this experiment. The relationship of the lingular deformations to the ad­
ministration of 2,4-D is dubious.
In a complementary experiment, a pregnant sow was fed 2,4-1) at a
dietary level of 500 p.p.111. during the entire pregnancy, and further
oil. During a protracted parturition, fifteen live and one stillborn pig­
let were delivered. The newborn piglets were underdeveloped and list­
less and showed a high mortality rale, ten out of fifteen dying within
24 hours. No malformations were observed. Autopsy findings in the dead
piglets were those of a generalized anaemia. On continued feeding of 2,4-D
at the same level to the survivors for another (5—7 months, growth depres­
sion was evident. On autopsy, no chracterislic gross changes were observed,
apart from multiple disc degenerations in the cervical and lumbar spine
in two animals. Heamatological, biochemical, and hislopalhological findings
were similar to those observed in the preceding experiment, thus confirming
the toxic action of the phenoxy acid. The dam developed weakness of the
hind quarters and was slaughtered at 0 weeks post partum because of a
complete inability to rise. Autopsy revealed excessive degenerative changes
in various regions of the spine, which provided a hislopalhological explana­
tion to the clinical signs. The toxicological significance of these changes,
however, as well as of the spinal degeneration in the young pigs, remains
obscure.

¿SSTZT

AlOo

Rats. Ten pregnant albino rats, initially weighing about 350 g, were
equally divided between an experimental and a control group. The
experimental group received 2,4-D in the drinking water, at a level
of 1000 p.p.m., during the gestation period and the following 10 months.
The estimated daily dose of 2,4-D was 50— 100 mg/kg body weight. No unequi­
vocal clinical or morphological changes were observed during this period.
On continued administration of 2,4-D to the second generation rats for up
to 2 years, growth depression and a reduced intake of feed and water and
also a slightly increased mortality, as compared with controls, were noticed.
Clinical-chemical and hislopalhological examination did not reveal any de­
finite abnormalities. This fairly high tolerance of rats to 2,4-D may be related
to the high plasma disappearance rate of the phenoxy acid in this species
(sec Distribution, aliove), which most likely is the result of an effective
renal excretion. In pigs a lower plasma disappearance rate was accompanied
by a lower tolerance to 2,4-1).
Chickens. Six while Leghorn chickens of both sexes, originally weighing
about 1 kg. were continuously Ted 2,4-1) at a dietary level of 500 p.p.m.
Except for one bird dying after 5 months or renal gout, no distinct clinical
33

0001401

5542

. «

symptoms were seen. The survivors were sacrificed ;il intervals helween 011c
and 18 mouths. The autopsy findings were mostly of a noiispeeirie character
such as waxy muscular degeneration, provcnlriculnr ulceration, peribronchilis, and generalized anaemia.
In another experiment, also extending over 18 months, sixty day-old

DOW
121538
F iy .
H ypertrophic k id n e y . Ik-fll of a .Viuonlhs oh! chirk jjiven 'J.-t-l) aiuinv 111)011 |>. p. in.
in tile drinking wider} since 3 days <rhl, as eiiiu|iari*d w ith norm al kill n rv s irijthll of
co n tro l chirk of Ilit- same aye.
F iy . It. Section u f h y p ertro p h ic kidney. T idm lar dilatation and hypertro p h y . I'A.S. .'1(10 X.
F iy . 7. Section o f iiirrinal cluck kidney. Norm al liilmles. I'A.S. .100 ■

:«

V

0001435

broiler chicks of bolli sexes were evenly divided between one experimental
group, receiving 2,4-1) ¡inline (1000 p.p.m.) in the drinking water. and one
conlml group. Apart from a reduced egg production in laying liens of the
experimental group, no specific clinical signs were observed. Klcvcn treated
and twelve control birds died or were killed because of disease during the

P-OW
121539

Fi<j. \S'. SertiiMi of h y p e rtro p h ic kidney. G lom erulus w ith exlreiiudy diluted capsular space.
PAS, aim .<.
Fiij. 9. Section o f norim il chick kiilncv. N orm al ¡{louirrulus. PAS, atXI

03

0001406
5544

I

. 4

r

experiment. The causes of death in both •'roups won- 111:1 inly coccidiosis,
leukosis, lymphomatosis, enlcrilis or unknown origin, or inanition of un­
known origin. Survivors went* sacrificed and aulopsied at intervals between
*2 and IS nioiiullis. Tlic 2,4-D-lrealed birds, dead as well as killed, displayed
a .spectacular kidney enlargement due to epithelial proliferation, a finding
which seems not to have been reported previously (Figs. 3—D).
The seemingly exclusive development in chickens of this lesion may
possibly be related to the fact, that of the species examined this is the only
one having a renal portal system which enables inlestinally absorbed substan­
ces to pass directly to the kidney. Interestingly, the renal hypertrophy deve­
loped only in those chicks that had been continuously exposed since a few
days old to the phenoxy acid. Apparently, this finding provides a parallel! to
the observation of Carr (ItkVi), that only very young chicks are susceptible to
leukaemia virus-induced renal carcinoma. In both instances the reactivity
may lie associated with the abundance of residual embryonic tissue in the
young birds.

DOW 121540

(

3fi

0001407 '

. 5545

Summary

DOW 121541

The investigations summarized lien- aimed at developing methods for
isolating, delecting and determining residues of chlorinated phenoxyaliphatic acids (phenoxy herbicides, "hormone weed killers”) in biological mate­
rials, and at studying the distribution and toxicity of these materials in
'experimental animals.
After examining the conditions for isolation of phenoxy acids from diffe­
rent types of biological materials, an analytical method was adopted in­
volving solvent extraction of the sample (the technique varying with the
type of material), purification of the extract by partitioning between im­
miscible phases, and separation and identification of the acids by thin-layer
chromatography. The eluted acids are determined photometrically.
The described method recovers phenoxyacetic acids from various biologi­
cal materials to the extent of 70—90 per cent, with a standard deviation
of 3—5 per cent (at the 4-p. p. m. level). The sensitivity is about 0.1 ,»g of
phenoxy acid.
Applying this method, the distribution of phenoxy acids was studied in
calves, pigs, rats, and chickens. After the administration of soluble salts of
2,4-D and of 2,4,5-T as single oral doses of 50—100 mg/kg body weight,
the plasma concentrations rose to a peak within 2—7 hours and then declined
to insignificant levels over a few days. The plasma half-lives ranged bet­
ween 3 and 12 hours, depending on species. Oral administration of a 2.4-D
ester resulted in considerably lower plasma levels of 2,4-D being attained
(no ester was delectable in plasma or tissues). 2,4-D apparently distributed it­
self rather evenly over the body water, the highest levels being attained in the
excretory organs (liver and kidneys). High levels were also found in ovaries,
adrenals, lungs and myocardium. The levels found in' brain were relatively
low, indicating a slow passage of the blood—brain barrier. Placental transfer
in swine apparently was less restricted. The disappearance rate from tissues
was fairly high, the average tissue half-lives for 2,4-D ranging between
5 and 30 hours, depending on the species. The highest disappearance' rale
was consistently seen in rats and the lowest in pigs. Elimination evidently
took place mainly be renal and. In a smaller extent, by biliary excretion of
the unchanged phenoxy acid.
Repealed administration of suhloxie doses did not lead to excessive accu­
mulation of phenoxy acid in any of the tissues examined. Except for a

00014 OP

DOW 121542

moderate increase in (issue levels relative lo plasma, (he distribution pattern
did not change on repealed intake. In pigs and chickens, the capacity for
excreting phenoxy acids was seen to rise during prolonged exposure.
In acute toxicity studies, reversible toxic effects from 2,4-D amine were
observed after single oral doses of 200 mg/kg in calves and of 100 mg/kg in
pigs, respectively, doses above 300 mg/kg causing severe poisoning in pigs.
Hals and chickens seemingly tolerated single doses of 100 and 300 mg/kg,
respectively. On repeated oral administration, daily doses of 50 mg/kg could
he toxic to pigs, whereas chickens tolerated 300 mg/kg/day for 2 weeks
without outward effects.
The results suggesl that clinical signs of acute or subacute poisoning are
likely to appear, at least in pigs, if the plasma concentration of phenoxy
acid exceeds a threshold value of about 200—300 /(g/ml.
In long-term studies, signs of toxicity from 2,4-D appeared in pigs at a
dietary level of 500 p. p. m. (corresponding to a daily intake of 25—50 mg/kg)
and in chickens at 1000 p. p. m. given in the drinking water (equivalent to
50—100 mg/kg/day). In rats administration of 2,4-D in the drinking water
at 1000 p. p. m. (50—100 mg/kg/day) over two generations caused a reduced
weight gain and an increased mortality in the second generation. No une­
quivocal morphological changes were observed.
In the pig experiments, the main symptoms were growth depression, loco­
motor}' disturbances, anaemia and albuminuria. On autopsy, degenerative
changes were seen in the liver and kidneys. On feeding 2,4-D at the level
of 500 p. p. m. to a pregnant sow during the gestation period and for 6
further weeks, no characteristic signs were observed. Autopsy did not reveal
any changes attributable lo the intake of 2.4-D. The newborn piglets, how­
ever, were underdeveloped and exhibited a high mortality rate (66 per cent).
Continued administration of 2,4-D lo the survivors resulted in growth depres­
sion, persistent anaemia and degenerative changes in the liver and kidneys.
In the chicken experiments, a spectacular renal hypertrophy was the main
finding, together with a reduced egg production. The renal lesion developed
only in those birds that had been exposed lo phenoxy acid since a few days
old.
The chronic oral toxicity of 2,4-D lo the species examined appears to be
moderate. Apart from the notable nephrotoxicity demonstrated in chicks,
the observed long-term effects seem lo be non-spceific. Attention should be
paid lo the high mortality rale in the newborn piglets and the reduced egg
production in the chickens, however, which findings, together with the de­
monstrated aecumulation of phenoxy acid in foetal tissues and in ovaries,
might be indicative of a phenoxy acid-induced interference with reproduction.
38

0001409

References
Aamiseii/i, .1.: The occurrence of 2,4-D in weds from cultivated plant* sprayed with
chlorinated plicnoxyacclic acids. K. Lantbr llögsk. Anntr ltlfil, 27, 44a— 431.
Anderssun, A. £ B. Berzins: N igra hcbiciders loxisku verkan p i risk och krfiftor. Svensk
l'isk —Tidskr. 1901. 70, 111— 110.
Antlrcnc, IV. .1. £ S . E. Cooil Studies on 3-indulcacctic acid metabolism. IV. Conjugation
with aspartic acid and ammonia as processes in the metabolism of carltoxylic acids.
P lant Physiol. 1957, J2, 500—572.
Audus, L .J .: The Physiology and Biochemistry of Herbicides. Academic Press, London A
New York 1904.
Bauer, A'.; Studien über Nebenwirkungen run Pflanzenschutzmitteln auf Fische und
Fischuährlicrc. Milt. biol. Bund Anst. Ld u. Forstw., Heft 105, Berlin-Dahlem 1901.
Berkley, .1/. C. & K .R . .1layer: Neuropathy following exposure to a dimethylaminc salt of
2,4-D. Archs intern. Med. 1903, H i, 351—352.
Beuenue, A., G. Zweig £ Nancy L. Nash: Residue determination of 2,4-dichloroplicnoxy«celic acid in dry crops and walnuts. J. Ass. off. agric. Client. 1902, 45, 990—993.
Brodie, B .B ., G.J. Cosmides £ D. P. Ball: Toxicology und the biomedical sciences. Science
1905. U 8, 1547—1554.
Brown, C.P. £ A. R. Mathicson: Dimerization of the chloroacclic a d d s in solution. J.
phys. Chera. 1954, 58, 1057— 1059.

121543

30

DOW

Carr, J. G.: Renal adenocarcinoma induced by fowl leukaemia virus. Brit. 3. Cancer 1950,
10, 373—383.
Crafts, A. S.: The Chemistry and Mode of Action of Herbicides. Inlerscicncc, New York £
London 1901.
Curry, A .S .: Twenty-one uncommon eases o f poisoning. Bril. mcd. J. 1902, 1, 087—GS9.
Dalgaurd-Mikkclsen, S. £ E. Ponlsen: Toxicology of herbicides. Pharm acol. Rev. 1902.
U , 225—250.
Davis, J .T . £ J .S . Hughes: Further observations on the toxicity o f commercial herbicides
to bhicgitl sunfish. Proc. Southern Weed Couf. 1903, 1ti. 337—340.
Prunk, P. .1. £ R. ii. Grigsby: Effects of hcrbicidal sprays on nitrate accumulation in
certain weed species. Weeds 1957, 5, 200—217.
Freed, V. II.: Qualitative reaction for 2,4-dichh>ruphcmixyacc|:ic arid. Science 19-18, 107,
08—99.
Goldmacher—u. Mallinckrodt, M. £ L. I.outcnbuclt: Zwei tödliche Vergiftungen (Suicid)
mit chlorierten Phcnoxyc.vsigsüurcn (2,4-1) und MCP). Arch. lox. 1900, 21. 201—278.
Goldstein. N .P ., P .ll. Jones £ J .R . Brawn: Peripheral neuropathy after rx|>miirc to an
ester of dichloruplicnoxyacclic a d d . J. Am. mcd. Ass. 1959, 171, 13(10— 1209.
Herbich, J. £ (1. Machutu: Vergiftung mil 2.4-l)icldiirphcuoxyessigsfiure (2.4-U). Rcitr.
gericht. Med. 1903, 22, 133— 139.
Hill, E .C . £ II. Carlisle: Toxicity of 2.4-dichkir<iphciH>\yacctie arid for experimental
anim als. J. ind. Hyg. Toxiocol. 1917, 20, 85—95.

j

• t''
.4

r

(

BOW 121544

Johnson, II. It. M. A 0 . Koumides: A further case M. C. 1* A. poisoning.. lirH. nied. J. 19G5,
2, C29—630.
Kldmht, II. I).: WuchsluuiMnduUion nml Wucli'i'loffmHabolivmus im Weizenkulcoptilzylimler 111. Sloffwi-chsvlproduklc der Xaphlhyl-l-csvigs:iurc und -M-Dichlorplienoxyossigsaurc uml dcr Vrrglcich mH jcncu dcr liidol-3-o*igsfuirc und Iienzoesuurc.
Pluula 11IGI, 57, 33'J—353.
l.r Tournean. U. A X. Krmj: The use of chromolropic a d d for (lie determination of 2.4dichlorophcuoxyucelic acid. Plant Physiol. 1952, 27, 822—827.
Mitchell, L. C.: Separation and identification of chlorinated organic pesticides hv paper
chrom atography. XI. A study of IN pesticide chemicals; technical grades produced
in 1957 and reference standards. J. Ass. off. agric. Client. 1958, i t , 781—80G.
Monarca, G. & G. Di Vito: SuM'iulussicazinirc acuta da discrlianlc (acido 2— t diclorofcnossiacelicd). FoHu nied., Napoli 19G1, 44, 480— 185.
Nielsen, K., II. Kuempe <C J. Jcnscn-llolm : Fatal poisoning in man by 2.4-dichlorophenoxyacetic acid (2,4-D): determination of the agent in forensic materials. Acta phnrmac.
tox. 19G5, 22, 224—234.
Po/ihum, R. D. A U. i l . Daoies: A cose of M. C. P. A. poisoning. Bril. nied. J. 19G4, 1,
677—G78.
Home, V. K. A T. A. Hymns: Summary' of toxicological information on 2,4-D and 2,4.5-T
type herbicides and an evaluation of the hazards to livestock associated with their
use. Am. J. vet. lies. 1954, la , G22—G29.
Schanker, L. S.: Passage of drugs across body membranes. Pharm acol. Rev. 19G2, 14,
501—530.
Shillinyer, Yu, I. A L .P . Naumova: Toxicological evaluation of a herbicide—the butyl
ester of 2,4-D. Gigiena, Toksikol. i KKn. Xovykh Iuscktofungilxidov, T rudy 1-oi
Vsesoyuz. Xauch. Konf., Kiev 1957, 297—305. (Through Chem. Abstr. I960, 54, 25514).
Slade, R .E ., IV. G. T em phm an A IV. .4. Sexlon: Plant-growth substances as selective!
weed-kVHcrs. Nature (Lond.) 1945, 155, 497— 198.
Sperber, I.: Secretion of organic anions m the formation of urine and bile. Pharm acol.
Rev. 1959. 11, 109— 134.
Stahler, L. i l . A E .J . Whitehead: The effect of 2,4-D on polaswium nitrate levels in leaves
o f sugar beets. Science 1950, 112, 749— 751.
Smanson, C. It. A IV, C. Shorn: The effect of 2,4-dichlorophenoxyacelic acid on the hydrocyauic acid and m im ic content of sudan grass. Agron. J. 1954, 4<S, 418— 121.
Teresi, J .D . A J . M. Luck: The combination of organic anions with scrum album in. IV.
Quantitative studies by equilibrium dialysis. J. biol. Chem. 1948. 174, 653—GG1.
Todd, R .L .: A case of 2,4-D intoxication. J. Iowa med. Soc. 19G2, 52, GG3—GG4.

40

0001411
5543

loi

5550

A L B E R T M. K L I G M A N . M. O.. P m. O.
M . *;P|TA t. O r

3 6 '«

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ANO

S p Q U r.c

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?rN N jri,V A N » A

ST^CCTS

Phii.A3£;Ph'A.pa *9iCa

J a n u a r y 23, 1968

CM
w

O

CM

H
O

o

M r . V. K . Rowe
B io c h em ic a l R e s e a r c h L a b o ra to ry
1803 B u ild in g
T h e Dow C h e m i c a l C o m p a n y
M id la n d , M ic h ig a n 48640

£o
a

D e a r V. K. :
T h is n o te is a f o ll o w - u p to m y r e p o r t of J u ly 8, 1966. In t h a t s tu d y ,
you w ill r e c a l l th a t 6 g r o u p s of h e a l t h y a d u lt s u b je c ts r e c e i v e d s m a l l a n d
i n c r e a s i n g d o s e s of t e t r a c h l o r o d i b e n z o - p - d i o x i n . We fo llo w e d a s p e c i f i c
p r o to c o l la i d down b y you. U n f o r t u n a te ly , not a s in g le s u b j e c t d e v e lo p e d
a c n e n o r w a s t h e r e a n y e v id e n c e of t o x ic ity .
T h is e n c o u r a g e d m e to p r o c e e d m o r e v ig o r o u s ly . We th e n a s s e m b l e d
a new p a n e l of 10 s u b j e c t s a n d a p p lie d 0 .0 5 m l of a 1% s o lu tio n in a lc o h o l
c h l o r o f o r m to a one in c h s q u a r e on th e b a c k . T h e s e a p p l i c a t i o n s w e r e m a d e
e v e r y o t h e r d a y f o r one m o n th . The t r e a t e d s i t e s w e r e c o v e r e d w ith a n o n ­
o c c l u s i v e g a u z e s q u a r e . E a c h w e e k f o r 6 w e e k s , the fo llo w in g l a b o r a t o r y
t e s t s w e r e d o n e : U r i n a l y s i s , C B C , BUN, SGOT, A lk a lin e p h o s p h a t a s e , a n d
C re atin in e c le a ra n c e .
C l e a r l y , th is e x p o s u r e w a s i m m e n s e l y g r e a t e r th a n th e f o r m e r o n e .
8 of 10 s u b j e c t s s h o w e d a c n e f o r m l e s i o n s u s u a ll y b e g in n in g 3 to" 4 w e e k s .
T h is b e g a n a s w ith a t y p i c a l d e v e l o p m e n t of c o m e d o n e s . I n 3 i n s t a n c e s ,
the l e s i o n s p r o g r e s s e d to i n f l a m m a t o r y p u s tu le s a n d p a p u l e s . T h e s e l e s i o n s
l a s t e d f o r 4 to 7 m o n t h s , s i n c e no e f f o r t w a s m a d e to s p e e d h e a li n g by a c ­
tiv e t r e a t m e n t . B io p s i e s w e r e o b ta in e d in 5 i n s t a n c e s a t v a r i o u s s t a g e s .
T h e h i s t o l o g i c a n d c l i n i c a l m a n i f e s t a t i o n s w e r e in e v e r y w a y c o m p a r a b l e to
c l a s s i c a l c h l o r a c n e . T h e l e s i o n s w e r e in d is tin g u is h a b l e f r o m th o s e o b ta in e d
b y Dow 6X a n d H a lo w a x .
I n no i n s t a n c e w a s t h e r e l a b o r a t o r y o r c lin ic a l e v id e n c e of to x i c i t y .
T h e s u b j e c t s r e m a i n e d w e ll t h r o u g h o u t th e s tu d y .

i

§551
0000450

9ZO STOO ^ ° °

M r . V. K. Rowe
Page 2
J a n u a r y 23, 1968

T h e s e r e s u l t s i m p l e m e n t the c o n c lu s io n s f o r m e r l y d r a w n , n a m e l y : i t
is m u c h m o r e d iff ic u lt to in d u c e a c n e in the h u m a n than in the r a b b i t e a r .
T h e p r o c e s s b e g in s m o r e s lo w ly a n d the d o s e s r e q u i r e d a r e v e r y m u c h
g r e a t e r . H o w e v e r , u n lik e the r a b b i t e a r , the c o m e d o n e s o fte n b e c o m e i n ­
f l a m m a t o r y . It i s a c e r t a i n t y th a t th e r a b b i t e a r is e x c e p tio n a lly s e n s i t i v e
to a c n e ig e n ic c h e m i c a l s a n d is a n e x c e l l e n t s y s t e m fo r the d e te c tio n of s u c h
c h e m i c a l s . F i n a l l y , it m a y be s a i d th a t c h lo r a c n e c l o s e l y m i m i c s a cn e v u l ­
g a r i s . T h e only d i f f e r e n c e is the p a u c ity of a n a e r o b i c o r g a n i s m s in the c o m ­
e d o . One m a y c o n c lu d e th a t b a c t e r i a a r e n o t v e r y s i g n if ic a n t in the p a th o ­
g e n e s i s of c h l o r a c n e .
V e r y s i n c e r e l y y o u rs

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GP 01

Nò. Urtai Site
Dose D ose

1 0.5
5 0.5

B

3GOT
18

__2

INITIAL LABORATORY
G reat.
Aik
BUN 7 * 3 0
C l.
Phos

11.5 9

7573

j 49
16L¿jcVMin

3GOT

FINAL U BORATO?Y
Hb. C reat.
Aik
SUIT
WBC
Phos
C’ .

