not harmful. You say you are darn sure it is harmful? Dr. STEINFELD. Yes, as far as teenage use, I think psychologically it is harmful. I do not think we can be sure of enzyme changes or long-term liver effects, this sort of thing. I do not think is is possible to be sure, but I would say it is harmful. Senator HART. What if you were unsure, then would you say let us go ahead, although I am not sure? Or would you say do not use it? You say with respect to the pesticides, you balance it and say since we are not sure it is harmful, go ahead? Dr. STEINFELD. I think we have some evidence in animals that 2,4,5-T is a teratogen and dioxins are present, and while we cannot be certain that women, mankind, behave similary to the mouse, yet pregnant women should not be exposed to this. This is a prudent action. , • Mr. BICKWIT. Do you know the date on which the National Cancer Institute received the first progress report raising the possible teratogenic nature of 2,4,5-T in mice ? Dr. STEINFELD. I have with me a chronology regarding 2,4,5-T. It is a few pages, but it is triple spaced. If you would like I could read it to you. 178 Senator HART. Was that a part of the insert that you presented!^ Dr. STEINFELD. We can provide it to you, and if you would like I can read it into the record. Mr. BICKWIT. We would like it for the record. Dr. STEINFELD. Maybe it would be useful to go through the chronology. With your permission, I will. Senator HART. Please. Dr. STEINFELD. In presenting the following chronology I should take a moment of the Committee's time to commend Dr. Kotin and Dr. Falk for their foresight and initiative in undertaking the studies which were conducted under their guidance by Bionetics Research Laboratories. This commendation extends also to the scientists .in, the National Cancer Institute who assumed responsibility for successful completion of the study after Drs. Kotin and Falk transferred to the National Institute of Environmental Health Sciences. It consumed large amounts of their time and energy without assurance that the investment would be rewarded. The total cost of this study approximated $3.5 million, and approximately 20,000 animals were studied. Summer 1963; The National Cancer Institute (National Institutes of Health) awarded a contract to the Bionetics Research Laboratories (Falls Church, Va.) to perform studies of the toxicology, carcinogenicity, teratogenicity, and mutagenicity of pesticides and industrial chemicals which were to be selected by scientists of the National Cancer Institute, according to protocols to be devised by the scientists of the Institute. During the fall, 1963, the chemistry and toxicology of the chemical compounds to be studied were examined and planning of the large-scale carcinogenicity screening operations was initiated. Fall and winter 1964: Large-scale screening activities in carcinogenicity were initiated and plans for teratology studies were drawn up. June 1966: First indication of possible teratogenicity of 2,4,5-T. At a dose of 113 mg/kg of body weight, 2,4,5-T, now recognized as containing substantial concentrations of dioxin impurities, produced an elevated incidence of cystic kidneys in one strain of mice. The 2,4,5-T had been administered by injection. At that point we did not know whether the results produced by injection were significant. The 2,4,5-T had not been fed. November of 1966: 2,4,5-T of a similar grade of purity administered by injection at a close of 133v./kg. body weight was found to be teratogenic in another strain of mice. The results obtained in June and November 1966, in the absence of information about rates of clearance of injected 2,4,5-T from the blood stream, were regarded as of uncertain significance. This route differs from human exposure and possible differences in metabolism could be very important. January 1968: Oral administration of 2,4,5-T of similar purity was initiated in mice. The data produced in this study indicated teratogenicity (cystic kidneys and cleft palate). May J968: Oral administration of 2,4,5-T of similar purity at a dose of 113 mg./kg. of body weight produced cleft palate in another strain of mice, itV September 1968: First draft of the final report of thedata on carcinogenicity arid teratogenicity was delivered to the N^fcial Cancer Institute by the Bionetics Research Laboratories. IK should be emphasized that these carcinogenicity data were in an incompletely analyzed state and required scrutiny for possible errors, plus numerous statistical analyses. The first evidence of teratogenicity obtained in rats fed 2,4,5-T was reported. October &£, 1968: The draft report of the "raw" data mentioned immediately above was provided to Dr. Fitzhugh in the Food and Drug Administration. October-November-December 1968: Scrutiny of the carcinogenicity data was undertaken by the National Cancer Institute scientists and report writing begun. January 30, 1969: At a meeting of scientists from the National Institutes of Health with representatives of the regulatory agencies, Consumer Protection and Environmental Health. Services, the National Academy of Sciences, and the chemical industry, attended also by Drs. Philippe Shubik and Samuel Epstein, the first two volumes of the final report of data on carcinogenicity, submitted by Bionetics Research Laboratories were made available. In addition a special preliminary report on the teratogenicity of 2,4,5-T, exclusive of data pertaining to the other teratogenicity studies, was provided to all participants in the meeting. The analyses of the carcinogenicity data had been given priority because of its volume and the apparent potential significance, based upon the indications of the raw data. It had been intended to completely analyze the teratogenicity data immediately following completion of the analysis of the carcinogenicity data. At the meeting of January 30 a number of uncertainties in the analyses of the carcinogenesis data were pointed up by Drs. Epstein and Shubik and one of the senior scientists in the National Cancer Institute. On this basis, it was decided to withhold publication of the data and findings until additional animal specimens had been examined and certain features of the study design had been reanalyzed. For the same reason, it was decided that a presentation planned for the March 1969 meeting of the Society of Toxicology would be withdrawn from the program. January-September 1969: Extensive statistical analyses of the teratology data were performed by the National Institute of Environmental Health Sciences. March 1969: In the course of the appropriations hearings, Dr. Kndicott promised to provide the results of the carcinogenicity studies to the Congressional Record just as soon as the analyses could be completed. This was accomplished in the last week of April or the first week of May 1969. June 1969: The preliminary report of the carcinogenicity findings was made in the Journal of the National Cancer Institute. June 1969: The Technical Panel on Carcinogenicity for the Secretary's Commission on Pesticides was appointed and included scientists from the National Cancer Institute and the National Institute of Environmental Health Sciences. June 1969: The intent to name a teratology panel to the Secretary's Commission on Pesticides was made known to the National 180 181 •^incer Institute liaison member of the Commission. The s ^roous offer by the Institute's liaison member of the commisslo? supply the Bionetics data on teratology was declined by a member of the staff of the Commission. July-September, 1969: Members of the staff of the National Cancer Institute and the National Institute of Environmental Health Sciences actively engaged in the work of the technical panels on carcinogenicity and terabology. Further analyses of the teratogenicity data were performed. August 15, 1969: Request made by the Teratology Panel for the Bionetics data on teratogenicity. /September 11, 1969: Data on teratogenicity provided to the Teratology Panel. Delay in part related to procedure involved in clearing permission for the data and in part related to putting the data into a condition suitable for examination by those who had not participated in their development. Fall 1969; FDA studies on embryotoxicity, and teratogenicity of 2,4,5-T and dioxins reinstituted, as described in Dr. Verrett's testiNovember 25, 1969: Meeting of Natipnal Institutes of Health scientists with those from FDA and DOAV Chemical Co. to plan further studies to clarify roles of 2,4,5-T and dioxin impurities in the production of teratological abnormalities. November and December 1969; Secretary's Commission reports published. January 1970: New teratological 'studies initiated at National Institute of Environmental Health Sciences using materials provided especially for the purpose by Dow Chemical Co. April JO, 1970: Above teratological studies completed. April 12,1970: Analysis of the above data completed. April 13 and 14, 1970: Interpretations of the above-mentioned findings by representatives of the regulatory agencies and parties to the interagency agreement for protection of the public health and the quality of the environment in relation to pesticides, and presen-tation of conclusions and proposed actions to members of the Cabinet. That is a long chronology. I am sorry. I thought it would be shorter. Senator HART. You have taken the words from me, it is a"lorig" time after that first bell was sounded before we got this morning's action. I am sure it is always easier to play it from the 20-20 vision of the grandstand up here than from the vantage point of the summer of 1966 when the first bell rang. But that is still a long time. Dr. STEINFELD. The studies were initiated at a time when this sort of thing was not ordinarily done. As we have more and more chemicals and materials put into our environment we must be more and more careful about the effects they produce. Senator HART. How can we compress the period between June of 1966 and April 15, 1970, in the future? What mechanism do you now visualize which will avoid this sort of lag from recurring? Dr. STEINFELD. If the procedures for registration of materials for use on food crops required teratogenicity studies as well as other long term chronic toxicity studies, as it is my undergfciding that they now do, we may be able to avoid this in the future. ^P The idea would be to prevent the introduction rather than react some years later, after the material was used, not only ubiquitously but in large quantities. I think this is the direction we must go, to prevent the introduction of materials rather than to react after they are used. Senator HART. Wouldn't this require the burden of proof to be on those who want a market ? Dr. STEINFELD. Yes. Senator HART. To make the affirmative case that it is not dangerous. That is correct, isn't it? Dr. STEINFELD. Yes, I think the thing we really need are good predicting systems for man. I think it would be ideal if we had some in vitro systems which would tell whether a compound is going to be toxic. This is what we need, a lot more research and correlation of animal data with human epidemiologic data. I hope we never do experiments on man but we can collect data in retrospect epklemiologically in individuals who may have been exposed to chemicals or certain diseases and so forth. Senator HART. Mr. Bickwit? Mr. BICICWIT. You obviously have done some thinking about how to patch up the system and I don't want to cry unduly over spilt milk, but do you have any idea why, when NCI received this first progress report, that it did not immediately pressure Bionetics to go into an all out effort to acquire further data quickly instead of allowing them approximately 21/2 years to complete their tests ? Dr. COURTNEY. The first statement NCI made was "Repeat the study and make sure it is right," and that is just what we did. We •went to a different strain of mouse, then we went to a rat. By the time we did all of these studies, it took a bit of time. Dr. STEINFELD. We were also studying similar chemical pesticide structures, so we could see if it was a larger problem than just this one, This was all going on at the same time. Mr. BICKWIT. Did the other pesticides that you were -studying exhibit the same kind of alarming data ? Dr. COURTNEY. I don't know how you describe it as alarming. Mr. BICICWIT. Would you not describe it as alarming? Dr. COURTNEY. Yes. We had some other pesticides that we were concerned with at the time and, of course, without repeated studies wo could not make a judgment. So some pesticides were not as alarming and some were more and as we repeated the tests we got our results. This pesticide seemed to give us a positive response every time we studied it. Dr. STEINFEU>. I would say we are not particularly pleased with the fact that it took so long to get all the data out. The first time around in one of these situations always takes longer and hopefully in the future we will be able to move much more rapidly. Senator HART. I was just thinking of all the things that have happened since that first alarm bell. We have elected two-thirds of the Senate, a new President, gotten further into Vietnam. Mr. BICICWIT. According to your chronology, if I read it correctly, the data from Bionetics were first made available to FDA on October 24-, 1968? 182 183 appeared to me it was the dioxin that was the lik|^ villain in Dr. S'fEiNFELD. Yes, the draft of the raw data was this piece, not the 2,4,5-T; the particular batch of the ^Jp-T used in >. Fitzluigh on October 24-, 1968. Hie experiments was heavily contaminated with dioxin. Our goal Mr. BICKWIT. Do you believe FDA, one of the government agon- | was to pin down the fact that it was dioxin and probably not 2,4,5-T eies responsible for the regulation of pesticides, should have known which was the teratogen and get rid of dioxins wherever they are about these preliminary indications prior to a time more than 2 found. years after the data first became available? So I think last week I would have said the same thing, Senator Dr. STEINFELP. I think in retrospect we could look at this and Hart. The data over the weekend have changed the picture comspeed everything up and inform everyone very quickly. I can't give pletely you the reasons why, (a), the information was not rapidly dissemiSenator HART. Yes; that will be made part of the record. nated as soon as it was confirmed and, (b) why things didn't move Well, then we all wind up saying it is a darn shame this past much more rapidly and on a larger scale. But I would point out weekend had to be the first time when you got the solid information, that the material used was heavily contaminated with dioxins. In which information was a result of an alarm bell that rang in June this interval we have identified the dioxins, and we are moving, 1 of 1966. think, on a broad scale to try to find out where else dioxins may be We all agree on that. found. I am not trying to look for a silver lining in a dark cloud Do you anticipate that the centralized clearinghouse which you but I do think we have a lot better data and a lot more information made reference to in your prepared testimony can assure that this as to just what did the job; it probably was the concentration of the kind of timelag no longer will occur ? dioxins used in the Bionetics experiments which was responsible for Dr. STEINFELD. I hope that that will help. Our other attempts at the teratogenicity. coordinating activities with regard to pesticides will also help. The Mr. BICKWIT. Then you do regard this as a dark cloud ? Secretary has a special commission: we have an interagency group Dr. STEINFELD. I Avould say the darkest part is that, whatever the of Agriculture, Interior, and HEW; we have Dr. Kussell Train, rules were, we permitted the utilization of the matei-ial without testEnvironmental Quality Council; I hope all of these will help us ing for what may be a significant hazard to man, teratogenicity. avoid problems such as AVO are-facing today. Senator HART. Doctor, I commented earlier on the fact that no Senator HART. 1 would ask our staff to obtain for the record the witnesses are scheduled today from the Department of Agriculture. announcement Avhich you anticipate the Department of Defense is My interest at that time bore on the action, if any, that would be about to make. You did indicate that they Avere taken to remove from retail channels and from shelves at home, perDr. STEINFELD. Idon't know if they Avill make an announcement haps, this product as a result of the announcement that you gave us If is my understanding that this is an action that Deputy Defense today. Secretary Packard has initiated this morning. Th'e Secretary of Agriculture participated with Secretary Finch Senator HART. If there is any announcement in connection with and Secretary liickel in this announcement suspending or canceling this, let it be a part of the record. I understand there is a big 2,4,5-T. I am' reminded and I must confess my own memory of this departmental request outstanding for a major purchase order for testimony is not clear, but it has been suggested to me that when 2,4,5-T. I Avould like to find out whether that contract request IIOAV witnesses speaking for the Deparment of Agriculture testified before Avill be withdraAvn in light of Deputy Defense Secretary Packard's this subcommittee last week, they took the position that the evidence position. I would assume it would. But let us make it a matter of did not warrant an action such as is taken today. record. I won't say that they promoted or advocated its use, but—Mr. Is there anything any of you would care to add, given the Bickwit, have you found any passage that bears on this? exchange we have had this morning ? Mr. BICKAVIT. Yes. Dr. STEINFELD, I Avould add one final statement. We used inbred Senator HART. From the transcript this sentence is cited. This is strains of animals and large doses of compounds in order to try to from a Department of Agriculture witness who addressed us on the find a particular phenomenon. The problem is that man is notseventh of this month. inbred; Ave don't breed brothers and sisters and so Aye can't predict. In view of all the information now available, we have not found that regisWe have a tremendous variation among people in this country; some tered use of 2,4,5-T without a finite tolerance on food crops warrants a suspenpeople may have missing enzymes of a particular type that may sion or cancellation of such registered use. make a chemical extremely hazardous at a very IOAV dose. NOAV, that testimony is April 7. You say that on April 13 the We have taken actions because we muct act prudently. We don't analysis which had been completed 2 days before were presented for want to alarm the public, but we do want to react prudently and proposed action. Whatever else you can say about it, it points up protect the public health. again the fact that on April 7, notwithstanding the patterns beginSenator HART. Amen. ning in June of 1966, indicating possible serious danger, this one Thank you very much, gentlemen. Department was still telling us, on the record, what I just read you. Dr. STEINFELD. Thank you. Dr. STEINFELO. I would have agreed with that position last week. (The information referred to earlier folloAvs:) I was surprised to see the data that developed over the weekend. It 184 185 necei The Secretary, whenever he deems it neTessary for effective administration of this Act, may require th« submission of the complete formula .of the economic poison. If it appears to the Secretary that the comREGISTRATION position of the article id such as to warrant the proposed claims for it and if the article and its labeling Sec. U.a. Every economic poison which is distriand other material required to be submitted comply with buted, sold, or offered for sale in any Territory or the requirements of section 3 of this Act, he shall the District of Columbia, or which is shipped or delivered for shipment frofli any State, Territory, or a register it. the District of Columbia to any other State, Territory,] •> c. If it does not appear to the Secretary that the or the District of Columbia, pr which is received frctt article is such as to warrant the proposed, claims for any foreign country shall be registered with, the . ,. : it or if ,the article and its labeling and other Secretary: Provided, That products which have the material required to be submitted do not comply with same formula, are manufactured by the same person, the provisions of this Act, he shall notify the applithe labeling of which contains the same claims, and cant for registration of the manner in which the the labels of which bear a designation identifying article, labeling or other material required to be the product as the same economic poison may be regissubmitted fail to comply with the Act so as to afford tered as a single economic poison; and additional the applicant for registration an opportunity to make names and labels shall be added by supplement statethe corrections necessary. If, upon receipt of such ments; the applicant for registration shall file with notice, the applicant for registration does not make the Secretary a statement including—• the corrections, the Secretary shall refuse to register the article. The Secretary, in accordance with the (1) the name and address of the registrant and the procedures specified herein, may suspend or cancel name and address of the person whose name will appear the registration of an economic poison whenever it on the label, if other than the registrant; does not appear that the article or its labeling or (2) the name of the economic poison; other material required to be submitted complies with (3) a complete copy of the labeling accompanying the provisions of this Act. Whenever, the Secretary the economic poison and a statement of all claims refuses registration of an economic poison or -deterto be made for it, including the directions for use; mines that registration of an economic poison should and be cancelled, he shall notify the applicant for regis(!»•) if requested by the Secretary, a full descriptration or the registrant of his action and the reasons tion of the tests made and the results thereof upon therefor. Whenever an application for registration which the claims are based is refused, the applicant, within thirty days after service of notice of such refusal, may file a petition requesting that the matter be referred to an advisory committee or file objections and request a public hearing in accordance with this section. A cancellation of registration shall be effective thirty days after service of the foregoing notice unless within such time the registrant (l) makes the necessary corrections; (2) files a petition requesting that the . matter be referred to an advisory committee; or (3), 187 186 ' to registration of the article and notify tn^applicant files objections and requests a public hearing. Each for registration or registrant. The applicant for regadvisory committee shall be. composed of experts, quali;> istration, or registrant, may, within sixty days from fied in the subject matter and of adequately diversified professional background selected by the National Academy ' the date of the order of the Secretary, file objections ' '._ thereto and request a public hearing thereon. In the of Sciences and shall include one or more representaevent a hearing is requested, the Secretary shall, after tives from land-grant colleges. The size of the comV due notice; hold such public hearing for the purpose of mittee shall be determined by the Secretary. Members receiving evidence relevant and material to the issues of an advisory committee shalj. receive as compensation raised by such objections. Any report, recommendations, for their services a reasonable per diem, which the underlying data, and reasons certified to the Secretary Secretary shall by rules and regulations prescribe, for by an advisory committee shell be made a part of the time actually spent in the work of the committee, and record of the hearing, if relevant and material, subject shall in addition be reimbursed for their necessary to the provisions of section ?(c) of the Administrative traveling and subsistence expenses while so serving Procedure Act (5 U.S.C. 1006(c)). The National Academy away from their places of residence, all of which costs of Sciences shall designate a member of the advisory may be assessed against the petitioner, unless the comcommittee to appear and testify at any such hearing with mittee shall recommend in favor of the petitioner or respect to the report and recommendations of such comunless the matter was referred to the advisory com: aittee upon request of the Secretary, the petitioner, or mittee by the Secretary. The members shall not be the officer conducting the hearing: Provided, That this subject to any other provisions of law regarding the shall not preclude any other member of the advisory appointment and compensation of employees of the committee from appearing and testifying at such hearing. United States. The Secretary shall furnish the committee with adequate clerical and other assistance, and ; As soon as practicable after completion of the hearing, shall by rules and regulations prescribe the procedures ;• but not later than ninety days, the Secretary shall • evaluate the data and reports before him, act upon such to be followed by the committee. The Secretary shall forthwith submit to such committee the application for ;.- objections and issue an order granting, denying, or registration of the article and all relevant data before cancelling the registration or reqxiiring modification of the claims or the labeling. Such order shall be him. The petitioner, as well as representatives of the United States Department of Agriculture, shall have the ; based only on substantial evidence of/record of such hearing, including any report, recommendations, underright to consult with the advisory committee. As soon lying data, and reason certified to the Secretary by as practicable after any such submission, but not later an advisory committee, and shall set forth detailed than sixty days thereafter, unless extended by the findings of fact upon which the order is based. In Secretary for an additional sixty days, the committee connection with consideration of any registration or shall, after independent study of the data submitted application for registration under this section, the by the Secretary and all other pertinent information Secretary may consult with any other Federal agency available to it, submit a report and recommendation to or with an advisory committee appointed as herein prothe Secretary as to the registration of the article, vided. Notwithstanding the provisions of section 3.c. together with all underlying data and a statement of (k), information relative to formulas of products the reasons or basis for the recommendations. After acquired by authority of this section may be revealed, due consideration of the views of the committee and all when necessary under this section, to an advisory comother data before him, the Secretary shall, within mittee, or to any Federal agency consulted, or at a ninety days after receipt of the report and recommendapublic hearing, or in findings of fact issued'by the tions of the advisory committee, make his determination Secretary. All data submitted to an advisory committee and issue an order, with findings of fact, with respect 45-362 O - 70 - 13 188 V V 189 support of a petition under this section shall ^F be ca»j•% «««idered on the record as a whole, includi^lany report recommendation of an adiri^ory committee. If applisidered confidential by such advisory committee: Pro is made to the court for leave to adduce addivided, That this provision shall not be construed ju , evidence, the court 1may order such additional hibiting the use of such data by the committee in con to be taken before the Secretary, and to be nection with its consultation with the petitioner or •; i;'*Wuced upon the hearing in, such manner and upon such representatives of the United States Department of Agri«| ®a and conditions as to the court may seem proper, culture, as provided for herein, and in connection with; such evidence is material and there were reasonable its report and recommendation? to the Secretary. Not- > withstanding any other provision of this section, the ••>: ^.gprounds for failure to adduce such evidence in the Secretary may, when he finds that such, action is neces- If"proceedings below. The Secretary may modify his findplifina as to the facts and order by reason of the addisary to prevent an imminent hazard to the public, by order, suspend the registration of an economic poison *•' ilional evidence so taken, and shall file with the court immediately. In such case, he shall give the registrant pfnach modified findings and order. The judgment of the prompt notice of such action and afford the registrant ; |;.wwrt affirming or setting ;aside, in whole or in part, order under this section shall be final, subject to the opportunity to have the matter submitted to an ; ^ ew by the Supreme Court of the United States upon advisory committee 'and for an expedited hearing under |«*rtiorari or certification as provided in section 125^ this section. Final orders of the Secretary under this |.of title 18 of the United States Code. The commencesection shall be subject to judicial review, in accord- : |«*nt of proceedings under this section shall not, unance with the provisions of subsection d. In no event , ||leos specifically ordered by the court to the contrary, shall registration of an article be construed as a ^operate as a stay of an order. The court shall advance defense for the commission of any offense prohibited ;0n the docket and expedite the disposition of all causes .inder section 3 of this Act, 'filed therein pursuant to this section. d. In a case of actual qontroversy as to the validity of any order under this section, any person who will be v e. Notwithstanding any other provision of this Act, ^registration is not required in the case of an economic adversely affected by such order may obtain judicial poison shipped from one plant to another plant operated review by filing in the United States court of appeals fj oy the same person and used sc-lely at such plant as a for the circuit wherein such person resides or has his | constituent part to make an economic poison which is principal place of business, or in the United States Court of Appeals for the District of Columbia Circuit, ^registered under this Act, within sixty days after the entry of such order, a f. The Secretary is authorized to cancel the regispetition praying that the order be set aside in whole tration of any economic poisofl at the end of a period or in part. A copy of the petition shall be forthwith transmitted by the clerk of the court to the Secretary, ji-; of five years following the registration of such or any officer designated by him for that purpose, and ti economic poison or at the end of any five-year period . ^thereafter, unless the registrant, prior to the expirathereupon the Secretary shall file in the court the : tion of each such five-year period, requests in accordrecord of the proceedings on- which he based his order, • ance with regulations issued by the Secretary that such as provided in section 2112 of title 28, United States registration be continued in effect. Code. Upon the filing of such petition the court shall have exclusive jurisdiction to affirm or set aside the order complained of in whole or in part. The findings of 'the Secretary with respect to questions of- fact shall be sustained if supported by substantial evidence when 190 Senator P!ART. Our next witness, as the Surgeon ready indicated, is Dr. Jacqueline Verrett. 191 . |>,,.' losses occurred after 3 weeks, and death ^fci weeks. The f>#y observed was congestion of the lungs anc^mottled livers. F fed 10 percent toxic fat in their rations lost hair on their STATEMENT OP DR. JACQUELINE VERRETT, POOD AND nnd shoulders (alopecia), and there was poor reproduction ADMINISTRATION, DEPARTMENT OP HEW lactation performance. Whelped pups were either dead or weak,, ,™ H»e mothers seemed to have an insufficient milk supply. Pups' Dr. VERRETT. Thank you, Mr. Chairman, for this opportunity w *•""—ed before weaning and fed a normal ration showed an immediscuss our investigations of the relationships between chlorinairf und dramatic increase in growth. Other litters maintained on phenoxy herbicides, chlorinated clibenzo-p-dioxins, and the chid „ ll« toxic fat ration postweaning demonstrated inferior growth peredema factors. 'fennance. p:•• Monkeys have demonstrated considerable sensitivity to toxic fat Chick edema disease was first recognized in 1957, when large mmr Materials. In one study nine monkeys received a toxic triolein at a bers of broiler flocks in the United States suffered what appeared level of 25 percent in their diets. One monkey died at 1 month, and to be an epidemic disease. The affected birds appeared droopy, will >four died at 3 months. At the 3-month period, corn oil was substiruffled feathers, and had difficulty breathing. In many flocks, more than 50 percent of the birds died as a result of the disease. Of tl*jg tuted for the toxic triolein, but the other four monkeys died from millions of birds affected, those autopsied consistently displayed* 9 Weeks to 5 weeks later, in spite of this substitution. Of the nine hydropcricardium (accumulation of fluid in the pericardial sac),'f iWHtlikeys fed the toxic triolein, eight were autopsied and showed irijrns of jaundice, pancreatic atrophy, and fibrosis, hemosiderosis, accumulation of fluid in the abdominal cavity, subcutaneous edeinfcj and additionally liver and kidney damage. \ •fatty liver with necrosis, bile duct proliferation, and gross hemorIn 1958 the investigations of a number of .laboratories indicated i , rJwgo in the intestinal tract. No such pathology was seen in the that the causal agent was contained in fats, and specifically in tin j i control -monkeys in this study. A second study with 36 monkeys given a toxic fat at levels from unsaponifiable fraction of fats in the commercial poultry rations. In; ^ laying hens the toxic fat caused a rapid drop in egg production.; fe 0.125 to 10 percent of their diet, demonstrated an inverse relationPullets receiving toxic fat during the full growing period did uol i |V' hip between the concentration of the toxic fat in the diet and their come into production, and mortality was very high. Hydropericar- *-•"•• 'mean survival time. Those given the highest level (10 percent) had ft mean survival time of only 91 days, while those given the lowest dium, the most common lesion found in young birds, was not found in birds of laying age. level (0.125 percent) had a mean survival time of 445 days. It has been estimated that the highest level provided approximately 728 The chick edema factor found in the toxic fat in the 1957 outbreak micrpgrams total chick edema factors, while the lowest level diet was presumed to have arisen as a byproduct of industrial production provided approximately 100 micrograms total intake. The toxic fat of stearic and oleic acids, since the unsaponifiable materials from this was lethal at all levels studied, and the animals were sacrificed when process were the components of fat in the poultry ration. Subpossible just before death. During the last 30 days of life, all monsequently, the toxic substance was found to be present in several Koys developed alopecia, generalized subcutaneous edema, accumuladifferent types of fats. It was demonstrated to be present in samples tion of fluid in the abdominal and thoracic cavities, and hydroperiof commercially produced oleic acids and triolein, in acidulated cardium. There were decreases in red and white blood cell counts, vegetable oils, and in inedible animal tallows. The demonstration of total serum protein values, and altered serum-protein ratios. There the presence of the chick edema factor in commercial fats led to the was also cardiac dilatation and myocardial hypertrophy and edema. ruling by the Food and Drug Administration in 1961 that higher Finally, the experimental monkeys had reduced hematopoiesis and fatty acids intended for food additive use must be free of the chick spennatogenesis, degeneration of the blood vessels, focal necrosis edema factors. The presence of the factor was to be ascertained by of the liver and gastric ulcers. a chick bioassay based on the volume of pericardial fluid in birds fed the fat under investigation. Limited experimentation with mice, pigeons, and turkeys, indicated that toxic fat in the diet led to reduction in growth without Beginning in 1958, fat that had first been proved to be toxic to hydropericardiiim or accumulation of abdominal fluid. Similarly, chicks was used by various investigators in experiments with other swine, fed toxic fat at a level of 9 percent of their ration, showed species, and was demonstrated also to produce deleterious effects in poor weight gain, but one pig sacrificed 6 weeks after the start of rats, mice, turkeys, pigeons, guinea pigs, swine, dogs, and monkeys. the study showed jno gross or microscopic lesions attributable to the Early investigations of feeding toxic fats to rats indicated that ration. they are more resistant than chicks in short term feedings, but when One important finding in the studies with chickens, is the apparent fed in sufficient dosage, extracts of the toxic fat produced definite storage of the chick edema factors in chick tissues. The unsaponideleterious effects as shown by growth depression, enlarged and fatty fiable fraction of carcasses (exclusive of intestines, head, and feet) livers, marked involution of the thymus, and enlarged adrenals. of chickens fed the toxic fat was very potent in producing hydroGuinea pigs fed 2^ percent toxic fat stopped growing at 6 weeks, pericardium in other birds when incorporated in their rations. Other and death losses occurred at 8 weeks. At a level of 4% percent the investigations of the distribution of the chick edema factors in the lias b Hfg-I;, •lis chick tissues indicated significant levels in bone, heart, 'kidney, liver, and skin. The liver contained more than 80 jj the total detected. A similar determination of the distnlmion it rats indicated the presence of chick edema factors only in liver in the feces, During the years that the previously described toxicity investigations were taking place, the toxic fats were undergoing intensiw chemical analyses to concentrate, purify, and finally determine tfrt nature of the compounds responsible for chick edema disease. At a!i steps of these procedures, the path of the toxic material was cpfi' firmed by assay in young chicks. This proved to be a time-consuniinf and difficult job because of the complexity of the fatty materials, A major breakthrough in this effort came when it was found thai t highly purified crystalline material possessing the properties of chick edema factor contained chlorine. This indicated that it was not i natural component of the fat in which it occurred. Work in several laboratories obtained similar results, and examii* tion of the purified material by a variety of analytical techniques suggested that chick edema factors could be highly substituted (chlorinated) derivatives of naphthalene, biphenyl, anthracene, or even structures common to the chlorinated pesticides of the DDT family. These latter compounds were ruled out when tested in the chick feeding assay, but some derivatives of the former classes of compounds were tested and found in some instances to be toxic, and indeed produce similar lesions to those observed with authentic toxic fat. PIpwever, none of these compounds demonstrated the high order of toxicity, or the complete chick edema syndrome when so tested, Finally, by means of single crystal X-ray crystallography, it was demonstrated that a pure compound isolated from a toxic fat wns a hexachloro-dibenzo-p-dioxin, This structure was Aerified by infrared, ultraviolet, and mass spectrometry data. Final confirmation came when this particular compound was synthesized and found to produce the same lesions in chicks as the compound isolated from the toxic fat. The finding that a chlorinated dibenzo-p-dioxin was a chick edema factor explained why different investigators had isolated materials - similar-in their capacity to elicit chick edema disease, but yet in their purest forms, had slightly different chemical properties. The large number of isomers possible (more than 60) in this family ranging from mono- to octa-chloro-dibenzo-p-dioxins illustrates the complexity of the problem. It then became a problem of determining whether some or all of these compounds are, in fact chick edema factors, and what their relative capacities in this regard might be. The chlorinated dibenzo-p-dioxin structures have been known in organic chemistry many years, and became particularly noteworthy, when in manufacturing processes with chlorophenols, their formation as byproducts posed serious occupational hazards., The most, potent in this regard seems to have been the symmetrical tetrachlorop-dioxin which was formed in the manufacture of 2,4,5-trichlorophenol. These chlorinated compounds were found to cause a serious and persistent disease referred to as chloracne. This disease was first described in, 1899. Associations of this disease with chlorinated dibenzo-p-dioxins were made by the Germans, who had several out- ittftka of this disease in their factories. There have also been similar ^ «mim';, clticc the chloracne in humans after the application of only 20 micro|; groins. The rabbit ear is especially sensitive, with concentration of g 0.001 percent to 0.005 percent producing severe reactions after local T application. This assay using the rabbit ear is apparently used as.an .'-• indicator in some plants of the content of this particular dioxin in *:• tho manufacturing process. Hence, the serious health significance of '•-•- those compounds for humans has, inadvertently, been clearly docu- ; mented. Research in Germany and Japan indicated that the magnitude of this problem was indeed large, since the formation of the chlorinated di-benzo-p-dioxins would be facilitated in the saponification procedures used in various processes involving chlorophenols. A further '•>'. complication is that a given chlorophenol preparation is generally \ contaminated with other isomers, increasing the possibility of formation of a wide spectrum of chlorinated di-benzo-p-dioxins beyond ;' those to be expected from the predominant component. Evidence that ; this does occur will be discussed shortly in connection with the chicken embryo studies of these materials. During the time the previously described investigations of the chick edema factors were underway, many of which were carried out by FDA investigators, methodology was developed for detecting the chick edema factors using sensitive gas-liquid-chromatographic (GLC) techniques. It became apparent that authentic toxic fats consistently gave peaks with specific retention times, and these peaks were used as an indication of chick edema factor in a suspect sample. Confirmation of this was obtained using the chick feeding assay. In the light of recent knowledge of the chlorodioxins as chick edema factors, it has been possible to establish that the materials being detected were hexa-, hepta-, and octa-chlorodibenzo-p-dioxins. Although toxic fat samples did indeed contain peaks corresponding to dioxins of lesser chlorine content, i.e., di-, tri-, tetra-, penta-, these are not detectable ,with this particular analytical procedure because their particular peaks are obscured by other components, A test However, in early 1969 there was another large outbreak of the including pesticide residues present in the samples. Other iliscaso in North Carolina and the toxic factor was tra^^to the fat }cedures are being developed to detect these latter dioxins. In the early 1960's the chicken embryo was being used in toxico- i| component of the feed. The toxicity was confirmed byWie chicken logical evaluations of a wide variety of materials. It was hoped to ]h embryo test with fractions of the hexa-, hepta-, and octa-chlorodevelop a rapid and sensitive screening system to pinpoint coin- j k dibenzp-p-dioxins from the crude fat. An investigation of the processing plant in which the toxic vegepounds with significant toxic and teratogenic effects for further ?p table oil products were produced revealed a proximate operation for study. In view of the demonstrated sensitivity of the chicken to chick ,Kthe manufacture of chlorophenol formulations. However, it is still edema factors, the chicken embryo was used to assay toxic fnt fe',. samples, and found to present the same syndrome as observed in the j|?, Hot possible to conclude that this accounted solely for the presence chick feeding assay. A high mortality was observed with toxic fnt |j: of dioxins in the fat, or whether they were at least to some extent in crude oil from a prior contamination. extracts, and additionally, hydropericardium, generalized and masSince that time FDA has initiated an investigation of the oils of sive edema, eye, beak, and leg defects, and necrotic livers were other manufacturers and processors and in a few cases GLC analysis apparent on gross observation. No microscopic studies have been lias indicated the presence of chloro-dioxins. These have not as yet conducted on embryos or hatched chicks in these investigations. been confirmed by chicken embryo bioassay; however, it is noteIn parallel with other investigations, the chicken embryo was used to test the toxicity of the chick edema factors isolated from toxic | worthy that in the case of these subject samples there is no known adjacent manufacturing operation that would give rise to direct fats. It was also found that the chlorinated biphenyls, naphthalenes, ; chloro-dioxin or chlorophenol contamination, so that entry of the anthracenes, and other compounds did indeed elicit a toxic response, chloro-dioxins from other sources must be considered. and in some instances, the chick edema syndrome was present. But This 1969 outbreak of chick edema disease, coupled with the quesin no case were any of these materials as potent as the toxic compotion of contamination of the herbicide 2,4,5-T by chlorinated dioxins nents isolated from toxic fats, and were generally less potent by a led us to renew this investigation. In the past 6 months the herbicides few orders of magnitude. 2,4-D and 2,4,5-T, as well as the particular tetrachlorodioxin purAfter the identification in 1966 of a hexachloro dibenzo-p-dioxiii ported to be the teratogenic agent responsible for the effects in the as a chick edema factor, studies were initiated in which various Bionetics study of 2,4,5-T have been under study. isomers of chlorinated dibcnzo-p-dioxin were prepared and tested in In an effort to assess the edema-producing capacity, the teratogenic the embryos. Although the investigation was not extensive or comactivity, and the acute toxicity of these materials, samples of the plete, it illustrated that isomers prepared by pyrolyzing selected original Bionetics 2,4,5-T, from Diamond-Alkali, were obtained for chlorophenols did give chloro-dibenzo-p-dioxins with GLC retention comparison with a sample representative of the current manufacture times duplicating those in the authentic toxic fats, and likewise, proof Dow Chemical Co. duced the chick edema syndrome in the treated embryos. It was also The Bionetics 2,4,5-T is reported to contain 27 plus or minus 8 apparent from this study that the various isomers .(that is, those parts per million of the 2,3,6,7-tetrachloro dibenzo-p-dioxin, with the with different chlorine content, and those with identical chlorine content of other dioxins unknown. The current production of Dow content, but with chlorine atoms positioned differently on the mole2,4,5-T has 0.5 parts per million of this dioxin, with no analysis for cule) varied in their toxicity, although in all cases only microgram higher chloro-dioxins reported, but does contain almost 5 percent of or less quantities were required to elicit the toxic response. It is not other impurities, mostly isomers of 2,4,-D 2,4,5-T, and chlorophenols possible to give exact figures for the toxicities obtained in this study, and chlorophenoxy compounds of undetermined structure. since most of the individual dioxins were contaminated with traces _ All investigations using the chicken embryo involved administraof others. Nevertheless, it was apparent that the symmetrical chlorotion of the compounds by injection through the air cell of the egg, dioxin prepared from 2,4,5-trichlorophenol (2,3J6,7-tetrachloro dieither preincubation or at the 4th day of incubation. benzo-p-dioxin) was more potent than any of the others tested, even A comparison of the Bionetics 2,4,5-T with the Dow 2,4,5-T indirecognizing its lack of purity. cates that with respect to the ability of the materials to produce During this investigation, samples of the chlorophenols, both techembryonic mortality, the Bionetics 2,4,5-T is more potent. The. nical and reagent grades, were examined by GLC to determine if Bionetics 2,4,5-T has an LDM—that is, kills 50 percent of the treated preformed chloro-dioxins were present. The presence of chloroembryos—of approximately 25 micrograms per egg, 0.5 parts per dioxins was demonstrated by GLC, and these materials, which can million, while the Dow 2,4,5-T LDGO is approximately 100 microbe_ removed by appropriate techniques, were then tested in the grams per egg, 2 parts per million. chicken embryo system and did indeed produce chick edema. A curWith respect to teratogenic effects, both samples produce chick rent study of similarly contaminated chlorophenols, containing from edema syndrome in the nonviable embryos, and hatched chicks, in18 ppb to 95 ppm of chloro-dioxins with six or more chlorine atoms cluding eye defects, beak defects—predominantly cleft palate—short are currently under test. and twisted feet—the result of tendon slippage—and diffuse and This investigation was not pursued further in view of the fact that localized edema in various parts of the body. there had been no known occurrences of chick edema disease since the late 1950s and, hence, such research had a low order of priority. 196 197 With both of these samples of 2,4,5-T these teratogenic o™ 'observed at levels inducing no significant embryonic mortality. The Dow 2,4,5-T still produces the chick edema syndrome at 50 micrograms, one part per million, a level where only 12 percent mortality is observed, while the Bionetics'sample has similar effect* as low as 6.25 micrograms per egg, 0.125 parts per million, a leve! inducing only 16 percent mortality. Both of these mortalities a« close to that induced by the solvent alone. It should also be emphasized that the chick edema syndrome is not observed in the embryos treated with the solvents only, at nny level, or in the control flock. A sample of 2,4,5-T from a chemical supply company was subjected to three recrystallizations before test. With the present GLC techniques no chlorodioxins are detectable in this purified sample. When tested in embryos it produced chick edema syndrome at 5, 10. and 25 parts per million, all levels which induced no more than 15 percent mortality in the embryos. This same sample was subjected to an additional purification by seven extractions to remove dioxins that might have been present, but were below the current detection levels. This repurified sample is still clearly teratogenic in the embryos since when tested at a level of 2.5 parts per million it produced 20 percent incidence of the malformations previously described, though no significant edema was seen grossly. The mortality induced was 24 percent, which is higher than that of the sample prior to the extensive extraction procedure. It is also noteworthy that the embryonic mortality occurred soon after treatment, and the hatched chicks had bleached down, indicative of an aberration in the normal pigment formation. With respect to the 2,3,6,7-tetrachlorodibenzo-p-dioxin, early investigations of this compound in a preparation containing some 2,3,7-trichlorodibenzo-p-dioxin indicate a high order of toxicity and teratogenicity. Whether prepared by pyrolysis of 2,4,5-trichlorophcnol, or direct chlorination of dibenzo-p-dioxin, the test preparations, which contained approximately 50 to 55 percent of the tetrachlorodioxin and 20 to 25 percent of the trichlorodioxin, produced significant mortality, that is greater than 20 percent, and chick edema syndrome in more than 40 percent of the treated embryos at levels of five tenmillionth^ of a milligram per egg, or 10 parts per trillion. More recent investigations, with two samples of the tetrachlorodioxin, both of purity greater than 95 percent, indicate edema and terata at 20 parts per trillion. These samples have only become available within the past month and additional testing is underway at lower and higher levels. It should also be mentioned that the herbicide 2,4-D as a commercially available sample, and a purified sample, a mixture of the K-butyl esters of 2,4-D and 2,4,5-T, and a sample of silvex, a related herbicide, have been tested. Terata and chick edema syndrome have been observed with all of these materials at levels of 10 parts per million and above. Lower levels are under investigation and the levels of dioxins in these samples are also being determined. .Studies have recently been initiated in the FDA tuaag pregnant golden hamsters, intubated on day 6 through 10 of^|anogenesis with the test compounds. The Dow 2,4,5-T, 0.5 parts per million tetrachlorodioxin, tested at 1W m./k. yielded about 80 percent fetal deaths and those pups born alive had gastrointestinal hemorrhages. The thrice-rccrystallized 2,4,5-T sample referred to earlier, with no detectable chlorodioxins, when tested at a level of 100 m./k. protlucd an average fetal mortality of 55 percent. Among 38 live pups, three abnormals were found: One with a deformed hind limb and two with inadequate fusion of the skull. At lower doses the fetal mortality was less, but still higher than that observed in control hamsters. When the extensively repurified sample of 2,4,5-T was tested in hamsters no gross terata were observed at 100 m./k., but the number of early fetal deaths was 70 percent, indicating a definite embryotoxic effect and corroborating the observation of increased mortality in the chick embryo studies. Additional tests with this compound arc underway. A dioxin preparation containing approximately 51 percent 2,3,6,7tetrachloro- and 21 percent 2,3,6 trichlorodibenzo-p-dioxin yielded 08 percent fetal deaths at 9.1 micrograms per kilogram. Gastrointestinal hemorrhages and eye anomalies—absence of lid—were present in many of the pups. • • Tests with the purer tetrachlorodibenzo-p-dioxins are underway. The numbers of animals in the hamster tests are too small to be considered statistically valid, but there are definite indications that alterations in fetal viability and gastrointestinal hemorrhages do occur at the levels tested. In summary, the chick embryo studies and additionally the preliminary hamster data indicate that the current production 2,4,5-T containing 0.5 parts per million of the 2,3,6,7-tetrachlorodibenzo-pdioxin is teratogenic and embryotoxic in these test systems. Further, an extensively purified 2,4,5-T sample, with no chlorodioxins detectable with the present techniques, has indicated significant embryotoxicity in the hamster and chick embryo, and addi-" tionally produced gross terata in the chicken embryos, making it impossible at this point in time to exonerate it of teratogenic or other adverse effects on the embryos that may have some health significance. The data for 2,4-D in chick embryos likewise demonstrate these effects in current production materials. These studies have in no way assessed another and perhaps more complicated aspect of this problem, and that is the interactions of the various chlorodioxin isomers with each other in the many combinations in which they are likely to occur, or the possible interactions, including potentiation or synergism, between the chlorodioxins and the chlorophenols, herbicides and other materials in which they are found. At this point, with your permission, Senator Hart, I would like to insert in the record the documents containing the data from which this testimony was derived. Thank you. That is the end of my prepared statement. 198 Senator HART. Yes, they will be received and placed in thej rewr after your oral testimony Dr. VERRETT. I realize I presented a rather lengthy and complicated piece. I will be happy to answer questions and elaborate il you wish, I also have some samples of embryos, if you would care to s« them, or chicks. Senator HART. Yes, I heard about them. The statement is not an easy one for one not trained in your discipline. I take it, Doctor, these are some of the chicks that have the deformities that you are talking about? Dr. VERRETT. That is right. These are chicks that have been treated with the tetrachlorodibenzo-dioxin. I would like to put them out on the table but it is not safe. As you can see, they cannot stand up. These chicks hatched early this week. They have noticeable edema. This is really the ankle, if you wish, of the chicken and this is really a result of the malformation, These are normal chicks; as you can see, they have no difficulty standing and walking. These are chicks that have been treated with the ethanol, the solvent alone or have been untreated. We always run some at the same time that have had no treatment and I think the comparison between that one and this one, if you wish, is quite obvious. Senator HART. Your last statement referred to a series of compounds. Dr. VERRETT. Yes. Senator HART. Exactly what are these ? Dr. VERRETT. It is a family of compounds which I may say very briefly is a series of phenols having a varying chlorine content. They go from dichlorophenol, which is the precursor or the compound from which 2,4-D is made, all the way up to pentachlorophenol. All of these materials are very widely used as herbicides. They also have a very broad use in industry and, in other words, are capable of being in the environment and if they are contaminated with dioxins, of also putting dioxins in the environment. What I was trying to say is that we have looked at these chloro•phenols,' apart from just the herbicides which are derived from them, and we know from past studjr, and there is a paper in the material that I have included in the record which demonstrates, that these materials are already contaminated with dioxin. The amount of the contamination at this point is Senator HART. It is very difficult to hear the witness. I do not know what the distraction is, but please be patient. Dr. VERRETT. We do know, at least by GLO (gas-liquid-chromatography) just chemical techniques—I will distinguish between chemical and biological techniques—by chemical analysis, we know these materials are contaminated with chlorodioxins. In addition to that, we know there are many' of these chlorinated' dioxins that are involved and not simply the single one which has been the subject of the 2,4,5-T. There are approximately 60 or more. 199 are We are aware from our previous work that these the point trendy in chlorophenols as they are being used. This'' I was trying to make. Senator HART'. The point I was trying to make by raising it is the nm>8sity that there be no more delay with respect to establishing the dangers, the hazards, the potentials for harm in this whole variety, this whole family of products, given the kind of damage that you |wvo so dramatically demonstrated here from one single element of ilw, environment. Dr. VERRETT. Yes. I might say—of course I could not bring very many—these are chicks treated with the tetradioxin in question, which is the most pertinent to the hearing. There are some here which have been treated with 2,4-D. Thes are 2,4,5-T treated chicks. \\'o do have underway an investigation of all the other dioxins. It in a question of having to synthesize these materials and test them in I>iiro form. But by inference at least, in our experience with the chick wlc-ma problem in the past, we know all of these dioxins are in fact toxic. We do know that. We have that information. As I said, at least in clucks, we know there is a storage and possible transmission from chickens that have been fed these materials to the human. The possibility does exist. Senator HART. By handling, as you just did? Dr. VERRETT. No, I hope not. The tetradioxin is potent. I do not know whether handling this bird will harm you, but chloracne is a very serious disease and persistent disease and there is no known cure. We are not aware that the other dioxins are as potent as that in this regard, but they have yet to be evaluated. To our knowledge the herbicides we have been discussing have not actually been tested to see if any other of the dioxins are also present. The concern, apparently, has revolved around the particular dioxin because it is extremely potent; even if the others are less potent, of course, they may still be important. Senator HART. I understood your explanation with the rats and the monkeys to indicate that this—that the dioxin in'2,4,5-T has an accumulative effect. Dr. VERRETT. The effects that are seen would indicate that. I would lie unable to say that has been proven, but the fact that, for example, in the one monkey study, when they were fed for 3 months and then the animals that did not die during that course were put on a ration free of it, still died in subsequent months, and that would indicate that either the damage had been done by the initial exposure or there was storage such that it finally did have its effect. But it was fatal in all cases. So the animal data would indicate— again we cannot apply that with certainty about the human—storage or persistence, if you wish to use that word. Senator HART. Then your attitude, which you say is not conclusive and certainly does not relate directly to humans, points to the persistence,'the cumulative character of the dioxin, rather'than in the other direction, that it is not cumulative? Dr. VERRETT. I would say it indicates it is probably cumulative. 200 Senator HAW. Do you know any experiments that point _ Ife direction that it is probably not cumulative ? "^ Dr. VERRETT. I am not aware of any, no. The human data I citw with respect to occupational exposure would also indicate that it tf probably cumulative in the human, of course. Senator HAKT. You used a word here which I take it means burning. Dr. VERKETT. Pyrolysis. Not exactly burning. In the sense I used it, it means reacting under conditions of elevated temperature, bill not the actual burning of the material itself. It indicates a high temperature reaction, perhaps, heat applied to the material in order to make the reaction take place, but not in the sense of actually igniting it. _ But it does indicate that heat, in other words, facilitates the formation of these compounds (dioxins), if that is what you are arriving at. Senator HART. What if some of this material is just put in the city dump and burned? Could that burning inadvertently produce dioxin ? Dr. VERRETT. I could only say that the likelihood is there; yes. Again, lack of actual experimentation does not give us any evidence or proof of this, but the fact is that these materials are formed when chlorophenols are subjected to heat and that would indicate to me that that is definitely a possibility. Senator HART. What common products, or what common articles contain chlorophenol ? Dr. VERRETT. There are so many that I wouldn't be able to name all. But, every piece of newspaper or paper of any kind probably has chlorophenol used in the manufacture of paper. I am sorry I am not in an area which would enable me to give you total usage figures, but they are considerable. This is washed out to some extent, but there probably are some chlorophenol residues, and paper would be one item and one which we can say is very widely used. Another example—well, leather is cured by using chlorophonols in the tanning process. So leather materials contain it. There would be any number of other eAreryday items that would possibly have it, Senator HART. Well, just as I am reluctant to have pictures taken, I am reluctant to make these contrary statement and yet this one is not inappropriate. If the materials that we customarily—and for generations, centuries I guess—have been throwing into a fire contain chlorophenol, when you burn them, it is your opinion that dioxins can result—Dr. VERRETT. Could possibly result; yes. Senator HART. Is it possible that some of the birth defects for which there has been no medical explanation to date are the result of this kind of thing, where we have always done it and it has never seemed to hurt us 1 Dr. VERRETT. I would say it is a possibility. It would be for others to assess this situation, but I think it is a distinct possibility because of the fact that these materials (chlorophenols) are ubiquitous, and if, in fact, chlorodioxins are formed in the environment, this is a possibility. 201 •'•* I should point out the studies clone in mammals ha^fcbeen done ;l'.^y feeding, while the largest exposxire may come from^Hialation or ; «f«rmdl contact. That was the source of exposure occupationally to ]; lU» tetrachlorodioxin. So here we have to be concerned not only tbrnit eating, but inhalation and perhaps contact exposure. This brings up the subject of other materials. For example, we are : »l«o investigating ,.'• Senator HART. I was just going to say that it is hard to visualize ' * jwbstitute for paper or leather, but can one have leather and paper without this material ? Dr. VERKETT. I would think so. I should point out some of the materials (chlorophenols) are washed out in the processing. The total amount used in the processing does not always remain in the products. I did not mean to imply that. That brings up another ( Duration: Are they washed out into the rivers et cetera ? :.• Nevertheless, I am not really sufficiently knowledgeable of the h technology to say whether something else could be substituted or not. ., Ibrahim, W., and Horwitz, W., JAOAC 46, 384 (1963)j^OAC (1965) xwtions 20.087—26.006. (• 11. 'The Injection of Chemicals into the Fertile Eggs Prior Wrncubation ns « Toxicity Test': Mclaughlin, J., Marliac, J. P., Verrett, M. J., Mutchler, M. K., and Fitzhugh, O. G., Tox, Appl. Pharm., S, 760 (1063). 12. 'Use of the Chicken Embryo In the Assay of Aflatoxin Toxicity': Verrett, M. J., Marliac, J. P., and McLaughlin, J., JAOAC 47, 1003 (1964). 13. 'The Role of Toxic Fat in the Production of Hydropercardium nnd Ascites in Chickens', Allen, J. R., Am. J. Vet. Res. 25, 1210 (1964). 14. 'Industrially Acquired Porphyria': Blieberg, J., AVallen, M., Brodkin, R., ami Appelbaum, I., Arch. Derm. SO, 793 (1964). 15. 'Electron Microscopic Alterations in the Liver of Chickens Fed Toxic Fat': Allen, J. R., and Carstens, TJ. A., Lab. Inves. 15, 970 (1966). 10. 'Chick Edema Factor: Some Tissue Distribution Data and Toxicologic Meets in the Rat and Chick': Campbell, T. C., and Friedman, L., Proc. Soc. Kxp. Biol. Med. 1%1, 1283 (1966). 17. 'Studies on the Metabolism of Chick Edema Factor: Distribution in Chick Tissue': Firestone, D., Higginbotham, G. R., Flick, D. F., and Ress, J., fc'DA Internal Preliminary Report, October 1966. 18. 'Light and Electron Microscopic Observations^in Macaoa mulatto, Monkeys Vcd Toxic Fat': Allen, J, R., and Carstens, L. A., Am. J. Vet. Res. 28, 1513 (1967). 19. 'Note on an Improved Cleanup Method for the Detection of Chick Edema Factor in Fats and Fatty Acids by Electron Capture Gas Chromatography': Neal, P., JAOAC 50, 1338 (1967). 20. 'Oils. Fats, and Waxes': Neal, P., JAOAC 51, 489 (1968). 21. 'Chemical and Toxicological Evaluations of Isolated and Synthetic Oliloro Derivatives of Dibenzo-p-dioxin': Higginbotham, G. R., Huang, A., Firestone, D., Verrett, J., Ress, J., and Campbell, A. D., Nature 220, 702 (1968). 22. 'Analysis of Commercial Chlorophenols for Trace Amounts of Their Condensation and Polymerization Products': Higginbotham, G. R., and Ress, ,T., FDA Internal Preliminary Report, November 1968. 23. 'The Identification and Crystal Structure of a Hydropercardium Producing Factor: 1,2,3,7,8,9-Hexachlorodibenzo-p-dioxin': Cantrell, J. S., Webb, N. C., and Mabis, A. J., Ohem. Eng. News 45, No. 5, 10 (1967), and Acta Cryst. U 25, 150 (1909). 24. ,'Clinical Picture and Etiology of Chloracne': Schulz, K. H., Arbeitsmedizln-Sozialmedizin-Arbeitshygiene 3 (2) : 25 (1968). 25. 'Report on Methodology for Chlorinated Aromatics in Fats, Oils, and Fatty Acids': Ress, J., Higginbotham, G. R., and Firestone, D., JAOAC, in press (1970). 26. Federal Register December 9, 1961, 26 F. R. 11828, 121.224; also Code of Federal Regulations, Title 21, sec. 121.1070. further amended Federal Register August 25, 1966, 31 F. R. 11215. 27. Table: Embryotoxicity of Chlorophenols, Dibenzo-p-dioxins (Chick Edema) FDA Preliminary Data, J. Verrett, 3/30/70. (Chick Embryos). 28. Table: Preliminary Report on Teratology Studies with Dioxin Using Golden Hamsters: FDA Preliminary Data, T. F. X. Collins, \V. H. Hansen, and C. H. Williams. 29. Table: Preliminary Report on Teratology Studies with 2,4,5-T Samples Using Golden Hamsters: FDA Preliminary Data, T. F. X. Collins, AV. H. Hansen, and C. H. Williams, 30. Letter: 1C D. Courtney, NIEHS to J. McLaughlin, FDA, of 12/4/09 re Bionetics sample of 2,4,5,-T. 31. Letter: G. E. Lynn, Dow Chem. Co., to M. J. Verrett, FDA, of 2/9/70 re composition of Dow 2,4,5,-T sample. 32. Memo: FDA Internal from D. Firestone to A. D. Campbell, of 3/2/70 re composition of chlorinated Dibenzo-p-dioxin Standard. 33. Memo: FDA Internal from D. Firestone to A. D. Campbell, of 3/20/70 re samples for Chicken Embryo Testing. 34. Memo: FDA Internal from J. Ress to A. D. Campbell, of 3/26/70 re chlorophenol samples for Chicken Embryo Testing. 45-362 O - 70 - 14 ir vitamin E) nor"it-Ionium had any cur^B effect. Sensitivity of different species to the toxic factor in these fats varies consideri t'lrie !•»» ably. \Vhen monkeys were fed a sample of ii- #ul^taiu'e was jircscnt in a triolpin that produced hydropericarduim in v e ? of fats. These indndcd a . . . ., ,-... i.(,Vi, ,ii ( , f i within three contained a toxic subnmahlv was present in of coiuair.i'd 25 per cent triolcin. The monkeys showed signs of jaundice, pancreatic atrophy and libro.-is, bemosiderosis, f n t t y liver f«Mrll>w;i« inriii|iuiuvv. with necrosis, and gross hemorrhage in the t * Vvcl oi 15 per cent, the, birds showed tract. w'yniptoms of hydropcricnrdium. f4im- intestinal Pigs fed a ration composed of equal parts jynploms were produced when chicks. of a highly toxic chick ration and a normal *«»? iVd rations containing distilhilcs or i-w'nip ration gained weight and appeared to %«*!'if! (ecured in commercial production show no abnormalities 'Sanger et n'.. !<>o. A fstiy ncid?. The toxic subs-lance (si was n'M. However, when a toxic fat was in•>•< frnmd in inedible animal tallows, aeicl- corporated into a swine ration at a level of <«',•>.! vegetable oils, and several com- 9 per cent, the. live shoats gained 0.72 »»T').'.!'y produced olcic acid* and irioU-in pounds per clay while the controls gained L •'Y'Mone, W, HorwitK, ]''ricdman, and G. 2 pounds (I.. C. Scott, J. Am. Vet. Med. M. flme, J. Am. Oil Chc.m. Soc. 38. .',1S .Issn. 137. ?.W U.'WO)). Despite the pom•)W\>. A report from another laboratory weight gain-, the one pig sacrificed about i«*f::mcil the presence of the. chick edema six weeks after ihc start of the <-Uidy showed f'nnr in various industrial fats (J. O. no gross or microscopic lesions attributable A'-xaiwk-r, R. J. Young, C. M. Burnett, and the ration. • II. I'l. Hnthaway, Poultry Sri. 41. S3 to The fat from the latter pig was rendered f^fjlt illlHI.t^vo "*.~ n-rt i|u,ihty" (A. YiirUoff ft til.. ./. A in. «*i,T'i. $,ie. 38. CO ( 1 / W l i i . When this THE CHICK EDEMA FACTOR i A toxic factor in tunic, teed fjmt't? tot* and /«//// uciilt product'* li;ttl<'uiifticara'iuin and asdics in'yo\\ng chicks u'hen 9 fig. per kilogram body weight ore. ft'.d per day. There are a manlier oj these toxic conii*ounds. containing large amounts aj cltlftrine. One has been diaraetcritctl and synthesized. In 1957, large numbers of chickens suf- ' cumulation of fluid in the pcncardial Ma 'fercd from what appeared to be an epidemic abdominal cavities of (he affected birds,tl* disease. Losses a t t r i b u t a b l e to this epidemic hematocrit, blood volume, and moisUlnr have been estimated in the millions of content of the heart, skeletal muscle, skin, dollars. The affected birds appeared droopy, and kidney were normal. However, the total showed ruffled feathers, and had difficulty body water content of the birds was signifibreathing' (L. Friedman, l'\-cilf:tiiljfi. March cantly increased. On the basis of these oil* 17, 1062 \. servations, it, was suggested that (.he clink In some flocks, over 50 per cent of the edema factor increaMid permeability of th( birds died with typical symptom* of the cardiac vascular bed (Flick, Douglass, mid disease (V. L. Sanger i t til,, J. . t m , Vel. Gallo, loc, oil.). A d d i t i o n a l support l'ov thi' Med. Assn. 133, 172 (JOSS)). Whi-n au- hypolhwis came from Ihe rinding that the topsied, the birds had pale hearts w i t h hy- toxic factor produced no appreciable clump 1 droperieardium, and livers that were pale, ii; the proportion or level of plasma prokin 1 mottled, and had an irregular granular sur- (Flick, D, Firestone, and .1. 1*. Marline, face. In, the advanced stages, the abdomens Poultry Kd. -14, 121.1, (IfiBi'i)). were distended, and contained 100 to 500 The type of diel had a marked effect on ml. of clear, straw-colored fluid. The pcri- the rate at which the chicks' bodies aceardial cavity is most, susceptible to fluid cumulated water. Chicks fed a natural accumulation, w i t h the abdominal cavity grain ration containing 4 per eent toxic f;it next, and then subcutaneous tissue l\">. F. showed an increased body moisture content Flick, C. D. Douglass, and L. Gallo, Paul-* (7S versus 72 per cent for the control') try Sc>. 42,.SflJ (IMS')). The kidneys were only at the end of the third week. Birds pale and swollen; the f a t t y tissue of the fed the same level of toxiu fat in a semigizzard was edematons; and the duodenum purified diet showed a marked increase in was swollen and soft (Sanger ct al, loc. body water content a f t e r seven days (70 n't,). Ilydropericardium was not as promi- versus 73 per eenl) (Flick, Douglass, anil nent in the older birds as in the broilers Gallo, he. cit.). (Friedman, loc. cit.), During 1958, a number of laboratories No acceptable explanation is available traced the disorder to the presence of a for the abnormalities associated with feed- toxic substance in the unsaponifiablc fracing the chick edema factor. Despite ac- tion of fats added to commercial poultry I'M presence of the chick edema factor •i roivniK-rcinl fats led to the ruling by the live' ration containing-the rendered lard der 9)). V'H-'l by Alexander and co-worUevs. T\\ey r/ifin. A more recent study of the response of •iVurved typical symptoms of the diMurb- different species, to the chick edema faclor :mri' only when the ration contained sodium utilized the unsaponifinblc fraction of a • 'nl'iriric; extra ^ilt accentuated the con- to'xic f a t . This was fed to young chicks and 'liiinn. Alihougli this syndrome had many nils iT. C. Campbell and Friedman. Prnc. 'it I!IP •.•amiiirk-- of a vitamin K dplicien"y, 206 Soc. K.rp. n\ol hied. .121, 1283 (HIGH)). I ' a r t o f these d i f f i c u l t i e s may he n.vnlu,! The w e a n l i n g rats fed I ho u n s a p o n i f i a b l e \ v i t l i c h a r a c t e r i s a t i o n and synthesis uf om f r a c t i o n nt a level of 9 //g. per k i l o g r a m body i'f t h e loxic. substances. By means of -in).'1' weight per day of the chick edema f a e i o r cry.-tal x-ruy c r y s t a l l o g r a p h y , .1. S. Cnln i showed a 3.7 per cent loss in weight over the X. C. U'fbb, and A. ,1. M a i n s rqm i ' six day feeding t r i a l . No gross t i s s u e altera(Abtlrficls, Aiif. ('ryxlnllorirajiltin .Iw tions were apparent on autopsy. The livers, Meelinii. 71. 37 (7.9671) t h a t the rnnipiiwi.) however, were 21 per cent heavier t h a n isolated in I heir laboratories was I .2,1! 7 . (hose of the controls when expressed on a S . 9 - h c x a e h l o r o - d i b e n x o - p - d i o \ i n . This \\ w body weight basis, while t h e adrenals were was supported by infra-red, ultra-violil, 50 per cent heavier. The heart, kidney, and and mass spectromclry p r i n c i p a l l y by ,1 C spleen were of normal sue. \Voolon and W. 11. Conrchene (,/. Ayr. I'uo'l Chicks fed a c o m p a r a b l e a m o u n t of con- Chnn. .12, 94 (19G.1,)). This struct urn < i c e n t r a t e developed h y d r o p c r i e n r d i u n i in six verified by synthesizing the compound. Tin days. The livers of these birds were 15 per l a t t e r produced the same, lesions, in chicki cent heavier than those of the c o u t r o U . The a* (he compound isolated from llie loxic l i t statement was made t h a t " t h e increase in S i g n i l i e a n t progress has. been made i l i v e r weight in the chicks was not due to i n d e i i t i f y i n g one of the l o x i c s u h s t a n f m o i s t u r e or fat." Such an observation present in some batches of f a t s i n t e n d e d iw should have been d o c u m e n t e d and cheeked' a n i m a l foods. There are s t i l l u immbci 1 ol by h i s t o l o g i c a l studies. , •' unanswered questions, the most impni'l iiil A few years ago, H. I1',, l l a r m a n and col- of these being: What is tin; possible si«n li laborators (J.- Am, Client. Soc. 82. 207S cam1!1 of these compounds, t o h u m a n h e a l t h ? (IflGO)) reported i s o l a t i o n of a c r y s t i d l i n e •Since substances w i t h physiological prnjisubstance from a feed-grade t a l l o w . This crties s i m i l a r to the chick edema f a c t o r i n compound produced h y d r o p c n c a r d i u m in be stored in the adipose (issue of chid < chicks when incorporated i n t o a commercial can t h e y also accumulate in h u m a n time ration at a level of 0.1 p.p.in, A report when i n i n j m a l amounts arc i n a d v e r t e n t l y (Yartzoff ct al., loc. tit.) indicated t h a t the ingested? crystalline substance c o n t a i n e d 47 per cent . A n o t h e r i m p o r t a n t q u e s t i o n is: What i' chlorine. t h e source of these compounds? A p p a r e n t l y , Apparently, there are a n u m b e r of com- the chick edema f a c t o r has been associated pounds t h a t behave l i k e (he c h i c k edema only w i t h f a t s and f a t t y acids subjected to f a c t o r . These compounds move closely w i t h a considerable a m o u n t of heat d u r i n g tli i t h e (o.\ie factor d u r i n g m o l r c i i l a r d i s t i l l a - processing. I f the source of t h e c h l o r i n e in t i o n , on thin layer e h r p n i a t o g r a p h y , and I lie l o x i c compound could In? i d e n t i f i e d , il show s i m i l a r peaks on gas chromato.!iraphy> might bo possible; |.o devise methods for it Xot all these compounds are toxic, and removal or e l i m i n a t i o n before m a r k e t i n g l u x i c i t y appears to vary in those t h a t arc. the f a t . 207 Reprinted .from Journal of the Association of Official Agricultural ChcmiS February, 1!10'9 Studies of the Chicken Edcifya Disease Factor* By I,. FRIEDMAN, D. FIRESTONE, W. UOIOTITZ, D. BANES, M. ANSTEAD, nnd G.8HUE (Food and Drug Administration, Washington 25, D.C.) we successfully produced the chniactcristic symptoms of the "chick edema disease'' inWhen the problem of "X" or "Edema" volved n sample of fat that had been colin poultry was brought to our attention in lected by one of Our Food and Drug inDecember of 3057, a considerable amount spectors at a feed manufacturing plunt and of work had already been done. It was es- which had been known to have produced tablished that drugs added to tho feed, as the disease, and also a sample, of a "larry" well as contiiniinnlion by drugs or heavy by-product from the manufacture of olcic metals such as lead or arsenic, was not rc- nnd stearic acid, which presumably had been n|Kinsible. Tho disease was not cruised by mixed into this fat sample to ihe extent of kiclcrial, viral, or parasitic infection, and about 40%. In this experiment HID v i t a m i n evidence was accumulating that tho only feed I?) hypothesis was tesletl. Also, tlio Miitabilingredient associated with all the various ity of the A.O.A.C. ration for this invc'-tig.'ioutbreaks was the fat. The symptoms have tion was checked against n feed. resembling been described in detail in the preliminary more closely a pructical commercial r a l i n n . report of Schmittlc, et. al. (J. Am. Yd. Mcd. Basal Nation and the Vitamin K JliipolhAsm., 132, 210 (1058)), which also summarized sonic of the work leading to the esis,—Table .1 shous: the composition of the iiicrimination of the fat added to tho ration. A.O.A.C. basal ration and the special basal ration. The major differences arc (hn subThe characteristic symptoms of this dis- stitution of Drackctt (an isolated soybean ease lire the presence of excessive, fluid in protein) for the casein of the A.O.A.C. diet, tho pcricardial sac, in the abdominal cavity nnrl the inclusion of alfalfa leaf meal ami (water belly), and less often subcutancous.ly, linseed oil meals. From the ri'.;alls shown Accompanied by high mortality beginning in Table 2, it is clear that: (/) the f a t t y approximately in the t h i r d week. The strik- field (F.A.) by-product is much more effecing resemblance of the "edema disease" tive in producing the disease thnn the. ^amsymptoms to those recorded for the exuda- ple of fat (INV. Fat), that had actually been tive diathesis syndrome of v i t a m i n 1C de- used by feed manufacturers;- (2) the special ficiency in chicks made a t t r a c t i v e the basal ration is to be preferred to the. hypothesis that the. disease outbreaks were A.O.A.C. diet, tincc. the severity of thn edema the result of an induced vitamin TO deficiency symptoms is incicascd, growth porfonnimco caused by the use of poor quality fals. . is improved, and gizzard erosion it elimiIt was convenient, because of other work nated; (;!) the feeding of vitamin K in bn;c Introduction in progress in our laboratory at the time, to use the A.O.A.C. vitamin D3 chic): test ration and (.'ingle comb white. Leghorn chicks in an attempt to produce the condition. T u h l u l . UHSU] chick r;il i n n s AOAC £|.rciltl 15 Whole yellow corn. uroinul ,r>R Id 2:> Wluvit1 lluur imiidli i>i;g 12 Cnidc pi'ccipit'ik'd njifcin 2.H Our first feeding trial with a sample of Calcium pho.^phnlc (piecipi- 2 tatiai) feed collected from one of the "disease areas" .lodiiU'l Pall, 2.01 2 was a false start, since a f t e r six weeks on Noii.irru'li.".tc(! yc:i Kt 2 H.2 0.112 0 . 02 the ration the ibidej were normal nnd showed MnSO,. 111,0 ,1 1 Coil I'lVLT oil no unuciuil symptoms «po:i poslmodem ex- ]3i;l>j mlh lard {•• X'ntnp. I.anl lnv. fat V. A. byproduct 0.35 3.8 HI. 7 f:its llurrlsan) rusiiivo* Dil'Uoiiiii lYcnpilrih)" 1 % Of UllMlp. % of U l \ M I | i , 23.0 70.11 02. ft 21.7 7.0 2.3 \ VOLATILE,IOO U C FATTY ACIDS 1.3% 88% I3grn. 880 gmUrea Fractionation i 1 UNSAP 10.7% ' I07gm. 5+ "u JO mji r/f-alpliii incnpliiM'nl per edick per diiy by month, Av. Miivivitl time, 29.2 days. dotes docs not prevent the occurrence' of the, disease, but docs seem to decrease, the severity of the symptoms. However, in an independent study by 1'. C. Underwood and C. 0. Diirbin at Ilie Bcltsville Laboratories of our Veterinary Medical Branch, w i t h Hhodo Island Reds, the oral administration of 1 ing per clay per chick of (W-alpha. locophcrol acetate had DO effect, the symptoms being slightly more pronounced in the supplemented group. The conclusion that some material associated with the "fatty acid by-product' 1 is responsible for the disease, and not n pimple v i t a m i n E deficiency, was confirmed in a subsequent test, when the fatty by-product was fed at 7% of the diet and all chicks showed marked to severe symptoms at autopsy; only 2 of 19 chicks survived the. twenty-third day, and the average survival of 17 chicks was 19.4 days, In n similar group fed 10 mg of rW-alpha tocopherol per chick per day, the average survival was 20.5 days for 15 chicks; 4 chicks survived to the twenty-third day. All showed marked to severe symptoms. BY-PRODUCT (lOOOom) f )«• Saponificalion No. NoriuM 20 12 4 free 1'alty acids c o n t a i n i n g nn unusunlly large amount of unsapouifiiible material. Furthermore, tho unsaponifiablc fraction is cumulatively different from t h a t ' f o u n d in fresh animal fat such as lard, by virtue of tho large percentage that reacts iii tho Licbcrmann-T'iurchai'd test, compared to the much smaller proportion that reacts with (ligitnnin. In the lard the proportion if almost one to one. Ure,a Fractionalion.—Tho f a t t y acids from the fatty by-product, nfter paponincation, were fractionated (J'ig. 1) with uvca into two portions: the, normally occurring f a t l y acids that form urea adducf, and modified or abnormal f a t t y acids that do not form urea adducts. About 70% of, tho f a t t y acids form urea adducts; the remainder ('<&%} comprise, the, urea, filtrate. K/Jcct of Urea Fractions on fiats wit . Chicks.—The urea, f i l t r a t e f.'ilty acid fraction was fatal to weanling rats (40-00 R 1 )'' upon oral administration. T\\o successive daily dopes of 0.4 nil resulted in marked loss of weight and death by tho fourth day. Two doses of O.'J nil caused a marked weight Joss from which recovery bepnn at the fourth day. This effect is similar to that observed in our laboratory w i t h the i.-tlty acids tli.nl do not, form urea adducts which were derived from healed cottonseed o i l , and to the observations recorded by pivmipton, ct at, (J. Nutrition, 14, ]77 (10.')!)) with heated linseed oil. Chicks react, similarly, although they RCfiin to be more resistant to tho l e t h a l effects of direct feeding than (he rats. However, not enough observations were made to establish the relative susceptibility of the two specie,". All polymon; .ind hydrop;ciin(ion products (sen Fie;, J) w c i o toxic. ADDUCT 76% (669gm.) M.W. Me. Esters: . 288 NON-ADDUCT 24% (2ll«jm) M.W. Me. Esters: 323 Molecular Disi II lotion l30°C.,7ju L....,_ POLYMERS/ 1% (2cjm.) ,,., .. MONOMERS " .1 99% 209qrn.) Ii . V V . R? 52 ' i Hydrocicnation 10% P d - C . Fig. J—Fmctionalion a\ fatly odd by-product. (Numbers fallowed by H- si<7» me le-xkil-v tee lootnate c, Table C.) Tho urea .ndrUict, fraction of the fatty acids of the liver markedly increase if., which was produced no unusual effects cither in mts or accounted for only in part by au increase in liver fat. No other signified)!!, differences in in chicks. the relative weights of organs (kidneys, K/Jcci oj Vnsaponifiitble I1'radian on Rats. thyroids, hearts, adrenals, etc.) were noted, —The significant, finding of feeding the un- although small differences caniwl be demonsaponifinbln fraction of the fatty ncid by- strated by such small groups. product to r a t s are conn in Table 4. One Although deleterious effects wi re produced ml of the un.'iapomfiublo fraction was fed to in rats both by the urea fihriU nnct by the each of f o u r rats every day for 22 days. unsaponifiablo portion, symptom.-: simihr to Control animals received no supplement to those of the "edema, disease" syndrome were I IIP. basal r a t i o n . The experimental group i.'fintainrd two inalo.s from different litter*, not observed in this species. Effect on C/ii'c/.-s.—When add;- ! to (he diet with lit lor mates in the control p.roup. The females of both the test and control groups of chicks at a level equivalent K> '<% "f the were, all from one litter. The growth rate original f a t l y by-product., the "ren l i l l i a l e was pRniMcmilly deputed— females were produced a fisznificant growth ilivvcvsion b u t , more drastically .ifi'cclcd t h a n males; (lie as Table 5 shows, very few eiT••tun difeasn H/C of thn tliyiruis decreased; and the t'm sym])tom?. Tho small amount of a c t i v i t y Tiiblo 4. Kvniilts of feeding uiitmponifinhlfg from "fully nolil ]>y-i\iro|>t. 2 2 2 2 2 4 •t 4 4 indicated in tho urea, filtrate was (shown, in occasionally we have useJ ,'ts few as im>, later experiments to reside in tho, unsaponifiwhen (he amount.of sampfc was limited. able residue that Imd not been eomplelely extra clod. It is clear that tho edema disCoiicenlrnlion of Twxlc Factor ease-producing material is associated with Alumina Chromaiography.—'Sl^ first, afp tho nnsaponifiablo portion of tho fatty by- m our attempts to isolate- the factor thai product. In Table 5 nro also shown the vari- caused (he "edema disease" from the tinations in severity of the disease produced by sapomfiablo portion of the fatty by-produc' a, series of graded doses. Although such a was chromatography on ahunina "(1:0). A t a b u l a t i o n as shown here cannot give a chromalogram . (Kig. 2 ) was obtained by complete picture of the range of observations plotting the weight of m.-Uorial eluted by encountered, it indicates how the pattern of increments of solvent, 1'cUoleum ether was symptoms seen in any one group depends ufed until the elution curve seemed to have upon the close of active material fed. In reached a minimum, then was followed by these ftudies the dose was usnnlly high 25% ethyl ether in petrol'mm ether. The enough to produce severe symptoms, and elution was completed v.i;h ]00% ethyl as we became more fnmiliar with the disease ether to give three distinct fractions of inwe became more confident of the result a ob- cnyiiig polarity, j,, „,„...,,,,_ j^,^ j tainable with smaller groups. Whenever pos- could be characterized as In diwarbon (I1C) sible we now use five chicks per gioup; Fraction If as containing carbonyl con25%ETjO IN PETR ETHER ETHYL ETHER FRIT FRJH 9.1% CO-tOH 7.6% CO la ' 54% t lib 16% 0.1 FRACTION NO. t>l 11 /•'if/. f—Ahiminn rJirmiKitaprn abltf /racliun jrtim toxic latin pounds, and Fraction III as. c o n t a i n i n g slcros. I ho ultraviolet absurj,!,-™ e])en(ra slltw (hat- I-rrtchou I has characlf ristio absorpfioi) peaks at 223, 227, 234, and M 3 m/,, suggestnig (he presence of significant amounts of cholestadiene, cither the 2,4 or 3,5 isomer or both. In anticipation of the possibility that the toxic.ty «-ould rondo in Traction I (S3"(i oM-hc u n s i i K H i i f i i i b l o i n a l L . r i t l ) a sample of d,u-choles[adie)ie was jirep/ued by \\ }, H a l in tune, to bo i n c l u / . d in tho third feeding trial. The result, of (hi, experiment indicated clearly that there was no response to choteiadicne but t h a t ,'-]| the activity responsible for (he chick e>i,,,,a disease was »> Fraction H, which c o n . ' i l u t e d apjiro.xiHialely &% of (he imsapor-.ifiable or about O.Su% of (he original of FfMlinx /'/.—Three differ- Saranle % >" Uio' 7 F.A. bj'-product F.A. by-productb + 7 • vitamin K 4.8 Urea adduct 1.6 Urea filtrate 0.70 TJnsnp. 0.50 Unsap. 0.25 Urisnr). 0.125 Unsnp. O.OC2 Unsap. No. Chicks pliowing Kdomn Symptoms Sovere Moderate M"a Dentil1 17(19.4) 2 15(20.5) 2 15 (20.2) 3 (20.3) —0 0 \ — 2 — i 5 — — 4 1 3 2 No. NorTon. Sroic* mol — on /I) 14 3 in 1U 4.9 4.7 0.2 4 . -~/ 4.0 2.4 2.0 • Flcuren in pnrcnUicscs indicate aycrniic! durvlvnl timo in dnys. i tnlHilot«l na tli<- loxirity scoro. «nt approaches were made simultaneously to Iho problem of further purifying Fraction II: ( / ) counter current distribution (iso-octane: mcthauol solvent system with 500 transfers 1 ), (%) chemical separation of carbonyl ronlnining compounds by the Girard Re*gent T, and (5) re-ohromatography on Alumina with a higher ratio of adsorbent to (ample and more gradual elution. potent, showing as much activity at a level of 4.2 mg per 100 grams of feed as had been observed with 5 grams of original product. It is of interest that the toxic fractions 5 and 6 did not react with digitonin and did not show the L.B. reaction. In preparing material for the chemical separation (Fig. f>) Fraction II was cut into two approximately equal portions; a charFor the counter-current distribution ex- acteristic yellow band was used aa the laudperiment 8.4 grams of Fraction II were mark for the separation. Each of the fracloaded into the first 10 tubes of a 500 tube tions (IIA. and IIB) was treated identically. Craig all-glass counter-current apparatus. First, after evaporation of the ether (he maAfter 500 transfers, the contents of every 10 terial was heated with methanol, (hen cooled. consecutive tubes were combined, and after When the hot methanol solution cooled, evaporation of the solvents the weight of white crystals were obtained. The infrared tolids was determined in each of the 50 spectrum for these crystals indicated a carfractions obtained, The resulting distribu- bonyl compound with a long aliph.ilic chain. tion curve, i.e., solute vs. tube numbers, is A synthetic compound, dipalmilcme (prepared by Jonas Carol), had a very similar fhnwn in Fig. 3. infrared spectrum. The 50 fractions wave, combined accordApproximately 16% of Fraction IIA was ing to the peaks indicated by the distribution curve into 10 fractions for biological insoluble in inelhanol. To the meth.inoltesting (Fig. 3). Each fraction was added soluble portion was added twice the weight to the test diet at a level equivalent to 7% of Girard Keagcnt T and to the mixture of the original fatty by-product. Figure 4 was added 10% its volume of glacial acetic chows the separation procedure diagrammati- acid. The mixture was allowed to stand for caliy, and indicates (he amount of material two hours at room temperature before the recovered in each fraction. Tho activity water-soluble derivatives were separated fcemcd to be equally distributed between from the unreactcd material ("non-kctonic"). Fraction 5 (tubes 240-320 containing 2.230 Tho "non-kotonic" portion was dissolved in grams) and Fraction 0 (tubes 321-3-1.3 con- hot acetic acid and heated with (wo times taining 0.000 grams), as indicated by the its weight of Girard Reagent T on the strain lom-itv scores. Fraction 0 was the mo.?f bath for 10 minutes, and the unrcacfcd material was separated from tho valer-t-oluWc 1 Wo K m l r f u l l y nrkiuwli-'lr!'- 1 ll«">' 'I''1'' <0 1'roclw & derivative.-'. Approximately 23% of sample Camilla Markers for fups«'tli'B « folvont syslfiii. UNSAP 10.7% 107 gm. Chromatography (1:6) I 88.2 gm. 100 200 300 Tube No. . 400 500 Fig, S—Counter-current partition ol Fraction II. Methyl alcohol ;iso-octane s-ystem, BOO lulia, SB hours. 1 Ea 4.2gm. Eb ' 3.8cjm. MeOH Soluble 3.53gm. MeOH Insoluble 0.67 gm. Girord -T Reagent (1:2) FATTY BY-PRODUCT lOOOgm. COLD KETONIC 0.8! gm. Saponification HOT KETONIC 0.28 gm. (UN) NON KETONIC 2.44 gm. 4.4* Fig. S—Further fractionaiion of Fraction II, COUNTERCURRENT DISTRIBUTION 500 Tubes MeOH ~ Iso octane I .01 5 2 3 . 4 .03 .26 67 .49 2.57 .69 .55 0.1+ 3.9* 3.8+ O.I* 8 9 10 1.13 1.97 -82gm, Fifl, //—Pfirtitinn o/ Fraction II wi/h methyl alaohol:iao-nctone coiinlar-eiirreni fyslcm. (Numbers foU'iwifd by + sign urn toxicity score.!,' sco footnote c, Table. 6.) utes. After the water-soluble derivatives had reacted in the cold and 8% more with were separated from the unrcaeted nonheat; 69% did not voxel under these con- ketonic material, two-thirds of the 11X ditions and was classified «" "non-ketomc fraction proved to have reacted. Tim non(UN). The various fractions derived from ketonic residue was again treated with exFraction IIA in Fig. 5, as well as ft similar cess Girard reagent and heat, and this time ret derived from Fraction 1IB, were also fed S0% of the "non-ketonic" material reacted. at a 7% equivalent level in the diet. 'Jhe When the original material from the Girard pynthetid dip.'dmitone was fed at a level of derivatives was regenerated and the reaction 10 mg per 100 grams. As indicated, there was repeated with excess Girard reagent, WHS no discose-prodxicing activity in any of again only 1 67% reacted to form ketonethe fractions obtained from IIB and only the ' Girard derivatives: It was clear that our "non-keloiiic" Frnclion UN was very active. active "non-kctonic" Fraction !1N consisted Siirpris'inKly, however, the infrared spectrum largely of compounds containinp; "hindered" of -Fraction UN definitely indicated the carbonyl prroiips that would react with Giranl presence of rarbonyl groups. This co-called reagent v/ith great reluctance. Of additional "noii-ketoiiitt" material was again trealC'l interest is the fact that th'>.e "hinrlci'-l with Girard Hengent T, this time with five ketone" fractions had characteristic xdli; 1 times the weight of reagent instead of two violel spectra willi a major ali-'orplion w::\times, and was heated at SO0 C for If. min- 214 215 UN NON KETONI c 2.44gm. 4.4+ Iso octane 0.41 gin. 2.2+ (3mg.% AUO-3, c. o Chromatoqraphy (l'.40) 1 2% Ether in 2% Ether in 4% Ether Iso octane Iso octane 0.27gm. I.l2qm. 0.63gm. 4.0 + Partition Chromatography I3mg.%) Silane-Treated Celite 80% EiOH 80% EtOH 0.22gm. 4,2+ 0.24 gm. (2ma,%) 5+ 1 90% EtOH 0.47 gm. 1 CHCI3 O.I9gm. -1 1 t2mg.%) 1 fig. G—Chromatography of Fraction UN. (Toxicily scores at the levels jcd appear below boxot.l iinum at .233 m /4 and a secondary maximum at 200 m/j,. The disease-producing activity of Fraction 11M \vas furthcv concentrated (Fig. 6) by chromatography, first on alumina. A ratio of one part sample to 40 parts alumina was used, and the column was chited successively with iso-octane, 2% ethyl ether in ifo-ootune, and 4% ethyl ether in iso-ocfane. The fractions were monitored by ultraviolet spoctrophotometry as they ware eluled and then were combined for feeding on the basis of spectral characteristics. The first part of (he material eluted with 2% ethyl ether in i.so-octane had a definite phenanthrene type of ultraviolet spectrum with characteristic maxima at 259 m/i and 300 m/i, and with no peak at 234 rry. The fraction immediately preceding, eluted with 100% iso-octane, did not show a phenanthrenc type of spectrum but did have a major absorption peak at 234 nifi and a smaller one at 200 m//, The phenanthrone spectrum disappeared upon further ohition with 2% ethyl ether in isooctane. Only the iso-octane and the first 2% ether fractions Khmvcd toxieity at a level of 3 mg per ]00 grams of feed. The fraction characterised by the phcnnnthrene ultraviolet spectrum' produced the more severe edema symptoms. Next, this "phcnaiithrcnc" material was further concentrated by partition chromatogrnphy on a column consisting of 5 prams of siUine-trcaled Celito plus 4 ml of isooclano. Mobile solvents were 80% ethyl alcohol saturated with iso-octane, followed by 90% ethyl alcohol saturated with isooolane. The first and second &()% alcolinl fractions were both very toxic at a level o( 2 mg per ]00 grams of feed. These two steps (Fig. 6) represent approximately nn S-fold concentration of the toxic material of Fraction UN. The first 80% alcohol cut had a typical phenaiithrcnc ultraviolet spectrum itnd no peak at 234 m/i, whereas tlio second SQ% alcohol fraction shows a major absorption maximum at 234 ny, m'additidn to the characteristic phenanthrene peaks al 250 and 300 m^. Simultaneously with the work just discussed, another.study of chroraatography on alumina was made with a higher ratio of adsorbent to sample (1001,)) snid move gradual clution (Fig. 7). We had learned thai Fraction II. obtained from the unonponififiblc fraction could be cut directly from the firrt alumina column into two parts (using (bo characteristic yellow band as a landmark) iiul that only the, first cut contained edema disease-producing material (Fig. 2). This Fraction IIA was clutcd from the second column with largo volumes of petroleum "I her (llircc arbitrary cuts), followed in succession by large volumes of 0.2%, 1%, '2%, "'id 5% and 25% ethyl ether in petroleum cilier. The seven fractions indicated in Fig. 7 were tested at a level of 3 mg per 100 grams of feed. Only Fractions 2 and 3 showed Activity. The methyl alcohol-insoluble portion from Fraction 3 showed activity. This finding did not agree with earlier experience, but njay bo explained by the larger quantities of mntorial involved in r,hese particular operations as compared to the earlier experiment. Apparently the active mater'ml has a more limited solubility in mcthanol than first supItosed. In one of our most recent experiments, the mcthanol-sohible portion of Fraction 3 from the second alumina column was subjected to partition chromatography on «!nno-treated Celite as previously described drig. 8). This time only 80% elhanol "was used as the mobile phase and the eluate was partitioned into forerun, tailings, and two major fractions, 82A and 82B, according to 4.4+ 3.0 nig % their ultraviolet spectra. Fraction Fract S2A consisted mainly of material with a plienanthvenc spectrum, and 8211 showed the major peak at 23-1 m/i. Fraction S2A was rcehromatographcd on the siliinc column, using 75% elhanol as the mobile solvtml, and again cut according to the ultraviolet spectra into a forerun, a fraction with a plicnnnlhvcne spectrum, an overlap fraction showing approximately equal absorption peaks at 2r>9 and 234 iii/i, and a fraction with a mnjor peak at 23-1- mp,, approximately 0 times as intense as at 259 m/i. All of the fractions were fed, and each of the major fractions was found capable of producing the chick edema disease when fed at a level of 0.5 mg per 100 grains' of diet with a severity approximately equal to that observed in chicks fed 5% of original fatty by-product. This is the. first instance where fractions with distinctly different absorption characteristics had approximately the same biological activity. Another recent experiment involves the reduction of one of our potent fractions with sodium borohydride. After (ha borohydride was separated, tbc treated material was 3.0mg% Fig, ?- Chrumalogrtiphy and clulion aj Pracl'wn IIA (1:100 column). MeOH Soluble 0.220 qm. Partition Chromalography on S i l a n e - t r e a t e d Ceiite ( 8 0 % EtOH} 4.4* 828 Tailings 24mg. 2.0 mg% 147mg. 82A 48mg. 1 mg, Partition Chromotoqraphy on Silane-treated C'eliie (75% EtOH) 1.2+ 0.25mg% A 6mg. 0.5mg% 0.5~mg~% p = phenonthrene Spectrum (U.V.) A=Absorplion Maximum a! 234mjj. -Keversc phase partition ch'ninalography licn:inthreiie type, producing fat was deposited in the flesh of If we were to speculate about the chemical chickens when they received it in ihcir diet. nature of tho disease-producing material This, of course, did not prove that the from the evidence available at this point, chicken meat contained tho toxic material, we would visualize a pair or a series of especially • after it was learned (Iwt the closely related compounds, with a 2-ring disease-producing activity was in Fraction II naphthalene structure, or more likely a 3: and not in Fraction I. In order (o settle ring nucleus, similar to phenanthrcne; a . this point, the unsaponifiable portion obc.'irbonyl group in a long aliphatic side chain; tained from freshly, ground chicken carcasses, not including intestines, head, or feet, and moleeulnv weights of 500-COO. was fed to chicks at levels of 1.0, 0.5, 0.25, Appearance of Poison in Flesh of Chiokcns 0.12C, 0.05, and 0.025% in the test ration. Once it had been 'established that thn As can be seen in Tfiblc 0, symptoms of nhickcn edema disease was caused by a toxic cdcma-diseaso were observed at all levels fed. principle,' tho possibility that edible chicken Tho unsaponifiable material from normal flesh might contain this toxic material had control chickens fed at 0.5% of the diet to be considered. As soon as we discovered produced no abnormal symptoms of any tiie unusual characteristics of the unsaponifi- kind. Tul>lc d. Results of feeding mi$upoiiifiiililo extract of chickens llml hncl boon fed fat. or fatty iicid by-product I'.xpt. No. 3 4 it (i ' J in Diet »cnth- 1.0 0.5 0.2", 0.125 2(13.5)5(10.0) 5010.0) 4 (21 .0) -— ' o.o:>o 0.02^ No. Oiick* Ph, 214-217 (1050). 'Drew, W B.. Dore, ,T. B., Benedict, J. H., Potter, G, C., and .Slpos, E., J. Assoc. Off, Aur. Cliem., 42, 120-128 (1050). „, _ T ' Kricdinan, L., Firestone, D., Horwitn, W., Banes, D., Anstead, M., and Shue, G., J. "w'ooten, j!'C.,leand Alexander, J. a, J. Assoc. Off. Apr. Chem., 1,2, 141-148 (1059). 'Amos, S R,, Swansea, W. J., and Harris. P. L., ''Studies on a Factor Causing I'crlcardlal Krtema in Chicks and Its Occurrence in Some Oleic Acids," Federation Proc., It. 323 (1000). STUDIES OF THE CHICK EDEMA FACTOK. II. ISOLATION OF A Toxic STJBSTANCE? The material which produces pericardial edema in chicks has been reported previously only in certain lots of fats subjected to special fat-processing operations (1, 2, 3). Therefore the finding that some U.S.P. oleic acids possessed a high degree of activity was unexpected. Feed -grade fats that contained tlio edema factor characteristically had high unsaponifiable levels ranging above 0% (2). However, in the present study, samples of the more active oloic acids had unsaponifiable levels ranging from 0.2 to 0.7%. This indicates a 10- to 30-fold concentration of the edema factor in the. unsaponifiaMe fraction and stiggests that the unsaponifiable fraction of an active oleic acid might be a starting material for isolation of the factor. Molecular distillation of a fat which contains the factor concentrated this factor in the more volatile fractions (2). Molecular distillation of an active glycerol mono-ester of oleic acid also concentrated the edema factor in the more volatile fractions, but separation from the monoglyceride fraction was not as complete as it was from the higher-distilling triglycerides. Molecular TABLE III.-COMPARISON OF PE (PERICARDIAL EDEMA) ACTIVITY OF OLEIC ACIDS AND THEIR CORRESPONDING GLYCEROL ESTERS ! Oleic Acid (from Table 1) 5 1 3 (75%) PE activity ratio PE activity score pos.ltotal percent 3/10 0/10 0/21 1/10 133) 0 133 0 33 Sample No. (from Table II) PE activity ratio pos./loM 2 6/10 3 4 5 25/31 0/11 1/14 PE activity score ! percent ', 230 ,! distillation of an active oleic acid concentrated the edema factor in the les« volatile fraction. Since fatty acids, such as oleic acid, distill at temperatures lower than the corresponding monoglycerides, the distillation range of tiic ortcma factor appears to lie above that of oleic acid and below that of the glycerol mono-ester of oleic acid. Andrew Yartzoff David Firestone, Daniel Banes, William Horwitz, Leo Friedman, and Stanley Nesheim, Bureau of Biological and Physical Sciences, Food and Drug Administration, Department of Health, Education and Welfare Washington, District of Columbia A crystalline halogen containing material producing chick edema symptoms at 0.1 part per million in the diet has been isolated from a sample of triolein which was toxic to monkeys. This material is similar to that reported by Harman et al. (4) but differs somewhat in iiltraviolet spectral properties. In a symposium on the chick edema disease in October, 1958, several laboratories (1,2,3) presented reports on their progress toward the isolation and elucidation of the toxic factor responsible for the occurrence of this unusual syndrome. It was established that the disease is caused by a toxic factor in the unsaponifiable fraction of a fatty by-product of industrial stearic and oleic acid manufacturing operations, and it was further suggested that the factor might posses a polynuclear or steroidal structure. A note, later published as an addendum to the contribution from this laboratory (2), reported that the toxic factor was associated with eVuates from alumina and "silane-treated Celite" chromatographic columns which exhibited the ultraviolet absorption spectra of polysubstituted naphthalenes (Xiuax at 236 mp, secondary XM-*wtnne in the immobile phase, and 80% alcohol saturated with iso-octane ** the mobile solvent. Vructions exhibiting maximum absorption at 245 m/i, but not at 235 mp or 2-Yi/i, were combined for two further chromatographic purifications on alumina. Merck alumnia was deactivated with 3% its weight of water, and tbe ratios •>f iilumina to sample and column size 2,000:1 in a 25 x 1-cm tube and 25,000:1 in a 25 x 2-cm. tube, respecively. Iso-octane, redistilled and chromatographed <»"or silica gel, was employed as eluent, and fractions were again monitored I')' ultraviolet spectral absorbance fractions devoid of inflection and 255 n>lt were combined and evaporated to dryness. Tbe solid residue was dissolved In a small volume of boiling iso-octane and stored over-night in the refrigerator. White crystals weighing 2.64 mg. were obtained. W) Milwtance sublimed on tbe hot stage about 239° 0. The infrared absorption Wctrmn of tbe substance, incorporated into a potassium bromide disc, showed evidence of aliphatic and aromatic linkages but no bands characteristic of oxygen or nitrogen functions. The reported presence of chlorine in the toxic substance isolated by the Merck group (5) prompted a Beilstein test, which showed the presence of Inilogen., The presence of halogen in the crystalline material being reported here was confirmed by examination in a microcoulometric gas chromatoKmph. 1 (In this instrument the sample is fractionated by gas-liquid chromatography, and, as they' are eluted from the column, the compounds are pyrol.vzed at 800° O. under oxidizing conditions. The halogen acid formed from halogenated materials is titrated in a microcoulometer.) In the chick biossay the substance, when fed at ca. 1 p.p.m. in the diet, produced severe hydropericardium, hydroperitoneum, and liver damage, with (loath occurring within 12 days. At 0.1 p.p.m. in the diet marked hydroperieardium was evident at autopsy after a three-week feeding period. The isolation of the toxic substance had been complicated by the presence of another material with an absorption maximum at 248 m/t. This second substance was isolated by the same techniques of chromatography, monitored by ultraviolet spectrophotometry, and a yield of white crystals was obtained. The ultraviolet spectrum of this material was identical with that of the toxic substance except that it was shifted 3 HIM so that the major absorption peak was at 218 m/». It behaved similarly to the toxic substance in the microcoulometric gas chromatograph, showing a similar retention time and a similar halogen content. However, it was completely inactive in the chick edema test when fed at 1 p.p.m. in the diet. In order to obtain additional crystalline material the portion remaining from nondestructive testing, fortified with the adjacent fractions from the final chromatography (of high potency, judging from the ultraviolet spectrum), was further chromatographed according to the previous isolation scheme. Small quantities of phenanthrene derivatives were separated with a consequent diminution in the ultraviolet absorption in the 250-300 m/i region. Furthermore, although there was no indication of nonhomogeneity by paper chromatography, tbe ratio of absorbances of the shoulder at 240 m/j to the peak at 245 varied from fraction to fraction, suggestion the presence of an additional component. The extinction values reported above may be in error because of inaccuracies In weighing on account of the difficulties in handling the small amount of material involved. There is no doubt however of the validity of tbe relative absorbances at various wave-lengths. The spectrum of the substance reported here differs somewhat from that reported by Harman ct al. (4), particularly in the ratio of the absorbance at the maximum (244 or 245 m/i).to the absorbance at the inflection at 238 or 240 m/n. 1 Dohrmann Manufacturing Company, Palo Alto, Calif, « DISCUSSION ^blf^ ^^^^•R.v ^^H1R ho toxic substance which we have isolated from triolein reseznbles tn^m iverod by Harman ot al. (4) from animal feed tallows. However the divergences in their properties suggest either that we are dealing with two different but closely related compounds, or that one or both of the preparations is still a mixture of related compounds despite the fact that only « single spot could be obtained on paper chromatography in a number of solvent systems. In view of the manifest difficulties involved in isolating a pure compound in minute quantities from a myriad of substances with similar properties it would be hazardous, as Harman warns, to infer chemical structure* from spectral data. Nevertheless it should be pointed out that the spectra obtained by us and by Harman et al. are strongly reminiscent of those exhibited by highly substituted naphthalenes (7). Furthermore the toxic factor occurs in association with a bewildering array of aromatic naphthalene anil phenanthrene derivatives, as we have previously noted (2). The detection of chlorine in large proportions in a toxic preparation, and the ultraA'iolet spectrum observed, suggest a possible relationship with chlorinated naphthalenes. Pontachloronaphthalene possesses an an absorption maximum at 243 m/i and a secondary maximum at 312 m/j (8), and it has been shown to cause hyperkeratosis in cattle (9) and several other species of animals including chickens (.10). Other chlorinated naphthalenes also are toxic (11). The possibility that the chick edema factor is a chlorinated napthalene derivative cannot be ignored. Samples of tetrachloronaphthalene and hexnchloronaputhalene, kindly provided by Bngel and BelU of the Virginia Polytechnic Institute, who had demonstrated that these compounds could produce hypcrkeratosis in cattle, were without effect in the chick edema test. Furthermore these compounds, despite the similarity of their ultraviolet spectra and their chromatographic behavior to the toxic substance, showed considerable difference in the microcoulometric gas chromatograph. For instance, chlorinated pesticides, aldrin and heptachlor, showed retention times of 10 min., tetrachloronaphthalene 9 min., and hexachloronaphthalene 14 min., whereas the toxic substance, as well as its inactive analogue with the absorption maximum at 248 ia.it, had retention times of 37-38 min. It is tempting to speculate that the greater retention-time of the toxic material is related to a greater molecular weight or to a substituent conferring different solubility and polarity properties. We are continuing our studies toward the isolation of the toxic factor. It is necessary that the chemical nature of this substance be elucidated to make possible a rapid chemical test for its detection, to clarify its origin, to verify the suggestion of its severe toxicity to primates, and to study its action in other species. ACKNOWLEDGMENT Numerous individuals in addition to those mentioned in the text have contributed directly or indirectly to this project. We are particularly grateful to O. TJ. Kline for his sustained interest, encouragement, and helpfulness in the course of these investigations; to Benjamin Webb, who conducted the extensive molecular distillations; to Donald F. Flick and Linda Gallo for the chick bioassays; and to Raymond .T. Gajan for the microcoulometric gas chromatography. The cooperation of the various commercial laboratories that were also working on this problem, in discussing their work with us, is also greatly appreciated. REFBIIENCES 1 W.B.,J,%,Dore, J.B., (1959), Benedict, J.H., Porter, G.C,, and Slpas, B., .T. Assoc. Offlc, Apr.Brow, Chemists, 120-128 Friedman, L., Firestone, D., Horwltz, W., Banes, D,, Anstead, M,, and Shue, G., Mel., 120-140. 3 Wooten, J.C., and Alexander, ,T.C., ibid., 141-148. •' ITarman, R.E., Davis, G.B., Ott, W.H., Brink, N.Q., and Kuehl, F.A., ,T. Am.' Chem. Soo., S3, 2078-2079 (1960). ' 5 Tlshler, M., Merck and Company, private communication, July 19, 1900. 0 S.K., Swanson, W.J., Ludwig, M.I., and Brokaw, G.Y., J. Am. Oil Chem. SI, 4(10)Ames, (1900). ' Abadlr, B..T., 7S, Cook, .T.W., and(1053). Gibson, D.T., J. Cliem. Soc., 19B3, 8 ; Mosby, W.L., J. Am. Clicm. Hoc.., 3348-3349 "DllckenntafP, U.T., and Callen, J.K., Anal. Chem., 2S, 1080-1589 (1954). "Slkcs, D., and Bridges, M.10., Science, US. GOO-507 (1052). "Kohler, H., Arclilv. Experimentelle Votcrlnarmedizln, 8, 163-198 (1954). "Bell, W.B., Vet. Med., J,S, 135-140 (1953). Reproduced by the U. S. DEPARTMENT*OF HEALTH, EDUCATION, AND Food and Drug AdminUttatlon 'ARE Collaborative Bioassay for Chick Edema Factor* By CARL D. DOUGLASS and DONALD P. FLICK (Division of. Nutrition, Food and Drug Administration, Washington 25, D.C.) A substance contained in certain processed fats and fatty products used in poultry feeds has' been implicated in the outbreak of the condition called "chick edema disease" which * Presented ns the report of the Associate Referee on Bionssay of Chick Edema Factor, Carl D. Douglass, at the Seventy-fourth Annual Meeting of the Association of Official Agricultural Chemists, Oct. 10-12, I960, at Washington, D.C. occurred in 1957 (1). A number of laboratories are actively working to isolate and identify the agent responsible for the disease (2-5) and each has developed its own bioassay, which in each case has admirably served its specific purpose. Ames and co-workers (6), early in 1960, reported this factor in oleic acid -samples destined for human consumption. The pres- 229 228 ence of this highly poisonous substance in human food products immediately resulted in a regulation from the Food and Drag Administration specifying that all such produc.ts must be, free of "chick edema factor" in order to be incorporated in food. From the point of view of the regulated industry and the regulatory agency, tho need to develop an assay method of adequate specificity and sensitivity, the results of which are comparable from laboratory to laboratory, can hardly be overemphasized. Since the chemical characterization of the substance has not proceeded far enough to provide the basis for a chemical or physical assay, it is necessary to use the bioassay. This method lias successfully proved itself capable of providing for the detection and semiquantitative estimation of small amounts of this factor. When the need for the standardization of a method became acute, investigators in eight laboratories that are currently engaged in some phase of work on the chick edema factor were invited to participate with us in a collaborative study and to submit suggested procedures. All graciously responded, and from the procedures which they forwarded, tho authors selected what they considered the most desirable features of each and com- Coll. 1 2 2 3 3 4 4 2 ^ 1 2 2 3 0 O 4 4 3 1 1 2 2 3 3 4 4 ^I 2 0/60 a MIXT. 15 14 9 8.S* 7.3 4.86 0.42 0.4 0.2 0.02 1.2 mg AMOUNT, a Folio acid (1.0% triturated in powd. glucose) 10 Biotin (0.1% triturated in powd. glucose) 7.5 Vitamin B,, (0.1% triturated in powd. glucose) 2.5 Niacin 2.0 Ca pantothennte 0.50 Thiomine 0.50 Riboflavin 0.375 . Pyridoxine-HCl 0.200 Menndione 0.025 Celluflour (Alphacel), to make 500 (c) Fat-soluble vitamin mixture.Vitamin A acetate, cryst. Vitamin Dj, cryst. o-a-Tocopheryl acetate Corn oil, to make No. oE No Early of' Pe^lS Diet 7.0 ml.—Insert sharp hypodermic needle on 10 ml syringe into intact pericardium. Aspirate as much as possible of the heart fluid (n-butyl alcohol rinse is unnecessary). Collect and measure remainder with blunt-end tuberculin syringe. Record total vol. heart fluid. (d) Other observations.—Observe and record other obvious changes such as peritoneal edema (ascites), liver changes, kidney changes, etc. Collaborator 5. , chicks of the age and w bioassay. From the observations reported by the collaborators it may be generalized that of the three anatomical sites in which fluid may accumulate, hydropericardium occurs with the greatest frequency, followed in order by peritoneal and subcutaneous accumulation. This is in agreement with earlier observations. The appended charts (Fig. 1) show graphically the response of the volume of peri- 21 Days 14 Days Score 0 + ++ 14 Days 21 Days 14 Days 21 Days 14 Days • Collaborator &. 14 Days Collaborator J, ! . 21 Days Collaborator 7. 21 Days Collaborator 9. 1 14 Days 21 Days 21 Days *Svsn i _ Seven of the nine cooperating laboratories submitted results in time for inclusion in this report. Their observations are compiled in Table 1 It is seen that mortality due to the toxic factor does not become appreciable until the third week on the diet containing the highest level of the toxic fat. Note that three of the in the Uw value /alls at (be high end of the normal ttnge of the volumes of pericardial fluid for • _ ft • , t . rfa R&3 bfel 1 2 3 —*-i K$fl B88 4 1 Diets fig. 1- 2 3 1liVi' A- 1 2 3 4 1 2 3 4 Diets •Volume of pericardial fluid (— S,E. of the mean) versus level of toxic fat for 14 and 21 day feeding periods. 232 —*—••• Coll, 9 No. Away No. of Period of Early Diet (Day.) Chick. Deathg 233 Table 1. (Continued) .."i Average Weight Gain Total (g) i Average Hydroperioaj-dium Sub- Hydracuta- peHwBS ""WJ-" £ A«oite« neous diua- 3 3 4 4 14 21 14 21 12 12 12 12 0 2 1 5 45.1 ± 2.8 13 + 0.203 ± .086 6 + 72.5 ± 8.3 28 + 0.878 ± .339 17+ 48.3 ± 5.4 28 + 0.903 ± .303 20 + 75.1 ± 6.4 26 + 3.44 ± 1.40 19 + 1 1 2 2 3 3 4 4 14 21 14 21 14 21 14 21 12 12 12 12 12 12 12 12 0 1 0 0 0 54.6 ± 3.3 0 0.09 ± .01 - 0 104.4 ± 9.6 0 0.10 ±.015 57.3 ± 5.6 1 + 0.15 ±.07 108.0 ± 7,1 |10 + 0.29 ± ,05 10 + 63.3 ± 3.6 4 + 0.19 ± .045 5 + 104.0 ± 11.8 36 + 2.86 ± .55 42 + 60.4 ± 4.8- 21 + 0.84 ± .24 22 + 89.6 ± 6 . 7 35 + 4.52 ±.86 34 + 1 0 5 Edema Incidence sJ 1/12 7/12 8/12 6/7 4/15 8/1 J 8/13 7/7 0/12 0/12 0/12 0/12 0/12 0/12 0/12 0/12 0/12 0/12 9/12 5/12 6/12 2/12 11/11 9/11 0/12 0/12 1/12 0/12 4/12 12/12 11/12 11/11 1/12 4/12 5/12 6/7 • Hydroperioardium incidence: baaed on measurement of pericardia! fluid. collaborators reported deaths of four birds Visual subjective scoring of the 'degree of on the control diet. Since these death's could not have been due to the factor, this and the hydropericardium according to the instruc-foregoing observation indicate that mortality tions given in the collaborative procedure is measurement' alone is not a reliable index of seen to agree fairly well with ft score calcuthe presence of the chick edema factor. It has lated from the actual measurement of peribeen found that signs of chick edema disease cardia! fluid as shown in Table 2. The aro tlie development of hydropericardium, figures given in Table 1 represent the sum hydropcritoncum, and subcutaneous edema, of the plus scores for individual chicks within in that order, as the-dose of toxic material is the group. It appears that there was greater increased. At higher dosage levels, death accuracy in visual scoring of large toxicity will occur from the 10th day. Birds dying responses than in the smaller responses. of chick edema disease invariably exhibit While visual scoring may be adequate in these signs. Deaths unaccompanied by any judging the presence of a large amount of of these signs cannot be attributed to the toxic material, it is inadequate in those borderline cases where controversy is most likely toxic agent. to develop. Weight gains of the chicks were somewhat We have arbitrarily adopted 0.2 ml as the depressed at the higher levels of toxic fat. In the control groups, the average weight upper limit of normal heart-sac fluid volume. gains at the end of the assay periods were This choice is justified on the basis of the quite variable'among the laboratories. This work of Shue and Gallo (7), who report that variability may be due to borderline nutritional inadequacies of the basal diet that possibly wore aggravated by hereditary or environmental factors or a combination of both, as may have happened in the case of Collaborator 7's group. Perhaps it would have been wise to follow the suggestion of certain of the collaborators and to have an antibiotic incoiporated in the test ration. Table 2. Scoring by Referee of pericardia! eclcrna <0.20 0.21-0.40 0.41-1,00 1.01-2.00 >2.01 0 + ++ ++ + + ++ + (Hud to the level of toxic fat in the <***• It is seen to vary directly with this fc«l- II is likewise seen that an appreciably t»**lfr response to the dose is obtained */'*f ft three-week feeding period than after » Cwo-weck period. Although there is a high evidence of negative responses in a group «f cliicks on a low level of toxic fat, the ivrage response of the group as measured by the volume-of pericardia! fluid is proporiKmnl to the dose level within the dose limits o( this experiment. Comments of Collaborators of sodium chloride was li^He the subject of question on the basis that 2% of salt is too high and subjects the chick to extra "stress." It has been found in our laboratory that this level favors the development of the edema. Selye and Stone (8) have found that extra sodium chloride in the diet of chicks accentuates the development of edema produced by certain steroids. Blester and Schwartz (9) state that 6-8 grama of sodium chloride per day is not harmful to 9 week-old chicks. We cannot see that a change here is justified. One collaborator reported that the ration was unpalatable to his chicks. The weight gains reported by him were much lower than in any other laboratory. We have no explanation for this effect. It is suggested that this laboratory may have some peculiar problem and that the performance of these chicks is not typical. Several of the collaborators preferred to use heavy breeds of chicks rather than leghorns. We have retained leghorns for the reasons of uniformity of response, widespread availability, convenience, and the demonstrated sensitivity of this breed. Most of the comments were directed at tlw nutritional adequacy of the basal ration. One investigator has suggested that dry, itnhilizod vitamins A, D, and E be used wthcr than the crystalline products used in the procedure, This suggestion appears to be meritorious and has been incorporated in the recommended procedure as optional. Another objected to the' mode of addition of rholine to the diet and suggested a 25% dry, free-flowing preparation which is commercially available. For the sake of convenience, this has likewise been'incorporated as optional. Objection was made by another eolSummary and Recommendation* lnlx>rator to adding the water-soluble vitaA successful collaborative study of a promins on Cclluflour as the carrier. Since many cedure for the detection and assay of the laboratories routinely use other of the dietary chick edema factor in fats and fatty mateingredients as carriers for the vitamin mix- . rials has been carried out. The results inditure, \ve see no reason why any of the major cate that the method as studied is satisfaccomponents cannot be used for this purpose. tory for the intended purpose. Although it Weight gains obtained on the diet are not is demonstrated here that the method studied optimal. This, however, is not critica-1 from is capable of differentiating contaminated the standpoint of the assay except, as one from uncontaminated fats, it would be decollaborator points out, that it is easier to sirable to carry out additional collaborative carry out the procedure of withdrawing the studies on samples of lower potencies than heart-sac fluid from a larger bird than from those used here. ft smaller one. Sinco i hr diet has given excelIt is recommended1— lent pericardia! -liu'id responses, major (1) That the method for bioassay of chick changes cannot be justified at this time. edema factor, presented in this report, be Other collaborators have suggested that adopted as first action. an antibiotic be incorporated in the ration, (2) That collaborative studies be conthat crude casein be substituted for the vita- tinued. min-free casein, and that commercially availREFERENCES able salt and vitamin mixtures be used. These (1) Editor's Note, This Journal, 42, 120 (1959). appear to be desirable from the standpoints 1 of economy and convenience and will form These repommedations were approved by General Kcfercc and by Subcommittee C, the basis of a modification of the diet to be tile and were adopted by the Association. See This used in further collaborative work. The level Journal, 44, 70 (1BG1). 235 > Brew, W. B., Core, J, B., Benedict, J. H., Potter, G. C., and Sipos, E., ibid.,- 42, 120 (1950). (3) Friedman, L., Firestone, D., Horwitz, W., Banes, D,, Anstead, M., and Shue, G., ibid,, 42, 129 (1959). (4) Wooten, J. C., and Alexander, J. C., ibid,, 42, 141 (1959). (5) Harman, R. E., Davis, G. E., Ott, W. H,, Brink, N. G., and Kuehl, F. A., J, Am. Chem.Soc., 82,2078 (1960). (6) Ames, S. R., Swanson, W. J., Ludwig, M. I., and Bookaw, G. Y., J. Am, Oil Chemists' Soc., 37, 10 (1960). (7) Shue, G. M., and Gallo, L., This Journal, 44, 456 (1961). (8) Selye, H., and Stone, H., Proc. Soc. Exptl, Biol. and Med., 52, 190 (1943). (9) Blester, H. E., and Schwartz, L. H., Diseases of Poultry, 4th Ed., The Iowa State University Press, Ames, Iowa, p. 112. PKOGBESS IN THK CHICK EDEMA PROBLEM Collaborators By Dr. Leo Friedman, Food and Drug Administnmon ;^TO>I J. C. Alexander, The Procter & Gamble Co,, Research Division, Cincinnati 39r Ohio Stanley It. Ames, Biochemistry Department, Distillation Products Industries, Rochester 3, N.Y, Carl D. Douglass, Food and Drug Administration, Washington 25, D.C. 0. F. Hixon, Laboratory of Vitamin Technology, Inc., 7737 S, Chicago Ave., Chicago 19, 111. Walter H. Ott, Merck Institute for Therapeutic Research, Runway, N.J. C. E. Poling, Swift & Co., Union Stock Yards, Chicago 9, 111. H. C. Sehaefer, General Research & Control Laboratories, Ralston Purina Co., St. Louis 2, Mo. It 1ms been almost four years since I first became aware of the problem that.we know today as "chick edema disease." Because of our activity on this problem, my colleagues and I have had the opportunity to become acquainted with Many scientific groups and individuals working in the same area with whom we have enjoyed a fruitful cooperation and pleasurable association. We hope that they feel as kindly toward us as we do to them, but sometimes, I known, they wish as we do that they had never heard of chick edema disease. This problem has been most difficult and progress frustratingly slow. Despite tlie meager amount of new information that can be added at this time, especially since the most recent advances were reported l>y Dr. Artman recently, it is nevertheless worthwhile to revie-\y the several aspects of this problem and see its present status in full perspective. As you recall, during 1957 an epidemic disease caused millions of dollars in losses among broiler flocks throughout a large part of the U.S. After elimination in succession of all other possibilities, attention was focused on the fat ingredient of the feed as the etiologic agent. A series of reports in 1958 from several laboratories described the manifestations of the disease and definitely implicated a toxic fat or a toxic substance in fat as the cause. The characteristic symptoms were droopiness, ruffled feathers, labored breathing and high morbidity and mortality. Autopsy findings revealed hydropericardium, abdominal ascites (water belly), subcutaneous edema, swollen liver, swollen and paie kidneys, etc. In laying hens the toxic fat caused a rapid drop in egg production. Pullets receiving toxic fat during the full growing period did not come into production, and mortality was very high. Hydropericardium, the most common lesion found in young birds, was not found in birds of laying age. SYMPTOMS MFFEBENT These differences in susceptibility and symptoms in different age groups of the same species should be noted. The feeding of toxic fat to other species has not produced such striking results as with young chicks, with the exception possibly of monkeys. However, every species that has been tested has shown evidence of deleterious effects. Very little work has been done with rats. Our very limited experience indicates that they are much more resistant than chicks in short-term feedings, but that when fed in sufficient dosage, extracts of the toxic fat produce definite deleterious effects as shown by growth depression, enlarged and fatty livers, and marked involution of the thyinus. I recall few reports of the effects of toxic fat. on swine, but again, in our own limited experience we have seen depressed growth . . . and have demonstrated the presence of toxic factors in the meat of .hogs that had been fed toxic fat. I am indebted to Dr. AVilcke of Ralston Purina for reports of studies on guinea pigs and dogs. Guinea pigs fed 2i/£% toxic fat stopped growing after six weeks, and death losses occurred after eight weeks. At a level of 4%% toxic fat weight Josses occurred after three weeks and deaths after four weeks. Control groups receiving non-toxic fats did not show weight loss or deaths. The only observed pathology at the conclusion of the experiment was congestion of the lungs and mottled livers. In experiments with three different breeds of dogs, using Purina Dog Chow in which 10% of toxic fat was substituted for the usual normal fat, there was poor reproduction and lactation performance. The females on the toxic fat ration whelped pups that were dead or weak, and, furthermore, the mothers seemed to have an insufficient milk supply. When the pups were removed before weaning and fed a normal ration the increase in growth was immediate and dramatic. Also, the females on the toxic fat ration tended to lose hair on their backs and shoulders. With the ration containing toxic fat, post-weaning growth tests (6-18 weeks) with five litters of pups demonstrated inferior growth performance using either weight gain or increases in body length as the criterion. WORK WITH OTHER SPECIES In these experiments with other species, fat that had first been proved- to be toxic to chicks had been used. In the case of monkeys, a sample of trioleln 45-362 0—70- -10 236 had produced irreversible toxic symptoms for no apparent reason 1; _ was proved toxic to chicks and was the source from which we isolated a highly purified crystalline "chick edema" factor. At the present time, then, "toxic fat." that produces "chick edema disease" lias been demonstrated also to !>«>• duco deleterious effects in rats, guinea pigs, swine, dogs and monkeys. It should be emphasized that the toxic fat undoubtedly contains many other substance* that may have effects in these other species. Definitive information as to tlie effect of "chick edema factor" (OBF) in other species must await tests vritli purified CEF. Triolcin-fed monkeys probably received the "purest" source of OEF. However, purified OBF should be administered to monkeys to verify tlic implication that the severe toxic symptoms observed with triolein were due to CEF. Relatively little lias been done to throw light on the mechanism of the toxic effect. Flick and Gallo in our laboratories have reported that in young chick." showing symptoms of the disease, the intra-cellular water was not, changed. Neither were the total blood and plasma volumes altered so that the observed edema was primarily interstitial. Hemoglobin and hematocrit levels were low and Wood glucose levels were decreased in advanced stages of the disease. Plasma sodium, potassium and chloride were not nffected. In the liver, neutral fat was decreased and phospholipid increased. Preliminary experiments by Flick give some indication of increased membrane permeability, but these must be repeated under much more rigorous conditions. DISSERTATION ABSTRACT A most interesting study in chicks has been reported by .T. It. Alien, Jr. Part of the results were presented at the federation meetings in March, 1001, and the complete study is available as the dissertation of ,T. It. Allen, Jr., University of Wisconsin, 1961. I will quote portions from the dissertation abstract [Dissertation Abstracts 22. [2], 645 (Aug., 1901)] : "Experiments conducted to determine the effects of 'toxic fat' on mice, pigeons and turkeys demonstrated a reduction in growth without hydropericardium or ascites." In chicks, "Microscopic examination of the tissues of the test animals revealed lymphocytic foci in the epicardium and myocardium. Jfldomatous fluid separated the myocardial fibers. Edema of the lungs was a frequent observe-' tion in the experimental birds. ". . . Blood pressure indicated the test birds had an elevated average mean pressure in the right ventricle of 6 cm. water and 2 cm. water in the vena «iva. Electron micrographs of the myocardium revealed shrunken, vacuolated mitochondria in the test animals. " 'Toxic fat' produces a reduction in growth rate of experimental animals. Tliis reduction depends on the age of the animal and the level of 'toxic fat' added to the diet. Hydropericardium and ascites are a frequent lesion in the animals receiving from 1.0 to 5.0% 'toxic fat." When this level was reduced to 0.25% in the diet, reduced testicular development was a more sensitive criterion than hydropericardium, ascites or weight gain for evaluating chronic toxicity. "The mechanism by which 'toxic fat' induces hydropericardium and ascites appears to be associated with degeneration and edema of the niyocnrdial fibers. These data would tend to eliminate the kidneys, liver and endocrinos as the primary cause of edema. The early development of hydropericardium, increased venous pressure, enlarged hearts, mitochondrial changes in the myocardium and generalized edema suggest that the myocardium may be directly inhibited; however, altered capillary permeability lias not been excluded. It is believed that cardiac decompensation and increased capillary permeability act together in producing the excessive extravascular fluid collection- and the demise of the animal." The wide range of susceptibility within and among species and the variety of toxic effects that have already been noted would make it appear logical that some primary unit of structure and function such as the mitochondrion, inny bo the target of Hie toxic factor and that the observed differences may be explained by factors such as absorption, specific binding, transport, detoxication, etc,, that determine the local concentration of any substance in n specific site. 237 Another possible mechanism that had suggested itself quite earlj^Bthat the toxic factor interferes with the normal regulation of electrolyte^mcl water tifUaiice. Selye and Stone, back in 1043, described the production of edema symptoms in chicks by certain steroids and the accentuation of these symptoms I)}' increasing the salt intake of the chicks. Alexander has shown that it is l"isnible to produce hydropericardium in chicks by increasing the NaCt in the ration and to prevent its occurrence even with OEF by eliminating NaCl from t)m diet. AVork in our own and other laboratories has illustrated beautifully flu> interaction of nutritional factors on the susceptibility of the chicks to a 1'ixic agent. For example, the present AOAO bioassay diet for "chick edema factor" is probably four times more sensitive than the assay diet we used originally, although both have approximately the same NaOl content. A point to remember is that hydropericardium as a symptom of toxicity in clucks is not new. In addition to NaCl and certain steroid hormones, chapter '10 in Blester and'Schwarte on Poisons and Toxins indicates that: (1) Zinc phosphide, used as a rodenticide produces "various degrees of congestion with the accumulation of some serous fluid in the pericardial sac as well as in the abdominal cavity in some cases." (2) Alpha naphthyl thiourea, the rodenticide ANTU, shows in poisoned chicks evidence of lung edema and excessive quantity of fluid in the pericardial BUC.(3) Sodium monofiuoroacetate, compound 1080, another very effective rodenticlde, produces in chicks distention of the pericardial sac with clear strawcolored fluid, in addition to other marked pathological changes on the heart and(4)lungs. Chlordane. "The primary lesions found in all fatal cases were in the heart. Excessive quantities of fluid were found in the pericardial sacs. . . ." The weed "corn cockle" and several species of Crotolaria produce seeds which are toxic, and in chicks the toxic symptoms include hydropericardium. However, in each case other characteristic pathology is usually present, and In no case is the purified toxic principle of the same high order of activity as the toxic substances that have been isolated from toxic fats. It is well known now that chicks can efficiently utilize large amounts of fat in properly balanced rations. The use of fat as a standard ingredient of poultry feeds grew as the price of fat calories dropped and became competitive with calories derived from corn. The chick edema disease epidemic of 1957 was a totally unexpected consequence of this growing practice. Many of you are familiar with the story of how the toxicity was associated with a fatty byproduct of stearic and oleic acid manufacture that had been blended with feed grade fats. Very large quantities of fatty acids are used industrially in the manufacture of lubricants, rubber, paints, asphalts, roofing, chemicals, and to a much smaller extent in foods. Relatively low grades of fat are split into fatty acids and glycerol at high temperatures and pressures, sometimes with the aid of catalysts. The glycerol is recovered and the fatty acids are distilled under vacuum. The first distillate may be used directly as the highest grade of mixed fatty acids or it may be. separated by a low temperature crystallization process into stearic (saturated) and oleic (unsaturated) fractions. The residue from this distillation is resplit and redistilled. The second distillate yields a lower grade of fatty acids. The residue from the second distillation is usually suitable for use on highways or in rubber manufacturing, hut occasionally it is again recycled to obtain a third distillate and another residue. It was this third residue that bad been blended with feed grade fat for use in feeds. Every sample of residue of this type from several manufacturers of fatty acids proved to be rich in chick edema toxicity. Our first impression, therefore, was that the toxic factor was produced during the splitting and distillation steps and that it was concentrated in the residue along with other non-volatile unsaponifiable substances. Closer study of the various stages of fatty acid production soon revealed that the toxic factor was distillable and was present to some extent in the first distillates which were used for the production of the best grades of fatty acids, and those intended for food purposes. This discovery was made independently and reported by Ames, et al., who had found several samples of oleic acid and a monoglyceride made from such an oleic acid to be contaminated. All tills happened just after the passage of the Food Additives Amendment. The fatty acid manufacturing firms were very cooperative in providing us « 238 i information and samples. However, they sincerely believed that tb e not part of the food business, that the bulk of their production wentf non-food industrial purposes. In our visits and talks with their technical we advised them to study the application of the new legislation to their industry. They realized that a substantial proportion of their highest grade of materials did enter food channels when their customers started asking for guarantees that the fatty acids met the requirements of the Federal Food and Drug I/aw, At that point the technical committee of the Fatty Acid Producers Council became actively engaged in a study of this problem, and we have enjoyed the whole-hearted cooperation of the fatty acid industry. While they made studies to determine what part of their processes were responsible for the production of the toxic material, studies were continuing on the isolation and chemical characterization of the active substances. Three years ago at the AOAC meeting a symposium on chick edema was held at which reports from the Purina laboratories, the Quaker Oats laboratory, the Procter & Gamble research laboratories and the Food and Drug laboratories described the progress made up to that time. The Merck group had recently entered the problem, and, although they did not report, they also had traveled a similar road. Every step and experiment had to be followed by the chick bioassay. However, during the first year all the groups bad made steady progress at about the same rate. First it was demonstrated that the "chick edema" toxicity was entirely in the unsaponiflable portion of the fat. Then, in our work, the unsaponifiable was separated by cbromatography on alumina into three fractions of increasing polarity by elution with petroleum ether alone, then with a mixture of ethyl and petroleum ether and finally with 100% ethyl ether. The first, or hydrocarbon, fraction contained 83% of the unsaponiflable, and its ultra-violet absorption spectrum indicated the presence of cholestadiene. The second fraction was characterized as ketonic and infrared absorption spectra suggested the presence of dipalmitone. The third fraction consisted of sterols and oxidized materials. Simultaneous bioassay of these three fractions and synthetically prepared samples of cholestadiene and dipalmitone showed that only fraction 2, but not the dipalmitone, was toxic. Several approaches were tried simultaneously to purify fraction 2. I will not take the time to describe our experiments with a 500 tube counter-current distribution between iso-octane and mothanol, or the separation of carbonyls with Girard-T reagent or the consecutive rechromatography on alumina and Rilane treated Celite. Suffice it to say that as our fractions became more potent we relied more and more on ultra-violet spectrophotometry as an indication of concentration and purity. Two distinct types of fractions were obtained, one with the ultra-violet spectra characteristic of naphthalene compounds and the other of phenanthrene compounds. Both fractions contained the toxic factor. However, the sum of their toxicities was not comparable'to that of the starting material in the final purification steps despite the fact that practically all of it was accounted for by weight in the eluates. Since only the fractions that had a significant weight had been tested biologically, the insignificant, hardly visible residue of less than 0.5 mg. in the practically "empty" beaker that represented the cut between.the two major fractions was rinsed into a chick diet and to our surprise was very active at approximately 0.1 ppm., representing the most potent material we bad obtained. At this stage we had practically exhausted our raw material, and we had to start from the beginning once again. During the course of these studies we had learned that Drs. Portman and Andrus iiv the department of nutrition at the Harvard School of Public Health had lost a number of monkeys in a nutritional study. They had used a synthetic triolein as the source of fat and had to abandon the experiment. At a Gordon Research Conference I had the opportunity to discuss this experience with Dr. Portman. Fortunately about 40 Ib, of the triolein was still available. It proved to be toxic in the chick edema assay. The triolein was of excellent quality, containing only 0.9% of unsaponiflable material. This unsaponiflable proved to be the richest source of the toxic material we had ever examined. The. toxic fraction was separated from the triglyceride by molecular distillation. The distillate from 17.0 Kg. of triolein was saponified, and the unsaponiflable was chromatographed on alumina to remove the cholostadiene fraction. The naphthalene and phenanthrene-containing cuts were collected, yielding 670 rag. of material. This fraction was rechronmtogrriphed on more retentive alumina with 5% ether in petroleum ether, and 240 mg. of naphthalone-phenanthrene material was separated. This was rechroinatojjaphed in the name system and three distinct types of U.V. spectra began tc^Bprge in the fractions: Naphthalene (235 m/j,), phenanthrene (260 m/j) andlPnew peak at 245 m^i. These materials were combined and chromatographed on a silnne treated Celite-iso-octane cohimn, with 80% alcohol saturated with iso-octane as the mobile phase. The cuts with maximum absorbence at 245 m/i were further purified by two more chroma tojrrnphic treatments on alumina at a very high sample: adsorbent ratio (1:2,009 and 1:25,000). The final fraction was completely free of absorption peaks near 235 and 200 m/i. This fraction was evaporated to dryness, dissolved in a small volume of boiling iso-octane und stored overnight in the refrigerator. White crystals were obtained, weighing 2.6 mg. and representing a concentration over the original triolein of three million-fold. This material at 1 ppm. in. the diet killed the chicks in 12 days and produced typical hydropericardium when fed at .05 ppm. in a 21-day test. During the last stages of this work the Merck group [Harmon, et a)., ,TACS 82, 2078 (1060)], announced the isolation of a crystalline chick edema factor from a toxic fat. On July 19, 1960. the Merck group informed us that they had found 47% chlorine in their crystalline material. The presence of chlorine in our material was quickly confirmed by the use of the Dohrmann microcoulometric gas chromatograph. In this test, a few micrograms of sample is injected into a gas chromatograph and the components as they emerge are pyrolyzed at 800° under oxidizing conditions. The halogen acid formed from halogenated materials is titrated automatically in a microcoulometer. Of interest also is the isolation of a crystalline material that had an ultraviolet absorption spectrum identical with that of the toxic material but shifted 8 mju so that the major peak was at 248 mp. instead of 245 m/t. It behaved like the toxic substance, in the microcoulometric gas chromatograph, showing a similar retention time and halogen content. However, it was completely inactive in the chick edema test when fed at 1 ppm. in the diet. The finding of chlorine in the toxic substance was a major breakthrough. It was no longer necessary to think in terms of a naturally occurring material that bad changed chemically under the conditions of industrial fatty acid production. The possibility of contamination with one of many familiar chlorinated hydrocarbons was obvious. Samples of tetracbloronaphthalene and hexachloronaphtbalene, kindly provided by Rngel and Bell of Virginia Polytechnic Institute, who had demonstrated that these compounds produced liyperkeratosis in cattle, were without effect in the chick edema test. We have tested a long list of chlorinated compounds including aldrin, dieldrin, lindane, DDT, DDE, BHC, chlordane, toxapliene, methoxychlor, and a series of Halowaxes, without any definitely positive indication. Furthermore these have all been heated with oleic acid at 250° C for long periods and then fed to chicks, with negative results. Also, on the theory that tallow may sometimes be bleached with active chlorine materials which may chlorinate a sterol nucleus, we have chlorinated cholesterol, squalene, estone and equilenin with negative results. To test the theory that the toxic substance is a metabolite of a chlorinated insecticide we have fed large doses of chlordane, methoxychlor, beptachlor, aldrin and dieldrin to rats for a month and are feeding the unsaponiflable extract of the rat carcass in the chick test. The results to date show that chlordane and methoxychlor produce no response. INDUSTRIAL EXPERIMENTS Similar experiments have been carried out and are still in progress by at least two industrial laboratories under conditions of fatty acid production. The results so far have been largely negative or at best only suggestive but not clear cut. Dr. Artman has reported that chlorination of naphthalene and phenanthrene by substitution reactions has produced CISF active products. The chlorinated phenanthrene is particularly promising, since preliminary fractionation experiments indicated the possibility that a highly toxic compound was produced. Dr. Boyd O'Dell and colleagues at the University of Missouri observed hydropericardium and other symptoms of chick edema disease in chicks housed in freshly painted cages. They traced the responsible agent down to one of the paint ingredients, a chlorinated biphenyl sold under the name 241 240 BIBLIOGBAPHY ft •Llol•!i of different chlorine content. Some were not toxic; others prod .isease but only at relatively high feeding levels, e.g., 200 ppm. It may that an impurity in these compounds is the toxic agent, or that they Innately toxic at the high levels fed. Of interest is the use of one of tlu>« Arochlors in some insecticide formulations. At the present time there has been no real evidence developed to implicate any product or compound. However, the circumstantial evidence is stimulating considerable speculation and activity. The feed industry has managed by careful control of ingredients to nvoW a recurrence of the 1957 epidemic. The color test developed by Brew, qt al., «l Purina has been very useful in screening out toxic fats. This test is not specific J but is useful since the presence of large amount of steroidal compounds thflt1 give this test usually indicates still residue that may be toxic. It is useless , J however, for fatty acids and other products such as triolein, monogylceride*, etc., that give no response in this test, but which sometimes are quite toxic. GEORGIA OUTBMCAK Last year, at this time, an outbreak of the chick edema disease occurred In Georgia. Although considerable chlordane residues were found in the feed, vrp do not believe they were responsible for the symptoms observed. Furthermore, the best information we have indicates that only rendered fat was used In this feed and no product of the fatty acid industry was involved. With cadi now development the scope of the problem increases. First we were concerned only with still residues, then also with fatty acid distillates and their derivatives. For a long time it was felt that only the fat derived from animals was involved. Recent evidence from sources in the fatty acid industry and our own studies indicates that some Vegetable fat sources may yield fatty acids contaminated with CEF. Still another development has occurred to further complicate the picture. ICarly this year in the course of our regulatory activities, we examined n sample of oleic acid. All the test chicks died by the end of the second week, with symptoms of severely stunted growth, ascites, jaundice and pathology of the liver and other organs, but with no hydropericardium. This sample lifl'l been tested by the manufacturer last year before the adoption of the present AOAO test procedure. The testing laboratory had used the procedure we ourselves had used in our earlier work, and had found the sample negative for chick edema. Repeat of the test in both laboratories by both procedures confinned both findings, The sample is free of chick edema disease factor when tested on a diet of natural ingredients, and the chicks survive in apparent good health. On the casein-sucrose diet of the AOAC chick edema test, tlie chicks fail to grow and die early with the described symptoms but no hydropericardium. Furthermore, the dose response curve for this effect is very steep, since the ratio of the dose that gives a maximum effect to the dose that produces a minimum effect is less than two, as compared to a ratio of four to five Cor chick edema factor. Evidence from preliminary fractionation studies also indicates that this is an entirely different substance. There is no other information as to its characteristics at this time. There is evidence that this toxic contamination has occurred in different places from time to time and in a variety of fatty acid samples and may occasionally occur together with C1W. Here again we must anticipate that this material may occur elsewhere independently of the fatty acid industry. For whatever comfort we may derive, it should be noted that chick edema disease has been observed in England. In a letter to the editor of the Veterinary Record of June 10, 1961, C. C. Wannop of the Houghton Poultry Research Station draws attention to a condition apparently identical with that reported by Sangcr, et al., and Schmittle, et al., in 1058, that has appeared in several broiler flocks. In a personal note dated Sept. 29, he says that the condition has disappeared for the time in his country, At the present time fatty acids can be used in the manufacture of foods or food ingredients only if they are free from CEF. This requirement made necessary the development of a bioassay which has been accomplished by collaborative work and is now being adopted ns official by the AOAC. I have tried to review the history of this troublesome problem, what little is known of its toxicology and its physiological aspects, the speculations as to the origin of the contamination, and attempts to track down its sources, 1 have sketched quickly our own attempts at isolation and identification of the toxic factor and have alluded to the most recent developments along this line that were reported at the AOAC Section of Fats and Oils by Dr. Artman. Chick "Edema" Disease 1. Sangor, V. I et ai. • ., 133, 172 (1058), " ^ Sci., 37, 1300 (1958), "Effect of a tox-c sub- , "Effect of certain toxic performance, embryon.c in fat on r development and health of djlckcji* Disease Besearch . r e m , s . chicken edema disease factor . Center, Athens, ^^ 42, 14i (1959), Some tot status " Manufacturers Ass,. d iRHues special report on edema hi chicken ^ in animal Asys^^'^s^ -* Meat Insti tute ****** toFoundation a tease." r 3 et al._pouury Science, 38, 579 (1959), "Relationship of oxlLMegVatton to . toxicityin ce^}n JateJ ^ (1M9)i ,.studies on toxic 1Q T^iiilflllOO "VV. &-t Gt ^' J, ULUH.J - ^ fat in rations of laying hens and pullet^ Science A Au^usf f^Tne S IOiacn , N.V, V AprU, 1000 ^ 2Q78 (1960). "The isolation products derived therefrom/' , Brink, N. O., and Kuehl, F. . - . . e Ohom. Soc., 38, 418-422 (1001). 