14

12.3 8

B

17
72
Cu/Min
6
1
9
7
15
9.7 13
13.7

15

0.5

1 0.5

B

0.1

5 0.5

B

16
93
8.8 12 7475 14.4 Du/Min 15

0.5

1 0.5

B

0.1

5 0.5

B

20

18

5009 15.4
67

12.3 9 7245 14.8 Cu/Mlr
10.2 12 6232 14.5
10.0 12 7173 15.0 67

Cu/Min

8.7 16 5680

9.1 13 7874 14.3 74
□u/Min

0.5

1 0.5

B

0.1

5 0.5

B

0.5

1 0.5

B

12

20

84

10.9 13 7121 15.7 Cu/Mln 14

9.4 11 5828

0.1

5 0.5

65
15.4 Cu/Min

17

10.( 12 8142 15.5

75
10.6 11 6819 15.7 Cu/Mln 18

10.2: 14 6927 13.9
—------ - r

i

14.8

14

B
GG< 1 4 3 3 Í ¡L-. _______ L

91

1r

i

11 9731 13.7| 96
Í ■*• ___ jCu/Mln;

Code

jubj. # A ge

Meg
Weight Pace D afly N o. Tbtal Cite
D ose Dose D ose
_____*

1

150

29

N

3

GP O

Forehead; Meg = M icrogram

1.0

1

1.0

F

0 .5

2

1.0

F

1 .0

1

1.0

F

0 .5

2

1.0

F

1.0

1

1.0

F

0 .5

2

1.0

F

1.0

1

1.0

F

0 .5

2

1.0

F

INITIAL LAB3RATOPY
G reat.
Alk
3GOT
7 '3 C it/.
C l.
Phos BUN

32

10.4

12

8155

69
15.5 Cu/Min

FINAL LABORATORY
Hb. C réât.
Alk
3GOT Phos 2UH WBC Kdb.
C l.

17

la 7

11

9346

15

20

10.4

13

7429

87
14.5 Ci/M in

26

10.7

10

6657

12.0

16

10.6

9

7405

12.4

24

9 .6

15

5481

I5 .O

20

12.S 14

7440

14.3

24

11.1

8

7757

12.8

27

11.9

11

7906

12.2

•
1

2

26

W

148

2

3

3

4

139

N

,

23

170

N

4

17

19

15

10.2

13.1

7726

11

7003

9

13.2

14.6

13

81
Cu/Mln

97
Cu/Mln

99
C i/M in

-----------r

555
CD

7690

9

11.1

5

28

w

156

J l.o
1
i

5
1

i

0 .5

1.0

F

— ------ r
!
l.o j
2

F

J 1

21

12.7 I 1°
1
------ /

J

1
Dii

6357

67
12.8 Cu/Min

15

"

19

H. c • 11

72
C i/ Min

74
Ci/Min

97
Ci/M in

!

5210 i 12.5
.. L

t ll.sjr ~13 j r8121

(j

J

12.
73
Jcu /M in ;
t ¥ /

)

n a s s

Code: P » Back; Meg = M icrogram

GROT

3

Code: F = Forehead; Meg = M icrogram

0 3 OU

4

Code: P = Back; M icrogram =* Meg

55fi2

GBOf

4

Code:

Meg
7/eigh t Race Daily
,
k
Doge

jubJ. # A ge

1

49

210

N

l

2

30

210

2

Sub
Die

je c t
charged

3

23

165

N

4

23

N o. Tbtal Site
Dose D ose

4 .0

1

4 .0

F

1.0

4

4 .0

F

4 .0

1

4 .0

F

INITIAL LAB3P.AT0RY
Q reat.
Alk
BUN 7 r3 C Kb*.
3GOT
C l.
Phos

18

25

12

9 .9

8 .4

11

7729

8312

FINAL L A B 03A T 03Y
Hb. C réât.
Alk
3GOT Phos 3UH WEC Refe.
C l.

83
129 Du/Min

62
15J5 Ct/M in

13

10.7

13

9978

14.0

16

11.0

12

9941

72
14.6 Cu/Mir

18

9 .9

10

9947

15.1

•

N

3

170

5

G ? Ol

Forehead; Meg - M icrogram

N

4 .0

1

4 .0

F

1.0

4

4 .0

F

4 .0

1

4 .0

F

1.0

4

4 .0

F

16

14

7 .7

6 .2

9

10

9982

9764

90
15kl Cu/M ir

76
1512 CVMin

i

12

9 .2

11

8873

14.5

14

9 .7 '

10

7781

77
14.9 Cu/Mtn

12

7 .9

11

9367

15.1

11

8.1

12

9074

l4.fe

l

5563

1

4

5

Sub ject
Dis chargee

5
1

Sub ject
D is chargee

•

__L..„
1

__1

9 J in
Ci/M

I

_______

CCI

"1-----------r
Î
m

ÏO —

_

«•

o < ^ \

k

\

i
!

Code: *" - Back; Meg = M icrogram

QBOi

5564
< i

Code: '

Forehead; Meg = M icrogram

QE 0 '

jubJ. # A ge

Meg
W eight Race D ally NO. Ibtal Site
P o s e Dose D ose

1

166

29

N

1

8 .0

F

15

11.8

il

9537

93
14.1 Du/Min

FINAL LABORATORY
*
Hb# C réât.
Alk
3G0T Phos BU1T V/BC X sb.
c i.:

16

10.3

12

7391

13.7

15

10.5

13

7446

81
13.9 CuAfin

72
Cu/Mip

16

8 .4

12

5695

15.0

»

16

8. Oj

11

5521

14.8

10

6591

15 *2

i
>

1
2

8 .0

INITIAL LABORATORY
C réât.
Alk
3GOT Phos BUN W 3C Jiïb.
C l.

22

164

N

2

1 . 0

8

8 .0

F

8 .0

1

8 .0

F

1 . 0

8

8 .0

F

12

9 .5

14

9524

16.4

t

1
3

3

4

Sub
D is

ject
charge*

Sub
Dis

ject
charge

1

38

201

W

(
i
î

I
i

i

8 .0

1

8 .0

F

13

11.1

15

8367

150

99
C iA ïln

9 .7

19

5565

72

•

;

4

fi

5

33

169

Sub ject
D is chargee

87
Cu/Min

W

1 . 0

8

8 .0

F

8 .0

l

8 .0

F

r
!*
1

11

10.2

18

9986

15.2

... J.
_ T ~ ..... p

0 0 0 4 3 4 2 i __

17

9 .5

18

6761

15.8 C i/ M in

20
68
Cu/Min i_

9 .a

13

9437

13.8

.1

!

_____

i

i
.

. 4.

k»

..
t l '\

Code: B - Back; Mcg = Micrograra

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• • • • • • • • • • « • • « • • • » • • • • • « • • • • • A * * * * * ’

/„ y ,

Bulletin
;..:i - A w r il L-ioS

• \ ' v.

¡'l- Q T ,'.' Vi

li.S. DEPARTMENT OF
HEALTH. FDÜCATION AND WELFARE
Public Health Service
Washington D.C. 20201

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to

S E A T K S . F R C M C H L O R IN A T E D PHI-lNOXYACETTC ACIDS
(2, 4-1); 2, 4 , 5 - Y; M C F A )

00

o

C i

in..: r. o i t >a ici '..T'/ ur-uti in-rbl* i ci vj -s a r c 2 , 4 - D , 2, 4, 5 - T ; and r e l a t e d c o r n . . . d a . V» n«:l i t e r o : n o t i}u-s<. a r e a c t u a l l y to x ic i s a m a -tr e r oi" c u n t r o •rsy.
- R. a *. h u i C.'- r * o u .
-.L.

'

i'I..- -*; m a y r.ot b e e b n t r o .-e rsy w h e n t h e s e . c h e m i c a l s a r e u s e d a s d i r e c t e d r , . T;• :>;.:d ?hc h ou s> ho lei o r in a g r i c u l t u r e , b u t th e M i c h i g a n f a r m e r w ho d e - Th."'1 T'';-. '

n v r l Lati‘,u !..t*rc*rt:,
iy b o th g i r l s ha«T a g e n e r a l i z e d e r y t h e m a o f ’
•- ik.ii., m i n i m a l e d e m a t o u s s w e l l i n g of. th e o r a l a n d v a g i n a l m u c o u s
:• n .! .r ;i:.e s , a lig h t s w e lli n g o f l i p s a n d e y e l i d s , i n f l a m m a t i o n o f th e r n i u t h ,
.. u-.:jr.u.’i.'i and o l i g u r i a .
^

->■
•

r e c e i p t .of a f a t a l c a s e h i s t o r y d u e to s u i c i d e in a 4 8 - , e a r - o l d C o n n e c t i c u t
;
a r e v i e w o f th e C l e a r i n g h o u s e s r e p o r t s w a s m a d e . In th e p a s t t h r e e " ' ' ’
•• i n , th e C l e a r i n g h o u s e lia s r e c e i v e d s e v e r a l r e p o r t s o f p e r i p h e r a l n e u r i t i s
-■A alt in r a s h .f l o w in g e x p o s u r e to tlie s e p r o d u c t s , in a d d i t i o n to t h o s e .
•areadv c i t e d : ^ ^ l > ? V - V - - ' . V "
;
.¿Lu w Vj'jTw.*•a
.
_ .• i - «4Tr.' if *^T*•

O t h e r syniptbn^p9Ta.t:w'<?r ^ a s s o c i a t e d •Wiiii th e ’ c h l o r i n a t e d p1ier.oxy a c e t i c .'i;E?TV5^'
• id derivntiv^i^:.ty.exe; w e a k n e s s o f c h e s t m u s c l e s a s s o c i a t e d w ith p n e u m o n i a Y.:*-: '
■
h<.u:.aiuri3|
^witii p o o r - m u s c l e to n e ; fw o b r o t h e r s w ith a t a x i a ,

•=' -.!I*tie ii and urinary freq u en cy;.irritab ility, lethargy; in c re a se or decrease;.-''-:^" '
•

pf r'*turv*V iwloKt o? th ^ v i c t i m s ..th a t 1 .id* s y m p t o m s ' h a d v o m i t e d . . T h e r e . ^ 4
r-.'j.'urt oi holiw ir.a iio iis n i u c i n h a l a t i o n o f Z, 4 , o - T .

A i Kltvli.i.-; oi i : :i D iv j ■■ion o ; D ir--, t H-- .It;;i h> r v h <
r.<
•: oi' H- .dll: rYi:r i :a --j

5568

c c c ç g o e

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'/•

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Zi
¿ , 4 - D a n d i t s d e r i v a t i v e s S e r v e a s a b a s e m a t e r i a l t r o m whirl-, s o lu b le
e s t e r s a n d s a l t s a r e p r o d u c e d . Tin: L D 5 0 o t t h e s e p r o d u c t s r a n jje i r o m
300 to 7 0 0 m i l l i g r a m s p e r k i l o g r a m o r a l l y .

►
;•*lrf*S.w>;

¿fír

■
^' . . . .
-jy
W hen th e c h l c r o p h u n o x y h e r b i c i d e s a r e i n g e s t e d in s i n a l e la ra n d o s e s
.‘"V."?
th e y m a y p r o d u c e d c-atn. F o u r e a s e s h a v e b e e n d e s c r i b e d in th e . l i t e m »
‘
J u r e a n d a 5 th c a s e h a s b e e n r e p o r t e d to the C l e a r i n g h o u s e . A C o n n e - n e u t
t r u c k d r i v e r in a s u i c i d e a t t e m p t i n g e s t e d a c u p f u l of w e e d k i l l e r w h ic h w a s
'■■Ti-ca
a p p r o x i m a t e l y 20 % 2 , 4 - D a n d 40% 2, 4, 5 - T . W ith in an h o u r , 'lie b e c a m e ' .
n a u s e a t e d a n d b e g a n to v o m i t . A p p r o x i m a t e l y o n e h o u r a f t e r th e in g e s t i o n ^ .'
he w a s fo u n d in a c h a i r c o n s c i o u s b u t in a d a z e d c o n d i t i o n . H e w a s a d m i t t e d '*rw to th e h o s p i t a l a n h o u r a n d a h a l f l a t e r , s t i l l c o n s c i o u s , b u t d i s o r i e n t e d .

O n h i s s e c o n d h o s p i t a l d a y h e w a s o n th e h o s p i t a l c r i t i c a l l i s t b e c a u s e h i s
g e n e r a l c o n d i t i o n w a s g e t t i n g w o r s e . H is t e m p e r a t u r e w a s 104° F , , h i s
b lo o d p r e s s u r e 7 0 / 5 0 , a n d a lt h o u g h h i s u r i n a r y o u tp u t w a s d e s c r i b e d a s g o o d ,
h i s BUN h a d r i s e n f r o m 16 m i l l i g r a m s p e r c e n t to 4 8 . He w a s h y p e r v e n t i l a t i n g
and had b a s a l a r r a l e s .

Late in the secon d day of h osp italization the patient's blood p r e ssu r e was
v ery low d e sp ite la rg e d o se s of Aram ine and a trial on Levophed. He had
sinus tach ycardia (150), a cen tra l venous p r e ssu re of m inus 3, tem perature
o f 104° F , h yp erven tilation , blood PH of 7 ,4 3 , and anuria. He a lso h a d t|
g en era lized e ry th e m a . v
'
'V
•. *.

■U.

.

.I* *

.

.w - .

iC
'

:
’ ■'

The p atient develop ed sca ttered c re p ita n t.r a le s in the right lung and died
o f cardiac sta n d still (no ven tricu lar arrh yth m ias w ere noticed), e a rly in :i
the m orning of h is third h osp ital day, approxim ately 46 hours after the
in g estio n .
T his c a se had s e v e r a l s im ila r itie s to the c a sé rep ort of poisoning by
MCPA (2 m e th y l-4 -c h lo r o -p h e n oxyacetic acid ). Vomiting occu rrin g
sh ortly a fter in g e stio n and u n co n scio u sn ess appearing in a few hours
Both had lo w ered blood p r e ssu r e but in the MCPA c a se it could be m ain -'
«
*
*7jy? Vv'.. ¿ *. - ? "*•*■•!
.••••■
■\ ■ . rtf*»*."'*'t*•*■-O'"•
tained w ith m e ta r a m in a l. H ow ever, ' none of the n eu rological sym p tom s
(fa cia l tw itcK m g ^ co n stricted pupils, d ecrea sed tendon r e fle x e s , c lo n ic
sp a sm o f u itib s) w e re m entioned in the p resen t c a s e ,

.
'

'■

E arly deaths in a n im a ls by la rg e d o s e s of chlorinated ohenoxvacctic acid
fVv>-r. ;
■r!.-*&SkiJi< r i
/d eriv a tiv esjw ere' attributed to’ ven tricu lar fib rilla tio n . |n delayed deaths^
tliere w as a^disinclination to m ove, p r o g r e ssin g to rigid ity of sk e le ta l
m u sc le s (m yotonia) and a taxia. The se v er e c a s e s had p r o g r e ssiv e apathy, " "-/V
m u scu lar w e a k n ess o f the hind lim b s, p a r a ly sis, clon ic sp a sm s and finally • com a.
‘
i -,

C004E07

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I n g e s t i o n s o f th e .s e h erbicide*-' - n :.. >r a ; / .i c a r c f i i l g a s t r i c l a v a g e and
s y m p t o m a t i c a n d s u p p o r t i v e t. :ra p ^ .
T o x ic o lo g y o f C o m m e r c i a l
P r o d u c t s r e c o m m e n d s Q u in iu .
S u 'fa ;. ,n;.
-M).2 g r a m s o r a l l y
n y o to n ia , if p r e s e n t ,
e v e r y tw o h o u r s f o r th e f i r s t j
.• ¡. }'
a n d to s u p p r e s s v e n t r i c u l a r c ti ■
ro , So r e - a s e s m a y r e q u i r e
r h -d to th e c h e s t .
c a r d ia c m o n ito r in g w ith d e f i b r u
TI*
ck s and alco h o l
F e v e r s h o u l d b e t r e a t e d u s i n g phy
s p o n g e s ) s i n c e a n t i p y r e t i c s m a y be in»
.a : . i n d i r r . t e d .

- '

■■ * 0

Crt

o
ÜX

P ark e - D avis PARCODE
P a r k e -D a v is has r ec en tly initiated a n u m erical product i.!enr ;
cion
sy stem known as PARCODE. The s p e c ia l number d esigi
ntifie s
each ca p su le, K apseal, tablet and E m p let. The in itia ls P -L id en tifies the
product a s a P a r k e -D a v is preparation and the im printed number id en tifies
the p a rticu lar product. Although the com pany's m anufactured products
w ill henceforth ca rry a PARCODE id en tification sym bol, there m ay be
con sid era b le tim e req uired fur u tiliza tio n o f the non-coded products
jj' ..-»y
■
cu rren tly in sto ck .
•

•

•

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V: •.,, ^

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ling o r id en tification , have alread y béén estab lish ed by E li L illy and
Coding
Company and the N orw ich P h arm acal Company .' The poison con trol cen ter s^
have been se n t a copy o f the P a r k e -d a y is recogn ition code and w ill be in form ed if any other p h arm aceu tical cpmpanieis adopt sim ila r s y s te m s .

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ACUTE OVERDOSAGE OF DORIDEN
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w r itin g 'to the.

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D E F E R O X A M IN E

D e f e r d x b i n i n e 'f a c h e l a t i n g ag en t," c o m p l e x e s w ith i r o n to f o r m f e r r i o x a m i n e . V
. w h i c h p r e v e n t s tb e / iTon f r p m e n te rifig V ih tp ^ -iu rth e r c h e m i c a l r e a c t i o n s . T h e
c h e l a t e i s ''s o l u b l e in w a t e r ; i s e x ’c r e f c d b y th e k i d n e y . : and p r o d u c e .« z
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c h a r a c f e r i s t i c . r e d d i s f i c o l o r tq .the i u ^ i n e ^ - ^ h e d r ' e t i c a l l y , d e f e r o x a m i n e w i l l
b in d 8 . 5 % 'o f i t s w e i g h t o f f e r r i c , x r o m
th e i n i t i a l r e c o m m e n d e d d o s e ;;
o f 1 g r a m w ould, bipid" 8 5 m i l l i g r a m s o f f e r r i c i r o n arid th e m a x i m a l 24 - h o u r,
d o s a g e o f '6 g r a m s 'w o u l d b in d o y e r o n e - h a l f , g r a m of i r o n , th e e q u i v a l e n t of';
2 1 /2 g r a m s of F e S O ^ . 7 1 1 ^ 0 .. ...c.b.. v' .r'
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F o r s e v e r a l - y e a r s th e C i b a C o r p o r a t i o n h a s b e e n c o n d u c ti n g r e » » ta r h on -i..^.
th e u s e o f d e f e r o x a m i n e ( D e s f e r a l ) , o l d e r n a m e d e sfc r r i o x a s m m - , . !:• the
t r e a t m e n t o f i r o n p o i s o n i n g . B e c a u s e t h e r e h a v e b e e n »ever-.:; --rib. ie»
p u b l i s h e d in th e m e d i c a l l i t e r a t u r e o n i t s e f f e c t iv e n e s s - , t h e r e l..i- b e e n a
s c r i e s o f r e q u e s t s f o r t h i s m e d i c a t i o n w h en 'p o i s o n i n g s h a v e o c c u r r e d .
S i n c e t h i s d r u g w a s . u n d e r ' i n v e s t i g a t i o n , ’i t w a s o n ly a v a i l a b l e to the
a u t h o r i s e d i n v e s t i g a t o r s . T h e l i s t o f i n v e s t i g a t o r s , w illin g to
w p t re- ' .
f e r r a l s o f i r o n i n t o x i c a t i o n s , c o u l d n o t be p u b l i s h e d . - T h e r e f o r . - , th e
N a t i o n a l C l e a r i n g h o u s e i n i t i a t e d a s e r v i c e to tin* P o i s o n C o n t r o l C e n t e r s
to p r o v i d e th e n e a r e s t i n v e s t i g a t o r ' s n a m e anti t e l e p h o n e n u m b e r w h e n an
i n q u i r y w a s m a d e f o r th e i m m e d i a t e t r e a t m e n t o f a n i n d i v i d u a l c a s e . We
f e l t t h i s m i g h t be o f b e n e f i t to a s e r i o u s l y i l l p a t i e n t , a s w e l l a s to th e i n ­
v e s t i g a t o r , by p r o v i d i n g h i m a l a r g e r n u m b e r o f o b s e r v a t i o n s f r o m w h ic h
to j u d g e th e u s e f u l n e s s o f t h i s m e d i c a t i o n . T h i s d r u g h a s b e e n r e l e a s e d f o r ;
g e n e r a l m e d i c a l u s e , a n d u n t i l th e M a n u f a c t u r e r can. c o m p l e t e i t s d i s t r i b.
b u tio n t h i s s e r v i c e w i l l b e c o n t i n u e d .
v1

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c u p l o v e e s o :i l t

GH-P 551
OATE

c :n ".:3 u rrs csPAarcssr

October 21, 1968

auummIS

Marguerite X. Long

-o
xj

GH-P 551

TITLE

IM-C^SUTIVE SWCURV UTM t~ ~ u i:iC K i

This g j ; tonalvo review w a s undertaken to determine whether phenoxy
horblcldoa undergo changes in plants or animals which would result
in olgnlficant rooiducs of metabolites which would have to be ac­
counted for in tho ootabllohmont of tolerances by FDA. The review
la dlvldod into sections as outlined in the attached Table of
Contonto.
Tho metabolic products shown to be formed in plants were short­
lived intorcodiates whose nature depended on the chemical structure
of tho phonony compound, tho rate and mode of application of the
herbicide, tho otago of growth and metabolic condition of the plants
and, moat importantly, on the species and varieties of the plants
atudiod. In oplto of very extensive work during the past twenty
years, no one has been able to identify the products, although
hydroxylation of tho ring is believed to occur in some plants. In
animals, each compound was excreted unchanged, virtually quantita­
tively within a short time. Traces of residues were found for only
two or throe days in milk from cows maintained in pastures during
and following treatment with 2,4-D.
It was concluded that there 13 no reasonable expectation that res­
idues of metabolltos of tho phenoxy herbicides could be present in
significant amounts in raw agricultural commodities of plant or
animal origin as a result of currently registered uses for 2,4-D,
2,4.3-T, silvox or HCPA. (This review was prepared for the NACA as
part of a supplement to pesticide petitions for negligible tolerancos of those phenoxy compounds in a number of crops. The total
compilation Included copies of the 73 references cited herein).
EXEC* WEI Pm MKCT KO.