20. Douglass, C., and KlicK, u. L : SS»ifciS!J Horwlte, W., —"<»' offlc _ Agri. chemists, 44 (3), , C. D, and Friedman, ,-, A e s Rex, ^^ 24. Woot'ton, ,T. C. Alexander, J O , 2 fed a chlorinated hydrocarbon a n d Friedman, I*- Qffic Iderstine, A.—Poultry Sci, 40, the edema producing factor m tox, fat." COMPOUNDS BY H. MAY'20, 1901 INTRODUCTION, QENEBAIa 242 243 an chemical works, the acne persisting over a long period and being ass with other effects on the health. Diseases in a group of 17 workers from a company in North Rhine/Westphalia were reported by BAADI3R and BAUER, as well as by BRINKMANN In 3O.TO/S.l. The workers in that company were engaged in the production of pentachlorophenol. Apart from comedoue acne with various degrees of secondary pustular infection and boils, most of the workers, whilst still in the flrst stages of the skin diseases, also experienced pain and weakness in tlic lower limbs, mild paraesthesia, heart complaints and indeterminate psychovegetative disturbances. Subsequent examination of the records of 17 cases' revealed the following findings : All 17 were suffering from an acno, 4 of these being very severe, 8 fairly severe, and 5 moderately severe to mild. In almost all cases there were extensive pustular infections and boils, 4 with bursitis on the elbow. Other disturbances amongst the workers included 11 cases of bronchitis, 5 of myocardinc damage, 2 of cirrhosis of the liver (one of which proved fatal), 0 of neuritis symptoms (severe pains in the lower extremities in 7 patients, sensibility disturbances in 4 cases, mild paresis without atrophy in 2 patients, and 2 cases of weakening of the Achilles' reflex). Seven \yorkers complained of physical conditions such as continuous fatigue, depression, lack of vitality, nervousness, slight headaches, disturbed sleep, and decrease in libido ami potency. A larger number (about 60 cases, Prof. Hergt) of similar conditions occurred in two Mid-Rhenish companies amongst workers who had been engaged for long periods, generally several years, in the production of trichlorophenol (snponiflcation of 1,2,4,5,-tetrachlorobenzene to 2,4,5-trichlorophenol by treatment with methanolic caustic soda solution). These trichlorophenol workers, like those in a third group of affected persons from the Hamburg region who will subsequently be dealt with in more detail, suffered from further disturbances to health, these often not occurring until a fairly long time after occupational exposure had ceased. In the course of a discussion on a paper by SP1EGELBERG, who referred briefly to our Hamburg cases in a lecture at the 1960 North-AVest German Neurologists' and Psychiatrists' Congress in Luueberg on psychopathological delayed and chronic damage following occupational intoxication. Janzarik described largely identical disturbances amongst workers from the Mid-Rhenish companies. The third group comprised 31 workers in a Hamburg company. Those of this group who were affected were engaged in the trichlorophenol department of the company, in which the herbicide 2,4,5,-trichlorophenoxyacetic acid was manufactured from technical 2,4,5-trichlorophenol by heating trichlorophenol together with caustic soda solution and monochloroacetic .acid in autoclaves. After completion of this esteriflcation process, the end product was purified by double recrystallization. The task of the workers consisted first of all in charging the autoclaves, for which purpose the trichlorophenol in flake form had to be removed by shovel from open barrels. In this operation, a fine dust formed and dispersed throughout the room. Other operations were concerned with filling and controlling centrifuges and regulating feed and outlet pipes. Since it was the workers most exposed to contact with trichlorophenol who suffered from the severest skin conditions, it was logical from the outset to suspect the causal noxa to be present in the trichlorophenol. The extent to which this assumption was valid is discussed later in this paper in connexion with etilogy. head, temples, chin and ears, after which folliculitis, pustules, boiisJ^Bl retention cysts occurred as a result of secondary infections. As the dnHlse processed, these symptoms spread in the majority of patients, especially to the "UK's of the neck, back of the neck, upper half of the back, chest, forearms, Kcnitala and thighs. Numerous boils formed, particularly on the back of the neck and on the back. The efflorescences were generally located so closely together that scarcely any follicles remained unchanged, In certain workers who had apparently been more strongly exposed, the development of these acne-like symptoms preceded a dermatitis associated with erythema and swelling, this extending to the region of the eyes, the cheeks find the forehead. At about the same time, blepharoconjunctivitis occurred in several patients, this, like the skin symptoms, becoming chronic in some cases. As the table of findings shows, spots or, in certain cases, patches of pigmentation occurred in the faces of some patients, these giving the skin a dirty, Kreyish-brown appearance. The overall clinical picture was identical to the symptoms occurring after working with chlorinated naphthalenes, diphenylenes and other aromatics as flrst described by HERXHEIMER (1899) and subsequently by several other authors (BETTMANN, HOLTZMANN, TELEKY, HERZBERG, BRAUN, GRIMMER, etc.). (For further details, see W. BRAUN and A. RISSESUNDKRMANN (I960)). Although not a completely exact description, the designations "chloracne" and "perna disease" have become the most popular for these forms of occupational intoxication. SOME CLINICAL OBSERVATIONS Of the 31 workers of this Hamburg company, 9 are still receiving medical attention 5 years after the termination of occupational exposure, this being due to residues of their acne, chronic neuromuscular weakness of the leg musculation, vaso-vegetative lability and, most especially, marked psychopathological disturbances. Details of the established complaints and damage to health are given in the table. The development of the skin conditions in the patients followed, by and large, a uniform pattern. Numerous comedones formed, first on the face, especially on the cheeks above the malar bones, fore1 Unr t h a n k n arc duo to the BcrufnuonosHcnsehaft dor Chcmlschen Industrie for jiliirliiK tlx-lr record* ntul other documents at our disposal, and also for their underxlniulhiii In the »omctlmc« lengthy clinical examinations. TABLE OF FINDINGS Skin and mucous»membranes: Dermatitis of the face in initial stage; comedones, retention cysts, nodules, pustules, boils; patches of pigmentation; blepharoconjunctivitis. Internal organs: Loss of appetite; abdominal complaints; loss of weight; reduction in general condition; altered acidity of the gastric juice; gastritis; damage to liver; pulmonary emphysema, dyspnea; myocardiac damage; blood pressure; edema; pathological urine finding (renal damage). Nervous system.—Neurological: Muscular pains; weakness in legs; (general) fatigue; increased sleep requirements; parestbesia; headaches; attacks of giddiness; orthostatic collapse tendency; paresis (implicit) ; coordination disturbances ; hypaesthesia; reflex irregularities; vegetative hyperexcitability; EEG findingJ; EMG finding.8 Nervous system.—Psychopathological: Decrease in initiative and interests; hyperaesthetic-emotional traits; pronounced fluctuations in intensity; disturbances in memory and concentration; disturbances in libido and potency; alcohol intolerance; depressions; decrease in impulsion; affective disturbances In the restricted sense of the term; experimental weakness in mental capacity; organic Rohrschach psychogram; individual neurotic traits. , n. = normal, aim. = abnormal, p. = pathological, n.s.p. = not definitely pathological, v.mU. = premature fatigue in the electromyographic series stimulus test. The course of the dermatological manifestations proved to be extremely obstinate in our cases. The therapeutic measures employed (drainage of the comedones, external keratolytic and antibacterial measures, as well as the Internal administration of antibiotics in severe cases) could not prevent the reformation of comedones, retention cysts and boils in the first year, or two, although there was no further contact with the causal noxae. Only after a long time did the tendency to relapses cease. A residual condition now to be found, particularly amongst the serious cases, is closely arranged pitted scars which have a disfiguring effect, especially where localization occurs in the face (pseudo-atrophodermia vermiculata). All affected workers reported pronounced fatigue and weakness in the legs, often with pain, especially in the region of the proximal leg musculation. These conditions were marked, even in the early stage of the disease, and in some cases even before the development of skin changes Paraesthesia was reported in the records or in spontaneous information in only 2 of'9 cases. 2 Our thanks nre due to Do/,. Dr. BOCHNIK and Dr. BTTSCHART for conducting theolcctro-encephalographlc investigations, and to Dr. PUFF and Dr. RUEDAS for the clectro-niyographic Investigations. -, ft; Implicit paresis or atrophy, weakening of the reflexes, or absence | expansion reflexes as a sign of toxic polyneuropathy was not esta~ any of the cases. Two of the patients examined indicated a decrease bility with isolated epicritical disturbances in the lower limbs. No deilnlt* signs of neurogenic damage that could have been expected with peripheral nerve lesions were established electro-myographically; premature fntlpt* which was recorded in the series stimulus test, requires further confirmntN regarding both the method and the raised findings. As these findings shnu, the neuromuscular disturbances do not fit in with the typical picture of toxk polyneuritis or polyneuropathy. The electroencephalographic examination produced an abnormal electroencephalogram in 6 cases, with frequency lability and dysrhythmie groups of * partly asymmetrical character. In one of the patients examined, aceentimtc) dysrhythmia and raised cerebral excitability were revealed after photostlinnli 1 • tion, The electroencephalographic changes found were uncharacteristic (tini afforded no diagnostic viewpoints of any real consequence. Some of the workers examined complained of headaches, attacks of gidclinw* and orthostatic collapse tendency. In 5 of the 9 patients examined, there were distinct signs of vegetative hyperexcitability, fine tremor of the hands, increased perspiration on the hands and legs, axillary perspiration, raised dermagraphism and suggestions of Chvostek's sign. The blood pressure value menuured during out-patient check-ups were all in the normal region, though «' the lower limit, of the norm in 5 of the 9 patients examined. Orthostatic collapse tendency was not established either during out-patient visits or diii'lnK in-patient observation by an internist. In 2 cases, myocardiac damage wfl* suspected. Abdominal complaints such as a feeling of fullness, pressure in the stomach and liver region, and slight pain, were reported by 5 of the 9 patients. There were 4 reports of disturbances in the gastric secretions, 3 of subacidity, one of hyperexcitability and one radiographic finding of gastritis. Very thorough investigations were conducted as part of repeated out-patient examinations and in-patient observation to ascertain any liver damage.'1 Whilst the liability reactions were uncharacteristic in all cases, the bromphthalein test indicated slight delay in the dyestuff excretion in 2 instances. In 3 eases, the liver biopsy produced pathological findings, these comprising 2 cases of slight perihepatitic changes and in one case a fatty liver with inflammatory symptoms and slight fibrosis of the liver. Owing to the clinical and histologic.nl findings, it was suspected that a condition following virus hepatitis existed in this instance. Deposits of ferrous and non-ferrous yellow-brown pigment were established in this case, although these did not correspond to the grey, non-ferrous pigment discovered by KALK and WI1VDHIRT in chlorophenol intoxication, The excretion of erythrocytes in the urine of one worker whose renal findings wore otherwise normal remained unaccounted for. The psychopathological changes in the chlorophenol workers who were nil psychiatrically and psychopathologically examined were especially remarkable. In 6 cases the course could be observed over a 2-year period and there \vns an opportunity for objective anamnesis investigation and experimental psychological examinations. 4 With a very large degree of agreement, n subjective syndrome of complaints was reported by the patients under investigation, this syndrome extending from the psychoneuropathic complaints in the region of the extremities, cardiovascular and abdominal symptoms to the mental/ spiritual sphere, especially in the modes of behaviour associated with the vital forces (BiiRG'ER-PRINZ). Considered in detail, there were reports of disturbances in the vital senses such as general .sense of weakness, feeling of fatigue, indisposition, sense of insecurity, inner restlessness and a feeling of illness. The basic mental mood was reported to be deteriorated and lowered towards behaviour characterized by dissatisfaction or sullenness and irritation. Not infrequently, a mood component of fear and unease was present. Changes in affectivity in the restricted sense of the term were reported by the patients .in the form of increased emotional reactions, irritability, tendency to fits of temper and also a certain hebetude. •Trot Dr. HORNBOSTEL and Dr. SCHONFELDEH, I. Mod. Univ.-KHnlk. HamburgKppcmlorf. 4 Our thanks arc dun nt this point to Dip], Psychologist W. von SCHUBERT, for conducting the tests (Rorschnch poychogram and Hamburg-Wcclislcr intelligence tent for nclulta). General loss of strength and reduced inner vitality and impulsion were *?ni[itoms noted in each of the cases observed. The probands d^feU)ed reduc6!f fluctuation occurring in favour of early morning, late morning or evening liuurs. in many probands, there were intervals of some days or weeks in which they were practically free from complaints; For completeness' sake, a further two less common phenomena are described. Two probands stated that the general symptoms and the polyneuroimthic symptoms were relieved for a varying length of time by the use of colrt media (cold showers and washing with cold water). One proband reiwted abnormal, constantly changing eating habits, such as a wish for nothing tint black bread, milk soup, or three litres of milk daily, there being no desire tor food of other types at these times. Compared with the multifarious polysymptomatic sxibjective pictures, the objective psychopathological signs can be recorded at less length. In exploratory conversations, the majority of the probands displayed a distinct, slightly depressed and subdued mood, which could be brightened only slightly or not nt all. So far as impulsion was concerned, the patients examined gave an impression of lif elessness ; their psychomotivity was feeble and fatigued. The Impression was rather one of slight cerebral organic impulsion reduction than »f inhibition. The affective modes of behaviour were occasionally notable for their reduced reactivity and oscillation capacity, though also because of lability nml decompensability. In 2 oases, pronounced hypochondria and, in one case, slight but distinct alienation of the total personality were recorded. The psychological tests are significant for the discovery of finer intellectual performance shortcomings and psycho-organic disturbances in affectivity. In Hie majority of the probands tested by the Hamburg-Wechsler intelligence tost for adults (HAW1E), there was a significantly raised percentage of deKcneration, this providing a certain indication of an acquired decrease in mental capacity. In the Rorschach psychogram, coartation of the experiential typo, signs of weakened emotional reactivity, poor concentration, reduction In tempo, sluggishness of the mental processes and a tendency to perservation point to cerebro-organically governed changes. DISCUSSION On the basis of the findings in three independent groups of chlorophenol workers, which together included more than 100 affected by diseases, a characteristic clinical picture is provided, the most important features of this being the following disturbances : 1. Following initial dermatitis of the face and symptoms of irritability on the part of the conjunctiva ; often together with gradually developing acne primarily in the region of the face, then the back of the neck, shoulders and upper trunk, and in severe cases on the entire body, with comedones, pustules, boils and patches of pigmentation. In several cases with severe irritation of the mucous membranes of the face and the upper respiratory tract ; sometimes with continuing blepharoconjnnctivitis, 2. In several cases, disturbances connected with the internal organs, especially damage to the liver, with deposits of a nonferrous pigment as a 246 .1 characteristic biopsy finding. In some cases, chronic bronchitis instances of inyocardiac' damage. 3. Ill all cases, general fatigue and weakness principally affecting the pro* iraal muscles of the lower limbs, often with pain in the muscnlation and IB some cases paraesthesia and slight hypaesthesia. In isolated cases only, more pronounced disturbances of sensitivity, slight paresis (implicit) and weakenim of reflexes. 4. A psychovegetative syndrome with the following disturbances: Subjective: Disturbances of the vital senses, disturbances in the basic mental mood nwl affectivity, disturbance in impulsion, weakness of memory and concentration, hyperaesthetic traits, vegetative dysregulation, tendency to orthostasis, slwp disturbances, much increased sleep requirements, disturbances of the instind sphere, reduction in libido and potency, and alcohol intolerance. Objective,—psychopathological: Reduction in impulsion, subdepressive trail' of u type characterized by genuine vital moments of depression, i disturbance* in affectivity in the sense of a certain levelling-out, increased' excitability: occasionally hebetude, hypochondria and personality alienation. Experimental psychological, HAWIE: Increased degeneration percentage; Rorschach psychogram: Coartation of the experiential type, signs of weakened emotional reactivity, weakness of concentration, reduction in tempo, sluggishness of mental processes, tendency to perseveration. The dermatological picture of the chlorophenol intoxication described shown extensive agreement with the disease caused i>y chlorinated aromatic hydrocarbons as first described by HERXHEIMER and later by several authors (see BRAUN, RISSE-SUNDERMANN). On the basis of the observation* that chlorinated naphthalenes were principally responsible, \VAUER, ami later TBLEKY, suggested the designation "perna disease" (.WJTZchlorinatcd A r 4phthalene). TELEKY pointed out that the chloracne already described by HERXflEIMER in 1890 was produced not by pure chlorine but by chlorinated hydrocarbons or the simultaneous action of chlorine and tar. Further observations on perna disease made by MITTELSTADT, FLINN and .TARW1K, DRINKER and collaborators, and GREENBURG and collaborators, indicated that not only the skin symptoms but also fatigue, loss of appetite, giddiness, and severe liver damage with acute yellow atrophy of the liver leading to death can result from work with chlorinated naphthalenes. BAADER mentions epidemics at American shipyards during the Second World War. Jn his description of the cases occurring in America and Great Britain, sometimes with a fatal outcome, TELKKY refers to the report of BROWN, President of the Halowax Co., New York (1937), that only the manufacture of the higher stages of chlorination and the combination with cb.lorinn.tod diphenyls and other substances led to severe damage to the health and in some cases to fatal acute yellow atrophy of the liver. TEIjEKY also refers to the animal experiments by 0.. K. DRINKER and collaborators to support the view that only the higher chlorinated diphenylamines produce serious damage. The general symptoms in occupational chlorophenol intoxications are apparently more pronounced than those occurring with the lower chlorinated naphthalenes employed earlier. This fact was also observed by TRUHATJT and collaborators amongst workers who had been using pentachlorophenol for wood preservation, as well as KUBOTA in Japan, who mentions multifarious disturbances of the autonomous nervous system and who observed several fatal cases. In all three German groups of chlorophenol intoxication, liver damage was established, the damage that was most pronounced and studied most intensely being that found amongst the cases of disease occurring in two Mid-Rhenish companies (HERGT, KALK and WILDHIRT). I" all the groups, several cases of chronic emphysema bronchitis and inyocardiac damage were found, although these disturbances did not occur yearly so regularly as the pronounced fatigue and neuromuscular weakness, which we observed in all our patients. The psychosyndrome described was equally regular, this being found not only by us but also by .TANZARIK and RTCHERT to a completely identical degree amongst the Mid-Rhenish workers. The psychopathological syndrome could be distinguished with a sufficient degree of certainty by differential diagnostics from endogenic psychosis, especially mild cyclothymia diseases, neurotic personality developments and organic psychosyndroines of different etiology, and somewhat presenlle or 247 Wclirosclerotic processes of degeneration. Phenomonologicall^Mhe relationi'ilpn to the pseudoneurasthenic syndrome, which is describecffn connexion with a large number of occupational intoxications such as those caused by toid, carbon monoxide, manganese, thallium, arsenic, carbon disulphide, tri'liloroethylene, etc. (for relevant articles, see BORBELY, von HATTINGSWKKU MEGGENDORFER, MOESCHUN, PBNTSOHEW, TELEKY)—and «^|K'cially the relationships to particular endogenic mood conditions—are "livlous. Lowering of the vital level, moments of depression, vegetative symptoms, and, not least, fluctuations in intensity can be observed predominantly in endogenic-depressive conditions. On the other hand, alcohol intolerance, bypnraesthetlcally excitable and polyneuropathic traits influence the differential diagnostic aspect more in the direction of an apparently exogenic condition. Tin1 somewhat older psychiatric literature should be borne in mind in this fwnexion (MEGGENDORFER, STBRTZ), this placing the neurasthenic M'wlrome quite definitely in the pattern of exogenic symptom complexes. Not li'iist, reference should be made to the phenomenological relationship of our '^nervations to the (exogenic) hyperaesthetic-emotional conditions of weakness of BONHOFFBR, which, from the psychopathological aspect, have significantly been designated by BWALD as no longer heteronomous but homonomous in the sense propounded by KLEIST. Despite the phenomenological relationships discussed, the psychopathological delayed syndrome of the chlorophenol workers scarcely corresponds completely with any of the known clinical pictures. In any case, the question of a special I'N.vchic-vegetative delayed intoxication syndrome, which was discussed by Nl'IlOGELBERG in connexion with observations on persons suffering chronic occupational damage from military poison gas, also demands consideration in view of the observations mentioned in this paper. It has been possible to rule out psychogenic-neurotic moments so far as our subjects are concerned, provided that individual neurotic conditions, i.e. tlmracterogenic and experiential situative data are involved. Two of the nine probands exhibited considerable psychopathic or neurotic structural elements. However, it was easily possible to separate these two probands from Hie other completely or largely non-neurotic cases. Certain "collective-neurotic" factors have, in our opinion, to be taken into account as an unfortunate but practically unavoidable fact in all group investigations but especially those Involvirig etiological evidence (SPIECELBERG). Reactions of this type have also been observed in our cases in the sense of a superimposed psychogenic accessory with, as it were, "physiological" but not inadequate, individualneurotic (complex-determined) idemnification wishes. The psychopathological analysis of the individual case and the comparison of the findings in each Instance with such independent collectives of the same etiology afford sufficient protection from authoritative and scientific false assessments. Despite the long course, the prognosis of the psychopathological intoxication results appears favourable. Although technical aspects of the pension situation have not yet been finally clarified, there was, on the whole, a certain subjective Improvement in the symptoms, or else they remained static. We have not observed any objective deteriorations, except for the momentary intensity fluctuations. The experience of the Mainz Nerve Clinic (RICHER) and the impressions of works medical staff (KNECHT) suggest a benign course of acute and chronic intoxications, provided no toxic parenchyma damage, as such, influences the prognosis unfavourably. It seemed appropriate to attribute the toxic action to the high-chlorinated chlorophenols, this view being supported by animal experiments conducted I'.v MACHINE and THOMAS, H. KITZMILLER, a series of other investigators (KEHOR, DE10HMANN, GRUEBLER, BOYD, McGAVACK, TERRANOVA, 1'ICCIONE cited ace. to von OETTINGEN) and also our own animal experiments. KTMM1G and SCHUL55 were, however, able to show that the use of non-industrial, analytically pure, high-chlorinated chlorophenols (trichlorophpnols, pentachlorophenols) does not lead to the characteristic symptoms of chlorophenol intoxication. Animal experiments were carried out with a view to discovering the noxae causing the symptoms. The rabbit's oar proved to be a suitable test object since it is possible to produce the changes on this with the substances causjng chloracne, these changes closely resembling those of human chloracne (HOFMANN and NEUMANN, BRAUN, LANDES, etc.). Brushing with a 248 substance which is active in tills respect leads at first to patches of in conjunction with reddening, swelling and flaking; then, some dlPrlntfT, hyperkcratosis linked with the follicles and also small cysts occur, the* being easy to record histologically, as well. In addition to the brushing exi*1''ments, tests were carried out on rabbits to determine the general toxicit;. whilst cats, too, were used for testing a number of substances. In these tod*, it was found, in corroboration of findings derived by OETT1QL and also IIOFMANN from similar cases of intoxication in a large chemical works !i southern Germany, that the substances producing chloracne possess inarM liver toxicity in rabbits. It was possible to trace effectively the damage t* the parenchyma of the liver intra vitam with the micro-modification of U*' bromsulphthalein test given by HOITMANN and OETTKL. In autopsies, dlf 1 fuse steatoses and extensive necrosis of the parenchyma of the liver vfin found. The investigations, which have already been reported (SCHULZ 1950; KIMMIG and SCHULZ 1957) led to the following results: The effective substances must have occurred in the alkaline hydrolysis of 1,2,4,5-tetrachlorobenzene to 2,4,5-tricblorophenol, this having been carried out technically under pressure at about 180°C in the presence of metlmnol and caustic soda solution. However, it was not the tricblorophenol itself Imt the by-products that formed in small quantities in the course of the pressurized phenol process that were regarded as the causal noxae; for it was not possible to produce any of the above-named changes on the rabbit's ear with pure, repeatedly distilled 2,4,5-trichlorophenol or with 1,2,4,5-tetrachlorobenzene, although they did occur with the tricblorophenol used technically. Since the isolation of defined compounds from the residue occurring in the distillation of technical tricblorophenol was not possible at first, compound* were synthesized by chemical means and given to us for testing on animiil* where there was a certain likelihood that these substances may occur as byproducts in the saponification of tetrachlorobenzene to trichlorophenol. The substances initially available were various chlorination products of tlie diphenyl ether and the dibenzofuran (diphenylene oxide). Although the diphenyl ether and its IX to 4X chlorinated derivatives, and also dibenzofnran and monochlorodibenzofuran were ineffective in experiments on animals, 3X and 4X chlorinated dibenzofnrans, even in concentrations as • low as '0.05%, produced the symptoms mentioned on the rabbit's ear1. Single closes of 0.5 to .1 mg/kg administered orally produced severe liver damage in rabbits, this leading to the death of the animals in most instances. The clinical observation of a laboratory assistant engaged elsewhere, wlio fell ill with severe chloracne after exposure to tetrachlorodibenzodioxine, indicated the chlorine derivatives of the dibenzodioxine. Tetrachlorinated dibenzodioxines, especially 2,3,6,7-tetrachlorodibenzodioxine, were highly effective on the rabbit's ear, even in low concentrations. Tlivoe brushed applications with 0.01-0.005% solutions (in polyglycol) were sufficient to cause severe areas of inflammation and follicularly arranged hyperkeratosis. When administered orally, single doses of 0.05-0.1 mg/kg body weight led to severe liver damage and generally the death of the animals. The assumption that 2,3,0,7-tetrachlorodibenzodioxinc is actually of considerable importance in causing the chloracne diseases'occurring in the chemical' works received further substantial support from the chemical angle. It wns possible to prove that this compound is formed from two molecules of sodium trichlorophenolate in association with the cleavage of NaCl under the pressure and temperature conditions prevailing in the autoclave. It was, 'moreover, possible to isolate the named tetrachlorodibenzodioxine from the by-product occurring in the technical pressurized phenol process (alkaline sanonification from tetrachlorobenzene to trichlorophenol). To prove that 2,3,6,7-tetrachlorodibenzodioxine is capable of producing alterations in the form of chloracne not only on the rabbit's ear but also on human skin, one of us (SCHULZ) carried out a test on his own body. Two brushed applications of a 0.01% solution on a circumscribed skin area of the forearm led within two days to a mild dermatitis, then some days later to a follicular hyperkeratosis and comedones, these also being easy to record histologically. The etiological significance of this substance for the diseases described here seems to us to be sufficiently evidenced by this experiment. However, it is not impossible that other chlorinated aromatic compounds with highly toxic characteristics may occur in this technical process, these possibly not having been so far identified or tested in experiments on animals. 249 The experimental, and clinical findings are an impressive ^•fcmp of the that, in works pathology, substances occurring in smt^Buaantities us 'ItrixhictR in chemical processes can be of importance. I n^Sper n p e r i m e n t s to ational disease, this constitutes an important prerequisite for successful 'iaxy. tn our own special case, it was possible, by changing the plant chemistry »*|i(>ct of the manufacturing process, to prevent the formation of the highly l»*k', multi-chlorinated dibenzodioxines and dibenzofurans. Since than, 2,4,5trlehloropbenol is again being manufactured in this works and processed into Hit1 herbicide 2,4,5-trichlorophenoxyacetic acid, without symptoms of intoxication of any kind occurring amongst the workers. A TECHNIC FOB TESTING ACNEQENIO POTENCY IN BABBITS, APPLIED TO THE POTENT AONEGEN, 2,3,7,8-TETBACHLOHODiBENZO-p-DioxiN* B. Linn Jones, M.D. and Helen Krizek, Ph.D. Pollicular hyperkeratosis is an important feature of the occupational disease known as chloracne, which is characterized by the appearance of papules, comedones and cysts after exposure to industrial materials containing highly chlorinated diphenyls, highly-chlorinated naphthalenes, and other chlorinated aromatic compounds. A characteristic epithelial byperplasia and hyperkeratosis can be produced on the inner surface of the rabbit ear by such compounds (1, 2), and a difference in intensity of response has been noted and suggested ns a basis for comparative tests (2). Experimental studies heretofore (1-7) have been, however, directed chiefly to the ability or the failure of various materials to produce this effect in experimental animals and in man, and in delineating its gross and its histological features; these studies have used cither material of unstated origin, or else mixtures (e.g. Halowax, 1014). We Imve attempted -to study the phenomenon of acnegen-induced hyperkeratinIzation on the rabbit ear in a quantitative fashion by recovering and weighing the keratin formed after applying known amounts of a single, well-characterized chemical compound under controlled conditions. To recover keratin a new Icchnic was developed based on the resistance of this material to digestion by pepsin. As test compound we have chosen 2,3,7,8-tetracluorodibenzo-p-dioxin, which has been reported to be so potent that painting the rabbit ear three times with a 0.05%-0.001% solution was sufficient to produce the acneform response (7). With a compound of such potency, the expected effects could be produced without the necessity of applying the material in an ointment or as a crust, circumstances which would have made very uncertain the quanity actually in contact with the skin. EXPERIMENTAL Preliminary gross and histologic observation indicated that 0.3 inierograms npplied to the rabbit's ear gave, by gross observation, a minimal follicular plugging whereas 0.02 micrograms caused no observable effect. Accordingly, 0.3 inierograms was chosen as the lowest dose; in addition dose levels of 1.0, 3.0 and 10.0 micrograms were studied. Seven mature, white, male rabbits were used for each dose level, except in the first studied (0.3 micrograms) for which only six were used. Seven days after wax epilntion t, of the inner surface of the ears, 1 ml. acetone solution of the compound was applied to one ear of each of the rabbits, and 1 ml. acetone to the other. Special effort was made to distribute the liquid uniformly over * From tho Section of Dermatology, Department of Medicine, University of Chicago, ChicaRO 37, Illinois. _ , .. This research was supported by the Research and Development Division. Office of the Surgeon General. Department of the Army, under Contract No. DA-40-007-MD-III and by United States Public Health Service Medical Training Grant No'. 2A-ti20,S(Cl). Presented at the Twenty-third-A-imnar-Meeilng of The Society for Investigative Dermatology, Inc., Chicago.. HlrTTune 26, 1962. t With "Improved Zip." k. 251 250 the entire inner surface, and to aid in securing uniformity the | The complete removal of all non-keratinized tissue from the bJM^y by pepsin :-*te«tUoii was confirmed by histological section of a keratin disc.^H. 10). divided into three applications, made on successive days. The righriif »t» •;'• In Kcneral, the keratin discs recovered from the treated amHils were less used as control for some rabbits in each group, and the left for others. K«' ;(irag)li> and thicker than the controls. At lower dosages the follicular keratin teen days after the first application, three biopsy samples, extending tlireisS ;..•*»* umially observed as tree-like forms representing casts of the multiolobated the cartilage, were taken under procaine anesthesia, with a 9 mm. pnncli. <** • *rfi»Cf>ous glands. (Figs. 5A, 6, and 7). These follicular projections were not sample was taken from the middle, one from the posterior and one from (1*, in the controls. (Fig. 5B). At higher doses the keratin in the follicles anterior area of the ear, at a level about 15 mm. distal to the notch of * Thews On1 dose. The averages vary approximately linearly with the logarithm of the with 10 ml. ether was made, after which the cup in the covered petri dish w»* 'lose, but individual (T — 0)/0 values at each dose deviate widely. The deviadried in vacuo overnight. The cup and sample were weighed to the nearest Found: O, 44.31%; H, 1.40%; 01, 44.18%," The melting point wan Control Treated ear 205-300° C.; literature values for 2,3,7,8-tetrachlorodibenz;o*?)-dioxin prepared Rabbit by chlorination of dibensso-p-dioxin: 205° C. (8),1 320-325° C. (9). The infrared 0.97 spectrum showed a doublet at 1310-1322 cm." in the range reported for n I 0.98 .1.07 series of clibenzo-p-clioxin derivatives (10). The ultraviolet absorption spectrum <9 .. 1 31 of the compound in absolute ethanol had a maximum at 233.m/t find one at 307 1.18 1.88 66 1 20 m/i; the respective molecular extinction coefficients were 46,500 and 4,250. . ... 1.13 1.31 RESULTS 1 Histological sections showed a characteristic hyperkeratosis of the folliculnr epithelium and a marked hyperplasia of the surface epidermis. At low dose level some sebaceous cells were present;. at high dose level, only a few were seen, and the follicle was filled with a keratinous mass. (Figs. 3 and 4). There was marked thickening of the epidermal keratin, although this was only rarely observed essing. on the slides, presumably because it was lost in the cutting and proc1 Following the practice of Chemical Abstracts we describe our command no 2,3,7, 8-tetraehlorodibeno-;)-dloxin. From a comparison of its preparation with the mode of oridin in the Industrial process discussed by Klmmig and Sehulz (7) we conclude that it IB the same as their 2,t),6,7-tetra-chlorodibenzo-p-dioxin. " Analyses by Micro-Tech Laboratories, Skokie, 111. Average of three samples from, respectively, anterior, middle, posterior. reveals some tendency for greater response to occur in animals for which control values were high, but this tendency is by no means clear-cut. We found no correlation between intensity of response and weight of the animal. There appears to be no consistent difference in the three positions, anterior, middle and1 posterior, along the line of section, either for the control or for the treated eaz , with respect to weight of keratin recovered. Average value for keratin recovered from the controls was remarkably constant: 1.11 ± 0.10, 0.85 ± 0.18, 1.02 ± 0.26 and 1.02 ± 0.16 mg. for the rabbits treated with, respectively, 0.3, 1.0, 3.0 and 10.0 micrograms. 45-362 0—70 17 253 h be used for of acnegonicity of various m^e^Nu'thi11?3'*mlsibst *^ * and 2,3,7,8-tetTachlorodibenzolfhovin tl H ™ «nce is applied ,_- „,. avoid the compltottoKff^^^totoe^auch a procednTUfc, number of animals necessary Control ' " l d u l , fences, and redllc(, lSrt, from untreated animals Three b i o n w . n i C°U d be secured taaepcnden*. ysis^of the data showed"S vS Sfflr^ot^..BO«««7?««|-»': ie (the nikl(!i+s on the nvcnvt' tlons. Thulaff O^an^n^n^"^^^^1- but ««re were"smne as showing folllcular dilatat of keratin. At 3.0 micr0g^7^T^^^ r.°.ur sh,owed increase in wolfM loiiIenHy 0.3 micrograms in acetone, died at the end of JMays, and two receiving, respectively, 30 and 2 micrograms died witl^^. week. How,' *»*r, tliere were undoubtedly other contributing factors, because in all animals «mii'i)tlf] recovered after careful pepsin digestion. , 'A When applied in acetone solution to the rabbit ear, 2,3,7,8-tetrachloro*f>vnzo-;>-(iioxin is effective at micrograin levels. Effect of dose and individual ittffi'rnnces in response are discussed. :. 'A. The new technic, using 2,3,7,8-tetrachlorodibenzo-p-dioxin, is suggested for , *Mni>nring acnegenicity of various substances. BEFKBENCE8 Milligrams of keratin per 9-mrn. biopsy sample 1. ADAMS, E. M., IEISH, D. D., SPENCEK, H. 0. AND HOWE, V. K.: The response of rabbit skin to compounds reported to have caused acneform dermatitis. Inc. Med., Ind. Hyg, Section, 2:1, 1941. 2. HOFMANN, H. T. AND NEUMANN, AV.: Eine Methode zur tierexperiruentellen ' Pi-titling der Hautwirkung chlorierter Naphthaline. Zbl. Arbeitsmed., 2: 169, 1952. .1. BBAUN, W.: Ohlorakne. Editio Cantor, Aulendorf i. Wurtt, 1955. 4. HAMBEICK, G. W., JK. AND BLANK, H.: A microanatonical study of the response of the pilosebaceous apparatus of the rabbit's ear canal. J. Invest. Derm., 26 : 185, 1956. 5. HAMBRIOK, G. W., JB. : The effect of substituted naphthalenes on the 1 pilosebaceous apparatus of rabbit and man. ,T. Invest. Derm., 28: 89,1957 . (1. SHELLEY, W. B. AND KLIOMAN, A. M.: The experimental production of acne by pentand hexachloronaphthalenes. A.M.A. Arch. Derm., 75: 689, 1957. 7, KIMMIO, J. AND SCHULTZ, K. H.: Berufliche-Akne (sog. clilorakne) durcli chlorierte aromatisclie zyklische Ather. Dermatologica, 115: 540, 1957. 8. TOMITA, M., UEDA, S. AND NABISADA, M.: Dibenzo-p-dioxin derivatives. XXVI Synthesis of polyhalo-p-dioxin. Yakugaku Zasslii, 79:186, 1969, Ohem. Abstr., 53 : 13152, 1959 0. SANDEBMANN, W., STOCKMANN, H. AND OASTEN, R.: Tiber die Pyrolyse des Pentachlorphenols. Ohem. Ber., 00: 600,1957. 10. NARISADA, M.: Infrared absorption spectra of aromatic ether compounds. II. Characteristic bands of dibenzo-p-dioxin derivatives. Yakugaku Zasshi, 79:183,1959; Chem. Abstr., 53: 10967,1959. DISCUSSION other, comedones were observed; but the response in terms of weight recovered was less than for two which gave the gross impression of follicular papules. Attention should bo called to the great toxicity of 2,3,7,8-tetrachlorodibenMp-dioxin. Kimmig and Schulz (7) reported that 0.5-1.0 mg. per kg. orally was lethal to most of their rabbits. In our preliminary experiments a rabbit receiv- DK. PETEH IfLESCH, Philadelphia, Pa.: Since the criterion of acnegenic activity appears to be the conversion of the sebaceous cells into keratin-forming cells, I would like to ask, what did you see in the histologic sections? Dn. E. LINN JONES, (in closing) : In the paper we will have histologic sections of treated glands and the digested keratin disc. In regard to histology there is varying response, depending on the amount applied. With lower doses there is conversion of the cells in the follicle to kerfitinizing squamous cells, with occasional remnants of selbaceous cells in pockets here and there. With larger doses no sebaceous cells can be found. The entire follicle is converted into a large keratin-filled papule. STUDIES OF THE CHICK EDEMA DISEASE ljy' 2. PREPARATION AXD BIOLOGICAL EFFECTS OF A CRYSTALLINE CHICK EDEMA FACTOR CONCENTRATE BY U. F. FLICK, D. FIRESTONE AND J. P. MARLIAC Reprinted (rom I'our/niY SriKM'R: Vol. XL1V, No. 5 September, 1965 (255) 256 m WH, . 257 phcnanthrene derivatives were collected, >,000-(old from a low potency commercombined and concentrated. The concentrlolcin produced hydropericardium trate was chromatographed on columns of. ) when fed to day-old chicks at 50 Cclite:H 5 SO,:fuming II-SO, (1:1:1) and ». in the diet, and resulted in death at 1 cluted with CCI4. The foregoing procedure :rr,n. WootlOQ and Courchcne (1964) was a modification of AOAC method 24.111 .band one fraction (designated a 3.02) (a) (Horwilz, 1.960). The CC1, eluates 5*Mch was highly toxic for the chick. These were re-chromalographett on Merck Alu}J»wkcrs estimated that ingestion of 5 \>.g. mina, and individual fractions were col£it the « 3.02 fraction was enough to kill lected and checked by ultraviolet speclroISM chick. Firestone et al. (1963) reported photomctry. Fractions eluting with 10% |$*l signs of the disease were elicited by dielhyl ether with absorption spectra of |«W fraction fed at 0.1 p.p.m., which subnaphthalenes (absorption maxima in the the report by Yartzoff et al. range of 240-250 my..) were combined and :,,.,«./. re-chromatographed on Merck Alumina I?' The purpose of this paper is to report with isooctane as the ekiant. Individual fcW recent studies on the purification and fractions were collected and bioassayed, ,|Wo1ogical' effects of a concentrate of the and a highly toxic crystalline fraction |chick edema factor (CEF) isolated from a (CEF concentrate) weighing 79 mg. was |J crude toxic fatty material (TFM) known obtained. 'f to produce the chick edema disease (Flick The CEF concentrate was examined by Itl al., 1962, 1963). Preliminary data are microcoulometnc and electron capture gas :-included on the effects of CEF-containing chromatography. A microcoulometric gas .; material on egg halchability and developchromatograph (Dohrmann Manufacturing ment of the chick embryo. Company, Palo Alto, California) was used at a column, temperature of 250°C. with a MATERIALS AND METHODS l Preparation of CEF Concentrate. Eigh- 6-foot X /\ inch (i.d.) aluminum colStudies of the Chick Edema Disease teen pounds of unsapom'fiable material was umn packed with 20% Dow-Corning High 2. PREPARATION" AND BIOLOGICAL'EFFECTS OF A CRYSTALLINE Mated from 180 pounds of toxic fatty Vacuum Grease on ackl-%Yashed ChromoCHICK EDEMA FACTOR CONCENTRATE material (TFM) by saponificatioi^and ex- sorb W. Details of this technique were detraction- of the unsaponifiable fraction with scribed previously by Firestone ct al. D. F. FUCK, D. FIRESTONK AND J, P. MAUI.I/U: petroleum etheridiethyl ether (1:1, v./v.). (1963). For electron capture detection, an Divisions of Ki/lrilioii, Food Chemistry, anil Taxicologlcal Kvaluatiow, Footl and Driip. Administration, The petroleum ether was redistilled and Aerograph Hy Fi (Model CiOOft) gas chroWashington, D.C. 2020J : the solvent boiling between 40-60°C. was matograph (Wilkins Instrument and Re(Rercirwl for puMirolioil February 23, 19fiS) collected and used. The .unsaponifiable search Inc., Walnut Creek, California) was INTRODUCTION' 1959; Wool (on and Alexander, 19S9; Ames | fraction was chromalographed on Fisher used. This instrument was equipped with a M)LLO\V)NG (he early report's of the ct- al., 19(50; Harinan ct at,, 1960; Yarlzoff | ; Alumina (Cat. No. A 540) essentially as tritium source electron capture detector, A chick edema disease (CED), Snngcr f.l al, 1961; Wool (on rf til., 1962; and described by Yartzoff ct al. (1961). Frac- stainless steel column, 5 feet X ?4 inch, 'tc™" HospUal, KC.. were anesthetized with diethyl ether, vol- burgh, Pennsylvania for detection of COHW. ?CUy lnl ° lhc *>»< ha. bm ume of hydropericardium (HP) was mea- stud j CggS (pcraon com on I'" ~ sured according to the procedure outlined .on rom T Dr. 1?'°°° McLaughlin) wi.l, "approximUcl, 00 chcmic,,s, lK,.a , nk anf) " by Douglass and Flick (1961), and postmortem observations were recorded. Blood d l'" , C ° njU " cti °" «'lth several differ! samples were withdrawn from the right ven- stud es. Reefed materfals were found to conMrt«ntly rema.n m the yolk nnd no great diflku'l'f. . I ride with needles and syringes moistened ^encountered which were ^ ^^ with heparin, and the following tests were performed: microhematocrit (Natelson, of fraction is a liquid residue fatty material which is a sli'J l»|. 1961), whole blood glucose (Somogyi, of 1952) and total plasma proteins and plasma ionoT protein fractions (Gornall el al., 1949). oT of , °;;H,hydrocarbon , • residue nn,| a(fattj-™ac Other blood samples were allowed to clot, tamed and copper determinations' were made on < a , o n products m addiMon ,o ( he 'chi the sera, ' Samples of scrum were colledcd in acidrinscd tubes, frozen under carbon dioxide and sent ,. ^., *.,*. uipiijjoim.ianic. material .contained <0.5% (v./v) of prcdpitable material. Sec Brew et al. (1959), Friedman ct al. (1950), and Woolton and Alexander (1959). MINUTES P- Fw. 1. Gas chromatogran. of (a) toxic concentrate, (b) Procter & Gamble a 3.0*• »»* J> P££r K'Cunhte a 3.1J. See text for details on instrumentation. The numbers adjacent to chrom.logi.phic pea lit retention times relative to aldrin. lion is shown in Figure l(a). The chroma- l(c). The chromatogram of our CEF contogram revealed that within a 70 minute centrate shows a. peak (retention time of nin at least eight components were present 10.S relative to aldrin) having the same in the preparation. Woollen ct al. (1962) retention time as the a3.02 rinactive isovner. The relative peak area ( /c of total area Isolated two components, which were desigof the chromaiographic peaks) of Ihe 10.5 nated o. 3.02 and a" 3.17 (gas chromatocomponent was estimated by using the regraphic retention lime relative to methyl arachidate). Each of ihese two component* tention X peak height method of Carroll contained a high-melting fraction that pro- . (1961). By this method the 10.5 peak obduced edema and a low melting fraction tained in the microcoulometric chromatothat did not produce edema. Our toxic con- gram represented about 20^ ot total comcentrate was chromatographically compar- ponents. ed to the low melting fractions of Woollon Chick Response. Results obtained on ct al.3 The peaks of the. latter two coin- weekly weight gain, food consumption, pounds, obtained with the electron capture feed/gain ratio and calculated consumption technique, arc shown in Figure l(b) and of the CEF are tabulated in Table 2. The weighl gains among control bird? increased 1 Kindly supplied by Dr. N. It. Artman, Procat a fairly constanl rate each week. Weight ter S; Gamble Company, Cincinnati, Ohi:>. 260 261 TA T/\fflWr,—Weekly weig/il gain, feed eoiisimip/io>i, feed/gain ratio and iiiecslion of crystalline CEF c± .10 sult in hypoalbuminemic edema (15cst and 3 .22 .08± .21 2 .12:1: . 1 7 0.')5± ,08 2.2'l± H'ret 3 Taylon, 1955; and Smith and Jones, 2 .20± .11 \.3<>x .11 1.70-' .20 3 .H8i .11 1 .'»* .20 I . S 7 ± ,.M 1.17± .18 3 .88t .37 1961), it may be that even slight decreases 1 .%± .16 ;.15± .12 1 . 7 7 ± .20 3 .15 + ,20 in albumin could enhance fluid transfer 1 S.K. of the menu. across endothelial membrane?'damaged by [thick. The difference between these values the chick edema factor. The degree of development of HP in re:< way be clue to a number of reasons. First, tWootton el. at. (1962) did not repnrt the sponse to the inrre.st.ion of the. CEF concenWiount of food ingested by ihcir birds fe:l trate is shown in Table 4, The (.oncentrate ; Ihe a 3.02 fraction. Second, they vised 'a fed at the 200 p.p.b. level prod need a mar1 heavy breed of chick, while we have used ked increase in fluid which accumulated » light breed. Third, though both diets were within the pericardia! sac. 'Hie magnitude *j'n(helic, they were not identical. Fourth, of this response is of the samrt order as retheir « 3.02 fraction was essentially homo- ported previously by Flick <.* al. (1963), geneous, while our prcparalion contained using 4% crude TFM in a similar diet. perhaps as many as eight components The HP group scores show (has. the SO p.p.b. (compare a, b and c, Figure 1). Never- level of CEF did not result in abnormal theless, the prediction of Brew ct al. volumes of pericardia! fluid according to (1959) that the chick edema factor may the ranges of volumes-reported by Shue be detectable when fed to chicks at levels and Gallo '(1961). The HP incidence of 2,0 p.p.m, or less has been confirmed shows that all the birds fed 200 p.p.b. CEF by our studies and by those of Woolton had abnormally high levels of fluid in the pericardinl sac. Also shown in Table 4 are rial. (1962), The results obtained for plasma albu- values for hcmatocrit, serum copper and min, globulins, A/G ratio and total'pro- whole, blood glucose. The' packed cell volteins are presented in Table 3. In general, umes were decreased by the t\vo levels of there were no appreciable differences in ei- CEF concentrate fed. These decreased liether the plasma protein fractions or the matoc.rits were not. so low as those reported total plasma proteins when considered on previously by Flick ct at. (1963) from r Uie group basis. Some chicks with severe feeding 5 /o crude TFM with a practical edema had marked hypoallnimincmia and type of ration. Scrum copper levels (Table 4) were elehypoproteinem'm, in confirmation of the observations of Alexander c.l al. (1962). vated at the SO p.p.b. level but no statistiFrom the data it seems unlikely that the cally significant change from normal ocmassive edema formed was the result of curred at the 200 p.p.b. level of CfiF concensuch modest alterations in circulating pro- trate. Since it had been observed that the teins. These data support the hypothesis ol liver, heart and kidney were adversely in. .—\\'telily Ictds of l>!a 5. C. Wliile !.Fgl,tmt cockerel* fa! CKI* 262 263 TAD IE 4.—Ilydropmcardiiim (111'), hcmattiml, .tenini cn/ificr and whole lilana i'/wotc values o/S.C, i Willie Leghorn cockerels fed CKl{ concentrate for 3 weeks CEF ' level P.P.». 0 50 200 • - • Hydropericardium : Mean volume Score' ml. 0.0 0.1J8iO.OI8(4)« 4.63 ±1.64(6) Incidence 0 o 10+ Hematocrit Observations Scrum Copper Glucose"' Untnp. bjetlcd nig. % '• 'Hi. i ill $& si'isl °/° 27.1 ± 1 , 4 (8) .j 1± g ( 5 ) 217±IO<» ij No. eggs ; . injected- 'L °' m, n _„ Embryo % 93' 40 20 0 20 20 20, . 5 - 10 ' ,. M 50 Hatch . • (Malformations of right {cerebral hemispheres, legs and beaks; small embryos Chick Normal [Weight retardation, {sparse and deformed feathers. •— m ' Controls were injected with 100 ,,1. of corn oil. |t ' Normal expected % hMch (Mclaughlin el al, 1963). . u, uiii ini-iin imimncr ol observations), sample of HP fluid lost from this group. volvcd in thn chick edema disease (.Allen, 1961; Alexander et al., 1962; and Flick ct vanced stages of the disease (see footnote^ 2, Table 4). .^ a/., 1963), and thai changes in these organs From what is known to date, the chick I had been involved in the mechanism of edema formation (Smith and Jones, 1961), edema factor elicits a number of signs of I it was thought that determination of the intoxication which not only accompany tk I levels of serum copper in the birds with the feeding of crude TFM but are more severe f disease might be of some diagnostic value. when the crude material is fed. The finding -1 Scheinberg and Sternlieb (1963) reported (hat . our CEF concentrate contained 8 ' that human patients with Wilson's disease peaks by electron capture gas chromatoghad severe liver disease, and many patients raphy (Figure 1) indicates that at least S had serum copper dyscrasias caused by ex- compounds were present in the purified cessive urinary excretion, , malabsorption preparation fed in these studies. It may be from the intestine, decreased protein syn- that only one, or' a few, of these comthesis (particularly in severe malnutrition) pounds possess the necessary molecular and severe hepatic dysfunction. I t may be configuration to produce signs of the disthat the elevated serum copper among the • ease equivalent to the estimated potency of chicks fed SO p.p.b. CEP'' concentrate was the « 3.02 fraction. associated with either decreased liver utilization of copper or decreased renal excre- Untcfiabilily St,,dy. The preliminary retion. The adverse effect on serum copper of sults obtained from injection of White CEF concentrate fed at SO p.p.b. is not Leghorn eggs with the unsaponifiablc fracclear, but may be indicative that CEF tion of TFM,-which conlained CEF, arc toxic activity is oligodynamic and more shown in Table S. The percent hatch of specifically oligof.oxic (more toxic at low control eggs was within the expected range reported by McLaughlin ct al. (1963) levels than at higher levels). Whole blood glucose levels were not ap- Chicks which hatched appeared to be norpreciably altered by feeding the concen- mal i" size and development by gross obtrate (Table 4), Crude TFM, however, fed servation. When fertile CW!S ,,ere ,fc.jn. in a purified ration, frequently elicited a jectcd with 10 ,,1. of the undiluted unsaponmarked hypoglycrtnia among chicks in ad- "liable fraction containing CEF, (he hatch was 40^; injection of 20 v.]. resulted in blood glucose; injection of CEF-containing unsaponifiable fraction and determination of its effects on embryonic development and on egg halchability. The following observations were made: 1) presence of approximately eight components (GLC separation) in crystalline material, 2) moderate growth depression at SO p.p.b. level of CEF concentrate, 3) essentially normal feed consumption, 4) moderately increased feed/gain ratio, S) increased HP at SO p.p.b. level o( CEF concentrate, 6) .severe HP at 200 p.p.b. level of CEF concentrate, 7) essentially no group changes in .plasma proteins, 8) moderate decrease in 'hematocrit, 9) no change or moderate elevation of' serum copper (SO p.p.b'. CEF concentrate), and 10) normal blood glucose levels. A preliminary hatchability study revealed that a CEF-containing material led to a SUMMARY decreased hatch of injected eggs and to deThe following studies were.- performed: velopment of embryonic deformities. gas-liquid chromatographic (GLC) separaACKNOWLEDGMENT tion of a purified crystalline concentrate • The isolation of CEF concentrate was containing the chick edema factor (CEF); largely carried out by Mr. ^dr.ej&.&'jirt* feeding of the concentrate at SO or 200 v phy was perparts per billion (p.p.b.) in a scmi-synthel- $~-~ ic d i e t . l o day-old S. C. White Leghorn 2™j£ . F T C K S S I s u y , lood and cockerels for three weeks and determinaDrug Administration, tion of growth, feed intake, 'feed/gain REFERENCES. ratio, mortality, total intakte of CEF concentrate, plasma proteins, hydropcricardi- Alexander, J. C., R. J. Young, C. :M. Burnett um (HP), hematocrit, serum copper, whole - ami II. D. HaUiaway, 1962. Hydropericard.um' hatch and SO p.l. completely inhibted hatching. Embryos which failed lo tolcli exhibited one or more of the followlng developmental anomalies: malformed . Courcheno, J964. A contribution to the knowledge of the structure of two hydropcricaidium-producing factors from a toxic fat. J. Agr. Food Chcm. J-2: 94-98. Yartwff, A., D, Firestone, 11. liancs, \V. HonviU, L. Friedman and S. Nosneiin, 1961. Studies of the chick edema factor. II, Isolation of a toxic substance. J. Am. Oil. Chemists Soc. JSi 6062. 265 Reproduced by the U. S. DEPARTMENT OF HEALTH, EDUCATION, AND WKLFARH Food and Drug Administration NUTRITIONAL ADJUNCTS Chick Edema Factor. III. Application of MicrocouEomc.fric Gas Chromalography to Detection of Chick. Edema .Factor in Fats or Fatty Acids By DAVID FIHKSTOXE, WALID JBJiAHIM, and WILLIAM HCfflYvTfZ (Division of lrood, Food nnd Drug Administration, Washington 25, D.C.) A rapid screening test for detecting ohick edema factor in ,falfi consists of adsorption cliromaiopraphy of extracted uiisapoiiiliablcK on alumina, followed liy HHMlysis of specific fraclions by a inicrocoulomctric gas chromnlogi'iiph M'liioli is ftr.imilivc only to )ifilogr.iiH. Tltls rliroinsitogrophic nirlhod nppears to be more stMisitive limn tlic cliick bioHssay. Toxic f a t yielded pas cliromaloRrapliic peaks vilii retention times relative to aldrin of 5 or more. AH samples which failed lo reveal those fliromato(jraphie peaks have been shown lo be nontoxie in the ohick bio;.iBsny, Tlin \vii|p.--)irfiui iicciin'oiice of chick c'dcin:i (lisciisc in 111")? rwnllcd in t h e ilcalhs of millions n f young chickens. The lox'u1 iiKilcrials (his disease have bron found to be clilorinatod tiroinnlic hyclrociuljons, occnrrinR in loNio fats in association with n lurpe iniinbpr of relatively nontoxic nromntic materials \vilh similur chemical ami physical propertics, This paper describes a screening procedure for detection of such toxic fals. Specific frjiclions of misnpoiiifiablr 1 m a t t e r isolaled from tlic fats arc examined by using a niimicotilmnclnc mii= rhromalouraph, ah mslniment. which can dclei-l siibmimiprum amuunls of lialogcn. r)'hc prosencc of f-lowcltiiin); snbslances is an indication of (lie chick cilein:i factor in the f.-i!. Tlic precise stniclnrc n! tlic Mih.-tance* CiiMsinu chick edema disc.-pc have yet to be determined. IVlimin.'iry work on the detection, isolation, and cli;ir:iclciiy:i(ion of the luxic aficnls \va-t rcjiorird in l!i.">!i by se\cral lalioraloriiv (I-.'!). ^idiM'iiiicntly, 267 266 Hnrmon and co-workers (4) isolated a toxic f a t t y acid fails to reveal the presence of subst:mcc in crystalline form • from .1 feed these gas chromalograpin'c peaks. grade (allow. A private coinnumicnlion from METHOD Tishlc.r of the same laboratory (5) disclosed Kxlrnciion o] wisapnnifiablc matter (modifi• that HIP. crystalline substance contained cation of AOAC method 26.06,4(5). — Reflux about >17% chlorine. \ 111 g sample w i t h 270 ml alcohol ami 55 ml YarUoff and co-workers (C) isolated a, fiO% (wAv) KOI! 'for 1 hour. -Transfer mixcrystallino halogen containing material that ture to 2 L separator, rinsing flnsk with 325 produced chick edema symptoms at 0.1 ppm ml U = 0, mid add rinsings to separator. Add in (ho diet from a sample of triolein. This 300 ml petroleum ether, A.C.S. (redistilled, retriolo.in was toxic to monkeys, producing taining cut w i t h b.p. •iO-CO°C), and shake vigorously. Let layers separate, breaking emulsions changes in the liver, kidney, pancreas, and that may have formed by adding 10 ml alcoother organs. More recently, Wootton and hol and swirling gently. Draw off lower layer, co-workers (7) isolated three compounds and transfer upper layer to Another separator. from a, toxic fat which produced chick edema Repeat extraction 3 times with 300 ml portions disease. IVfiw spectra of two of the, com- of petroleum ether and combine extracts. pounds indicated a molecule which has a Wash extracts twice with 60 ml portions molecular weight of 3JU and contains six of H:0 by swirling gently. .Wash petrochlorine atoms. Ultraviolet spectra wore leum elhcr extracts first w i t h 60 ml H.,0 and consistent with the concept that these mate- tlien with CO ml of an alkaline dilute alcohol rials arc highly chlorinated aromatic com- soln (dissolve 28 g anhydrous KiCOi in 600 ml H,0 and then add 400 ml alcohol), and pounds. repeat washings in same order. WnHi extracts Ames and co-workers (S) and Firestone with 60 ml portions of H.-O until neutral to and co-workers (9) reported (he occurrence phenolphthulein. Transfer extract to a 2 L of chick edema disease factor in oleic acid crlen'moyer and dry by adding 20 g tuihydroua samples destined for human consumption. NtisSOi, swirling vigorously, and letting the A food additive regulation 1 -of the Food and solution stand a half hour. Decant solution Drug A d m i n i s t r a t i o n now requires that food through a'glass funnel, containing u pledget of grade f a t t y acids he "free of chick edema or cotton in the neck and holding 20 g nnhydrouti other toxic factor." At present, the detection Na:SO.. into another 2 J, crlenmeyer. Wash and assay of chick edema factor in fats is first erlcnmeyer 'and funnel with lined 10 ml portions of petroleum ether, transferring washcarried out by n bioassay procedure (10-12). ings from the erlenmcycr through (lie funnel t h a t requires 21 days to complete. and into the filtered solution. Evaponito most We observed t h a t unsaponifiable matter of solvent on steam bath, imd transfer extract from toxic f a t s contained a number of chlo- to 100 ml tared fat flask containing several rinated 'components which had greater re- boiling chips. Evaporate solvent on ."Loam bath tention 'limes t h a n chlorinated pesticides and complete drying under a gentle current of when examined in a. microeoulometric gas air, or by evacuating flask to 0.5 cm of merchromalograph;' 1 our observation prompted cury while swirling on steam bath, Determine this investigation of the use of microcoulo- weight of unsaponifiablc matter. PraclioiiHlinn uj Miuttpmiifiablc matter by metric gas elu'om.ilogniphy for detecting the alumina cliromalograpliy. — To a chromntopresence of chick edema factor in fats. Chick graphic column, 25 mm o.d. X 300 mm long, edema factor is presumed to be present if fitted lit the bottom with a coarn; porosity one or more gas chromatographic peaks with fritted glass disk and Teflon stopcock, ndd rcr e t e n t i o n times relative to aldrin of !> to 20 dislillod petroleum ether, dried prior to use are f o u n d ; its absence is presumed if analy- with anhydrous Nit:SOi, u n t i l column is % sis of the. equivalent of 100 g of a fat or full. Weigh 50 g aluminum oxide (Merck reagent, No. 71707), and transfer lo column. Store tlin alumina in tightly closed I'otllu, nml ' Cmli' of I'Vdi'riil !(cK"liifimiH, Tillo 21, ficc- How boll IP «s «>on us possible after weighing li.m out portions for chromalography. Let alumina 1 121.1070. 1 inliriiwnu M a n u f a c t u r i n g Company, I'alo settle, and when air bubbles slop riv.ng lo the All.i. < ' n l i f . jig Aldrin = (peak area, in.3) X [recorder sensitivity (min./in.)(mv/in.)] X (35.5g/eq.) X (OOuoB/min.) X (10' /»g/g)(10-'v/mv) (10)/(scnsilivity range, ohms) X (% chlorine in compound) X (96,500 coulombs/eq.). For n 0.1 mv/in. recorder sensitivity, 2 min/ in. chart speed, and 12.8 oiims sensitivity range resistance, the equation alxove reduces to: jig Aldrin = (area X 34J>)/% chlorine. surface of the solvent, place a disk of coarse filler paper on top of the alumina.. Cover the disk with 20 g nnhydroua NojSOi. Drain the excess petroleum other so that it is level with the upper surface of the NoiSOi. Transfer unsaponifiable matter to the chroirmlographic column, using u total of 20 ml petroleum ether. Allow liquid level to fall so that it is just below the top of the NaiSO,. 151ute sample with 400 ml portions of each of the following solvents (dried prior to use by shaking with anhydrous NiuSO.): Petroleum ether (fraction 1), 5% ethyl ether in petroleum clhi.'r (fraction 2), and 25% ethyl ether in petroleum ether (fraction 3). Collect eluatcs in GOO ml erlcnmoyer flasks, add several boiling chips, and evaporate, lo small volume on steam bath. Transfer residues to tared fat extraction flasks, evaporate solvent, and weigh. Transfer to 2 g short stylo vials having screw cap with tin liner, and evaporate solvent. 'Aficrocaulomclric 0os c/tronm/or/rnp/ij/.—Dissolve 4 g silicon'; grcnno (Dcnv Corning High Vacuum Grcuw) or Dow Corning DC 200 silicouo fluid (12,.r>00 cunlislokcs) in 200 ml chloroform on sloani bath. Add 10 g acidwashed Chromosorb W (Jolms-Manville Co.), and stir coutiuiiotisly until most of solvent evaporates (about half an hour). Let stand on steam bath 1 hour, and place in vacuum oven at 60°C overnight to remove residual solvent. Pack the coated Chromosorb W into a 3' length of 0.25" o.d. aluminum tubing plugged at one end with glass wool, using a Burgess Vibratool. (Two 3' columns may be prepared from 20 g coaled Chromosorb W.) Add a plug of glass wool to the open end of the column and bend it into a light spiral, using a 3" diameter mandril. Condition the column at 275°C for 48-72 hours, passing nitrogen through at 20 ml per minute. Prepare a 1.00 X 10-'% solution (10 mg/L) of aldrin in hexane or benzene and chromatoRraph 100 /d portions in n Dohnnann microeoulometric gas chrnmatograph at 2<16-2-18°C, »"ing a nitrogen flow rain of 60-100 ml per minute so that aldrin elutcs in 2.3-3 minutes. Use the 128 ohm range setting. a Determine area of aldrin peak by (riangulution, or with a disc chart or electronic integrator installed en Die strip chart, recorder, nnd calculate recovery of aldrin using the following equation (applicable to chlorinated compounds): The number of strokes of a disc chart integrator coupled to a chroronlograpliy recorder 4 equivalent to each square inch of urea can be determined as follows: (a) Remove the fuse from (he. strip chart amplifier; (6) move the pen upscale on the strip chart a known distance from the baseline; fc) run the chart a known distance; and (rf) divide the calculated area (height X distance traversed by the pen) by the number of strokes obtained. By using the formula, cafrculalcd as described above, n recovery of at least 70% of the aldrin injected nliriiild bo obtaiuwl. Dissolve fractions 2 un. = 6-20. Peaks in this ranglt is used with a I niv slrip chart retonler, ' MiMaeapolis-ITnnu.vwcIl Model T ]fi3X (Mimn'npnliK-lIom'ywcll Kcf-ulalor C»., I'hiladl'lpliin, I'll.), or equivalent. *' 48-362 O - 70 - 18 268 Kciiulu and Dincusaions at 250"C on silicone columns. When a !<>«•• Relative Retention Times ol Chlorinated melting inactive isomer" having the win* Pesticides awl Chlorinated Materials from retention time (/JA = C.O) as one of (I* Toxic, Fats.—A number of chlorinated pesti- toxic factors was chromatographed in ll* cides iincl several chlorinated materials iso- Dohrmann instrument, the following Rf, vallated from toxin fats were chromatographcd ues were obtained at 2-10°, 248°, and 250* in the Dohrmnnn instrument at 24S°C with respectively: 0.0, 0.4, and 0.2. the 3' column. Retention times 'relative to Preliminary Analysis of a Group oj Totk aldrin (#A) ^re shown in Table 1. The and Nontoxic Fats. M icrocoulomctric Andpesticides arc representative, of the whole ysis of Uivsaponifiable Matter Without Prioi range of retention, times displayed by chlo- Fractionalion on- Alumina.—A group of 7 rinated pesticides. A toxic factor isolated toxic, and 7 nonloxic fats were examined from triolein (G), an inactive analogue, and initially. The fats are described in Tables J a concentrate prepared from a, toxic fat, all yielded chrqmatogrnms with peaks of R.\'~ Tnble 2. Data on toxic fate 5 or greater whereas the pesticide peaksOrmnio were all less than ]{A = 4. The toxic factor Clln Un««from triolein as well as the toxic fat concen% Unsn- ponidjvbl' trate produced chick edema when fed to Manu- ponilmbln Mailer, Component facturer Alattcr ppra young chicks at a level of 0.1 ppm in the diet. 1. Tullow acids, 1 18.3 10 still distillate 2. Tallow acids, Table ]. Relative retention limes of . 1 14.8 47 chlorinated pesticides und materials still distillate isolated from toxic fats 3. Tallow ncidtt, 2 U 10.1 (3 foot, }4 in. diameter column, 20% silicone still distillate grouse, 80% Chromosorb W) carrier gas flow 4. Tallow acids, 3 2.0 25 " " i• ' ' still distillate rate, about CO ml/min.; column temperature, 248°C; injection block temperature, 270°C) 5, Tallow acids, 1 4.5 2000 still residue Retention 6. Tallow 2 5.1 39 Time r«. Aldrin 7, Fat from broiler Sample 4 2.5 8000 feed Clilordano 1.0 Ileptachlor 0.0 and 3, respectively. Presence of chick edema Kcpone 2.2 disease wns determined by bioassay (2) using Mircx 3.0 a special basal ration. Organic halogen in Strobnnc ' 0.6-3.5 Tcdioii the unsaponifiable matter (assumed to be 3.4 Toxnplicnc chlorine) was determined by microcoulomc0.0-3.8 Toxic factor from triolciii 5.0 trlc gas ohromatographic analysis of 50 ing Innctivc analogue from triolein 9.0 portions of unsaponifiable mailer without Concentrate from u toxic fnt 2.3, 3.G, 5.4 prior fractionation by alumina chromatograpliy. Both toxic and nonloxic fats conThe chick edema-producing /actors iso- tained widely variable amounts of unsaponlated by Wool ton and co-workers (7) had ifiable matter and organic chlorine. Sources retention times relative to methyl arachi- refer to individual manufacturers. Gas chrodatc of 1.17, 3.02, awl 3.17 when chromato- matograms of unsaponifiablo matter from graphed at 2f>0°C on a 20% silicone column. two of the toxic fats (Nos, 1 and 2) show Since aldrin chiles (wire as fast as methyl peaks with R\ values of 14. Gas chromatoarachidate under these condition.1', it would grams of Unsaponifiable m a t l e r from the be expected .that these toxic factors would * Ptipjilicd by Dr. N, K, ArUnnii, T'roctcr nml have Rf. values of about 2.4, (5.0, and 0.3 Sainfoin Co,, Cincinnati, Ohio, 269 solvents h priately largor nmounts o T eelutmg were iilw> nuwl. Fractions.' obtained by admrptinn rhromaOrcnme Cl in * togrnphy on alumiiiii of uiisiipnnifmhle matIJnan* % UlWl- inniiinhln ter were, .'maJymMl liv itiirrocoulinnetrir gas Mnnu- Hjmliabln MnUcr, lirnii Multnr fn.,|il"'r ehromnliifti.ipliy. ( or mnre urn usually present in ples 1, 2, and 7) failed to reveal chromatothe unsaponifiable matter of toxic fats at gniphic j«!!ik.s with }tA values greater than 3. very low levels, n concent ml ion stop by alumina chrimmtn|'.rai>hy ix iici;r-s:«ir.v prior Taljlc 4, Slow-clutitiR peulis in inicroto microcoiildiiictric analysis. cnulonicti'ic KHS chromntnitrnmg from Frnctionaliun oj llnsapnitifiMc Matler nuulsis nf taxic fnt«» on Alumina- Prinr ta .\firroriiiihntrtrk Annli/.tis.—-Tho "I toxie and 7'nonloxic fats were MnnuHA SB lar!-yer Ihoii iiiiiilyzi-d liy procedures essentially a* Toxic. Vat Nn. described'nl'ove. A t fust, 0-fuoi, and later, 0, 9, 12,21 j Tn'loH' acids. 1 d-foot cliriiinatuKi'iipliii' col'iiMii." won: uwil Still distillate in the. miruicoulomclric gap ohromatograph. 6, 9, 10, 14 . ] Tallow acids, 2 With shorter columns, f.'isler I'ltiliou per1 slil residue mitted analysis of 12 wimples cadi working 0, 9, 10, 18 > 3 Tullow acids day, and results were comparable to those 6, 9, 12 2 4 Tallow oblained with (he ctmvpntion.il 0-foot col2 6, 9, 10, 18 Tullow acids, 5 umns. Jieciiise 10-10(1 mj: portions of samstill distillate ple were rcpratrdly injeetrd, all romponents 6, 10 3 Tallow acids G of Hie imrrocoiilomrfric gas chromatugr.iph were rlcatn.il every 2-4 weeks as required. Table 4 lists the slow-eluiing peaks found C.Jis chromaloftrapliic columns were replaced in gas chromatograms from 0 of the 7 toxic each 1-2 months. I n CIIEI-K wheru samples fills. Figure 1 shows chroimilogram.s of aluconluined large nmounls of unsaponifinblc mina fruction 3 from three still distillates, matter, larger alumina column? were used each of which was obtained from a different for the column chromrifoKrapliy TO Hint the n i a n n f a e l u r e r of commercial f a t t y ncids. A ratio of alumina lo miwipnmfuililc mailer similar p.'iUern of slow-ehuing peaks SURwas at Icfisl 20 to 1, l u thwe eaww approTnlilo 3. Hula y the bionssay. In comparing hydropericardiuni activity and gas eliromatographie response (relative peak area), no parallel relationship was found between the bioassay response of the' toxic oloic acids and the eliromatographie response. It should be emphasized, however, Hint the sltivv'-eluting compounds in toxic samples represent doth toxic and relatively iiontoxic materials, and most of the gas chromalogr'aphic "response is probably due (o relatively nonloxii! substances. Nevertheless, microooiilonietrie pas ehromatographic detection of slow-fluting compounds appeal* (o be an c.fiVotivo scriieninf; tool for segregating (jiidiilionnble sampk;s and diverting them to noitudiblc. uses or for lurlliur testing by bioassay. The R«S chronia(ogr/ij)liic response of the /?A = 9 peak present at widely different levels in 2 o!eic nrids was checked by analyfis of individual samples on diHermit days, using different gas eliromatographie columns for each run. Results are shown in*Tnblo 7. Sample I contained a low level of material responsible for (he Rf, = 9 peak. The differences in integrnlnr resp(jn>e are due both to variation in coulomcter response and to in(iccurac'ies of disc integrator ri'spoiise at low halogen levels. The larger a m u u n l s of maU'rial in sample 2 responsible for I he J?A — (1 peak were distributed in alumina fractions 2 nnd 'A to varying extents depending upon the alumina activity. The total integrator response from both alumina fractions was fairly constant, however, varying from 136 to 169 integrator pen strokes. In addition to the oloic acids, a number of derivatives of oleio arid (triolein, glycerol monooleate, etc.) were analyzed. Nine of ten samples were negative by both the bioassay and chromatographic analysis. One toxic sample, a triolein, gave gas chromatograms'with peaks of 7?A = 0, 10, and 19. Ten steitric acids and derivatives examined were negative by the bion.isny and chromatogrtiphic mxfliod, Anuliisit of Animal find Vegetable Fats ami (,'uin'i»vri;iul Yi'gelalilii Oil Falty Acids.— Fifti'i'ii animal and vegetable fats were examined. Three lo,\ic animal tallows were positive by the chroniiitographic method. Table. 7. KcsponRc" of KA. •= 9 peak from 2 oleio uculs Bmt Simple 2 Hun No. Frnolion 2 Fraction 3 Fraction 2 Fraction 3 I 0.2 O.S 0.0 5.0 0 20 Hfl 0 0 0 H(i 48 0.5 SO ',. 2 3 4 10S 100 « J)isn|n-i' w:\K pvownlml :il tin- RovcntyJuiirniil, 4 4 , 4 4 0 (1901). u i x l l i Auniiiil Mrolitij; of Hie Ast-oci:iUn» of Olii(11) "Cliiiiigcs in Mi'lliods," ibiil., 44, 146 ciiil ApriciilUirnl Clicinists, Oct. lfl-37, 3062, at Wnsliint-ton, !).(.'•. (1901). 278 279 Reprinted from TOXICOLOGY AND APPUKD PHARMACOLOGY, Volume 5, Number 6, November 1963 Copyright © 1M3 by Academic Preti Inc. Printed in V. S, A. TOXICOLOGY AND API-LIED PHARMACOLOGY 5, 760-771 (1963) The Injection of Chemicals into the Yolk Sac of Fertile Eggs prior to Incubation as a Toxicity Test JOSEPH MCLAUGHLIN, JR., JEAN-PIERRE MARLIAC, M. JACQUELINE VERRETT, MARY K. MUTCHLER, AND O. GARTH FITZHUGH Division of Pharmacology., Food and Drug Administration, Department of Health, Education, and Welfare, Washington 25, D. C. Received January 24, 1963 The increasingly large number of food additive chemicals introduced into the market each year has necessitated the development of rapid and reliable methods for the evaluation of their toxicity. Toxicologic studies of all these chemicals by the usual methods using animals are very difficult, and such studies sometimes give inconclusive results. The toxicity of some chemicals, and especially of food additives, may be determined by injection of the chemical into the yolk sac of fertile eggs prior to incubation and subsequent observation of the effects of the chemical on the embryonic development of the chick, This appears to be a promising method in that it may be carried out much more economically in terms of money and space than would be possible with larger animals. Hundreds of chicken embryos .may be observed in a minimum of space, and over a comparatively short period of time: The feasibility of using such large numbers is valuable also in the statistical evaluation of toxicity data. A review of the literature shows how little work has been done in this field except in a fragmentary way on isolated cases. Most of the reports refer to injections of chemicals made after the fourth or eighth day of incubation and examination of the embryos killed before they hatch. The earliest work that we have found in the literature was by Fere (1893). During ten years after this date he published about sixty-seven papers; a review article (Fere, 1899) contains a summary of many of his studies. His work consisted mainly of injection before incubation, but the eggs were usually opened on the third day of incubation. His interest was mainly in the teratogenic effect of chemicals. contain a wealth of information on the avian embryo. Preliminary reports of this investigation have been presented by Marhac (1962) and McLaughlin and Mutchler (1962). EXPERIMENTAL ' 7a« of *e y«ar. No important variation »as detected^ atles 95% A further restriction is based on the waght of the eggs aU t?ose weeing less than 52 g or more than 63 g are reacted. After 1 Truslow Eggs, Chestertown, Maryland. |fc needle (hypodermic linen long ei^r n, « ^^«J Ilk viscosity of the liquid to be injected) i m s withdrawHV Car The initial experiment with a given chemical is for range-finding IH A cell into the yolk (Fig. \ ^^ ^ce such damage could , to avoid damaging the vi elhne^^^rane sm ^ ^ ^^ performed at two or more concentrations of the chemical with 10 e&o'r^t ~ the yolk to spread ou m the>to™^ The needle shouW be level. On the basis of this information, 20 or more eggs are injected ll|| ifc yolk on it, the injection is not satlst™ the appropriate amount of the chemical. As soon ^ the egg a sterile gauze pad ^^^^ a small piece of If the chemical proves to be nontoxic, the experiment is repeated -^ andling, the eggs are randomized in order to avoid series of infertile i any one experiment. the minimum number of eggs that will give a reliable and reprodutilft| value for the hatchability. In the case of a toxic chemical, additional are injected to determine the specific effects of the chemical. The .—u number of eggs used for a chemical depends upon the data obtained initu%;, and upon the kind of information desired. Hence, data for some chemkAl are based on less than one hundred eggs, whereas data for others are b*8*l-f on several hundred eggs. "| Technique of injection. The injections of pure chemicals, chemical wit"! tions, or suspensions are made at volumes up to 0.10 ml. When necessary! dilutions are made with solvents such as water, propylene glycol, corn peanut oil, or other nontoxic solvents. , In order to avoid contamination, the injections are carried out in MM Isolator Box2 with a sterile atmosphere created by using formaldehyde j vapors (produced by mixing 2 g of potassium permanganate and SO mi ij of 37% formalin). During the period of a year, more than fifteen hundred;^ noninjected eggs were exposed to formaldehyde vapors; no toxic effect was !i noticed. After exposure to these vapors for 30 minutes, the eggs are ready for injection. \. the entire air cell. GERMINAL DISC V1TELLINE MEMBRANE ALBUMEN CHALAZA SHELL MEMBRANE FIG. 1, Diagram of egg and position for injection. Incubation and hatching. The injected eggs are put into the incubator The large end of the egg is wiped with a sterile gauze pad moistened | trays with the large end up; the trays are placed in the incubator,4 which with a 70% alcohol solution, and a hole is drilled in the shell in the center J automatically rotates hourly and is maintained at an optimum temperature of the surface over the air cell (Fig, 1). Care must be taken not to daraag* | of 38°C and a relative humidity of 60%. The eggs are candled on the fifth 8 the shell membrane with the point of the drill ; this is to avoid, if possible, day of incubation and every day thereafter. Clear eggs and dead embryos contact of the air with the egg membrane. Fine particles of shell are reare removed for examination. On the seventeenth day of, incubation the moved with an aspirator to prevent the needle from carrying them into fertile eggs are transferred to the hatcher5 and kept at a temperature of the yolk. 37°C until they hatch. Immediately before the injection, each egg is shaken with a quick twist EVALUATION OF DATA of the wrist. Since the germinal disc occasionally sticks to the air cell and The injection of the chemical into the egg may produce one of four it is possible to damage it with the needle, this movement will allow the disc to float free in the egg. possible results'. (1) the chemical is highly toxic at the level injected, and 2 8 Kewaunee Scientific Company, Adrian, Michigan. Burgess Vibrocrafters Inc., Grayslake, Illinois. * Humidaire Incubator Co., New Madison, Ohio; model no. SO, capacity 4SO eggs. 5 Brower Manufacturing Co., Quincy, Illinois. 283 i . all ^Jembryos are killed during the first 20 hours of incubation (before Qj emmra *Hn, and this coagulation may decrease the availability of the chemlPFas the two-somite stage); (2) the chemical is toxic but allows a numberet,jj **!! as some yolk nutrients, and thereby alter the response of the embryo. embryos to develop only up to a certain point, and some possibly even W. | If the pH is highly acidic or basic, a pH effect differing from the true hatch; (3) the chemical has little effect on the hatch; and (4) the chct8-|| toxic effect of the chemical may be obtained due to interference with the ical has no effect on the hatch or on the posthatch development of the chid ||| normal acid-base equilibrium in the egg. ("no effect" level). ^ The ionic concentration is also important for a similar reason. The obIf the chemical appears to be highly toxic and all the embryos are killed,; lervation of increasing toxicity with increasing concentration of a chemical the experiment is repeated with smaller doses of the chemical until sow should be interpreted cautiously, since highly concentrated solutions may hatch is obtained. If the chemical is toxic, but allows the embryos to deUpset the physical equilibrium of the yolk by causing osmotic effects. velop for a longer period of time, dead embryos are examined pathologically and the chicks that do hatch are examined for eye damage, color of the Finally, the introduction of a chemical into the yolk may cause a special feathers, weight, length of the legs, form of the beak and of the rump, type of toxicity because it destroys, alters, or combines with essential hematologic changes, and condition of the internal organs (liver, kidneys, nutrients such as vitamins and minerals. heart, gall bladder, and spleen). EXPERIMENTAL DATA It is advisable in all cases to observe the chicks for a period of a few Twenty-five thousand eggs have been used in our laboratory during weeks in order to detect any delayed effects. Since as much as 30°/o of the the past three years to test more than 100 chemicals with the following yolk remains at the time of hatching and is absorbed during the first 7 days thereafter, effects of a toxic chemical may first be observed at this TABLE 1 time. There may be weight retardation, death during the first week, or the NONTOXIC CHEMICALS appearance of nerve damage occurring as late as 2-6 weeks after hatching. Solution injected The toxicity of a chemical is evaluated mainly from the percentage Per cent Quantity hatch of hatch at varying dosages of the chemical as compared to noninjected (ml) Concentration Chemical (control) eggs, from a study of the embryonic development of the eggs 95, 0.05 Water (boiled) r\t — 95 that fail to hatch, and from a study of the appearance and development 0.05 Undiluted Propylene glycol 90 of the chicks that do hatch. However, several other factors must also be O.OS Undiluted Corn oil 90 considered in this evaluation: these are specific gravity, solubility, coagulat0.05 Undiluted Peanut oil 90 ing effect, pH, and the ionic concentration of the chemical tested. 0.05 0,9% in water Sodium chloride 70 0.05 If the chemical has a high specific gravity, there is the possibility of its 5.0% in water 30 0.10 5.0% in water settling out in the bottom of the egg and thereby giving a value of ap20 0.05 10.0% in water parently low toxicity. 90 0,05 5.0% in water Dextrose The solubility is quite important since the availability of the chemical for utilization in the chick embryo is partially dependent on its solubility results: (1) nontoxic chemicals injected at an appropriate level allowed in the egg. However, since egg yolk is an emulsion, solubility problems are the embryo to develop and to hatch as did the controls; (2) toxic chemicals somewhat minimized. In the case of insoluble chemicals that are injected produced effects at dose levels which may be compared to those producas suspensions, it is also necessary to consider particle size in the evaluation ing effects in feeding experiments using animals; and (3) this technique of toxicity data. often provided toxicologic information which had not been shown by conIn order to have a toxic effect, the chemical must come in contact with ventional methods. the embryo either directly or indirectly through the bloodstream. Chemicals Table 1 lists the results obtained with some chemicals in common use such as the lower aliphatic alcohols have a coagulating effect on the proin food and shows the dosages used and the percentages of hatched chicks. , 49-362 O - 70 - 19 285 \ s !• •S •& H f **-< <*) O g £ u B 1 - .B^ ^ nj i/i 51 * -00 P nj •a •8-S •av T3 £ A 1*8 1> /—s gl '•« fe ^3 N ^ So V- Jin, r Bc»k deformity (»hor! . i*Wnwt, »Mwtll»*<< o Beak deformity • 1i W Most of the developin;g embryos died on the 15th day o f incubation Hatched chicks showed growth re- 284 " tx *-< §3 o o (u j^ Ui H rt o o CS o o CN o \o »n V} »O q •' q O O o O O g o o ^4 o o o 0 'J3 S in ui q Q S«1 ^ -5 O O U M <«! w s< q q qO 0 q 0 -^ rvj •* qqq o q P o 6-0 0 O0 w "2u C) rt »d •<-* (U u, rt Ui tn il ,S ^J cj oj ! > £ > £ > p |S [S _g ^ .S .S .S &tfc& *"* cj d o O (M »-l *H Undiluted Undiluted Undiluted _aj ^ rt oj ? p ' Undiluted aj ^> rt rt ? p Undiluted -S 3% in wai 4) c nj S3 'g J «v o s « ' ."S £' a ••.••» -r . •3 • §a OJ u rt J? J a 1 U U (U S Q, aS3 ^ a *>>? O) -SD, -11 I <(i. lj '- • chloride showed no hatch even at a level of 0 5 mg per egg. .mourea is a known carcinogen with a basic effect ^^^ f The hatch time was delayed with increasing amounts of this chemical At | IteTof™egg no chicks hatched and the embryo require 35 days """ to develop to the stage normally attained at 20 days. At 2 5 m g _ p e r e g g some chicks hatched, but most of them had to be helped out of the shell. This effect has been reported also by Yushok (1950) Cavanagh (1954) reported that triorthocresyl phosphate (TOLF). a weUkno'n plasticizer fo'r nonfood use, causes paralysis when.fed£> aduU chickens. We observed this paralysis in some chicks which hatched from eggs injected with 10 mg of the undiluted chemical. °° J . . . . T . I 1.~.«.*.U«**n V»«1C 1 5c 5 .aj 4J S ' d) 1i 1« «i. '*s 13 1 T! 0. 0 V3 3 JS ifl ~ ^a >, U •a •i s! DibutylAroclor (chlori O tS ja Triortho O e Thioure. So S Concentrat 3 .2 ! nw Q M t questions as to the safety to the consumers of any food contaminated with C 8 • o bending of the tibia and a general shortening of the bones of the l egg, Sodium selenite proved to be highly toxic. At a level of 0.1 mg no embryos developed to more than the 5-day stage. Drbutyl-tin-dilaurate showed no hatch at a level of 10 mg per egg The majorhy of the embryos, which did not live more than 15 days at th,s 286 "rt ( Iw -H t "« be S 13' S K O,_rt oO ttj D >\ Sll JQ +f ^ g a; | 2 -g ^i *** *S3 3i «! rt 4J O flj * ^ x fe < CS'l * 287 &s a, 0 rt rt •g S "§ *•* p vj 0 0 0 os 1^ r-* S ° 8 3 3 •H o^ *o IH rt 1 have been shown to be toxic in some degree to animals, and which may be w£ ^ 1* found in some processed foods. Included in this group are various solvents, plasticizers, and insecticides. Some of the chemicals on this list require /—*—. further study, including observation of the chicks until they reach ma55 ^o o^ **•• • *o So fi TH o o g 3 Si turity, before they may be classified as to low or high toxicity. i»* 1' K k £•' '* * •3 £• q q o o oTt- S 2 2 2 '5 2 o 2 ° o 2 o o S o o S wo o•-< 55o 23 ^ rt « ^ fa ro o o o o o o 6 6 d 6 oooo'oo'do'ooo'o ddci oe 0""" The injection of chemicals into the yolk of fertile eggs prior to incubation is a method which can be advantageously used as an element in the evaluation of the safety of food additive chemicals and drugs, and which ^ could be used to screen new products-and eventually to correlate their toxicity with that of similar products already tested. If one considers that a chemical injected into the yolk may be comi¥ pared to a substance which has the power to cross the placental barrier, this technique, in addition to being an embryonic feeding study, assumes further importance in that it is also a reproduction study. The unfortunate experiences recently suffered with chemicals that have teratogenic effects |"4 in humans, and the failure of conventional testing methods to produce this effect in animals, emphasize the urgent necessity for new methods of analysis. Preliminary work that we have done in this area has given satisfactory results (Verrett and McLaughlin, 1963). f Since this represents a system in which the chemical is in direct contact with the embryo throughout development, it is more than likely that any 1 toxic or teratogenic effects would be readily observed. However, there is always the possibility that the chicken will not be a species susceptible to a i?i particular compound, just as it has been shown that the other commonly i used species of animals do not respond to all chemicals in a similar manner. 1 It is also possible for the chicken to be more sensitive to a chemical than other species. f Finally, this technique may be applied also to the study of the synergistic effects of chemicals. Results of experiments in our laboratory on T H * f •d "3 B 0 1 "0 T3 O £ JB £! i H3 i-g q ^5 a .g 13 c s ii K3 c P "O o «J fe' 'S 13 S i! '^ I I a3 s 0) "§.' "o Ethyl acetate 'a , 5rt •s Diethylene glyc o fl s is* il 4) S «> T3 « 13 r-« a T3 « Undilu-ted ^ OJ tj C O Methanol 1/3 I "S u C _w Undiluted ^ fl Malathion •fl J3 Carbon tetrachl c 0 S Acetone «-Butanol 1 '5 ""* " •9 8 « •R ^ " W -a "S>•> . &s • .a« „ "3> t*> w. t o S t ^ ^ W "S. S 5? " S 7 o. « wi Q PM £ a -8 & S 2o. -p .b S SQ i l t t w DISCUSSION l!i y | F 288 -;S> ;w I n•AM the potentiation of a few pesticides have been very encouraging (Marfi«| and Mutchler, 1963). SUMMARY An evaluation of toxicity by injection of the chemical into the yolk sac of eggs prior to incubation gave the following results: Water, propylene glycol, corn oil, peanut oil, isotonic saline solution, and isotoaic '|5|i glucose solution showed no toxicity or a very low order of toxicity. ";|if Mercuric chloride, lead acetate, selenium, triorthocresyl phosphate, £,#'-diam!< diphenylmethane, thiourea, Aroclor 1242, and dibutyl-tin-dilaurate showed a W order of toxicity and/or teratogenic effects at certain levels. Acetone, methanol, ethanol, n-butanol, diethylene glycol, ethylene glycol, ii* ' propanol, di-2-ethylhexyl phthalate, hydrochloric acid, carbon tetrachloride, etijf \ acetate, malathion, heptachlor, and styrene showed an intermediate order of toxidiy, REFERENCES CAVANACH, J. B, (3954). The toxic effects of tri-ortho-cresyl phosphate on li* nervous system: an experimental study in hens. /. Neural. Neurosurg. Psychial. il, 163-172. DE FRANCISCIS, P., and BOCALATTE, F. (1962). Lead acetate and develqpment of If* chick embryo. Nature 193, 989-990. FERE, C. (1893). Note sur 1'influence, sur 1'incubation de 1'oeuf de poule, d'injectiom pre'alables dans 1'albumen, de solutions de sel, de glucose, de glycerine. Compl. Rend, Soc. Biol, 45, 831. FERE, C. (1899). Teratogenie experimentale et pathologic gene>ale. Cinquanlenoin de la SocUte de Biologic, Vol. jubilaire, pp. 360-369. GOERTTLER, K. (1962). Der 'teratologische Grundversuch1 am bebruteten Hiihnchenkeim seine Mo'glichkeiten und Grenzen. Klin, Wochschr. 40, 809. HAMBURGER, V., and HAMILTON, H. L. (1951). A series of normal stages in the development of the chick embryo. /. Morphol. 88, 49-92. KARNOFSKY, D. A., and RIDCWAY, L. P. (1952). Production of injury to the central nervous system of the chick embryo by lead salts. J. Pharmacol. Exptl. Therap. 104, 176-186. KEMPER, F. (1962). Thalidomid und Entwicklung von Huhnerembryonen. Arzneimittel-Forsch. 1 2 , 640. . , ' , , . , , . , MCLAUGHLIN, J., JR., and MUTCHLER, M. K: (1962). Toxicity of some chemicals measured by injection into chicken eggs. Federation Proc. 21, 450. MARLIAC, J. P. (1962). Injection of chemicals into chicken eggs as a toxicity test. Federation Proc. 21, 450. MARLIAC, J. P., and MUTCHLER, M. K. (1963). Use of the chick embryo technique for detecting potentiating effects of chemicals. Federation Proc. 22, 188. PLATT, B. S., STEWART, R. J. C., and GUPTA, S. R. (1962), The chick embryo as a test organism for toxic substances in food. Proc. Nutr, Soc. (Engl, Scot.) 21, XXX. RIDGWAY, L. P., and KASNOFSKY, D. A. (1952). The effects of metals on the chick embryo: toxicity and production of abnormalities in development. Ann. N.Y. Acad. Sci. 66, 203-215. 289 A. L. (I960). The Avian Enoryo: Structural and Factional Develop- mtnt, 1st ed. Macmillan, New York. , A. L., and ROMANO^ A. J. (1949). The Awn ff M. J., and MCLAUGHLIN, J., JR. (1963). use 01 me t« iff compounds on the rat liver. IMi. York * ^ / 290 Use of the Chicken Embryo in the Assay of Aflatoxtn Toxicilj' By M. JACQUELINE VERRETT, JEAN-PIERRE MARLIAC, and JOSEI'lI McLAUGHLIN, JR. (Division of Toxicological Evaluation, Food and Drug Administration, Washington, D.C. 20204) The possibility of using the chicken of aflaloxiii-producing mold cultures inembryo as a test organism for the assay dicated that the chicken embryo mi of fiflaloxin toxicity lias been investisensitive to these compounds. A dosegnled and found to he feasible. The injections of test solutions were made response was exhibited in that the toxicity of the samples was related to the before incubation, in fertile White Leg- mortality at Uie j t m e of hatching. horn eggs, by either of iwo routes: Extracts of nflatoxin-free pcnnul yolk or air cell. The development of products were found to Le nontoxie lo the embryos was observed for the full the chicken embryo. The addition of 21 day incubation period. The vehicle aflatoxin B, to such ui.coiiiuminatcd for all injections was propylene glycol. The injection of solutions of pure extracts produced the expected toxicily nflatoxiiis B l nnd G |t and of extracts in the embryos. The i.ijeclion of extracts from contaminated peanut prod- 291 fef »«« resulted in a toxic response that injected at two or more levels when there ft*f«*rr*latod well with that obtained by was sufficient material available. More than fjkjedion of pure aflaloxin B, solutions 8,000 eggs have been used in these studies to "$JH llic same dose levels, nnd in most date. The injections into the eggs were made i the chemical analysis was conThe presence of aflaloxins G,, by one of two routes: into the yolk, or into ,,-„ ond G,, had no apparent effect on the air cell. The technique for injection into 1*0 loxicily due to aflatoxin B,, at the the yolk has been described previously (2). * Itvels nl which they occurred in the The volume injected into the yolk was 0.05 ml or less in all cases. For injections into particular samples tested. The separation of aflaloxin B, from the air cell, n hole of about 5 mm diameter ftmlnmiiuitcd extracts by thin-layer was drilled in the shell over the air cell. (bromnlograpliy, and its subsequent The solution was then deposited on the egg tlulion from the plates and injection membrane by a syringe, and the hole was Into the eggs, confirmed lhat the toxicily scaled with adhesive cellophane tape. The «f these extracts was due primarily to eggs were allowed to remain undisturbed in a vertical position (air cell up) for about Uielr aflatoxin Bj content. an hour to let the material disperse. The volume injected into the air cell was rer^., The sensitivity of the chicken embryo to stricted to 0.04 ml or less. The solvent used for all injections was JS tflstoxins was reported in 1962 by Platt.-et IF' «/. (1) who observed that preparations of propylene glycol, which was known, from $': "groundnut toxin" injected into the yolk of previous investigations (3), to be nontoxic in 6-day old chicken embryos caused deaths, the eggs at the levels used. However, eggs and'that the quantities required were about were periodically injected with this solvent l/200th of those required .for a positive re- in appropriate amounts and incubated with the aflatoxin-injected eggs. Noninjected conwit in the day-old duckling. An investigation of the feasibility of using trols and drilled-only controls were also inthe chicken embryo for a bioassay of afla- cluded in the experiments to provide the loxin toxicity is currently underway in our necessary data on the background mortality. The eggs were incubated (2) and candled Inboratories. The preliminary results reported here consider only the general sys- daily from the fourth inc.ubation day on, at temic toxicity of the aflntoxins lo the chicken which times all nonviablc embryos were reembryo. At the present time, the studies moved and examined grossly. are not sufficiently complete to verify whether the aflatoxins produce any specific pathological lesion in the embryos. Experimental The sensitivity of the chicken embryo to aflatoxins was. studied by injecting the following: ()) solutions of pure aflatoxin "S>1 and pure aflatoxin G t ; (2) extracts of aflatoxinproduciug mold cultures; (3) extracts of raw peanuts, roasted peanuts, peanut meal, and peanut butter; and finnlly (4) aflatoxin B, obtained from crude extracts by clution from (lun-laycr chroinfttographic. plates. The solutions were injected into fertile While Leghorn eggs before incubation. Groups of at least 20 eggs were used at each dose level of a sample, nnd each sample was Results and Discussion 1'nrificd Aflatoxin Solutions and 1(1 old Culture Extracts.—The toxicity of solutions of crystalline aflatoxin B, and aflatoxin G, 'to the chicken embryo was first determined. The toxicity of aflatoxin B, to the chicken • embryo was greater when injected via the air cell route than when injected into the yolk. Figure 1 contains plots of the mortality at 21 days due to the injection of several dose levels of nflaloxin B, by both the yolk and the air cell routes. The LD5ns obtained were 0.048 nnd 0.025 /ig for the yolk and air cell routes, respectively. Allatoxin (!,, which was injected inlo the yulk only, showed a lower loxicily to the chicken embryo than that obtained "with 292 aflaloxin 33,. The injection of 1.0 us of nflatoxin 0, produced ft mortality of 00% (nt 21 days), while 2.0 //.g wns. required to produce a mortality of 00%. fig- ?—Toxicity of aflatoxin Bi in Ihe chicken embryo; mortality at 21 clays, LOsoi yolk, 0.048 /ig; air cell, 0.025 fig. Open squares: yolk injection, Closed squares: air cell injection. Examination of nonsurviving embryos from eggs injected with nflatoxin B, by either route revealed a severe growth retardation in most cases, In addition, edema, hemorrhage, undordevfilopmciit of the moseneephnlon (in embryos that died before the seventh day), mottled and granular liver surface, short logs, and slight, clubbing of the down were also observed in many of those embryos. Extracts of several cultures of nflntoxinproduciug molds were used to determine the sensitivity of the chicken embryo to combinations of nflntoxing B n G,, B,,, and G.,. The concentrations of these four constituents in (he exfracts were known from prior chemical analysis. The extracts were injected into eggs by both routes, in amounts designed to contain .specific levels of aflaloxin B,, to compare their loxicities to I how of solutions of (ho pure nflnloxin B, at the same dose levels. 293 and air cell injections were made in embryos route, and tlmt the toxicity was not The toxicilics of these extracts to ll* from 1 to 18 days old. iiliy different from that obtained bryos showed a good correlation wi& These experiments revealed that, with both jj*i* UK* solution of pure adatqxin U t of the standard solution of nflatoxin B,. Tl*ff injection routes, the embryonic, sensitivity to M* concentration. no apparent alteration in the toxicity «< (A* the aflatoxin decreased rapidly as the emAfntoxin-conlaminated Peanut Products. toxin Bj due to the presence of nftiWHW? bryo age increased, and the maximum toxic A c l s ' from raw or roasted peanuts, B,, G,, and G 2 at the levels at wliiefc tf effect was obtained with pre-inc.uba.tion inmeal, and peanut butter, which were occurred in these extracts. n to contain aflatoxins by chemical amil- jections. Aflatoxin-free Extracts of I'camil Evaluation uj Sumjilc. Toxicily.—In the were injected into eggs by both routes. nets.—The usefulness of the chicken course of these studies it was also observed amount of extract to be injected in the in a bioassay of aflatoxins in peanut that the toxicity of aflatoxin B, injected at was calculated on the basis of the nflanets depends on whether the corisfiltr the higher dose levels was apparent early in teun Jl, content determined chemically, irreaflaloxin-freo peanut product extract* the incubation period, since most of the om'livo of the amounts of aflatoxins U , G,, 5 inherently toxic to the embryos. • bryos did not survive beyond the eighth to . present. In most instances the. results To determine the toxicily of these u»i*< tenth day. With lower dose levels', it is necflwitolxmited the chemical analysis, since the rials, extracts of raw • peanuts, roasted p»j essary to continue observations for the duraftaieity was comparable to that obtained nuts, peanut meal, and peitnut butter, »fc«ii tion of the 21 day period, since many emWlh the injection of equivalent amounts of were found to be free of afiatoxins by diw* bryos survive longer than the tenth dny but UK pure aflatoxin B, solution. eal analysis, were injected into eggs 1>) Wfe fail to hatch. In these instances an evaluaAfatoxin B, Obtained by Thin-Layer the yolk and air coll routes. In most of tin* tion of the toxicity of a sample on the basis Cknmatoornphy (TLC).-ln order to conexperiments the equivalent of original pac, of survivors at 8 or 10 days would be preInn Hint the toxicily of the contaminated nut product injected ranged from 1 l o J f mature, and the true toxicity of a sample fcrtrncts wns primarily due to their aflatoxin per egg. The toxicity was low for nil rf might be underestimated. B, content, separate portions of some cxthese extracts; in general, it was equal I* IrtcU were subjected to TLC and the roor only slightly higher limn that of Ifcr Acknowledgments wlliiiK aflatoxin B, spots were removed from background, which ranged from 0 to SC^ We wish to thank Mary 1C. Mutchler and U»e plates, eluted, dissolved in propylene mortality. William F. Scott for their technical assistance fljcol, and injected into eggs. One experiment was carried out with w in this work. Separate experiments were performed to extract of aflatoxin-frco raw peanuts, *• The Division of Kood Chfinittry, Food and confirm that no toxic materials were derived jccted by both the yolk and air cell roiih Drug Administration, supplied the pure, aflafrom the TLC process itself. TLC "blanks" in quantities corresponding to a raw pe.im* toxin B, and G t and the extracts of mold injected into eggs in a similar manner had equivalent of 1, 2, 4, and S g per egg. Tbt cultures and peanut products, nnd per* very low toxicity and .were comparable to toxicity observed for the S g level wns «* formed the chemical analyses of these exbackground. significantly higher than dial observed f(* The toxic.it.ies of these eluted aflatoxin D, tracts. the ] g level pr that of the background, •pots from more than 20 extracts of a variIn the same experiment, known qnantilM HRFUBKNCKS ety of peanut products correlated very well of the pure aflatoxin B, were added to lira (1) Pln.lt, B, S., Stewnrt, U. J. C., nnd Gupta, with that of standard nflatoxin B! solution raw peanut extract, and injected at the same I?.., Proc. Niilr. ,Soc., 30, 21 (t%2). injected at the same dose level, and verified (2) Mclaughlin, J., Jr., Marline, J. P., Vcrlevels mid by both routes. This was done l» (hat the toxicity of these extracts was, .in vctt, M. Jacqueline, Mulchler, Mary K., determine whether the nfinUixin B, toMnty am! 1'iUhMRh, 0. G., Toxicol. Appl. Phur(act, primarily due to aflatoxin B,. could be masked or enhanced by the presEmbryo Age and Aflatoxin Toxicity.—The , macol, 5, (CO (1903). ence of increasing amounts of peanut innlftoxicity'of aflatoxin I}, to the chicken em- (3) Mclaughlin, J., Jr., Marline, J. P., Vervetl, rial. Separate groups of eggs were injected M. Jncr|iK'line, Mutclilcr, Mnry K., nnd bryo n't various stngcs of incubation was also with corresponding amounts of the st.iwl.inl • FilzlniRh, 0. G., Am. Ind. llyg. Aasnc. J., investigated. Single injections into the yolk aflatoxin B, solution for comparison. Tltc 25,282 (UIQ-i). wore made up to the fourth inco.ba.lion day, results of this experiment, indicated that ni 1 little as- 2. ) pph of aflatoxin B, in the original raw peanuts could be oasily detected by Reprinted from the Journnl of the Association of Official Agricultural ChenM,, Vol. 47, administration of the extract by either inDecember 1964. . Tills impel- wns prcwnhil nt tliu "sw7n'lyijitflilh Aimiiiil Mwliiii; nf llic Asmrinlinn nf Ollicml Agrii'iiltm-iil ClirmiKts Oct. 10 -"2 111(11 nt Wn'shiiiKlnii, U.C.. 294 295 THE BOLE OF "Toxic FAT" IN THE PRODUCTION OP HYDBOPEBICAKDIXJM AND ASOITES IN CHICKENS ie remainder renu Mood counts were conducted on a portion of the sample, and the was allowed to clot. After centrifugation the serum was saved for total protein,' 7 electrolyte concentration, and nonprotein nitrogen determinations' and electrophoretic studies.21 In experiment 1, surviving birds were used for vascular perfusion studies. Immediately after they were killed, the thoracic aorta was perfused with 5.0% dextrose to remove blood, and 1.0% silver nitrate was injected to outline the "cement substance" between the endothelial cells of the mesenteric capillaries. Slides were prepared of the mesenteric vessels and examined for alteration of Interendothelial silver precipitate in birds consuming toxic fat. Five control and 5 test birds given (he ration containing 1.0% toxic fat were "elected for hemodynamic studies after 150 days on trial. Venous pressures were determined by making an incision through the skin over the jugular vein. The vein was dissected away from the surrounding tissue, and 2 ligatures were placed around the vessel. The superior legature was made secure, and a small incision was made into the vessel. A No. 5 cardiac catheter was placed In the vein and made secure by the inferior ligature. The birds were placed under a fluoroscope and the exact position of the catheter determined. The catheter was attached to a 3-mrn, water manometer filled with physiologic saline solution, thus enabling pressures to be determined in the venae cavae flnd right ventricles. At the termination of experiment 2, tissues were obtained for electron microscopy by severing the cervical spinal cord and exposing the heart by removal of the sternum. Sections were taken from the left ventricle and cut into blocks of approximately 1 mm. with a razor blade. The tissues were immediately fixed in15 1.0% osmium tetroxide, dehydrated, and embedded as outlined by Palade. Thin sections2 were cut on a cicrotome.1 Tissues were examined with an electron microscope opera/ting at 50 kv. James E. Allen, D.V.M., Ph.D. SUMMARY When "toxic fat" was added to the diet of experimental birds at concentrations from 0.25 to 5,0% for 35 to 150 days, edema of the myocardium, skeletal musculature, and lungs; hydropericardium; ascites; and foci of lymphoytes in the myocardium and epicardium were observed. Appreciable changes were not observed in the total serum protein levels, albumin: globulin ratio, electrolyte balance, or in the nonprotein nitrogen levels of the blood. There was dilation, edema, and lymphocytic infiltration of the heart. The myocardial mitochondria were vacuolated and shrunken. An increase in venous pressure was also noticed. The fluid imbalance observed in birds that consumed toxic fat' did not result from a decrease in total blood proteins or from an alteration in the albumin: globulin ratio, but was associated with cardiac decompensation and increased capillary permeability. Schmittle et al.™ were the first to incriminate some fats as responsible for the production of hydropericardium and ascites in young chickens. Subsequent studies 3i" have demonstrated a reduction in growth rate, retarded sexual development, and increased mortality in pullets that have consumed toxic fat A marked reduction was observed in the hatchability of eggs from hens fed toxic fat." Turkeys and ducks appeared to be4 less susceptible than chickens to the detrimental effects of this fat in the diet. Fat-soluble tissue extracts from chickens fed toxic fat were capable of producing hydropericardium and ascites when uadded to the diet of other birds." Associated with the transudate, Sanger at al. observed necrosis of the liver, subepicardial hemorrhage, and lymphocytic infiltration of muscle fibers in chickens consuming toxic fat. In addition to these changes, Simpson et al.™ also noticed bile duct hyperplasia and proliferation of the endothelium in the parenchymal tissues. The unsaponifiable portion of some batches of fat was found responsible for the toxicity.2'18'17'2* Repeated passages of this fraction through alumina and silica gel columns led to the isolation of a crystalline factor of unknown structure which was able to produce anasarca.8 The primary aim of this study was to investigate the mechanism by which toxic fat produces anasarca. Preliminary studies suggested that a vascular or cardiac injury may be responsible for the transudation of fluid into tissues. In the hope that some clarification of the mechanism might be obtained, arterial perfusions, recordings of hemodynamic changes, and ultrastructural myocardial studies were undertaken. MATERIALS AND METHODS In experiment 1 (acute), 100 White Leghorn cockerels, 1 day old, were separated into 5 groups of 20, placed in heated batteries,- and fed rations containing 0, 0.5, 1.0, 3.0, and 5.0% toxic fat* for 35 days. Because the exact chemical nature of toxic fat is not known, the amount of the toxic material undoubtedly varies in fats from different sources. The fat used in these experiments was from the same source, identical shipments, and had the samd LDso when f e d t o day-old chickens. ' . . . . , In experiment 2 (chronic), forty-eight 4-week-old cockerels of comparable weight were given, for 150 days, a ration** containing 0, 0.25, and 1.0% toxic fat The birds were fed and watered daily. Throughout the trial period Hie general appearance, food consumptions, and mortality were spleen, pancreas, kidneys, adrenal glands, skeletal muscle, brain, and bone marrow were fixed in 10.0% neutral formalin, embedded in paraffin, sectioned at 7 p, and stained with hematoxylin and eosin. Frozen sections were prepared from the livers and kidneys and stained with Sudan IV to demonstrate neutral fats. At the termination of the trials, blood was obtained from the cephalic vein, complete Received for publication .Tan. 8, 1084. Krom the Depnrtment of Pathology, University of Wisconsin Mecllcnl School, Madison. This Investigation was supported in part by Public Henlth Service research grant nF-898!) from the Nntionnl Heart Institute, Public Health Service. The author Is Indebted to Mr. Homer Montague for the photography. • Emery Industries, Cincinnati. Ohio. •• McMlllen Feed Mills, Fort Wayne, Ind. RESULTS i In experiment 1, birds given rations containing 0 to 1,0% toxic fat survived, whereas those consuming rations with 3.0 and 5.0% toxic fat had a 25.0 to G5.0% mortality, respectively (Table 1). As the level of toxic fat in the diet TABLE 1.—EFFECTS OF TOXIC FAT CONSUMPTION ON CHICKENS No. birds 0 0.5 20 20. 20.. 20 Toxic fat In diet (percent) 20 12 12.. 12.. 12... :; 1.0 3.0 5.0 0 0,25 0.5 1.0 Days on trial No. died 35 35 35 35 35 150 150 150 150 0 2 11 0 1 4 6 Ascites Hydropericardium 0 0L . 20 20 0 1 0 2 20 20 0 4 8 11 was increased, there was a corresponding decrease in growth rate. The control birds averaged 348 Gm., whereas those given rations with toxic fat averaged 176 Gm., at the end of 35 days. Hydropericardium and ascites were not observed in the chickens fed rations containing 0 and 0.5% toxic fat. Perionrdial fluid volumes ranged from 0.5 to 5.0 ml. in the birds given rations with 1.0 to 0.5% fat. Ascites was also observed in these groups, but the volume was not determined due to the partial coagulation of the transudate. In experiment 2, 6 birds fed 1.0%, 4 fed 0.5%, and 1 fed 0.25%.toxic fat in the ration died. The mean survival time for the groups was 80 ± 20, 100 ± 14, and 135 days, respectively. The control group gained an average of 10.5 Gm., whereas the survivors of the group fed the ration containing 1.0% toxic fat gained 14.9 Gm. per day. The weight gain of the surviving birds of .the 1 Porter-Blum m)cro f ome, Ivan Sorvall. Inc., No-rwalk. Conn. 1 RCA, BMU-3G, Radio Corporation of America, Cnmden, N.J. 296 297 fed rations containing 0.5 and 0.25% toxic fat was almost to that of the controls. Hydropericardium was observed in 11 and ascites birds fed rations containing 1.0% toxic fat. Eight of 12 birds on the 0,5% toxic fat ration had hydropericardium but were free of ascites. The average volume of pericardial transudate in the test birds was 13.5 ml. No appreciable difference was noticed in the organ weights of the test and control birds, With the exception of the testes and hearts of the birds in experiment 2. The average weight per testis of birds in the control group was 14.9 Gm., whereas that of the 0.25% group was 6.6 Gm., the 0.5% group was 4.1 Gm., and the 1.0% group was 3.9 Gm. The hearts of birds of the control group averaged 15.0 Gm., and those of birds in the test groups averaged 23.0 Gm. and were markedly dilated in most cases (Fig. 8). Tissue sections of the various organs were examined microscopically. Tlie birds with hydropericardium had fibrinous deposits and foci of lymphocytes on the visseral pericardium. The muscle fibers were separated by edematous fluid. A number of the small myocardial artieries were surrounded by lymphocytes. There were also foci of lymphocytes between the myocardial fibers (Fig. 12). The lungs were congested and had a moderate amount of peribronchial lymphoid hyperplasia. Pulmonary edema was found in those birds which died during the experiments. Livers from birds .with marked ascites frequently had thickened capsules. In many cases there was coagulation of the transudate on the convex surface of the liver which formed a false capsule (Fig. 3). There was moderate fatty infiltration of the hepatic cells in birds given toxic fat in experiments 1 and 2 (Fig. 4). Extensive lymphoid hyperplasia around the perlportal areas was found consistently (Fig. 5). Five of the test birds in experiment 2 also had myeloid hyperplasia scattered throughout the parenchyma! tissue of the liver. Microscopic examination of the testes of the birds given toxic fat in experiment 2 revealed a reduction in size of the seminiferous tubules. The Sertoll cells and spermatogonia appeared normal, but there was a reduction of the primary and secondary spermatocytes. These maturing cells were reduced to the point where no spermatids and spermatozoa were observed. Cells in tlie testes of the test birds appeared normal. The major difference between testes and 143 mEq./llter in the control birds. The potassium levate were 5.3 raEq./liter in the test birds and 5.5 mEq./liter in the control birds. ^A When 1.0% silver nitrate solution was perfused in the thorac^aorta and the mesenteric capillaries examined microscopically, a difference was found in the cement substance located between the endothelial cells of the test and control birds in experiment 1. The control group had uniform diamond-shaped bands of silver precipitate between the capillary endothelial cells, whereas the bands in the test birds were irregular and indistinct (Fig. 9,10). Catheterization of the heart revealed tlie mean right ventricular pressure of the control birds to have an average value of 15.3 Cm. of water, whereas the test birds "had an average of 21.3 Cm. of water. The pressures in the inferior vena cava averaged 5.5 cm. of water in the control birds and 7.1 cm. of water in the test birds (Table 3). TABLE 2.—SERUM PROTEIN STUDIES ON CHICKENS FED TOXIC FAT Toxic fat in diet (percent) 0 .. 0.5... 1.0... 3,0.. 5. 0... Serum protein (Gm./lOO cc.) 3.36 3,24 . 3.14 . 3.08 . 3.06 Albumin (Gm./lOO cc.) Globulin A/G (Gm./lOO cc.) Nonprotein nitrogen (mg./100cc.) Sodium (mEq./liter) Potassium (mEq./lilsr) 1. 06 2. 30 46 21.7 143 5.5 0.94 2.12 44 20.7 146 5.3 of birds in test and control groups was a lack of spermatogenesis and n marked reduction in tubular size (Fig. 6,7) in testes of test birds. Total blood proteins were determined by the micro-Kjeldahl method. Tlie control group of experiment 1 had an average value of 3.3C Gm./lOO cc. The 0.5, 1,0, 3.0 and 5,0% groups had values of 3.24, 3.14, 3.08, and 3.00 Gm./lOO cc. of protein, respectively (Table 2). In experiment 2, it was found that the groups given rations containing 1.0% toxic fat had an average of 4.1 Gm./lOO cc., the 0.25% group averaged 4,5 Gm./lOO cc., and the controls had an average value of 4.8 Gm./lOO cc. of serum protein. The protein level of the ascitic fluid of the experimental birds was 1.7 Gm./lOO cc., with no appreciable difference in the levels of the various groups. The albumin: globulin ratio was determined by paper electrophoresis, and little difference was found in the protein ratio of the various groups. Sodium, potassium, and nonprotein nitrogen levels were determined on serum Rumples from 6 birds given rations containing 5.0% toxic fat and 6 control birds. No appreciable difference was found in the nonprotein nitrogen levels of the 2 groups, with the controls averaging 21.5 mg./lOO cc. and the test birds 20.7 mg./lOO cc. The sodium values were 146 mEq./liter in the experimental TABLE 3,-HEMODYNAMIC ALTERATIONS DUE TO TOXIC FAT CONSUMPTION IN CHICKENS Toxic fat in diet (percent) 0 . .. o 0 0 0 5,0... 5.0 5.0 5.0 5.0 „ " ". ." Mean right ventricular pressure Mean superior vena cava pressure Mean inferior vena cava pressure (cm. HjO) (cm. HsO) (cm. HsO) 5.6 5.4 5.2 5.8 5.4 7.4 7.0 7.2 6.5 6.8 5.2 5.3 5.6 5.8 5.7 7.0 7.1 6.9 7.2 7.4 15.4 15.8 15.2 15.0 15.0 22.2 20.0 21.0 25.5 20.4 Preliminary data obtained from electron micographs of heart muscle from 10 test and 10 control birds of experiment 2 indicted that the major changes occurred in the mitochondria of the cardiac muscle cells. In some cases the mitochondria were markedly shrunken and vacuolated with indistinct cristae. In the birds with more advanced cases, many of the mitochondria had disappeared, leaving large areas devoid of any organelle. DISCUSSION There was normal development of all organs with the exception of the testes of birds in experiment 2, -which were markedly reduced in size. Kumaran and Turner10 outlined the tubular and spermatogenic development in chickens at various states of maturity. It would appear that the consumption of toxic fat In these experiments retarded testicular development by approximately 2 mouths in the 6-month-old roosters. Despite the reduction in testicular size, secondary sex characteristics such as comb size and body conformation were unaffected. In earlier studies,1 young birds were killed every other day for 3 weeks to determine when hydropericardium or ascites developed. Hydropericardium was usually noticed about the 16th day after feeding toxic fat in the ration was Initiated, without ascites or pulmonary edema. Fluid accumulation in the lungs and peritoneal cavity invariably developed later. When the concentration of . toxic fat in experiment 2 was reduced to a low level, the birds failed to develop ascites, but hydropericardium was a consistent observation. The most prominent lesions at necropsy were hydropericardium, ascites, and pulmonary edema. A number of procedures were conducted to resolve what caused the anasarca. Birds with ascites and hydropericardium in many cases had higher serum protein levels than those in the control group. The albumin: globulin ratios of experimental and control birds were approximately comparable. The nonprotein nitrogen and electrolyte levels of the blood were within normal range in the test groups. There was a reduction in the cement substance in the experimental birds with hydropericardium and ascites. Since a large quantityu of fluid escapes between the endothelial cells rather than through them any alteration in the cementum would affect the permeability o£ the vessels." Whether this alteration in the cement substance of the capll- 299 298 larics wa$ associated with liypoxia of the endothelial cells resulting from cardiac decompensation or from a direct effect of toxic fat on the capillary mem- • brane remains to be clarified. Hemodynamic studies revealed an increase in right ventricular and vent cava pressures in the experimental birds. Associated with these pressure changes were dilatation and hypertrophy of the right side of the heart of birds given toxic fat. Pulmonary edema, cardiac dilatation, and increased venous pressure are indicative of cardiac decompensation. What do these data reveal in regard to the causes for the excessive extravascular fluids? The lack of any appreciable change ia the total blood protein and albumin: globulin ratio would indicate that the liver was producing adequate amounts of albumin. There were no marked alterations in kidney function, because the serum albumin and nonprotein nitrogen levels were comparable in the 2 groups, which suggests that the kidney tubules and adrenal cortex were not affected by toxic fat ingestion. The microscopic observations fortify the biochemical data, because the alterations in the liver, kidneys, and adrenal glands of test birds did not appear of sufficient magnitude to account for the anasarca. Since myocardial fibers are very active, there is a constant demand on tlie respiratory enzymes located in the numerous mitochondria between the myoflbrils. A rather high metabolic demand is implied, - because the ratio of mitochondria in cardiac muscle to skeletal muscle is approximately 500:1." Any significant alteration in the mitochondria would affect, the .respiratory enzymes and sooner or later lead to cardiac failure. Certainly this would appear to be a logical explanation for what occurs in birds consuming toxic fat. As the heart becomes less efficient, there will be an increase in hydrostatic pressure in the veins and capillaries, with a predisposition for the extravasation of fluid into the tissues and body cavities. REFERENCES 1. Allen, J. R.: The Role of "Toxic Fat" in the Production of Hydropericardium and Ascites. Ph.D. Dissertation, University of Wisconsin, Madison, June, 1001. Dissertation Abst, 22, (1961) : 545. 2. Brew, W. B., Dare, ,T. B., Benedict, J. H., Potter, G. C., and Sipos, E.: Characterization of a Type of Unidentified Compound Producing Edema in Chickens. ,T. Assoc. Off. Agric. Chem., 42, (1959) : 120-128. 2. Dunahoo, W. S., Edwards, H. M., Schmittle, S. C., and Fuller, H. S.: Studies on Toxic Fat in the Ration of Laying Hens and Pullets. Poult, Sci., 38, (1959) : 663-067. 4. Edgar, S. A., Bond, D. S., Melius, P., and Ingram, G. R.: The Effect of a Toxic Substance in Fat on Poultry. Poult. Sci., 37, (1958) : 1200. 5. Folin, 0., and Wu, H.: Revised Colorimetric Method for Determination of Uric Acid in Urine. J. Biol, Chem., 38, (1919) : 469-460. 6. Friedman, L., Firestone, D., Horwitz, W., Barnes D., Anstead, M., and Sime, G. : Studies of the Chick Edema Factor. J. Assoc. Off. Agric. Chem., 42, (1959) : 129-140. 7. Hald, P. M.: The Flame Photometer for the Measurement of Sodium and Potassium in Biological Material. J. Biol. Chem., 167, (1947) : 499-510, 8. Harman, R. E., Davis, G. E., Ott, W. H., Brink, N. G., and Kuehl, F. A.: The Isolation and Characterization of the Chicken Edema Factor. J. Am. Chem. Soc., 82, (1960) : 2078-2079. 9. Kingsley, G. R.: The Direct Biuret Method for the Determination of Serum Protein as Applied to Photoelectric and Visual Colorimetry. .T. Lab. & Olin. Med., 27, (1942) : 840-845. 10. Kumaran, J. D. S., and Turner, 0.: The Normal Development of Testes in Plymouth Rocks. Poult, Sci., 28, (1949) : 511-520, 11. Kuschner, M., and Lordell, D. H.: The Pathology of Congestive Heart Failure. ,T. Chron. Dis., 9, (1959) : 424-441. 12. Machlin, L. .T., Gordon, B. S., Meisky, K. A., and Maddy, K. H.: Relationship of Exudative Degradation to Toxicity in Certain Fats. Poult. Sci., 38, (1959) : 579-585. 13. Marches!, V. T.: The Passage of Colloidal Carbon Through Inflamed Endothelium. Proc. Roy. Soc. Biol., 156, (1962) : 550-552. Lin 'el14. Naber, E. C., Bletner, J. K,, and Touchburn, S. P.: Effect of Toxic Fats and Their Derivatives on Growth, Reproduction, Embryoni> opment, and Health of Chickens. Poult Sci., 37, (1958) : 1229. 15. Palade, G. E.: Study of Fixation for Electron Microscopy. J. Exptl. Med., OB,16. (1952) : 285-288. •J. R.: Passage of Molecules Through Capillary Walls. Pappenhelmer, Physiol. Rev., G. 33, C., (1953) : 387^23. 17. Potter, Brew, W. B., Patterson, P. L., and Sipos, B.: Current Status of the Toxic Fat Principle Causing the Chick Edema Syndrome. J. Am. Oil18. Chem. Soc.,V.36,L.,(195ft) 214-217. Sanger, Scott,: L., Hamdy, A., Gale C., and Pounden, W. D.: Alimentary Toxemia in Chickens. J.A.V.M.A., 133, (Aug. 1,1958) : 172-176. 19. Schmittle, S. C., Edwards, H. M., and Morris, D.: A Disorder of Chickens Probably Due to a Toxic Feed—Preliminary Report. J.A.V.M.A., 132, (March 1, 1958) 216-219. C. F., Pritchard, W. R., and Harms, R. H.: An Endotheliosls in 20. :Simpson, Chickens and Turkeys Caused by an Unidentified Dietary Factor. J.A.V.M.A., 134,21.(May 1, 1959) : 410-416. Williams, F. G., Pickels, E. G., and Curren, E. L.: Improved Hanging-strip Paper Electrophoresis Technique Science, 121, (1955) : 829-830. 22. Wooton, ,T. C., and Alexander, J. C.: Some Chemical Characteristics of the Chick Edema Disease Factor. J. Assoc. Off. Agric. Chem., 42, (1959) : 141148. , , SUMMARIO IN INTERLINQUA |; !_ f |,1 I, • < Le Rolo de "Grassia Toxic" in le Production de Hydropericardio e Ascites in Gallinas Quandro "grassia toxic" esseva addite al dieta de aves experimental a concentrationes de 0,25 a 6,0 pro cento durante 35 a 150 dies, le sequente alterationes esseva observate: (1) Edema del myocardio, del musculatura skeletic, e delpulmones; (2) hydropericardio; (3) ascites; e (4) focos de lymphocytes in le myocardio e le epicardio. Appreciabile alterationes non esseva observate in le nivellos de proteina total in le sero, in le proportion albumina a globulina, in le balancia electrolytic, e in le nivellos de nitrogeno non ligate a proteina in le sanguine. Esseva notate dilatation, edema, e infiltration lyrnphocytic del corde. Le mitochondrios myocardial esseva vacuolate e contrahite. Un augmento del tension venose esseva etiam notate. Le imbalancia de Hquido observate in aves que consumeva grassia toxic non resultava ab un decline in total protelnas de sanguine o ab un alteration in le proportion albumina a globuina sed esseva associate con dlscompensation cardiac e un augmento del permeabil- ; itate capillar. ]'.' i: 5 \; ; >i INDUSTRIALLY Ai;^uin^ INDUSTRIAL!* PORPHYRIA * Twenty-nine patients working in a chemical factory engaged in the manufacture of 2,4-dichlorophenol (2,4-D) and 2,4,5-trichlorophenol (2,4,5-T) exhibiting features of chloracne were studied for the presence of porphyria cutanen tarda. In 11 cases urinary uroporphyrins were elevated. Two of these patients who shpwed evidence of acquired porphyria with chloracne were hospitalized. The features of chloracne as well as the clinical and laboratory features of acquired porphyria have been discussed. There appeared to be an etiologic but not quantitative relationship between the chloracne in workers 'engaged in the manufacture of 2,4-D and 2,4,5-T and porphyria cutanea tarda of the acquired type. It is our feeling that either the finished chemicals or some intermediate are responsible for both diseases. Since Waldenstrom first implied that porphyria cutanea tarda might be acquired, a growing number of chemials have been implicated in the pathogenesis of this disease. These chemicals have included alcohol, sedatives, fungicides, etc.1"1' While treating a severe outbreak of chloracne in a factory which manufactures 2,4-D and 2,4,5-T, a number of workers were noted to have hyperpigmentation, hirsutlsm, fragility of the skin and vesiculobullous eruptions on exposed areas of skin, together with cutaneous findings of chloracne. Investigation revealed evidence of porphyria cutanea tarda of varying degrees of severity in 11 out of 29 workers investigated. Porphyria cutanea tarda has never before been described as related to chloracne, nor has it been ascribed to 4B-362 0—70 20 301 300 iwsltesj; PI | industrial exposure in the United States. This outbreak is therefore of int*H*:Sjj iSSlinotelns, albumin-globulin in adding more evidence to the growing concept that porphyria cutanea Unfaj ^twculiitlon, and thymol may be an acquired disease occurring after various insults to the liver, TWwtl "Igwer, as well as serological cases were studied in detail. ' -j$% From the Departments of Dermatology and Medicine, Newark Beth »-—*•''•' Hospital. ' ,,„, Chief of Dermatology, Newark Beth Israel Hospital (Dr. . Bleiberg) ; SenWJst r : »sident PllVBirfnn Merlini'tip. Newark Nuwnrlr Beth TSoth Israel Tc,.ool 7T/u,nUni /T-I- Woll»n»«- .« Resident Physician in in Medicine, Hospital (Dr. Assistant in Dermatology, Newark Beth Israel Hospital (Dr. Brodkin); tor of Medical Education and Consultant in Medicine, Newark Beth In»tli! Hospital (Dr. Applebaum). (jK^fft =«•£ SStSS "SSHs 4 mum ioi oi rf $«*r u. 8 wood' . mm. I'sj? jssStie «» ; ', ^i;;troi""i"8Mn fe^",si°. xri»KJ^i»«!*,rs^Sin ^«»»- A. ii three years as a chemical operator. His work brought him into intimate cosvj tact with the suspected chemicals. His past history included two attacks of bO- .' iury colic prior to 1953. He was never a heavy user of alcohol. A diagnosis ot'$ cholecystitis had been made and a cholecystectomy was performed early i»'*| 1953. After this he came to work at the factory in question. In 1956 he bcgaa || to notice some darkening of his skin and suffered right upper quadrant pala. ' A diagnosis of common duct obstruction was made, and this patient was opcr-'. ated on again in January of 1956. An unsuccessful attempt was made to prol* . the common duct, and no further operative procedure was done. During the postoperative course, this man received 2 gm of barbiturates. The patient stated that his urine had turned "the color of Coca-Cola" at least one year prior to the second operation. That spring, an eruption of bullae appeared on the face, ears, and hands. These lesions could be produced either by exposure to the sun or by pressure. In addition to the vesicular eruption, the patient noted progressive darkening of his skin and marked hirsutism, especially over the temples. Inspection of the urine revealed a Coca-Cola coloration and, under the Wood's Light, a brilliant red fluorescence. The exact laboratory data on this patient are no longer available except for the presence of quantitatively markedly increased excretion of urinary porphyrins including uroporphyrins, coproporphyrins, and porphobilinogen. This man is now alive and well and apparently is suffering minimal if any symptoms of porphyrim cutanea tarda. His present job does not entail the use of any chemicals. He has failed to present himself for further testing. CASE 2.—This is a 60-year-old white male who has been employed in the factory for seven years as a welder. In the course of his work, which consisted of welding tanks and pipes, lie was brought into frequent and prolonged contact with chemicals. He was admitted to the Newark Beth Israel Hospital for investigation. He stated that three months prior to admission, he had noted an increased darkening of the skin, thickening of the eyebrows, and a darkening and reddening of his urine. His family history and his past medical history were unrevealing, except for moderately heavy alcohol intake for some years. Physical examination revealed numerous comedones and small epidermoid cysts and furuncles the face, chest, and shoulders. There was intense grayishbrown Jiyperpig-mentation with a purplish tint on the exposed surfaces of the face, neck, chest, and hands and moderate hypertrichosis of the temples. The scalp hair showed a lusterless, dull silver color change. The liver edge was palpable about 3 cm below the right costal margin and was smooth and nontender. The remainder of the physical examination was within normal limits. A casual urine specimen revealed a strong tea color with a deep fluorescence, reddish, under the Wood's light. Laboratory studies revealed increased urinary uroporpliyin, coproporphyrin,' and urobilinogen excretion. There was no demonstrable porphobilinogen. The foc.es showed increased uroporphyrins and coproporphyrins. All porphyrin determinations were qualitative and done by the Watson-Schwartz method. Other significant findings included an elevated serum glutamic oxaloacetic transaminase ranging between 41 and 51 units on five different days. Serum glutamic pyruvic transaminase on corresponding days ranged between 53 and 04 units. The sulfobromophthalein retention was 6% in 30 minutes. The erythrocyte sedimentation rate (Westergren) was 94 mm In the first hour. All other Htudies, which included complete blood count, bleeding and clotting time, urirmlysis, glucose tolerance test, serum bilirubin, blood urea nitrogen, total tlfeUy. About four days aftei the^onse^ ^ ^^ appeared. ^g^ tlme " " i eruption on i stopped, but finer i t .in tu« ov.«**" ~ -.. .I: o,«»» - ——-^i^^l — htlent :t J 12.. 13.. H.. 15.. 17. 18. 19. 20. 21. 22 23 24. 25 26 . Mild ... Moderate Severe "„ Miid None " .. .do " Mild . None . . ..do ."..."...do lo'."." 11 is:. :. " . mffiion mentation Mild — . Severe Mild do . Severe . Mild do ; do Severe . Mild .. Moderate .. Severe : None do do Moderate • «V." s t I I ; Chloracne' do Hirsutism ••••- Urine Utoporphyrins pos None do .do . Pos None . Pos None ^j^ Contact Moderate Skin FwgllHl P°s- Do. None... None -.do Mild : Do. "."Mild..: ...do do Mo'd'efaYe-— - Nes None Do. "None Moderate .do Do. " do do Mild Do. 1 ..do Moderate—. "Severe ..do Do. " Mild Severe . Mild ..do — Do. Moderate— . • Moderate— "". Moderate do Do. ..do Severe ' ...do do— . Do. None ' do " None Do. ".".".'.-"-do— ..do Moderate do Po ... Pos do do "" do Do. None ""Moderate— . Severe N " Mild .do Ne 8 Moderate... " Marked ..do— "" Mild '00. None ....do " Mild Do. """" ..do— "" ....do Marked ModerateDo. "" Pos None "" do Marked-- " Moderate— Do. Pos "•"• . . .do Mild Do. None -, " None..... . None . .do Do. """ —do— ...do.... .do ..do—. ..do— Do. .do "....do "" ..do.... .do— Do. ..do ido ..""—do..., Do. """"Pos —do — """.""..do do— "",".". -do— ",. Pos— — :--:&--><»• i Brief period of employment. OH S3SSBS3SS5S3H** 302 303 defect of ingestion of hirsutism which involved the temples. The dull silvery tint of the taSf ible. He stated that in the past he had had episodes of blistering of IS* exposed skin. He also had noticed that his urine was dark on voiding. IW family history was noncontributory. The physical examination revealed t*1 laiysis 01 uie mmc. "- -— -intense hyperpigmentation of the face, neck, and hands. There was severe U* sutism involving the eyelids, eyebrows, and lateral aspects of the forelini p,,^,,-,.,, of chloracne does not usually. w™?^"" 8 "ioT2 ~13,T6). — (Fig 2). Oomdones and small epidermoid cysts were very prominent, and tliw IS^micals (patients 1, 8, 11, 10 or patients 2 ^ '^r^ 4> g, physical examination was within normal limits except for prolapsed hemW" p-fLUenta 7, 20, 25, 26 or patients rhoids. The following laboratory studies were within normal limits: complete fej ,wne does not usually correj*nT1 ^ to ™° Prft would appear that there is some or patients 5, 20, 25, 26). ^refoie^ wom^TO fl, thflt . blood cell count, urinalysis, bleeding and clotting time, prothrombin (lux. . mil. 1 . Individual susceptibility to these disease. It lias been oose _ _ _ _ * „ , „_,„„ Mood glucose tolerance test, urea nitrogen, cholestrol, bilirubin, alkaline pl«* H*' '4... » , _ , . , _ _ _ i _ iii, ,,/iniacuiaiir. time tend to phntase, total protein and albumin-globulin ratio, ceplialin flocculation, tliyidd turbidity, serum electrolytes including sodium potassium, chlorides, COi com*' bining power, calcium, and phosphorus. The serum glutamic oxaloacetic trans*minase on five successive days ranged between 39 and 56 units while II* serum glutamic pyruvic transaminase on corresponding days ranged betweffl i 47 and 72 units. The sulfobromophthalein retention was 8% in 30 minutes. Ttif electrocardiogram was normal. The chest x-ray revealed a diffuse nodular ; infiltration of both lungs due to pneumoconiosis. This was consistent with th» patient's history of having worked a number of years as a coal miner. T!«, plain film of the abdomen was negative. The urine revealed a negative Watson-Schwartz test The urine failed to fluoresce under the Wood's light Th* * «annot be overlooked. ., h , offered by Dr. Donald 3> P MC erythrocyte sedimentation rate (Westergren) was 24 mm in the first hour. . A liver biopsy was performed and the specimen immersed in saline. Under the Wood's light the specimen and saline in which it was immersed Buoreswd , 40 Union Ave, Irvington, NX faintly. On microscopic examination, the liver biopsy showed evidence of liver cell regeneration and hemofuscin deposition. A skin biopsy showed brown granBEFEBENCES ular pigmentation as the basal margin of the epidermis. There was a mild chronic inflammatory infiltrate scattered through the dermis. No sebaceous glands were visible in the sections. -W.M~.tro., Since the man's chloracne has-been so severe, he had been removed from contact with chemicals two years prior to his admission to the hospital. Till* probably was responsible for the failure to prove qualitative chemical evidence j, A . : UUOC4 vu^u»u ^..- v of porphyrins in the urine. It also may indicate that acquired porphyria cute.: The Porphyrias, Advances In Internal Medicine, Chicago: Year Book nea tarda is reversible. .»«. < » — >. " to SCREENING TESTS Twenty-six additional men working at this chemical factory were studied on on ambulatory basis. In addition to routine urinalysis, each urine specimen was tested for uroporphyrin by the Watson-Schwartz method. Bight out of tlie 26 manifested significantly increased excretion of urinary uroporphyrius by tlie Watson-Schwartz method. If the three cases described in the case reports above are added, this is a total of 11 cases of porphyria cutanea tarda of varying degrees of severity out of 29 patients tested, or 37+% (Table). COMMENT Hyperpigmentation in these workers was limited to the sun-exposed I areas of the head, neck, and hands. It was more frequently observed in the Negro patients involved. The degree of hyperpigmentation was roughly proportional to the severity of the chloracne. The hyperpigmentation varies from mild redness in extremely fair individuals to dark gray intense dusky bronzing of the skin. The degree of hirsutism was also proportional to the severity of the chloracne. This too was quite variable in degree but always involved the temples between the lateral half of the eyebrow and the temporal hair of the scalp. The hirsutism in a few cases, notably case 3, extended beyond this and involved both the upper and lower eyelids. The hair was of approximately tlie same texture and density as that of the eyebrows. The occupational environment of these men consists of a group of basic chemicals including acetic acid, phenol, monochloracetic acid and sodium hydroxide, plus the finished products 2,4-D and 2,4,5-T as well as many unknown intermediary products. It is known that one of the intermediaries ia a highly volatile chlorinated phenolic ether which contains six chlorine atoms. Thin particular compound, because of its volatility, in strongly suspected-of being a possible causal agent. Porphyria has been described in many cases as a - Dirin (I8tanbul) "warnes, JEI. u.. iuM,..j..^ In Bantu Races on the Wltwatersrand, S Afr Med J 7 20:781-784, 1955. Schmla, U., and Schwartz, S.: Experimental Porphyria: III. Hepatic Type Produced by Sedormld, Proc Soc Exp Blol Med 81:665-G89, 1952. * Goldberg, A., and Rlmlngton, G.: Experimentally Produced Porphyria In Animals, Proc Roy Soc A., (Blol) :257-279, C.: 1955. • Goldberg, and 143 Rlmlngton, Diseases of Porphyrln Metabolism: American Lecture 10 Series, Springfield, 111.: Charles C Thomas, Publisher, 19R2, p 194. Solomon, H. M., and Klgge, IT. H. J.: Disturbance In Porphyrin Metabolism Caused i... Woo/iiv,^ niotiivi l.4r-Dlhydro-2,4,6"Trlmethypyrldlne-3,5-Decarboxylate, Proc Soc Exp I. 3. E.; and Rimlngton, C.: Nervous and Blochemlci Intoxication, Nature (London) 191:363-_.., •JIIB ri-uutcui -^ Porphyria—Some Facts and Questions, New 5, il.:1960. Acquired Porphyria From Liver Tumor, Clln Scl 16 :517-527, 1957. ELECTRON MICHOSCOPIC ALTERATIONS IN THE LIVER OF CHICKENS , FED Toxic FAT* Toxic fat is the name applied to certain fats that produce hydropericardium, hydrothorax, and ascites when added to the diet of chickens.'1 "•" It has been demonstrated that10 the toxic fraction fe associated with the unsaponiflable portion of the fat. -" The toxicity can he increased by repeated passages through silica and alumina gel columns."' * Crystalline preparations of the toxic fraction have been prepared, but the chemical nature of the compound remains to be identified." Accepted publicationo£March 21, IflOfl. From the for Department Pathology and the Regional Primate Research Center, University of Wisconsin, Madison, Wlnconsln. ,' * This research -was supported in part by grants HB-08081 and FR-0167 from the National Institutes of Health. 304 1 ^, -ae following alterations that resulted from the consumption of toxic .-;!, 10 have been reported, ganger, Scott, Hamdy, Gale, and Pounden observed ff&t 1 GROSS AND MICROSCOPIC OBSERVATIONS jUj- A large quantity of colorless, semiclotted fluid accumulated in the subcutaft; neons tissue and the pectoral, thigh, and lower leg muscles were pale and edeIjjmatous. The abdominal cavity of each experimental chicken contained approxil^vWntely 40 ml. of ascitic fluid. The liver was somewhat mottled with rounded W < margins and a thick gelatinous material resembling coagulated plasma was EXPERIMENTAL PROCEDURE ||, Brmly attached to Jthe capsule in 25 per cent of the chickens that had received One hundred sixty-eight 1-day-old White Leghorn-New Hampshire chicken* ; '^toxlc fat. The kidneys were pale and swollen. All of the organs within the were divided into groups of 48 and 120 chickens. One-half of the chickens frota , i abdominal cavity appeared edematous. There was marked distention of the each group were given a commercial diet containing 3.0 per cent toxic M jjlpericardial sac with a clear, slightly yellow fluid. Five to 10 ml. of pericardial (Emery Industries, Inc., Cincinnati, Ohio), while the remaining chicken*-, flktfluld were obtained from each chicken. There was a noticeable dilation and received a comparable diet which contained 3.0 per cent corn oil. In tlie group ; 1;: i hypertrophy of the right side of the heart and the myocardium was pale, with of 48 chickens, three control and three experimental chickens were killed every P; no gross lesions evident. The lungs were extremely edematous and the tracheoother day for 16 clays and sections of the liver were obtained for electron , |f .bronchial tree was filled with blood-tinged, foamy fluid. The brain was edemamicroscopic evaluation. i i|' ,tous but free of other gross lesions. The group of 120 chickens remained on the toxic fat diet until the LDw wa» j p/ The major microscopic alterations were observed in the liver. Its capsular established. From the survivors, blood was obtained for hematocrit," henioglo, ; {§;' surface was covered by a thick layer of homogeneous, eosinophilic material bin,8 total serum protein,13 serum electrophoretic pattern,23 and electrolyte studies." ' |i .containing numerous fibrin strands. Although the general architectxire of the The ascitic fluid was also collected for total protein and electrophoretic pattern |u; Hver parenchyma was maintained, numerous foci of necrosis were observed determinations. All chickens that died during the course of the experiment n» :$ J > ! (Fig. I), These lesions varied in size, from only two or three cells to a major well as those sacrificed at its completion were necropsied. Portions of the liver 5'ji portion of the lobule. There was a fairly sharp line of demarcation between were fixed in 10 per cent buffered formalin for 24 hours, dehydrated, embedded 5;:, the viable arid dead cells. Immediately adjacent to these necrotic foci, the cytoin paraffin, sectioned and stained with hematoxylin and eosin. Other portions jj: plasm of the parenchymal cells was quite vacuolated, but otherwise not of tlie liver were sectioned on a cryostat and stained with Sudan IV for neu;S~• remarkable. tral fats. Additional paraffin-embedded tissues .were stained with aniline blue THIN SECTIONS FOB LIGHT MICROSCOPY and phosphotungstic acid-hematoxylin.5 Better visualization of the histologic alterations was obtained from tissues Small sections of liver from the 48 chickens of the first group plus 20 conembedded in the Araldite-Epon mixture than was possible using the conventrol and 20 experimental chickens of the second group were 7 obtained at tlie tional paraffin embedded preparations. The parenchymal liver cells of tlie contime of death, cut in small cubes, and fixed in Oaulfield's and Millonig's trol chickens stained uniformly with toluidine blue. Their nuclei were dark fixatives.10 These tissues were dehydrated through a graded series of ethanol blue and the cytoplasm was much lighter and granular. The degenerating paand embedded in an Araldite-Epon mixture." Thin sections of approximately renchymal liver cells of the toxic fat chickens were of two types on the basis of 0,5 ft were cut on an 3ultramicrotome for light microscopy and stained by tlie their affinity for toluidine blue. One cell type had a decided affinity for the dye toluidine blue method. " Ultrathin sections were placed directly on 400-mesli, so that both the cytoplasm and nucleus were very dark (Fig. 2). Many of uncoated copper grids, stained with urany! acetate, and examined with an these cells were shrunken, distorted and contained large morphologic features RCA BMTJ-3G electron microscope. of the organelles were fairly well maintained in the cells obtained during the first 5 days of the experiment. However, the organelles were in very close TABLE l.