(*

HW rt)

CHARGE NO.
m e 0E REPORT

DEPARTMENT

*eair

pachi

STAGE

psoaLCM no.

56

DOW 763478

ÜS
30

ro
oo
o

and Animalo

•

2n

Reviow on tho Kotabolion of Phenoxy Compounds In Plant3

NSW PHYSICAL PROPERTIES
Q y is

CJno

Summary

' executivi research * biqprooucti research manaoer
*SIOPROOUCTt INPORMATKM CENTER

* eioprooucts department manage*

«registration SECTION

(b b ta j

«Corporate Res. & Dev. (CWH,JEJ)
«Product Team Manager (DDD)
♦Ag. Products Dept. Manager (REN)
Puerto Rico PS Field Res.
*Ag. Products Res. & Dev. Manager (EHB) Davis PS Field Res. & Dev.
«Vegetation Control Bus. Manager (JHG)
Oklahoma City PS Dev.
«Crop Protection Bus. Manager (AJ.B)
San Francisco PS Dev.
«PS Res. & Dev. Director (KCB)
«Pesticide Wildlife (EEK)
*AgT Products Chemistry Director (RDM) «Biochem. Investigations (WRB)
♦Ag.. Products Information Center (RJS) «Biomechanisms (GNS)
«Walnut Creek PS Lab.
Winter Park PS Dev.
«Midland PS Research (FHD)
Wayside PS Field Res. & Dev.
«Field Research Manager (ERL)
Animal Ag. Prod. Res. & Dev.
«Product Technical Specialist (JHD)
__ BiocTlem. Res. Lab.
Distribution list continued on back
DISTRIBUTION CONTINUED ON REVERSE SIDE IF REQUIRED.
EOMIC.n»<OPR»lTEOINU.tJI. A t-it

557 3

^

'4

Distribution continued:
♦E. C. Britton Res. Lab. (GDJ, DUO)
Ag. Products Formulations Lab.
Formulations life. Department
Lako Jackson Lab.
Patent Department
Pitman-Uoore
PS Dov. Manager
U.S. Dov. Uanacor
Dow Canada Aroa Dov.
European Area Dov.
Latin Acorlean Aroa Dov.
Pacific Aroa Dov.

♦Western Division Research
Midland Division Res. & Dev.
♦Analytical Uothcds (MEG)
♦Screening Formulations (CEC)
♦Jonoon, D. J.
♦Williams, C. S.
♦Watson, A, J.
♦Raynor, R. N.
♦Hoaly, U. J.
♦Lynn, G. E.
♦Southwlck, L.

i
i

i

CG04347

<

i
REVIEU ON THE METABOLISM OF FHENOXY COMPOUNDS
IN PLAINTS AND ANIMALS

DOW763480

Table of Contents

Summary Statement
Glossary of Terns
Review of the Literature
I. Metabolism in Plants
A.
B.
C.

Introduction
Tabulation of Conditions Used in Studies with Plants
Metabolism of 2,4-D
1.
2.
3.
4.
5.

D.
E.
F.

Hydrolysis of Esters and Salts of the Herbicide
Decarboxylation and Formation of the Phenol
Conjugation into Water Soluble Detoxication Products
Transformation into a Different Compound
Beta-oxidation of the Side Chain in 4-(2,4-DB)

Metabolism of 2,4,5-T and 2,4,5-TB
Metabolism of MCPA and MCPB
Metabolism of Silvex (2,4,5-TP)

*

II.

Metabolism in Animals

III.

Recent Residue Studies by Newer, Methods
!

IV.

Significance of Metabolites as Potential Residues in
Foods and Feed
j
i

V.

References

!

i

557 5
CC04343

5576

£

T able 30.— Sum m ary o f dotage» o f various organic herbicides that cause significant weight loss, reduced weight gain, or poisoning in
cattle, sheep, and chickens1

B erbicida

0 1

OÌ
■»nJ

00028C3

Cblorophenoxy compounds:
2.4- DichIor<mhenoxyacctie
acid (2,4-D), alkanolamine
•alts (of the ethanol and
isopropanol aeries).
2.4- Dichlorophcnoxyacetlo
acid (2,4-D), propylene
glycol butyl ether cater.
2,4,5-Trichlorophcnoxyacctic acid (2,4,5-T),
propylene glycol butyl
ether ester.
2-(2,4,5-Trichlorophcnoxy)«
propionic acid (eilvex),
propylene glycol butyl
ether eater.
2-Methyl-4-chlorophcnoxyacctic acid (MCPA),
alkanolamine salts (of the
ethanol and iaopropanol
scrica).
Amide compounds:
Af, Af• D imet hy 1-2,2-dipheny 1acclamide (diphennmid).
2-Cldoro-N,N-diallylacctamidc (CDAA).
2-Chloro-Af.Af-diallylacctamide (CDAA) and
trichlorobcnxyl chloride
(TCBC).

Dosage Least number of dosages for—
rate
Cattle Shcop Chickens

Dosage Least number of dosages for—
rate ■
■ ■ ■Cattle Sheep Chickens

Herbicide

Mg./kg.

Ms-Ike-

500 ............... 7
10
250
1
10
10
200 . 25 .............................
100
85
8 ______
250
3 - 2
4
100 ........................

10

500 ................................
6
250
4
4
10
100 ............... 3 .............
250 ............... 5
10
100

10

0

Phenyl urea compounds:

3-(p-ChlorophenyI)-l,1-

aimethylurea (monuron).

3-(3,4-DlchlorophenyI)-l*
methoxy-l-methylurea
(linuron).

8-Phenyl-l.l-dlmethylurea
(fonuron).

10

500
8 ......
1
250 ............... 0
10
100 ............... 4 .............
500 ...............................
10
250
3
7
10
250 ...............
1 ......
100 ...........
1
1
50
1
1
0
25
1
4
10
250 ...............
1
1
100
1
1
10
50
2
2 ........... .
25 ............... 6 ........... .

3-(3,4-DtchlorophenyI)-l,ldlmethylurea (diuron).

Thlocarbamate compounds:
-2.3-Dichloroallyl dilaopropylthlocarbamato (distiate).

-2,3,3-Trlchloroallyl
diisopropylthlocarbamate
(triauate).

n r r - r n o

aaoh

1
500
250 .
100 .
50 .
25 .
250 .
100
0
50
10
25 .
10 .
500
2
250 .
100 .
250 .
100
10
60 .
25 .
10 .
250 .
175 .
150 .
125 .
100 .
50
3
1
25
250 .
200 .
175 .
100 .
50
3
25
5

2
4
3
4
1
3
10 .
8
1
2
1
3
3
1
2
1
2 .

0
8
10
10
8
10
10
10
10
8
10
10
10
10
7
10
10
10
7
10
10

PRODUCTION RESEARCH REPORT NO. 106, U.S. DEPT. OF AGRICULTURE

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STATIONS VirnCUlNÂUMI’.lJK'.IXSKA ANSTAIT
S-1 0 4 O'j ST(K-.K!IOI.M r.o
Chcnictry Department
RECEIVED
. dec 2 2 12Ô9
r e g is t r a t io n

December 1C, 1969.

Marguerite 1 . Long, Ph.D.
B eg istra tio n Section,
A gricultural Department,
The Dow Chemical Company,
P .0 . Box 1706
MIDLAND, Michigan

USA

Dear Dr. lon g,
Thanking you for your le t t e r o f December 11, 1969, I am
en closin g some rep rin ts including those you requested. Also
included i s a ta b le o i tissu e d istrib u tio n data fo r 2,4-D and
2,4,5-T in chickens, taken from a forthcoming paper (l7.-S.
••B.iSDaLund and K. Erne, Acta Yet. Scand. v o l.
k970?T
<

/a }

, J974-

In a p liy sio lo g ica l study under way we have found chlorophenoxya c e tic acid s to be excreted in chickens by the proximal con­
voluted tubules in e sse n tia lly unmodified form (acid -hydrolysable conjugates making up le s s than 10 fo o f the excreted
phenoxy a cid ) and apparently by a transport mechanism in common
Y/ith u ric acid and a few other aromatic acid ic compounds
(K. Erne and I . Sperbcr. to be published). 2 ,4 ,5-T was found
to ex h ib it a higher a ffin ity fo r the carrier than did 2,4-D .
I should g rea tly appreciate i f you could le t ’ mo have one copy
o f your fin ish ed review .
S in cerely yours,

121504

X. Erne
Ph.D. (V et. S c i.) , A ssoc. Prof.

0001371

5579

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gable 2 . Phsaoriy acid le v e ls in tiaouoo o f b r o ile r chickens fod 2,4-D or 2 ,4 ,3 -T a t 1,000 p .j .n .
in the drinking r/ater fo r 104 days.-

Chicken
"o.

Pkcr.oxy
a c id

Sex

0 21S4/67

2,4-3 .

0 21 05/67
C 21S2/67

C 21 33/67

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Kidney

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4 .0

5.0

3 .6

20.0

10.0

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2,4-3

9

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10.0

7 .2

28.0

15.0

0 .4 - 2.0

2 ,4 ,5 -T

o

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8 .5

8 .0

85

18.0

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2 ,4 ,5 -0

9

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1

Epidemiology, Prevention and Treatment
ees which wore •'•.-¿'laced by aqueous suspensions and
cquent replacement of the organoclilorine compounds with
-fttnophosphorus or.ci. These efforts included also an
.jiicationa! campaign on the veterinary and medical, etc.
. peels o f pesticide application. The first reassuring results of
J i efforts are now available. The need in 1967 for chemical
-eatment of the potato crops, however, precluded achieving
- ,e desired results in preventing residue accumulation. In
,-i66. residues were found in 125% o f sugar beet samples,
*5~ of potato haulms and 19.4% o f food product samples. In
>67, their corresponding values decreased to 6.71 and 6.3
:<ic| • In 1967 as compared to 1966, there was a significant
:.vp in the number o f mass pesticide poisonings of animals.
..-terinary specialists played no little part in achieving these
uals.

r*■■* ':v..
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.

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.

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'

•extent recall the course of infectious dheas.'s such as plague.
Aujeszky's disease and paratyphoid, so that mercury poisoning;
should be eliminated during a differential diagnosis.

7I-050S. Rusin, K. N.; Tokarev, N. F .; lAroshcnko, N. M_
(Min. of State Farms. Ukrainian SSR, USSR). Cases of swine'
poisoning with Granosan. Veterinariya A o. 9: 61-3; 1969.
(Russian)
Cases o f severe Granosan poisoning in swine arc
reported. In the fall of 1967 on the “V ictory” State Farm ia
Nikolaev Province, all the Granosan-treated wheat not used in
sowing was stored next to forage grain. The treated, thrashed
grain was used as swine feed from November I I , 1967 at 2 .2
kg/day. The contaminated grain was fed up to 26 days, since
the initial poisoning symptoms were lacking. At 1 month, th e
2- to 5-inonth-old piglets began to display clinical signs; refuse?
of food, depression, muscular tremors, etc. The oral mucosa
was hypercmic; the feces were o f a yellow color and in m any
eases liquid, covered with a thick, clastic coating, etc. S o m e
swine liad temperatures below normal for 5 to 6 days and, as a
rule, these animals died (76% o f those affected). The autopsar
findings included marked plethora, hypercmic oral m ucosa,
enlarged lymphatics, uueven coloring o f die lungs i r o n
grey-red to dark red which were o f a doughy consistency, e tc .
The diagnosis was based on the pathologicoanatomical
findings, the clinical picture and laboratory analysis, all o t
which excluded infectious disease. Mercury was found i i
samples of- feed and of the parenchymatous organs. T h e
treated grain, for example, contained 0.18 to 21.7 mgffcg
Granosan. The therapeutic regimen, once the diagnosis waoc
established and the feed chang.’J, ir.ciudcd glue™ aa.5
caffeine i.v. and various doses of unithiol s.c. and Lm. But o n ly
34% o f the affected swine were saved. Follow-up over the next
5 months showed that poisoned swine w ere retarded in weight
and the mercury salts also had a negative effect on sow
productivity (premature, stillborn and aborted young). As a
result of this incident, the “Victory” State Farm was noc
allowed to supply animal husbandry products to the m arket
for 5 months.

ii ■

j |. An

i.1.*’

tt ;";
\\
•:

.

:hi î. L)J" •
' '•

■Jatier.
.V.. V
■vit***
'

71-0507-9

*1-0507. Birbin, S. S.; Alekseeva, A. A.; Bulatov, A. A.
■jtishchcvo District Veterinary Station o f Saratov Province,
,jtislichcvo, USSR). The poisoning of swine treated with
dranosan. Veterinuriyj Av. 8: 60-1; 1968. (Russian)
Mass poisoning o f swine with the organomercury
.erbicide Granosan was seen on two farms in Saratov Province,
tin the “October” Collective Farm in Tatishchevo District,
- j of 414 swine of various ages were affected during August
¡1967], the acute period; 121 died and 145 in the agonal state
*.jQ to be slaughtered. By November, another 44 animals had
:d. On the “ Fcdou.v'' State Farm in Marx District, 211 o f
¿44 swine were affected. The cause of the toxicosis was the
-olonged feeding (up to 30 days) of ground barley that had
*.en treated with Granosan and o f granary grain sweepings
•hich contained a b.:a? percentage o f the preparation. The
. rit signs of poisonir.a appeared in suckling pigs and failing
.'.is 20 to 25 days after beginning feeding on the treated grain;
" sows, the signs appeared in 30 to 40 days. At first, the
a-mals refused food -ml water and became restless. There was
~>ie nasal mucous secretion. Then weakness in the hind limbs
rpeared, with different types o f movement coordination
order. Some animals showed spinal involvement. Signs of
rural disorders were quite clear, including muscular tremors,
nvulsional jerking of the extremities and tetanic contrac­
t s of the pelvic musculature: As their conditions worsened,
'' -• animals lay on their stomachs or sides, developing varying
rrccs o f paralysis with loss o f pain sensation, rapid
rjthing, etc. The younger swine almost all died within 3 to 6
-.- > after the symptoms started; the sows' condition persisted
-■i-t until death (6 to II days) and 40 to 45% o f the
- : m ed animals died. In some instances, the swine suffered
: above symptoms, including partial or complete loss of
>u. for 2 to 3 months. The autopsy findings were initially
■' icme in all the animals, the most constant changes being
b-l in the intestines. The intestinal mucous membrane was
- '-'red with dryish, dirty yellow or brownish-green deposits
■'ciectcd to the undeilying tissue. Liver and kidney findings
also described. 'Ihciapy with uuithiol, cardiac agents,
• :-icilliii .uni glucose were ail ineffective. The fact of a delay
'■he appearance o f symptoms following Granosan inloxica■must be taken into account in diagnosing organomercury
"mugs. The clinical symptoms and pathologicoanatomica!
•: b<-'s in mass poisonings o f swine with Granosan to a great

-a '

71-0509 J Bashirov, A. A. (Azerbaijani Res. Inst, o f La bo*
Hygiene and Occupational Diseases, Baku, Azerbaijani SSFL.
USSR). The state of health in workers manufacturing th e
herbicides, the amine salt and the butyl ester of 2,4-D acicL
Vmchcbnoe Dclo No. 10: 92-5:796g^( Russian)
A medical examination was done o f 292 persons engaged
in the manufacture o f the title herbicides; 24S were men a n d
44 women. Their ages ranged tram under 20 (nine) to over
(19). Professionally, die workers' jobs ranged from laboratory
worker to machinery npciator; their tvork exposure ranged
from under 5 years (194) to 6 to 10 years (98). The workers^
general complaints included general weakness, rapid fatig­
ability. frequent liojiiaehes <>f a dull nature mainly localized i a
the fromal-p.nictal area which intensified at the end of th e
work day (63%), vertigo (33%). etc. Objectively, arterial!
hypotension was* found in 59 persons (20.2%). Frequent
complaints were noted in 51.7% of those examined w hich
were typical of digestive function disorders: dyspepsia, poor

40

jr

n
Ô
139

GGGGÎ-34

71-0510

Epidemiology, Prevention and Treatment

71-0510. Lobzin. V. S. TSinovpi, P. E. (Dept, o f Nervous
Diseases, Kirov Military Medicine Acad., Leningrad, USSR).
N eurological disorders' in chloroplios poisoning. Zh.
NcvmnatuL i i'sr.hiatr. 69(5): 679-83; 1969. (P re fe re n c e s).
(Russian)
-See also

71-0439
71-0444
71-0445

71-0447
71-0451
71-0474

71-0489
71-0520
71-0526

Neurological disorders in acute chlorophos poisoning
described based on 41 case histories, 11 o f which were •
severe (two patients died), 14 o f median and 16 of he:.:
intoxication. The severity and character of the clinical >¡; ,
depended on the dose and route of the poison into the Ive-.
In most instances, chlorophos had been taken interHalh. ]•
the qcutc poisoning phase, the predominant symptoms
central nervous system lesion and vegetative disorders. L..
frequency of individual symptoms (muscular wejío*
disorder of consciousness, headache ar.d vertigo, tiem :>
delirium and hallucinations, sleep disorder, etc.) is shown l:;.
table. Two individual ease histories arc detailed, illustrative. ;
severe chlorophos poisoning. Both patients had respira!.--,
disorder requiring intubation and were comatose. In one
a 52-year-old man, the poisoning was so severe that in spite. •'
various therapeutic measures (atropine i.v.. gastric lavage, et, >.
the pafjcnt died of respiratory and cardiac arrest. In i!..
second ease, a 46-year-old man, 3 days-aftcr treatment u
i.m. TMB-4, i.v. atropine and'glucose and regaining conseiu.:
ness, the patient developed a. delirious syndrome. Aft ;
successful therapy for" tliis, he complained of muscular pa::.,
and difficulty in walking. Objectively, decreased left knee «.c
Achilles reflexes were noted; clumsiness in .the finger,
appeared. A diagnosis o f toxic polyneuritis was made. Im­
patient developed intense pains in the distal parts of V»
extremities. After 5 months of therapy, only_ slight impr^c
ment occurred. The movement disorders and disorder r
sensitivity, in the distal parts of the extremities'proved ver
resistant to treatment. O f the 41 chlorophos poisoning caweight developed toxic polyneuritis: the .¡alter appeal,,
unexpectedly and anywhere from 10 days to 1'A months at:,
the poisoning. Muscular atrophy was detected usually a'to ■
weeks after the first signs of polyneuritis. A discussion of ti;
problems o f therapy is included.