-ALTERATIONS IN THE PERIPHERAL BLOOD OF CHICKENS CONSUMING TOXIC FAT apposition as a result of what appeared to be shrinkage of the cells and loss of the cytoplasmic matrix. Although the external mitocliondrial membranes were quite irregular, the shape and distribution of the cristae remained unaltered. The membranes of the endoplasmic retieulum were in close apposition and had abundant ribosomes along the outer surface. Free ribosomes were dismEq.lliter mEg./liter ffm.periOO persed throughout the cytoplasm. In the dark cells, the Golgi complex, lysosomes, and microbodies were usually sparse and only vaguely discernible. Only isolated areas within the cytoplasm contained glycogen granules. Numerous myelin-like structures appeared to arise from the cytoplasmic membranous systems of these cells. The nuclei of these dark cells had approximately the same electron density as the cytoplasm. The paired nuclear membranes appeared as one wide, irregular dense band containing few discernible pores. Except for the When 3.0 per cent toxic fat was added to the diet of young chickens 50 per extreme density of the nucleoplasm, the chromatin material and nucleoll cent died within 15 days. Approximately 24 hours prior to death, the chickens became listless and moved only when agitated. Because of the marked abdomiresembled those in the control cells. From the 7th through the 15th day of the experiment, the dark parenchymal nal distention the chickens assumed a ducklike gait when forced to walk. liver cells became increasingly electron-dense, the intercellular spaces wider Moist rales were present in the lungs and considerable amounts of clear to and myelin-like bodies more abundant along the plasmalemniae. The cytoblood-tinged fluid were observed in the oral cavity of the experimental chickens at the time of death. plasmic organelles became more difficult to visualize. Numerous oval to oblong clear vacuoles of variable size developed in the cytoplasm. Myelin-like bodies, On the 15th day, blood studies were made on the surviving chickens. Those identical to those along the plamnalemma, were present throughout the cytofed toxic fat showed a reduction In several of the blood components (Table 3). plasm. At this time it was difficult to visualize the existence of a distinct Hemoglobin levels decreased from 10.0 gin. per 100 ml, to 0,0 gm. per 300 ml., nuclear and cytoplasmic separation because of the electron density and the and hematocrits wore reduced from 31.0 per cent to 18.0 per cent. There was a decrease In total serum protein from 3.4 gm. per 100 ml. to 2.3 gm. per 100 ml. t 306 ^ 307 absence of any distinct nuclear membrane. Eventually these cells d* into dark, shrunken, fairly homogeneous masses which exhibited little i WM also be related to altered «*«»»* ^ toxic effect of this fat upon logic resemblance to liver parenchymal cells (Fig. 6). The second type of parenchymal cell in the degenerative areas was i«n« »•»»,{ ipMota confirm a previous report regarui 6 very electron-lucent. An isolated cell of this variety was seen most freqwWfrij ISfWiKmlature of chickens. BimMAKY within a group of the previously described dark cells. The plastnaleuiiwf'rf| these cells were in close apposition to the adjacent dark cells. The mlewWjl 16 projecting into the space of Disse were shorter and less abundant than tUfty the chickens died rent of the dark cells (Fig. 7). Many of the organelles were markedly dWwfct] Small segments of the endoplasmic reticulum were scattered throughout =. CHICK EDEMA FACTOR : SOME TISSUE DISTRIBUTION DATA AND §li««»tui«, columnga., oven60W-O. M f«*<°L"efl«ne purge PM „„»„„„ 180 cc/min. TOXICOLOGIO EFFECTS IN THE BAT AND CHICK.* (31020) *l».. Mlum carrier cc/mm«rgo»,MA « o][iiii|iWy fc The chick edema factor (CEF), responsible for a large number of deattattjj! the broiler industry in the fall of 1957, was traced to the unsaponlftri&tjl P r matter (unsap) of the fat used in the broiler rations. It since has been cij*r^j tallized (1,2,3) and its structure proposed as that of a hexachlorohexalij;| phenathrene (2). It is known that in the toxic fat, a mixture of related w*;;^ pounds can be found, some toxic and some relatively nontoxic. Y1* To lay the groundwork for a study of the specific physiological effect* **; If«k8 1 through 8 as shown. Peak pure CEF compounds, a few short studies have been completed to learn IS* li?'>wparation.§ rr n i>io L TT distribution of the toxic material in the body and which organs were priniarfil W( Results and discussion. -Lau The results j.ui «.^ --»_ f .e „ fnr the rats. ^. • -d-feedconsumptionj affected. rnthenr|^ Eatperimcntal. Adult rats and day-old White-Rock chicks were used. Bccaittf the pure material was not available in sufficient quantity, we used, from tin* toxic fat, the unsap which represented 38% of the original toxic fat and wtt. estimated to contain at least 10 ppm CEF. The unsap was force fed because tb* animals' food intake was drastically curtailed when it was mixed in the dlA; AH animals were offered water and commercial feed ad libitum. . Table I shows the experimental plans and dosage levels employed for nil I TABLE ll.-GROSS EFFECTS IN RATS Trials. Feed consumption and fecal and urinary excretion were measured fw the rats. Body weights were recorded in all experiments. All animals, upca sacrifice, were examined grossly for pathology and selected organs were weighed and frozen. Hydropericardial fluid (HPF) volume was measured I* the chicks. In addition to the examination for gross effects, the presence of CEF mate-'/J rial in various tissue was determined. Adrenals, kidneys, and livers wew'J; assayed in the rats; livers only have been analyzed in the chicks. To obtain » picture of the amount of material being absorbed and perhaps excreted, analyses of the feces and urine of rats and of the combined chick excreta wew made. £.,RL°o',tt«r.-S,,"K. szsfgSSSZS**. «- ftsy^raTSMt? rei?-'. s^sxx H e (s> ferr»n?x s.™~n — - * ^jrs-, TABLE 1.—EXPERIMENTAL PLAN OF TRIALS L _ Dosage (per kg) Trial 1. H days Species Rat animals Group High ! High Control ' III. 6 days Chick High ! Low Control' 6 1 r M TABLE III.-ORGAN WEIGHTS EXPRESSED -' Unsap CEF (Mil'.-•; (ml/day) nated (ntlttfl v 2.0 1.0 2.0 1.0 0 5.4 1.1 0 •H H ; 0 41 . 10 0 Control animals were not intubated. The assay method for CEF is being reported in detail elsewhere.t In short, the sample is homogenized with water and saponified with alcoholic KOH. The unsaponifiable portion is extracted with petroleum ether and chromatographed first on an alumina column. The eluate obtained with 25% ethyl ether in petroleum ether, following prior elution with petroleum ether and 5% ethyl ether in petroleum ether, is concentrated and chromatographed on 500 fi thin layer silica gel plates with 3% ethyl ether in petroleum ether. The silica gel in the area of Kr 0.80-1.00 is removed and eluted with ethyl ether, the solvent removed, and the residue is redissolved in isooctane for gas chromatography We used an F & M Model 400 gas chromatograph with an electron capture detector and a U-tube column, 3 ft X 6 mm (o.d.) X 4 mm (i.d.), packed with * Supported by TJSPHS Grant EF 00305. Contribution 858 from Dept. of Nutrition and Food Science. Mansnchiiflfttts Inst. of Technology. t T. C, Edema Campbell nn monkeys given the lowest level and the mean survival time of 445 days, f During the last 30 days of life, the monkeys developed generalized subcutn- , neons edema, ascites, hydrothorax, and hydropericardium. There were decreases in erythrocytes, leukocytes, total serum protein values, and altered albumin :globulin ratios. There was also cardiac dilatation and myocardlal hypertrophy and edema. Experimental monkeys had reduced hematopoiesis and spermatogenesis, degeneration of the blood vessels, focal necrosis of the liver, and gastric ulcers. It was proposed that toxic fat exerted its injurious effects upon the parenchymal cells of the liver, endothelium, and myocardium with subsequent development of generalized anasarca. Fats from plant and animal sources have been used to increase the caloric level of diets for animals. As a result of increased demands by feed manufacturers for low-cost fats, almost every available source of these products has been utilized. Certain fats were found to be extremely toxic to poultry, and hundreds of thousands of chickens died or were killed after they were fed diets containing these fats. Results of experiments indicated that young chickens developed hydrothorax, pulmonary edema, ascites, and subcutaneous edema in 1 or 2 weeks when there was, toxic fat in their diet.1'8'15"17 The accumulation of large quantities of extra'vascular fluid in chickens seemed to result from altered permeability of the vascular bed, cardiac decompensation, and liver necrosis'.1'2 Chemical studies on toxic fat indicated that the toxic fraction was located < in the unsaponifiable portion.8'20 By repeated passages through alumina and silica gel columns, crystalline preparations of the toxic fraction were prepared"; • however, the chemical composition of the compound was not determined. Before the cause of this intoxication of poultry was established, many chick- ; ens that had been fed toxic fat were processed for human consumption. Since . that time, the clinical, histologic, and electron microscopic1 15changes that occurred in the intoxicated chickens have been enumerated. ''' '" Data are not available, however, concerning the effects of toxic fat on primates. Since the chemical composition of this fat is unknown, the possibility exists that it may once again adulterate various edible fats. This is a report on experiments undertaken to determine the effect of toxic fat on lower primates. The results of the experiments may be helpful in postulating the effects that toxic fat might have in man. MATERIALS AND METHODS Since the chemical nature of toxic fat was unknown and chemical procedures were not available to determine the toxicity, a biological assay was performed on the fat used in the experiments. When the diet of 1-day-old chickens contained 3.0% toxic fat, 50.0% died within 15 days. In the initial experiment, 16 Macaca mulatta monkeys (av. weight, 4.2 kg.) were allotted to 4 groups and fed diets containing 0 (control), 1.0, 5.0, and 10.0%, respectively, of toxic f a l i . T n the 2nd experiment, 20 M. mulatta monkeys (av. weight, 6.0 kg.) were allotted to 4 groups and fed diets containing 0 (control), 0.125, 0.25, and 0.5%, respectively, of toxic fat. The toxic fat was combined with corn oil to obtain similar fat intake levels in all groups of monRocelvod for publication .Tune 23, 1966. From the Department of Pathology and the Regional Primate Research Center, University of Wisconsin, Madison, Win. 53706. This research w»n supported In part by grants HE-08681 and FR-0167 from the National Institutes of Health. The authors thank Mlns Karen Welke and Mrs. Adricnno Cuppas for technical aiiHistance, TABLE l.—TERMINAL HEMATOLOGIC CHANGES IN MONKEYS FED DIETS CONTAINING TOXIC FAT Group Controls F«d toxic fat Total serum No. of protein monkeys (Gm./lOO ml.-) 9 27 7.5 5.4 Serum albumin (%) Packed cell volume (%) White blood count xlO»/cmm. Red blood count xlO«/cmm. 61 35 41 16 6.8 3.0 6.5 2.5 placed on 400-mesh uncoated copper grids stained with uranyl acetate, and examined with an electron microscope.* RESULTS In the initial experiment, monkeys in the groups fed diets containing 5.0 and 10.0% of toxic fat had the mean survival time of 01 days. The monkeys in the group fed the diet with 1,0% toxic fat had the mean survival time of 100 days. In the 2nd experiment, the monkeys survived for a much longer period. The monkeys in the groups fed 0.5, 0.25, and 0.125% toxic fat' had mean survival times of 202, 274, and 445 days, respectively. There were considerable differences in survival times of monkeys in the various groups; however, the major clinical and pathologic changes were similar and occurred during the terminal 30 days regardless of whether the monkey survived for less than 4 months or longer than 1 year. Therefore, the data from the experimental monkeys will be presented collectively. HEMATOLOGIO EVALUATIONS Total serum protein values of the monkeys were reduced approximately 2.0 Gm./lOO ml. during the experiment (Table 1)'. The mean percentage of serum albumin in the monkeys fed toxic fat on the last determination prior to death was 35%, whereas that of the control monkeys averaged 61%. There was a gradual decrease in the cellular elements of the blood during the experiments. Packed cell volumes were reduced from 32% to 16% during the last 30 days of life. These findings were substantiated by the total red blood cell counts which averaged 2.5 million/cmm. of blood immediately before the monkey died. The hemoglobin values followed a similar course, with the mean value of 6.0 Gm./lOO ml. of blood being obtained in the last 30 days of life. Comparable observations were recorded for white blood cell counts; these averaged 3,000 white cells/cmm. of blood on the last hematologic evaluation of the experimen* RCA KMU-3G Electron Microscope, Hadlo Corporation of America, Camden, N.J. 45-362 0—70 21 316 Jtl monkeys. Prothrombin times, serum bilirubin values, serum electrdl^K :|1 mood urea nitrogen values, and cholesterol levels of serum were not chanpiYfj appreciably during the experiment. • • 317 tnyelold and erythroid series (Fig. 3). Most of the bone marrow i^Pcomposed of fatty tissue and proteinaceous fluid. The blood vessels and sinuses contained only a limited number of cells. Skeletal Musculature.—The skeletal muscle was pale and edematous. MicroCLINICAL OBSERVATIONS •coplcally, the muscle bundles and fibers were widely separated by fluid, but otherwise seemed normal. The major changes in the monkeys were the development of generalized nlsTcstes.—Grossly, the testis seemed normal; however, when examined micropccia and subcutaneous edema 1 to 2 months before death, Edema, first notiwd , ^ scopically, active spermatogenesis was not found. The seminiferous tubules had around the lips and eyelids, progressed to the remainder of the face and even- • -j abundant spermatogonia and Sertoli cells, but only a limited number of pritually involved the subcutaneous tissue of the trunk and extremities. Es|*-^| mary spermatocytes. There were no spermatids or mature spermatozoa. Intercially obvious was the marked edema of the scrotum and sheath wltlcfc "fKtltial tissue was moderately edematous, but the Leydig cells did not seem developed during the last few weeks of life and, in some monkeys, partially :| affected. obstructed the flow of urine. During the last month of life there was decreased j| Gastrointestinal Tract.—In 18 of the 27 experimental monkeys, marked food consumption and the subsequent loss of body weight was frequently u-.ty hypertrophy of the gastric mucosa occurred in the fundic and pyloric regions. much as 1 kg. Diarrhea developed in 75% of the experimental monkeys during fti the same areas, small gastric ulcers penetrated the mucosal layer. (Fig. 4). the last few days of life. Results of bacteriologic cultural examinations of |In In 6 monkeys, inflammatory changes in the intestinal tract were seen. Results feces were negative for pathogenic enteric organisms, of bacteriologic evaluations of these lesions indicated no pathogenic organism that could have been associated with the gastrointestinal disturbance. MicroGROSS AND MICROSCOPIC FINDINGS .',;: scopically, the hyperplastic gastric mucosa was seen to form many large, inter(Hgitating folds. Adjacent to these proliferative areas, the mucosal lining was The findings at necropsy substantiated the presence of extensive subcuU- :; eroded, and the underlying tissue was necrotic. Large numbers of polymorneous edema in over 75% of the monkeys fed toxic fat. 5 phonuclear leukocytes were in the necrotic tissue and underlying musculature. Heart.—Dilatation of the heart was especially obvious on the right side- ii Blood vessels of tjie edematous submucosal and muscular layers of the stomThis was further clarified when the circumference of the valves was deter- f| ach adjacent to the ulcerated areas were free of obstruction. mined. The mean tricuspid and mitral valve circumference of the experimental i| Enteritis in 6 monkeys had caused moderate denudation of the intestinal monkey hearts was 55 and 45 mm., respectively; in contrast, the tricuspid and :| mucosa and considerable hemorrhage Into the lumen. The mucosal lining near mitral valves of the control monkey hearts averaged 40 to 36 mm., respec- ,-| the base of the crypts was intact, and the underlying musculature was normal. tively. Hypertrophy of the cardiac muscle was also apparent in the experimen- J; Skin.—There was marked edema .of the dermal layer of the skin, causing tal monkeys. The hearts of the experimental monkeys were 0.56% of the body -i weight, whereas hearts of the control monkeys were 0.30%. Microscopically, .|| disarray of the collagen fibers. The epidermal layer was comparable in the control and experimental monkeys. There was an absence of any detectable the muscle fibers were distinctly separated by fluid. Individual muscle cells change in the hair follicles of the monkeys given toxic fat. were hypertrophic, and their nuclei were enlarged, distorted, and hyperchromlc The adrenal gland, pancreas, kidneys, cerebrum, cerebellum, pituitary gland, (Fig. 1). There were no distinct valvular lesions in hearts of the experimental thyroid gland, and 'urinary bladder of the experimental and control monkeys monkeys. .;fM Lungs.—Lungs of experimental monkeys were not altered appreciably. Iso- ;|§ were comparable grossly and microscopically. lated areas of atelectasis, congestion, edema, and flbrosis were observed. The / ELECTRON MICROSCOPIC CHANGES proliferation of fibrous connective tissue was associated with the presence of j lung mites (Pnewnonysis simicoli). , , . , , ,.| The extent of electron microscopic change in the liver correlated well with Liver.—Livers of experimental monkeys were small, firm, and moderately .the level of toxic fat in the diet and the duration of toxic fat consumption. An yellow. On microscopic examination, moderate distortion of the architecture , early change in the parenchymal cells was the disruption of the orderly was found. Many parenchymal cells were enlarged, multinucleated, and had ' arrangement of the granular endoplasmic reticulum. The cisternal spaces were only moderate affinity for stain, whereas other cells were small and markedly dilated, and the loss of ribosomes from the outer surfaces of the cisternae hyperchromic. There was also focal necrosis of the parenchymal cells in the resulted in an apparent increase in the smooth endoplasmic reticulum. As a centrilobular zone (Fig. 2). Many parenchymal cells contained vacuoles in result of mitochondrial swelling, the cristae seemed shorter and less abundant their cytoplasm which stained positively for neutral fat when frozen sections than those of the control monkey hepatic cells (Fig. 5 and 6).. Cytosomes of were prepared. Small areas of fibrous connective tissue occurred in the peri- " variable content and size were moderately prevalent in the cytoplasm. Small portal area; however, they did not alter the architecture appreciably. vesicles and flattened lamellae comprised the relatively small Golgi complex. Spleen.—Spleens of experimental monkeys averaged only 0,074% of the body ITat vacuoles were abundant throughout the cytoplasm (Fig. 6). The nuclei weight, and those of the control monkeys were 0.13%. Microscopically, the gercontained distinct nucleoli and abundant chromatin dispersed throughout the minal centers were surrounded by only a narrow zone of lymphocytes, the nucleoplasm. blood sinuses were practically devoid of cells, and the trabeculae were espe- \ Results of hepatic biopsies made a few weeks before death, and experimencially prominent. tal tissues obtained at the time of death, indicated distinct alterations. Many Mesenterio Lymph Nodes.—Lymph nodes were light tan and edematous. parenchymal cells were shrunken and electron dense (Fig. 7). These dark cells Microscopically, the germinal centers were surrounded by a narrow band of were seen in various stages of degeneration. In some cells, the cytoplastnic lymphocytes, The medullary cords were indistinct, and the sinuses were filled organelles were still visible despite the extremely dense matrix. Vacuoles were with proteinaceous fluid. dispersed between the cellular organelles. Myelin bodies were abundant in the Sternal Bone Marrow.—Grossly, the bone marrow resembled coagulated cytoplasm, along the plasmalemma, and in the intercellular spaces. The nuclei plasma. Microscopically, only a small number of hematopoietic cells were seen . were also electron dense, and the nuclear envelope was only vaguely discerniin the marrow, and those were approximately equally divided between the ble. Other dark cells had lost all resemblance to normal parenchymal cells, 319 318 - along the plasmalemma adjacent to ...„ ,„„*,<= yiuuess. u.ue cytoplasmic organelles were quite i abundant matrix, There was marked disruption of the endopla .--.I, with only short fragments being scattered throughout the Abundant free ribosomes were quite evenly dispersed between the urpuwtn* The external contours of the mitochondria were frequently irregiilnr, »s* m matrix was moderately electron dense. Occasionally, bulblike projection* «w*f formed by the external mitochondrial membrane. The plasmalemma? tmfc irregular, and the microvilli were short and sparse. Occasionally, thcr* «w*C. myelin bodies along the plasmalemmal surface. In some instances, the K04 cell plasmalemmae extracellular space. had ruptured, and organelles were dispersed througliWiiSt ons have been reported in PSfe by which toxic fat exerts its im,^^,, -research wiu be IfeMilOKenoRls has not been determined, and fmttwl to elucidate these points. monkeys and the development relationship of toxic fat consumption'^^^^ ioA i C My, under $»*mlerate to extensive alopecia has not "f^^ monkeys. However, only in %*Ml conditions, there is a loss £!£« ^^Ha that of the monkeys fWW«l Instances has alopecia become as extensive ppm toxic fat. and not well-understood ^Btnnd lesion of gastric ftl*a Interesting lesion was was the developmentmicroscopic $*»« in more than 66% °f the monkey fedJo^cja^ ^ ^ MjtNKwrniice of the ulcers was similar toi tnat; oob« equaUy evasive. The |tS* and determination of the etiologk factors ™s ^ ^ vasculature ^gS^SXSZSfi^^™ «*the ulcers were JBtsi^ra^^ff ^^—ra ^vS Bile duct epithelium was affected markedly in monkeys fed diets conti ' toxic fat. Many of the epithelial cells were so electron dense that tlic plasmic organelles were difficult to visualize, and their nuclear ineinbB*48 gpwUtlon of large quantities of fluia.^. t1ileB_ decrease de e in l total serum protein were irregular and extremely dense. The interlocking plicae of adjacent <*8*| Bftkcnthe blood protein values were tested a ™ fo und (Tabie i). The fpri a reversal in the albumin :tfojuj* ratio were lou ^ ^ attrlbuted were widely separated. As a result of the shrunken condition of the epHWtitr'' 0 foftcraiBe in serum protein values, J^ ™?"J/tacrease i cells, microvilli on the luminal surface of the plasmalemmae seemed thta iMfaj in b 00d urea nitrogen : elongated. '|| gto altered renal function ^^^"^^a^tton for the decrease in m st og : 1 Endothelial cells in some areas of the liver had a distinct resemblance to fltf fe# albumin in the urine. The J ? .^ " 1fn Tir£ observed in Focal i the liver. ^ ^^ {_ dark parenchymal and bile duct epithelial cells. The dark endothelfal «W; liltrum protein would be ^ated to the chan^bserveci ob degenerative were shrunken, and their internal structures were distorted. There were *W* |l*m»8 of necrosis and degeneration -were^ ^rvea m o£ the ened fenestrations between the endothelial cells. Changes in the cytoplawftfj I/awntnl monkeys. Examination of electron nucrogru^ organelles were comparable with those observed in the parenchymnl wB*: ^.*wu, substantiated the light .mi«.o«°P£ ^Symal lells, there was a *H«ed functional status of the liepatic pa«^ »• ^^ pressure Occasionally, large cytoplasmic sequestra from the endothelial cells wwf$ observed in the lumen of the vessels. ''^f Crease in albumin production. This wouia, in mru^ extravascular spaces. Heart.—The main differences between hearts of control and experlment*i'j| ;«t the blood sufficiently to produce ^"^"le^ect of toxic fat unon the dark) shrunken, monkeys were the dilatation of the intercellular spaces and the wide dlsix?r«i;r! r Another important aspect of the pH)ble.n vw the^£ .'wsculature. Many of the hepatic »^ mj ocara^ these vasc ular of the myofibrils in the latter. Between the widely separated groups of m)'«&fi Qed ^tu tue Hght brils (Fig. 9), there were mitochondria, occasionally a segment of sarcopltt«ml* fj il«l seemed to have undergone degenerative cnanb thnnges were not detected when the tissues• ™™ fl b d nad undergone reticulum, and abundant matrix. Usually, only the Z lines could be readily Ti*-:.:,| the vascular ualized. Gytosomes were much more abundant in the muscle cells of expcrf-,;"! Microscope, it is likely that other•vessel^f^ r g^ ^y^tth w c 1 mental monkeys and were usually found near the nucleus (Fig. 10). In mow,'v* i-rimllar changes. These observations °"^u °gd toxic fat. As a result of celthan 75% of the hearts of the experimental monkeys there was a dlstlnrt '| :Hmnges recorded in chickens *at ^™ c^umrf t«u at ^^ ^ & would eventually swelling of the mitochondria. The cristae were widely separated, and the nilto-1 s lular shrinkage, the intercellular BparoB were wia chondrial matrix was abundant (Fig. 11). Large myelin figures were observed••% iqucnt increase in the l'0108}*^ ?* ^uJfSd predispose to the accumula ion fend to an increase in vessel peimeability ana^ p P hypertrophy in all of within and surrounding many mitochondria. Myocardial nuclei, components fit ••;{ "of extravascular fluid. There was ^/^^^he^yocardium was examined the transverse tubular system, as well as elements of the sarcoplasmic retlc«-';"| n m : the monkeys that consumed toxic fat Whe ™e y°cft ^ and lum, seemed unaffected. There was a noticeable separation of intercalnted % n U microscopically, there was hyper rophj' oi'.to^ ™ c£mmonly reported in disks in many of the experimental monkeys (Fig. 12). The various bnwlt ; | the a marked interstitial edema. Both of . **"" ? Jecame less efficient, there was formed were often masked due to the abundance of edema-"'-'' tous fluidbyin the the myofibrils tissue. . ;, patients with cardiac insufficiency. As \he h«art^ame less ^^ ^ Many of the endothelial cells appeared electron dense and shrunken, with ,'.' likely a gradual increase in ^°^ep^Se spaces. An increase in right distortion of the nuclei and cytoplasmic organelles (Fig. 13). In most ', instances, the organelles were in close apposition as the result of the cell '; tUat P StUlated £OT ,: ^^^^*~^^^°» monkeys has been reported in chickens fed toxic^at °permeability, 'and ma shrinkage. There were cytoplasmic myelin bodies in many of the cells. Occa- .1 sionally, intercellular stromal cells had changes similar to those in the endo- . Reduced osmotic pressure of themblood, ^^"-TYhe causes for development of thelial cells. C cardiac insufficiency have been ^ «iaiJjdd]a^cSt *"etermine at this time c anasarca in monkeys fed toxic fat It ^ ™ g^\ikel expianation is that all DISCUSSION '""' """ ' entity at a particular Toxic fat consumption had a decided effect upon hematopoiesis and spennn-togenesis. Myeloid, erythroid, and lymphoid elements of the peripheral blood stage of the disease. were markedly reduced. Germinal centers in the lymph nodes and spleen and the islands of hematopoietic cells in the marrow were extremely sparse. Inhibited spermatogenesis was also observed in the monkeys fed toxic fat. Similar 321 320 REFERENCES 1 Allen, J. R.: The Role of Toxic Fat In the Production of Hydroperlcardium Ascltes in Chickens. Am. J. Vet. Kes., 25, (July, 1964) : 1210-1219. 3 Allen, J. K., and Carstens, L. A.: Electron Microscopic Observations in the Chickens Fed Toxic Fat. Lab. Invest., 15, (1900) : 970-979. s Allen, J. R., and Lallch, J. J.: Response of Chickens to Prolonged Feeding of Toxic Fat. Proc. Soc. Exptl. Biol. & Med., 109, (1902) : 48-51. „ ., ™ « Bowman, R. E., and Wolf, R. C.: A Rapid and Specific Ultramlcromethod for T«W ;'|;3 Serum Cholesterol. Clin. Chem., 8, (1902): 302-309. ' Gornall, A. G., Bardawlll, C. J., and David, M, M.: Determination of Serum Prolriw by 8 Means of the Bluret Reaction. J. Biol. Chem., 177, (1949) : 751-766. , --, Hald, P, M.: The Flame Photometer for the Measurement of Sodium and Potas»!«» 'ij,| in Biological Material. J. Blol. Chem., 187, (1947) : 499-511. -.'.'4 "Harman, R, E., Davis, G. E., Ott, W. H., Brink, N. G., and Kuehl, F. A.: Tw ,,-M Isolation and Characterization of the Chicken Edema Factor. J. Am. Chem. Soc., f* /.-f (1960) : 2078-2079. .. •'.' 10 Mallory, H. T., and Evelyn, K. A.: The Determination of Bilirubln with tw Photoelectric Colorimeter, J. Btol. Chem., 119, (1937) : 481-490. „,» 11 Mlllonig, G.: Further Observations on a Phosphate Buffer for Osmium Solution* I* :'i Fixation. Vol. 2. Proc. 5th Internatl. Cong. Electron Microscopy. Academic Press, pr* ,'gjj York (1902) : 8. 13 Mollenhauer, H. H.: Plastic Embedding Mixtures for Use In Electron Microscopy. Stfiln Tech., 39, (1904) : 111-114. " "4! 11 Quick, A. J,: Hemorrlmglc Disease. Lea & Febiger, Philadelphia, Pa., 1957. Rosenthal, H. L. : Determination of Urea in Blood and Urine with Diacetyl MonoiImc. Analyt. Chem. 27, (1955) : 1980-1982. irt " Sanger, V. L., Scott, L., Hamdy, A., Gale, C., and Pounden, W. D.: AllmenUrj.« Toxemia in Chickens .T.A.V.M.A., 133, (Aug 1, 1958) : 172-170. , * ia Schmlttle, S. C., Edwards, H. M., and Morris, D.: A Disorder of Chickens ProliaMj ; Due to a Toxic Feed — Preliminary Report. J.A.V.M.A., 132, (March 1, 1958) : 216-219. " Simpson, C. F., Prltchard, W. R., and Harms, R. H.: An Endothelosls In Chicken* -,, and Turkeys Caused by an Unidentified Dietary Factor. J.A.V.M.A., 134, (May 1, 1059)! 410-416. 18 Williams, F. G., Jr., Pickets, E, G., and Durrum, E. L.: Improved Hanging Strif , Paper-Electrophoresls Technique. Science, 121. (1955) : 829-S32. 10 Wintrobe, M. M.: Clinical Hematology. 5th ed. Lea & Febiger, Philadelphia, Ft. 20 Wootton, J. C., and Alexander, J. C.: Some Chemical Characteristics of the Chltl Edema Disease Factor. J. Assoc. Off. Agrlc. Chem., 42, (1959) : 141-148. CALCULATED DIETARY INTAKES OF CHICK EDEMA FACTOR(CEF) FROM DATA PUBLISH ED BY ALLEN AND CARATONJ (AM. J. VET. RES, 28, 1513-26 (1967)) Dietary level of toxic fat (Percent) ToW Ctf Moan survival time (days) CEF intake per animal per day (ug.) Inlikl 445 • 274 202 169 90 0.225 0.45 0.90 1.80 8.0 1(0 123 182 ZM 721 (ut) MONKEYS (MACACA MULATTA)' . 0.125 0.25 0.50 1.0 5.0 10,0. l/HH;iv 1.^1*. * i » « -• "-" " t. Capture Gas Chroinalograpby CHICKS (DAY-OLD)» 3.O.. 15 0.36 5.4 ( w.nrl riiomi«trv Food and Drug Administration, liy PAUL NKAL (Division of Food Uioinisir>, I" 1 Calculations based on average daily food consumption of 225 gm. for a 5.0-kg. monkey (F. Sperling, personal, communication, June 12,1969). ' Calculations based on average dally food consumption of day-old chicks (personal studies, see Flick, et al., Poultry Science, 45,630-36 (1966)). i |I Washington, D.C. 20201) , f Tho elytron cupUirc GLO scmen.ng test for rhi.'k cduma factor 0, « lias Lie-on nf HIP wtrolcum ellier extract from Die s\il° J™^11^,,,,,^ on „„ alumina column, ,' ' f • ftH( | H(.,M1,,p „/ ll,e tl.iul «l'»»;'«l •! - w/, ,.c.lnclion in simple plonnvip i""pm i l s a f i O / r ic'Midio" ' , , „ (....-imcnt Tlie inodifiofl procedure i m n n f ! " I.IL.IUIII, v1 "" • f in 2i ire indicative of the vw « llln " "' 10 , vn,.p o[ chick c drma fnflor. 322 323 Table 1. Comparison of sulfitric acid cleanup with saponifieation for detection of chick edema factor by electron capture gas chromatojjraphy (ECGLC) ECGLC Analysts* Sample" HjSO< Cleanup Saponlflcation 10.1 (18), 11.8 (14), 17, 6 (40), 20.4 (34) Low positive reference fat (1.0% toxic fat in USP Cottonseed Oil) 10.1 (45), 11.8(48), 17.6(65), 20.4(60) Toxic animal tallow 10.2(74), 11.9(82), 18.8(94), 20.G(98) Toxic ololc acid 10.1 (147), 11.8(23), 17.8 (>500), 20.4 (200) 10.1 (98), 11.8 (10), 17.8 (>500), 20.4 (154) Toxic glyceryl monooleate 10.1(50), 11.8(23), 17.8(200), 20.4(82) 10.1 (48), 11.8(14), 17.8(258), 20.4(98) Vegetable oil soapstock (nontoxic) 10.4 (trace), 13. 5 (trace) 10.4 (trace), 13. 5 (trace) Oleicacid (nontoxic) 10.4 (trace), 13.5 (trace) 10, 4 (trace), 13. 5 (trace) Cottonseed oil (nontoxic) 10.4(23), 13.5(22) 10. 4 (trace) Blank 10. 4 (trace), 13. 5 (trace) 10.4 (trace), 13 .5 (trace) . 10.2(56), 11.9(4?), 18.8(85), 20.6 (90) " Toxic and nontoxic refer to results of AOAC chick bioassay (AOAC Official Motlmcls of Analysis, 10th Ed., 1965, 26.087-26,091). 6 The first values (without parentheses) refer to retention time of peaks at 200°C vs. aldrin; the values In parentheses rafer to peak area which is equal to retention time- (cm) X peak height (cm). Method IteaKcnIs uittl -Apparatus 'Hinsc all glassware with appropriate solvent before use. Do not use polyethylene containers to store solvents (6). (a) Petroleum ether.—Reagent grade; redistill in glass between 30" and 60°C (available from Burdick. and Jackson Laboratories, Muskngon, Mich.). (b) Carbon telrachhride.—Distillcd-in-glass, (c) Celile,—Johns-Mansville #645, acidwnshed. Wash well with petroleum ether and dry. (d) filler paper.— #5-19 S&S Blue Ribbon, or equivalent, /)«rermiiintj«iii Std/uri'c acid cleanup.—Dissolve 2.5 g fat in 10 ml CGI., in 100 ml beaker (heat, if necessary). Add 10 ml concentrated H 2 80j and thc-n 20 g Oolite; mix with heavy glass stirring rod during additions and stir until homogeneous mixture is obtained. Adrl 125 ml petroleum ether, mix well, let solids settle, and ftltr>r the supernatant liquid through filter paper in 00 mm conical funnel. Repeat with additional 125 ml porlion of petroleum ether. I'lVttporalo combined petroleum ether filtrate to 5 ml for alumina column frni-tioiia.tic.in. Complete dclerminalion as outlined in the method of lligginbothani f t al. (1). Kcsull* iitid Discussion GLC retention limes and prmk areas for negative, positive, and blank samples were compared for both the sulfnric acid and tlic saponificalion methods; sec Tab'.c 1. Results are comparable as indicators of toxic material. Gas chromatographic peak heights were lower in some ea^es with the sulfuric acid cleanup; however, Hie presence of toxic factor win clearly indicated in the low positive reference material, The nontoxic cottonseed oil samples would have been judged toxic by the saponipnu ll ficalion mollieid because of Hie relatively largo GLC peak of R,, 13.5, ft ponk detected at low levels in gas chroinalograms from blanks and other nonloxic samples; see Tnblo 1. Small peaks of It,, 10-25 wore observer! in both procedures in the blank mil] nonloxic samples. However, they did not interfere with identification of the toxic fats and differentiation of toxic from nonloxic samples. Gas ehromalograms of blank and low positive reference samples aflor saponificatiou and preliminary culturie acid treatment are shown in Figs. 1 and 2. Acknowledgments The author expresses sincere thanks to David Firestone for guidance and encouragement throughout the development of this project and to Richard Staaf who performed many of the analyses. • REFERENCES (1) Higginbolhnm, G. R., Firestone, D,, Chavez, Linda, and Campbell, A, D,, This Journal 50, 874-879 (1907). (2) Higginbotham, G. R., Ross, J., and Firestone, D., ibid. -SO, 884-885 (1607). - Ftg. 2—Gas chromatograms of (a) blank and (b) low positive reference fat after cleanup with sulfurlc acid treatment. See Fig. i for GLC condition!. (3) Davidow, B., tbirf. 33, 130-132 (1MO). (4) Dingle, J. H. P., Analyst 90, G38 (IOCS). (5) Burke, J., , and Giufl'rida, Laura, This Journal 47, 326-342 (1964). • LOW POSIWf KerfRENCC ""V"~'J -—;,-—• 4- •-/.,-" •# • jj, - ' fl« W»WTI1 fig, J—Gas chromatograms of (a) blank and (b) low positive reference fat after cleanup with saponifieation. GLC conditions: T X 4 mm i.d. glass column packed with 2.5% StT-52 on GO-SO mesh GaJ Chrom Q at 205°C. Amount injected: 1/SOth of alumina column fraction 3, Reprinted from the Journal oj the Association of Official Analytical Chemists, Vol. 50, December 1967. - 325 26. OILS, FATS, AND WAXES [) The official, first.action GLO-microcoulometric method for chick edeS factor, 26.092-26.096, was changed by adding the following to 26.092: (g) Ethyl ether for alumina chromatography.—Ether (not >2% alcohol) or absolute ether (not >0.01% alcohol) (available from Burdick and Jackson Laboratories). (2) The official, first action electron capture method for detection of chick edema factor, This Journal 50, 216-218(1967) was changed by addition of the following to (b) in the Determination section: After ". . . 26.094" in line 6 add "(using ether specified in 26.092(g)" (item (1) above). (3) The following rapid screening method for detection of chick edema factor was adopted as official, first action : PRINCIPLE Samples are subjected to preliminary HaSO( cleanup and extd with petr. ether. Ext. is purified on A12O3 column and examined by electron capture GI/C, after addnl H2SO« cleanup. Gas chromatographic peaks with retention time relative to aldrin of 10-25 are indicative of chick edema factor. REAGENTS AND APPARATUS (a) Petroleum ether.—Eedistd in glass, b.p. ,30-60° (available from Burdick and Jackson Laboratories, 1953 S. Harvey St., Muskegon, Mich. 49442). (b) Ethyl ether for alumina chromatography.—Ether (not >2% alcohol) or absolute ether (not >0.01% alcohol) (available from' Burdick and Jackson Laboratories). (c) Carbon tetrachloride,—Bedistd in glass (available from Burdick and Jackson Laboratories). (d) Celite.—No. 545, acid-washed. Wash well with petr. ether and dry at room temp. (e) Alilrin standard soln.—0.1 jug/ml. See Reagents and, Apparatus, section (a), JAOAC 50, 216(1967). (f) Chick edema -factor low positive reference sample.—1.5% reference toxic fat in TJSP cottonseed oil. (Available from Division of Food Standards ami Additives, Food and Drug Administration, Washington, D.O. 20204). (g) Activated alumina.—See Reagents and Apparatus, revised 26.092(1)), JAOAC 50, 216(1967). (h) Alumina chromatographic column.—To dry chromatographic tube, 17 mm o.d. (14.6 mm i.d.) X 250 mm long, fitted at bottom with coarse porosit}' fritted glass disk and Teflon stopcock (tube without fritted disk but holding glass wool plug in bottom may be used), add redisd petr. ether, dried before use with anhyd. Na2SO<, until column is % full. Weigh 15 g A1203 and transfer to column in small portions, tapping tube as AUOa settles. When last portion of AlaOa settles and air bubbles stop rising to surface, add 5 g anhyd. Na3SOi. Drain excess petr. ether so that it is just above upper surface at Na2SO(. (i) Oas chromatoffraphic column.—Glass, 5-7' X %" i-d., packed with 2.5% SE-52 silicone gum rubber on 60-80 mesh Gas Chrom-Q (Applied Science Laboratories, State College, Pa. 16801). Coat support with substrate as follows: Weigh 2.5 g silicone gum rubber stationary phase and dissolve in 300 nil CHsCls-toluene (1 + 1), heating to dissolve. Add 97.5 g Gas Chrora Q and let stand 10 mm with occasional gentle stirring. Dry in rotary evaporator held in 50° bath. Pack coated material into chromatographic column by adding small amts while vibrating column at packing level with Vibro-graver tool (Fisher Scientific Co., Pittsburgh, Pa, 15219). Fill to within I" on exit side and 3" on entrance side, and fill remaining space with silanized glass wool. Condition column at operating pressure 2-5 days at 250'. (j) Gas chromatoyraph with electron capture detector.—See Rear/cuts anil Apparatus, (.e), JAOAC 50, 216(1967). DETERMINATION (a) Preliminary sufurie acid cleanun.—Dissolve 2.5 g fat in 10 ml CCt< in 400 ml boaker; mix with heavy glass stirring rod while adding 10 ml H»SO<. Add G g anhyd. Na2SOi and stir well while adding 20 g Celite, until homogeneous uiixt is obtained. Add 125 ml petr. ether, mix well, let solids settle, and filter supernatant thru paper in 90 mm conical funnel. Repeat with^idnl 125 ml petr. ether. Evap. combined petr. ether filtrate to 5 ml for AliC^Humn fractlonation, (b) Fractionation of petroleum ether filtrate T>y alumina ehromatography.— Dry solvents prior to use by shaking with anhyd. Na8SO». Transfer petr. ether nitrate from (a) to A1803 chromatographic column, using total of 15 ml petr. ether. Let liquid level fall to just above top of NaaSO(. Elute sample with 100 ml petr: ether (fraction 1), 50 ml 5% ether in petr. ether (fraction 2), and 100 ml 25% ether'in petr. ether (fraction 3). Relatively fast flow rates of ca 8-0 ml/min give satisfactory results. Keep liquid level above top of NaaS04 at all times. Discard fractions 1 and 2, and collect fraction 3 in 125 ml erlenmeyer. Add several boiling chips and evap. to dryness on steam bath. Transfer residue with petr. ether to 10 ml g-s. graduate and evap. petr. ether soln to 3 ml. (c) Sulfurie acid cleanup of alumina fraction 8.—See Determination, section (c), JAOAC 50, 217(1967). (d) Electron capture gas chromatographif of petroleum ether extract.—See Determination, section (d), JAOAC 50, 217(1967). (4) The official, first action method for methyl esters of fatty acids, 26.055-26.059, with changes in This Journal 49, 231-232 (1966), was revised ns follows; (A) 26.058(c), revised third paragraph, TMs Journal 49, 232(1966), after ". . . to obtain calibration factor." insert "Reference mixts simulating most fats and oils may be obtained from Applied Science Laboratories, Box 440, State College, Pa. 16801; Supelco, Box 581, Bellefonte, Pa. 16823; and Lipids Preparation Laboratory, Hormel Institute, Austin, Minn. 55912)." (B) 26.059, change to read as follows: "Two single detns of major components (>6%) performed in 1 laboratory shall not differ by >1.0 percentage unit, Two single detns performed in different laboratories shall not differ by >3.0 percentage units." (5) The official, final action lead-salt ether method for determination of saturated and unsaturated fatty acids, 26.040, was changed as follows: (A) Change first paragraph, first sentence to read: "Accurately weigh 10 (for plant fats used in common household cooking oils) or 20 g sample into 200 " (B) Add the sentence "Reserve ether filtrate (contains ether-sol. Pb soaps)." to the end of the second paragraph. (C) Fifth paragraph, line 8, after ". . . HCl-free", insert "(no ppt with AgN0 3 )." (D) Revise the sixth sentence of the fifth paragraph to read: "Distill ether, avoiding any loss of fatty acids, and heat, over steam bath to constant wt under controlled flow of N to prevent oxidation of fatty acids. Cover steam batli with towel to prevent splashing 11,0 into erlenmeyer." (E) Paragraph 6, line 1, insert "reserved" after "Transfer". Line 6, add "Repeat HC1 hydrolysis until no more PbCU is pptd." after ". . . into beaker." (F) Change paragraph 7, lines 2 and 3 to read, ". . . until HC1 is removed (no ppt in wash H2O with AgNOa). Dehydrate ether with ca 2 g anhyd. Na2SOi and transfer ether soln . . . ." (G) Change paragraph 8 to read: "Det. in duplicate I numbers of 0.2-0.3 g oil from unsatd fatty acid fraction, and from entire satd fatty acid fraction. (I number of satd acid fraction is due to presence of some unsatd acid.)" (6) The following gas-liquid chromatographic method for butylated hydroxyanisole (BHA) (121006) and butylated hydroxytoluene (BHT) (128370) in corn and rice breakfast cereals was adopted as official, first action. APPARATUS (a) Gas ehromatoffraph.—Barber-Colman Model 5000, or equiv., with H flame ionization detector and strip chart recorder. Establish following operating conditions: temps—column 160°, detector 210°, flash heater 200°; N flow rate, sufficient to elute BHT in 3-4 min from QF-1 column and elute BHA in 3-4 min from Apiezon column; H flow rate, ca 40 ml/min for Apiezon L and ca 25 ml/min for^QF-1; air flow rate, ca 340 ml/min; electrometer sensitivity; 500 X (5 X 10 lo) amp full scale deflection) with 5 mv recorder. Adjust H and air flow rates, if necessary. Adjust electrometer sensitivity so 0.1 /ig BHA gives ca 50% deflection. Repeat injections until constant peak heights are obtained on successive injections of identical vol. of std mixt. Order of appearance on Apiezon column (4 f ) : BHA, BHT, di-BHA. Order of appearance on QF-1 column (6') : BHT, BHA, di-BHA. 327 326 l from Nature, Vol. 220, Wo. S16S, />/>. 7ft2-^OJ, W<'mi)-ji-ill"*l n-o 17'4 20-0 Chemical and Toxicologicai Evaluations of Isolated and Synthetic Chloro Derivatives of Dibenzo-p-dioxin utcaric acids, has bcon u«ed as a reference in nil our prc viou« chemical and biologicnl work1. Tnlilo 1. RXAMINATIOH OF CIUOKOI'MBNI ONK of tlm toxic compounds Unown ns hydropericnrrlimn / r a n t snir c of com 3niii|)tR pyrntyfed* |ionciHi| In pyrolynfl fuclor (clticU ocdcmin factor) its l,2,3,7,(i,0-hoxflchlorodi1 boirAo-ji-dioxin . llccftufio it is highly improbablo th'it ' 2,.|'))tclil»rnol (111 ?•» pononls of tho fats frnm which they have U«m isoluted, • 2Xo-Trli'lilurmik>no1 ,!M,0-Tc(riu>!|Wan1wnol (T) fl'0,10-0, 11-2.17'4, 100 ao o. nfl'O PII tho qtifHtifin oi' l,)icir origin baa aroused Bpi'Diilation. 27 I'rulfH'lilortiplU'iiol (K) SJHt li'O A clnn to the possiblo origiu of hydroporicartlUim factor Clilfirlnnl-'rt illlii'ii/n.ji-rtioxln 1 -0,1 'ft, 3'5, fl-G 0'05 is found in a report by Tomiin c( tiL* of tho ayntlicftin of llcrcrcncii toxic fnl comuoiiPiiti 1-0. 1-8, 3-It, 0-fi, 8-0, polyh»loclibenxo-j>-(lioxins: chlorophcnols nnd their miltR, 10-0, 11-2, 1 7 - 4 , when healed, nnrlcryo condensation reactions imd form chlorinated dorivptiveH of (lib(iii70-;;-dioxin. BOCEIUHO of 1 j|... r r l r n f l o i i Untr» nl'i^Un Hr.llvn tn Uio rAttnllon time of hWrln. . tliis obsoi'vntion, \\~f.\ studied tho pyrolysis of n number of 1 1 /in -20 n.ii.h, In tin; i-fin, Stinutle InjTlcil Into (he n\t cell nf frr*h coinniorciftlly avnilnblu ohloroplienols which nro widely ferUln uffl* Iwroro IncuimiloH. " Solvents usod: ctlmiiol, nccUnm and cliloroused in agriculture HIK! industry, nnd present vesulu of The morlnllly of non-lnjcclPd and S Embryonic niorlnlllv iU 21 riny HOITUI pi'olinu'nai'y pxp^riininitB \\hicJi tuip^est Umt hydro- ftolvrnt-lit^'Cind controls Viiu 10-ln rent. pcrirnrdinni fnctor could arise fi'om cerlnin ohlorophenols. Tho chromntO[;rnm of components from tho toxio fntty Tlio commeiTiul clilnrophennU u.scd in this work \veve pyrolyfefl in neconlnneo w i t h tins gcnci'al proccclurcH acid nirttci'ial conUiinvd ft nnmbor of pea UK of widely described3. Heuwno extracts of thu reaction mixture* varying retention limes; four of Ihoflo pcutt« had H» wero fiiH'tionnted by passing tbeni through an alumina vahiee grcaler than4 H). According to n current cJcetrcm column. Residues of the beii'/.euc effluents wore oxl.rne.tcd capturo OLO lest , the presonco of ono or more GLC with pctroloum el her at room tempcnUviro, and tho pcatiH with /in values of 10 or more indicates that hydro* poll-oleum el her \\nft then removed. The resultini; pro- periciii'dinm factor is jimse-nt in a fat. Tlio jjyi'oiysftlfl ducts wni'o sufticienily pure in most CHRCK for uxnininnlion from Luclim'eal grado S.^.-J^-lctmchlorophcnol was llto by electron capluru gns--lic|iiid chvomutonra])liy ( t J l » ( 3 ) only product Mmt showed u peak pattern iiidiciiling tlie and liinlogieftl ie»linjr hy (ho chicken embryo nwsny*. presenco of hydrupcrii-ardium factor; tho peak pntlcrn Tablo 1 hhoxvrt retention limes of Iho Ol.C peaks on a rtiHcmhlud that UiKplnyod by the long rolentinii time com7 font, a-5 per emit W;.52' column at S'OO* C nml iho poiiLMils of th<* refereiino toxic, fat. \Vhen tested l)y tlw.' resuUHof lljccliickei) emhryo ansny. I'lHlividimlly isolated ollicinl cbifk hioasfiny* for hydrojH-ricardinm factor, llic hydnmericni'ilium fnc!|.oi's of known chemical utruduiva mixture produced I be rjiso-fisu a(. a tfteltuy lord of 0-1 p.pJiiIn addition, dilK'n/.o./)-din\iu was chlorinated directlyl wiicu not lu'ailidilo for Ut*n as Klandnn.!:!; cnnKetjuent ly. a cimcenlrale IViim tho nininponifialil" fmcl i"ii of a certain in order In prepan- y,;t,V 1 R-li'lni(ihluKnUl>eM/.o-/)-dioNin . Thin malurinl jirovr-d to he cxtrenvly Uixie in llrti chic.ltcit eiHumerc.ia) li»xic f a t l y ncid mati'rinl u m uned as a (!!.(.' reference. The unsapnniliablu fraction of Ihe f a t t y ucid cml.M-jo anrtny (Tfthle 1). and provided attnuivn for i^lofioii of u t'riehloi'o Hf well a-i tlio tuirnchlonxlibeiv/.o./j.diitx-iii. nuilorial \vt\» known to contain trneo (imouiUs of the The tee.hniual ^rad-: a.H.'l^l-tulrachlorophriiol pyrolyti* hydnipei'iciiRliiim factor. Thu toxic fatty ncid innlcriiil, a hy.pnuluot obtniitcd from tlio mannfucturn of oleie and ale, ehlorinated dil»',ii/,o-/'-di'ixin, nnd c!i>mpoui?ntfi fVoia Infrared spectra of individual components exhibited characteristic absorption bnndu in the return of J,3301.280 cm p l attributed to asymmetric titrotehin^ vibration of C-*-0---CJ in the flibeu/o-;j-(lioxiu rinf/. The- two principal compouiulH isolated from the cihlorinntcd dibcu/.o-;)dioxtn were '2,3,7-trichloi-odibcnx.o-/).dioxiii ami 2,11,7,8toLmohltiifxlibettv.o-jj-dioxm. The infrared Kpeelrum of tbn trichloro compound luw briuds f\t 870 em- 1 and 805 cm-1 (1,2,-1-Hubstiluted brii/.cne). and Sfid cm-1 (1,2,4,,'}. Huhslituted hen/cuu), Tho infrared t*}i:*e|rum t>f tho tdtrne.hloro compound has a strong band at 8fiS cm-' (l,2,4,fi-Hul'9titule(l bmxcnn). Tho infrared fipectrum of tln % letrachloro compound matched (ho fcpcutrum of 2,.'l,7,S.t[itracblorotlibcir/,o-/)-dioxiii published by 'J'omitn fit (tl,*. Chicken cmljryo assay showed ilitit the tetrnchlovo compound was more toxic t h a n the trichloro conipoxitid, The fir«t four pcnka in t!ie chromnloft-rajii from tho telrachlorophetio! pyrolysalo were caused by positional isomei'fl, each containing six cblorino atomR,; tho fifth and tiixth peaka were caused by positional isomcrs, o«ch contnining sovcn chlorine atoms. A seventh ponk in the olij-omntogriim, observed when Ihe snmpl'.i injection wns overlonded, had a retention time icleuficul to (hat of a anmplo of ochtuhlnrodtbenxo-;}-r]io\in prepared by pyrolysis of pentacililorophonol. The presence of pentaolilfrophcniil in tcclnu'cul ^I'iido 2,3,4,0-tctrachlgrophenol nccountu for Ihn formntion of tho two hrptacliloro ifiomera nnd tho oclnchlorodibew.o-p-dinxin. A fourth hoxaehloi'o ifiumer, in addition to the ihreo oxpcctcel from pure 2,3,4.li-tetrachlorophenol, is prohnbly cnufiecl by the iiresenco of ft tctrnohlorophpiiol other than the 2,3,'1,0-iaomrr in the otortinK material. Infnu-ed npcotrn of peaks Ii and 4 from tho 2,ii.4,(i-teinH'hloruphenot pyrolysato M'ore very Hiniilnr (o pubd'sln/cl K|jcctrn- of two liydroperieardium factors iKuhvlcf! from a contamitifited fnl«. Tho conmonenlff isnlated by preparative C!IX: from the refercnci! tuxit 1 f a t . tbe ehloriutited dihen/O'/t-dioxin. and the 2,3,-1,0-leirachlnrf.pheiiol pyrol.vsnle Mere mialy.si.-il by electron e i i p t u r e Of.C,' at 2ilnlw > niililji>r.ti-innli>cnclir|lir. t SI-R rooliioitB3nnO 4,Tithlc t . t ComiKineiilsrroincWi.flimM ill\«>n/o-j)-(lltfxlii. ond ono lieptn-i^oniei 1 ure less toxic than the ollmr iKOincrs. Work IB now under wny tu jJi-epin'O indivjdun) eomponndH of high purity in sufficient tjiiBuHticB Tor ndditionnl e.lioimcul nnd bioloRicul Icsling. Refcrenco toxic fa I components with low /?• vnlucn (less timn ffa 8-U) cmilcl !«• Huo (o oJiloro-orgnnir pesliRides nnd/or lower cliliiro(lilwn/o-;).tiioxina. Additional work is required to identify iho conijiovmds remwins)blo for the penkH with low Ite vnlnpB. Tho pof\U» with high }{„ vnlnc-s .(tlioso cqunl to nr ^renter than (Ml) correspond to timso of the liexneliloro nnd heptixchloro positionnl ismners ifjolnleil from ihc pymlj-Mite of 2,3,4,0letmehlnropbeiiol. Tlie overall ro«ulln of thin work Rupf-L-Bt tbut chtnmphcnoU conUt lie precursor;* of hydropci'iRwrdinm ft\rlor. Comnicicinl chloroplirnols nnj widely nnf'd for such divo.rsc npplienfions as contiiet lierbiciclcs. defoliant s nnd lermitc control njieiUfi, n« Mi'U us for control of miorobinl nttnek in (tin jmmufacfurt' of n number of products. There me ninny opportunities for fata nnd fatty acids, the only products in which hydropericardium lac tor bns bt-on found , to bccumo contaminated. "When crude ftxts and tflt[o\\P nrc subjected f.o heating operation!* (hyilrojyms, (listiltntion) in tho production l»piiola during production of connnemul f a t t y acids. This laboratory i^ developing methods ftir detecting cblomphenola in fats and fnlty neifln. \Vo thank Dr Jo-Vun Chen, Dr Donald P. Flick, Mr Robert Barrcm, Mr Jamca K)ilum, Dr 1C, K. llcynnldo, ML- William V. Scott niul Mra Mnry K. Mutchlcr for tccbnicnl npsistanpc, G. H. HKiGINHOTIMM 1 ANITA HUASG DAVID FIRKSTONK jArqUKi.iKK VKUIIBTT JOJIN KKSS A. U. CATilVnKM. ]Jnrt*ftti of Science, Food and Drug Arbninisti-alion, Deprtrlnicnt of Dealt)*. Kdueutiun and \\'elf«re.. Wellington. I'M.'. ,! AiiRiitt b; r ri 27. 1 1 C/JI-IH, Km], ,V(., Ynl,i,,nla Zattfti. 79, IfO (1050): '' ' iiin[<-l.iiiGl.liri,J.,.;. X'i"^ (I • V i T n - t l . M .K.Miiil ^7, KHIII (iflrtt). 1 Xi-til. )'., ./. A***, OJlic. Anal. i'l.= 9-379 + 0-01, <: = 9-433+ 0-0.1 A, « = 92-35°±0-20°, 0=92-39°±0-20", >>= 10!l-92°±0-30°, The calculated density is 1-958 s.cm-» for Z<=1, compared with 2-01 B.cm-> measured for (lie bulk material. A statistical treatment of the 1158 measured reflections indicated a center of symmetry; the space group was therefore assumed to be /*!. The structure was solved by the symbolic addition method of Karle & Kane. The nearly planar molecules arc almost parallel to the (Oil) crystallogrnphic planes. No unusual bond lengths or angles were found. The structure was rclincd to R= 10-5%. cc-3-17 material, docs indeed produce the hydropcricardium condition in chickens.* Kodak No-Sctcciv). Intensity data were recorded for both crystals, reduced separately, then compared, edited, and averaged. Absorption corrections were made separately for each crystal using Busing & Levy's general absoiption correction program as modified by Jeffrey (1964). Owing to the very tiny ciyslals, and in part to the . integration, very long exposures of approximately 1150 hours were required to obtain satisfactory multiplefilm data. The entire Wcis;.enbcrg camera was placed inside a plastic bag and a helium atmosphere was provided to reduce background due to air scattering. Of the 3030 possible reflections, 115S (38%) were recorded; 397 of these reflections had intensities less than a minimum threshold value and were classified as 'Icss-thans'. The intensities of most of the reflections were measured by a Joyce .Locbl microdcnsitometcr scanning at right angles to Ihc longer integration direction. The weakest reflections were estimated visually. A standard intensity strip wns prepared and used for the visually estimated intensities. To ensure that both types of intensity data were on the same scale, a sufficient number of medium intensities were measured both visually and by the dcnsitometer. Radiation damage effects were found to be negligible by retaking data for earlier crystal settings. Statistical treatment of the intensity data by K.tmachandran & Srinivasan's (1959) modification of the method ofHowclls, Phillip & Rogers (1950) indicated a center of symmetry. The s.pace group was assumed, therefore, to be I' I (Cj) and this assumption was confirmed during the direct method calculations. Experimental Two types of crystals were isolated from a warm bcnzcne-hcxanc solution of the K-3-17 material. The bulk of the crystalline material appeared to differ in phase from the material used for this study. No crystals of the bulk phase were found to be satisfactory for single-crystal studies, and only two crystals of the studied phase were isolated. Measured r/-spacings of X-ray powder patterns taken of the bulk phase material did not match rf-spacings calculated from the unit cell of l,2,3,7,8,9-hexachlorodibcnzo-/)-dioxin. However, when the bulk phase was heated to just below the melting point (230"C) n phase change occurred. Measured rf-spacings from X-ray powder patterns of the transformed bulk phase match the calculated r/spacings of l,2,3,7,8,9-hcxachlorodibcnzo-/;-dioxin reasonably well. Therefore, it was assumed that the material used for this crystal structure determination was a high temperature phase of the bulk crystalline material known as ot-3-17 H1TF. The single crystals used were diamond shaped and had the approximate dimensions 0-18 xO-10x0-08 mm (a x b x c). The uni [-cell parameters were determi ned from singlecrystal data using a General Electric single-crystal orienter and Mi-filtered Cu Ka radiation (A= 1-5418 A). The parameters of the triclinic cell chosen according to Dirichlct (Balashov & Ursell, 1957) are as follows: ff=7-952 ±0-005 A «= 92-35±0-20° es = l-958g.cm-3 Solution anil refinement of the structure 6 = 9-379 i 0-01 /!= 92-39 + 0-20 e» = 2-0l g.cnv' c = 9-433+ 0-01 • v'= 109-92 ±0-30 Z = 2 Initially we knew the weight of the molecule and the 1'=662-8 A» number of chlorine atoms per molecule, and we knew where t>0 was measured for the bulk phase by flotation. that the molecule possessed some aromatic character. Two-dimensionally integrated equi-iuclinatiori Wcis- Attempts to solve the structure from the three-dimensenberg data were collected for the ir-axis /ones, 0/c/- sional Patterson map were not successful. The symbolic 4W, and for the /j-axis zones /tO/-/i5/ using the multiple- addition method of Karlc & Karle (1963, 1966) was film technique (one pack each of four films, Eastman. then employed. The phases were determined for the 251 most intense * The composition for the structure reported here, namely reflections in terms of four algebraic quantities, n, b, <•,#. CuOzHzClg, agrees well with unpublished niicrochemical A summary of the calculation of the unitary structure analyses performed by Professor Wolfgaiiu J. Kirslen, University of Uppsala, Uppsala, Sweden, at a very curly stage of factors or £-valucs used for tlus determination is compared with theoretical Values and is as follows: tills structure work. Quantity (1964). One of the active fractions of material isolated was that called a-3-17, where this nomenclature refer* The isolation, chemical analyses, and spcctroscopic to the vapor phase chromalographic behavior as dedata on the hydropcricnrditim toxic factor (1IPTF) scribed by Wootton cl til. (1962). Wootton and his colmaterial have been described by Wootton, Artman & leagues proposed that lll'TF was a chlorinated teaAlexander (19(0), and by Wootlon & Courehcnc hydrophciwnthrcne with the empirical formnli C M H|oCI1 Itcprinl rcquc.sK sluniltl beflcUla-sscdto this author lit the ical properties arc remarkably similar to the isolated Procter & Gimihle address. Introduction 45-362 O - 70 - 22 Noil-centrosymmetric 0-886 0-736 1-000 Ccn (rosy in metric 0-798 0-968 1-000 0-3% 5-0% 32-0% Kartert n/. (1964) . J-lndolj-la-cctic acid 0-772 0-769 0-970 0-934 1-000 1-031 0-J% 02% 4-SV. 3-3% 36-1% 30-8% 1-158 rclleclions 1-28-9 reflections 761 non-zero 8W non-7cro 397 unobserved 424 zero Clcss-than') C^HjOiCltf 333 332 overa tempcnilure temperature rnctor actor wns was 2-24 '4. The os'cnill A summary of the sixteen cases that resulted from thc sign permutation of Ihc four algebraic assignments is as follows: • No. rcD. ycneialcd No. incortcct signs % \viong sijin Objections lo choice Ctisc 1 a H.4. 1} c — c •t- — 2 -14+ 3 •1•1- 4 + 5 .). — — •i142 109 — 119 132 117 — 183 68 132 125 4—-f 153 98 132 46-6 52-6 49-8 52'6 53-0 v/ •1 J '< •/ — •1114 137 133 smnc some changes ang made mae in carbon a r o and chlorine o r n assignassgn . r . _ :. - I . ii.. I i 1 -i tl n r. ,j,,j_ mcnls resulting in, ideally, a p.1. l a n a r 1,2,.1,7,8,9-tara chloroanthraccne with mm symmetry, This change iill the structure dropped the R value to 35%. The plan1C 8 129 118 133 13D 251 0 129 121 130 121 10 II I?. 13 14 15 121 130 128 115 136 •I125 12S 121 130 122 129 6] 128 123 120 16 H- 122 129 111 / Klcctron density peaks were found nl ccnlcrs or symmciry wlicn llic special scclionfi .v«0 nnd ,v« 1 \vcrc compuled. 1 ll is very unlikely llial nil 251 most inlense rcHcclions would be positive, * This correct cusc was selected after examining fhrcc-dimensioiiiil electron ) 9503 (22) 8539(17) 57V8(I9) 5629 (19) 7461 ( I K ) 7591 (17) 6496(13) 671.1(13) Kor «,, 58V (20) 362 (23) 487 (26) 372(23) 395 (24) 282(21) 354(41) 262 (35) 3S6M3I 370 (-12) 321 (39) 219 (3-D 427 (45) 249 (35) 325 (39) .160 (421 240 (3-D 213 (32) 355 (27) 334 (27) «J! 376 (291 378 (30) 411 (30) 457 (3d) 375 (29) 421 (29) B»" fln flu Ba 487 (26) 417(7.71 3Vfi (26) 347 (24) 309 (23) 473 (27) '149 (21) 025 (22) 163 (24) 17-1 (23) 016 (22) 148(21) -035(17) -162(19) -056 (21) -018(18) 034 (18) 028(18) -OS8 (22) -043 (22) -084 (24) -105(22) -024 (21) -102(23) sifylng sonic of the 'Icss-Uians' to observed reflections on the basis of visutil estimations or the weakest reflect ions. , The It value droppccPto 13% during the next leastsquares refinement. Fixing the chlorine atoms and refining only the carbon skeleton lesnlted in an R value of 11-8% at which time (he anisolropic temperature refinement ou the chlorine atoms was undertaken and gave (lie final R of 10-5%. Of the 397 'Icss-tliun' reflections only 42 calculated were larger than their threshold values and none by more than 3d%. A weighting function similar to that given by Hughes (1941) was chosen so as to have l i t t l e dependence on /v, it was taken as )'«• = *•/(/-„+ 53-4) for !•,> I-,,, nnd \'w=-KI(l'm + tt-4) for F, < Tin, where Fm- 21 '4, and the quantity minimised was i'liVo-Fr)3.'Less-Hum' reflections having l"c> lr, (thrcshnid) contributed like an ordinary reflection, but those having /•"„, and the maxima and minima did not show any chemically or structurally significant features. The final position and thermal parameters are given in Table 1. The observed and calculated structure factors are listed in Table 2. In this work, the scattering factors.arc taken from International Tables for X-ray Crystallography (196?.); those for carbon arc by FreeTable 3. Distances of dlants from Icftst-squares planes Distances for atoms not defining Ihc plane arc maikccj with an asterisk; s.d. is the standard deviation of the atoms defining the plane. Under the (044) heading are listed lite deviations from the (034) plane containing the molecule. The plnnc* are defined in direct space by equations /'.vH-Qy f Rz^S, am Cl(2) Cl(3) Cl(4) C1(S) Cl(S) CO) C(2) C(3) C(4) C(6) C(7) C(8) CX9) C(ll) C(I2) C(13) CO 4) 0(5) 0(10) Origin s.d. f Q li. S All -0-10 -0-11 O'OO -0-16 -O'OS 0^13 -0'02 -0-06 -0'02 0-OS O'OO -O'OS -O'OI -0-01 006 0-05 005 0-06 0-OS O'OO 1'57« 0-07 -1-1589 -5-1655 6-8500 1-5675 C+0 -0-10* -0-12* -0-03' -0-1S* '-0-06* 0-13* -0-03 -0-08 -O'OS 0'03 -0-03 -0-07 -0-03 -0-02 005 0-03 0-02 0-05 0-05 0-08 1-55* 0-05 -I-IOS6 -6-0031 6-M04 1-5497 (05(4) 0-33« 0-10' -0-25* -0-45* 0-12* 0-54' 0-15' 0-02' -0-16* -0-17* -0-25* -0-21* 0-03* 0-13* 0-13* -0-09' -0-10' 0-12* -0-17' 0-25* • 1-67* 0-00 O'OOOO -6-6618 6-6618 1-6655 man (1959); those for oxygen by tlcrghuis, I laanappcl, I'otlers, Uiopslra, MacGillavry & Vtcncndaal (IV55); and those for chlorine by Uawson (1960). Discussion The molecule is nearly planar and the molecular plane is tilted 8° to Ihc (Oil) plane. Deviations of atoms from the (034) plane essentially containing the molecule and Fig. I. Molecular packing in the (03-1) plnnc conlp.inmp the molecule. Large shaded ciiclcs aie Cl, solid ciicles me C, atul open circles Rrc O. Cl(3') Fig. 2. Projection onto Y7. (ij.iii 334 from the least-squares planes of t h e entire molecule nml of t h e carbon-oxygen skeleton are given in Table 3. The molecule' appears to he s l i g h t l y bowed in the middle :ind s l i g h t l y twisted about a line from Cl(3) to Cl(5). The packing arran.ccmcnt of chlorines 4, 5, and 6 appears to be more crowded t h a n t h a i for chlorines I , 2, and 3. This packing dillcrcnee could account for the slight twist of the molecule. Fig. I pictures the molecular packing in the (O^T4) plane c o n t a i n i n g the molecule, and Fig.2 gives a projected view of two adjacent molecules related by the center at (1, !,•?)• Intermolecular distances in this (034) plane of less t h a n 4-0 A are shown in Fig.3. Between ccnlrosymmetriciilly related molecules there are a number of CI(»}-Cf(w') and equivalent CI(m)-CI(V) distances of 4'0 A of less. I-'rom t h e parent molecule to the one related by the center at QvM) the distances are CI(2)-CI(4')-3'S5 A, CI(?)-CI(5')"3-S6 A, and CI(3)-CI{(/) = 3 ; K 3 A ; by the center at (0,0,.J)-CI(1)CI(3') = 3-84 A; by the center at (0,-J,-J)-C!(l)-CI(r) = 3'39 A nnd CI(2)-CI(6') = 3'98 A; and by the center at \\ / \V" »/\V» A,\A' ,W o , V -''Z..U,_,.J\..--CCI(T| vii"~5~w~~"''w>™ ^-v : .cit4 335 (1,J,1)-CI(4)-CI(6') = 4'00 A. The least-squares planes of the two adjacent molecules related by the center at (J,-J,-J) arc 3 ' J 3 A a p a r t ; between these two molecules the shortest, interatomic disumcc is 3-30 A from a C(I2) to an O(IO). Fig.3 indicates the bond distances and angles. The mean standard deviations arc as follows: (TC-C~ O025A, o r .oi'19 A, n-c o = 0'022A; for angles n~2'0°. The bond distances are not significantly different from those found by Davydova & Slruchkov (1962) and Gafncr & Hertistcin (1962) foi 1,4,5,8letrachloronaphthalenc where molecular over-crowding results front the presence of m a n y chlorine atoms s u b s t i t u t e d on adjacent aromatic positions. This compound belongs in group (I) according to the classification d u e lo l l a r n i k , Mcrbslcin, Schmidt & Hirshfcld (1954) for compounds that are affected by molecular over-crowding. An electron density map shotted in the (034) plane containing the molecule is shown in Fig.4. The authors wish to acknowledge their appreciation to Dr J, M.Stewart of the University of Maryland, who furnished the X-ray 63 computer program and provided much information on its use. In addition, we wish to thank Dr Lyle Jensen for a number of helpful discussions on the use of the X-ray (53 computing system and on approaches to the solution of the structure in general. We wish lo express our appreciation to Drs Jerome and Isabella Karlewho provnkd assistance in applying the direct method for determining the phases of a number of the most intense reflections. Thanks are.due to Mr Robert Gloss who obtained part of (he data and provided the computer programs used in generating the relations between reflections necessary for applying the direct method. References BAUSIIOV, V. & UnsiiLL, M. D. (1957). Ada Crysl. 10, 582. BEftOlllilS, J., HAANAI'Pin., U, M,, 1'OTTUtS, M., LOOPSTRA, MR..I. C") Inieraloiviie ctiM;im:e<;. IVimcil atoms .ire on ni-iyhboring molecules in ihc same pl,ino, ffo-c**0-025 A; ffr-t.i ---O'OI'J A ; ou-ii'--0-022 A. View corresponds to l;'ig. j . B. O., MA'CCILLAVUY, C. H. & VECNKNHAAL, A. L. (1955). Ada Cryst. 8, 478. DAVVDOVA, M, A. & STRUCIIKOV, Yu, T, (1962). Zli, Slrukl. Khlmil,\ 184, DAWSON, 11. (I960). Ada Crysl. I.I, 403. Fltl.niAN, A. J. (1959). Aria Crytt. 12, 261. GAI-NCR, G. & HERIKIEIN, F. I I . (1962). Aeta Crysl. 15, 10SI. G A I X I R , G. A HhRii.siF.iN, F. II. <1963). Nnlm-c, Lontl. 200, 130. HARNIK, 13., I IrmisiniN, F. H., SrjiMrirr, G, M. J. £ Hmsitl i : r n , F. L. (I9.M). J. Client. Sic. p. 3288. Howu.is, 1:. R,, I'lin i.ii-s, 13. C. & KCKM-KS, D. (1950). Ai-la('rnl.\,lW. , .. Hucillt.s ]•;, W. (19(1). J. Amcr. Client, Sue., 61, 1737.' Inlcrnalhtiml Ttihlwfar X-ray Cry>>l, 589 (1957). Schulz, K. H.: Unpublished experiments, 1965. Schmidt, W. and W. Boslet: Contribution to a knowledge of permanent skin changes in chloracne patients with demonstrable insurance claims. Berufsdermatosen //, 109 (1956). Shelley, W. B. and A. M. Kligman: The experimental production of acne by penta-und hexachloronaphthalenes. Arch, of Dermat. (Chicago) 75, 689 (1957). Spiegelberg, U.: On the question of delayed and permanent psychopathological damage following occupational intoxications. Med. Klinik 56, 436 (1961). Teleky, L.: Perna disease (chloracne). Klin. Wschr. 845 (1927); Klin. Wschr. 897 (1927) ; Klin. Wschr. 214 (1928). REPORT ON METHODOLOGY FOB CHLORINATED AEOMATICS IN FATS, OILS, AND FATTY ACIDS By JOHN RBSS, G. R. HIGGINBOTHAM, and DAVID FIRESTONE, (Division of Food Chemistry and Technology, Bureau of Science, Food and Drug Administration, Consumer Protection and Environmental Health Service, Public Health Service, Department of Health, Education, and Welfare, Washington, D. C. 20004) ABSTBACT The official, first action electron capture GLC (EC-GLC) method for chick wleina factor (polychlorodibenzo-p-dioxins) have been reviewed. This general procedure, which underwent collaborative study in 1967, has undergone several minor modifications which result in better recoveries of polychlorodibenzo-pdioxins and increased specificity in interpretation of the gas chromatographic results. The EC-GLC method can be used as a screening test, or where a typical pattern of GLC peaks is obtained as a preliminary test, but confirmatory tests are needed to demonstrate structure and toxicity of polychlorodibenzo-p(lioxins. Preliminary work with combined GLC-Mass Spectrometry indicated that this technique might provide a suitable test, if adequate sample cleanup can be accomplished. A chicken embryo assay has been developed to the point where toxicity can be observed in three to five days after injection of eggs. A preliminary procedure has been developed for isolation and gas chromatography of chlorophenols in fats and fatty acids. Polychlorophenols have been found to be precursors of chlorodibenzo-p-dioxins. The use of a non-specific microbiological test for chlorophenols employing the Bacillus megaterhtm was evaluated. Chlorophenols were found to produce uniformly graded growth inhibition of the test organism in the range 1-100 /tg. The widespread use of toxic organochlorine compounds in agricxilture and industry requires development of sensitive methods for their detection in a wide variety of commodities. In addition, it is equally important that methods be developed to^detect toxic breakdown or conversion products of organochlorine compounds. One of the most urgent needs in the fat and oil industry is for a rapid and specific method for polychlorodibenzo-p-dioxins (chick edema factors) in fats, oils, and fatty acids. The official, first action, microcoulometric and electron capture methods for chick edema factors (CEF) are essentially screening procedures (1,2,3), Both methods, at present, require a rather time consuming three-week chick bioassay (4) for confirmation. The purpose of this report .is to review the current status of chemical and biological methods for chlorophenols and chlorinated dibenzo-p-dioxins in fats, oils, and fatty acids. CEF consists of a mixture of chlorinated dibenzo-p-dioxius which occur occasionally as ,1 trace contaminant in fats. Recently, a communication (5) from this laboratory reported the results of a preliminary study which demonstrated the possibility tiiat CBF could arise from residues of pentachlorophenol and 2,3,4,6-tetrachlorophenol in fats and fatty acids. Chlorophenols and their salts, when heated, undergo condensation reactions and form chlorinated derivatives of dibenzo-p-dioxin. The following equation illustrates this condensation reaction. Technical grades of pentachlorophenol contain ca 10% of 2,3,4,6-tetrachlorophenol which also undergoes thermal condensation reactions and forms hexachloro derivatives of dibenzo-p-dioxins. The condensation of 2,3,4,6tetrachlorophenol with pentachlorophenol forms two heptachloro derivatives of dibenzo-p-dioxin. An electron capture GLC method has been developed for pentachlorophenol and 2,3,4,6-tetrachlorophenol in fats, oils, and fatty acids (6). However, recov- 342 343 orios of the two volatile polychlorophenols were low and varied over a wic range; nevertheless, the method appears to be satisfactory for qualitative measurements at the 0.5 ppm level, Several samples of oleic acid known to contain (JjiJF were analyzed and were found to be contaminated with residues of pentachlorophenol. The method requires further study. The official electron capture and microcoulometric methods for CEF were developed before their chemical structures were known. The methods are screening procedures and are based on the observation that toxic fats contain a number of chlorinated components (now known to be polychlorpdibenzo-pclioxins) which have greater retention times than chlorinated pesticides. The electron capture method has received wide acceptance. It is approximately 2000 times as sensitive and requires less sample than the microcoulometric method. In addition, electron capture gas chromatographic equipment is simpler and is in general use in many laboratories. Recently, it has come to our attention that a number of laboratories that routinely use the electron capture method for control work are not aware of improvements (3) that have been made in the original procedure (2). Kecent changes which have not been published include a minor modification of the HaSOi cleanup step and a slight modification of the procedure for packing the GLO column. The modified method, which includes these changes, replaces all existing GLC methods for the chemical assay of CEF. stiHr iso that it is solvent, add 5 g anhydrous NaaSOj. Drain excess petroleum etc just above upper surface of NaaSOj. (11) Gas chromatographic column.—Glass, 0-7' long x %" i.d., packed with 21/2% SE 52 silicone gum rubber on 60/80 mesh Gas Chrom Q (Applied Science Laboratories, State College, Pa. 16801). Coat the support with substrate as follows : Weigh 2.5 g of the silicone gum rubber stationary phase and dissolve in 300 ml of 1:1 methylene chloride-toluene, heating to dissolve. Add 97.5 g of support material to liquid mixture and let stand 10 minutes with occasional gentle stirring. Dry in rotary evaporator. Apply vacuum to the chromatography column, and pack the coated material into the column by adding small amounts while tapping the column at the packing level after each addition. Fill to within 1" on the exit side and 3" on the entrance side, and fill the remaining space with silanized glass wool. Condition the column at operating pressure at 250° C for 2-5 days. (12) Gas chromatograph with electron capture detector.—A tritium source concentric type detector is recommended. Operate instrument in accordance with instructions of manufacturer, and obtain a stable baseline before carrying out analyses. Choose an operating voltage (ca 50-80 volts) that will cause between 0.6 and full scale deflection for 0.1 mg of aldrin (2 ftl of standard aldrin solution) at a sensitivity setting of 1 x 10"° AFS. Keep the column temperature at 200° ±1°C, and adjust nitrogen flow rate so that aldrin elutes in 1-1.5 min. (3-4 min. per in. chart speed). Inject 2 jA. of the standard aldrin solution before injection of each reference or test sample. METHOD Reagents and Apparatus Rinse all glassware with appropriate solvents before use. Do not use polythylene containers to store solvents. (1) Concentrated HaSOi.—Reagent grade. (2) Petroleum Ether.—Reagent grade, redistilled in glass between 30 and 60°C (Available from Burdick and Jackson Laboratories, 1953 S, Harvey St, Muskegon, Mich. 49442). (3) CCU—Distilled in glassl (Available from Burdick and Jackson Laboratories, Muskegon, Mich.) (4) Anhydrous Na2SOi.—Analytical reagent grade. (5) Ethyl ether.—Analytical reagent grade (not >2% alcohol) or absolute ether (not >0,01% alcohol). (6) Iso-octane.—Distilled in glass (Available from Burdick and Jackson Laboratories, Muskegon, Mich.) (7) Standard aldrin solution.—Dissolve aldrin in iso-octane to make 0.05 /ig/ml solution. (8) Chick edema factor low positive reference sample.—1.5% reference toxic fat in USP cottonseen oil or other suitable vegetable oil. (Prepare from reference toxic fat available from the Division of Pesticides, Bureau of Science, Food and Drug Administration, Washington, D.C. 20204). (9) Activated AUOa (Fisher No. A-540, do not substitute).—Activate 100 S portions by heating 4 hours at 260° C. Transfer without cooling to dry container and close tightly. Check activity of AlsO,i by analysis of the low positive reference sample, examining A1203 fractions 2 and 3. With sufficiently activated AlaOa, chick edema factor elutes predominantly or entirely in AlsOa fraction 3 as indicated by the gas chromatograms. (Chromatogram should show n series of GLO peaks with Ra values between ca 8 and 45). (10) AlaOu chromatographic column.—To a dry chromatographic column, 17 mm o.d. x 2BO mm long, fitted at bottom with coarse porosity fritted glass disk and Teflon stopcock (a column without the fritted disk but holding a glass wool plug in the bottom may be used), add redistilled petroleum ether, (fried prior to use with anhydrous NazSO*, until column is % full. Weigh 15 g AUOi and transfer to column in small portions, tapping the column as AlaOa settles. When last portion of AlaOa settles and air bubbles stop rising to surface of DETERMINATION (a) Analysis of 1.5% reference toxic fat in USP cottonseed oil.—Dissolve 2.5 g of the 1.0% reference toxic fat in 10 ml of C01< in a 500 ml glass stoppered Erlenmeyer flask. Proceed with determination as described below in sections ( b ) , (c) and ( d ) . Take up residue from (d) in 250 /tl iso-octane and inject 5 microliters of reference solution (equivalent to 50 mg of the original sample) into calibrated gas chromatograph. The resulting gas chromatogram should exhibit a series of GLO peaks with Ra ca 8-45, depending on operating conditions. Peaks at Ra S-13 are due to hexachlorodibenzo-p-dioxin isomers, 2 peaks at Ra 17-22 are due to the 2 heptachlorodibenzo-p-dioxin isomers, and a peak at Ra 35-45 is due to octachlorodibenzo-p-dioxin. (6) Preliminary sulfuric acid cleanup.—Dissolve 2.5 gm of fat in 10 nil CC1( In 600 ml glass stoppered Erlenmeyer. Add 10 ml cone. HzSOi, stopper, and shake for 30 sec. Add 125 ml of petroleum ether, stopper and shake vigorously for ca one minute. Allow layers to separate and decant supernatant liquid into a 500 ml erlenmeyer, avoiding transfer of lower layer. Repeat extraction with additional 125 ml portion of petroleum ether. Evaporate the combined petroleum ether extracts to 5 ml for AljOa column fractionation. (a) FractionaUon of Petroleum ether filtrate 'by alumina chromatography.— Solvents must be dried prior to use by shaking with anhydrous NaaSO4. Transfer petroleum ether filtrate from (b) to AlaOa chromatographic column using total of 15 ml petroleum ether. Let liquid level fall to just above top of NaaSOv Keeping liquid level above top of NaaSOj at all times, elute sample with 100 ml of petroleum ether (fraction 1), 50 ml of 5% ethyl ether in petroleum ether (fraction 2), and 100 ml of 25% ethyl ether in petroleum ether (fraction 3). Relatively fast flow rates of ca 8-9 ml/min give-satisfactory results. Ground glass stoppered separatory funnels (250 ml) are satisfactory reserves for eluting solvents. Discard fractions 1 and 2, and collect fraction 3 eluate in 125 ml Erlenmeyer. Add several boiling chips and evaporate solvent to dryness on steam hath. Transfer residue with petroleum ether to 10 ml graduate cylinder equipped with a ground glass stopper, and evaporate petroleum ether solution to 3 ml. 344 (A] Sulfurio acid cleanup of alumina fraction 3.—Add 2 ml of concentrated HsSO< to graduated cylinder containing 3 ml petroleum ether solution from (c), stopper and shake vigorously for 30 sec. Allow layers to separate nnd decant petroleum ether layer into 10 ml beaker avoiding transfer of H»SOi layer. Add 2 nil petroleum ether to cylinder, swirl vigorously, allow layers to separate, and decant petroleum ether layer into beaker. Add ca 0.5 g of solid naHOOii to beaker and stir ca % min. Let stand five minutes and decant petroleum ether layer into clean 2 or 4 dram vial. Wash NaHCOa with 2 ml petroleum ether and decant washing into vial. Evaporate solvent under N. (c) Electron capture gas chromatography of petroleum ether extract.—Take up residue in 250 pi iso-octane (redistilled in glass), stopper vial tightly and rotate so that solvent wets sides of vial. Inject 1 microliter of sample solution (equivalent to 10 mg fat) into calibrated gas chroma tograph. Gas chromatographic peaks with Ea S-45 are indicative of the presence of chick edema factor. Compare Ka values of sample peaks with Ka values of peaks from reference toxic fat. See (a) for identification of peaks. If peaks indicative of chick edema factor are not observed, inject 5
    .inol .0.7 1.5 2,4-Dicr.lcroph.enoxyacetic Unsaponifiable* Fraction (Dose in rig) 100 50 0 Pyrolysis* Products (Doss in ing; 100 50" 0 Phenols (Doss in ag) >0.5 2.5xlO~ 5 0.01 2.5xiO~6 0.005 2.5x10-3 - 2x10-* l.OxiO' 4 6xKr3 1.2xlO"3 6xlO~4 2 3 ixlfi-3 0.05 IxlO"3 0.4 _ 2 - ' r 2.5xlo' i.o ^o.i >-, 5x10-3 l.SxlO"2 SxlO'1- 0.25 5x13"° " 2Kl3~3 .05 .->lxlO" SxlO" - 2 , 4 , 5- Tr ich lorophar.oxyacetic acid Eior-etlcs Dow £123449 0.25 1.0 1.0 co en bO < .05 —0.5 Polvchlorodiben3o-p-3ioxins * 0.5 Diberv^o-p-dioxin Chlorinated dibonzo-^-dioxin 2.5:-:10"D IxlO-5 2.5x10 <> Tetrachloro diber.so-p-dioxln 2xlO"4 5xlO"5 - 5xUT4 - :" 1.25xlO"4 2xlO~3 Ho::achlcro iscniers (4) z.. Xcst toxic Hcr^tachl^ro iso:r.ers(2) a. Xost toxic h. Least toxic Uapublls-.-ad data of J. Varrctt * >'aziy of c'aesa ara z;i:cturas Code: - =3 " 2O pp= 1 jit - 20 jpb 3 1.25x.lO- 2.5xlO' 4 • - IxlO"3 ~ 1.25X10'3 Air- Call Iroactions Pra-incubation PS,~LBii:-liSY RHFCST CH Tr5AXOI.CC? SVUDIT.S ma FICXIN USISG GOI2a? ^.I-lSrEF.S Oocpoun'J .Amou.:it Intubateil try..-, f, - 10 ?c-i i titter 'Iota! i1 of Fo.ti *•• Bcra Alive Ccatrol 0 a.s 43 41 Ecus 2 - 1 ED, 1 ID' 1.55 10.3 62 11 eye arsffl^slics 5 1 - 4 ME, 47 ID 1.33 Srtr,tro- 2 - 2 2S 1.47 1 > 1 O< 1.6S 2 - 2 CD 1.53 C'hlovod Lbcr.ro-p9.1 .'g/!-.g/r. C.E. Uillie=3 Kictcua ™ox:".color>,y Srcnch r=:vi;lca o£ ?^r.-:i.cidc ',tic;-ictry •!- T '.'fficc; of ?'":tictdr:i 6 Prucuct Seicsty co CTI CO 354 355 «H * I,"' 4.) |j 1.3 w '-', g 5 K S S S rrf rt iH r« M i-l ^ O DEPARTMENT Off HEALTH, EDUCATION, AND WELFAR| PUBLIC HEALTH SERVICE, Research Triangle Park, N.G,, December 4, 1969. P !?? I F)H. J. MCLAUGHLIN, i and Drug Administration, " *if! '!-'- Food ^ 1 '"lip Washington, D.O. :T' tj *• ml "» fji'^1 |>; DEAH DR. MCLAUGHLIN : I tun sending you under separate cover a sample of 1 k?''T (2,4,5-trichlorophenoxy acetic acid) from the supply that was used at fciBlonetics Research Labs, Inc. for the Teratogen Screening Study for NCI. Sincerely, « &**1I fy "' ^* u S^'tf? K. DIANE COURTNEY, Ph.D, Pharmacology and Toxicology Branch, 3 »•* , 9 S 3 9 ' fi 0 S t t f"!llll ' iii r*4 (/) ft' [-1 R 6 $ *« 1 1 » , ' "' 1 C» ^O O '*", t*t ^< vt ^< ' 1 t** in M *"' 13 v, J-) cl TO hi w <•< U"! S •» " j O •'.-" IN j«'.. ~" 'ji fi o [?, o to w to e! « B rj 1 •* s* t ;-i ,rj 2 ^? C « » '<^ ." O O M O U O Al V) t.| l'i V U W H AJ ci n 4j f] o o « w 0 <>! *J u d « F. w rj |j 0 o to -M ^i to -ri ji f;; •>». ril fn CO 0*1 O in O r^» O IH >iJ »-< ^ d 4.> o o if 4-J J-J w f! cj to •• ' & "^•. 03 t^v •t! '|J H "' H f> c! o W - ^^n5! TH T-i V4 C? ^* ** ,5 M ^ w B !J •• 5i "fl t| "i lit 1 ' f,o S 1^1 '=1 »rf <-*x tj K VO co v* y? TO (^ . i^t fH Jj 4J O 4J ! J' -it : . v*> i .'fi , 'y«iJ,| ?, "'*- S 1 ','^i r.-j ft t;n ; sssiS ,- 5°-'*il 9 ci ts i •< i.^ THE DOW CHEMICAL COMPANY, Midland, Mich., February 9,1310. •'). M. JACQUELINE VEHRETT, Bureau of Science, Division of Toxicology, Department of Health, Education, and Welfare, Food and Drug Administration, Washington, D.C. A,"i''*« j DEAR DR. VEBBETT :The complete assay on the sample of 2,4,5-trichloro^*t ^ I'lionoxyacetic acid (Dow production batch 120449) which we recently sent to '• ™ * ion is as follows: !M <> ", a, i f> i~* M u fl <":« fc;; ' f' u J>'-4' *l* Vl \4 *'' E s '* "•(J <^ CM t-l 1 "d VJ "^ til "* •U I :z { ", 1. i M ft VO ^ W ^v rj t*! R t-T t=*l (^ •O ,o c.'; t.l^x' K) ... § 0 02 B I>> f? to-.-- 0 !-: 0 «tf l^ t*\ ci 5.' (=j 5. ''' " ' " « , - c% - 1 U (.J — W v- o m o o i-I o o O O 'H ji t- i'i" ia "1-4 1 O *"*« a "^M 0 fi ^^ •v) *O K U w4 ;1 r-l (^ ^ •~^ rj JJ '.i O o . 'i N P ;j . ( o f. (" v^ t. J in i* - U "^ t'i i •: ^ ^'. r'« CNi • •• fij U J.J *.' t-l p' '. 1^.) 11 f'J. S .'•". t " « •£rt ;'1 if. • 0 ' w •ri-r ' "" o o *-4 f "* (V* L f!C) *'"' fc s •ill .01 * '- H C.I " _ • : ' " ' | ' ^> ,!,' jf- 2,3,7,8-tetrachlorodibenzo- p-dioxin = (0.5 ppm) 2,0-dichlorophenoxyacetic acid = <0.02 2,5-dichlorophenoxyacetic acid = '0.42 2,4-dichloi'ophenoxyacetic acid = 0.05 2,3,6-trichlorophenoxyacetic acid = 0.55 2,4,6-tichlorophenoxyacetic acid = <0.1 Bis-(2,4,5-trichlorophenoxy)acetic acid = 0.4 3 isomers of dichloromethoxy phenoxyaeetic acid = 2.9 2,4,5-trichlorophenol = 0.23 (Max.) sodium chloride = 0.035 2,4,5-trichlorophenoxyacetic acid = Balance Freezing Point = 152.9" 0 Total assay by acid titration = 100% Sincerely, GEORGE B, LYNN Government Regulatory Relations, Dow Life Sciences. 357 356 MARCH 2,1971 CHLORINATED DIBENZO-P-DIOXIN STANDARD (F768) 1. We have supplied Dr. C. Billiams with a mixture of tri- and titrncliloi*-HJIJJ dibenzo-p-dioxins (F768) prepared by direct chlorination'of dibenzo-p-dloxln »( ;-^lf room temperature for two and one-half hours (see memo of Ress to CuuipWJ,/{% 1/30/70 and memo of Firestone to Campbell 2/5/70). f !||( 2. This mixture, previously analyzed by EC-GLC and found to contain aboul J\jif| f50% tetrachlorodibenzo-p-dioxin (GLC peak area), was reexamined by GUJ:pj,||| with flame ionization detection (6 foot glass column; 200°C; 3% C '* Packard GO model 871), which gives a more accurate determination of sition. By this latter analysis, it was determined that the chlorination mixturt~';t;|j| F768 consists of two components as follows: ff' (a) 38% 2,3,7-trichlorodibenzo-p-dioxin *'•"< ( 6 ) 62% 2,3,7,8-tetrachlorodibenzo-p-dioxin ,C; •, . MABCH 26, 1970. ;,ii SAMPLES FOU CHICKEN EMBRYO TESTING '•''.'•';. 1. The following samples were delivered to Dr. Verrett on 3/17/70 for chicken ; embryo testing: '.;.j Our. No. Identification F871 2,3,7,8-tetrachlorodibenzo-p-dioxin, Dow, pure (ca. 95% by GLC) F877 Remarks „1 0.0878ms in 10 ml of acetone. 0.3806 mgin 10ml of acetone. .-.; 2,3,7,8-totrachlorodibe(izo-p-dlo)iin, FDA, prepared by Dr. Pohland (99.5% 0.1735m«in 10ml pure by GLC). of acetone. 2,4,5-tricdlorophenoxyacetic acid; Dr. Williams' purified sample; further ex- 1.712 grams. tracted 3 x with petr. ether and 4 x with 1+i petr. ether—diethyl ether. SylvexiDr. Williams'purified sample; further extracted 3x with petr. ether and 2.519 grams. . 4xwkh 1+1 petr. ether—diethyl ether. 2,7-dichlorodibenzo-p-dioxin, Dow, pure(ca, 99% by GLC) F883..... F881-A-1 F881-B D. FIEESTONE, Head, Fats and Oils Section. '' ' CHLOROPHBNOL SAMPLES ' MARCH 26, 1970. ' '•' 1. About 3 grams each of the following chlorophenols were delivered on 3/23/70 to Dr. Verrett (at her request) for chicken embryo testing. Our No. F888-sub A F888-sub B F888-subC F888-subD F888-sub E F888-SUD F Identification EC-GLCanil PPB'CEF Pentachlorophenol (purified) Aldrich Chemical Co 2,3,4,6-Tetrachloropnenol (technical) Eastman Chemicals 2,4-Dichlorochlorophenol (purified) Eastman Chemicals Ortho-chlorophenol (purified) Fisher Scientific 2,4,5-Trichlorophenol (technical) Eastman Chemicals 2,4,5-Trichlorophenol (reagent) Dow Chemical Co 167. 96,500. 18. No analysis. Trace. Trace. •Hexa-, hepta-, and octachlorodibenzo-p-dioxine. J. RESS, Fats and Oils Section. I. Cleft Palate. 21-day Embryo. 0.02 micrograms 2,3,6,7-Tetrachloro-dibenzo-pdioxin. j 358 359 II. Abnormal (incomplete) Development of Eyelid. 21-day Embryo. 0.002i ;'^ mierograms 2,3,6,7-Tetrachloro-dibenzo-p-dioxin. ;' : i III. Edematous Cyst Covering Rump. 21-day Embryo. 0.02 ^ (micrograms), 2,3,6,7-Tetrachloro-dibenzo-p-dioxm. 361 360 Senator P!ART. The hour being 12:25, I suggest we recess until 2:15. (Whereupon, at 12:25 p.m., the hearing was recessed, to reconvene at 2:15 p.m. this same day.) AFTERNOON SESSION Senator HART. We resume this afternoon to hear first from Dr. Julius Johnson of the Dow Chemical Co. If a corporate entity can have a spirit attached to it, the comparing is a distinguished corporate constituent of mine. STATEMENT OF DR. JULIUS E. JOHNSON, VICE PRESIDENT AND DIRECTOR OP RESEARCH, THE DOW CHEMICAL CO.; ACCOMPANIED BY ETCYL BLAIR, DIRECTOR OP DOW AGRICULTURAL CHEMICAL RESEARCH; V. K. ROWE, DIRECTOR OP THE DOW TOXICOLOGICAL LABORATORY; AND 'GEORGE LYNN, DIRECTOR OP GOVERNMENT REGULATORY RELATIONS OP THE DOW CHEMICAL CO. Dr. JOHNSON. Thank you, Senator Hart. I have with me Dr. Blair, director of Dow Agricultural Chemical Research, Mr. Howe, director of our Toxicological Laboratory, and Mr. George Lynn, director of our Government Regulatory Relations. Senator HART. Thank you. You are all welcome. Doctor, you have given us a statement. We will order it printed in full in the record and as you go along, if there is any extension or summation you care to make, the record will contain the full statement in any event. Dr. JOHNSON. Senator Hart, the policy decision has already been made and announced this morning. I will, however, with your permission, read my testimony, all except the last part which deals with some historical matters that already appears in the record, Then, if you would permit, 'I would like to make some additional comments which may be appropriate to the process of shortening the time interval between the discovery of a suspected toxic phenomena and taking appropriate action. I would_ also like to refer back to some earlier work done under Senator Ribicoff's guidance in his committee and quote at least one passage from that work published in 1966, which I think is appropriate to the issue, if I may do so. Senator HART. By all means. Dr. JOHNSON. Thank you. Mr. Chairman, I am Julius E. Johnson, vice president and director of Research and Development of the Dow Chemical Co., Midland, Mich. I also served as a member of the Secretary's Commission on Pesticides and Their Relationship to Environmental Health, May 8, 1969, to November 7, 1969, chairman, Emil M. Mrak. I have with me George Lynn, director of Government Regulatory Relations of the Dow Chemical Co. V. K. Rowe, director of the Dow Toxicological Laboratory and Etcyl Blair, director of Dow Agricultural _ Chemical Research, are also present to assist if necessary. This statement is concerned with the herbicide 2,4,5-trichlorophenoxyacetic acid, which has often been referred to as 2,4,5-T and I 1 •4 the chemical "intermediate 2,4,5-trichlorophenol used in the manufacture of 2,4,5-T. An announcement was issued October 29, 1969, by Dr. Lee Dubridge of the Office of Science and Technology which referred to birth defects observed in tests by the Bionetics Laboratories using 2,4,5-T in various dosage ranges in mice and rats. This announcement preceded the final report of the Panel on Teratology of the Mrak Commission appointed by Secretary Finch which, since May 8,1969, had been reviewing the effects of pesticides upon health and the quality of environment. At the time, October 29, 1969, members of the Mrak Commission had not seen the Bionetics report on teratology. Following the announcement by the Office of Science and Technology, I became particularly concerned because Dow is a manufacturer of this herbicide. Consequently, I made a diligent effort to trace the source of samples used and learned that the 2,4,5-T sample came from the Diamond Alkali Co. (which no longer makes 2,4,5-T). Moreover it was learned that '2,4,5-trichlorophenol also tested by the Bionetics Laboratory came from Coleman-Mathison-Bell who had obtained the sample from McKesson-Robbins who in turn had procured it from the Dow Chemical Co. 2,4,5-trichlorophenol is used as an intermediate in the manufacture of 2,4,5-T. Hence, the quality of 2,4,5-T is related to the quality of its intermediate 2,4,5-trichlorophenol. The chemical process used by Dow for manufacture is as follows: 1,2,4,5-tetrachlorobenzene is hydrolyzed in a solution of methanol and sodium hydroxide in water to form sodium 2,4,5-trichlorophenate. This is in turn reacted with sodium monochloroacetate to form sodium 2,4,5-trichlorophenoxyacetate. The solution is acidified to precipitate and recover the 2,4,5-trichlorophenoxyacetic acid. Since 1950 we have been keenly aware of the possibility of a highly toxic impurity being formed in 2,4,5-trichlorophenol as a side reaction under conditions • of elevated processing temperatures. The most sensitive toxic reaction observed in humans to this impurity was manifested by a condition known as chloracne, a skin disorder mostly prevalent on the face, neck, and back. It is similar in appearance to severe acne often suffered by teenagers. We also knew that if the impurity was present in the 2,4,5trichlorophenol it could be carried forward to the end product, 2,4,5-T. It is not formed during the manufacture of Dow 2,4,5-T from the 2,4,5-trichlorophenol, nor does it form on storage even at high temperatures. To avoid the impurity in 2,4,5-T it is necessary to keep it out of the 2,4,5-trichlorophenol. Our early control test was a bioassay. This consists of applying a solution of the material to the inner surface of a rabbit's ear and observing for the typical skin response described in a paper published in 1941 by Dow scientists. I wish to insert in the record at this point the paper entitled "The Response of Rabbit Skin to Compounds Reported to have cause Acneform Dermatitis," by E. M. Adams, D. D. Irish, H. C. Spencer, and V. K. Rowe, published .in Industrial Medicine, January 1941. Senator HART. It will be printed. (The information follows:) 363 362 The Response of Rabbit Skin to Compounds Reported to Have Caused Acneform Dermatitis E. M, ADAMS, D. D. Inisii, H. C. SPENCER. and V. K. ROWE, Biochemical Research Laboratory, The Dow Chemical Company Midland, Michigan HOSE of us acquainted with the industrial lion of some hundreds of test substances arc field have recognized the need of an experi- easily arranged according to type. Certain of the strongest irritants produce a mental method lor studying skin irritation. We would profit greatly by knowing the potential rapid destruction of the tissue (necrosis), without skin hazards of a substance before it is put into the skin having an opportunity to show an active use; we would be able to take proper precautions response. Irritants with milder and slower actions In the cheapest and most satisfactory manner and lhan this have some effect upon the tissues, as • result of which we see certain responses on tinmany undesirable incidences could be avoided. In the literature there arc many instances of part of the tissue. Most irritants have resulted irritation tests upon the skin of animals, but ap- ir responses in the rabbits' skin which tend l"> parently there has not been a comprehensive develop rapidly and to subside in a short time. relatively rapid response, which we have study. In an attempt to develop an experimental • This method, we began about six years ago to study the termed a simple irritation or reaction, may independing upon the severity, any of the responses of rabbits' skin to various types of sub- clude, hyperemia, congestion, inflammation, stances. We considered the possibility that if • following: exfoliation, edema, blistering, sloughing, exudaenough were known of these responses to different tion, crustation, necrosis, induration, hair loss. types of compounds, particularly to those with Microscopically one may see hyperemia, congeswhich there has been considerable human experi- tion, hemorrhage, edema, leucocytic 'ence, then these responses could be organized to infiltration, sloughing, and blistering, various degenerative form the basis of an experimental method. Acneform dermatitis, characterized by such changes. One type of response has been observed, howlesions as folliculitis, comedones, nodules, papules, ever, which requires a somewhat longer interval pustules, and inflammatory changes, has been re- in which become apparent, and which has D ported arising from exposure, to quite varied sub- much moretoprolonged course. This latent reaction stances including petroleum oils and greases, is a proliferative response may possibly shale oil, paraffin, zinc oxide, chlorine, tars, occur in any of the structureswhich of the skin, but that pitches, chlorinated diphenyls, chlorinated naph- about which we are particularly 1 concerned now thalenes, and crude chlorinated phenols.' '• '• '• '• '• is epithelial hypcrplasia, with its resultant thick-1 I. 8. 10. 11. IS, H. 15. Jfl. IT. IS. 10, 20, U. 13, It. IT. !l. 10, 90. 33. K. !H. ening of the skin, follicle enlargement and si ™ The recent occurrence in this country of such nil acnct'orm eruption, '• "• l a ' 1 8 t UB sometimes called quellae. Naturally responses vary to some extent, anil "chloracne," lias attracted particular interest, and we have observed various combinations of these iv c included in our animal studies five types of reactions, depending upon the substances applied substances known to cause the reaction. Today to the skin and the intensity of action. we wish to describe the unusual response of the For purposes of classification we have arhitarily rabbits' skin to these materials and to consider its divided the prolifcrativo response into the followpossible significance, ing five groups according to intensity; 1. Least detectable. Expevimcntnl Part 2. Very slight. . , , N OUR experiments, materials have been ap3. Slight. plied to the inner surface of the ear of albino 4. Moderate. rabbits and to the shaven belly. The undiluted 5. Severe. materials have been used as well as solutions of While there are naturally no sharp breaks bevarious concentrations in olive oil, paraffin oil U. S. P., propylenc glycol, ethanol, and water. tween these, and some overlapping occurs, diwas rather easy and has brcn very useful. Liberal applications were made on the ear with- vision Least detectable epithelial hi/jiorplasia: This out any covering. The applications on the ab.degree of response is manifest as an increased domen were made in a small cotton pad which prominence of the hair follicles on the inside nf was covered by a large bandage of filter cloth held in place by adhesive tape. Applications were the ear. The little dots t h a t one sees on the insirip made mice a day. live days a week, for four of the car simply become slightly larger. After exposures arc ended this enlargement regresses weeks or u n t i l a marked reaction resulted. The responses obtained following the applica- in a short time, leaving the skin apparently normal. T I lium cracks and lifts off in la^^fiioci-s like porThis degree of response is commonly seen as part tions of a cast. Often beneath these is a soft, of a mild simple irritation which is maintained cheesy, foul-smelling material, which soon drcs by repeated exposures. Thus far we have been and comes off revealing a markedly exfoliating unable to attach a particular significance to this skin beneath. The exfoliation often has a granular consistency intensity of reaction. Venj slight epithelial hyperplasia: This reaction at first, which later becomes flaky. There is a appears on the ear as a slight enlargement of the complete hair loss. hair follicles, which protrude and become hard, This proliferation of the epithelium seems to causing the ear to feel rough. The thickness of progress only to a certain extent, even with rethe ear may be increased. A very slight scaly peated applications of thi1 provoking agent. The exfoliation may accompany this degree of re- slowness and persistence 'Of this latent reacli-in sponse, but seldom is there any detectable is to be emphasized. Thr maximum of a severe hyperemia or hair loss. On the abdomen one sel- hyperplasia usually has occurred in the neighdom sees any gross evidence of hyperplasia. borhood of two weeks, the largest amount of exSlight epithelial hypcrplasia; In this reaction foliation around four weeks, and a scaly exfoliation the ear increases in thickness to about twice and hyperemia have persisted for months. normal and feels slightly stiffened and "leatherAlthough wo make exposures upon both ear like." There is some hyperemia, scaly exfoliation, and belly, the skin of the car appears to respond and hair loss. The hair follicles become slightly in the most satisfactory manner. There the mildest enlarged, raised and hard. On the abdomen there reactions are more apparent and the enlarged may br a slight thickening ol' the skin and an ex- follicles are more easily seen. As a rule the abfoliation, but enlargment of the follicles is not dominal skin shows a more marked simple irapparent. ritation. Moderate epithelial hyperplasia: This reaction consists of a thickening of the ear to 3 to 4 times Histology normal as a result of which it is quite stiff and ICROSCOPIC examinations were made usleathery. The follicles on the ear become modering 10% formalin as fixative, paraffin for ately enlarged, raised and hard, causing the sur- imbedding, and hematoxylin-eosin as stain. face of the ear to feel like the coarsest of sand The slightest hyperplastic response is shown by paper. After a time the protruding hard masses a very slight increase in thickness of the epithelium can be easily expressed by the finger-nail or by and the development of small projections (like bending the ear. At limes the enlarged follicles papillae) of but a few cells in size. The early are not apparent until after considerable exfolia- stages of more severe responses show increasing tion has occurred. A moderate hyperplasia is degrees of thickening of the surface and follicular usually accompanied by a slight to moderate epithelium. Numerous projections reach downhyperomia. Exfoliation of a granular or scaly type ward from the surface epithelium, nearly to the is of modenili' intensity and hair loss is nearly cartilage of the ear. The follicular epithelium complolc. After a number oi weeks the oar is spreads outward and downward, often completely completely deuuilod ol' hair, slightly pitied, with engulfing hair follicle and sebaceous glands. Apa slight or moili-nitc- hy|,ori'mia and possibly some parently there is also a hyperplasia in the corium. I'Nt'olialion. Tlu> abdominal skin may show a Accompanying this hyperplasia, one may see conUR'iilrv ximplr irritation than docs, the car; hy- gestion, even occasional h .-morrhagos, edema, and peromia, edema, mid even sloughing and exuda- leucocytic infiltration. tion have occurred, llypcrcmia is usually mainLater the rate of proliferation apparently lestained during the course ol' thickening. The ab- sens and those changes resulting in keratinization dominal skin finally becomes hard and stiff, fol- become more evident. As those changes leading lowed by a marked scaly and granular exfoliation, to keratinization progress from the lowermost layer of the epithelium, which constitutes a basal which persists for weeks. Severe hyperplaftta: This reaction is usually layer markedly displaced from the original, largepreceded by a marked simple irritation, including masses of material are thrown off. Thus in one even necrosis; however, there may be only section of abdominal skin we see a thick layer hyperemia and edema. As a severe hyperplasia of partly keratinized and degenerate tissue being progresses, a 1 marked hyperemia is evident until thrown off above a flat, normal-appearing stratum obscured by the thickened epithelium. The thick- vorneum. At the hair follicles most of the tissue ness of the ear is increased to many times normal, undergoes complete keratinization, forming the ears at least 1 cm. thick having been formed. As hard plugs that may be expressed. Completely a result they become very stiiV, hard, and heavy. engulfed follicles and glands are destroyed as the Fixfoliation at first has a granular consistency, hyperplastic epithelium is keratinized and thrown later flaky, and persists for months. The enlarged hair follicles are buried under the thickened epi- off.The sebaceous glands have seemed to bo inthelium and become apparent only after con- active. One sees them, apparently normal, being siderable exfoliation has occurred. From them engulfed by proliferating epithelium. Some glands, large masses of keratin may be expressed leav- of normal size and appearance, arc scon opening ing pits that may reach 2 to 3mm. in width. into the pits or cysts; others arc seen with their On the abdomen the hardened mass of epithc- M 43-362 O - 70 - 24 364 rhicts extending through large masses of kera- cysts in descriptions of the ucnoform ilorm»liH; tinized epithelium. only two instances were found of the specific n** Sections taken at a late stage show an ntropliic lion of sebaceous glands in doscriplinnti "I ft; or Very .slightly thickened surface epithelium and microscopic picture. Jones nnd Alden" rrj»«W# luimorous large pits surrounded by slightly hy- slight cdemato.iK elianges in n few glands UMf prrpliislic epithelium. The curium may still be saw; Curgil and Acton" snid that Hie srlwivw' thicker than normal. glands were unaffected. Bornemann 3 fell that IkU l t i m a t e l y there is a tendency tor the pits to cysts were of sebaceous origin hut udmilliil (l»< broaden nut and become shallower, mill one sees d i f f i c u l t y of proof, and his description shows (I** n very irregular atrophic epithelium. to be essentially epithelial structures. These facts, together w i l h our experimental itDiscussion suits, indicate Hint the so-called "sebaceous cj-><«' '"pHERE are certain points which indicate a of the acneform dermatitis in man are dlricO)? JL relationship between this reaction observed the result of an epithelial hypcrplasia. Their tjusin the rabbit and the acneform dermatitis of man. First, the reaction in the rabbit was produced by tent of sebaceous-like material is probably tat to the occurrence of inflammatory and dcgoncr»5 types of substances known to cause an acneform tive changes in the mass of epithelial tissue. W dermatitis in man. They were chlorinated di- course, the retention of sebum may also occur, phenyls, chlorinated naphthalenes, chlorinated and influence the picture to some extent, but Iba diphenyloxides, crude chlorinated phenols, and appears to be a secondary reaction. Bacterial inpetroleum oils. A few other types of substances fection may be a factor influencing the imtuif have produced the epithelial hyperplasia,but there of the reaction. has been no exposure of these on man. Wacker and Sohmincke'-' reported the experimental pro- Conclusions duction of epithelial hyperplasia with various oils, ,rr,n-n fats, and paraffin, Sachs,2'' and others, apparently, 'PHERE have been a number of hypotheses conhave produced I he identical epithelial hyperplasia *~ cel'ni"S 'he formal ion of this iicneform cru|>W(1 fed lhi in rabbits with a number of dyes. In his review tlon '" man ' " evidence shows llm of the pertinent literature, Sachs states that the acllefo ™ dermatitis to be :he visible response »< the skm to a most common dermatosis arising from exposure to " '"•'la"! acting upon it from UKaniline and coal tar dyes is eczema; however, exterior, and that I his response takes the form i)( warty growths and acneform dermatitis have also first epithelial hyperplasia, second inflammalorjr occurred. Thus it appears probable that the de- and degenerative changes, and finally regencravclopment of an outstanding hyperplastic response live processes. of the rabbits' skin is specific for those substances And in conclusion, it is possible that this aj>capable of causing an acneform dermatitis in man, parently unusual response of the rabbits' skin ofand possibly, the related papular and warty erup- fers us an experimental method which will indicate the ability of substances to produce an Secondly, by gross and microscopic examina- acneform dermatitis in man. tion, the enlarged follicles produced in the rabbit •escmble the comedones, nodules, and cysts of the Bibliography: lormatitis in man. In both cases there is a rela1. BEVTMANN: "Chtor-alu.e.' 1 oinc bcKoiidpir Form von ively large pit or cysl whose walls are composed pi-o/essinriollE'r Itnulcrknmkunij, Dc'lif, mrd. Wwhlixflir. 27, 437-440 (1001). if e p i i h e l i u m and which contains varying ainnunls 2. BI.ASCHKO: DcrtaaM. Xi'il. In, 70-72 (1011). if kcralini/.cd epithelium, and at times hair, hair 3. BOIINEMANN. W.: Ucbor • H " ProsJ. Med. Res. 12, 257-201) (1924-5), ser White 3 * describes acanthosis in the "primary lildion 7. DimoN, W. F\: Petroleum Dennntitis, M«dirnl Recpapule" and considers one important factor in the ord, 140, 650-552 (1834). production of oil folliculitis to be the chemical 8. DUVOIB, M.I ApropuH des d'^'mnloses protesslnnirritant causing auxetic cell growth. The ability nelles par le triclikirniinpMhiili'Mr. Aintuli'x ik> tnpd. Itg. 1J de rrlnilii, H, 539-544 (11134). of tar to cause active mitosis is well known. 3 9. FUI.TON, W. H.. nnrl J L. MATTIKWH: A Prcliminury Bornemann's nt stage, Report ornemanns first rst case, case, examined e amn nt aa late late stage, ot Iho DormnliiinKkiiV ii'nri "sysinnic 'rcM"e'd's""af showed more mitoses than normal and slight D Exposure to Hc.vHchlom-miplifhjiicne nnd o.lordiphenyl, Sprrinl , No, 43 thickening; but in his second case, examined at an Pennn. °"-" Di'pl, ""•" 'I-nbnr, ........" .....' H"u l l..... ~ (1D30V 10. HrnxiJLiMKi?. K.: Urhpr C'hloi'nUnt'. A/iiiirrln'ii. Mcif. earlier stage, the thickening of the epithelium was Woelniiichr. 46, 27fl (1890). much more marked. H; JAMIJON: Trcniinc-ni rif rOcxcmn with ('mil Tnr, Ann, Although menlion is often made of sebaceous rtc dernt. cl de suph,, .Inn,. 1900, pngo 22. Quoti-d by R. 365 The Dormnlcrg.ises. H. K. Lewis iiWVu., Ltd., I/ondon, 1034. ^5. SACHS, (V: KliiiiKcho und oNiM-rlmentelle Unlcrsuchunt,'.'ii iK-bci- die EU.wirkunfl vtm AnilinfnrbsttjETen nut die r.inisrliliclii 1 und Tieiisi'lle Hunt, ^rclt. /. Dvrtn. u. Si/ph. I in. 555-684 (1913). 26. SroTT. A.: The Occupuliunal Dcrmaloses of the I'urnltiii Workers (if the Scottish Shnle Oil Industry, Brit. Med. J. 2, 381-3B5 (1922), 27. SKZAHY, P. VAI.I.KBV-HAIKIT, and BENOIST: Mclanose ' ("MACKENZIE, S.: A Case ol Tnr Eruption, Brit. J. de Riehl, bullion d'huile, hypcrkdrntose folllculnlre chez un ouvrler tuumeuv f:ur mutiuix, Bull. aoc. ftdnf. dp. tttim. 10, 417 (1898). , 18. MAVEBS, M. R., and M. G, SiLViBBF.no: Skin Condi-, rtcnunt. el d.' »|/nh. 34. 139-143 (1927). Won, Resulting from Exposure to Certain Chlorinated 211. SULZBEBGER. M. B., A. ROSTENBERCJ, J R . , i.nd J. J. Hydrocarbons, J. Indiut. H|/». * To.t. 20, 244-258 (1938). RFIEH: Acneform Eruptions, N. Y State J. Met. 34, 899-908 17. N.COI.AS and J. LACASSAONE: Un^eas d ««"* ?hlOT" (19341. 29. TEI.EKY: Die Pernnkmnkhcit, Klin. Wrhnichr. 6. • IB. NOC.UEH-MORE, S., and M. C,BA«-BAHBraA| ContriJ'u- 845-8411 (1927)! 6. 897-901 (1827); 7, 214 (1928). 30. TOTSUKA, R.: Study o( the So-cnlled "Oclkrntzo," Ikm ft I'ctude des polkllodermles t. propos do trow CM OL Pi-oduL-rd by IhQ Induslvinl Use of Mineral Oil and Turpcnlh.e, Jn|i. Xeil. f. Dorm. ... l/rol. 17, 395 (1917). Ab19 OOSTON, O.: On the Local EtVocls ot Crude Paraflin, stnii'lod in Brit. J, Dormnlol. 29, 227-228 (1917). 31. TounAlNE, SOI.ENVB. MKNr.TRB... nnd AUBRUN: Clqu, Idhi M.'d J 17. 544-547 (1B71-2). % O rEN,i,IM, M.: Aftcctinns ot '*« Skin Cjiujed * iintp-quatiT ens d. di-rmntilis pjir Irtchloronnpl.Oiulrne, Hmipnllon nnd P.-ofosaion, The Mcdlcnl ]Vw» 169 (N1I8>, fli.H. tar. tram: de derm, el A? svph. 41, 265-208 (1934). 32. TUHNEII, ,T. A.: An O::cupntionul Dermntoco..iosis "il" iC" C «., nnd L. n. Bmhndl: Oil Folllc-ulllis, J. iimimK Zinc Oxide Workers, U. S. Public Hcnlth Reports :16. 2727-2732 (1921). 33. WABBE. W.: A Cose of Oil Folllculitls, Bril. J. Dcrinntiil. 12,212-213 (1900). 34. WHITE. R. PBORSEH: Tbe Dermatcriioses, H. K. Lewis nnd Co., Ltd., Ixjndon, 1934, pp. 195-241. ' 35. Occupation nnd Henlth, lnlcrnation.nl Labour Office, , H. C., nnd J. W. O,or««: The Problom o£ the . Genevn. 1930. Sec Cl.lorlne, Con'. Tars. Paraffin. PetrolOwwoi-ks PUch Industries and Cancer. The J. H. Me- ' cum Oils. Pitch, Shale Oil Industry. Fedden Researches, 1913. Quoted toy R. PBOSBEH WHITE, IWcr Wliitc, The Dcrrmitoi-BOsoB, H, K. Lewis nnd Co., IM, Inrndon, 1034. _, IJ JONES, J. W.. and H. S. AU.EN: A n .^;»j'"7" ° trm •IrrjMls, Arch. Derm, nncl Suph. 33. 1022-1034 (1036 . 13 I.EWIN. b.: UcbC'i- nllgomelno und HmitvcrMitlun* *nch I'cti-oloum, Virclwiu's Arch. 1. Palliol., Annlonur u. f»»,. 112, 35-119 (1888). H. MCEWEN: Acne due to Tiir, J, Cut. DH. 35, 1153 367 366 Dr. JOHNSON. Thank you. -:;m THE DOW CHEMICAL COMPANY MIDLAND, MICHIGAN 48B4O ~~. — r^ulJCU uiuoracne and our bionssav ¥ "*rs&?fflri^r^&?rtf by incineration S ANALYTICAL .June 22, 1965 METHOD MLW.65.11 THE DETERMINATION OP 2,2,7,8-TETRACHLORODIBENZO-p-DIOXIN IN 2,4,5-TRICHLOROPHENOXYACETIC ACID BY GAS-LIQUID CHROMATOGRAFHY . We confirmed to the point where method for the P.p.n, in Dow Chemical Co. Soaator HAHT. It will be printed< ( I he information follows :) ' 1. Scope by early' 1905 | as chromatographic This 'method i's applicable to the determination of 2,3,7,8- tetrachlorodibenzo-p-dioxin in 2,4,5-trichlorophenoxyacetic acid. The dioxin can be detected at the one ppm level with a lower limit of 0.5 ppm possible at optimum operation conditions. _, 2,4,5-tri- .,w Ohromatography," by the ,si fl '-B Principle The 2,3,7,8-tetrachlorodlbenzo-p-dioxin is separated from the 2,4,5-trichlorophenoxyacetic acid by means of an extraction with chloroform. The chloroform extract is concentrated and then chromatographed. The 2,3,7,8-tetrachlorodibenzo-p-dioxin in the sample is measured and compared to a known standard. 3. Safety Precautions 2j3j7,8-Tetrachlorodibenzo-p-dioxin is capable of causinp a severe .delayed skin response (chloracne) upon minimal contact. Samples suspected of containing any of this compound should IK-handled so aa to prevent all skin contact and inhalation. Wealimpervious gloves (rubber, polyvinyl chloride, etc.) at nil times when contact is a possibility. Clean all equipment with acetone followed by a chloroform wash. Dispose in such-a manner as to prevent all skin contact, any potentially contaminated equipment or materials which are not readily cleaned with chloroform, i.e., towels, gloves, etc. ty. Apparatus (a) Gas chromatograph, Aerograph A-600-D with flame ionization detector, Wilkins Instrument and Research, Inc., Walnut Creek, California, or equivilent. (b) Recorder, -0.05 to +1.05 millivolt, full span, one-second full response time. (c) Syringe, Hamilton microliter. No. 701N, or equivalent. (d) Syringe, Multifit 5 cc, Becton, Dickinson and Company, or equivalent. (e) Syringe, Yale 1/i) cc, Becton, Dickinson and Company, or equivalent. 368 369 (f) Centrifuge •: •}: (g) Injector •••-•• — 'Insert, Pyrex glass for A-600-D. Available ."3 vo/ from Wllkins Instrument and Research, Inc., Walnut Creek, ' ,j California (Note lla). \v; (h) Column, 1/8-inch O.D., 0.081-inch I.D., stainless steel .-*,? ; tubing, five feet in length .packed with reagent 5(c). ;y 5. Reagents (e) Add 25 ml. of I N sodium hydroxide to the chloroform txtract and shake for 15 minutes (Note lie) . (f) Centrifuge for five minutes. •• . (g) Using a five-milliliter syringe, draw off as much of the Bottom chloroform layer as is possible into a small vial. Note Mils volume. . • (h) Evaporate to dryness in a hood. (i) Take up with chloroform to 5-0$ of the volume noted in step ig) . This final solution represents ten grams of sample per ml, of chloroform. (j) Inject 1.0 microliter into the chromatograph and measure the response of the 2, 3,7,8-tetraohlorodibenzo-p-dioxin . Figure II •hows a representative chromatogram. fa) Solid support, Chromosorb W, 60/80 mesh, Johns-Manville. .:; Partitioning agent, SE-30, Silicone gum rubber-methyl -:• (Note (b) lib). (c) Column packing, five percent by weight of SE-30 on 60/80 ';; mesh Chromosorb W. Available from Wilklns Instrument and Research*]' Inc., Walnut Creek, California. •-}?. d) Carrier gas, nitrogen, commercial grade. . . %; 9. Calculations e) Chloroform, ACS grade, ' ,| f j 2,3 J 7,8-Tetrachlorodibenzo~p-dioxln, available from +':/! Let: •• • The Dow Chemical Company, Midland, Michigan. .;-•:' A = The area of the 2, 3,7,8-tetrachlorodibenzo-p-dioxin in (g) Sodium hydroxide, 1 N solution. Dissolve 40 grams of \\i reagent grade sodium hydroxide in one liter of water. '•* the sample. 6. Chromatographic Conditions B - The attenuation of the chromatograph for the sample. Oven temperature, 225°C. Inlet temperature, 260°C. ( c j Carrier gas flow rate, 75 ml. per minute as determined by the moving soap bubble technique, (d) Attenuation, such that a response of at least 50$ of scale is obtained from a 1.0 microliter sample of a standard containing 100 micrograms of 2,3.>7,8-tetrachlorodibenzo-p-dioxin in one milliliter of chloroform. C = The .micrograms per milliliter of the 2, 3,7,8-tetrachlorodibenzo-p-dioxin In the standard. 7. Thon. D = The area of the response from the 2,3,7,3-tetrachlorodibenzo-p-dioxin in the standard. E = The attenuation of the chromatograph for the standard. Preparation of Standard (a) "Again read Section 3(b) Weigh, using a micro-balance, one milligram of 2,3,7,8tetrachlorodibenzo-p-dioxin into a ten ml. volumetric flask. (c) Dilute to the mark with chloroform. (d) Inject a 1.0 microliter sample Into the chromatograph, See Figure I for a typical chromatogram. 8. Procedure 'a Weigh 10.0 grams of the sample into a four-ounce bottle, b Add 20.0 mllliliters of chloroform and shake for one hour. c Place the solution in a centrifuge tube and, with proper balancing, centrifuge for five minutes., ...... (d) Using an eye-dropper, draw off as much of. the clear chloroform layer as possible into a two-ounce bottle. li pin of.' 2",3,7,8-tetrachlorodibenzo-p-dioxin = n\JA X,•X,c.'?B xX ,?Xv.' m 10, Accuracy The accuracy of this method is - 5$, or less, relative, 11, 1,'otes (a) Glass inlet liners have been found "o be necessary to provide reproducible results. 371 370 FIGURE I (b) Silicone FS-1265 ( f l u r o ) has been f o u n d - t o work well as a stationary phase. It also is available from Wilkins Instrument and Research, I n c . , Walnut Creek, California. (c) Any 2,^.,5-trichlorophenoxyacetic acid which has dissolved in the chloroform extract must be removed as it will interfere with the chromatographic analysis of the 2,3,7,8-tetrachlorodibenzo-pdioxin. Attenuation 1 x 1 2,3,7,8-Tetrachlorodlbenzo-p-dioxin 114.4 mierograma/ml. # * # * * # # # # * * * The analytical procedures given herein have been adapted from literature sources or developed upon the basis of experimental data which are believed to be reliable. In the hands of a qualified analyst they are expected to yield results of sufficient accuracy for their intended purposes. However, The Dow Chemical Company makes no representation or warranty whatsoever concerning the procedures or results to be obtained and assumes no liability in connection with their use. Users are cautioned to confirm the suitability of the methods by appropriate tests. Anyone wishing to reproduce or publish the material in whole or in part should request written permission from The Dow Chemical Company. 0 Start 4 6 8 Retention Time (minutes) 372 373 Dr, JOHNSON. Thank you. Attenuation 32 x 10 FIGURE II Attenuation 1 x 1 2,3>7 ( 8-Tetraohlorodibenzo-p~dloxin 2.5 ppm v 0 Start Attenuation 32 x 1 2 Retention Time (minutea) Senator Hart, the 2,4,5-trichlorophenol plant was redesigned to insure, insofar as possible, the production of a product containing a minimum of the tetrachlorodibenzo-p-dioxin. By so doing we were able to control the quality of Dow 2,4,5-T. By May 1965 we had the technology to establish a manufacturing specification of no detectable 2,3,7,8-tetrachlorodibenzo-p-dioxin in 2,4,5-trichlorophenol and 2,4,5-T, using an analytical method sensitive to 1 p.m.m. While the plant was being rebuilt, we purchased 2,4,5-trichlorophenol and 2,4,5-T on the basis of this specification. The now plant came on stream in 1966 and since that time Dow 2,4,5-trichlorophenol and 2,4,5-T have met this specification, and most has contained less than 0.5 p.p.m. of the 2,3,7,8-tetrachlorodibenzo-p-dioxin. • I apologize for repeating these long chemical names but the position and number of chlorines is important. Senator HAUT. The day will come when I can pronounce them, even if I can't understand them. I can't do either yet. Dr. JOHNSON. When the difficulty was encountered in 1964 we notified the Michigan Department of Health, the Institute of Industrial Health, University of Michigan, and various other health oriented individuals in private medicine and industry. In addition we called a meeting which was held in March 1965 to notify other manufacturers of 2,4,5-T of the difficulties encountered. We described to them the nature of the health hazard and shared our test procedures and analytical standards. With this background—and firsthand experience—it was only natural that my associates and I would inquire about the identity of the sample used for the Bionetics tests. The 2,4,5-trichlorophenol tested was Dow material and the 2,4,5-T was a Diamond Alkali sample. It is important to emphasize that 2,4,5-trichlorophenol was reported to show no significant increase of anomalies by the Bionetics Laboratory, but the sample of 2,4,5-T did display a significant increase of anomalies. This prompted examining our past records of tests run in 1964. The records of analytical determinations of different supplies showed that samples of Diamond Alkali 2,4,5-T in fact did contain tetrachlorodibenzo-p-dioxin up to levels of 16 ppm. It should be emphasized at this point that Diamond Alkali has since stopped manufacturing 2,4,5-T. I presented the essence of the above information to the Mrak Commission November 7, 1969, and showed pictures of the chloracne observed in humans and pictures illustrating the rabbit ear test. Moreover, I stated that the Bionetics test with 2,4,5-T may have been complicated by an impurity in the 2,4,5-T. • I further emphasized the importance of tests using procedures recognized among experts as being valid and meaningful; the importance of representative materials which could be better obtained by consultation witli industry; and the importance of knowledge.of composition and purity of the materials tested. These points were made in the course of writing the final draft or recommendations of the Mrak Commission. 374 In view of , 2,4,5-T and the «.«, defects in cattle or &u we found it difficult to UBUBV the registered uses of 2,4,5-T. 375 I'vtAformation has been supplied primarily through Dr. Burger of the '* rfiii'fi °f rreased incidenco rangelands s ra ed P y On December 11, 1969, Dr. V. B. Robinson and V. K. Rowe met itli Drs. Falk, Courtney, and Gaylor at the Research Triangle and '^tscussed with them the design of teratological study to be conducted T+ i • jT ' '" ""ra : 6 ,i) *tt Dow regular production 2,4,5-T. Agreement on the design of the as iSjBperiment was easily achieved and was followed in our studies. tested Bionetics taminated |if At this meeting Dr. Courtney of the NIEHS Laboratory provided if the If 4 two gram sample of the 2,4,5-T used by the Bionetics Laboratory. BThis sample was examined at Dow with the following results: I?' L Rabbit ear tests showed a positive reaction characteristic of the |4' contaminant. if" 2. Analysis by gas liquid chromatography indicated the presence < v v to disc for tests which would be z,c™r,tM« f their A • B sclenfcis . "ss protocol I Of 27 plus or minus 8 p.p.m. of 2,3,7,8-tetrachlorodibenzo-p-dioxin. asked Dr. McLauo-hlin of S?M ts- Dr. LimMj |K In late December Dr. Burger of the OST requested a review of the ^bera, l^lr^^t^nSg1^^ ^ ™ ^1 i-dicmistry of 2,4,5-T production to be presented to Dr. BaldeschDrD J.^SSSiS^Jft^J?^ of a? fational Cancer Instill |weiler, a consultant of the agency. The information for this report k'Wiis organized by Dr. Blair of Dow and presented at a meeting with lister; flio OST on December 29, 1969 in Washington. ''". Dr. Blair is on my right. ;•••' By January 12, 1970, we had made enough progress in the teratological study in rats with Dow production grade 2,4,5-T to make a would bo tli , . . ' report to Dr. Egeberg, Assistant Secretary for Health and Scientific that, for {lit if Affairs, HEW. Copies were sent to other involved persons in DHEW USD A. This report showed that the Dow 2,4,5-T of regular the kforlrL^ Sf^r? ?, fSory experiments m' r and production grade did not cause birth defects as determined.by gross Health in tlle tories of a third'party ?iX^ ^ ^ > labom-.^ examination of fetuses. The dosage levels used were selected in conG nment or Dow o r in the Dow laboraE o^ToS °J °r ) fl with Drs. Falk, Courtney, and Gaylor of NIEHS. nel of the Department PIEW °bservatlon afc any time by perW"4 sultation Furthermore, we were able to report to Dr. Egeberg that a pilot study with pregnant rabbits fed the same 2,4,5-T had not caused it 0 h> ™ birth defects. Dr. H. L. Richardson, pathologist, FDA, observed the results of both of these tests. T -i *>-***w ouuu.j' yieiueu. DOS These preliminary observations were followed by the more time consuming microscopic examinations of the tissues and detailed ifc id necess-aryt'rmi feKesS *« Jffl ,™ ^^ ™' ^ skeletal examinations, This work confirmed the preliminary findings. and on refined 2 4 5 T Ttl? #raded levels of the contaminant The final report of the study was presented before the Society of Dow wo Id samples of 2,4,5-T and 2 S 7 s /f^ll ^ » P^vide Toxicology in Atlanta, Ga., on March 17, 1970. I wish_to insert into to National Institute of^vSS^ff^Pfl°™ «'« nmental Health the record at this point an abstract of this report entitled "TeratoResearch Triangle N 0 Science laboratories at genic Study of 2,4,5-trichlorophenoxyacetic Acid in the Rat" by J. L. Emerson, D. J. Thompson, C. G. Gerbig, and V. B. Robinson, The laboSTesT:?^rl^Sl^f^ ™ld ^ ^ ™HS them conce Dow Chemical Co. the test methods to be used ™ing tlie details of Senator HAKT. That will be received. D rid and B k (The information follows.) h1lhfcy 5f a contaminant in A study to determine the embryotoxicity or teratogenieity of 2,4,5-triicholorthe sample tested by Bionetics onr? P]°SS1?the ophenoxyacetic acid containing less than one part per million of 2,3,7,8Dow. At this meetijo- the samp ^ f \nfo™^^ known to tetrachlorodihenzo-p-dioxin has been completed in Sprague-Dawley derived rats. 8 as to the Mrak Commfeion Thl ^CfJTi^ ^ Panted" Five treatment groups, each consisting of 25 females were administered 1, 3, 6, for cussed with Drs.1HCJjau McLauo-hli^ additional testing as dis12 or 24 mg/kg/day of the compound via gavage in 0.25% METHOCEL® on hIm B7?nf also presented. S > ates, and Mitchell of DHEW was days 6 through 15 of gestation. A single group of 50 females received the sus. practical hazard existed ^offiTi^T ^?.^ of »Tt'•apS'tiroflS ?• ^ ? *• - % 6 nterested in M for^atFo^lfdotltKd'^T-n'" i - «^hen it was availabl,?^:?^^^^^ pending vehicle and served as controls. The following parameters were examined: clinical observations, maternal body weights (prebreedtng and day 20), number and position of fetuses and resorptions, number of corpora luten, imp weight and aex, gross external examination of pups, and macroscopic examination 'for intestinal hemorrhage in pups. Two-thirds of each litter were fixed in Bouin's solution and one-third was prepared for alizarin red-S staining 376 377 ; ff and skeletal examination. Examination of Wilson sections under the clissortktB, microscope or of alizarin stained skeletons of all fetuses from the 24 mg/kg/dWf'| group and an erjual number of control fetuses was performed. KepresentfttW f stained lustologic sections through the head, thorax, and abdomen of 10 conltsi and 10 high level fetuses were studied for histopathologic changes. No clinical or gross pathologic signs of adverse chemical effect were o in treated dams during the period of treatment or gestation. Similarly size, number of fetal resorptions, birth weights and sex ratio of pups tipixw to be unaffected by chemical treatment. Skeletal and visceral examination *« high level and control fetuses as well as histopathologic examination of certain H fetuses failed to reveal any terntogenic or embryotoxic effects. •! The results of this study fail to substantiate the findings reported reecnll}••':j (unpublished data: Bionetics Besearch Laboratories, Bethesda, Maryland) «f>> serious effects in fetuses obtained from dams given comparable daily doses of 2,4,5-T containing approximately 27 parts per million of the contaminant' 2,3,7,8-tetrachlorodibenzo-p-dioxin. • , ' ' "., Dr. JOHNSON. Thank you. '.'" In accordance with the plan discussed with the DHEW in Decent her, as soon as the preliminary results of the 2,4,5-T study on rat* indicated no fetal anomalies, we proceeded to conduct a teratology' study in rats with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Dosages were used which bracketed the levels of the contaminant which were given ; inadvertently to the rats in the Bionetics study. The results of this experiment indicated that a high of maternal and fetal toxicity WKS, associated with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Dr. H. L. Richardson of FDA and Dr. C. T. G. King, National Institute of Dental Research, NIH, participated in the observations made on.these animals at necropsy at the Dow Laboratories in Midland. We concluded that the presence of the tetrachlorodibenzo-pdioxin in the sample tested in the Bionetics Laboratories could have accounted for the observations reported and attributed 2,4,5-T. At this point I wish to insert into the record an abstract of the report as presented to the Society of Toxicology, March 17, 1970, Atlanta, Ga., entitled "Teratogenic Study of 2,3,7,8-Tetrachlorodibenzo-pdioxin in the Bat"1 by G. L. Sparsc'hu, F. L. Dunn, and V. K. Howe, The Dow Chemical Co. Senator HART. That will be inserted. Dr. JOHNSON. Thank you. The detailed results of these tests were also presented by Dow personnel to the scientists of FDA, NTH, NIEHS, in Washington on February 24,1970. In addition to our investigations with laboratory animals, we have also utilized the medical records of Dow employees accumulated throughout their Dow careers. Our physicians have made an in-depth evaluation of the health of male employees who have been exposed to 2,4,5-T in manufacturing operations for from 6 months to approximately 20 years. From the medical data available, over 50 clinical parameters were selected for statistical evaluation. The control population for this evaluation consisted of 4,600 other individuals for whom similar data were available. After careful study of this information, it was the conclusion of our medical staff that there was no evidence that exposure to 2,4,5-T had resulted in adverse effects. 1 Scop. 470. It is our belief that the adverse effects reported by the Bionetics •Laboratories were the resivit of a contaminant—2,3,7,8-tetrachloro5-T until 1964. 4B-302 0—70 2ti 380 , _Senator HART. Did those tests include any tests to determine W] carcinogenicity of dipxin in the formula that you were producing!,? Dr. JOHNSON. No, sir, they did not. Senator HART. Then in June of 1964 you were concerned nboe$ the chloracne, and in your testimony you say I think that you nolified a number of people. You notified the Department of Heallfe of Michigan and the Industrial Health Institute in Ann Arbor and various other health oriented individuals in private medicine ana industry, and you had the meeting in March of 1965 notifying other manufacturers. Why not the FDA and the U.S. Department of Agriculture? Dr. JOHNSON. At that time, Senator Hart, we considered our obligation discharged by removing the dioxin from our product, by notifying health authorities in the State and we thought we hud the problem solved. In retrospect it would have been much preferred had we notified the U.S. Department of Agriculture, the agency that has statutory authority for the registration. Senator HART. I would agree. It would seem to me, and as I sny, it is easier second guessing, that it would have been more appropriate and foremost to notify the agency that registers the product. But what about the 2,4,5-T that you learned in June of 1964 hnd this contaminant? Is it a practice to make an effort to remove tlio contaminated product from the shelves ? What about the product in the houses call back? and retail channels ? What retrieval effort is made ? What Dr. JOHNSON. Senator Hart, according to the procedures we wero using at the time we did not produce or sell contaminated 2,4,5-T within the limits of sensitivity we had available for measure. Senator HART. What was it you were notifying people about in 19(14? Dr. JOHNSON. This was the chloracne problem. May I add a comment ? Senator HART. Yes. Dr. JOHNSON. As indicated in the formal testimony, the manufacture of 2,4,5-T, when pushed by temperature or heat, will produce this contaminant; it builds up as a caustic insoluble oil. This problem produced the chloracne which initiated the actions we took. Senator HART. What percentage of dioxin was in the 2,4,5-T that you produced prior to the correction made at this time? Dr. JOHNSON. It was—now you are asking about the 2,4,5-T? Senator HART. I beg your pardon ? Dr. JOHNSON. You are asking about the 2,4,5-T. At the time there was an undetectable amount using the rabbit ear test as an indicator. It might be important to point out that chromatographic proce-dures for analysis were developed during the late 1950's and early 1960's and applied with increasing sensitivity. This is a changing background of analytical capability. At the time we were using the rabbit ear test and we did not know that dioxin was present, if any. Senator HART. As of now, do you believe that the earlier 2;4,5-T that you were producing was safe or unsafe? Dr. JOHNSON. Safe, because we were monitoring the intermediate 2,4,5-T-chlorophenol and similar products since 1941. Senator HART. But you really do not know how much dioxin was in it. How can you say that ? 381 ; Dr. JOHNSON. It was, according to our ability to determine dioxin, at that time, Ave thought zero. , Senator HART. But you know better now, don't you ? Dr. JOHNSON. We know better now because we have more sensitive methods. Senator HART. How can you say it was safe earlier when we know now it was not? Dr. JOHNSON. In our firm, Mr. Chairman, the matter of safety is considered to be related to the dosage. The product as we sold it and monitored it; and, as it was used, according to the label, we are convinced it was safe. There was not sufficient exposure to the tetrachlorodibenzo-p-dioxin, even if it had been there in the amounts known today, one part per million or a half part per million. We are convinced it was safe. - Senator HART. Did you feel any requirement at any time to engage others in making the judgment which you just made about the earlier formula? Dr. JOHNSON. The answer is no. Senator HART. Now, do you agree with the position that has been taken, as announced this morning by the three Secretaries? Dr. JOHNSON. In a matter of practical hazard, an an imminent hazard to health, I do not agree. Under the climate of pressure today, it was a wise decision. Senator HART. That sounds like you are planning to run for reelection, but you do not want to announce it. I am reminded that the action taken today was to cancel, not to suspend the nonliquid 2,4,5-T and that means, as I understood the Surgeon General's testimony this morning, the Secretaries do not regard that form of 2,4,5-T as imminently hazardous to health. Do you want to rephrase your answer so as to respond specifically to the finding on the nonliquid ? Dr. JOHNSON. The nonliquid form I consider to be safe under labeling registrations. Senator HART. I think the Food and Drug Committee finds it has potential hazard to health and therefore cancels rather than suspends it. Do you agree with that ? Dr. JOHNSON. I dp not agree that cancellation is necessary. Senator HART. Is it your intention to appeal the action? Dr. JOHNSON. This must be considered. I cannot answer at this time. We have few, if any, products of our own that are nonliquid formulations of 2,4,5-T. Mr. BIOKWIT. Do you have any evidence on the degradeability of 2,4,5-T? Dr. JOHNSON. Yes. Just one moment, please. The evidence is present in the literature published by the land grant colleges and the U.S. Department of Agriculture, predominantly. A publication by Dr. P. C. Kearney, E. A. Woolson, J. R. Plimmer, and A. R. Isensee, reviews the subject of degradation in a chapter entitled "Decontamination of Pesticides in Soils." Page 139 indicates the persistence of 2,4,5-T to last 5 months. There are additional references and review articles that, if you like, I could submit for the record. It would take quite a bit of time to read these, but I could do so, Mr. Chairman, if you like. Senator HART. They will be received. 382 List of References on Degradation of phenoxy herbicides, including 2,4,5-T The Dow Chemical Company April 15, 1970 Audus, L. J. (1960) Microbial Breakdown of Herbicides in Soils, p. 1-19 in Herbicides and the Soil, edited by E. K.Woodford and TTT R. Sagar, Blackwell Scientific Publications, Oxford. (45 references) Sheets, T, J. and L, L. Danielson (1960). Herbicides in Soils, p. 170-181 in The Nature and Fate of Chemicals Applied to Soils, 'Plants, and Animals, ARS 20-9, USDA (62 references) Tliiegs, B. J, (1962). Microbial Decomposition of Herbicides, Down to Earth, Fall 1962, (31 references) Freed, W-, H. and M. L. Montgomery (1963). The Metabolism of Herbicides in Plants and Soils, p. 1-18 _in Vol. 3 of Residue Reviews, edited by F. Gunther, Springjer-Verlag, Inc., New York. (115 references). Kearney, P. C. (1966). Metabolism'of Herbicides in Soils, p. 250-262 in Organic Pesticides in the Environment, Advances in~5hemistry Series 60. (42 references) Kearney, P, C., E. A. Woolson, J. R. Plimmer, and A. R. Isensee (1969). Decontamination of Pesticides in Soils, p. 137-149 _in Vol. 29, Residue Reviews, edited by F. Gunther, Springer-Verlag, New York. Loos, M. A. (1969). Phenoxyalkanoic Acids, p. 1-50 _i£ Degradation of Herbicides, edited by P. C. Kearney and D, D. Kaufman, Marcel Dekker, Inc., New York. (166 references) Midwest Research Institute (1967). Herbicide Residues and Their Persistence, 'and Some Factors Determining the Fate of Herbicides, p. 208-231 and 232-249 in Assessment of Ecological Effects of Extensive or RepeaTed Use of Herbicides, final report processed for Defense Documentation Center, Defense Supply Agency, AD 824 314, U. S. Dept. of Commerce. Alexander, M. and M. I. H. Aleem '(1961). Effect of Chemical Structure, on Microbial Decomposition of Aromatic Herbicides. J. Agr. Food Chem. 9_, 44. Bell, G, R. (1960). Studies on a Soil Achromobacter which Degrades 2,4-Dichlorophenoxyacetic AcidTCan. J. Microbial. 6, 325. Bollag, J, -M., C. S. Helling, and M. Alexander (1968a). Enzymatic Hydroxylation of Chlorinated Phenols. J. Agr. Food Chem. 16, 826, .m , 383 Bollag, J. -M., G. G. Briggs, J. E. Dawson, and . (1968b). Enzymatic Degradation of Chlorocatechols. Food Chem. 16, 829. J. Agr. Tiedje, J, M., J. M. Duxbury, M. Alexander, and J. E, Dawson (1969). 2,4-D Metabolism: Pathway of Degradation of Chlorocatechols by Arthrobacter sp. 'J, Agr. Food Chem. 17, 1021. — Duxbury, J. M., J, M. Tiedje, M. Alexander, and J. E. Dawson (1970). 2,4-D Metabolism: Enzymatic Conversion of Chloronaleylacetic Acid to Succinic Acid. J. Agr. Food Chem. 18 (2), 199. . — Rogoff, M. H. (1961). Oxidation of Aromatic Compounds by Bacteria, p. 193 in Volume 3 of Advances in Applied Microbiology, edited by W. W. Umbreit, Academic Press, New York. Aly, 0: M. and S. D. Faust (1964). Studies on the Fate of 2,4-D and Ester Derivatives in Natural Surface Waters. J. Agr. Food Chem. _12, 541. Crosby, D. G. and H. Tutass (1966). Photodecomposition of 2,4-Dichlorophenoxyacetic Acid. J. Agr. Food Chem. 14, 596. Crosby, D. G. and M. -Y. Li (1969) . Herbicide Photodecomposition, p. 321-363 jln Degradation of Herbicides, edited by P. C. Kearney and D. D. Kaufman, Marcel Dekker, Inc., New York. (102 references) Brown, E. and Nishioka, Y. A. (1967). Pesticides Monitoring J. 1(2), 38-46. Pesticides in'Water. Norris, L. A, (1968). Stream Contamination by Herbicides after Fall Rains on Forest Land. Western Society of Weed Science Research Progress Report, p. 33-34. Montgomery, M. L. and L. A.- Norris (1970). A Preliminary Evaluation of the Hazards of 2,4,5-T in the Forest Environment, USDA Forest Service Research Note PNW-116. Sheets, T. J. and J.. F. Lutz (1969). Movement of Herbicides in Runoff Water. 'Presented at the Dec. 1969 meeting of the American Society of Agricultural Engineers, Chicago, Illinois. Bailey, G. W., J. D. Pope, Jr., andD. R. Cochrane (1968). The Degradation, Kinetics, and Persistence of Silvex Under Impound Conditions. Abstracts p. 43, WSSA Meeting, New Orleans, Louisiana, February 1968. 384 ^aurfi ^i I^Mu*..-.Iu, i^A'.A.^iSKi2«w».ii^iisii Ux2/ll' PIUaM L tkMOa n.. Urea, triazine, and piclorarn herbicides i' : 7T^?!5!W ^^Pfip{|^>^i ^ - - . Benzole acid and amide herbicides ' - ^ •, tv^i^ji ^'ti t.... Phenoxy, toluidine.and nitrile herbicides u RESIDUE Carbamate and aliphatic acid herbicides Decontamination of pesticides in soils By P. C. K EARNEY) * E. A. WooLsoN 18 Fig. 1. Persistence of pesticides in soils J. R. pLIMMER>* and I. Introduction ', senfe one or more .'.' j'.^ Atra/ine, Monuron Fig. 3, Processes that determine fate of a pesticide in soil Diphenomide Diuron Linuron, Fenuron Prometryne I I I 0 3 4 CDAA, Dicambo 4 I . I i I 6 8 10 12 Months L 8 10 iz 14 is re Months Phenoxy, foluidine.and nitrile herbicides .i^ito" "f!(?./ffi')'.'I'i> :'J>1' ^.ii-v- '- [- J; '^.^"V'vjj TCA Trif lurolin i:rtiiLKIiiiA&*i' i.?.'? i-.r teii&.a"!Li:j^ii3 2, «*,*!-T Dolopon.CIPC ^'iiki^ri^'X^'^ '••'; '^'1 CDEC Oichlobenil MCPA IPCJEPTC 2,4-D 0 1 2 Corbarnofe and aliphatic acid herbicides ,_3 4 , 5 6 Borban l 3 2 ! Months 1 4 1 6 Weeks l 8 1 10 , 1. 12 Fig. 2. Persistence of individual pesticides in soils Now that we have defined the problem and the need for decontamination in soils, what methods are available for removing persistent pesticides? The fate of a pesticide in soils' is determined by a number of processes which come under the general heading of physical, biological, and chemical (Fig. 3). Under physical, they include photo- decomposition, volatility, leaching, and adsorption. Under biological, they include root uptake and microbial metabolism and under chemical, they include oxidation, reduction, and hydrolysis. Several of these are responsible for decomposing pesticides. For example, soil enrichment techniques for the proliferation of specific microorganisms effective in metabolizing foreign substances have been a favorite method for microbiologists. It is conceivable that "catch plants" or plants with a high affinity for certain pesticides could be grown on contaminated soils and then removed after taking up some part of the residual pesticides. It is possible that a combination or several of these methods could be employed to reduce pesticide concentrations in soils. If each of these processes were active on a pesticide, then soil residues would not exist. If soil microorganisms could be induced to rapidly metabolize DDT to the level of carbon dioxide, then soils would be an ideal medium for decontamination. They don't, and therein lies the problem of reducing soil residues. The work to be reported today deals with two approaches to reducing pesticide residues. The first concerns the use of light to decompose transported pesticides in water and hence its application to irrigation waters and soils. The second concerns the decontamination of field soils containing DDT by flooding and inoculation with microorganisms. II. Pesticide decontamination in water A major source of environmental contamination is caused by the movement of materials from their site of application. Pesticides move primarily in the liquid or vapor phase. Pollution of water by organic 388 389 rorn impounds is undesirable, contamination by biologically active com- pounds is potentially dangerous. Two particular situations in which pesticides in water cause concern relate to the waste problem encountered in static or lagoon operations and to irrigation systems. The danger of the latter situation is best illustrated with the water-soluble, mobile herbicide picloram (4-amino-3,5,6-trich]oropicolinic acid). Minute amounts of this potent herbicide irrigated oa sensitive crops could have disastrous results. Concentrations as little as 10 p.p.b. in soils have a lethal effect on such sensitive crops as soybeans. What methods are available for removing pesticides in water? The use of energetic radiation (ultraviolet or gamma ray) has been suggested (MARCUS et al. 1962) for fragmentation or destruction of organics in water. This method should be effective on a large number of pesticides especially in dilute aqueous solutions. Unfortunately, the technology has developed little beyond die experimental stage. Large scale ultraviolet and gamma irradiation techniques are in early technological stages and wider industrial application is the needed stimulus for further development. We have determined the periods of exposure required to destroy the biological activity of a number of herbicide solutions in a smallscale ultraviolet irradiator. The method may be applicable as a pretreatment for waste waters or as a treatment for contaminated irrigation systems. The reaction system is a borosilicate glass vessel and holds 250 ml. of the solution to be irradiated (Fig. 4). A quartz, water-cooled, double-walled tube is fitted into this well and is immersed in the solution. A 450 watt Hanovia lamp is suspended in the well. The quartz well transmits a large part of the available energy down to the shortest wavelengths emitted by the lamp. Solutions of herbicides in water wore irradiated (250 ml. at a time) for periods of 5, 10, and 15 minutes. Picloram,' 2,4,5-T, bromacil, " diphenamid, and 2,3,6-TBA were die herbicides used in the initial experiments. These compounds were chosen because their solubility . and persistence are sufficiently high for them to be potential contaminants in irrigation water. Oats were used to bioassay picloram, 2,4,5-T, and diphenamid and cucumber was used for bromacil and 2,3,6-TBA. The treatments consisted of zero, 5, 10, or 15 minute irradiations of the solutions at 1, 5, or 10 p.p.m, concentrations and a control in which no herbicide was added. The time required to destroy the five herbicides is shown in Figure 5 (PUMMER 1968). A five-minute irradiation greatly reduced the phytotoxicity of picloram and 2,4.5-T at five and 10 p.p.m. and bromacil at one p.p.m. Diphenamid and 2,3,6-TBA required a 10-mim;te exposure. These initial results indicate that five minutes or less exposure to ultraviolet irradiation of solutions in the range of one p.pm. would significantly lower their phytotoxicity to plants. More pesticides, under conditions Water Lamp Quartz gloss well Solution irradiated" Fig. 4, Photochemical reaction vessel 5 Picloram 2,4,5-T 5 and 10 p.p.m. Sp.p.rn. Bromacil 2,4,5-T I p.p.m. lOp.p.m. Diphenamid 10 p.p.m. Bromacil 2,3,6-TBA lOp.prn. lOp.p.m. 10 Fig. J5. Times required to destroy herbiddal activity by irradiation 15 391 390 approaching large, volumes of water on flow systems need to be investigated before wide application to pesticide decontamination is attempted. mtamn of f-ve percent SE 30 sorb W, Column temperature was 210 ml/minute, Detector temperature was 21& |j ined consider- III. DDT decontamination Turning our attention to field soils, one of the most serious residue problems occurs with orgnnochlorine insecticides. As previously mentioned, DDT persists for several years in most agricultural soils. Complete removal of these residues may be impossible. However, lowering existing residues below some arbitrary threshold level may result in minimal plant residues. Our objective, then, was to find some agent in nature that could attack the DDT molecule. This agent did not necessarily have to cause complete destruction of DDT, but perhaps alter it to a more biodegradable or labile form. Obviously, organisms indigenous to most soils do not possess this agent. Intestinal microorganisms in the rat, however, are able to alter DDT extensively. Whole cells or cell-free extracts of Aerobacter aerogenes catalyze the degradation of DDT in vitro to at least seven metabolites (WEDEMEYER 1966), previously reported from rats given DDT orally (PETERSON and ROBISON 1964). These reactions proceed by dechlorination, elimination, oxidation, and finally decarboxylation to yield dichlorobenzophenone. Therefore, it occurred to us soils inoculated with A. aerogenes may be capable of metabolizing residual DDT. To test this hypothesis, three soil types were amended with zero, 5, 10, and 20 p.p.m, of DDT, The soils were Lakeland sandy loam, Hagerstown silty clay loam, and Sharkey clay. Four-hundred g. of soil were weighed into pots and DDT was applied in chloroform solution. Since metabolism of DDT by A, aerogenes appears to occur most rapidly in still cultures or under partially anaerobic conditions, two-thirds of the soils were flooded to simulate partial anaerobiosis. The water covered the soils to a depth of approximately one inch. One-third of the DDT-treatcd soils was maintained at field capacity, one-third was flooded, and one-third was flooded and inoculated with A. aerogenes. Cells of A. aerogenes from slants obtained from the American Type Culture (ATC 13048) were mass cultured in three percent trypticase soy broth at 36° C, for eight hours, The cells were harvested by highspeed cenlrifugation, washed, and resuspended in the original volume of fresh broth solution, The cells were incubated for three days in still cultures, harvested again, washed, and concentrated 10-fold in a one "percent yeast-extract solution, Aliquots (10 ml.) were added to the flooded soils and mixed into the surface layers. All soils were sampled at weekly intervals. Residual DDT and products v/erc measured by electron-capture ga,s cbromritography. Moist soil samples were extracted with a 3:1 mixture of hexaneiisopropanol and injected on to a Flooded Flooded- A. aerogenes Fig. 6. DDT decomposition in three soils (see text) Flooded - A. oerogeneS' Flooded Lakeland Fig. 7. DDT decomposition in three soils (sec text) disappeared more rapidly in the inoculated soils. Second, complete loss occurred in the Sharkey clay, while lesser amounts were lost from Lakeland and Hagerstown in both Hooded series. DDD (TDK) was observed to occur in most soils, but its appearance did not parallel 392 393 Tosses in-DDT. In other words, there was a net loss of DDT-DDD in. this system and no other products were detected by gas chroma!