BOW ’J167974

-appetite, radiating pains in the right epigastrium, etc. A slight
increase ii< the liver was found in 12 persons. From these
findings, SO workers (41 men and 9 women) were selected for'
detailed clinical studies o f the stomach and liver as well as of
the cardiovascular system. Their case histories showed no
particular diseases prior to their present work with the 2,4-0
acid herbicides. A control group of 20 persons with no toxic
chemical contact was included. Bradycardia was found in 20%
which varied from a, lying to a standing position. Arterial
oscillography demonstrated that the mean dynamic pressure
while lying was within normal limits but while standing this
index showed asymmetry. Fourteen of the SO persons
examined had a lowered acid-forming function in the stomach;
in eight there was a histamine-resistant achylia; etc. Liver
dysfunction was often found: 22 of the 5 0 (versus four of the
20 controls) had disordered antitoxic function expressed as a
decreased hippuric acid excretion in the urine. Liver protein­
forming function was also disordered in 19 of Lite SO: lowered
albumin level, etc. Four o f the workers had a lowered
prothrombin index. Blood sugar was lower than 70 mg% in 16
(versus two of the controls). The various liver dysfunctions
found were significantly more pronounced in workers with
longer work exposure to the herbicides. The results o f the
survey show that in the manufacture o f the title herbicides,
various disorders o f the cardiovascular system and o f the
organs of digestion may occur. They point up the need for
checking the cardiovascular system and the digestive organs m
those entering work and in those already working in the
production o f 2,4-0 herbicides.

/
71-0527
71-0535
71-0562

* 71-0569
71-0573
71-0580

71-0583
71-0605

5584

0C0G23U

lo i

5585

Residue Reviews 26, 37-62 (1969)

MN069799

Toxicity and hazards to man,
domestic animals*, and wildlife
from some commonly used
auxin herbicides
By
J. M . W a y *

Contents
I. Introduction .................
II. Toxicity nnd hazards to m an............................................................
III. Toxicity and hazards to domestic and ta'.rraiory animals...................
a) Acute and chronic toxicity...........................................................
b) Indirect toxicity........................................■.................................
IV. Bees and other insects.......................................................................
V. Soil animals-.....................................................................................
VI. Fish and aquatic organisms................................................................
VII. Wildlife in general............................................................................
Summary....................................
Itdsumd ..................................................................................................
Zusammcnfassung.....................................
References...............................................................................................

37
38
41
41
46
48
49
50
53
54
55
56
58

L Introduction
This review has been compiled from searches in the literature for
references to the toxicity and other hazards that may arise from the
use of auxin herbicides, and specifically of formulations of MCPA
(4-chloro-2-methylphenoxyacctic acid), S.S. 2,4-D (2,4-dichlorophc-noxyacctic acid) and of their butyric acid analogues MCPB and
2,4-DB, of 2,4,5-T (2,4,5-trichlerophcnoxyacetic acid), mecoprop
[± tt-(4-chloro-2-methylphenoxy) propionic acid], and 2,3,6-TBA
(2,3,6-trichlorobenzoic acid).
Assuming 70 percent of the eight million acres of cereals grown in
the United Kingdom to be treated annually with herbicides ( W ood * Monks Wood Experimental Station (The Nature Conservancy), Abbuis
Ripton, Huntingdon, England.

37

f

0007424

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e :'

J.M.Wat

38

ford 1964), it is apparent that the tonnage (circa 2,500 tons) of the
compounds mainly used for this purpose is similar to the total tonnage
of insecticides and fungicides used annually on all crops in the U.K.
(circa 2,660 tons) (Strickland 1966). In 1962, Dadd (1962), from a
survey of eastern England, found that over 90 percent of cereal Helds
treated with herbicides were treated with auxin compounds based on
chlorinated phenoxy acid derivatives, notably salts of MCPA and to a
lesser extent of mecoprop. Although the use of straight formulations of
MCPA has been gradually declining (Woodford 1964) in favour of
mixtures of compounds, many of these mixtures still include MCPA
or other phenoxy compounds and increasingly include 2,3,6-TBA. In
addition to its agricultural use on arable and pasture crops, 2,4-D to*
gether with 2,4,5-T, is used, or is likely to be used on an increasing
scale, for scrub control and control of unwanted broad*leaved tree
species in conifer plantations. 2,4-D is also used to a limited extent for
control of submerged aquatic weeds. On a world-wide basis 2,4-D is
probably used to a greater extent than any other herbicide and this is
reflected in the greater number of references to this compound. The
extensive use of a single biologically active family of compounds
clearly presents a considerable potential hazard in respect to their
direct and indirect toxicities to animals including man. It is a measure
of the comparative safety of the auxin herbicides that, in spite of
their increasing use throughout the U.K. since the mid-1940*s, they
continue to be applied much in the same routine way as are inorganic
fertilizers, and with as little regard for any possible toxic hazard.
During the many years of their use throughout the world there have
been notably few authenticated incidents of poisoning of domestic
animals or wildlife resulting from the proper application of these com­
pounds. The fact that fully authenticated incidents of poisoning have
not been reported cannot oe taken as an absolute guarantee of safety,
particularly with regard to indirect or chronic toxicity, because of the
frequent difficulties of attributing the underlying cause of an illness
or of death, especially in wild animals. Nevertheless all the direct and
circumstantial evidence to hand at the present time seems to indicate
that incidents of poisoning have been extremely few and generally
under exceptional circumstances.
n. Toxicity, and hazards to man
The principal routes of toxicity to man are either orally or by in­
halation; there appears to be little hazard of transport through the
skin although individual allergies can develop leading to dermatitis
( V a l l e t 1965). Eyes may be directly but are usually only temporarily
affected. Hazards to man may occur from the concentrated chemical
before dilution, from inhalation of spray or dust during application, or
from ingestion of the chemicals in food or in water. Because the greatf

0007425

cn

CD
a /t

CD
CD

I

Toxicity and hazards of auxin herbicides

C
C

39

1 546794

est hazards arc from the concentrated chemical and because man is
handling the chemicals in this form at all stages from manufacture to
dilution, it follows that he is at greater potential risk than any other
organism. However, there are very few reports in the literature of
tests or incidents of poisoning of man by these compounds; the
majority of these reports refer to accidental poisoning of children. As
a result it is now generally accepted that auxin-type herbicides do not
present a direct toxicity hazard to man (Bailves 1963) when correctly
handled or used for weed control.
Kraus in 1945 (in Kepilart 1945) reported that he had taken 0.5 g.
of 2,4-D per diem for 21 days with no demonstrable ill-effects. A clini­
cal study was made in Denmark by Nielsen et al. (1965) on a 23-year
old man who had committed suicide by apparently drinking 125 ml.
of 50 percent w/v 2,4-D dimethylaminc salt The total weight of
2,4-D in his body was calculated as being not less than six g. (the
equivalent of 80 mg.Ag.), about 10 percent of the total weight of
active material ingested. The principal damage appeared to be to
nerve tissues and the central nervous system.
Vallet (1965) gives acutely toxic oral doses of 2,4-D to man of
50 to 500 mg.Ag. body weight, of 2,4,5-T of 500 to 1,000 mg.Ag.,
and of MCPA of 50 mgykg. No indication is given of the source of
these figures which are probably extrapolated from data on laboratory
animals. Freireic2 I et at. (1966) have shown that a number of drugs,
on a mg.Ag. basis, are more toxic in man than in the mouse, when used
as a laboratory test animal, by a factor-of 10 to 15. There are clearly
dangers therefore in assuming that a toxic dose to man is the same as
the toxic dose to a laboratory animal multiplied by some factor based
on the mean differences in weight. In fact these latter authors show for
a number of anticancer agents that a very much more accurate assess­
ment of the ratio of animal to human toxicity can be obtained on a
mg./m.* of body surface area, than on a mg.Ag. basis. For this reason
Vallet’s (1965) figures may be suspect, particularly in respect of the
range of 50 to 500 mg.Ag. for 2,4-D, although the lower figure would
be of the correct order of magnitude when compared to the residue of
80 mgTkg. found by Nielsen (1965) in his suicide case. Nevertheless,
this figure represented the amount actually found and it is likely that
a greater proportion of the total 2,4-D ingested was necessary to
produce death. In this context it is noteworthy that the acute oral
LDgo’s to rats of the most common organochlorine insecticides range
between 10 and 135 mg.Ag. (Martin 1963J.
Edwards and Ritter (1953) have discussed the hazard to operators
from inhalation of sprays or aerosols during application of herbicides
with particular reference to methods of protection. Monarca and
Di Vrro (1961) have described a clinical study of an acute
of
accidental poisoning of a man in Italy. In this instance a farmer became
ill after applying a 40 percent aqueous solution of 2,4-D by habdpump
i

00074SF»

5588

40

J.M.W ay

against the wind. He was admitted to hospital, suffered a relapse after
18 days, and recovered sufficiently to be discharged after 40 days.
Initial symptoms of muscular weakness, vomiting, perspiring freely,
and oliguria were noted in the Held whilst a diagnosis of bradycardia,
respiratory difficulties, and urinary abnormalities was made after ad­
mission to hospital. However the authors report that the case was
exceptional. F e t is o v (1966) has reported similar field symptoms in
Russian workers engaged in field applications of 2,4-D. This author
concluded that a range of formulations of 2,4-D was "highly toxic to
animals in different ways of introduction.’* Whilst reports of minor
discomfort following exposure to auxin sprays during field application
are rarely reported in scientific literature, there is no doubt that a
proportion of workers so exposed do suffer a degree of transitory dis­
comfort Whether this is of any significance as a long-term toxic hazard
has not been determined for man.
From the very nature of their use it is unlikely that auxin herbicides
will appear as significant residues in food crops. Williams ( 1964 ) was
unable to detect any residues of auxin herbicides in a number of total
diet samples down to the limit of sensitivity (0.01 p.p.m.) of his
analytical techniques. D uccan and Weatuerwax (1967) calculated
pesticide chemical residues in 'total diet' samples collected on 46 days
in 25 American cities during a 699 day period from June 1964 to
April 1966. Each sample represented the total amount of food and
drink consumed by one person over a two-week period. The total
samples represented in all a food and drink supply sufficient for 644
days. Herbicide chemicals were found infrequently and averaged about
0.01 mg./day of which one third was 2,4-D, and half was MCPA and
pentachlorophenol (PCF) combined. 2,4-D was found in oils and fats
(0.001 mg. in 1964/65) and sugars and sugar products (0.004 mg. in
1964/65,0.002 mg. in 1965/66), whilst MCPA was found in grain and
cereals (0.002 mg. in 1964/65), in dairy products (0.003 mg. in 1965/.
66), and in leafy vegetables (0.001 mg. in 1965/66). These amounts
are substantially below the limits set for acceptable daily intake by the
World Health Organization and United Nations committees. It seems
probable, therefore, that toxic hazards from auxin herbicide residues
in food are very small Faust (1964), in a survey of water pollution
hazards to man from organic pesticides, came to the conclusion that
there did not appear to be danger to health at the present time from
the background concentration of pesticides believed to be in ground and
surface water. However, 2,4-D could persist in lake water and bottom
mud for long periods under certain environmental conditions. Work
in Russia, quoted by Faust, suggested that the threshold taste and
odour concentrations auxin compounds, especially of phenolic deriva­
tives such as 2,4-D, that would prove unacceptable to the consumer
were very considerably below the threshold concentrations for toxic
effects. A particular risk might be supposed to lie in contaminated milli

0007427

BOW : 1546795

I

I

DOW 1546796

Toxidly nnd hazards of auxin herbicides

'll

drawn from cows feeding in treated pasture, but no residues of either
2,4-DB or 2,4-D were found in the milk of cows that had been fed
these compounds (Gutenmann ct al. 1963a and 1963b). The beasts
were fed 5.0 p.p.m. (based on a daily ration of 50 pounds) for a
number of days; the limit of detection for 2,4-DB was 0.2 p.p.m. and
for 2,4-D 0.1 p.p.m. Whereas milk might be a source of contamination
these results indicate that there is no apparent hazard.
ITT. Toxicity and hazards to domestic and laboratory animals
a) Acute and chronic toxidly

The toxicity of agricultural chemicals to land fauna is normally
quoted in terms of the dose that kills 50 percent of a population of
test animals (LDS0). Whilst this figure gives a useful indication of the
comparative toxidties of different compounds to a given test spetics,
the figures obtained in different tests may be influenced by a number
of factors. Thus the age and sex of the test animal, method of dosing,
and general conditions of the test may have an important bearing on
the susceptibility of the animal to the compound being studied. The
formulation of the active compound has a considerable influence: for
instance 2,4-D acid has an LDfio to rats of 375 mg.Ag. but the sodium
salt has an LDso of 805 mg.Ag., the propylene glycol butyl ether
ester of 570 mg.Ag., and the iso-propyl ester of 700 mg./kg. (Rowe
and Hymas 1954). It should be noted, however, that Bjorklund and
Erne (1966) do not regard these differences as being appreciable. In
addition, the LD» for different test species may vary quite widely: for
example, 2,4-D acid has an LDso of 375 mgAg. for rats, of 100
mg.Ag. for dogs, 469 mg.Ag. for guinea pigs, and 541 mg.Ag. for
chicks (Rowe and Hymas 1954).
Bearing in mind these reservations, the acute oral LDSo to rats of
the acid formulations of the compounds under consideration are:
2.4.5- T 300 mg.Ag., 2,4-D 400 mg.Ag., MCPB 680 mgVkg., MCPA
700 mg.Ag., 2,4-DB 700 mg.Ag., 2,3,6-TBA 705 mg.Ag., and mecoprop 930 mg.Ag. (Woodford and Evans 1965). Thus, 2,4-D and
2.4.5- T fall in the moderately toxic and the other five compounds in
the slightly toxic class according to the widely accepted classification
of toxicity of chemicals quoted by Frazer (1963). In a comparison of
the relative toxicities of a number of commercial formulations of 2,4-D
and 2,4,5-T,- Rowe and Hymas (1954) concluded that the ‘inert* con­
stituents of the formulations did not add to the toxicity of the active
compound, nor did they exact any potentiating effect. Similar con­
clusions were reached by Dalcaard-Mixxelsen and Pooisen (1962)
on the basis of this and other work. However, it is possible for sur­
factants and other additives to enhance- the toxicity of active, com­
pounds. Whilst there is some evidence with insecticides that mixing
f

0007428
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J. M. Way

' |546797

of compounds leads to synergistic effects on the acute toxicity of the
constituent compounds (Keflincer and Deichmann 1967), effects of
this sort have not been reported for herbicide mixtures in common use.
However, die possibility of dieir occurring should not be overlooked,
nor, also, the possibility of antagonism where the effect of one com­
pound upon another in a mixture is to depress its biological activity.
Descriptions of the clinical symptoms of poisoning by MCPA,
2,4-D, and 2,4,5-T of a range of experimental animals have been sum­
marized by Rowe and Hymas (1954) and more recendy reviewed with
comments by Dalcaard-Mikkelsen and Poulsen (1962). Bjorklund
and Erne (1966) have subsequently further reviewed and reported
on their own results on the effects of feeding 2,4-D to calves, pigs, rats,
and chickens. A range of symptoms was produced that was generally
similar to those described by the previous authors. Erne ( Iwitia and
b) studied the distribution and elimination of 2,4-D and 2,4,5-T in
these animals. Amine and alkali salts of both compounds were readily
absorbed and completely distributed in the body, but 2,4-D ester was
incompletely absorbed and reached only a low level in the plasma
and tissues. The highest tissue levels of the two compounds were
found in liver, kidney, spleen, and lungs and the levels found in these
organs sometimes exceeded the level found in .the plasma. In blood
cws some 10 to 20 percent of the plasma level was found. Penetration
of 2,4-D into placental tissue of pigs was recorded but there was little
or no evidence of penetration into adipose tissue or the central nervous
system. Elimination of the compounds was rapid, the plasma half-life
being about three hours in rats, eight in calves and chickens, and 12 in
pigs. The tissue half-life values ranged between five and 30 hours. No
retention of the compounds was noted in the tissues. There was no
accumulation after repeated dosing and in pigs there was an increase
in the rate of elimination after repeated administration. In all species
the main excretory route was via the kidneys. Khanna and Fanc
(1966) traced the metabolism of C“-labelled 2,4-D in rats dosed at
rates from one to 100 mg./animaL Radioactivity was found in all the
organs studied together with some accumulation as early as one hour
after dosing. At the one mg. dose rate a concentration peak of radio­
activity was demonstrated after six to eight hours but decreased there­
after and was non-detectable by 24 hours. At the SO mg. dose the
peak occurred at eight hours and persisted for 17 hours. Extracts of
the tissues were shown to contain mainly undianged 2,4-D residues.
No radioactivity was found in the expired carbon dioxide, but elimina­
tion in urine and faeces was dose dependent. At die one to 10 mg.
doses 93 to 96 percent of the ingested 2,4-D was excreted unchanged
in the urine in the first 24 hours. At the 20 to 100 mg. doses greater
amounts of 2,4-D were found in the second 24 hour period after dosing,
with a linear decrease in percentage recovery with increase iq dose.
In experiments with cattle Gutenmann et al. (1963a and b) were unf

000742.9
5591

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<•

I

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Toxicity and hazards of auxin herbicides

43

546798

able to detect any residues of 2,4-DB or 2,4-D in milk or faeces of
cow's fed five p.p.m. of either compound in a 50 pound daily ration.
In these experiments there was no evidence of beta-oxidation of 2,4-DB
to 2,4-D. Disappearance of 2,4-D was thought to occur as a result of
dilution in the rumen, some absorption on the gut wall, and by de­
composition. In subsequent experiments Bâche et aL (1964) and
St John et aL (1964) studied the fate of MCPA, MCPB, 2,4,5-T, and
a number of other herbicides in cattle. All the MCPA fed to a single
steer (113.5 mg. single dose based on five p.p.m. of a 50 pound daily
ration) was accounted for in its urine over the four days after admin­
istration. No MCPB was found in the urine of MCPB-dosed cows (total
dose of 56.75 mg. and 113.5 mg. based on 2J5 and 5.0 p.pan. of a 50
pound daily ration) on the first day after administration, but subse­
quent recoveries in their urine showed that 7.2 to 9.2 percent of the
MCPB was converted to MCPA. Analysis of MCPB itself, however,
was said to be unsatisfactory. It is of interest that a similar conversion
of 2.4-DB to 2,4-D can be performed by bluegill sunfish ( Lcpomis
machrochirvs) (Gutenmann and Lise 1965). Urine and milk samples
were also analysed from cows fed a total of 454 mg. of 2,4,5-T over a
period of four days. It was shown that the compound was excreted
exclusively in the urine over a period of six days, a total of 430.7 mg.
being recovered. Clauxe et al. (1964) investigated the fate of C14labclled 2,4-D in sheep fed a dose of four mg. of 2,4-D/kg. calculated
as the minimum daily dose likely to be ingested from grazing treated
pasture. The level of radioactivity in the blood rose to a peak 1.5 hours
after dosing but fell to the normal background level in 24 hours. All
the material was excreted within 72 hours, with over 98 percent in the
urine and only 1.5 percent in the faeces. Balayaknis et aL (1965)
dosed a rabbit and six mice with 2,3,6-TBA and showed that a pro­
portion of the compound was recovered after passing through the
animals. These authors discussed the significance of biologically active
residues of auxin compounds in animal excreta that might become
incorporated in manure or straw. It was shown that 2&6-TBA residues
in particular could remain active for a period of months and affect
susceptible crops to which the contaminated manure was applied. The
same hazard does not normally exist with the phenoxyacetic acid
derivatives, where the compounds are broken down and become bio­
logically inactive in a relatively short period of time. However, Lise
(1966) has pointed out that the excretion of 2,4-D in the urine of cows
docs present the admittedly remote possibility of active 2,4-D being
transferred from a treated pasture to a susceptible crop.
Mitchell (in Kepiiart 1945), Dalcaaud-Mikkelsen et aL (1959),
and Coldstein and Lonc (1960) all reported that there were no ap­
parent ill-effects in cattle, sheep, or horses from grazing pasture
sprayed at hcrbiddal or two times hcrbicidal rates of 2,4-D or MCPA.
CmcsBY and FAnwrxL ( 1950) (in Spiunceh 1957) reported that there
i'

0007430

5592

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44

1546799

was no significant difference in the amount of feeding of horses, cows,
sheep, ana pigs in untreated plots or plots sprayed with the sodium
salt or the isopropyl ester of 2,4-D,-or the isopropyl ester of 2,4,5-T.
However, there did appear to be less feeding in plots sprayed with
the alkanolamine salt of 2,4-D. There was no effect on milk production
of cows feeding on sprayed vegetation. Goldstein and Lonc (I960)
found no ill-effects on two cattle rrom adding 0.25 pints of a 1.5 percent
w/v 2,4-D/2,4,5-T mixture to every five gallons of their drinking water
for 41 days, Diese authors also reported spraying the skins ora calf,
of a cow, of sheep, and of pigs with doses ranging from 0.002 to 0.003
pounds of 2,4-D or 2,4-D/2,4,5-T mixture, with no ill-effects. These
dose rates would be of the order of those that might occur in an in­
stance of spray drift
D obson (1954) sprayed MCPA, 2,4-D, or 2,4,5-T on grassed
chicken runs daily for 14 days at normal and ten times normal dose
rates. 2,4^3-T significantly reduced egg production and the -weight of
the birds; MCPA and 2,4-D also affected egg production, mainly in
the second week of spraying or during the week after spraying had
stopped In all instances there was no effect on the fertility of the
eggs and all the progeny reared well, although the dose rates and
. frequency of application in this trial were much more severe than are
likely to be found in practice. Erne (1966) showed that some of the
2.4- D fed to hens could be excreted in their eggs. Donachie and
Fletcher (1967) injected hen’s eggs with MCPA, 2,4-D, MCPB,
2.4- DB, mecoprop, and 2,3,6-TBA amongst a range of other herbicides.
Dose rates were 10, 100, and 200 p.p.m. equivalent to 0.5, 5, and 10
mgyegg. The percentage hatch was recorded. At the lowest dose there
was 100 percent hatch from the MCPA- and MCPB-treated eggs; 90
percent rrom the mecoprop- and TBA-treated eggs, and 80 percent
from the 2,4-D-treated eggs. At the highest dose mere was 80 percent
hatch from mecoprop, 50 percent from 2,4-D and TBA, and 20 percent
from MCPA and MCPB. None of the chicks that hatched was de­
formed although some feather blanching was noted from the MCPA,
MCPB, 2,4-DB, and mecoj >treatments. Roberts and Rocers (1957)
reported on various feeding experiments on turkeys with alfalfa
sprayed with a low volatile ester of 2,4,5-T at herbicidal rates. No
deleterious effects were noted. Calculations were quoted to show that
for a one kg. chicken to acquire a lethal dose of 2,4-D from an applica­
tion rate of one pound/acre, the bird would have to consume in two
days all the 2,4-D applied to the vegetation over an area of 72 square
feet Similar calculations by Route and Hymas (1954) for cattle sugested that for a 770 pound animal to acquire an acutely toxic dose of
,4-D of approximately 1,000 mgAg., it would have to graze com­
pletely ana ingest all the 2,4-D from an area of 0.17 acre (one pound/
acre es 10.41 mg./ft* *a 93.7 mg./yard2«a 112.1 mg./m.*).
Accounts are given of direct oral dosing or dermal applications of

t

0007431
5593
*

/ M r J ft /o o o s u y /A

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Toxicity and Lizards of auxin herbicides