*' graphy. Somewhat the same picture is encountered at five p.p.in. of DDT (Fig. 7), with complete loss in the Sharkey clay, more accelerated disappearance in the inoculated soils, and a general trend for total loss of DDT-DDD with time. Recovery values for DDT during the early sampling periods on Lakeland were high and explain the values obtained above five p.p.m. in the flooded soils. Additional studies were initiated to determine the fate of the DDT in these systems. The experiment using Lakeland soil at five p.p.in. plus DDT-1d ondrin f rom soil, A gt > '• by Acrobacter aerogcne, Science 1* Mr. BICKWIT. Do you have any evidence on the degradeability of jj-wxm? ^^ ; Dr. JOHNSON. In terms of photodegradation, we have somd^Bdence j *"tl)is is not exhaustive—but nevertheless some evidence that would ^'indicate a half life of—excuse me, I will get that in just a moment. "•• This is the type of experiment run in a laboratory in a solvent. lHi& half life was 21/2 hours under a typical type of sun lamp. This » only an indicator type of test. The prognosis is in the presence ,*f light, dioxin will degrade. Degradation in soil we do not have information on. We are diligently, however, preparing carefully totaled—radiochemically labeled—2,3,7,8-tetrachlordibenzo-p-dioxin, mul supplying this to the U.S. Department of Agriculture for tests M quickly as possible, because this is the fastest way to get the answer. Mr. BICKWIT. Do the light conditions you are using exist in nature? Dr. JOHNSON. Primarily in ranges and pastures, yes. Because 2,4,5-T is intercepted on the upper surfaces of weeds and brush more deposits in exposed than in shaded conditions. The predominance would be exposed to light conditions. Obviously some is going into the shade. Under those conditions of lesser light intensities, I can't reply. Mr. BICKWIT. Is it ultraviolet light you are using? Dr. JOHNSON. Yes, with a typical sunlamp. Mr. BICKWIT. Do I summarize your findings correctly when I say you believe 2,4,5-T is degradeable in a matter of months, and witli respect to dioxin there is some evidence it is degradeable, but we doDr. not JOHNSON. know whether or notI itshould is? again emphasize if specifications Correct. are low, the minute amount presents an extremely small exposure. Mr. BICKWIT. With regard to the calculations that you offered us earlier, you have assumed that a person who ingests 2,4,5-T is ingesting one part per million dioxin. Isn't it possible Dr. JOHNSON. May I clarify the statement for the assumption? Mr. BICKWIT. The Sure.0.2 parts per million referred to a hypothetical Dr. JOHNSON. situation. Dr. JOHNSON. People do not ingest that much, and that is the total dietary intake, and moreover, 2,4,5-T has not been a residue in food. It is, in effect, zero tolerance. Mr. BICKWIT. I understood that. What I still understand to be your assumption was that any amoxmt of 2,4,5-T, no matter how small, that was ingested, would have one part per million dioxin in it. That was the basis of your calculation. Dr. JOHNSON. The specification we have set for our own product is less than one part per million. Actually, it is 0.5. The assumption was to make it easy mathematically, on the high side, and some day I will learn not to draw these mathematical analogies. They never makeWell, the mark. Mr.quite BICKWIT. I hope you learn right now. What I was suggesting as a possibility is, that since 2,4,5-T is degradeable, and since we do not know whether or not dioxin is degradeable, that although your product, when sprayed, has 0.5 parts per million of "' :' Dr. JOHNSON. I think Mr. Bickwit is talking about ^^possibility *f the 2,4,5-T degrading in the food consumed, whereas the degradation question only applies to environment. Mr. BICKWIT. No, I am not. .Senator HART. Try it again. Mr. BIGKWIT. You are assuming that the maximum amount that '*o would ingest would be 0.2 parts per million in every amount of iood, in every iota of food, that we eat. From that you conclude - that the amount of dioxin in that food would be one-millionth of ! 0.5} parts a million, which I admit is an extremely small amount. I am questioning your assumption of whether the amount of dioxin, compared to the amount of 2,4,5-T that is ingested, would be onemillionth merely because dioxin is one-millionth of the 2,4,5-T amount when it is sprayed ? What I am suggesting is that the dioxin consumed may actually l» more than the amount of 2,4,5-T consumed, even if it is only one 1 part per million when sprayed. Dr. JOHNSON. Is the situation that you have in mind that the 2,4,5-T is sprayed? Mr. BICKWIT. Yes. Dr. JOHNSON. The 2,4,5-T is sprayed into the environment. This contains one part per million. Then the 2,4,5-T degrades. The dioxin does not. And over a period of time, is there is a buildup of dioxin ? Is this the problem? Mr. BICKWIT. I have another way of getting at this, perhaps. If one gram of 2,4,5-T is sprayed on a blueberry—an unlikely assumption—and the 2,4,5-T degrades so that it complies with the tolerance of 0.2 parts per million, you may still have one microgram of dioxin sitting on that blueberry, without any violation of the tolerance of 2,4,5-T, And that one microgram may well be toxic. Dr. JOHNSON. I would like to find a degradeable material like that. Theoretically, obviously, if the one gram of degradeable material contained the one microgram or one part per million of nondegradeable material, then the one gram degrades, that one microgram would still bo sitting there. Is that the point you are trying to make ? Mr. BICKWIT. That's right. Dr. JOHNSON. That's right, Mr. BICKWIT. Well, how then does a 0.2 parts per million tolerance for 2,4,5-T protect us from dioxin ? Dr. JOHNSON. Because the ratios of those two during the process of growing and supplying the food are going to remain essentially the same. Mr. BICKWIT. You have no evidence for that statement. Dr. JOHNSON. I know I don't, but you have no evidence for the hypothetical question, either. Mr. BICKWIT. I do have some evidence, by your own statement, that dioxin is more likely to be nondegradeable than 2,4,5-T. Dr. JOHNSON. But these are matters of relative rates. You are suggesting an instantaneous degradation. Mr. BIGKWIT. I am using a hypothetical situation, as you were. • : ._. _,~^WJ.T. uertamly not. , '> Mr. BLAIR. So your question as phrased has, no meaning as,I triwj to interpret it. Unless you mean that possibly as 2,4,5-T degrade*,-, that it would degrade through dioxin, or into a dioxin product Then, the percentage would go up, the exact concentration ; has not while gone up. Mr. BICKWIT. What I am saying is that your calculations nre based on the view that the hypothetical view—that we would allow a 0.5 parts per million tolerance for 2,4,5-T. You then conclude, if 0.2 parts per million of 2,4,5-T were ingested, that an extremely minute amount of dioxin would be ingested. What I am questioning is whether the conclusion follows from that premise? Dr. BLAIR. Yes, it would be an extremely small amount, and it would not increase with time. Mr. BICKWIT. What I am suggesting is that the 2,4,5-T could break down so that it complies with the 0.2 parts per million tolerance. In fact, it might even disappear. Yet, we would be ingesting a good deal of dioxin for which there would be no tolerance. Dr. BLAIR. It is not possible. Mr. BICKWIT. I don't understand why not. BLAIR. How could you ingest 2,4,5-T that contained a tenth of Dr. a part per million Mr. BICKWIT. It contained that much when it was sprayed. Dr. BLAIR. Yes, and with time the concentration of dioxin in that environment has not increased one iota. Mr. BICKWIT. That's right. The amount has not increased. Dr. BLAIR. is not possible to ingest more than what is there, It doesn't makeBut sense. Dr. JOHNSON. Mr. Chairman, could I make a comment? Senator HART, I wish I could be helpful. I would like to see if a rephasing of the question might not elicit the point that bothers Mr, Bickwit. Dr. JOHNSON. rephrased first? Do you want to comment first, or have the question Senator HART. Make your comment. 399 400 -Jr. JOHNSON. My purpose in bringing up what appears to ha* been a rather foolish example was merely to give a feeling for the magnitude of how much dioxin we are talking about. Mr. BICKWIT. And my contention is that once the assumption on which the hypothesis was based is removed, then it does not give that feeling. Dr. JOHNSON. I am sorry. I don't agree. Senator HART. That is one of the fortunate features of having_a reporter here. We can all grab the record in the morning to see if we can count out an understanding and agreement of yesterday. Mr. BICKWIT. You state in your statement that members of the Mrak Commission had not seen the report prior to Dr. DuBridge's October 29 announcement. Last week Dr. Kotin of NIEHS told us as work was completed it was promptly passed along to the Commission. Do you mean to imply that the Bionetics report was not complete until this time? Dr. JOHNSON. I did not receive a copy of the report of the Panel on Teratology until very late in the deliberations. Mr. BICKWIT. I think that is unfortunate. I wonder if you have knowledge as to why that was so ? Dr. JOHNSON. No. Mr. BICKWIT. We have have been told that whenever you burn a polychlorinated phenol, dioxin prodiiction is possible, or even likely. Could you enumerate a few of your products that contain such phenols ? Dr. JOHNSON. There's trichlorophenol, tetrachlorophenol and pentachlorophenol, and sodium salts thereof. Mr. BICKWIT. What is a Dowicide product ? Dr. JOHNSON. The products I just mentioned. Since you related it to the phenols, I assume that is what you are talking about. "Mr. BICKWIT. Yes. I wonder if you could furnish us with a list'of all such products Dr. JOHNSON. Mr. Howe clarified a point here that our Dowicide trademark applies not only to chlorophenols, but to other antimicrobials. Mr. BICKWIT. I wondered if we could have for our files a list of the products which you produce which contain polychlorinated phenols ? Dr. JOHNSON. Yes. I prefer the term chlorophenols. Polychlorinated phenols would indicate a polymer. Chlorophenols. Senator HART. Now I am getting into something that you don't have to be anything to understand except efficient.' ' Do you have anything to do with Lake St. Claire? Dr. JOHNSON. I am not really prepared to discuss that in detail, I don't have any direct responsibility for the Sarnia Plant. Senator HART. I would be disciplined severely by my outdoor friends if I didn't ask what you are going to do about the mercury that has found its way into Lake St. Claire. Dr. JOHNSON. I might say this, we intend to exercise responsible action. Senator HART. That is like the lawyer admitting his client is innosnt. But I won't push you if you are not prepared. ^^ cent. Dr. JOHNSON. I am not prepared to say any more. ^J Senator HART. Thank you very much. Are there any further items that you have ? Dr. JOHNSON. Yes, if I can take a few more minutes. Senator HART. Time is not a problem. Dr. JOHNSON. I would like to make a comment or two, again on dioxin before getting into the last part of my testimony. There have been questions raised about the decomposition of 2,4,5-T and dioxin. 2,4,5-T is unstable at elevated temperatures. We were able to get degradation when 2,4,5-T or dioxin were burned on paper. We were unable to detect any dioxin residue in the smoke. Now, these are very early bits of information, strictly preliminary but I thought I should mention it at this time. This morning you asked a question about shortening the time interval between the early indication of some possible difficulties and the learning of sufficient truth about a situation so that we can take appropriate action prior to an imminent hazard to health. Hopefully, if we can improve some of our combined procedures between Government and industry, we can shorten that time. I have a few remarks to make that apply to the scientific community as well as to this interface with Government. Screening tests, as we have heard, (and I commented on some of my own,) I think are of value. As early indicators. But screening tests alone, without the consideration of quantitative data, can be quite misleading and if necessary Senator HART. Can be what 1 Dr. JOHNSON. Quite misleading and I think it is necessary to develop understanding in meetings where open reports are published early and where scientific information is submitted to the challenge of other scientists; scientists of the universities; government; industry and professionals so these things can be considered in the caldronI of openwe technical debate. think can operate more quickly to shorten this gap if there is more openness about reports. Moreover, I think another procedure for shortening the time is better to understand test procedures. The screening tests are simple but those tests which are substantive enough to justify intelligent action require, the consideration of scientists outside of the generating laboratory, and the review of appropriate methods. Test methods for regulatory action are—or for clearance of new products, in particular, require consideration. I don't mean we sho.uld lock every clearance procedure into a lock step because the development of techniques is a mutable, changing thing. But the confirmation of tests prior to official acceptance as this relates to public policy important. of Official Agricultural Chemists has established The isAssociation a useful procedure. When these gentlemen and this association are considering new tests, the topic is identified, the first scientists to work on the test are identified, collaborators are found to_ work out objective tests and these tests are run in separate laboratories so that confirming methodology can be established. 402 As a result the test must be reliable; that is, it miist give accifll< Precise and reproducible results when used by qualified analysts^!* must be practical; simple. It must be available to all analysts and !l must be substantial; that is, supported by collaborative study. Now, I will say these types of tests apply primarily to cleaninc* procedures or to regulatory tests. I am not saying extreme tests are not in order. But those which support the authority of regulatory backup need this type of scientific consideration. The American Standards Society also has procedures for standardizing tests. These are physical tests. They involve one or more laboratories in the preliminary study and interlaboratory studio.1*, maybe as many as 10, in the more refined studies, and this results in a standard test method which can be used. The Department of Commerce, National Bureau of Standards, does an excellent job in developing standards. Moreover they have an industrial associates program which permits the access of industry into these laboratories, for a period of time to work, so thai industry representatives can better understand the problems of standards and go back to their companies better informed. Protocols for clearance of new and existing products, I think are important for the Food and Drug Administration to consider seriously. Again I don't mean standard tests would remain forever, but protocols. Industi'y needs to know where it stands, industry also need to participate; appropriately and at arm's length, to be sure, nevertheless; to participate with specific information. The period of time between 1966 and now could have been shortened by sharing appropriate information, and I admit we could have assisted by volunteering earlier. But there seems to be some reverse of togetherness and there seems to be great concern that communication between governmental agencies and industry is suspect and would be misused. I would hope that one valuable result to come of this would be a continuation of developing science fortified by interest in this important matter of communication, On the point of developing protocols (and I frankly think the whole subject of teratology requires the vigor of scientific debate among professional peers involving industry, university and government) I would hope the National Academy of Sciences could provide such a forum, if not the National Academy perhaps the New York Academy of Sciences and I intend to help encourage this because it is important. It is important that these ideas are traded openly and published. I would mention that the World Health Organization, in a document entitled "Principles for the Testing of Drugs for Teratogenicity," the World Health Organization Technical Report, Serial No. 364 or 1967, has the following to say on test animals for teratology, on the subject of the chick embryo, page 7. The chick embryo contributes greatly to basic embryological knowledge. Hownver, for the screening of drugs for teratogenicity, its use is not recommended. It is too sensitive to a wide range of agents and affords no parallel with the anatomical and physiological relationship existing between pregnant mammals and her conceptus. 403 .1 I will support the idea of the use of a chick embryo^P screening tests, early indications, but from that point on additional protocols and understanding, I think, are necessary. Again in the line of helping to shorten this interval, I think strict standards are necessary in defining matters of safety and public health. No question about it. I don't object to tought regulations. But industry needs to know where it stands and the dialogue is important. The dialogue between agencies can help to shorten this time and I am glad to see distinct improvement in communications between the Department of Health, Education, and Welfare and the Department of Agriculture. Again I would like to say we are not talking about a locked-in procedure that could be used as an excuse to avoid fxirther responsibility for progress. Another point; millions have been spent on cancer' research. Yet to this day, adequate experiments have not been devised or supported on an adequate scale by government to establish thresholds. By that I mean levels below which no response occurs. Without that type of information—there will be continuing public fear as analytical methods become more and more sensitive. Senator HART. May I interrupt you there? Dr. JOHNSON. May I finish the point and come back? With teratogens, mutagens, radioactivity and teratology, this all applies. In other words, if the dose is low enough can any of these effects be avoided. This is the big question today. It is the question which coupled with sensitive analytical methods makes the Delaney clause a difficult thing to live with. I would like to make the comment that vitamin A, an essential nutrient, is a teratogen at high doses. So this matter of developing test procedures and a big enough experiment with carcinogens, mutagens, teratogens or low levels of radioactivity, needs to be done on a national scale and supported so that some of this public fear can be avoided and so we know where we stand. Scientists should put limitations on their speculations and preconceptions and get the facts but also realize we have a job in serving the public policy that does not exactly follow the scientific method. I hope we can work out quicker procedures to go from early indications to reliable decisions by involving some of the procedures I have talked about. After responding to your question, I would like to make that final statement. Senator HART, I am not sure I need to ask the question now. I agree completely on the desirability of doing that which our best minds can suggest to shorten the time. The question I intended to interrupt you with I think you did answer. I was going to ask to what extent in developing these thresholds and having broader sampling and more reliable data, to what extent is that the responsibility of the Federal Government? Dr. JOHNSON. On the matters of carcinogenicity, teratology, radiation, and mutagenesis, where such public concern is involved, and since this involves a wide variety of suspected material, it is my opinion that the Federal Government should play a major role,in supporting this research, both in its own laboratories and in extra- 404 4U5 iRRESTATEMENT OF DR. SAMUEL S. EPSTEIN, CHILDREN'S C ;OOL, ^•ural research done in universities and in industry to help g ; SEARCH FOUNDATION, INC., AND HARVARD MEDICO ^uversity of methods and the best thinking of the nation. ' ''flf BOSTON, MASS. Senator HART. All right, then to the extent that the story tlm( *<*|||| have heard today and heard last week reflects delay and a failure«*^if| Dr, EPSTEIN. Mr. Chairman, it is a privilege to testify before you communication, I would hope that the suggestions you have ma*!*'-" ' today. The subject of my presentation is teratogenic effects of 2,4,5-T and the discussions that we have had all will insure that there wjR be no delay, there will be no barrier to free communication, aiv4 ,, { formulations. My professional background and experience, as stated in the there will be no suspicion attached as agency and producer try tt»''' attached appendix 1, broadly relate to the study of hazards due to drive through to the answer. _ .'.',5 chemical pollutants in the human environment. Relevant to the presNow, you wanted to remind us of what the Ribicoff comrniUrt';4| ent testimony is my recent role as Cochairman of the Advisory Panel said. on Teratogenicity of Pesticides, and as Chairman of the Advisory Dr. JOHNSON. Yes, if I may. Panel on Mutagenicity of Pesticides, of the Secretary's Commission This is read from Report No. 1379 of the 89th Congress, 2d fison Pesticides and Their Relationship to Environmental Health, HEW, sion "Interagency Environmental Hazards Coordination, Pesticides December 1969. and Public Policy", a report of the Committee on Government Oper- SI GPO, The Panel on Teratogenicity was composed of outside consultants ations United States Senate, made by its Subcommittee on Reorganiand representatives of HEW agencies—CPEHS, FDA, NCI, zation and International Organizations, I will read from page 50. NIEHS, and NIDR. The HEW Panel report on teratogenicity is And this puts the scientists on the spot, including me. included in the appendix (II). The heading of the paragraph is "The Responsibilities of the SciA. Teratogenicity as a public health hazard: Potential hazards entists" and we are talking about industry scientists, governmental, posed by environmental pollutants and drugs include toxicity^ or poiand university. soning, carcinogenicity or induction of cancer, mutagenicity or "The committee asked the scientific witness for meaningful induction of genetic damage, and teratogenicity or induction of advice for the Congress, but much of the testimony was inhibited bv developmental abnormalities in the growing embryo. defense of past positions, employer loyalties, and lack of authority. Although teratogenic effects of various agents have been recogScientists should do as thorough a job of preparing answers on nized for several decades, it was only as a reaction to the thalidoaspects of research administration and planning as they do on the mide episode of 1962 that a requirement for teratogenicity data technical details of the work. became established. The maker of public policy must have alternatives from which to Teratology is the study of congenital malformations. These are choose. There are always strong vested interests which resist change. generally denned as stmctural abnormalities which can be recogUnless, the technological situation (in this case, ecology) can be nized at or shortly after birth and which can cause disability or clearly explained and related to public policy issues, the decisiondeath. Less restrictedly, teratology, also includes, microscopical, biomaker is hardput to recommend any new course. This understanding chemical and functional abnormalities of prenatal origin. must be extended also to the citizen. No great social issues have ever Congenital malformations pose incalculable personal, familial and been decided until the needs were clear to the man in the street. social stresses. The financial cost to society of one severely retarded Scientists cannot assure that their knowledge will reach the decichild, computed on the basis of specialized training and custodial sionmaker through the normal channels of publication and review in care alone, approximates to $250,000 (Oberle, 1969), This figure the scientific community. Without shortcutting the classical methods excludes further costs to society due to deprivation of earnings. of assessing the truth, there is still an obligation to interpret what is In the absence of a comprehensive national surveillance system, known and replace emotion, rumor, and misconception with a clear the precise overall incidence of congenital malformations is explanation of the facts. unknown. This incidence has been variously estimated as ranging The role of the scientist in relation to the legislator is limited to between 3 and 4 percent of total live births. an area somewhere short of the decisionmaking point. Proper use of Three major categories of human teratogens have been identified scientific advice requires considerable effort on both the part of the —viral infections, for example German measles; irradiation, for scientific community and the body politic. example X-rays; and chemical agents, for example thalidomide and This is the end of my statement. mercury. Senator HART. I am glad you reminded all of us of it. B. Methods for teratogenicity testing: teratogenic effects of chemDoctor, thank you very much. icals and other agents should of course be identified in experimental Do your associates have anything to add in light of the statement ? animals rather than in human beings following accidental or unreAll right, thank you very much. cognized exposure. Test agents should be administered to pregnant Dr. JOHNSON. Thank you. animals during active organogenesis of their developing embryos. Senator HART, Next we have Dr. Samuel Epstein, Children's Shortly before anticipated birth embryos should be harvested by Cancer Research Foundation. caesarean section and examined. Doctor, we welcome you. Parameters to be considered in test and concurrent control anil ^ The report of the advisory panel on teratogenicity states unambigsh^k include the incidence of abnormal litters, the incideiw* ;|»ously * * * . ^^ abrrarmal fetuses per litter, the incidence of specific congeri*»{| h Pesticides should be tested at various concentrations including le^^rsubw abnormalities, the incidence of fetal mortality, maternal *J|P*$j ,, *teiiUally higher than those to which the human population are likely to be gains in pregnancy, and maternal and fetal organ/body wei^{|5 m ratios. ."•'!$ |!;,«|K)sed, ; The,report also emphasizes the insensitivity of standard test sysAdditionally, some pregnant animals should be allowed to p»*r, tems imposed by the relatively insufficient numbers of litters convenbirth in order to identify abnormalities that may otherwise mnnun* .;• tionally tested. only in the perinatal period. '•' The report further states * * * Agents and their known metabolites should be administered to tw1 5: Thus, compounds showing no increase (in birth defects) cannot be considor more mammalian species under various nutritional condition* ' ered nonteratogenic. during active organogenesis and by a variety of routes reflecting.; Epidemiological surveys of human populations may provide post possible human exposure. hoc information on geographical or temporal clusters of unusual Of interest in this connection is the lack of data in the availabw types or frequencies of malformations following exposure to undeliterature on teratogenicity testing by the respiratory route. Respiratected or untested teratogens in the environment. However, logistic tory exposure is particularly important for pesticide aerosols uw « considerations, quite apart from inadequate current surveillance sysvapors. Agents should be tested at higher dose levels than might be untici- ; tems, limit the utility of this approach. It should be emphasized that no major known human teratogen pated in humans following high-level accidental exposure, as well as < ; , such as X-rays, German measles, mercury, or thalidomide, has been following extensive low-level exposure. This is essential to attempt identified by retrospective epidemiological analyses, even in industo reduce the insensitivity of conventional test systems based on very ; trialized countries with highly evolved and sophisticated medical small numbers of animals compared with the millions of humans al presumptive risk. facilities. Prospective epidemiologic surveys on agents previously shown or To illustrate this further, let us assume that at actual human suspected to be teratogenic, by experimental studies or by retrospecexposure levels, a pesticide induces teratogenic effects or cancer in as tive population surveys, are clearly inappropriate. many as 1 out of 10,000 humans, then the chances of detecting this C. Bionetics studies on teratogenicity of 2,4,5-T: Bionetics in test groups of less than 50 rats or mice exposed at these actual Research Laboratories, Inc., of Litton Industries, under a contract levels would be very low. Indeed, many more than 10,000 rats or from the National Cancer Institute, tested 48 pesticides, including mice, depending on their spontaneous incidence of teratogenic effects 2,4-,5-T and related compounds, for teratogenic effects during or cancer, would be required to demonstrate a statistically significant effect, if we assumed that rats and humans have similar sensitivity 1965-68. Although the bionetics studies were originally designed for purto the teratogen or carcinogen being studied. poses of large-scale screening 2,4,5-T was tested more extensively For some teratogens, humans may be less or may be more sensitive than any other pesticide. Thus the data on 2,4,5-T may be regarded than test animals. Meclizine—a drug used for morning sickness in pregnancy—for example, is teratogenic in the rat, but not apparas more definitive. The Bionetics Research Laboratory report is included in the ently in a restricted number of humans studied (King, 1965; appendix (III). A revised and more detailed statistical analysis of Yerushalamy and Milhovich, 1965). these data is summarized in tihe report of tihe Advisory Panel on TeraWith thaiidomide .conversely, the lowest effective human teratogenic dose is 0.5 mg. per kg. a day. Corresponding values for the togenicity of Pesticides (appendix II). 2,4,5-T was tested on repeated occasions from 1965-68 in three mouse, rat, dog, and hamster are 30, 50, 1.00, and 350 mg, per kg. a day (Kalter, 1968). strains of mice and in one strain of rats by subcutaneous and/or oral administration over a dose range from 4.6 to 113 mg. per kg. The Thus humans are 60 times more sensitive than mice, a hundred total numbers of litters tested at each dose level, by each ro\\te in all times more sensitive than rats, 200 times more sensitive than dogs, 700 times more sensitive than hamsters. strains and species, excluding C3H mice in which only one litter was Clearly, attempts to determine a safe level for thalidomide, based tested, were as follows, and I list these in a table enclosed. As can be seen, the bulk of the data was obtained with BL6 mice. on animal teratogenicity data, would clearly expose humans to significant teratogenic hazards. Accordingly, it is routine practice to Due to control variability, the BL6 data have been considered for test for teratogenicity and carcinogenicity at a range of concentrathree time intervals—prior to September 1966, from September to tions, including those higher than human exposure .levels,, and November 1966, and from November 1966 to August 1968. extending to maximally tolerated doses (MTD). Data on AK mice were considered for two time intervals—prior to Even at MTD levels administered to mice from day 7 of life until November 1966, and from November 1966 to August 1968. sacrifice at 18 months, less than 10 percent of the 1J/.0 pesticides Data for BL6 mice, AK mice, and Sprague Dawley fats, as tested in the recent Bionetics study were shown to be carcinogenic. derived from the bionetics report, are as follows: 408 JP3L6 mice: 2,4,5-T administered on days 6-14 or days 9-17, a mice sacrified on day 18 of pregnancy. BL6/AK/imce: 2,4,5-T administered on days 6-14, and mice sacrificed on day 18 of pregnancy. AK mice: 2,4,5-T administered from days 6-15, and mice sacrificed on day ID of pregnancy. Sprague Dawley rats: 2,4,5-T administered from days 10-15 and rats sacrificed on day 20 of pregnancy. In the 4 tables that follow dealing with a wide dose range, the results are expressed as percentages of abnormal fetuses. I would point out those occasions where statistically significant incidence of results was noted. Major abnormalitities in mice were cleft palates and cystic kidneys, and in rats, cystic kidneys and gastrointestinal hemorrhages. Increased fetal mortality was generally concomitant with these abnormalities. It is of particular interest that 39 percent abnormal embryos with cystic kidneys were seen in rats even at the lowest dose tested. Thus the no effect level was not reached even at 4,6 mg./kg. _ _ _ Teratogenicity data on 2,4,5-T, as summarized in the bionetics report (appendix III) are quoted in extenso below. Some critical sentences are italicized: This compound was given by the oral route to BL6 mice at dosages of 40.0 und 113 nig/kg and to AKR mice nt 113 mg/kg. It was given by subcutaneous injection to BL6 mice nt dosages of 21.5 and 113 mg/kg and to AKR mice and B6AK hybrids at 113 mg/kg. It was also given subcutaneously to 03H mice ut 215 mg/kg, but there were too few of these to merit inclusion in the discussion which follows. Administration was for eight days (6th through 14th) in most cases; for nine days (6th through 15th) in some; and for five days (10th through 14th) in one case—the details are indicated in the tabulated results. Subcutaneous administration used DMSO as a vehicle; oral used 50 percent honey. With the single exception of the lowest dosage used (21.5 mg/kg to BI/6 subcutaneously) all dosages, routes and strains resulted in increased incidence of abnormal fetuses. The incidence of cleft palate was high at the 113 nig/Us dosage, but not at lower levels. The incidence of cystic kidney was also high except in the AKR strain and in the BL6 mice which received 46.4 mg/Jtg orally. Fetal mortality was increased in all groups given 113 mg/kg for eight or nine days, but not in mice (BL6) given this dosage for only five days nor in the two groups of BL6 mice given lesser dosages (46.4 mg/kg orally and 21.5 mg/kg subcutaneously. Most fetal and maternal measurements showed inconsistent changes from which no conclusions can be drawn. In contrast, there was a highly consistent decrease in maternal weight gain in BL6 mice given 113 mg/kg by either route. Tjower dosages and the AKR strain showed either no change or a slight increase. All dosages, strains, and routes showed an increase in the maternal liver weight and this led to a further study discussed separately below. These results imply a hazard of teratogenesis in the use of this compound. The problems of extrapolation preclude definition of the hazard on the basis of these studies, but Us existence seems clear. The observed influence of 2,4,5-T on maternal liver weight as mentioned above raised a question as to its effect on the fetal liver. This was answered by a study carried out in Bt/6 mice using subcutaneous injections of DMSO solutions at a dosage of 113 mg/kg only. The period of administration was lengthened to cover the period from the 9th through 17th day of gestation. Separate control groups were used concurrently. Except for the inclusion of fetal liver weight, measurements were made as previously described. The fetal livers of the 2,4,5-T treated mice weighed significantly more than those of controls given DMSO only and the weights of the whole fetuses were 409 liver nignificantly less. Correspondingly, there was an increase in weight expressed as percent of body weight. Other observations were consistent with those reported above. The incidence o/ abnormal fetuses was unusually high as were those of cleft palate and cystic Udney. Because of the potential importance of the findings in mice, an additional ntudy was carried out in rats of the Sprague-Dawley strain. Using dosages of 21.5 and 46.4 mg/kg suspended in 50 percent honey and given by the oral route on the 6th through 15th days of gestation, we observed excessive fetal mortality (almost 80 percent) and a high incidence of abnormalities in the survivors. When the beginning of administration was delayed until the 10th day, fetal mortality was somewhat less, but still quite high even when dosage was reduced to 4.6 mg/kg. The incidence of abnormal fetuses was threefold that in controls even with the smallest dosage and shortest period used. Fetal and maternal measurements showed only occasional instances of significant differences from controls except in the case of maternal liver weight which was consistently increased in all 2,4,5-T treated animals. It seems inescapable that %,4,5-T is teratogenic in this strain-of rats when (liven orally at the dosage schedules used here. These findings lend emphasis to the haeard implied by the results of studies on mice. I). Recent reanalysis of the Bionetic data on teratogenicity of 2,4,5-T. More refined and more appropriate additional statistical analyses of these data were presented and discussed in the report of the Advisory Panel on Teratogenicity of Pesticides (appendix II). These are clearly confirmatory of the original conclusions of the Bionetics report on the teratogenicity of 2,4,5-T. Some relevant portions of the HEW panel report are quoted in extenso below: Tested more extensively than other pesticides, 2,4,5-T was clearly teratogenic as evidenced by production of statistically increased proportions of litters affected, and increased proportions of abnormal fetuses within litters in both DMSO and honey for both C57BI//6 and AKR mice. In particular, cleft palate and cystic kidneys were significantly more prevalent. In addition, a hybrid strain resulting from a C57BI//6 female and AKR male showed significant Increases in anomalies, in particular cystic kidney, when administered at 113 mg/kg of body weight in DMSA, Additionally, 2,4,5-T was tested in Sprague-Dawley rats. AVhen given orally at dosages of 4.6, 10.0, and 46.4 mg/kg on days 10 through 15 of gestation, an excessive fetal mortality, up to 60 percent at the highest dose, and high incidence of abnormalities in the survivors was obtained. The incidence of fetuses with kidney anomalies was threefold that of the controls, even with the smalleat dosage tested. E. Recent studies on teratogenicity testing of relatively pure 2,4,5-T. In view of the fact that the Bionetics study was conducted with a sample of 2,4,5-T which was subsequently shown to contain a relatively high concentration, 27 ppm, of a tetrachloro dioxin contaminant, testing has been recently repeated with relatively pure samples containing less than 1 ppm of this particular dioxin. * The results of these studies were presented by the FDA and NIEHS at a recent conference of February 24, 1970, at the FDA; the Dow Chemical Co. data were presented at the 9th annual meeting of the Society of Toxicology, Atlanta, March 17,1970. As can be seen from the data summarised ~below, purified tl^fi-T is teratogenic in three species—rats, mice and hamsters. These data should be regarded as preliminary. Confirmatory data on chick eggs are not presented here. 1. Dow Chemical Co. studies (Emerson et aL, 1970). 2,4,5-T with 0.5 ppm dioxins, as a probable contaminant, was tested in pregnant A 411 410 rats by repeated oral administration at doses of 1, 3, 6, 12, and 24 mg./kg.; the maximal dose tested was 24 mg./kg. No embryo deaths or weight losses were noted within the dose range tested. However, at 24 mg./kg. there was a sevenfold increase in the incidence of fetuses with defective ossification of the fifth sternebra; poor sternebral ossification was noted in four out, of 103 control fetuses, and in • 29 out of 103 fetuses of 2,4,5-T treated groups. Defective sternebral ossification has been described in the rat as an expression of the teratogenic effects of drugs such as protamine zinc insulin and tolbutamide (Lichtenstein et ah, 1951; Dawson, 1954). 2. NTEHS studies: Using the purest sample of 2,4,5-T, made available by Dow Chemical Co., teratogenic effects were induced in Swiss-Webster mice. Cleft palates were noted at dose levels of 150 mg./kg. and scattered abnormalities at 100 mg./kg.; the cleft palate incidence in control mice was essentially zero. 3. FDA studies: Hamsters were injected with five doses of 100 mg./kg./day of various batches of purified 2,4,5-T between days 6-10 of pregnancy. In one of these studies, there was a 66-percent incidence of mortality in 50 fetuses. Of the surviving fetuses, 17 percent had congenital abnormalities—crooked tail, missing limb, and defect in skull fusion. No data was presented on possible effects induced by doses less than 100 mg./kg. Of additional interest was a report also presented at the same conference on purified 2,4-D, which produced a 22-percent incidence of congenital abnormalities in hamsters at a dose level of 100 mg./kg./day. > . . , , 1 should like now to address myself to the toxicity of dioxins. Toxicity of dioxins: Rabbit ear skin is highly sensitive to dioxins, repeated application of which can produce chloracne, as a cumulative manifestation of local toxicity. Approximately 0.3 micrograms of the tetra isomer will produce a positive response; "more than 10 micrograms on a surface wipe sample indicates acute hazard" (to man) (Silverstein, 1970). The acute oral LD-50 dose of tetra dioxin in male guinea pigs is 0.5-1.0 micrograms/kg., and in male and female rats, 22.5 and 45 microgram/kg,, respectively. Feeding chicken edema factor diets, containing dioxins, produced cumulative toxicity in monkeys (Allen and Carstein, 1967). Storage of hexa, hepta and octa isomers, as identified by GLC, has been reported in chickens and rats fed chicken edema factor diets (FDA, unpublished). Chronic administration of 2,4,5-T or 2,4-D to dogs produces cumulative toxicity with gastrointestinal haemorrhage, suggestive of cumulative dioxin effects (Drill and Hiratzka, 1953). TERATOGENICITY OF DIOXINS 1. FDA studies (FDA Conference, Feb. 24, 1970) : A mixture of dioxins, 21 percent trichloro and 53 percent tetrachloro isomers, were injected in hamsters between days 6-10 of pregnancy over a dose range from 0.5 to 9.1 microgram/kg. per day. At the highest dose, the incidence of fetal mortality was 82 percent and the incidence of congenital abnormalities, 82 percent. At the 0.5 microgram/kg. dose, there was a 5 percent incidence of abnormalities. The no-effect level was thus not reached at 0.5 micrograms per kg. ,f isomer 2. Dow studies (Sparsclm et al., 1970) : The tetra dk^fc — - - - . ' • » « ^ w n ^^V was fed to Sprague Dawley rats between days 6-15 of ^&t over a dose range from 0.03 to 8.0 micrograms/kg. per day. There was a marked increase in resorption sites at the 2 microgram level. Gastro-intestinal hemorrhages occurred over a range from 0.125 to 8 micrograms, dose-dependently. Additionally, at the 0.125 microgram/kg, level there was a decrease in male fetal weights. It should be emphasised that cystic kidneys were not seen at the 0.125 microgram/kg. dose of the tetra isomer or even higher levels. In the Bionetics study, 2,4,5-T at 4-6 mg./kg., containing 25 ppm of the tetra dioxin isomer equivalent to 0.124 microgram/kg., produced a 39 percent incidence of congenital abnormalities with, cystic kidneys. There is thus a clear discrepancy between the teratogenic effects of 2,4,5-T containing 25 ppm of dioxin, and the effects of the equivalent concentration of the same dioxin. It is, however, conceivable that this discrepancy may reflect synergistic interactions between dioxin and 2,4,5-T. SOME UNRESOLVED PROBLEMS RELATING TO 2,4,5-T AND DIOXINS 1. Chemical composition of 2,4,5-T formulations: Currently used 2,4,5-T formulations contain about 5 percent of known impurities, largely polychlorophenols. Analytic data on a sample of 2,4,5-T (Dow data, on production batch 120449) in the following table substantiates the approximate 5 percent of polychlorophenol impurities in 2,4,5-;T formulations as currently used. There are no available data on the presence and concentration of the more than 60 positional isomers of dioxin, other than the 2,3,7,8-tetrachloro dioxin isomer, in this batch of 2,4,5-T, or in other batches produced for food crop or other purposes in the United States and abroad. In view of the relatively high concentration of polychlorophenol impurities in 2,4,5-T, it is likely that a wide range of dioxins are also present. 2,4-D and other phenoxy herbicides are similarly chemically uncharacterized. . The higher positional dioxin isomers, hexa, hepta, and octa, have been identified in 2,4-dichlorophenol, a precursor of 2,4-D. Apart from the presence of dioxins in polychlorophenols, heating of polychlorophenols will produce additional and very high yields of dioxin. Illustratively, heating 5 g. of pentachlorophenol at 300° C. for 12 hours yielded 1.5 g. of the octa-dioxin isomer (Cowan, 1970). There are no available data on the possible production of dioxins from combustion of 2,4,5-T or 2,4-D. While improved production techniques may well reduce the levels of polychlorophenols and the levels of the 2,'5,7,8-dioxin isomer, apart from other isomers, in 2,4,5-T and other phenoxy herbicides, the degree to which this is practical does not yet appear to have been clearly defined. 2. Stability and persistence of dioxins: The extent of• usage of 2,4,5-T and other phenoxy herbicides on food crops and for other purposes in the United States and abroad dictates the scale of resulting environmental contamination with 2,3,7,8-dioxin and other 45-362 0—70 -27 413 412 isomers. The following data are illustrative and (Agricultural Kco* nomic Eeport No.-131, IJSDA, 1968). These data reflect deliberate applications of phenoxy herbicides on crops, and do not reflect unintentional crop contamination following the more extensive application of herbicides for brush control or .other purposes. There are no available data on the extent of such unintentional contamination. It is, however, well known that phenoxy herbicide dusts may drift for miles, even on nonwindy days, following routine application (Federal Register, 1969). The concentration of phenoxy herbicides in the air in Washington in 1964 reached /™ 3a, with a maximum maximum of 01 "' 3.4 —--«~~«or>-, microgram/m. „»„ n,n averaee „ of 0.046 3 microgram/m (Bamesberger and Adams, 1966). These figures probably underestimate the proportional concentration of atmospheric dioxins, in view of their high stability relative to phenoxy herbicides. I now present a table on calculated dioxin contamination of an acre of soil, following 2,4,5-T application. CALCULATED DIOXIN CONTAMINATION PER ACRE FOLLOWING 2,4,5-T APPLICATION Based on 0.5 ppm Dioxin Basod on ppm "ioiin 0.57 5.70 2M Application 2,4,5-T (Ibs/acre) 2.5 (domestic .use). 25.0(export use)... n Now, these calculations are based on the 2,3,7,8-dioxin isomer alone, and ignore additional contamination due to other dioxin isomers. The figures for export use should be adjusted to reflect varying concentrations of 2,4,5-T in different formulations. The high concentration of polychlorophenolic impurities in 2,4,5T, approximately 5 percent, apart from other sources of polychlorophenols, may resiilt in extremely high yields of dioxins. As mentioned previously, heating of 5 grams of pentachlorophenol at 300° C. for 12 hours results in a yield of 1.5 grams of the octadioxin isomer. Combustion of shrub, brush, timber, or other materials exposed to phenoxy herbicides or other polychlorophenols, may thus liberate high concentrations of dioxins in the atmosphere. It is thus of interest to examine the data on stability and persistonce of dioxins in the environment. The 2,3,7,8-tetra isomer is known to be heat stable up to 800° C. There are, however, no available data on the heat stability of other dioxin isomers. There are also no available data on the stability and persistence of the 2,3,7,8- and other dioxin isomers in soil, water, crops, milk, and animal or human tissues. Most importantly, there are no available data on the possible accumulation and transmission of 2,3,7,8- and other dioxins in the food chain—air, soil, and water—to plants, brush and crops—to fish, birds and cattle—to man, with attendant accumulation in man. The heat stability of the tetra isomer, the general lipid solubility of the dioxins, and their cumulative toxicity in experimental' am1 smismals, all serve to enhance the possibility of food chain tr sion of the various dioxin isomers. ' 3. Teratogenicity of relatively pure 2,4,5-T. The relatively contaminated 2,4,5-T used in the Bionetic study, containing about 27 p.p.m. of the tetra-dioxin contaminant, induced congenital abnormalities in mice and rats, particularly cystic kidneys, that were not produced by the pure tetra dioxin. Illustratively, the contaminated 2,4,5-T at 4.6 mg,/kg./day oral dose level in rats produced a 39 percent incidence of congenital abnormalities. This dose of 2,4,5-T is equivalent to 0.124 micrograms/kg./day of the tetra-dioxin. However, as reported in the Dow studies, presented at the Society of Toxicology meeting in Atlanta on March 17, 1970,_ 0.125 micrograms/kg./day of the tetra dioxin did not produce cystic kidneys' in rats. This discrepancy may however conceivably reflect synergistic interactions between 2,4,5-T and dioxins. Additionally, as indicated above, relatively pure 2,4,5-T, containing one p.p.m. of the tetra-dioxin contaminant, was teratogenic in preliminary studies with three species—mice, rats, and hamsters, over a dose range of 24^-150 mg./kg. In some of these studies, for example, in hamsters at 24 mg./kg./day, no effect levels were not reached. Thus, recent studies on relatively pure 2,4,5-T clearly confirm its teratogenecity and lend further emphasis to the following conclusions of the report of the HEW Teratogenicity Panel: "The use of currently registered pesticides to which humans are exposed and which are found to be teratogenic by suitable test procedures in one or more mammalian species should be immediately restricted to prevent risk of human exposure." All these considerations confirm the following conclusion stated in the Bionetics reports (App. Ill, p.19) : "These results imply a hazard of teratogenesis in the use of this compoxind, 2,4,5-T. The problem of extrapolation preclude definition of the hazard—but its existence seems clear." 4. TOXICOLOGY Or DIOXINS The toxicology of dioxins is of particular interest in view of the previously reviewed data on the very high acute toxicity, embryotoxicity, cumulative chronic toxicity of 2,3,7,8-dioxin and related isomers, and also the stability, widespread environmental distribution, and likelihood of accumulation and transmission of any dioxins in theThere food chain. are no data, in the available literature on the carcinogenicity or mutagenicity of positional isomers of dioxins. Recent studies on the teratogenicity of dioxins have been largely restricted to the 2,3,7,8-isomer; there are no available experimental data on behavioral or psychopharmacological effects due to dioxins; this would be of interest in view of the possible psychiatric effects described in humans exposed to dioxins. There are no data in the available literature on any toxicological studies on any dioxin isomer following acute or chronic administration by inhalation. 414 415 The extreme inadequacy of toxicological data on dioxins dearly precludes consideration of potential human hazards .due to, dioxin* in air, food or water, and consideration of possible safety margini following exposure to dioxins. I would like to reemphasize the conclusions of the HEW panel on teratogenicity: "The use of currently registered pesticides to which humans are exposed and which are found to be teratogenic by suitable test procedures in one or more mammalian species should be immediately restricted to prevent risk of human exposure. Such pesticides, in current use, include Gaptan; Garbaryl, the butyl, isopropyl, and isooctyl esters of 2,^,-D Folpet; mercurials; PGNB; and H The teratogenicity of 2^.,-D, the other salts and esters of both ,i and 2,4,5-T, and that of IPO should be investigated further." Finally and critically, available data on tlae toxicology of the dioxins, and more importantly on the lack of data on the toxicologyacute and chronic toxicity, carcinogenesis, mutagenesis, and teratogenesis—of the numerous positional isomers of dioxins, indicate an urgent need for restriction of human exposure to dioxins. Similar restrictions should extend to polychlorophenols, polychlorophenolic containing formulations, and their combustion products. I thank you, sir. (A list of the references to the statement follow:) ties in the hamster. What is your opinion as to the teratogenicity of 2,4-D in comparison to 2,4,5-T ? Dr.' EPSTEIN. The findings of the Bionetics study in relation to 2,4-D, were certainly not as conclusive as for 2,4,5-T. But, on the other hand, in the wide range of tests performed in the Bionetics study there were strong suggestions of teratogenic effects. That was as far as one could go. For these reasons the HEW Panel on Teratogenicity clearly recommended that further work should be done on 2,4-D and that a high degree of suspicion should be attached to it. Moreover, the recent FDA studies on hamsters would seem to confirm the earlier suspicions of the Bionetics studies in mice and would indicate to say the least the need for a high degree of caution and minimally, indicate the need for restriction of further human exposure to 2,4-D pending clarification of these problems. I think to await further definitive studies before taking action would not be appropriate in the light of the available data. Senator HART. That gets \is back, and we return here all the time, to this balancing of principle. On the point that Mr. Bickwit was developing with Dr. Johnson, on the business of the cumulative effect of dioxin I take it you regard them as cumulative; is that correct? Dr. EPSTEIN. On the basis of the available data in the literature, one could say there is a strong suggestion of cumulative effects. The rabbit ear skin test depends on cumulative toxicity and the chick edema effect is cumulative. One can demonstrate the pickup of dioxins in rat and chicken liver. So available data indicate they are persistent and cumulative, although I would regard these data as far from definitive. Senator HART. What is your feeling as to the adequacy of the evidence? What is the extent of the evidence on the degradability of the dioxins in ultraviolet light ? Dr. EPSTEIN. I would submit that data on strong sunlight and shortwave TJV is probably irrelevant. If you expose dioxins to strong sunlight you are exposing material to light which contains short wave ultraviolet in a manner not closely related to normal conditions. I would like to see studies on conditions of photo-degradability of dioxins on conditions of shade and normal daylight. This is not available. Senator HART. Well, we always seem to run into the lack of available data in pursuing answers to these questions. Now let's get to the availability of substitutes. What alternatives exist, or are you in position to know, for 2,4,5-T and 2,4-D ? Dr, EPSTEIN. T am obviously not an agricultural expert and we are now talking about crop use, I presume. Does your question relate to crop use of herbicides or total use ? Senator HART. I guess I should ask it totally and if there are areas where there is an alternative, identify that. Dr. EPSTEIN. With the qualification that I am no expert in this field and not qualified to comment on the relative merits of various BEFERENCES Oberle, M. W. Science 165, 991-992, 1069. King, 0 J. Pharm. JStsp. Therap. J.Jf7, 391, 1965. Yerushalamy, J. and Milkovich, L. Am. esearch ' Institute and T u m o r ' P a t h o l o g i s t ' a t ' t h e H o s p i t a l for Sick C h i l d r e n , Great Orrcond Street, London. Consultant in Pathology, The Memori?! 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Socle ty Mejnbarsh 1 ps: Committees: British Medical Association Society of Clinical Pathologists Society for Pathology and Bacteriology Society for General Microbiology Society of Protozoologists Air Pollution Control AssoclAtion American Association of Pathologists and Bacteriologists American Society for Experimental Pathology American Association for Cancer Research American Board of Microbiology Society of Toxicology Environmental Hutagenesis Society (a) (b) (c) (d) (e) (f) (g) (h (i j k) Cong r_es r. i onal> Tes.tj ngny: Military Awards in Royal Army Medical Corps, 1953 (a 'Montefiore Go'ld Medal in Tropical Medicine b Montefiore Prize In Tropical Hygiene c Ranald Martin Prize in Military Surgery Society of Toxicology, 1969 Achievement Award Member, Committee on the Relation of Protozoology to Public Health. The Society of Protozoologists, 19S2--1969. Chairman, Committee on Biological Effects of Air Pollution, Air Pollution Control Association, 1963-1969. Member, Technical Council of the Air Pollution • Control Association,, 1963--1969. Executive Secretary, Environmental Mutagenesis Society, 1969. Chairman, Committee on Cyclainates and Caffeine, Environmental Mutagen Society, 1969. Chairman, Committee on Liaison, Environmental Mutagen Society, 1969. Chairman, 1969, HEW Pa-nel, Hutagenicity of Pesticides " Chairman, 1969, HEW Panel, Teratogenicity of Pesticides Member, 1969, HEW Panel, Pesticide Interactions Member, 1969, HEW Panel, Cardnogenicity of Pesticides Chairman, NIMH Panel on Chronic Non-psychiatric Hazards of Drugs of Abuse, 1969. On "Cancer and Mutation-Producing Chemicals in Polluted Urban A i r " , at Hearings before the Sificommittee on Air and Hater Pollution of iho Committee on Publia Harks, July 2931, 1968, presided by Senator Edmund S, Muskie. 1. Epstein, S. S., and Winston, P.: Intubation granuloma. 0. Laryntjol. and Otol., 7j_: 17-38, 1957. 2. Epstein, S. S., Winston, P., Friedmann, I., and Ormerod, F. C.: The vocal cord polyp. J. l.aryngol. and Otol., 7J_: 673-688, 1957. 3. Epstein, S.' S., and Shaw, H. J.: Metastatic cancer of the larynx as a cause of carotid-sinus syndrome. Cancer, 1_0_: 933-937, 1957. 4. Epstein, S. S.: An intra-oral inoculation technique for the production of experimental pneumonia in mice. J. Hygiene, 56: 73-79, 1958. 5. Epstein, S. S., and Stratton, K.: Further studies in the mouse Intra-oral inoculation technique. J. Hygiene, 56: 81-83, 1958. 6. Epstein, S. S., and Shaw, H. J.: Multiple malignant neoplasms in the air and upper food passages. Cancer, Vl_: 326-333, 1958. 7. Winston, P., and Epstein, S. S . : Papilloma of the larynx: A clinico-patholoqical study. .... J. Laryngol. and Otol. j 72: 45Z-464, 1958.' 8. Epstein, S. S . , and Friedmann, I.: j<]ebs_ij:l_]_a_ serotypes in infections of the ear and upper respiratory tract". 0. Clin. Path., 2_: 359-362, 1958. 9. Epstein, S. i.: A "stripping" technique for the examination of the total epithelial surface of the larynx. J. Path, and ISact. , 7.5: 472-473, 1950. 10. Fropnian, T., Wakefielcl, G. S., nnd Epstein, S, S . : Platelet-agglutinating factor in glandular fever complicated by jaundice and thromSocytopciria. The Lancet, 383-385, October 25, 1058. 422 423 Publ PuM ications 11. Epstein, S. S. , and Brcidboar, T. I.: A case of primary diphUir-rti.io otil.fs media. J. Laryiifiol. anJ Otnl., 72; 1001-1003, lflf.8. 21. Epstein, S. S . , Weiss,, 0. I!., IHisli, P . , and Causey, I).: Vit-iiiiii! B,/, tiiitl growth of Cug'lena G r a c i l l s . Fed. Proc., _20_(l): 4bO, Mroxli "Te'MV 12. Epstein, S. S.: 22. Epstein, S. S . , ai:d Tii,v,,i1s, G. II.: "Simplfl" Vit.fniiin B 1? antiire-Ubo'litos. Proc. Amor. A s s o c . Cantor R e s . , 3 ( 3 ) : 223, 19C1. 23. Epstr.-iti, S. S. , and Burrouijhs, M. : Some factors Influencing the photodynai'ilc rusponse of Paj'anisc'li'iii caudt.tuii! to 3,4-b2itxpyi'crifi. Hnturo',"]_9.3..: 337-33(1, 1962. 24. Epstein, S. S . , Burroughs, M., Small, M., and Verbrugghsn, M.: The pho'todynamic toxi.dty of polycycllc hydrocarbons. Proc. Aniar. Assoc. Cancer Res., 3_(4): 316, 1962. 25. Epstein, S. S . , W e i s s , 0. B., Causeley, D., and Bush, P.: Experimental K'lebsjella pneumonia in mice with particular reference to peri arterial changes. J. Path, and Bact., 78; 389-390, 1959. 13. Epstein, S. S. The biochsmistry and antibiotic sensitivity of the Klobsi.ella. J. Clin. Path., 1_2: 52-58., 1959. 14. Epstein, S. S., end Payne, P. M.: The effect of some variables on experimental KlcbsJ.ella infections in mice. J. Hygiene, 57; 68-80, 19D9. 15. Shav), H. J., and Epstein, S. S . : Cancer of the epiglottis. Cancer, 1?: 246-256, 1959. 16. Epstein, S. S. , and Weiss, J. B . : The extraction of pigmants from Euc|luna_ cjracjllis_. 'Biochem. J., 75: 217-250, 1959. 17. Influence of Vitamin B,, on the size and growth of Euglena qracillis. J. Protocol., 9_, 336-339, 1962. " 26. Epstein, S. S . , and Timmis, 6. M.: Effect of Vitamin B,, antagonists and other compounds on the C1300 tumor. Biochsm. Pharrnacol., 11: 743-746.. 19S2. Timmis, C. M., and Epstein, S. S . : New antimetabolites of Vitamin B,,,. Nature, 184: 1383-1384, 1959. 27. Epstein, S. S . , and Timmis, 6. M.: Simple antinrotabolites of Vitamin B,,. • l£ - .J. Protpzool., JO; 63-7.3,. 1953. 18. Epstein, S. S . , Payne, P. M., and Shaw, H. 0 . : . Multiple"primary malignarit'neoplKsms in the air and upper food passages. Cancer, 13.: 461-463, 19GO. 28. 19. Epstein, S. S . , and Weiss, 0. B.: Measuring the size of isolated cells. Mature, 187.: 461-463, 1900. Epstein, S. S . , Burroughs, M., and Small, M.: Tho photoclynamic effect of the carcinogon ; 3,4-ben?.pyrene on Paramecumi cauclatuin. C"a"nceir"Re~s".7"2S: K-44, 1963. 29. Epstein, S . S . : L'ffectsof some benzinvidazolc.'S on a Vitamin B^-requirinrj alga. Nature., 188: 143-144, 1960 Epstein, S. S . , Small, M., Koplan, J., and Mantel, N.: Photodynaiiiic bioassay of t>eiizo[a'lpyreno using Paijjmiecluin amdatuii). J. Nat. Cancer Inst., 31; 163..168', 1S63. "" " 30. Epbioin, S. S . , Small, M., Jones, II., Koplan, 0. , and Ifankel , N . : A photodynaiiiic bioassay of atmosphurlc pollulanls. Proc. Amor. Assoc. Cancer lies., _4(1): 18, 19C3. 20. 425 424 Pub! ica'tions j'ubVi.catipns 41. 31. Acta Unlo internsi., i,um..« 32. Epstein, S. ~S., Small, M., Koplan, J., Mantel, H., Falk, It. 1., and Sawicki, V. Photodytisuic bioassay of polycyclic. air pollutants. A.M.A. Archives of Environmental Health, 7: 53'l-b37, 1SS3. 42. Small, M., Brickman, E,, and Epstein, S. S.: Uptake of polycyclic compounds by phagotrophic protozoan. Fed. Proc. , ?A_: 684.. 196b. 43. . Small, H. , Jones, I!., and Epstein, S. S.: I'hotodynavAic activity of polycyclic compounds. Fed. Prpc., 22: 316, 19G3. 34. tpstein,On S.theS.,association Small, M.,between Falk, H.photodynamic I., and Mantel, M.: and carcinogenic activities 33 in polycyclic compounds, Cancer Res., 24: 855-862, 1964. E p s t e i n , S. S , , Saporoschetz, 1. I!., S m a l l , M . , Park, W . , and Mantel, f ! . : A s i m p l e bioassay'for a n t i o x i d n n t s based on protection of Tetjjihyiiiei p y r i f o r a i s from the photodynamic t o x i c i l y of b(inj:o[a](iyrone. KaUir"e', 200: 655-C58', 1965". Epstein, S. S.., Forsyth, J., and Bulon, I.: A simple bioassay for antioxidtnts. Fed. Proc., 24j ,623, 1965. 44. Epstein, S. S.: Bioassay for polycyclic atmospheric pollutants and for antioxidants based on photodynamic response of protozoa. Abstract from Second International Conference of Protozoology, London, August, 1965. Reprinted from Excerpta Msdlca International Congress, Series 91. 35. Foley, G. E., and Epstein, S. S,; . Coll culture and cancer chemotherapy. in "Advancfts in Chemotherapy", ]_: 196-1, Academic Press, New York. 45. 36. Epstein, S. S., Bulon, I., Kaplan, 0., Small, H., and Mantel, N.: Charge-transfer complex formation, carcinogenicity and photodynamic acti.vlty in polycyclic compounds. Nature, 2U4_: 750-754, 1964. 46. Epstein, S. S . , and J o s h i , S. R . : Obstructive renal f a i l u r e in random-bred Swiss mice. Fed; P r o c . , 25: 237, 1966. 37. Epstein,Charge S. S,,transfer Gulon, I., and Kaplan, J.: carcinogenicity and photodynamic complex formation, . - . activity in polycyclic-compounds. Fed. Proc., 23_(2);' 287, 1964. - 4.7,..Eps.tein, S. S . , Saporoschstz, I. B . , and M a n t e l , N , : Interactions between antioxidant and photosensitizer in the photodynamic bioassay for a n t i o x i d a n t s . Life Sciences, 5; 783-793, 1966. 38. Epstein,Photoactlviition S. S.:' of polynuclear hydrocarbons. A.M.A. Archives of Environmental Health, 1_0: 233-239, 1965. 39. Epstein, S. S., Small, M., Sawicki, E., and Falk, H. l.\ Photodynawic bioassay of polycyclic atmospheric pollutants. J. Air Poll. Control Assoc., 15: 174-176, 1965. , ' 40. Epstein,A S. simple S..: photodynamic assay for polycyclic atmospheric p o l l u t a n t s . World Health Organization Report, UHO/BL/S1, 1965. E p s t e i n , S, S. : The l u n g as a transplant site for m a l i g n a n t tumorsin rodents. Cancer, 19_: 454-157, 1966. 48. Epstein, S. S . , Forsyth, J . , Saporoschetz, I. li., and M a n t e l , N . : An exploratory investigation on the i n h i b i t i o n of selected photosensitize by agents of v a r y i n g a n t i o x i d a n t a c t i v i t y . Rad. Research, £8: 322-335, 1966. 49. E p s t e i n , S. S . , and Tabor, F. B, - . P h o t o s e n s i t i z i n g conipounds in extracts of U . S . A . d r i n k i n g water. Science, 1.5_4.(3746): 261-263, 1966. 50. E p s t e i n , S. S , : Two s e n s i t i v e tests for carcinogens in the a i r . J. Air P o l l . Control Assoc., 1 6 ( 1 0 ) : 546-546, 1966. 427 426 Publications 51. 52. 53. 54. 55. Publications Epstein, S. S,, Joshi, S., Andrea, J,, Mantel, H., Sawicki, (.., Stanley, T . , and Tabor, E. C , : Carcinogenicity of organic parti cul ate pollutant;, in urban air after administration of trace quantities to neonatal mice. Nature, 212: 1305-1307, 1966. Small, A . , Mantel, N., and Epstein, S. S . : The role of cell-uptake of polycyclic compounds in photodynanric injury of Jetrajiy_mena_ pyrvformis. ExperimentaT'Cel 1 'Research ,~Wr""206-217, 1967. Epstein, S. S. , Joshi, S . , Andrea, J., Forsyth, J., and Mantel, N.: The null effect of antioxidants on the carcinogenicity of 3,4,9,10dibcnzpyrene to mice. Life Sciences, (5: 225-233, 1967. Epstein, S. S., and Niskanen, E. E.: Effects of Tween 60 on bsiuo[a]pyrene uptake by Tetrahymena £JZ-!!Pr"v'.s. and by isolated rat liver mitochondria. Experimental Cell research, 46: 211-234, 1967. Epstein, S. S., Ooshi, S., Andrea,J., Clapp, P., Falk, II., and Mantel, N.: The synergistic toxicity and carcinogenicity of Freons and piperonyl butoxide. Nature, 21_4: 526-528, 1957. 56. Epstein, S. S., Saporoschetz, I. B., and Hutner, S. H.: • - - - . - - • Cytoxicity of antioxidants to Tetrahyaiena- pyrlforrr,is; J. Protocol., J_4_: 238-244, 1957. '" 57. 58. 59. Nagata, C . , Fujii, K. , and Epstein, S. S . : Pliotodynamic activity of 4-n1troC|Uinoline-l-oxide and related compounds, Nature, 21_5: S72-973, 1967. Epstein, S. S., Andrea, J . , Joshi, S . , and Mantel, N.: llepatocarcinoganicity of cjriseofulvin following parenteral administration to infant mice. Cancer Research, 27_: 1900-1906, 1967. Epstein, S. S., Andrea, J., Mantel, II., and Fall;, II.: Carcinogenicity of the herbicide uialeic hydrazido. Nature, 215: 1308-1390. 1967. ! " 60. Epstein, S. S . : Cjrcinogonici ty of organic extracts of atmospheric pollutants. J. Air Pollution Control A s s o c . , 17, 7?fi-7!>(i, 1967. 61. Epstein, S. S . , Andrea, J., Clapp, P., and Mackintosh, D . : Enhanceii'iSnt by piperonyl butoxide of acute toxicity due to Freons lien?.o(a)pyreno, and Grisoofulvin in infant mice. Toxicology and Applied Pharmacology, VI, W-448, 19G7. 62. McCarthy, R. E., and Epstein, S. S.: CytocheiTiical and cytoofinetic effects of maleic hydrande on cultured mammalian cells. Life Sciences, 7_, 1-6, 1968. 63. Epstein, S. S , , Mantel, N., and Stanley, T. W . : Photodynaiivic assay of neutral sub-fractions of organic extracts of particulate atmospheric pollutants. Environmental Science and Technology, £, 132-141, 1968. 64. Epstein, S. S . , and Mantel, N,: Hepatocardnogem'city of maleic hydroxide following parenteral administration to infant Swiss mice. Intel-national Journal of Cancer, .3, 325-335, 1968. 65. Rondia, D . , and Epstein, S, S . : The effect of antioxidants on photodecomposition of benzo(a)pyrene. Life Sciences, 7_, 513-518, 1968, 66. Epstein, S, S . : Carcinogenicity of Tetraethyl lead, Cxperlentia, 2_4_, 580, 1968. " ' 67. Epstein, S. S. and Shafner, H.: the of mammals in a practical screening test for chemical mutagens in the human environment. Mature, 2_19, 385-387, 1968. 68. Jaffe, J . , Fujii, K. , Sengupta, M., Cuerin, II., and Epstein, S. S . : In yi_vo inhibition of mouse liver microsomal hydroxylating systcmr, by wetiiyloii(:UioiVpl\fjiiyl insectitidal syneryists and related compounds. Life Sciences, 7_, 1051-1052, 1908. 428 429 Publications 69. Epstein, S. S.: Cancer and mutation-producing chemicals in polluted urlmn air. Air Pollution (Air Quality Criteria) Hearings before the Subcommittee on Air and Water Pollution of the Committee on Public ' . Works.. U. S. Senate, 91st Congress. Washington, D. C., 1968. Publications 78. Lpslcjin, S. S. : Introduction to sympovia on toxicologic and epidemiologic bases for air quality criteria. Journal of Air Pollution Control Association, 19, 629-630, 1969. 11 70. Epstein. S. S.: Irradiated roods Science, 161_, 739, 1968. 71. Fujii, 1C.,'Jaffe, H. and Epstein, S. S.: Factors influencing the haxobarbital sleeping time and zoxazolamine paralysis time in mice. Toxicol. Appl. Pharmacol., 13, 431-138, 1968. 72. Epstein, S.'S., and Saporoschetz, J.B.: On the association between lysogeny and carcinogenicity in nitroquinolines. Experientia, 24_, 1245-48, 1968. 73. Jaffe, H., Fujii, K.. Sengupta, M., Guerin, H., and Epstein, S, S.: The bl-modal effect of piperonyl butoxide on o- and p-hydroxylation of biphenyl by mouse liver microsomes. Biochem, Pharmacol., 18, 1045-1051, 1969, 74. Epstein, S. S.: Chemical mutagens and the Environmental Mutagen Society -"Current Opinion" editorial Medical Tribune and Medical News,"10., pp IT-IS, June ?.', 1969.' 75. Pagnatto, L. [K , and Epstein, S. S.: The effects of antioxidants on ozone toxicity in mice. . Exporientia, 25, 703-704, 1969. 76. Epstein, S. S.:A oaioh-all toxicological screen. Exporientia, £!j_, 617-618, 1969. 77. Epstein, S. S., and St. Pierre, J. A . : Mutagenicity in yeast of nitroi|uino1inos and related compounds. Toxicol, Appl. Pharmacol., 15, 451-4GO, 19C9, 79. L'pstoin, S. S. : ' • Chcmicali Iwards in the human environment. Ca-A Cancer Journal for Clincians, J_9, 277-281, 1969. 80. Epstein, S. S., Hollaonder, A., I.ederberg, J., Leoator, M., Richardson, II., and Wolff, A. H.: Cyclainate Ran Science, 166, 1575, 1969. 81. Lijinsky, W., and Epstein, S. S.: liiti'osann'nes as environmental carcinogens, Nature, 225, 21-23, 1970. 82. Epstein, S, S., Fujii, K., Andrea, J., and Mantel, H.: Carcinogenicity testing of food additives and antioxidants by parenteral administration to infant Swiss mice. Toxicol. Appl. Pharmacol. 16, 321-334, 1970. 83. Fujii, K., Jaffe, H., Bishop, V., Arnold, E., Mackintosh, D., and Epstein, S. S.: Structure-activity relations for methylenedioxyphsnyl and related compounds on hepatic microsomal enzyme function, as measured by prolongation of hexobarbital narcosis and ?.oxazolai»1ne paralysis in mice. Toxicol. Appl. Pharmacol. 16., 482-494, 1970. 84. Epstein, S. S., Arnold, E,, Steinberg, K., Mackintosh, D., Shnfner, H., and Bishop, Y.: Mutagenic and antifertility effects of TEPA and METEPA in mice. Toxicol. Appl. Pharmacol, 85. Epstein, S.. S., Joshi, S. Pv,,_ Arnold, E., Page, E. C., and Bishop, Y. Abnormal zygote development in mice after paternal exposure to a chemica] mutagon. Nature 86. Epstein, S. S., Csillag, R. G., Guerin, H., and Friedman, M. A. In vitro effects of methylonedioxyphenyl insecticidal syncrgists on hydroxylations of biphenyl by mouse liver microsomes. Biochem. Pliannatol. 87. Epstein, S. S. , Bass, W., Arnold, F., and Bishop, Y. The failure of caffeine to induce mutagenic effects or to synergizc thfc offe.cts of known mutagens in mice. Fd. Cosmot. Toxicol. 430 431 APPENDIX II Publications •88. Epstein, S. S., and Lederbcry, J. Chronic non-psychiatric haxards of drugs of abuse Science 89. Friedman, M., and Epstein, S. S. Stability of piparonyl butoxido. Nature 90. Epstein, S, S.s Bass, W., Arnold, E,, and Bishop, Y.' The imitauenicHy of trimethyl phosphate in mice. Science 91, Epstein, S. S. , arid Fujii, K. Synorgism 1n carcfnogenesis with particular reference to synorgistlr. effects of piperonyl butoxide and related insacticidal synergists (Chsp coy*) In, Chemical Tumor Problems, ed. Nakahara, W., Tokyo, 1970, 92. Epstein, S. S. The failure of caffeine to induce mutagenic effects or to synerqize the effects of known mutacjens in mice. (Chapter) In, Chemical mutagens. ed. Vogel, F., Heidelberg, 1970. 93. Bateman, A . , and Epstein, S. S. Dominant lethal mutations in mammals. (Chapter) In, Environmental chemical mutagens. ed. Hollaender, A . , Plenum Publishing C o . , New York, 1970. 