45

1. Sheep tolerated 481 daily doses of 100 mg.Ag. of the aOcanolamino salt or
propylene glyuol butyl ether ester of 2,4-D.
2. Cattle suffered from chronic tympanites after 88 daily doses of 100 mg./
kg. of the alkanolamine salt of 2,4-D. One animal died after 34 daily doses of
200 mgAg.
S. Sheep tolerated 481 daily doses of 100 mgAg. of the triethylamine salt
of 2,4,5-T but succumbed to 389 doses of 100 mg./kg. of the propylene glycol
butyl ether ester.
4. Sheep were killed by 383 daily doses of 100 mg.Ag. of MCPA amine.
The toxicity of 2,3,6-TBA in these trials was said to be slightly
greater than that of 2,4-D or 2,4,5-T. Strach and Bohosiewicz (1964)
reported that no abnormal behaviour in pigs had been noted following
40 daily doses of 15 to 100 mg.Ag. of 2,4-D, nor from single doses
of 2,4-D of 200 to 800 mg.Ag.
In short term trials by Bjorklund and Erne (1966), calves and pigs
showed definite though reversible symptoms of poisoning after single
doses of 2,4-D of 200 and 100 mgAg., respectively. Rats and fowls
did not show any sign of distress after single doses of 100 and 300
mg.Ag., respectively, and fowls tolerated daily doses of 300 mg.Ag.
daily in their feed for several weeks without visible effects. Repeated
daily doses of 50 mg.Ag., however, led to toxic symptoms in some
pigs. In longer term studies (Erne 1966a), five young pigs were fed
500 p.pjn. of 2,4-D for up to 12 months but, although various toxic
effects were noted and their growth rate was affected, none of the
animals died. When 2,4-D was fed to a sow throughout gestation and
for a further six weeks, 10 of the 15 underdeveloped and apathetic
piglets she produced died within 24 hours and the mother subsequently
had to be slaughtered because of abnormalities that developed in her
spine. Heavy dosing of pregnant rats, however, with 1000 p.p.m. of

f

0007432

/546800

auxin herbicides to a variety of domestic animals by Kephart (1945)
(cow), Rowe and Hymas (1954) (laboratory animals and cattle),
Dalcaard-Mikkelsen et a i (1959) (heifers), Palmer (1963) (cattle),
Palmer and Radelefk ( 1964) (sheep and cattle), Clarke et a l ( 1964)
(sheep), and Strach and Bohosiewicz (1964) (pigs). Palmer (1963)
gave daily oral doses of 2,4-D alkanolamine salt to steers for five days
in every seven. He recorded signs of poisoning in animals dosed at
250 mg.Ag. after 15 administrations as opposed to 88 administrations
of 100 mg.Ag.; at 50 mgAg. no ill-effects were recorded over a period
of 112 administrations. From these results he concluded that although
animals could probably ingest enough 2,4-D from concentrated solu­
tions at any one time to produce illness or death, the chronic toxicity
of the compound was sufficiently low to make it unlikely that an animal
would pick up enough of it over a period of time to cause any serious
ill-effects. Further work by Palmer and Radeleff (1964) using single
animals gave the following results:

J. M. W ay

40

I546S01

2,4-D in their drinking water over 10 months and of their off-spring
for up to two years, whilst leading to retarded growth and increasea
mortality, did not produce unequivocal signs of toxicity. Continued
administration of 500 p.p.m. of 2,4-D in feed or 1,000 p.pjn. in the
drinking water of fowls led to reduced egg production and kidney
abnormalities. These results led the authors to conclude that the
chronic toxicity of 2,4-D to the species studied was moderate. They
were, however, concerned about the mortality of new-born piglets,
with evidence of movement of 2,4-D through the placental tissues, and
the reduced egg production in fowls which they thought might indi­
cate a possible interference with reproductive processes.
In general the findings of other workers support these conclusions
on acute and chronic toxicity. In all the work quoted the amounts
administered to the test animals for effect, have been well in excess
of the amounts they might be expected to pick up from a treated
pasture, or in feed derived from crops that had at some time been
treated with auxin herbicides at normal dose rates.
b ) Indirect toxicity

Indirect effects of herbicides on grazing animals have been associ­
ated with increased toxicity of toxic plants, increased palatability of
normally non-palatable toxic plants (e.g., ragwort, Senecio jacobaea ),
and induced toxicity in normally nontoxic plants (e.g., temporary in­
creases in nitrate content) ( Wellard 1950). However, Fame (1953)
claimed that, up to 1953 in America, in all cases where poisoning of
livestock from herbicides had been reported, the effects noted could be
attributed to some other cause. '
■Examples have been given by Willard (1950) of cattle eating
■wild cherry ( Prunus serotina), of pigs eating Cocklebur (Xanthium
sp.), and of lambs eating thistles after herbiddal treatment with
auxins. Instances have been reported of ragwort becoming 'sweeter'
from two or three days after application and being preferentially
grazed by cattle for a short period. Giucsby and Ball (1952) and
Lynn and Barrons (1952) investigated the hydrocyanic acid (HCN)
content of the leaves of wild cherry from untreated trees and trees
treated with 2,4-D and 2,4,5-T. Their conclusions were that the foliage
was no more toxic to cattle after treatment and that there might even
be less HCN in the leaves of the treated trees than in those of the
untreated ones. Buck et ai. (1961) fed the alkaloid-containing plants
Delphinium barbeyi (tall larkspur) and Helenium hoopscii (sneeze
weed), after treatment with 2,4-D ester or 2,4,5-T ester, to calves and
ewes. No increased toxicity of the plants attributable to application
o‘f the herbicides was noted. Williams and Cronin (19G3) analysed
D. barbetji, treated with 2,4,5-T amine at various growth stages, and
showed lliat the alkaloid content of the plants was increased for several

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weeks after treatment at the vegetative and early bud stages. It was
noted, however, that the bitter taste of the alkaloids might make the
treated plants even less palatable to animals than untreated ones.
Swanson and Shaw (1954) showed that 2,4-D affected the HCN
content of Sudan grass ( Sorghum vulgare ssp. stidanense). Initially
there was a decrease in the content of IICN but four days after treat*
xnent there was an increase over the controls which was maintained
for a further 12 days. Similar effects were shown to occur with the
nitrate content of leaves. Buck et al. thought that there might be a
relationship between HCN and nitrate metabolism in Sudan grass, an
increase in one leading to a decrease in the other.
The clinical aspects of nitrate poisoning in stock, conditions under
which nitrates are likely to accumulate in the leaves of certain plants,
and lists of these plants have been reported by Bradley et a l (1940),
Davidson e t a l (1941), Gilbert e t a l (1946), Case (1957), and
Sund et a l (1960). The toxic effects of nitrate are caused bv a re­
duction of nitrate to nitrite and the conversipn by nitrite of haemo­
globin in the blood to methoglobin: the animal dies from asphyxia.
Intravenous iniection of methylene blue in doses of two g./500 pounds
of body weight gives immediate relief. Nitrate in plants is generally
resent in the form of potassium nitrate and increases in nitrate content
ave been associated with drought conditions and high soil nitrogen
(Gilbert e t aL 1946, Case 1957). Sund et a l (1960) noted a high
nitrate content in Urtica spp. and Rubus spp. after heavy rains,
followed by preferential grazing of these and other weed species by
cattle. A number of abortions in these cattle was correlated with oc­
currence of high nitrate rather than grazing of the weed species per se.
Recent increases in vitamin A deficiency in North American ruminants
has been associated with ingestion of nitrates occurring in herbicidetreated plants by PuiLLirs (1964).
The accumulation of nitrates in the leaves of sugar beet is well
known (e.g., Savace 1949). Increased levels of nitrate in the leaves
of this crop as a result of herbicide application have been reported by
Willard (1950), Stahler and Whitehead (1950), and Whxiekead
et aL (1956). Isolated incidents have been reported of nitrate poisoning
of cattle in America as a result of feeding on sugar beet that had
previously been sprayed. In one incident in N. Dakota, the nitrate
content of sugar beet leaves after spraying was found to vary from
1.81 to 8.77 percent of the dry weight, as against 0.22 percent for un­
treated plants and a toxic level of 1.5 percent (S tahler and Whttehead 1950). Studies on forage crops (B erg and McElroy-1953) and on
a range of weed species (F rank and Cricsby 1957) have shown which
of these may contain high levels of nitrates after auxin application.
Cell-free extracts of maize and cucumber from plants that had been
previously sprayed with 10 and 100 p.p.m. of 2,4-D were investigated
by S ervers and H aceman (1962). Tlic level of nitrate reductase was

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increased in maize but reduced in cucumber. Studies on the formation
and breakdown of nitrates in plants (Fame 1952, Freiberc and Clare
1952, Whitehead et al. 1956) have shown that 2,4-D causes more rapid
increases in nitrate content than MCPA, that levels rise to a peak soon
after spraying and subsequently decrease with time, and that increases
in light intensity hasten decreases in nitrate content
It is clear from these reports that nitrate poisoning in stock does
occur from time to time and that it is possible for the hazard .to be
increased by application of auxin herbicides to nitrate-accumulating
plants.
IV. Bees and other insects
Herbicides affect bees (Apis mcllifera ) and other insects if they
kill the plants on which the insects feed. In addition, Wahlin (1950)
has reported that 2,4-D and MCPA were toxic to bees, not only from
visiting the flowers but also as a result of drinking contaminated water
trapped on treated plants. Antoine (1966) reported that MCPA para­
lysed and killed bees which ingested doses that corresponded to those
recommended for weed control. Other workers have reported effects
on bees after application of auxin herbicides to plants in flower but
not at other times (Haracsimova 1962, Palmer-Jones 1964). PalmerTones (1964) and Antoine (1966) have suggested that 2,4-D might
nave some effect on nectar which made it toxic to bees. Kinc (1960a)
has shown that radioactive 2,4-D can be translocated to the nectar of
Poinsettia and red clover plants and may be detectable there for two
to three days after treatment Feeding trials of auxin herbicides to
bees have been reported by Glynne Jones and Connell (1954),
Palmer-Jones (1960), King (1960b), and Byrdy (1962). PalmerJones (1964) found no effect on bees that had been directly dusted
with 2,4-D or when they were made to crawl through 2,4-1) dust in
order to enter the hive. Glynne Jones and Connell (1954) classed
2,4-D and MCPA as stomach/contact poisons of low toxicity to bees,
with LDgo values of 0.105 mg. compared to insecticides in the range
0.00004 to 0.002 mg. Byrdy (1962), on the other hand, reported total
mortality of bees within four days of feeding 30 pg. of 2,4-D and 10
percent mortality within three days rising to 20 percent in five days of
feeding 20 pg. Johansen (1959) reported that 2,4-D and related com­
pounds were not toxic to bees, except when formulated as the alkanolamine salt or the isopropyl ester. Haracsimova (1962) found that a
30 percent calcium/potassium salt formulation of MCPA was toxic to
bees and caused mortality up to 13 days after ingestion. However,
part at least of the loss was attributed to cresol impurities, which led
to loss of smell amongst the bees with consequent loss of sense of
direction.
Occasional observations on other insects have been reported.
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Mellanby ct al. (1959) observed that a spray of mecoprop on oats at
the time of a heavy frit fly attack had no effect on the fly ( Oscinclla
frit). Maxwell and Harwood (1960) treated broad bean (Vicia faba)
plants with sublcthal doses of 2,4-D. and recorded a marked increase
in the reproduction of the pea aphid ( Macrosiphum pisi) feeding on
them. The longevity of adult aphids was unaffected. Robinson (1959)
also recorded increased fecundity in another pea aphid ( Acyrthosiphon
pisum ) after caging on broad bean plants treated with 2,4-D. Adams
(I960) and Adams and Drew (1965) showed that the application of
2,4-D amine could enhance aphid infestation in New Brunswick grain
fields, probably as a result of depressing the activities of cocdnellid
beetles predating on the aphids. In laboratory experiments with cocdnelh'd larvae treated with 2,4-D amine, there was a fourfold increase
in mortality and an increase in time to pupation. There was little
mortality amongst the adult beetles, which usually recovered after a
few hours inactivity. Ismi and Hxrano (1963) concluded that increases
in the growth rate of the larvae of the rice stem borer (Chile suppressalis ) feeding on 2,4-D treated rice plants, was a consequence of in­
creased nitrogen content of the plants rather than a direct effect of the
chemical itself.
In general, it appears that there is a real hazard to bees—and pos­
sibly other nectar feeding insects—from applications of auxin herbi­
cides to plants in flower. Otherwise there would seem to be little
hazard to insects from direct toxidty of the compounds at normal
herbicidal rates of application. However, the work of Adams and Drew
(1965) does suggest that some insects may be more susceptible at
particular stages of their life cycle than at others.

i

V. Soil animals
Amongst soil arthropods, Davis (1965) was unable to show any
differences in poDuluticns of Aeari or Collcmbola sooci^s between un­
treated plots and plots that had been sprayed with MCPA in 10 out of
13 yean. Davis (1965) was unable to find any reports of significant
effects on soil animals in other experiments of a similar nature. Bollen
(1961) concluded that auxin herbicides, based on phenoxyacedc and
propionic acid, were the most susceptible to breakdown by micro­
organisms of the many pesticides applied to the soil. The importance
of soil microorganisms in the breakdown of these herbicides is well
known from the work of Audus (1964) and othen. Webster (1967)
has briefly reviewed the literature on the influence of plant growthregulator auxin herbicides on the host/parasite relationships of nema­
todes, in which 2.4-D has been shown to increase nematode reproduc­
tion in plant callus cultures. In addition, plant cell hypertrophy and
proliferation, which is a common effect of 2,4-D in many plants, pro­
vides highly suitable conditions for development of nematodes. In

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this way susceptibility of a normally nematode-resistant variety of oats
could be induced, although there did not appear to be any greater
susceptibility of a non-resistant variety.
VL Fish and aquatic organisms
Under field conditions the toxicity of a pesticide in water is affected
by a number of factors in addition to those that affect its performance
on land. Thus acidity, hardness of the water, and the sorbent qualities
of suspended organic matter in the water may directly effect the
toxicity. The trophic nature of the ecosystem, the oxygen status of the
water in respect of both producers and demand, and the amount of
movement of water both within the system and in terms of flow will
affect the concentration of the chemical, its persistence, and its possible
toxic side effects. Because of these, and many other interacting factors,
the toxicity of a given formulation of a given chemical compound to
an individual species will vary under field conditions depending upon
the nature of the water body and the immediate environment. For this
reason toxicities to fish and aquatic organisms are usually estimated
in terms of median tolerance limit for exposure to a given concentration
of the pesticide, for a given length of time (TLmx).
In addition to direct or indirect toxicity, the effects on aquatic
organisms of the removal of the substrate that gives them food and
shelter must also be considered. For instance, in one of the Tennessee
Valley Authority’s reservoirs two applications of 2,4-D controlled con­
siderable acreages of Eurasian water milfoil ( Mtjriophijllum spicatum).
The eradication of the plant eliminated the substrate that might have
been colonized by large populations of epiphytic insects such as the
larvae of midges, mayflies, and dragonflies (Smith and Isom 1967).
Although under some conditions this might have a very serious effect,
it has been shown by Way et aL (1968) in an ecological investigation
of the use of paraquat in lakes, that although individuals and popu­
lations of animals may be severely affected in the weeks after applica­
tion of a herbicide, the species affected may be present again in the
season after treatment. It has also to be recognized that very heavy
infestations of submerged or floating aquatic plants may interfere with
the passage of nutrients and considerably reduce the temperature and
dissolved oxygen values of the water (Fisn 1966). Thus, any possible
hazards from the use of a herbicide may be outweighed by the advan­
tages gained from the removal of the vegetation. ■
The danger of significant amounts of 2,4-D appearing in ground
water or streams as a result of local applications seems to be slight.
Aldiious (1967) measured residues of 2,4-D in drainage channel water
after aerial spraying of a Scottish forest at four pounds/acre active
ingredient in 12 gallons of water. Up to two p.p.m. of 2,4-D was
measured in the seven days after application but none was detectable

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by 2S days. Brown and Nishioka (1967) analysed samples of a watersuspended sediment mixture from 11 streams in the western United
States at monthly intervals. Amongst the analyses made were gas
chromatograph determinations for 2,4-D and 2,4,5-T. Neither com­
pound was found in any of the samples. The absence of the compounds
was attributed to some extent to their susceptibility to degradation.
Reviews of toxicity hazards to fish of a range of pesticides, in­
cluding auxin herbicides, have been made by Bauer (1961), Bandt
e t al. (1962), and Cope (1965 and 1966). Cope (1966) noted that
variations in formulation gave rise to greater differences in toxicity
than the differences in toxicity between the basic compounds. Ester
formulations were often more toxic than amine or metallic salt formu­
lations. Similar observations were made by Lhoste (1959) who re­
viewed effects on a number of crustaceans, aquatic insects, and
molluscs.
Trout ( Salmo trutta ) are normally regarded as being amongst the
most sensitive fish to water pollution. Alabaster (1958) lias given
median tolerance limits for 24 and 48 hour (TLm-i and TLm«) ex­
posures of trout to 2,4-D or 2,4,5-T, or to mixtures of these two
compounds, of 9.5 to 250 p.p.m., depending on formulation, compared
to 1,150 to 2,000 p.pjn. for sodium chlorate or 0.005 p.p.m. for phenyl
mercuric acetate. Holden (1964) devised a formula for comparing the
likely toxic hazards to trout from a number of pesticides1 applied at
agricultural rates. The following comparative estimates of hazard were
given: aldrin - 70, PCP - 7, MCPA -1.5, 2,4-D -1 , 2,4,5-T - 0.5, para­
quat -1/12, simazine -1/27, diquat - 1/40, dalapon * 1/46, TCA 1/120, and aminotriazole -1/150. It should be emphasized that these
figures refer to agricultural rates of application: hazards from the con­
centrates might be different.
Perch (Perea fluviatilis) and roach ( Rutilus rutilus) are unlikely
to be affected by MCPA, 2,4-D, or 2,4,5-T (Bandt 1957) at rates of
application used for aquatic weed control, although a commercial mix­
ture of 2,4-D and 2,4,5-T was more hazardous. In later trials Bandt
e t al. (1962) found threshold values for toxicity to perch and roach of
MCPA of 200 to 215 mg./litre, of 2,4-D of 75 mg./Iitre, of 2,4,5-T of
55 to 60 mg./litre, and of 2,4-D + 2,4,5-T mixtures of 5 to 12 mg./litre.
These results show rather greater toxicities of 2,4-D and 2,4,5-T com­
pared to MCPA than were shown in Holden’s (1964) calculations
(see above). The much enhanced toxicity of the 2,4-D + 2,4,5-T mix­
tures may have been an effect of the particular formulations used, or
1Aldrin ■ l t2^3,4,10,10-hexaclilorx>.l,4,4a^>,8,8a-hexahydro-«o-l,‘i-<m<£o-5,8dimcthanonaphthalene; PCP » pentachlorophenol; paraquat ■ l,l'-dimethyl-4,4'bipyridylium dichloride; rimnzlne ■ 2-chloro-418-biscthylamino-l,3,5-triazine; di­
quat ■ 9,10-diliydro-8a,10a>diazonaiaplienanthrene dibromidc; dalapon » sodium
2,2-dichloropropionatc; TCA « tricldoroacctic add; and aminotriazole « 3-amino1,2,4-triazole.

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could demonstrate a synergistic effect of one compound on the other.

Davis and Habdcastle (1959) established median tolerance limits

over a 24 hour period (TLm24) for bluegill sunfish ( Lepomis macrochirus) to a number of herbicides.. Values obtained when the com­

¿089PS/

pounds were added to relatively pure water were 2,4-D - 39 p.p.m.,
MCPA - 20 p.p.m., 2,4-DB - 20 p.p.m., and 2,3,6-TBA -1,800 p.p.m.
Cofe (1966) noted delays in spawning of bluegill sun£sh of up to two
weeks after treatment of water with the propylene glycol butyl ether
ester of 2,4-D at five and 10 p.p.m. However, no other effects were
noted on reproduction or on survival of fry. In pond experiments, death
of some fish as a result of 2,4-D treatment led. to increased size in the
survivors, probably as a result of the greater food supply available
to the individual fish. In further trials with bluegill sunfish, Huches
and Davis (1963) and Davis and Huches (1963) reported on effects
of different formulations of 2,4-D and other auxins. Their tests showed
2,4-D and 2,4,5-T esters to have TLmMranging from 1.8 to 10 p.p.m.
depending on the ester used. Dimethylamine salts of 2,4-D and 2,4,5-T
had TLm5 4 of 162 to 542 p.p.m. and 144 p.p.m., respectively, com­
pared to the alkyl amine salt of MCPA of 163.5 p.p.m. and of 2,4-D
acid of 8.0 p.p.m. This work (which is referred to in Cope 1966, see
above) shows the wide differences in toxicity that can occur in differ­
ent formulations and the care which must therefore be taken in assess­
ing the toxicity of an individual product before recommending it for
use as an aquatic herbicide. •
In addition to work on fish, Walker (1962) has reported effects
on a variety of bottom-feeding fish food organisms following applica­
tion of 2,4-D to plastic enclosures at 1.0 to 4.0 p.p.m. Lhoste (1959)
has reported that ester formulations of 2,4-D or mixtures of 2,4-D and
2,4,5-T affected crustaceans, aquatic insects, and molluscs in the range
of 0.1 to 3.3 p.p.m. In their investigations into the effect of the use
of 2,4-D at rates from 40 to 100 pounds/acrc in Tennessee Valley
Authority reservoirs. Smith and Isom (1967) found no measurable
toxic effect on benthic fauna or significant changes in mean numbers
of burrowing mayflies ( Hexagenia) before or after treatment There
were some deaths of caged fish but no dead or distressed free fish
were found. Observations of free living fish indicated that they ap­
peared to move out of the treated area, whilst analysis of a number of
fish for 2,4-D residues gave no measurable results at the limit (0.14
p.p.m.) of detection. However, analysis for residues in mussels (mainly
Elliptio crassidens) indicated that they concentrated the chemitkl from
the surrounding water although no mortality was recorded. Analysis
of mud samples showed that significant amounts of 2,4-D (up to 58.8
p'.p.m. butoxycthanol ester) were present in isolated sediment samples
up to 10 months after treatment. These fairly high levels in the en­
vironment of a sedentary animal such as the mussel might explain the
residues found in these animals. Rawls (1965) has reviewed literature

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on the toxicity of herbicides to estuarine fauna, principally Crustacea
and mollusca, and described experiments from his own laboratory. A
variety of formulations of 2,4-D affected several species of animals at
concentrations of one to 10 p.p.m. Larvae of oysters ( Crassostrea
virginica ) were killed over a period of time by one p.p.m. of butoxyethanol ester and dimethyl salt of 2,4-D, and though oyster eggs were
less susceptible, their development could be affected by 2,4-D butoxy-'
ethanol ester at 10 p.p.m. These effects might give some cause for
alarm when rates of application of 20 to 30 pounds/acre arc required
for weed control in estuaries. Doses of this sort would give 7.34 to
11.02 p.p.m. if evenly distributed throughout one acre of water one
foot deep. Because of tidal duxes and uneven distribution only very
local concentrations at toxic or near toxic amounts may occur in
practice.
■The results of these various investigations suggest that at hcrbicidal
rates of application of auxins the hazards from acute or chronic toxidties to aquatic organisms are low. Nevertheless in some instances
the dose rates required for effective herbicidal action, for example in
estuaries or where the chemical is likely to be rapidly dispersed, may
give rise to local and perhaps short term concentrations not far re­
moved from those required for toxic effects on some organisms at sus­
ceptible stages of their life history.
VIL Wildlife in general
Hazards to wildlife from auxin herbicides have been reviewed by
Rudd and Genelly (1956), Sfbincer (1957), and Mellanby (1967).
Whilst with any material having biological activity a risk of acute or
chronic toxicity is always present, authenticated incidents of widcscale
poisoning of wild animals by these herbicides have not been reported.
However, there is the furtner hazard to all forms of wildlife from
herbicides of altering the habitat. Whilst herbicidal applications of the
compounds to agricultural land undoubtedly modify tne habitat there
for wildlife, it is unlikely that individual instances of spray drift of the
compounds at sublethal doses, although undesirable, have any really
significant effect on habitat of adjoining non-cultivatcd land. This is
not to say that some food plants for animals are not regularly killed
in hedgerows and other field margins from spray drift, nor that in
isolated instances of gross mishandling more severe effects do not occur
over larger areas. It is probably true, although there is little or no
scientific evidence, that tne continued use of herbicides in the U.K. is
gradually leading to the impoverishment of the flora‘of the field
margins, and consequently to the gradual impoverishment of agri­
cultural land as a whole as a habitat for wildlife.
H e principal problem from the use of auxin herbicides in relation

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to wildlife, is not toxicological but ecological in terms of scale and
intensity of use, especially on non-agricultural and industrial land.
Thus the total destruction of a small area of vegetation on one occa­
sion might be less serious for the wildlife of a region than the selective
destruction of vegetation over a wide area at regular intervals. The
hazard from the use of herbicides lies in the fact that they can now
be used to affect vegetation over very wide areas in a short period of
time, thus eliminating 'reservoirs’ of wildlife and wildlife habitat that
would have otherwise survived.
Summary
Toxicities and hazards to man, domestic and laboratory animals,
wildlife, bees, and other insects, soil animals and fish from MCPA,
MCPB, 2,4-D, 2,4-DB, 2,4,5-T, mecoprop, and 2,3,6-TBA are discussed.
In man only one authenticated instance of death by poisoning has been
noted in West European and North American literature. Authenticated
case histories of sublethal effects are also very rare. However, com­
plaints of transient dizziness, sickness, and other symptoms are made
rrom time to time by workers engaged in field applications, especially
under conditions- where the spray is inhaled excessively. The possibility
of man acquiring toxic doses of these compounds in food, milk, or
water appears to be very low.
Toxicity tests on a range of laboratory and domestic animals with
a wide range of formulations of the compounds tend to show greater
differences Detween formulations than between compounds. Amongst
the domestic animals studied dogs appear to be more susceptible than
• the others. Acutely toxic doses of the compounds to farm animals and
fowls are shown to be greater than the animals could normally ingest
from treated vegetation or water following an agricultural application.
Hazards to stock from the concentrated materials are emphasized, but
the offensive and unpalatable nature of the undiluted chemicals would
probably preclude any normal animal from ingesting a toxic dose. The
danger of chronic toxicity is also shown to be very low and continuous
high dosing over periods of months rather than weeks are required
to produce severe symptoms of poisoning. In many animals a high
proportion of the active chemical is voided in the urine within 24 to
48 hours of ingestion. Secondary hazards to animals as a result of
changes in the chemical constituents of food plants may occur, for
instance toxicity has been associated with temporary increases in the
nitrate contents of some plants after spraying. Current recommenda­
tions for stock to be excluded from treated pasture for a period after
treatment should be observed.
Hazards to wildlife exist from the use of herbicides but are also
associated with other changes in agricultural practices. The effects
of herbicides on habitat ore serious for wildlife and should not bo
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underestimated. Deaths of bees have occurred following spraying of
nectar-producing plants when in flower. It is possible that similar
hazards exist for other nectar-feeding insects. Where other insects have
been studied, increases in population have been noted more often than
decreases. With some aphids this has been associated with reduction
in numbers of predators. No effects have been noted in studies of
populations of soil arthropods. Increased activity by nematodes has
been associated with the favourable conditions for invasion of the
host plant created by auxin induced teratomas and other effects.
Toxicity to fish and other aquatic organisms has been shown to depend
on the formulation of the compound used. In general, toxicity to fish
is low and hcrbicidal applications to water do not present a severe
direct hazard. The dangers of secondary effects must be assessed in
relation to other methods of control and the environmental char­
acteristics of the water body to be treated. Some aquatic organisms
other than fish may be directly affected at dose rates not very much
in excess of those recommended for hcrbicidal use in water.
Résumé *
Sont discutés les toxicités et les dangers, pour l’homme, les animaux
domestiques et de laboratoire, la vie sauvage, les abeilles et autres
insectes, la faune du sol et les poissons, des composés suivants: MCPA;
MCPB; 2.4.D.; 2.4.DB; 2.4.5.T; mecoprop et 2.3.6.TBA. Chez l'homme,
un seul cas authentifié de mort par intoxication a été relevé dans la
bibliographie de l’Europe occidentale et de l’Amérique du Nord. De
même, les cas authentiques d’effets subléthaux sont très rares. Néan­
moins, des sujets affectés à l’épandage de ces produits se plaignent, de
tempÿ à autre, particulièrement lorsque les conditions ae dispersion
exposent à une inhalation excessive, ae vertiges passagers et ¿’autres
symptômes et se sentent malades. L’éventualité d'absorption par
lîiomme de doses toxiques avec les aliments, le lait et l’eau semble
très limitée.
Les épreuves de toxicité sur une grande variété d’animaux de la­
boratoire et d’animaux domestiques tendent à montrer que les diffé­
rences sont beaucoup plus grandes en ce qui concerne les formulations
qu’en ce qui concerne les matières actives. Parmi les animaux domes­
tiques, le chien paraît être le plus sensible. Les doses susceptibles de
irovoquer des effets de toxicité aigüe chez les animaux de ferme et
es volailles sont supérieures à celles quë ces animaux pourraient
ingérer à partir des plantes traitées ou de l’eau éventuellement polluée
à la suite des applications en Agriculture. L’accent est mis sur les
risques pour le bétail des formulations concentrées, mais la nature
agressive et le mauvais goût de ces formulations rendent très impro-

{

* Traduit par R. Taciuur.

0007442
i

ObU4

I

J. M. Watt ’

50

MW I 546811

bable l’ingestion d’une dose toxique par un animal normal. Les risques
do toxicité chronique sont également très faibles, car des doses élevées
doivent être répétées, non seulement pendant des semaines mais
endant des mois, pour pouvoir provoquer des symptômes graves
’empoisonnement. Chez beaucoup ¿’animaux, une forte proportion des
matières actives est excrétée dans l'urine dans les 24 à 48 heures qui
suivent l’ingestion. Des risques secondaires pour les animaux peuvent
résulter de modifications dans les constituants chimiques des végétaux
consommés; par exemple, des effets toxiques ont été associés à des
augmentations temporaires des taux de nitrates de certaines plantes
consécutives aux traitements. Il convient de respecter les recommanda­
tions relatives à l’exclusion du bétail des pâturages traités pendant une
certaine période après le traitement.
Des dangers pour la vie sauvage peuvent résulter de l’usage des
herbicides, mais ils peuvent également être causés par d’autres varia­
tions dans les pratiques agricoles. Les effets des herbicides sur l'habitat
sont graves pour la vie sauvage et ne doivent pas être sous-estimés.
Des morts se sont produites chez les abeilles à la suite de traitements
de plantes mellifères à l’époque de la floraison. Il est possible que des
risques analogues existent pour les insectes qui se nourrissent du nectar
des fleurs. Les études relatives à d’autres insectes ont révélé des aug­
mentations de populations plus fréquemment que des diminutions.
Dans le cas de quelques especes de pucerons, cet accroissement était
associé à une réduction du nombre des prédateurs. Aucun effet n’a
été observé dans les études sur les populations d’arthropodes du sol
L’accroissement d’activité des nématodes était lié aux conditions favo­
rables pour l’invasion des plantes hôtes créées par les tératomes et
autres effets dûs aux auxines. La toxicité pour les poissons et les autres
organismes aquatiques s’est révélée dépendre du type de formulation
utilisée pour l’épandage de la matière active. En général, la toxicité
pour les poissons est basse et les applications d’herbicides à l’eau ne
présentent pas de risques directs graves. Les risques d’effets se­
condaires doivent être évalués en liaison avec d’autres méthodes de
lutte et les caractères de l’environnement des eaux à traiter. Quelques
organismes aquatiques autres que les poissons peuvent être directement
affectés à des doses ne dépassant pas beaucoup celles recommandées
pour l’emploi des herbicides dans les eaux.

S

Zusammenfassung *
Toxizität und Gefährdung bei Mensch* Haus- und Laboratoriums­
tieren, Wild, Bienen und anderen Insekten, Bodcnticrcn und Fischen
durch MCPA, MCPB, 2,4-D, 2,4-DB, 2,4,5-T, Mecoprop und 2,3,6-TBA
* Übersetzt von F. Baa.

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Toticity and hazards of auxm hcrbicidcs

57

DOW 1 546812

werden diskutiert. Beim Menschen wurde nur ein verbürgtes Beispiel
eines Todesfalles infolge Vergiftung in der westeuropäischen und
nordamcrikanischcn Literatur berichtet Verbürgte Fälle von sublcthalcn Effekten sind auch sehr selten. Jedoch werden von Zeit zu
Zeit Klagen über vorübergehendes Schwindclgcfühl, Übelkeit und
andere Symptome von Arbeitern vorgebracht, die in der Anwendung
auf dem Felde tätig sind, besonders unter Bedingungen übermässiger
Spray-Inhalation. Die Möglichkeit für den Menschen, toxische Dosen
dieser Verbindungen in Lebensmitteln, Milch oder Wasser zu er­
reichen, scheint sehr gering zu sein.
Toxizitätsteste bei einer Reihe von Laboratoriums- und Haustieren
mit einem weiten Bereich von Formulierungen der Verbindungen
neigen dazu, grössere Differenzen innerhalb der Formulierungen als
zwischen den Verbindungen zu zeigen. Von den untersuchten Haus­
tieren scheinen Hunde empfindlicher zu sein als andere. Die akut­
toxischen Dosen der Verbindungen für Vieh und Geflügel waren höher
als sie die Tiere normalerweise aus behandelten Pflanzen oder aus
Wasser nach Anwendung in der Landwirtschaft aufnehmen können.
Die Gefährdung des Viehbestandes durch konzentriertes Material wird
hervorgehoben, aber die agressive und widerwärtige Natur der un­
verdünnten Chemikalien würde wahrscheinlich jedes normale Tier von
der Aufnahme einer toxischen Dosis abhalten. Die Gefahr der chro­
nischen Toxizität erwies sich auch als sehr gering und eine kontinuier­
liche hohe Dosierung eher über Monate als über Wochen ist erforder­
lich, um schwere Vergiftungssymptome zu erzeugen. Bei vielen Tieren
wird ein hoher Anteil der aktiven chemischen Stoffe im Ham 24-48
Stunden nach der Aufnahme entleert. Sekundäre Gefährdung bei
Tieren als Ergebnis von Veränderungen der chemischen Bestandteile
von Nahrungspflanzen kann Vorkommen; zum Beispiel war die Toxi­
zität begleitet von einem vorübergehenden Anstieg des Nitratgchaltes
gewisser Pflanzen nach dem Besprühen. Laufende Empfehlungen für
die Fernhaltung des Viehbestandes von der behandelten Weide für
einige Zeit nach der Behandlung sollten befolgt werden.
Eine Gefährdung für Wild besteht durch die Anwendung von
Herbiziden; sie ist jedoch ebenso verbur ' ~i mit anderen Änderungen
in der Landwirtschaftspraxis. Die Effe...e der Herbizide auf dem
Standort beim Wild sind ernst und sollten nicht unterschätzt werden.
Tod von Bienen erfolgte nach dem Besprühen von Nektar-erzcugenden
Pflanzen in der Blüte. Es ist möglich, dass eine ähnliche Gefäiirdung
für Nektar-fresscnde Insekten besteht Beim Studium anderer Insekten
wurde ein Populationsanstieg häufiger als eine -abnahme festgestellt
Bei Blattläusen war dies verbunden mit der Herabsetzung der Anzahl
der Raubinsekten. Keine Effekte wurden fcstgestcllt in Untersuchungen
von Populationen von Bodenarthropoden. Eine gesteigerte Aktivität
durch Nematoden war verbunden mit günstigen Bedingungen für den
Befall der Wirtspflanze, erzeugt durch Auxin-induzierte Teratome und

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References

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Adams,

t

000744S

flOIV I 546813

andere Effekte. Die Toxizität für Fische und andere Wasserorganismen
zeigte eine Abhängigkeit von der Formulierung der angewandten
Verbindung. Im allgemeinen ist die Toxizität für bische gering und die
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lenen liegen.

Toxicity and hazards of auxin herbicides

SO

546814

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+

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546815

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Toxicity and hazards of auxin herbicides

81

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00
C3

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Swanson,

546817

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in water by micro..,.3 . J. Amer. Water

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Toxicity and hazards to man,
domestic animals, and wildlife
from some commonly used

o
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sri
. <*•

436190

,..r in s e c t ic id e

auxin herbicides
By
J. M. W ay •

Contents

Resumi ................................................................................

Zusammcnfassung
References.........

fcssgsssgBsass

I. Introduction ......................................................................
II. Toxicity and hazards to m an............................................
HI. Toxicity and hazards to domestic and laboratory animals
a) Acute and chronic toxicity...........................................
b) Indirect toxicity............................................................
IV. Bees and other insects........................................................
V. Soil animals ........................................................................
VI. Fish and aquatic organisms................................................
VII. Wildlife in gener»!..............................................................
Summary .....................................................................................

L Introduction
This review has been compiled from searches in the literature for
references to the toxicity and other hazards that may arise from the
use of auxin herbicides, and specifically of formulations of MCPA
(4-chloro-2-methylphcnoxyacetic acid), S.S. 2,4-D (2,4-dichlorophenoxyacetic acid) and of their butyric acid analogues MCPB and
2,4-DB, of 2,4,5-T (2,4,5-trichlorophenoxyacetic acid), mecoprop
«-(4-chIoro-2-methylphenoxy) propionic acid], and 2,3,6-TBA
(2,3,6-trichlorobenzoic acid).
Assuming 70 percent of the eight million acres of cereals grown in
the United Kingdom to be treated annually with herbicides (Wood-

* Monks Wood Experimental Station (The Nature Conservancy), Abbots
Ripton, Huntingdon, England.
37

5613

C
f"
k.«..