94. Joshi, S. R., Paye, E. C., Arnold, E. , Bishop, Y . , and Epstein, S. S. Fertilization and early embryonic development subsequent to mating with TEPA-treated male mice. . • Genetics 95. Oaffe, II., Epstein, S. S., and Neumeyer, 0. L. _• , Comparative effects of .piperonyl butoxide and N-(4-pentynyl) phthalimido on mammalian microsamal enzyme functions. .J. Mecl. Chem. 96. Epstein, S. S., and Rohrborn, G. Recommended procedures for testing genetic hazards due to chemicals based on the induction of dominant lethal mutations in mammals. Nature SUMMARY AND CONCLUSIOKS Teratology deals with the etiology and development of congenital malformations. Congenital malformations are generally denned as gross structural abnormalities of prenatal origin, present at birth or manifesting shortly after, which kill or disable. In a broader sense, teratogenesis is considered to include histological, biochemical, and functional abnormalities of prenatal origin. Congenital malformations present obvious personal, medical, and social stresses. Additionally, it has been recently estimated that the costs to society of one severely malformed child, in terms of medical and other care and deprivation of potential earnings, amount to several hundred thousand dollars. There are now well over 400 substances that, in various forms and combinations, are currently used as pesticides. Pesticides may represent an important potential teratogenic hazard. Therefore any teratogenic pesticide to which the population is exposed should be promptly identified so that appropriate precautions can be taken to prevent risk of human exposure. It is feasible to test these substances for teratogenic effects in test animals so that potential hazards to human health can l>e evaluated. For these and other reasons detailed in the report, we conclude that: a. All currently used pesticides should be tested for tetratogenicity in the near future in 2 or more mammalian species chosen on the basis of the closest metabolic and pharmacologic similarity to human beings possible. Pesticides should be tested at various concentrations including levels substantially higher than those to which the human population are likely to be exposed. Test procedures should also reflect routes related to human exposure. Apart from the obvious route of ingestion, attention should be directed to other routes of exposure, including inhalation exposures from pesticide aerosols and vaporizing pesticide strips used domestically and exposures from skin absorption. Parenteral administration is an appropriate test route for pesticides to which humans are exposed by inhalation, or for pesticides which are systemically absorbed following ingestion. b. The use of currently registered pesticides to which humans are exposed and Avhich are found to be teratogenic by suitable test pcocedures in one or more mammalian species should be immediately 432 433 restricted to prevent risk of human exposure. Such pesticides, in current use, include Captan; Carbaryl; the butyl, isopropyl, and isooctyl esters of 2,4-D Folpet; mercurials; PCNB; and 2,4,5-T. The teratogenicity of 2,4-D, the other salts and esters of both 2,4-D and 2,4,5-T, and that of IPO should be investigated further. c. Pesticides found to be' inactive after appropriate testing can be considered as provisionally safe, unless other evidence of teratogenicity develops. d. No new pesticide should be registered until tested for teratogeuicity by suitable procedures. Any pesticide found to be teratogenic should only be used in circumstances where risk of human exposure is minimal. e. Efforts should be made to improve and standardize procedures for teratogenicity testing and population monitoring. A scientific group or commission should be charged with responsibility for continued surveillance of the whole problem of pesticide ' teratogenesis. ment is apparently undisturbed, and above which delM in utero results. Most agents are teratogenic only in the developmentally labile early period of gestation, during which active organogenesis occurs. In humans, this sensitive period extends aproximately from the end of the first week of pregnancy to the 12th week. Other circumstances may also influence the effectiveness of human teratogens, such as maternal nutritional, demographic, socioeconomic, and cultural factors, physiological states, and temporal and seasonal situations. Thus a potential teratogen may manifest its effect only when particular conditions conjoin. The relationship between human exposure to a teratogen and subsequent induction of congenital abnormalities is generally not obvious. Any one teratogen may produce a multiplicity of effects and any specific effect may be produced by various teratogens. In test animals, the teratogenetic response may differ from species to species. In humans, differences in genetic, metabolic, and environmental influences may contribute to a variety of specific effects from exposure to a particular teratogenic agent. Induced and spontaneous effects may be difficult to distinguish. The teratogenicity of thalidomide might have been missed had it not produced malformations rarely encountered; additionally, only a fraction of the pregnant women who took thalidomide had defective children. Consequently, further data on the possible teratogenic effects of pesticides in experimental animals are urgently needed to provide a basis for evaluating potential hazards to human health. Ancillary methods Preliminary screening can be accomplished by the use of nonmammalian species, particularly the chick embryo. These tests may give useful ancillary data prior to further testing in mammals. However, negative results in these systems alone should not be considered proof of safety. METHODOLOGIES FOR TERATOGENICITY TESTING Introduction Prior to 1963, the Food and Drug Administration did not require evaluation of teratogenicity. As a result of the thalidomide disaster, the need for data on teratogenicity became evident. In 1963, the President's Science Advisory Committee on "Use of Pesticides" recommended that toxicity studies on pesticides include effects on reproduction through at least 2 generations in at least 2 species of warmblooded animals. Observations to be included were effects on fertility, size and weight of litters, fetal mortality, teratogenicity, and growth and development of sucklings and weanlings. Such toxicity studies including the three-generation procedure were not designed primarily to detect teratogenicity and thus may not be appropriate. The potential teratogenicity of chemicals may be detected by two complementary approaches. First, chemicals or other agents may be administered to experimental animals to determine whether they induce prenatal damage. Secondly, and on a post hoc basis, human populations may be epidemiologically surveyed to detect geographical or temporal clusters of unusual types or frequencies of congenital malformations. Combinations of these approaches are likely to ensure early detection and identification of teratogenic hazards. Experimentally, a complex of factors are needed to elicit teratogenic effects. These relate to gestation period, genotype of the pregnant animals, dosage, mode of administration and metabolic transformation of teratogen. For example, teratogens may be effective only at a certain dose range, whether high or low, narrow or wide, below which develop- Use of lower mammalian species a. Purity, composition, stability, and source of compounds under test should be determined. b. At least two mammalian species should be tested. These should be chosen on the basis of metabolic and pharmacokinetic similarity to humans. If possible, commercially available inbred strains should be used; if not, intra-species variability must be recognized. Species commonly used include mice, rats, hamsters, rabbits, dogs, cats; sheep and swine have also been used. c. Preliminary mammalian experiments should determine--the amounts of the compound and its appropriate metabolites necessary 434 to produce serum levels comparable to ranges likely to be found in humans after high level accidental exposure as well as potential exposures assuming extensive use of that pesticide. Multiples of these dosages, up to the mammalian maternal LDBO should be administered to determine the lowest dosage causing a significant increase in fetal death, or resorption. Dosage in this critical range should be tested for teratogenic effects .with care to distinguish these effects from other embryotoxicity and to determine dose-response relationships. d. Compounds should be administered, by appropriate routes, within the critical dose range determined by preliminary tests. Parenteral administration is an appropriate test route for pesticides to which humans are exposed by inhalation, or for pesticides which are systemically absorbed following ingestion. Compounds should first be tested by single administrations of a range of doses at various times during the phases of active organogenesis. The substance should be administered at discrete times throughout the period of organogenesis as various organs are developing, since some substances have specific effects on the development of particular organs. By this technique, the possibility of inducing hepatic microsomal or other enzymes facilitating metabolic detoxification or activation of the substance is also minimized. If no teratogenic effects are detected by this technique, subsequent testing should be based on repeated administrations of the substance at daily intervals or if feasible, intervals of less than 24 hours during the entire period of organogenesis. e. When appropriate, metabolites should also be tested for teratogenic effects. f. Additional investigations should include— i. Determination of appropriate plasma and fetal levels of compounds; ii. Determination of the biological half-life of the compound in test animals; iii. Metabolic studies to identify mechanisms of detoxification or activation of compounds when appropriate; and iv. Determination, when appropriate, of the possible potentiating effects of protein deprivation or concomitant exposure to other pesticides or other environmental agents. ' ' g. All procedures, including those relating to animal.breeding, housing, handling, feeding, husbandry, methods for examining fetuses for congenital malformations, defining the onset of pregnancy, and classifying congenital malformations should be rigorously standardized. Numbers of pregnant animals and offspring must be adequate for statistical significance. All tests must be replicated on independent occasions and with contemporaneous controls. 435 Nonhuman primates . Results from lower mammalian species may warrant subsequent testing in nonhuman primates The following considerations should be noted: a. Records of menstrual cycles are essential. Primates whose reproductive history is known and have previously delivered normal young should be selected for testing. Timing of ovulation, and therefore gestation, should be accurately determined by allowing the males and the females to be together for no more than 3 consecutive days. Vaginal smearing, to determine the presence of spermatozoa should be avoided; the use of Tullner's method for determining chronic gonadotropin levels and rectal palpation is preferable. b. Compounds should be carefully administered in controlled dosages. i c. Pregnant animals should be handled only minimally. d. Compounds should be administered during the various phases of organogenesis. Embryos can be obtained by laparotomy any time after the first 100 days of gestation; the mother may be subsequently used for other experimental procedures. Additionally, some young should be allowed to go to term to identify possible teratogenic effects detectable only in the neonatal period. Population monitoring It has been shown (see Literature Review) that some pesticides induce congenital malformations in experimental animals providing a critical dose, is appropriately administered at critical times. "When animal experiments indicate that a pesticide is teratogenic, human effects should be retrospectively evaluated, when possible, by study of pregnancies during which the mothers were inadvertently exposed to the pesticide, such as a result of farm work, accidental ingestion, or industrial exposure. Prospective epidemiologic approaches may involve follow-up of large numbers of people over long periods of time, and be slow, tedious, expensive, or difficult to implement. It is not appropriate to conduct prospective epidemiological studies on human populations with pesticides previously shown to 'be teratogenic by experimental animal studies or retrospective human data. Human exposure to such compounds must be minimized by appropriate regulatory preventive action. Prospective epidemiological approaches for pesticides in current use may provide important information, however, it should be realized that no major teratogen has yet been recognized in this way. The malformations induced by X-ray, German measles, thalidomkle, and mercury—Minamata disease, Avere each recognized by an alert medical practitioner who observed a cluster of cases and then traced the cause tojjs source. m ^PLat can be done to enhance prompt recognition of such clusters should they occur from previously unsuspected teratogens in the future? A variety of existing data resources can be used for this purpose. In each, the occurrence of congenital malformations in substantial segments of the population is being recorded in a standard fashion. The best of these resources are local, rather than statewide or national. The prepaid medical program of the Kaiser-Permanente Hospitals and Clinics in the San Francisco Bay Area are of particular interest. A detailed study there of the occurrence of malformations among 16,000 births represents a good model for additional investigations. A similar study has been made by the Health Insurance Plan of Greater New York, but its 30 or more cooperating clinics are less easily coordinated than the Kaiser system. A citywide surveillance, known as the Metropolitan A'tlanta Congenital Defects Program (jointly directed by Emory University School of Medicine, the Georgia Department of Public Health, and the National Communica,ble Disease Center, USPHS), involves reports on all children with congenital malformations born to residents of the five-county Atlanta area.1 As yet, no cluster of cases has suggested an environmental influence since the program began in October, 1967. In a substantial number of States, birth certificates contain an item concerning congenital malformations. The completeness and accuracy of such reporting varies considerably and depends on the physician's interest and diligence and on the conspicuousness of the abnormality. Birth-certificate data on malformations in New York State are more extensive than those of many other States and have been effectivel}' used in several research studies. Nationally, however, no attempt has been made to collect and evaluate all data on malformations that are available on birth certificates. A select committee convened by the National Center for Health Statistics (NCHS), has recommended, in an excellent but little known report, that efforts be made to improve and use information on congenital malformations recorded on birth certificates (Vital and Health Statistics, Documents and Committee Reports, NCHS Series 4, Number 7, March 1968). Implementation of this recommendation would bo of great value, for monitoring to detect the teratogenic effects of newly introduced or geographically localized environmental chemicals or other agents. To enhance our ability to recognize significant changes in congenital malformation rates, a systematic collection of data from concentration points should be established. Specifically, a surveil- lance 'should be made of claims submitted to private, State, or local agencies for the medical care of children with birth ded^ks. Because the Children's Bureau, DHEW, has so much experien^r with these agencies, its assistance should be sought in planning the surveillance network. Data from foreign countries should also be evaluated as part of a national effort to study possible relationships between pesticides and congenital malfunctions. In studying the possible relationships between exposure to pesticides and the occurrence of diseases, statistical associations, if present, will provide important information. However, when possible it is important to secure additional information concerning the following: a. Dose-response relationships. b. Absence of alternative explanations. c. Biological plausibility. d. Consistency with other knowledge from clinical, laboratory, and epidemiologic research. e. Disappearance of the effect when the presumed cause is removed. In particular, as clusters of specific anomalies are recognized, through whatever resources that presently exist or may be developed, any possible relationships to pesticides would be clarified by the use of laboratory techniques to measure the maternal, fetal, or neonatal body burden of suspect chemicals. There are national units engaged in teratologic research, but each is following a set method. There is a critical and immediate need to establish a national or international center to study congenital malformations in man not by a single method but by whatever techniques are most appropriate for testing or generating hypotheses. The center should be diversified and fast moving, ready to use local, national, or international resources in order to determine the significance of laboratory or clinical data. LITERATURE REVIEW Animal studies For convenience, detailed results of the Bionetics study are presented in a subsequent section. Much of the total available literature and data reviewed by this Panel were methodologically inadequate to support definitive conclusions. Additionally, the authors of many reports tended to confuse or equate em'bryotoxicity and other adverse effects on reproduction with teratogenicity. It is also apparent from the literature that insufficient attention has been directed towards problems of interactions in testing for teratogenesis. The Panel considered the following information to be of a. Captan and Folpet.—These pesticides have been shown to be terntogenic in chicken embryos (Verrett et al., 1969). Captan was also shown to be teratogcnic in rabbits (McLaughlin, 1969), although other rabbit studies yielded negative results (Kennedy et al., 1968; Fabro et al., 1965). The enhancement by protein deprivation of the acute toxicity of captan to rats (Boyd, 1968), was noted with particular interest. The teratogenicity of captan and Folpet in mice was demonstrated in Bionetics studies. Unpublished data on captan in monkeys were evaluated and found inadequate; in these studievS, the duration of organogenesis was not entirely covered mid controls were not appropriate. However, the 3/7 abortions observed at the highest dosage given, 25 mg./kg., may be indicative of an em'bryotoxic hazard due to captan. b. Carbaryl.—This was tested at 66.7 and 200 p.p.m. in the diet of pregnant mice (FAO/WHO, 1967). In tivo litters at the 200 p.p.m. level, a total of seven instances of skeletal malalignment, nonfusion, incomplete ossification, and one case of cleft palate and gross facial malformation were noted, as opposed to no malformations in the lowlevel group and-two cases of cleft palate in controls. Teratogenetic findings for carbaryl are also reported in the Bionetics study. In a. study in beagle dogs fed carbaryl during gestational periods at levels of 50, 25, 12.5, 6.25, and 3.125 mg./kg. body weight daily, teratogenic effects were found at all but the lowest dose level (Smalley, 1968). c. Mercurials.—Organomercury compounds: Various mercury containing pesticides were evaluated under, the heading "phenylmercury acetate (and other organomercury compounds)" by the 1966 Joint Meeting of the FAO Working Party and the WHO Expert Committee on Pesticide Eesidues (FAO/WHO, 1967). The results of additional experimental work have been reported in the 1967 Evaluations of Some Pesticide Kesidues in Food. Additional information on "Methylmercury" was published by the Ecological Research Committee, the Swedish Natural Science Research Council (1969) Bulletin no. 4. by Goran Lofroth, where embryotoxic effects in mice (reported by Fro'len and Ramel) were discussed along with other data. When given subcutaneously, in doses of 0.11 mg. on day 7 of gestation, phenylmercuric acetate was reported to cause fetal malformations in mice. Eye,'tail, and central nervous system defects were noted (Murakami et al., 1956). d. Organ&chlorine,—Embryotoxicity in rats and dogs has been reported for organochlorines including dieldrin, chlordane, and kepone. In the absence of convincing data, kelt'hane has been claimed to be teratogenic in mice (An Der Lan, 1964); see also Bionetics studies. ^^ e. Organophosphates.—The cholinesterase-inhibitn^p organophosphate insecticides, guthion, parathion, diazinon, Bidrin, Trithion, and EPN, have been shown to be teratogenic when injected directly in the yolk sac of chick embryos. The malformations were nonspecific or common to all organophosphates (Fish, 1966). It was also claimed that these compounds are teratogenic in mice. The data reported, however, suggested that organophosphates, like the organochlorines, act by reducing litter size and producing embryotoxicity rather than by producing specific teratogenic effects. See also Bionetics studies. /. Thiram.—Thirarn was reported to be teratogenic in hamsters at 250 mg./kg. (Ilobens, 1969). In the Bionetics study it Avas not found to be teratogenic. In a study of three generations of rats, no toxicological effects were observed at a dietary level of 48 p.p.m. (FAO/WHO, 1967). However, Thiram should be further investigated for possible teratogenic effects. g. Miscellaneous reproductive effects,—Placental transfer of dieldrin and incidence of stillbirths have been studied in cows (Braund, 1968); increased stillbirth rates have been claimed in cows fed with DDT (Labon, 1965). The estrogenic activity of o,p'DDT has been related to reproductive effects in chicken, quail, and rats (Baseman, 1968, Wurster, 1968; Porter and Weimeyer, 1969). Diminished population size and reproductive failure have been produced in sparrow hawks by DDT and dieldrin (Porter and Weimeyer, 1969). These resulted from a decreased eggshell thickness, increased breakage of eggs, and increased egg eating by parent birds. Other studies of interest include the following: Finnegan, 1949; Tauber, 1950; Fisher, 1952; Narpozzi, 1956; Swann, 1958; Cottrell, 1959; Marliac, 1964; Backstrom, 1965; Hathaway, 1967; Ware, 1967; Weike, 1967; Carlton, 1968; Keplinger, 1968; Khera, 1968; Verrett, 1969; Legator, 1969. Bionetics animal studies Bionetics Research Laboratories of Litton Industries, during 1965-68 under a contract for the National Cancer Institute (NCI Contracts PH 43-64-57 and PH 43-67-735), tested various pesticides and related compounds for teratogenic effects. These studies were designed as large-scale screening tests. The Bionetics data were reanalyzed statistically to account for litter effects. The results of this statistical re-evaluation are presented in this section. More detailed material on these pesticides will be published in the future. a.. Summary of findings from Bionetic animal studies.—Tested more extensively than other pesticides, 2,4,5-T was clearly teratogenic as evidenced by production of statistically increased proportions of 440 441 , and increased proportions of abnormal fetuses within litters in both DMSO and Honey for both C57BL/6 and AKR mice. In particular, cleft palate and cystic kidneys were significantly more prevalent. In addition, a hybrid strain resulting from a C57BL/6 female and AKR male showed significant increases in anomalies, in particular cystic kidney, when administered at 113 mg./kg. of body weight in DMSO. Additionally, 2,4,5-T was tested in Sprague-Dawley rats. When given orally at dosages of 4.6, 10.0 and 46.4 mg./kg. on days 10 through 15 of gestation, an excessive fetal mortality, up to 60 percent at the highest dose, and high incidence 6f abnormalities in the survivors was obtained. The incidence of fetuses with kidney anomalies was three-fold that of the controls, even with the' smallest dosage tested. PCNB produced an increase in renal agenesis between litters, and within litters, when administered orally from days 6-14 or days 6-10 of pregnancy. However, renal agenesis was not produced when PCNB was administered only from days 10-14 of pregnancy. These effects were produced in only the C57BL/6 strain of mice. Other pesticides producing a statistically significant increase in the proportion of litters containing abnormal fetuses and in the increased incidence of abnormal fetuses within litters were: Captan, Folpet, 2,4-D isooctyl ester, 2,4-D butyl ester, 2,4-D isopropyl ester, carbaryl (Sevin), and IPC. These pesticides produced elevated incia.nd in one solvent only. The resuHs for carbaryl and for IPC were less consistent than for other compounds. (The pesticides 2,4,5-T, PCNB, captan, Folpet, carbaryl, IPC, and the butyl and isopropyl esters of 2,4-D were statistically significant at the .01 level, for one or more tests. This criterion is similar to that adopted by the Technical Panel on Carcinogenesis, Chapter 5, to identify "positive" compounds. The isooctyl ester of 2,4-D was significant at the 0.05 level.) Compounds inducing only an increase in the proportion of abnormal fetuses within litters were: a-naphthol, and 2,4-D methyl ester. The statistical significance of these results was relatively weak; further study is required before any conclusions can be reached. Similarly, 2,4-D produced only an increase in the proportion of abnormal litters during 1965 in AKR mice. Due to the teratogenic activity of certain of its esters, 2,4-D should be studied further. Carbaryl plus piperonyl butoxide did not show an overall increase in nonspecific anomalies, but resulted in significantly more cystic kidneys for doses above 10 mg./kg. carbaryl plus 100 ^,l./kg. piperonyl butoxide. It must be emphasized that failure to detect statistically significant increases of .anomalies may be due to insensitivity resulting from experimental variation and small numbers of littesRested. In addition, higher fetal mortality among some of the "negative" compounds may be selectively eliminating abnormal fetuses. b. Methods.—Four strains of mice were used: C57BL/6, AKR, C3H, and A/Ha. Most of the studies were performed with the C57BL/6 strain. A hybrid fetus resulting from mating a C57BL/6 female with an AKR male was used to study a few compounds. More restricted studies were also made on Sprague Dawley rats; results of these with reference to 2,4,5-T a.re considered separately. Most compounds were administered subcutaneously in 0.1 ml. solutions of dimethylsulf oxide (DMSO). Water soluble compounds were administered in saline, and some times also in DMSO. Compounds administered orally were given by gavage in 0.1 ml. in a 50-percent honey solution. Groups of positive controls and untreated controls were included, as well as controls receiving only DMSO, saline, or honey. While controls were run periodically throughout the duration of the study, compounds and controls were not matched with respect to either route or date of administration. Virgin females were used in these studies. The onset of pregnancy was determined by detection of vaginal plugs. Compounds were administered daily from the sixth to the 14th day of pregnancy (15th day for AKR mice). Mice were sacrificed on the 18th day (19th day for AKR mice) of gestation. On sacrifice, fetuses were examined for anomalies. Approximately two-thirds of the fetuses were then stored in Bouin's solution until necropsy. Remaining fetuses were stained with alizarin red S after proper processing. Numbers of resorption sites and dead fetuses were also scored. G. Statistical analysis,'—All analyses were performed on a per litter basis rather than a per fetus basis, since initial investigations indicated that the occurrences of anomalies among fetuses within litters were correlated. The large litter-to-litter variation may reflect some maternal effect-, an indication of the effective dose level of the compound actually reaching the fetuses, experimental variation, or, as is most likely, some combination of the three factors. While there were no statistically significant time trends within the various control groups in terms of the onset of fetal anomalies in the C57BL/6 mice, the incidence of fetal mortality was certainly timedependent in this strain, with 1965 being characterized by a low incidence of prenatal deaths. Furthermore, there was a period of approximately 6 months, extending from the latter part of 1965 into early. 1966, during which no control animals were tested. During this period a change in .the substrain of C57BL/6 mice used in the study took place. Finally, among abnormal litters, as defined by litters con- 442 443 at least one abnormal fetus, there was some suggestion that' the distribution of abnormal fetuses per litter was stochastically larger in the DMSO controls than it was in the untreated controls. Thus, the possibility exists of a time/strain/solvent interaction that is undetectaible in the controls because the level of background teratologic activity is relatively low. This potential interaction effect could either enhance or dissipate the effect of any given compound, depending on the conditions under which it was administered. Thus, the data were necessarily separated by both time period and solvent for the purposes of analysis. Similarly, an increase in fetal anomalies in the DMSO controls of the AKR mice was noted after November 1966. Thus, the AKR data were analyzed separately in two time periods. It should be noted that not all compounds were administered on more than one occasion or in more than one solvent or strain. Thus, in general the compounds in the study cannot be compared for teratogenic potential, since those that were tested extensively were more likely to show some adverse effect and, perhaps, less likely to appear consistent over time, solvent, and/or strain, As noted, approximately two-thirds of the fetuses were stored in Bouin's solution until necropsied; the remainder being stained with alizarin red. However, in many instances the proportion of necropsied fetuses was slightly higher for the compound under investigation than for the corresponding controls. It is doubtful if this discrepancy could have any appreciable effect on the conclusions since the incidence of anomalies detectable only by necropsy among control animals was relatively low. Furthermore, if all of the control and test mice had been necropsied, the significance of the differences observed in this study would be intensified. Thus, no effort was made to correct for inequalities in the necropsy/stain ratio in the present analysis. Additionally, no attempt was made to correct for differences in litter sizes or sex-ratios within litters, since both of these factors may, at least in part, reflect effects of the Compound under test. (I, Itcvults.—Data for pesticides yielding a statistically increased level of anomalies in C57BL/6 and AKR mice are listed in tables 1 and •2, respectively. The proportion of abnormal litters gives the proportion of litters containing one or more abnormal fetuses, as a measure of the prevalence of anomalies across litters. The proportion of abnormal fetuses per litter gives a measure of the prevalence of anomalies within litters. The proportion of abnormal fetuses per litter for litters containing abnormal fetuses gives a measure of the prevalence of anomalies within effected litters. A significant increase of dead fetuses and resorptions is also listed. Some tests were conducted on only one par- tioular day or on adjacent days as listed. Eye anomalies, j^fcfily microphthalmia and anophthalmia, accounted for approximate^St) percent of the individual anomalies in C57BL/6 mice. To a large extent, results in table 1 reflect changes in the incidence of eye anomalies. Yet, when the data were analyzed excluding fetuses with microphthalmia only, there were no striking changes in the results. In the last column of table 1, statistically significant increase in various types of anomalies other than eye anomalies are listed. The .positive controls, trypaii blue and ethyleneimine, table 1, and 6-aminonicotinamide, table 2, showed elevated levels of anomalies, although the latter control did not yield consistent results over all dose levels. Only those test conditions which resulted in statistically elevated incidences of anomalies are listed in tables 1 and 2. Some compounds gave no increase in anomalies (based on the overall incidence if tested in both time periods) when tested in other solvents, strains, or dose levels (table 3). It must be emphasized that failure to detect a statistically significant increase in anomalies may only be a reflection of experimental insensitivity due to experimental and biological variaton and insufficient number of litters. Thus, compounds showing no increases cannot be considered nonteratogenic. For example, trypan blue in DMSO at the highest dose level tested, 3Y.5 mg./kg., did not show an increase in anomalies, possibly due to higher fetal mortality. Standard corrected 2 X 2 chi-square tests (1) were used to compare the proportion of abnormal litters for the compound with the controls in the same solvent. In the cases where tests were conducted in two time periods, the results from the two chi-squares were combined (1). The levels of statistical significance for the combined tests are listed under the total column for proportion of abnormal litters. The distribution of the proportion of abnormal fetuses per litter (tables 1 and 2) for compounds were compared with the appropriate control distribution by use of the nonparametric Mann-Whitney litest (#). This test requires that the proportion of abnormal fetuses per litter is independent from litter to litter, but requires no assumption about the frequency distribution of these proportions. Again, where litters were run in both time periods, the significance level for the combined tests is given under the total column. Bracketed data include groups which were combined before statistical tests were conducted. STATISTICAL REFERENCES ( / ) SNEMicoH, G. W. AND CocHRAN, \V. G.: Statistical Methods, 6th ed. Iowa State Univ. PresH, Ames, Iowa (1967), (2) STEEL, K. G. D. AND TOBKIE, ,T. H.: Principles and Procedures of Statistics. McGraw-Hill Book Co., Inc., New York (1960). 45-362 O - 70 - 29 TABLE 1.—Tests which displayed significant increases of anomalies (C57BL/6 mice) Compound Dose per Solvent kg of body Proportion of abnormal litters Proportion of abnormal fetuses per litter 1965 Negative controls: .42 Untreated . None Controls . ... DMSO... .53 Do .52 . . Saline __ _ Do.... Honey Positive controls: Trypanblue. .. DMSO- 5-Omg .60 Do. _. DMSO- 12. 5mg .86 Do DMSO.. 37. 5 mg .60 Do Saline... 5.0 mg LOO Do. ...do 12. 5 mg .71 Do ... .. do. 37. 5 mg . 71 Proportion of abnormal InTests No. of live fetuses per litter in abnormal creased repeated litters litters mortalover 1966-68 Total 1965 1968-68 Total 1965 1966-68 Total .39 .41 .37 .40 .46 .43 .08 .16 .13 .11 .12 .10 15 .10 .13 .11 15 .18 .33 .24 28 28 28 32 25 29 26 32 26 70 31 32 54 52" 5 7 46" 54 Yes 52** Yes 60 Yes 61** 69*** Yes 46" Yes .49"* 49*** Yes f . •".60 ... .86 .60 .LOO .71 - .71 32 44*** .36 . 61*** .49" 33* 44*" .36 .61*** .49** 33* .49*** 49*** .60 .61" .69"* Increased anomalies other than 1965 1966-68 "'" 69 112 46 32 1 iHydro1 cephaly. 5 5 7 7 cephaly. Ethyleneimine . Experimental: 2,4,5-T.. 2 4 5-T PCNB (days 5-14) PCNB (days &- 14) PCNB (days 6-10) Folpet ...do 4. 64 ;sl LOO* 1. 00* DMSO_. 113 mg . 79** 46. 4 mg 113 mg 1 00* 1 00" do . 79** LOO* 1.00" .88* .88* 67] ' 67") LOO] 1 OOJ 61 . 71*** 77 ** .77 ** LOO * 67 .67 .75 ** .75 ** .70 ** .70 ** -.56*" .56*" 71*** 71*** 37** 70*** 37** 70*** 37 70*** .37 .70*** Yes .25** .25" 25l 25) 38) 27 .29 *** 381 35"* .29 *** .28 ** .25 *** .26 *** .28 ** .25 *** .26 *** - .29 .29 38 38 .37 37 .49 ** Yes 44 38 * ' .38 * 24 .41 * .41 * .34 . ... i .34 37 * .37 * do do do DMSO DMSO DMSO DMSO DMSO DMSO 215 mg 464 mg 464 mg 100 rag 1 00* 48 A*! 130 pi 100 ftl 94^1 1. 00 * DMSO 100 mg 1 00 * 54 71 ** 46 **' 16 26 ** .46 * DMSO DMSO DMSO DMSO 850 mg 10 mg 106 mg 1. 00 ** 86 .43 .46 *** 33 * .09 30 * 13 .27 ** 39 * 30 * 13 .46 * .38 83 50 .71 * .86 83 50 -50 .50 .10 .10 r 58*** 24 24 .58" ""24" " 7 No 14 Yes - No . Cleft palate cystic kidney. 6 .. 9 .. Cleft palate cystic kidney. 8 .12 .. . 10 .. No No 6 IS 13 .1 , J Renal agenesis. 6 -. 15 20 Agnathia. 20 ester. IPC Butoxide. Do Significance level: DMSO 100Ml 46 4mg4464 ;il *(.10). "(.05). "(.01). 29 .37 6 11 7 7. 36 26 -.46 * .38 .36 .26 .21 .21 Hydrocephaly, skeletal 7 .. fi No '... fi 1 [Cystic kidney 12 ^ (^, 446 447 TABLE 3.—Tests which showed no significant increase (with particular doses, solvents, or test strains) S.I oo Compound s a 2v >, ^SSS a ss s .8 "3 e . es fig S8 8 S 88 •I s_: I "S. I °< - .§ S8 >e 88 • M to S S "Si °Q o to ft S I 23 XO 2J ^ ss« R tt |'? i. •v" 4" I 5 ±M ^-S. ce Le 11/66 With t £ S 2 •3 -g 8 'Sig fTh Not $o 8 Strains 2,4,5-T C57 PCNB (days 10-14)... CB7 PCNB AKR Captan C57 Do AKR Folpet. C57 Do AKR 2,4-D Isooctyl ester C3H Do A/Ha Do AKR 2,4-D Butyl Ester C57 Do AKR 2,4-D Isopropyl Ester.. C57 Do AKR Carbaryl C3H Do C57XAKR Do ... . AKR IPC 03H IPC AKR 2,4-D Methyl Ester AKR Do C57XAKR o,p'-DDD... C57 Do AKR 2,4-D 057 Do... 057 Do C3H Do 057 X AKR Zectran 057 Do AKR C57 Thiram--AKR Do Ferbam C3H Do 057 Monuron C3H Do 057 Do > . 057 Do AKR Diuron C3H Do 057 2,4-D Ethyl Ester 057 Do AKR Atrazine C3H Do 057 Do AKR Solvent Doso per kg. bodywt. DMSO Honey Honey Honey DMSO Honey DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO Honey DMSO DMSO 21.5 mg. 464 mg. 464 mg. 100 mg 100 mg 100 mg 100 mg. 48 M!24 jd. 130 M!. 46 M!. 100 /ul. 46 M!. 94 M!. DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO Honey DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO 10 mg. 10 mg. 10 mg. 115 mg. 4.64 mg. 4.64 mg. 215 mg 215 mg ; 215 mg 215 mg. 215 mg 215 mg 86 M! 86 M! 46.4 mg 46.4 mg 46.4 mg Increased mortality (G67BL/6) Total number of litters 6 9 100 mg 100 mg 464 mg. 850 mg 850 mg. 106 mg. 106 mg. 100 mg 100 mg. 100 mg , Yes 100 mg 100 mg 98 mg. 7 7 8 7 .: - 9 12 13 5 13 6 5 8 6 10 6 6 8 6 13 11 13 7 5 13 12 16 12 6 11 .- ...... .a.. 6 6 7 13 9 13 6 6 7 7 6 13 15 448 449 TABLE 3.—Tests which showed no significant increase oj anomalitt (with particular doses, solvents, or test strains)—Continued mothers showed transplacental passage of DDT and DD^T (O'Leary, 1969). Low placental and high vernix levels were noted; ietal blood levels were one-half maternal levels. In a similar study on premature infants (O'Leary, 1969), high fetal levels were noted; no relationship between maternal blood levels of DDE and DDT and the incidence of first trimester spontaneous abortion were found, although the num-' ber of pregnant women reported on was inadequate for firm conclusions. Organophosphates.—Evidence of teratogenic potential of organophosphates in humans has been reviewed and found inconclusive (Khera and Clegg, 1969). Mercurials.—Consumption by Japanese pregnant women of fish and shellfish contaminated by methylmercury produced a high incidence of infantile cerebral palsy (Matsumoto et al., 1965). This condition has been termed fetal Minamata disease. Compound Strains Piperonyl Butoxicle C3H Do - 057 Do.C57 p,p'-DDD „' C57 p,p'-DDT C57 Carbffryl + Nicotinamide C57 Nico Unamide C57 CIPC C57 Nabam '. C3H Do C57 Do 057 Do AKR Do AKR Propazine. C3H Dicryl C57 Perthane.' C57 Ovox AKR Tedion AKR Amitrol C57 Do C57 Do -. AKR Solvent Dose per kg. body wt. DMSO 1000 Ml DMSO DMSO , DMSO ' DMSO DMSO 1000 M! 21.5 M! 46.4 mg. 46.4 mg. 100+61 mg. 61 mg. 1000 mg 21.5 mg. 46.4 mg 46.4 mg 46.4 mg 46.4 mg. 464 mg. 21.5 mg. 100 mg 185 mg 217 mg. 464 mg. 215 mg. 464 mg. DMSO DMSO DMSO DMSO Saline DMSO Saline •DMSO DMSO DMSO DMSO DMSO Saline Honey Saline Increased mortality . (C67BL/6) Total number o( llttera 0 6 6 — 0 Yes -. -'. -- .- 6 0 10 '.. - Yes •. e 6 6 14 5 14 6 6 6 7 6 13 8 14 Human studies Epidemiologic data on possible effects of pesticides on human reproduction and teratology are grossly inadequate. Prospective studies on this subject are difficult to design and almost nonexistent, except for the community pesticide program of the Food and Drug Administration. Chlorinated hydrocarbons.—In a recent review (Khera and Clegg, 1969), no adverse human reproductive effects were attributed to DDT and other chlorinated hydrocarbons. Studies on 240 pregnant women indicated that 21 percent had significant first trimester pesticide exposure, and that 52 percent were exposed during their entire pregnancy. No statistical difference in numbers of patients with anomalies existed between these exposed groups (Nora et al., 1967), Low values of DDT residues have been found in a small number of human placentas (Rappolt et al., 1969). Sharply reduced tissue levels were also found in 68 newborn infants (Zavon, 1969). Pesticide levels in human milk have not shown any relation to perinatal toxicity (Laug et al., 1951; Lofroth, 1969; Curley and Kimbrough, 1969). Studies on 152 CITED REFERENCES AL-HACHIM, G. M.: Development of progeny of mice given DDT or parathlon during gestation, Diss. Abst. 26: 6768,1966. AL-HACHIM, G. M., AND FINK, G. B.: Effect of DDT or parathion on the minimal electroshock seizure threshold of offspring from DDT or parathion-trented mothers, Psychoparmacologia 13: 408-412,1968. AN DEB LAN, H.: TJMSCHAU: TJber Die Fortschritte in Wissenchaft .Und Technik. 21: 649,1964. BACKBTROM, J., HANSSON, E., AND VILLBERG, S,: DDT and dieldrin in pregnant mice, Toxic. Appl. Pharmacol. 7: 90-96,1965. BAOKBTROM, J., ET AL. : Distribution of 0"-DDT and C"-dieldrin in pregnant mice determined by whole body autoradiography. Toxic. Appl. Pharmacol. 7: 90-6, January 1965. BERNARD, R. F., AND GAERTNEH, K. A.: Some effects of DDT on reproduction in mice, ,T, Mammal, 45 : 272-276,1964. BIONETICS RESEARCH LABORATORIES: Evaluation of the Teratogenic Activity of Selected Pesticides and Industrial Chemicals in Mice and Rats. Unpublished Report, 1969. BITMAN, J., et al.: Estrogenic activity of o,p'-DDT in the mamalian uterus and avlan oviduct. Science 163: 371-2,1968. BOYD, E. M., AND KRIJNEN, C. J.: Toxicity of captan and protein-deficient diets. J. Olin. Pharmacol. 8:1225-234,1068. BRATJND, D. G., et al.: Placental transfer of dieldrin in dairy heifers contaminated during three stages of gestation. J. Dairy Sci. 51:116-8, 1968. CARLTON, W. W., AND KELLY, W. A.: Reproductive response of female rats receiving a mulitipestielde mixture and diets containing deficient, marginal, or excess amounts of copper. Industr. Med, Surg. 37 : 547,1968. COTTRELL, T.' L., and HECKEL, N. J.: Effects of DDT on rat testes. J. Urol. Bait. 81(4) : 551-3, 1959. CURLEY, A. AND KIMBROUQH, R.: Chlorinated hydrocarbon insecticides in .plasma and milk of pregnant and lactating women. Arch. Environ. Health. 18: 156164, 1969. 450 FAIIIIO, S., SMITH, R. L,, and WILLIAMS, E. T.: Embryotoxic activity of some pesticides and drugs related to phthalimide, Food Cosmet. Toxic. 3: 587-590, 19(i5. FINNEHAN, J, K. ET AL. I Tissue distribution and elimination of DDD and DDT following orul administration to dogn and rats. Proc. Soc. Exp. Biol. Med. 72 ; 357-00, 1949. FiSHKii, A. L. KT AL. 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TANIMURA, T., KATSUYA, T., and NISHIMURA, H.: "Embryotoxicity of acute exposure to methyl parathion in rats and mice." Arch. Env. Hlth. 15: 609-813, 1967. . . TAUBER, O. E., and HUGHES, A. B.: "Effects of DDT ingestion on total cholesterol ' content of ovaries of white rats." Proc. Soo. Emptl. Biol. Med. 75: 420-2, 1900. VEBRETT, M. J., ET AL : "Teratogenic effect of captan and related compounds in the the developing chicken embryo." Ann. N. Y. Acad. Sci. 160:334-43, 1969. WARE, G. W., and GOOD, E. B.: "Effects of insecticides on reproduction in the laboratory mouse." //. Minm, telodrin, and DDT, Toxicol. Appl. Pharmacol. 10: 54-61,1967. WARE, G. W., and Goon, E. E.: "Effects of insecticides on reproduction in the laboratory mouse." ///. Tranid and OC-9160, J. Econ. Entomol. 60: 530-632, 1907. WEIKE, JI.: "Effects of DDT on reproduction in hens." Acta. Pharmacol. 25: Suppl. 4 : 5, 1967. WELCH, R.: "Sex organs under DDT attack." Med. World News: 5 February 7, 1909. WURSTER, C. F. ET AL : "DDT residues and declining reproduction in the Bermuda petrel. Science 159: 979-81,1968. ZAVON, M, R. ET AL : "Biological effects of pesticides in mammalian systems: chlorinated hydrocarbons insecticide content of the neonate." Ann, N. Y. Acad. Sci. 160(1) : 190-200, 1969. 452 453 Senator HART. Before adjourning, I should add for the record^P'6 nad been scheduled Dr. DuBridge of the Office of Science and Technology, 'but Mr. Bickwit advises that in the face of a 5:30 appointment, he has asked not to be heard, but instead submits his statement for the record. (The statement follows:) defects in mice and rats if administered in sufficiently Iq^^doses at nn appropriate stage of pregnancy in these animals. These resUr were available in 1908 and they were subsequently further analyzed statistically by the National Institute of Environmental Health Sciences. No further action was taken on the findings of the Biouetics study after August 1908 nor was the information on teratogenesis publicly available. However, copies of the study reports did find their way to members of Congress, to journalists, and to some members of the scientific community. Coincidentally, in May or June of 1969, a number of anecdotal articles appeared in the Vietnamese press which reported an unusual incidence of congenital abnormalities and abnormalities of pregnancy in certain parts of Vietnam. In some cases, these reports were linked to defoliation operations. The evidence for this is, however, extremely doubtful. It was with this background that I met in October of last year, in my capacity of Executive Secretary of the Environmental Quality Council, with representatives of the several Federal agencies which were most concerned with the use of this herbicida It was the consensus of these representatives that this research information from the Bionetics study warranted serious consideration including certain restrictions on the use of 2,4,5-T. The announcement of these intended actions occurred on October 29. One of these actions was a limitation of defoliation operations in Vietnam. The limitation, which did occur subsequently, took the form of restricting defoliation to non-populated areas. Another announced action was aimed at the Government's own use of 2,4,5-T domestically in programs of brush and weed control. These programs were mainly pursued by the Department of Agriculture and the Department of the Interior. Here the Government did restrict the application of this herbicide so as to reduce possible exposure to uinn. TinState Department, which to some extent had been a party to the use of 2,4,5-T along our border with Canada, took steps to reduce human exposure here and make available to foreign countries technical data about the subject. The Department of Agriculture agreed to cancel the registration Of 2,4,5-T for use on food crops by the first of the year unless the Food and Drug Administration could, by that time, satisfy itself that it had enough evidence to establish a negligible tolerance limit for human exposure. The food crop uses of 2,4,5-T incidentally represented a minority of its total domestic use. At the time of the announcement of concerted Government actions, I also assembled a panel of experts within the Office of Science and Technology to review all that is known about of 2,4,5-T. This Panel has prepared a report on the subject which I expect to make available within a few weeks. During the course of this review, it became known that an impurity of 2,4,5-T was of potential importance. The impurity, a polychlorinated dioxin, was apparently very toxic and had been identified in batches of 2,4,5-T as early as 1957. It arose partly as an impurity of the chlorphenol starting material and partly as a result of the temperatures and pressures of certain of the reactions in the manufacturing process. It had provoked severe skin irritations among workers in 2,4,5-T plants in Germany and in the United States. The discovery of this industrial hazard had led one U.S. manufacturer to curtail his process until he was able to reduce the dioxin content to less than 1.0 ppm in the 2,4,5-T product. This eliminated the skin irritation problem. Within weeks after my announcement, some additional animal experiments were begun in two laboratories simultaneously. These experiments were directed towards confirming and extending the results of the Bionetics studies. In addition, they were aimed at finding out whether the apparent teratogenic agent was 2,4,5-T itself, or a potent impurity. Fortunately, the experiments needed to test for teratogenesis are essentially acute, short-term studies. With an expectation of meaningful results from these experiments in a fairly short period of time and. in view of the potential STATEMENT OF DR. LISE A. DUBBIDGE, DinECToa, OFFICE OF SCIENCE AND TECHNOLOGY SENATE COMMERCE COMMITTEE, APBIL 15, 1970. Mr. Chairman, Members of the Subcommittee, Let me say at the outset Unit I am pleased to have an opportunity to discuss with this Subcommittee certain aspects of the herbicide, 2,4,5-T. An examination of the subject illustrates a number of important issues relating to the Federal Government's involvement with pesticides. I believe that these deserve some discussion and I am glad to have the privilege of exploring them with you. The herbicide, 2,4,5-trichloropherioxyacetic acid is a member of a family of pesticides which have served mankind very well for a long period of time. This group of compounds, known as phenoxy or auxin herbicides, have been vised since the late 1940's and resulted from research work performed during the 1940's on herbicides and defoliants for military as well as civilian use. The toxicity of 2,4,6-T was studied in line with the requirements for its registration by the Department of Agriculture. The toxicology required for this registration of 2,4,5-T was aimed primarily at determining its acute toxicity. In this regard, it is now quite clear that the experiments performed for this purpose (almost all of which were done by or for the manufacturing industry seeking the registration) revealed that 2,4,5-T was relatively non-toxic This herbicide demonstrated a pWsistence in soil and water which was very short (on the order of three months for total disappearance). It is true also, ns you heard last week, that only rare instances of 2,4,5-T residues have been rliscovered in the food surveys performed by the Department of Health, Education and Welfare. As a result of these findings, plus its proved utility as an herbicide and as a defoliant, 2,4,5-T was considered a very beneficial and safe herbicide and with good reason. As evidence for this, it is plain that the demand for 2,4,5-T has risen, especially in the last several years. The production of 2,4,5-T in the United States increased from 7.0 million to 42.5 million pounds between 1960 and 1968. Domestically, it has proved its worth ns a valuable adjunct in the clearing of range and pasture lands of brush, in the clearing of roadsides and rights-of-way, in the suppression of aquatic weeds, in the limited use for control of weeds in croplands, • and for altering physiological responses of crops. The increase in production apparently has reflected the demand for 2,4,5-T both domestically and as a defoliant for military operations in southeast Asia. In fact, the domestically used quantities actually decreased between 1964 and 1966. In 1964, the National Cancer Institute of the National Institutes of Health undertook on contract with the Bionetics Research Laboratories, Incorporated, a screening study of a large number of economic poisons. As you have heard, the general purpose of this study was to ascertain the potential for cancer, for genetic alteration, and the potential of producing birth defects for this long list of pesticides. All who have been concerned with this subject recognize the value of the Bionetics study as a screening mechanism for these potential hazards. 2,4,5-T was among the list of materials screened. One of the results of this study was that a particular lot of commercial grade 2,4,5-T provolced birth 454 of t l m o x i n impurity, the Department of Agriculture in consultation with Interior, HEW, "and my office delayed any action toward cancellation of the food crop registrations for 2,4, 5-T as you heard last week. One of the sets of confirmatory studies was undertaken by the Government, itself. The results of these experiments, pursued at the National Institute of Environmental Health Sciences, were made known to you by Dr. Steinfeld this morning. Essentially, these results implicated both 2,4,5-T (in the purest form available) and its dioxin impurity as potential teratogens. This story, I think you will agree, represents an example of some appropriate Government actions. As you have heard and as Dr. Steinfeld pointed out, the results of these confirmatory studies were translated into immediate actions in the form of a series of announced Government restrictions on uses of 2,4,5-T. Let me now turn to what I believe are some important lessons to be learned from tliis fascinating case study. First, let me review. We have here an example of a chemical substance intentionally placed into the environment by man for the betterment of his welfare. Where the aim has been to exchange capital ' for labor in land and waterway management, there can be no doubt that this herbicide has proved its worth. In view of what we have now learned, I am persuaded that we must consider some changes in our procedures and we must be willing to submit our regulatory systems for pesticides, as for other chemicals in the environment, to examination. The very excellent report of the Secretary's Commission on Pesticides, headed by Dr. Kmil Mrak, will, of course, serve to point up this issue. From a number of indications it is quite apparent that we, as a society, have relatively recently begun to ask more sophisticated questions about adverse effects on health of a variety of chemical substances. In part, this has come about because of some additional scientific knowledge and investigative tools. In part, it has arisen simply by virtue of some increased concern about the safety of environmental chemicals. For example, the realization that environmental agents may be major contributors to the incidence of cancers in the epidemiological sense is a fairly recent observation. In brief, then, we have set our sights higher in terms of the questions we would like the scientific community to ask about pesticides. As I reminded you at the outset, the total amount of background toxicology performed on 2,4,5-T had been limited to studies of acute toxicity— performed essentially by industry as directed by the Federal Government. No one had seriously suggested that the hazards of birth defects, genetic change, or cancer be tested for in the case, of 2,4,5-T, nor were there tools to screen for these diseases. The Bionetics study represented a step up in degree of sophistication of research. Certainly this evolutionary process is a highly commendable situation and is one which is to be encouraged. There do exist some dilemmas, however. The major dilemma accrues from the fact that there is no real end point to this questioning process. The more research that is performed,1 the more new questions will be raised about the chemical under investigation . That is, it is quite obvious that decisions virtually always will have to be made on admittedly Incomplete information. Perhaps the goal we should seek is a sufficiently flexible system to allow us to change our minds (when confronted with new information), coupled with an explicit acknowledgement of the perpetually interim state of our scientific knowledge. Again, the Bionetics study is illustrative. The 455 Bionetics' results were new and unexpected findings—albeit tABlve findings. It is the nature t>f science that experimental results are alwayWrobject to further confirmation and refinement. The discovery of teratogenesis in experimental animals required confirmation and further investigation to make that finding meaningful. Fortunately, the experiments to do this were begun almost immediately, as you know, and the results have just now become available. What these results have done is to sustain our earlier concern about this herbicide. At the same time, scientific logic would dictate that we should continue to apply more research effort to better understand these findings. . Then too, the more sophisticated the scientific investigations become, theV more expensive they are. The cost of the Bionetics study was approximately \. two and one-half million dollars. Remember, this was only a screening study.r__J Much more extensive studies would surely be desirable. A related question that is raised concerns the distribution of these costs. As I have noted, in the case of pesticides, the tradition has been for the Government .to impose on industry the obligation of proving that a material is safe and of performing the toxicology necessary for that proof. As the cost and the time required for this background research rises, the manufacturers may be less and less inclined to pursue the development of new products of limited or uncertain marketability. Since we depend on the manufacturing industry for this development, we may be discouraging innovative and improved products, i Hence, I submit that additional public investment may have to be made in the \ future in background research relating to health and other effects of environ- 1 mental agents—including pesticides. The President's Science Advisory Commit-' tee is studying these issues presently. Let me touch now on the subject of the translation of research findings into policy decisions and regulations. I have made the point that the heart of the Federal Government's control over pesticides resides in the process of registration with the Department of Agriculture. This registration is based, in part, on toxicological information supplied by the manufacturer. There has been relatively little thought given to the subject of how to incorporate new, unexpected information which is collected outside the registration process into the regulatory process. This was clearly demonstrated after the Bionetics study. Finally, let me raise the question of the latitude available for regulation of pesticides. Under the existing Federal Insecticide, Fungicide 'niid Rodentlcide Act (which law regulates pesticides), the burden of proof of safety resides with the manufacturer. In the case of an existing registration, the options for action available to the Government, however, are relatively few.- These are cancellation or suspension of the registration. Both of these are relatively drastic actions and are not supposed to be entered into capriciously. If a registration is cancelled (which was the uigge«*ion made for 2,4,ti-T), the decision nitty Lie appealed by the manufacture r ami it then befalls the Government to prove that a hazard exists, rather than the industry to establish its safety. In short, there does not exist a mechanism whereby the Government may exercise prudent and unequivocally effective restraint temporarily on the receipt of new, unexpected information, and while awaiting more definitive results. There are now under discussion a series of proposed amendments to the Federal Insecticide, Fungicide, and Rodenticide Act. These matters are seriously being considered in these discussions before your Committee. 457 456 Watershed Studies with 2,4-D, 2,4,5-1, and Picloram' Description of Watersheds The two experimental watersheds are located in the southern Appalachian near Waynesvi1le, North Carolina. Watershed 1 contains 4.64 acres, and watershed 2 contains 3.66 acres. The slopes of both watersheds averaged about 35 to 40 percent. The predominant soil is Halewood clay loam. The watersheds are delineated and enclosed so that no surface or subsurface flow'can enter, Each is equipped with a Weir i n s t a l l e d to bedrock, and total flow from the watersheds Is measured. Three 0.05-acre plots with catchment devices for surface runoff determination are superimposed on watershed 2. The vegetative cover was a mixed grass sward containing discontinuous infestations of herbaceous weeds and small woody plants. Experimental Procedure The map of watershed 1 (Figure 1) shows the nine 0.05-acre plots sprayed In 1967 and the three large plots (1.16 acres) sprayed in 1968 and 1969. There are three replications of three treatments in I9&7. Large plots A, C, and D were sprayed w i t h the same herbicides in 1968 and 1969. The a p p l i c a t i o n rate was 2 Ib/A. When large plots were used, there was one replication per watershed. The map of watershed 2 (Figure 2) shows the small plots sprayed In 1967 and 1968. The plots ware sprayed as shown in 1967. In 1968 herbicide treatments ware rotated to so that each plot received a different herbicide. The a p p l i c a t i o n rate was 2 Ib/A in 196? and 1968. In I9&9 the large plots were sprayed at a rate of 4 Ib/A. The treatments were adjusted so that the herbicide assigned to each surface runoff plot had not been applied to that plot in 1967 or 1968. There-were three replications of treatments when small plots were used. Treatments on large plots were unrepllcated w i t h i n a watershed. The chemical 'and common names of the herbicides were 3,6-dichloro-o-anisic a c i d [dicamba], 2,4-dlchlorophenoxyacetic acid [2,4-0], 2,4,5-trIchlorophenoxyacetlc acid [2,4,5-T], and 4-amino-3,5,6-trichloropicolinic acid [picloram]. The herbicides were applied in September, 1967.and in August, 1968 and 1969. All applications were made with a Knap-sac sprayer. The herbicldal formulations were as follows: dicamba (dimethylamine salt) 2,4-D (alkanolamine salts) picloram (potassium salt) 2,4,5-T (propylene glycol butyl ether ester in 1968) 2,4,5-T (trlethylamine salt In 1969). • • , , A contribution of the North Carolina State University Agricultural Experiment Station. This research was supported by the U, S. Department of Agriculture under Contract No. 12-14-100-8938(3*0. For several months after spraying, grab samples of water were collected at the flumes during storms, and runoff samples were removed from the surface-runoff tanks at the end of each rain storm. Water and soil samples were shipped to R a l e i g h for analysis. Soil samples were frozen before shipment. Water samples were shipped as soon as possible after collection and were stored at 4°C on arrival in the laboratory. Usually, analysis of water samples began w i t h i n 3 to 4 days after collection. An electron-capture gas chromatographtc method was developed for simultaneously measuring residues of the four herbicides. Low l i m i t s of detection of 2,4-D in water was 0.002 to 0.003 ppm; for picloram, 2,4,5-T, and dicamba the l i m i t was 0.0005 to 0.001 ppm. Results Water samples collected from flumes at the base of each watershed during end after rain storms in 1967 contained 2,4-D, but concentrations of picloram and dicamba were below the l i m i t s of detection. The highest concentration of 2,4-D (0.028 ppm) occurred shortly after peak runoff of the first storm after application. The level decreased with each subsequent storm and was below the l i m i t of detection in samples taken between September 27, 1967 and June 17, 1968 when sampling was discontinued u n t i l the 1968 application. Although one-fourth of watershed 1 was sprayed with each of three herbicides In 1968, neither 2,4,5-T nor picloram was detected in flume water, and only low concentrations of apparent 2,4-D (0.003 to 0.005 ppm) occurred sporadically (Table 1). A small interference peak with a retention time in the gas chromatograph equal to that of 2,4-D raises some doubt about the authenticity of 2,4-D values In the 0.002 to 0,004 ppm range (Tables 1 and 2). Concentrations of the herbicides in flume water samples collected In 1969 from watershed U.were below the detection l i m i t i n . a l l cases (Table 3). After the 1969 applications, 2,4,5-T was detected in water samples taken at the base of watershed 2 during the first and second storms (Table 4). The highest concentration was 0,048 ppm in a sample collected while runoff was increasing during the second storm. The concentration was less In other samples and decreased to less than 0.001 ppm when flow returned to normal. Low concentrations of picloram were detected in flume samples during the second storm also. The maximum concentration was 0.003 ppm in a sample collected while flow rate was decreasing. Picloram was detected at 0.002 ppm In the first base-flow sample taken after the storm, but levels were less than 0.001 ppm in all samples thereafter. Residues of 2,4-D, 2,4,5-T, and picloram in soil at several times after application are shown in tables 5, 6, and 7, respectively. The 2;4-D disappeared rapidly from soil. Although picloram persisted for several months, none was detected 1 year after application. A very small amount of 2,4,5-T was present In t;he 0 to 6-inch soil depth at 3 and 7 months after application, but none was found 12 months after. 458 Table 459 Concentrations (ppm) of 2,4-D, 2,4, 5-T, and picloram in water from the flume of watershed 1 over a 4-month period after a p p l i c a t i o n of 2 Ib/A of each herbicide to 25% of the watershed area on August 21, 1968. Flow condi t ion 2,4-D (ppm) 2,4,5-T Peak Base <0.002 <0.002 Peak 1 Table 2. I Flow condi t ion 2,4-D (ppm) 2,4,5-T (ppm) Picloram (ppm) Half-up Peak Ha If -down <0.002 0.002 <0.002 <0.0005 <0.0005 <0.0005 <0.0005 <0.0005 <0.0005 8-31-68 Peak 1 <0.002 <0.0005 <0.0005 9- 1-68 Ha If -down Peak 2 Peak 3 Base <0.002 <0.002 <0.002 <0.002 <0.0005 <0.0005 <0.0005 <0.0005 <0.0005 <0.0005 <0.0005 <0.0005 9- 6-68 Base <0.002 <0.0005 <0.0005 10- 3-68 Ha If -down Base <0.002 <0.002 <0.0005 <0.0005 <0.0005 <0.0005 10- 6-68 Ha If -down Base 0.003 <0.002 <0.0005 <0.0005 <0.0005 <0.0005 <0.0005 10-16-68 Base <0.002 .—_—_____ Soi 1 depth (i nches) 0-3 _ , : Months after a p p l i c a t i o n 0 Months after application Soil depth (inches) 0 1.5 3.0 0-3 1.14 0.15 3-6 3.0 7.0 12.0 0.03 0.04 <0.01 0.01 <0.01 <0.01 <0.01 <0.02 <0.02 ' 0.03 0.02 3-6 <0.02 <0.02 6-12 0.04 <0.02 6-12 <0.04 <0.04 12-18 0.08 <0.02 - 12-18 <0.04 18-24 0.02 <0.02 - <0.04 18-24 ' <0.04 0.0k 0.30 0.03 0.03 0.06 Total 0.79 1.5 Residues of 2,4,5-T (lb/A) in soil at 0 to 12 months after application of 2 lb/A August 20, 1968, to Watershed 2. 0.79 . Total 1.14 0.04 <0.04 • Table 7. Residues of picloram (lb/A) in s o i l 0 to 12 months a f t e r application of 2, lb/A August 20, 1968 to Watershed 2. Months after application Soi 1 1 depth ( i nches) 0 1.5 3.0 0-3 1.27 '0.52 3-6 7.0 12.0 0.37 ' 0.08 <0.02 0.11 0.08 0.06 <0.02 6-12 0.18 0.04 0.06- <0,04 12-18 ' 0.10 0.06 - 18-24 0.04 0.06 - 0.95 0.61 Total- 1.27 ' 0.20 <0.08 WATERSHED MOUNTAIN I RESEARCH STATION WAYNESV1LLE, N.C. A PICLORAM B DICAMBA C 2,4-D D 2,4,5-T Fiqure 1. a Map of watershe-' I ihowinc t L l e r.ine O.C>-c-t_re p l o t s (sr-.all p l o t s A, B, and C) sprayed in 1967 and ire I.l6-acre plots (A, C, and 0) sprayed in 1553 and 1563. FLUME WATERSHED MOUNTAIN RESEARCH ' 2 STATION WAYNESVILLE, N.C. A PICLORAM B DICAMBA C 2,4-D D 2,4,5-T FLUME ! ~ 7 a r i IJ6^ Figure 2. K3P of .jarer'-ed - =fi -jnj :'••-- ' - a c r e -'-J'.s 466 467 •e™ror HART. May I thank all who assisted in the development of SeHlor this very informative hearing. We stand adjourned. (Whereupon, at 5:15 p.m., the subcommittee was adjourned.) (The following material was subsequently received for the record:) spoiled areas which remain in our industrialized country to l^freserved as natural regions. I was dismayed to learn recently that a 20-foot wide strip through the middle of one of our most beautiful national parks has been defoliated with another herbicide, picloram. Mr. Chairman, it is incredible to me that Glacier-Waterton International Peace Park should be subjected to defoliants for the creation of a "North American DM55" through its center. It is an unpleasant irony that a park which was established to commemorate the peaceful relationship between two nations and •to preserve a unique glacial wilderness must be divided in two by a senseless and ugly defoliated strip. Moreover, even less is known about the possible teratogenic and carcinogenic effects of this chemical picloram than about 2,4,5-T, the agent under immediate suspicion. Our lack of a ""national policy in the area of herbicides and pesticides reaches its ultimate absurdity in this defoliated strip in Glacier Park. I urge the Subcommittee to consider the far-reaching measures which are necessary to control all aspects of the use of pesticides. Only a comprehensive approach to the problem will enable us to avoid mistakes such as DDT and 2,4,5,-t. STATEMENT OF HON. LEE METCALF, U.S. SENATOR FBOM MONTANA Mr. Chairman, I appreciate the opportunity to outline briefly a few of the special problems which arise from the use of herbicides such as 2,4,5,-T in Montana. I will not go into the potential health dangers involved with the widespread use of 2,4,5,-T and other insufficiently tested herbicides. Others, far better qualified than I to assess these dangers, have already presented their professional opinions to the Subcommittee. What I would like to bring to the Subcommittee's attention, however, is the particular impact the use of herbicides has upon n vast agricultural State such as Montana. Montana, as you know, is in the unique situation of lying at the head of three major international drainages which carry waters into the Gulf of Mexico and the Pacific and Arctic Oceans. The river systems whose headwaters lie in Montana, the Columbia, the Missouri-Mississippi, and the Saskatchewan, pass through over half of the land area of our nation and part of that of Canada. Into these drainages enormous quantities of herbicides and pesticides are sprayed each year. Forests, grazing lands, grain fields, power line rights of way, and highway shoulders are sprayed with a wide variety of chemical poisons. Herbicides are used by Federal agencies, State agencies, private companies, farmers, and home gardeners. The State undergoes a veritable deluge every year. I hardy need to point out the potentially dangerous effects of such chemicals, if the.y are found to persist and collect in water supplies and in the food chain. Kcologists have pointed out that the little understood processes of accumulation, biological magnification, and synergism can bring about completely unexpected results from commonplace chemicals. One would assume because of the pervasive use of 2,4,5,-T and other herbicides and because of the harmful side effects which have already been discovered to result from some group of these cheimicals, that truly exhaustive tests would be required to be run on any new candidate for herbicidal use. Unfortunately, this is not the case. To our astonishment we have been told in effect that the burden of proof is on the public to prove that a proposed substance is dangerous rather than on the supplier to prove that it is not. The result of this convoluted notion of regulation is that enormous amounts of potentially dangerous herbicides are spread about our land, they eventually find their way into water systems and merge with others as they travel their way to the oceans. Once the Department of Agriculture places its seal of approval on a chemical, the substance can be sprayed with impunity. Whatever detrimental effects may result are thereafter extremely hard to ascertain. The unfortunate results may in fact only become visible years from the time they are used. The Montana State Legislature Is now considering a new Pesticide Commission which would provide much better regulatory machinery at the State level. However, due to the problem of human and financial resources, such State commissions will always have to take their cue from the Federal regulatory bodies. And as it now stands, the Federal regulating process is a very confused and weak one. What is vitally needed, Mr. Chairman, is a clearly delineated Federal regulatory policy in the field of pesticides, herbicides, and defoliants which would: 1. Place the burden of proof of the safety of a product upon the manufacturer ; 2. Provide for thorough, independent testing in government laboratories; 3. Place the sole responsibility for the approval of the health dangers of a product with the Secretary of the Department of Health, Education and Welfare; 4. Substantially strengthen the enforcement machinery in the agencies which control pesticides; and fi. Create a national policy for the use of pesticides by Federal agencies. Concerning the final point, I think it is extremely important that the use of these chemicals in national parks be critically examined. Our national parks and primitive areas were set up specifically in order to sector off the few small, un- OFFICE OF THE SECRETARY OF DEFENSE, Washington, D.O., April 21,1910. Hon. PHILIP A. HART, Chairman, Subcommittee on Energy, Natural Resources, and the .Environment, Committee on Commerce, U.S. Senate, Washington, D.C. DEAR MB. CHAIRMAN : The following information is provided for the record in response to a staff request from your Subcommittee : Deputy Secretary of Defense David Packard on April 15 ordered the immediate suspension oC the use of 2,4,5-T within the Defense establishment pending a more thorough evaluation of the system. Sincerely, J. F. LAWRENCE, Brigadier General, USAIC, Deputy Assistant to the Secretary for Legislative Affairs. DEPARTMENT OF AGRICULTURE, OFFICE OF THE SECRETARY, Washington, D.C., April 31, Mr. LEONARD BICKWIT, Staff Co/tnxcl, Subcommittee on Energy, Natural Resources, and the Environment, U.S. Senate, Washington, D.C. DEAR MR. BICKWIT : The following comments on the April 15 joint U.S. Department of Agriculture, U.S. Department of Health, Education, and Welfare, and U.S. Department of the Interior release "Home Uses of 2,4,5-T Suspended" are offered for the record of the Hearings of the Committee. The release is not clear on the cancellation of registered uses of 2,4,5-T ou food crops. The action to be taken is the cancellation of all registered uses of 2,4,5-T on apples, blueberries, barley, corn, oats, rice, rye, and sugarcane. The term "cancellation" as here used means that each registrant will be notified that his registered products subject to this action shall cease to move in interstate commerce 30 days after notification of cancellation. During this 30-day period, the registrant may correct the labels on his cancelled products to delete the cancelled uses. The registrant may appeal the cancellation action and ask for an advisory committee or public hearing. In such case, the registrant's cancelled products may move in interstate commerce until action on the appeal is completed. The term "suspension" as used in the release with reference to action to suspend all registered uses of liquid formulations around the home and all registered uses of 2,4,5-T on lakes, ponds, and ditch banks, means that such suspended products shall cease to move in interstate commerce upon receipt of notice of the order of suspension by the registrant. The registrant may appeal the suspension action but in such case the suspended products cannot move in interstate commerce while action on the appeal is in process. Sincerely, T. 0. BYERLY, Assistant Director, Science and Education. 468 CENTER FOB STUDY OP RESPONSIVE LAW Washington, D.G., April 80, Senator PHILIP HABT, U.S. Satiate, Washington, D.G. DEAB SENATOR HAM : May we commend yon and the Senate Commerce Committee for investigating so thoroughly the hazards presented by weed-killer 2,4,5-T. It, was encouraging to see USDA relent and prohibit certain uses of the chemical. However, after this brief commendation, we would like to make clear' our feeling that the bans on 2,4,5-T were inadequate, and the press release announcing the ban misleading to the public. According to the official press release, "In exercising its responsibility to safeguard public health and safety, the regulatory agencies of the Federal Government will move immediately to minimize human exposure to 2,4,5-T and Us impurities. The measures being taken are designed to provide maximum protection to -women in the child-bearing years by eliminating formulation, of 2,4,5-T use in household, aquatic and recreational areas (emphases added)." We contend that the measure taken reflect an utter disregard on the part of the USDA for public health and safety and represent minimal rather than maximal protection. 1. The use of Silvex (also called 2,4,5-TP ; 2-(2,4,5-trichlorophenoxy) propionic acid) has not been suspended. Silvex is as closely related to 2,4,5-T as pancake are to waffles. Because of this similarity one can predict with confidence that Silvex will prove to be about as teratogenic as 2,4,5-T. Furthermore, the series of chemical reactions by which Silvex and 2,4,5-T are made are almost identical. These reactions lead unavoidably to the formation of the dreaded tetra-dioxin contaminant. If any serious attempt were being made to "minimize human exposure" to hazardous weed-killers and the impurities contained therein, the home use of Silvex would certainly be banned. Silvex, incidently, is a more common ingredient in garden products than is 2,4,5-T. 2. "Minimizing human exposure to impurities in 2,4,5-T and its impurities" would demand an immediate, total recall of 2,4,5-T and related compounds from retail stores and homes (with the manufacturers reimbursing stores and consumers). Although the USDA has called for recall in a private communication to manufacturers, no recall will be complete unless the announcement is published in the Federal Register and a list of products containing the dangerous substances is published in newspapers so that consumers may return what they had previously purchased. 3. "Minimizing human exposure" to impurities in 2,4,0-T would necessitate suspending the use of 2,4-D (the most widely used weedkiller), 2,4-DP (found in Scott's Turf Builder Plus 4), and pentachlorophenol (present in Ortho Triox Liquid Vegetation Killer). The processes by which all three of these compounds are made lead unavoidably to formation of dioxins. 4. "Minimizing exposure to 2,4,5-T and .its impurities" would necessitate suspending' all uses and formulations of all chlorophenoxy and chlorophenol weedkillers : (a) The possible persistence of dioxins represents a very real danger, as pointed out by Drs. Verrett and Epstein, Mr. Bickwit and ourselves at the Commerce Committee hearings. (6) Accidental exposure to the weed-killers or their contaminants due to direct spraying, drift or residues on food is possible, indeed inevitable, when these weed-killers are used on crops, ranges, rights-of-way, neighbors' yards, etc, In testimony before the House Subcommittee on National Security and Scientific Developments on December 2, 1069, Dr. Arthur Galston of Yale University said, "I suggest that its (2,4,5-T) teratogenicity is such that even its use in such apparently innocuous domestic manners as clearing brush near powerlines is undesirable. Such chemicals could find their ways into water supplies and could be Ingested in teratogenic doses." If the regulatory agencies truly wish to exercise their responsibility to safe-., guard public health and safety, particularly that, of women of childbearlng age, by minimizing human exposure to 2,4,5-T and it.H impurities they will certainly have to go far beyond the recently announced minimal actions, If possible we would like this letter made part of the hearing record. Thank you once again for your interest in this important matter. Sincerely yours HAHBISON WEIXFOED. JAMES TUBNEB. 469 STATEMENT OF R. L, CUSHINO ON BEHALF OF THE HAWAIIAN S^^ PLANTERS' ASSOCIATION, HONOLULU, HAWAII ^J The Hawaiian Sugar Planters' Association, an association representing all of Hawaii's 24 sugar-producing companies, wishes to present its views