38

O

i,>

CO
a

J. M. Way

ford 1964), it is apparent that the tonnage (circa 2,500 tons) of the
compounds mainly used for this purpose is similar to the total tonnage
of insecticides and fungicides used annually on all crops in the U.K.
(circa 2,660 tons) ( S trickland 1966). In 1962, D add (1962), from a
survey of eastern England, found that over 90 percent of cereal fields
treated with herbicides were treated with auxin compounds based on
chlorinated phenoxy acid derivatives, notably salts of MCPA and to a
lesser extent of mecoprop. Although the use of straight formulations of
MCPA has been gradually declining ( W oodford 1964) in favour of
mixtures of compounds, many of these mixtures still include MCPA
or other phenoxy compounds and increasingly include 2,3,6-TBA. In
addition to its agricultural use on arable and pasture crops, 2,4-D to­
gether with 2,4,5-T, is used, or is likely to be used on an increasing
scale, for scrab control and control of unwanted broad-leaved tree
species in conifer plantations. 2,4-D is also used to a limited extent for
control of submerged aquatic weeds. On a world-wide basis 2,4-D is
probably used to a greater extent than any other herbicide and this is
reflected in the greater number of references to this compound. The
extensive use or a single biologically active family of compounds
clearly presents a considerable potential hazard in respect to their
direct and indirect toxicities to animals including man. It is a measure
of the comparative safety of the auxin herbicides that, in spite of
their increasing use throughout the uJC. iltttig the mld-1940's, they~_
eonLiuue to be applied much in Tli<±Sama juuUne way us an* lUCTganic
fertilizersTand WitVm lilllt regard for nnv possible tu.iii. luzant
Liunng the many yeuis uf their use tlnuughuut the wona mere have
been notably lew authenticated incidents ot poisoning of domestic
animals or wildlife resulting from the proper application of these com­
pounds. Tiie tact that fully authenticated incidents of poisoning have
noFbeen reported cannot be taken as an absolute guarantee of safety,
particularly with regard to indirect or chronic toxicity, because ot the
frequent dilticulties oi attributing the Underlying cause ot an illness
or of death, especially in wild animals. Nevertheless all the direct and
circumstantial evidence to hand at the prfefiHt tlmfe seems to indicate
that incidents ot poisoning naVfe bfeen 'extremely few and generally
under exceptional circumstances. ~ "
"
’

ril liar.'..
handlin'.:
d ilu tio n .

ergannn
tests or
majority
ii re su lt i
p rese n t

handled

K mai

of 2,4-D
cal study
old man
of 50 pi
2.4-D in
cquivale:
active in
nerve tiv
V a i .i.:

50 to 5(i
and of N

these litv
animals,
on a mg.
as a Iahe
dangers t
the toxic
on the m<
a ntimhi-i
ment of (
mg./m.5 <
V alle r's

range of
lie of the
80 ing./k'.
this figiiu
a greater
produce •
I.l)iu,S to
between 1

IL Toxicity and hazards to man
The principal routes of toxicity to man are either orally or by in- '
halation; there appears to be little hazard of transport tlirough the
skin although individual allergies can develop leading to detmatitis
(Vallet*1SG5). Eyes may be directly but are usually only temporarily
affected. Hazards to man may occur from the concentrated chemical
before dilution, from inhalation of spray or dust during application, or
from ingestion of the chemicals in food or in water. Because the great-

Lll'VAI

from iuli:*
with part
Di Vno ■
accidental
ill after nj

0002640
^

^ ft

Hfc./v--4'1“!" Qojb ^ ^
Toxicity and hazards of auxin herbicides

ly or by inihrough the
> dermatitis
temporarily
•d chemical
olication, or
e the great-

est hazards are from the concentrated chemical and because man is
handling lire chemicals in this form at all stages from manufacture to
dilution, it follows that he is at greater potential risk jthan any other
organism. However, there are very few reports in the literature of
tests or incidents of poisoning of man by these->mmpounds; the
majority of these reports refer to arridcntal poisoning of children. As
a*result it is now generally accepted that auxin-type herbicides do not
presenTa direct toxicity hazard to man ( B arnes 19oo) when correctly
handlpd nr used-for weed C o n tr o r ~ ~
K raus in 1945 (in Ke h ia r t 1945) reported that he had taken 0.5 g .
of 2,4-D per diem for 21 days with no demonstrable ill-effects. A clini'cal study was iriade 111 Denmark by N ielsen et al. (1965) on a 23-year
old man who had committed suicide by apparently drinking 125 ml.
of 50 percent w/v 2,4-D dimethylamine salt. The total weight of
2,4-D in his body was calculated as being not less than six g. (the
equivalent of 80 mgVkg.), about 10 percent of the total weight of
active material ingested. The principal damage appeared to be to
nerve tissues and the central nervous system.
V allet (1965) gives acutely toxic oral doses of 2,4-D to man of
50 t(T5^ mg./kg. body weight, of 2,4,5-T of 500 to 1,00Q mg./kg.,
arid of MCPA of 50 mg./kg. No indication is given of tbe source of
these figures which are probably extrapolated trom data on laboratory
gnimnls KnruwirH pt at. M966) have shown that a number ot drugs,
on a mg./kg. basis, are more toxic in man than in the mouse, when used
as a laboratory test animal, by a factM Of 10 1615. There"are clearly
dangers therefore in assuming that a toxic dose to rasa is the same as
the toxic dose to a laboratory' animal multiplied by some factor based
on the mean differences in weight. In fact these latter authors show for
a number of anticancer agents that a very much more accurate assess­
ment of the ratio of animal to human toxicity can be obtained on a
mg./m.1 of body surface area, than on a mg./kg. basis. For this reason
V allet ’s (1965) figures may be suspect, particularly m respect of the
range of 50 to 500 mg./kg. for 2,4-D, although the lower figure would
be of the correct order o f magnitude when compared to the residue of
80 mg./kg. found by N ielsen (1965) in his suicide case. Nevertheless,
this figure represented the amount actually found and it is likely that
a greater proportion of the total 2,4-D ingested was necessary to
produce death. In this context it is noteworthy that the acute oral
LDsa’s to rats of the most common organochlorine insecticides range
between 10and 135mg.Ag. (M artin 1963).
E dwards and Ripper (1953) have discussed the hazard to operators
from inhalation of sprays or aerosols during application of herbicides
with particular reference to methods of protection. M onarca , and
Di V ito (1961) have described a clinical study of an acute case of
accidental poisoning of a man in Italy. In this instance a farmer became
ill after applying a 40 percent aqueous solution of 2,4-D by handpump

DOW 43C192

i Ions) of the
total tonnage
is in the U.K.
1962), from a
>f cereal fields
jnds based on
CPA and to a
irmulations of
) in favour of
ldude MCPA
2,3,6-TBA. In
ops, 2,4-D toan increasing
d-leaved tree
ted extent for
basis 2,4-D is
de and this is
mpound. The
f compounds
pcct to their
is a measure
, in spjte of
!-l£ , they
are inorganic
nzard.
Id there have
of domestic
>f these comisoning have
:ee of safety,
cause of the
of an illness
e direct and
s to indicate
id generally

'

39

5615
0002641

o

40

c:

J.M .W ay

^

against the wind. He was admitted to hospital, suffered a relapse after
IS days, and recovered sufficiently to be discharged after 40 days.
Initial symptoms of muscular weakness, vomiting, perspiring freely,
and oliguria were noted in the field whilst a diagnosis of bradycardia,
respiratory difficulties, and urinary abnormalities was made after ad­
mission to hospital. However the authors report that the case was
exceptional. F etisov (1966) has reported similar field symptoms in
Russian workers engaged in field applications of 2,4-D. This author
concluded that a range of formulations of 2,4-D was "highly toxic to
animals in different ways of introduction." Whilst reports of minor
discomfort following exposure to auxin sprays during field application
are rarely reported in scientific literature, there is no doubt that a
proportion of workers so exposed do suffer a degree of transitory dis­
comfort. Whether this is of any significance as a long-term toxic hazard
has not been determined for man.
From the very nature of their use it is unlikely that auxin herbicides
will appear as significant residues in food crops. W illia m s (1964) was
unable to detect any residues of auxin herbicides in a number of total
diet samples down to the limit of sensitivity (0.01 p.p.m.) of his
analytical techniques. D uggan and W eathebw ax (1967) calculated
pesticide chemical residues in ‘total diet’ samples collected on 46 days
in 25 American cities during a 699 day period from June 1964 to
April 1966. Each sample represented the total amount of food and
drink consumed by one person over a two-week period. The total
samples represented in all a food and drink supply sufficient for 644
days. Herbicide chemicals were found infrequently and averaged about
0.01 mg./day of which one third was 2.4-D, and halt was MCi'A and
Dentacniorophenoi ( fL -i 1') combined. 2.4-D was found in oils and fats
(O.Otil mg. m lMt>4/tio) and sugars and sugar products (0.004 mg~in
1964/65, 0.002 mg. in 1965/66), whilst MCPA was found in grain and
^cereals (0.UU2 mg. in 19t>4/6o ). in dairy products ( 0.003 mg in 1965/
fed), and m leafy vegetables ( 0.001 mg. in 1965/661. T^iese am ounts
are substantially below the limits set for acceptable dailyIntake by the
World Health Organization and United Nations committees, it seems
probable, therefore, that tuxiC hazards irom auXln herbicide-1residues
iirtood are very small, r aust (lid-l), in a survey ot water pollution
hazards to' man from organic pesticides, came to the conclusion that
there did not appear to be danger to health at the present time from
the background concentration of pesticides believed to be in ground and
surface water. However, 2,4-D could persist in lake water and bottom
mud for long periods under certain environmental conditions. Work
in Russia, quoted bv F aust , suggested diat the threshold taste and
odour concentrations auxin compounds, especially ot pncnOllC dCHTg^
tives such as 2,4-L>, that would prove unacceptable to the consumer
were yen' considerably below the threshold concentrations for toxic
effects. A particular risk might be supposed to lie in contaminated milk

drawn from
2,4-DB or 2
these compr
were fed 5.
number of c
for 2,4-D 0.1
these results
HI. To.\
The toxi
quoted in ti
test animals
comparative
the figures c
of factors. 1
and general
the susccpli
formulation
O.1
salt has ail
ester of 570
and H ym as
E rne (1966
addition, tin
example, 2,
mg./kg. for
chicks (Rov
Bearing
the acid fi2.4.5- T 300
700 mg./kg.
prop 930 n
2.4.5-T fall
the slightly ■
of toxicity c
the relative
and 2,4,5-T.
stituents of
compound,
elusions wo
on the basi
factants an;
pounds. W1
<*| .

•5 6 ^

02S42

rrt-d a relapse after
after 40 days,
pirspiring freely,
.isi.s of"bradycardia,
v.ts made after ad<h.if die case was
iield symptoms in
This author
,-.:s "highly toxic'to
t reports of minor
u* iield application
'."no doubt tliat a
of transitory' dis^-terni toxic ha2T.»rd
at auxin herbicides
(1964) was
a number of total
03 p.p.m.) of his
(1857) calculated
!!acted on 46 days
.cm June 1964 to
our’ nf food and
pe» - The total
sufficient for 644
:d averaged about
i was MCPA and
.d in oils and fats
cts (0.004 mg. in
vend in grain and
003 mg. in 1963/
'i. These amounts
r.ily intake by the
miltees. If seems
erbicide residues
‘ water pollution
conclusion that
resent time from
he in ground and
atcr and bottom
onditions. Work
shold taste and
phenolic derivao the consumer
-ations for toxic
itaminated milk

. l ia m s

41

drawn from cows feeding in treated pasture, but no residues of either
2,4-DB or 2,4-D were found in the milk of cows that had been fed
these compounds ( G
c t a l. 1963a and 1063b). The beasts
were fed 3.0 p.p.m. (based on a daily ration of 50 pounds) for a
number of days; the limit of detection for 2,4-DB was 0.2 p.p.m. and
for 2,4-D 0.1 p.p.m. Whereas milk might be a somce of contamination
these results indicate- that there is no apparent hazard.
u t e n m a n x

OOV/436194

T o x i c i t y a n d h a z - ir d s o f a u x in h e r h fc k U - s

III. Toxicity and hazards to domestic and laboratory animals
a) Acute and chronic toxicity

The toxicity of agricultural chemicals to land fauna is normally
quoted in terms of the dose that kills 50 percent of a population of
test animals (LDW). Whilst this figure gives a useful indication of the
comparative toxicities of different compounds to a given test species,
the figures obtained in different tests may be influenced by a number
of factors. Thus the age and sex of the test animal, method of dosing,
and general conditions of the test may have an important bearing on
the susceptibility of the animal to the compound Being studied. The
formulation of the active compound has a considerable influence; for
instance 2,4-D acid has an LDMto rats of 373 mg.Ag. but the sodium
salt has an LDsn of 805 mg./kg., the propylene glycol butyl ether
ester of 570 mg./kg., and the iso-propyl ester of 700 mg./kg. (Rowe
and Hymas 1954). It should be noted, however, that Bjorbluxd and
Erne (1966) do not regard these differences as being appreciable. In
addition, the LDjj for different test species may vary quite widely: for
example, 2,4-D acid has an LDW of 375 mg.Ag. for rats, of 100
mg./kg. for dogs, 469 mg.Ag. for guinea pigs, and 541 mg.Ag. for
chicks (Rowe and Hymas 1954).
Bearing in mind these reservations, the acute oral LDjo to rats of
the acid formulations of the compounds under consideration are:
2.4.5- T 300 mg.Ag., 2,4-D 400 mg.Ag., MCPB 6S0 mg./kg., MCPA
700 mg.Ag., 2,4-I>B 700 mg.Ag., 2,3,6-TBA 705 mg.Ag., and mecoprop 930 mg./kg. (Woodford and Evans 1963). Thus, 2,4-D and
2.4.5- T fall in the moderately toxic and the other five compounds in
the slightly toxic class according to the widely accepted classification
of toxicity of chemicals quoted by Frazer (1963). In a comparison of
the relative toxicities of a number of commercial formulations of 2,4-D
and 2,4,5-T, Rowe and Hymas (1954) concluded that the ‘inert’ con­
stituents of the formulations did not add to the toxicity of the active
compound, nor did they exact any potentiating effect. Similar con­
clusions were reached by Dalcaaed-Mikkelsen and Poulsjen (1962)
on the basis of this and other work. However, it is possible for sur­
factants and other additives to enhance the toxicity of active com­
pounds. Whilst there is some evidence with insecticides that mixing

5617
■VB"

0002S43

o

CD
42

C
ZJl
H

J . M . W ay

of compounds leads to synergistic eiFects on the acute toxicity of the
constituent compounds (K eplincer and D e ic h m a n n 1967), effects of
this sort have not been reported for herbicide mixtures in common use.
However, the possibility of their occurring should not be overlooked,
nor, also, the possibility of antagonism where the effect of one compound upon another in a mixture is to depress its biological activity.
Descriptions of the clinical symptoms of poisoning by M C P A ,
2,4-D, and 2,4,5-T of a range of experimental animals have been summarized by R o w e and H y m a s (1954) and more recently reviewed with
comments by D a l g a a r d -M ikkelsen and P o u l s e n (1962). B j o r x l u n d
and E r s e (1966) have subsequently further reviewed and reported
on their own results on the effects of feeding 2,4-D to calves, pigs, rats,
and chickens. A range of symptoms was produced that was generally
similar to those described by the previous authors. E r n e (ls»o6a and
b) studied the distribution and elimination of 2,4-D and 2,4,5-T in
these animals. Amine and alkali salts of both compounds were readily
absorbed and completely distributed in the body, but 2,4-D ester was
incompletely absorbed and reached only a low level in the plasma
and tissues. The highest tissue levels of the two compounds were
found in liver, kidney, spleen, and lungs and the levels found in these
organs sometimes exceeded the level found in the plasma. In blood
cells some 1.0 to 20 percent cf the plasma level was found. Penetration
of 2.4-D into placental tissue of pigs was recorded but there was little
or no evidence of penetration into adipose tissue or the central nervous
system. Elimination of the compounds was rapid, the plasma half-life
being about three hours in rats, eight in calves and chickens, and 12 in
pigs. The tissue half-life values ranged between five and 30 hours. N o
retention of the compounds was noted in the tissues. There was no
accumulation after repeated dosing and in pigs there was an increase
in the rate of elimination after repeated administration. In all species
the main excretory route was via the kidneys. hanna and anc
(1966) traced the metabolism of C M -labelled 2,4-D in rats dosed at
rates from one to 100 mg./animal. Radioactivity was found in all the
organs studied together with some accumulation as early as one hour
after dosing. At the one mg. dose rate a concentration peak of radioactivity was demonstrated after six to eight hours but decreased thereafter and was non-detectable by 24 hours. At the 80 mg. dose the
peak occurred at eight hours and persisted for 17 hours. Extracts of
the tissues were shown to contain mainly unchanged 2,4-D residues.
N o radioactivity was found in the expired carbon dioxide, but elimination in urine and faeces was dose dependent. At the one to 10 mg.
doses 93 to 96 percent of the ingested 2,4-D was excreted unchanged
in the urine in the first 24 hours. At the 20 to 100 mg. doses greater
amounts of 2,4-D were found in the second 24 hour period after dosing,
with a linear decrease in percentage recovery with increase in dose.
In experiments with cattle G u t e n m a n n et d . (1963a and b) were un-

K

F

ajjjc .
cous
jn th.
j0 2 , 1
(liluii>
coin;:
S t Jo .

'

a nu:;
stccr
ration
¡strilli­
dose 1
pounc’
quent
M C Pi
was s.
of 2.-1
machz
were :
period
ox-Hun
being
label!'
as the
pastur
after <
the mi
urine .
dosed
portioi
animal
rcsidi:<
incorp
in par'
suserp
same 1.
deriva:
logical
(1966)
docs p
transit
Mi
and Cparent
sprayp
Cnicsi.

5618 0 0 0 2 6 4 / J

<

’ of the
Feels of
ion use.
luoked,
ic comlctivity.
MCPA,
:n sumed with

RKLUNB

sported
is, rats,
■nerally
'6a and
,5-T in
readily
:er was
plasma
s were
i these
blood
s 1>
ervous
alf-life
i 12 in
rs. N o
/as no
crease
pecies
F ang

;ed at
>11 the
hour
radioiheree the
:ts of
dues,
nina1 mg.
nged
eater

sing,
dose,
i un-

96T9EÊAAOQ

Toxicity and hazards of auxin herbicides

43

able to detect any residues of 2,4-DB or 2,4-D in milk or faeces of
cows fed five p.p.m. of either compound in a 50 pound daily ration.
In these experiments there was no evidence of beta-oxidation of 2,4-DB
to 2,4-D. Disappearance of 2,4-D was thought to occur as a result of
dilution in the rumen, some absorption on the gut wall, and by de­
composition. In subsequent experiments B a c h e et al. (1964) and
St Jo h n et al. (1961) studied the fate of M C P A , M C P B , 2,4,5-T, and
a number of other herbicides in cattle. All the M C P A fed to a single
steer (113.5 mg. single dose based on five p.p.m. of a 50 pound daily
ration) was accounted for in its urine over the four days after admin­
istration. N o M C P B was found in the urine of MCPB-dosed cows (total
dose of 56.75 mg. and 113.5 mg. based on 2.5 and 5.0 p.p.m. of a 50
pound daily ration) on the first day after administration, but subse­
quent recoveries in their urine showed that 7.2 to 9.2 percent of the
M C P B was converted to M C P A . Analysis of M C P B itself, however,
was said to be unsatisfactory. It is of interest that a similar conversion
of 2.4-DB to 2,4-D can be performed by bluegill sunfish ( Lepom is
machrochirus) (G u t e n m a n n and L isk 1965). Urine and milk samples
were also analysed from cows fed a total of 454 mg. of 2,4,5-T over a
period of four days. It was shown that the compound was excreted
exclusively in the urine over a period of six days, a total of 430.7 mg.
being recovered. C larke et a t (1964) investigated the fate of C 14labelled 2,4-D in sheep fed a dose of four mg. of 2,4-D/kg. calculated
as the minimum daily dose likely to be ingested from grazing treated
pasture. The level of radioactivity in the blood rose to a peak 1.5 hours
after dosing but fell to the normal background level in 24 hours. All
the material was excreted within 72 hours, with over 98 percent in the
urine and only 1.5 percent in the faeces. B a l a y a n n i s et aL (1965)
dosed a rabbit and six mice with 2,3,6-TBA and showed that a pro­
portion of the compound was recovered after passing through the
animals. These authors discussed the significance of biologically active
residues of auxin compounds in animal excreta that might become
incorporated in manure or straw. It was shown that 2,3,6-TBA residues
in particular could remain active for a period of months and affect
susceptible crops to which the contaminated manure was applied. The
same hazard does not normally exist with the phenoxyacetic acid
derivatives, where the compounds are broken down and become bio­
logically inactive in a relatively short period of time. However, L isk
(1966) has pointed out that the excretion of 2,4-D in the urine of cows
does present the admittedly remote possibility of active 2,4-D being
transferred from a treated pasture to a susceptible crop.
M itchell (in K ephart 1945), D alcaard -M ikkelsen et al. (1939),
and G oldstein and L ong (1960) all reported that there were no ap- '
parent ill-effects in cattle, sheep, or horses from grazing pasture
sprayed at herbicidal or two times herbicidal rates of 2,4-D or M C P A .
G iucsby and F a rw ell (1950) (in S pringer 1957) reported that there

5619
0002645

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u
-c n
CO
44

J. M. W ay

was no significant difference in the amount of feeding of horses, cows,
sheep, and pigs in untreated plots or plots sprayed with the sodium
salt or the isopropyl ester of Z,4-D, or the isopropyl ester ot 2,4,o-T.
However, there did appear to be less feeding in plots sprayed with
the alkanolamine salt of 2,4-D. There was no effect on milk production
of cows feeding on sprayed vegetation. G oldstein and Lose (I960)
found no ill-effects on two cattle irom adding 0.25 pints of a 1.5 percent
w/v 2,4-D/2,4,5-T mixture to every five gallons of their drinking water
for 41 days. These authors also reported spraying the skins of a calf,
of a cow, of sheep, and of pigs with doses ranging from 0.002 to 0.008
pounds of 2,4-D or 2,4-D/2,4,5-T mixture, with no ill-effects. These
dose rates would be of the order of those that might occur in an in­
stance of spray drift.
D o b s o n (1954) sprayed M C P A , 2,4-D, or 2.4,5-T pr
chicken runs daily for 14 days at normal and ten times normal dose
rates.-2,4,5-T-signifieantIy redneed egg preductiuu and'~tlie wtdgliljof
the~birdsr MCPATffld 2,4-D also affected egg production, mainly"in
the second week of spraying or during the week alter spraying had
stoppedr~In~,
Jil Imlaiices llicie m s uu effeet on ihn fyililii/ uf~lhe
eggrund all the progeny reared well, allliuugli the duse wies^Juid
frequency cf application
ere severe than”arc
likely to-be found in pun.1liefe. E r n e ^1500) sliuwed Lhai some ru rhs
2.4- D fed to hens~cöuld be excreted in their eggs. D un a c h ie an d
FLETCHEH~t 1907) iu jm fcd liffll's eggs with MCl'A, 2,4-D, M C P B ,
2.4- DB, mecoprop, and 2,3,6-TBA amongst a range of nt-W hAphieirU-c
Dose rates wefe~10, 100, and 200 p.p.iri. equivalent to 0.5, 5, and 10
mg./egg.-The-percentngc hatch w as recoidid. At the luwtisrdflse therewas 1UU percent hatch from the M C P A - and MCPB-treated eggs; 90
percent from the mecoprop- and TBA-treatcd eggs, and SO percent
from the 2,4-D-treated eggs. At the highest dose mere was SO percent
hatch from mecoprop, 50 percent from 2,4-D and TBA, and 20 percent
from M C P A and M C P B . None of the chicks that hatched was de­
formed although some feather blanching was noted from the M C P A ,
MC PB, 2,4-DB, and mecoprop treatments. R oberts and R ogers (1957)
reported on various feeding experiments on turkeys with alfalfa
sprayed with a low volatile ester of 2,4,5-T at herbicidal rates. N o
deleterious effects were noted. Calculations were quoted to show that
for a one kg. chicken to acquire a lethal dose of 2,4-D from an applica­
tion rate of one pound/acrc, the bird would have to consume in two
days all the 2,4-D applied to the vegetation over an area of 72 square
feet; Similar calculations by R o w e and H ym as (1954) for cattle sug­
gested that for a 770 pound animal to acquire an acutely toxic dose of
2.4- D of approximately 1,000 mg./kg., it would have to graze co m­
pletely and ingest all the 2,4-D from an area of 0.17 acre (one pound/
acres 10.41 mg./ft.5as93.7 mg./yard-*= 112.1 mg./m.a).
Accounts are given of direct oral dosing or dermal applications of

auxin b
(cow),
D a l c a .Pa l m e :
(sheep
gave d
in ever
250 m u
of 100":
of 112
animaltions a*
of the c
would
ill-effc<.
animal

1. :

propylc:

2. ■

ks. of t

nnn__
r*
O.

of 2,4,5
butyl c!
4.
Th<
greater
report;
40 dai
of 2.4In:
showe*
doses
did nt
mg./k:
daily i
daily i
piss. 1
500 p.
effects

anima
for a
piglethad ti
spine.

5620
0002S46

d o w

45

auxin herbicides to a variety of domestic animals by Kefhart (1945)
(cow), R ow e and H ym as (1954) (laboratory animals and cattle),
D alcaard -M ikxelsen cf al. (1959) (heifers), P alm er (1963) (cattle),
P alm er and R adeleff ( 1964) (sheep and cattle) , C larke ct al. ( 1964)
(sheep), and Strach and Bohosiewicz (1964) (pigs). P almer (1963)
gave daily oral doses of 2,4-D alkanolamine salt to steers for five days
in every seven. He recorded signs of poisoning in animals dosed at
250 mg./kg. after 15 administrations as opposed to 86 administrations
of 100 mg./kg.; at 50 mg./kg. no ill-effects were recorded over a period
of 112 administrations. From these results he concluded that altnough
animals could probably ingest enough 2,4-D from concentrated solu­
tions at any one time to produce illness or death, the chronic toxicity
of the compound was sufficiently low to make it unlikely that an animal
would pick up enough of it over a period of time to cause any serious
ill-effects. Further work by Palm er and R adeleff (1964) using single
animals gave the following results:

1. Sheep tolerated 4S1 daily doses of 100 mg.Ag. of the alkanolamine salt or
propylene glycol butyl ether ester of 2,4-D.
2. Cattle suffered from chronic tympanites after 88 doily doses of 100 mgV
kg. of the alkanolamine salt of 2,4-D. One animal died after 34 daily doses of
200 mg./kg.
3. Sheep tolerated 481 daily doses of 100 mg./kg. of the triethylamine salt
of 2,4,5-T but succumbed to 369 doses of 100 mg./kg. of the propylene glycul
butyl ether ester.
4. Sheep were killed by 383 daily doses of 100 mg./kg. of MCPA amine.
The toxicity of 2,3,6-TBA in these trials was said to be slightly
greater than that of 2,4-D or 2,4,5-T. Strach and Bohosiewicz (1964)
reported that no abnormal behaviour in pigs had been noted following
40 daily doses of 15 to 100 mg./kg. of 2,4-D, nor from single doses
of 2,4-D of 200 to 800 mg.Ag.
In short term trials by Bjorxluxd and Erne (1966), calves and pigs
showed definite though reversible symptoms of poisoning after single
doses of 2,4-D of 200 and 100 mg.Ag., respectively. Rats and fowls
did not show any sign of distress after single doses of 100 and 300
mg.Ag., respectively, and fowls tolerated daily doses of 300 mg.Ag.
daily in their feed for several weeks without visible effects. Repeated
daily doses of 50 mg.Ag., however, led to toxic symptoms in some
pigs. Jn longer term studies (E rne 1966a), five voung pigs were fed
500 p.pjn. of 2,4-D for up to 12 months but, although various toxic
effects were noted and their growth rate was affected, none of the
animals died. When 2,4-D was fed to a sow throughout gestation and
for a further six weeks, 10 of the 15 underdeveloped and apathetic
piglets she produced died within 24 hours and the mother subsequently
had to be slaughtered because of abnormalities that developed in her
spine. Heavy dosing of pregnant rats, however, with 1000 p.pjn. of

436198

Toxicity and hazards of auxin herbicides

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J. M. W ay

2,4-D in their drinking water over 10 months and of their off-spring
for up to two years, whilst leading to retarded growth and increased
mortality, did not produce unequivocal signs of toxicity. Continued
administration of 500 p.p.m. of 2,4-D in feed or 1,000 p.p.m. in the
drinking water of fowls led to reduced egg production and kidney
abnormalities. These results led the authors tr> rnnrlnd» that »T»a
chronic toxicity ot '2a -u to tiie species studied was moderate. They
Were, hoWéVW, concchied about the mortality of new-bom piglets,
with evidence ot movement ot 2,4-D through trie placental tissues, and
the“Teduced"5gg pTUÜUétion in towis which they thought might indicate~â possible lMterferaiiCe with reproductive processes.
’
•In-general the fanclings of other workers support these conclusions
on 'Scute and chronic toxicity. In all the work quoted the amounts
adminisfe'red”tô'the test Ullimals foi' effttil, luvt uuuti well in lüfuiss
of-the amoumsthey might bè è>Tpecléd tu pick up ir6m a treated
pasture^or-in -fced-dcrivcd h em cious tint had at smile hmtT 'been
treated ulth aaxil "herbicides at normal dose rates.

b) Indirect toxicity
Indirect effects of herbicides on grazing animals have been associ­
ated with increased toxicity of toxic plants, increased palatabiliiy of
normally non-palatab!e toxic plants (e.g., ragwort, Senecio jacobaea ),
and induced toxicity in normally nontoxic plants (e.g., temporary in­
creases in nitrate content) ( W illard 1930). However, F ehtic (1953)
claimed that, up to 1953 in America, in all cases where poisoning of
livestock from herbicides had been reported, the effects noted could be
attributed to some other cause.
Examples have been given by W illard (1950) of cattle eating
wild cherry (Prtmvs serotina), of pigs eating Cocklebur (Xanthium
sp.), and of lambs eating thistles after herbicidal treatment with
auxins. Instances have been reported of ragwort becoming 'sweeter'
from two or three days after application and being preferentially
grazed by cattle for a short period. G rigsby and B all (1952) and
L y n n and B arrons (1952) investigated the hydrocyanic acid ( H C N )
content of the leaves of wild cherry' from untreated trees and trees
treated with 2,4-D and 2,4,5-T. Their conclusions were that the foliage
was no more toxic to cattle after treatment and that there might even
be less H C N in the leaves of the treated trees than in those of the
untreated ones. B uck et al. (1961 ) fed the alkaloid-containing plants
Delphinium barbeyi (tall larkspur) and Hclenium hoopscii (sneeze
weed L after treatment with 2,4-D ester or 2,4,5-T ester, to calves and
ewes. N o increased toxicity of the plants attributable to application
of the herbicides was noted. W illiam s and C ronin (1963) analysed
D. barbeyi, treated with 2,4,5-T amine at various growth stages, and
showed that the alkaloid content of the plants was increased for several

weeks aft
noted, hotreated p!
S w ans

content o ’
there was
ment ther
for a furl
nitrate co
relations!)!
increase h
The cl
which nit:
and lists c
D a v id s o n

S und ct c
duction cl
globin in
Intravenot
of body v.
present in
‘have been
( G il b e r t

nitrate co
followed 1
cattle. A r
currence o
Recent inc
has been ;
treated pi
The ac
known (r.
of this pro
W lLLAHD (

et al. (195(
of cattle i
previously
content of
LSI to S.7
treated pi.
h e a d 1950
a range of
of these n
Cell-free c
previously
by B eeviu ;

ir off-spring
id increased
. Continued
p.m. in the
and kidney
le that the
crate. They
om piglets,
tissues, and
might indi­
conclusions
ne amounts
11 in excess
'l a treated
time been

eer 'sociital y of
jacobaea ),
iporary in-

tic (1953 )

lisoning of
d could be

ttle eating
'Xanthium
nent with
l ‘sweeter’
ferentially
1952) and
d (HCN)
and trees
he foliage
light even
>se of the
ng plants
i (sneeze
alves and
.^plication
analysed
ages, and
or several

47

weeks after treatment at the vegetative and early bud stages. It was
noted, however, that the bitter taste of the alkaloids might make the
treated plants even less palatable to animals than untreated ones.
S w anson and S h a w (1954) showed that 2,4-D affected the HCN
content of Sudan grass (Sorghum vulgare ssp. sudancitse). Initially
there was a decrease in the content of HCN but four days after treat­
ment there was an increase over the controls which was maintained
for a further 12 days. Similar effects were shown to occur with the
nitrate content of leaves. B uck et al. thought that there might be a
relationship between HCN and nitrate metabolism in Sudan grass, an
increase in one leading to a decrease in the other.
The clinical aspects of nitrate poisoning in stock, conditions under
which nitrates are likely to accumulate in the leaves of certain plants,
and lists of these plants have been reported by B radley et al. (1940),
D avidson et al. (1941), G ilbert et al. (1946), C ase (1957), and
Sund ei al. (1960). The toxic effects of nitrate are caused by a re­
duction of nitrate to nitrite and the conversion by nitrite of haemo­
globin in the blood to methoglobin: the animal dies from asphyxia.
Intravenous iniection of methvlene blue in doses of two g./oOO pounds
of body weight gives immediate relief. Nitrate in plants is generally
present in the form of potassium nitrate and increases in nitrate content
have been associated with drought conditions and high soil nitrogen
( G ilbert et al. 1946, C ase 1957). S und et al. (1960) noted a high
nitrate content in Urtica spp. and Rubus spp. after heavy rains,
followed by preferential grazing of these and other weed species by
cattle. A number of abortions in these cattle was correlated with oc­
currence of high nitrate rather than grazing of the weed species per se.
Recent increases in vitamin A deficiency in North American ruminants
has been associated with ingestion of nitrates occurring in herbicidetreated plants by P hillips (1964).
The accumulation of nitrates in the leaves of sugar beet is well
known (c.g., Savage 1949). Increased levels of nitrate in the leaves
of this crop as a result of herbicide application have been reported by
W illard (1950), S tahler and W hitehead (1950), and W hitehead
et al. (1956). Isolated incidents have been reported of nitrate poisoning
of cattle in America as a result of feeding on sugar beet that had
previously been sprayed. In one incident in N. Dakota, the nitrate
content of sugar beet leaves after spraying was found to vary from
1.81 to 8.77 percent of the dry weight, as against 0.22 percent for un­
treated plants and a toxic level of 1.5 percent (S tahler and W h ite ­
head 1950). Studies on forage crops ( B krc and M c E lroy 1953) and on
a range of weed species ( F rank and G ricsby 1957) have shown which
of these may contain high levels of nitrates after auxin application.
Cell-free extracts of maize and cucumber from plants that had been
previously sprayed with 10 and 100 p.p.m. of 2.4-D were investigated
by B eevers and H a c e m a n (1962). The level of nitrate reductase was

DOW436200

Toxicity and hazards of auxin herbicides

5623
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J. M. W at

increased in maize but reduced in cucumber. Studies on the formation
and breakdown of nitrates in plants ( F eiitig 1952, F reiberg and C lark
1952, W hitehead et al. 1956) have shown that 2,4-D causes more rapid
increases in nitrate content than MCPA, that levels rise to a peak soon
after spraying and subsequently decrease with time, and that increases
in light intensity hasten decreases in nitrate content.
It is clear from these reports that nitrate poisoning in stock does
occur from time to time and that it is possible for the hazard to be
increased by application of auxin herbicides to nitrate-accumulating
plants.
IV. Bees and other insects
Herbicides affect bees (Apts meUifera) and other insects if they
kill the plants on which the insects feed. In addition, W aulin ' (1950)
has reported that 2,4-D and MCPA were toxic to bees, not only from
visiting the flowers but also as a result of drinking contaminated water
trapped on treated plants. A ntoine (1966) reported that MCPA para­
lysed and killed bees which ingested doses that corresponded to those
recommended for weed control. Other workers have reported effects
on bees after application of auxin herbicides to plants in flower but
not at other times ( H aracsimova 1962, P a lm er -Jones 1964). P alm er Tones (1964) and A n t o in e (1966) have suggested that 2,4-D might
have some effect on nectar which made it toxic to bees. K ing (1960a)
has shown that radioactive 2,4-D can be translocated to the nectar of
PomsettUi and red clover plants and may be detectable there for two
to three days after treatment. Feeding trials of auxin herbicides to
bees have been reported by G ly n x e Jo nes and C onnell (1954),
P almer -Jones (1960), K ing (1960b), and B vrdy (1962). P a lm er Jones (1964) found no effect on bees that had been directly dusted
with 2,4-D or when they were made to crawl through 2,4-D dust in
order to enter the hive. G l y n n s Jones and C o n n ell (1954) classed
2,4-D and MCPA as stomach/contact poisons of low toxicity to bees,
with LDjo values of 0.105 mg. compared to insecticides in the range
0.00004 to 0.002 mg. B yrdy (1962), on the other hand, reported total
mortality of bees within four days of feeding 30 jig. of 2,4-D and 10
percent mortality within three days rising to 20 percent in five days of
feeding 20 /»g. Jo h ansen (1959) reported that 2,4-D and related com­
pounds were not toxic to bees, except when formulated as the alkanolaminc salt or the isopropyl ester. H aracsimova (1962) found that a
30 percent calcium/potassium salt formulation of MCPA was toxic to
bees and caused mortality up to 13 days after ingestion. However,
part at least of the loss was attributed to crcsol impurities, which led
to loss of smell amongst the bees with consequent loss of sense of
direction.
Occasional observations on other insects have been reported.

li
l-

p

ii
t!
a.'
V
(

2,

ii<
b.
ci
in
m
fe
in
so

cr<
Ui
sil
cid
ha:
he;
(P
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dit!
troa
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die.
(1C

proj
org:
of s
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tin»

proli

0002550

4

DOW 436202

Toxicity and hazards of auxin herbicides

49

M ellanby et al. (1959) observed that a spray of mecoprop on oats at
the time of a heavy frit fly attack had no effect on the flv ( Oseinr’lla
frit). M axw ell and H arwood (1960) treated broad bean ( Vida faba)
plants with sublethal doses of 2,4-D and recorded a marked increase
in the reproduction of the pea aphid ( Macrosiphum pisi) feeding on
them. Tire longevity of adult aphids was unaffected. R obinson (1959)
also recorded increased fecundity in another pea aphid ( Acyrthosiphon
pisum) after caging on broad bean plants treated with 2,4-D. Adam s
(I960) and A dam s and D rew (1965) showed that the application of
2,4-D amine could enhance aphid infestation in New Brunswick grain
fields, probably as a result of depressing the activities of coccinellid
beetles predating on tire aphids. In laboratory experiments with coc­
cinellid larvae treated with 2,4-D amine, there was a fourfold increase
in mortality and an increase in time to pupation. There was little
mortality amongst the adult beetles, which usually recovered after a
few hours inactivity. Isnn and H e u n o (1963) concluded that increases
in the growth rate of the larvae of the rice stem borer ( Chilo suppress
salis ) feeding on 2,4-D treated rice plants, was a consequence of in­
creased nitrogen content of the plants rather than a direct effect of the
chemical itself.
In general, it appears that there is a real hazard to bees—and pos­
sibly other nectar feeding insects—from applications of auxin herbi­
cides to plants in flower. Otherwise there would seem to be little
hazard to insects from direct toxicity ot the compounds at normal
lieibloldftl i ales of application. However, the work ot Adam s and D rew
(1965) decs'suggest ¡.Inti, some insects may be fflOtft susceptible~at
particular"stages of their life cycle than at others.V
.

V. Soil animals
Amongst soil arthropods, D avis (1965) was unable to show any
differences in pooulaticns of Acari or Collembola sn<*n<*s between un­
treated plots and plots that had been sprayed with MCPA in 10 out of
13 years. D avis (1965) was unable to find any reports of significant
effects on soil animals in other experiments of a similar nature. B ollen
(1961) concluded that auxin herbicides, based on phenoxyacetic and
propionic acid, were the most susceptible to breakdown by micro­
organisms of the many pesticides applied to the soil. The importance •
of soil microorganisms in the breakdown of these herbicides is well
known from the work of Atjdus (1964) and others. W ebster (1967)
has briefly reviewed the literature on the influence of plant growth- .
regulator-auxin herbicides on the host/parasitc relationships of nema­
todes, in which 2,4-D has been shown to increase nematode reproduc­
tion in plant callus cultures. In addition, plant cell hypertrophy and
proliferation, which is a common effect of 2,4-D in many plants, pro­
vides highly suitable conditions for development of nematodes. In

5625

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J.M .W at

this way susceptibility of a normally nematode-resistant variety of oats
could be induced, although there did not appear to be any greater
susceptibility of a non-resistant variety.
VI. Fish and aquatic organisms
Under field conditions the toxicity of a pesticide in water is affected
by a number of factors in addition to those that affect its performance
on land. Thus acidity, hardness of the water, and the sorbent qualities
of suspended organic matter in the water may directly effect the
toxicity. The trophic nature of the ecosystem, the oxygen status of the
water in respect of both producers and demand, and the amount of
movement of water both within the system and in terms of flow will
affect the concentration of the chemical, its persistence, and its possible
toxic side effects. Because of these, and many other interacting factors,
the toxicity of a given formulation of a given chemical compound to
an individual species will vary under field conditions depending upon
the nature of the water body and the immediate environment. For this
reason toxicities to fish and aquatic organisms are usually estimated
in terms of median tolerance limit for exposure to a given concentration
of the pesticide, for a given length of time (TLmx).
In addition to direct or indirect toxicity, the effects on aquatic
organisms of the removal of the substrate that gives them food and
shelter must also be considered. For instance, in one of the Tennessee
Valley Authority’s reservoirs two applications of 2,4-D controlled con­
siderable acreages of Eurasian water milfoil ( Mtfrioplujllum svicatum).
The eradication of the plant eliminated the substrate that might have
been colonized by large populations of epiphytic insects such as the
larvae of midges, mayflies, and dragonflies (S m it h and I so m 1967).
Although under some conditions this might have a very serious effect,
it has been shown by W ay et al. ( 1968) in an ecoloeical investigation
of the use of paraquat in lakes, that although individuals and popu­
lations of animals mav be severelv affected in the weeks after applica­
tion of a herbicide, the species affected may be present again in the
season after treatment. It has also to be recognized that very heavy
infestations of submerged or floating aquatic plants may interfere with
the passage of nutrients and considcrablv reduce the temperature and
dissolved oxygen values of the water ( F ish 1966). Thus, any possible'
hazards from the use of a herbicide may be outweighed by the advan­
tages gained from the removal of the vegetation.
The' danger of significant amounts of 2,4-D appearing in ground
water or streams as a result of local applications seems to be slicht.
A ldiious (1967) measured residues of 2.4-D in drainage channel water
after aerial spraving of a Scottish forest at four pounds/acre active
ingredient in 12 gallons of water. Up to two p.p.m. of 2.4-D was
measured in the seven days after application but none was detectable

by 23 day?
suspended
States at
chromator.
pound wa
was attrib
Review
eluding ai
et al. (IS
variations
than the c
formulatie
lations. Si.
viewed e!
molluscs.
Trout ■
most senv
median to
posurcs oi
.cc:r.pct:::d
to 1,150 tv
mercuric a
likely toxii
agricultur.
given: aid
quat - 1/1
1/120, am
figures ref
centrates ;
Perch
to be alfe>
applicatio:
turc of 2.-’
ct al. (19u
MCPA of •
55 to GOir
These rest
pared to
(see ahov
hires ni.iv
i Aldrii
dlmetiuHi’n
bipyriiUliv
tju.il i 9.11

2,2-<l;, lili’ti
1.2,-MriJ.v!

C 0 0 Z S 5 2

51

by 28 days. Brown and Nisiiioka (1967} analysed samples of a watersuspended sediment mixture from 11 streams in the western United
States at monthly intervals. Amongst the analyses made were gas
cliromatograph determinations for 2,4-D and 2,4,5-T. Neither com­
pound was found in any of the samples. The absence of the compounds
was attributed to some extent to their susceptibility to degradation.
Reviews of toxicity hazards to fish of a range of pesticides, in­
cluding auxin herbicides, have been made by B auer (1961), B and t
et al. (1962), and C ope (1965 and 1966). C ope (1966) noted that
variations in formulation gave rise to greater differences in toxicity
than the differences in toxicity between the basic compounds. Ester
formulations were often more toxic than amine or metallic salt formu­
lations. Similar observations were made by L hoste (1959) who re­
viewed effects on a number of crustaceans, aquatic insects, and
molluscs.
Trout (Salmo trutta) are normally regarded as being amongst the
most sensitive fish to water pollution. A labaster (1958) has given
median tolerance limits for 24 and 48 hour (TLmn and TLniig) ex­
posures of trout to 2,4-D or 2,4,5-T, or to mixtures of these two
compounds, of 9.5 to 250 p.p.m., depending on formulation, compared
to 1,150 to 2,000 p.p.m. for sodium chlorate or 0.005 p.p.m. for phenyl
mercuric acetate. H olden (1964) devised a formula for comparing the
likely toxic hazards to trout from a number of pesticides1 applied at
agricultural rates. The following comparative estimates of hazard were
given: aldrin - 70, PCP - 7, MCPA - 1.5, 2,4-D -1, 2,4,5-T -0.5, para­
quat - 1/12, simazine - 1/27, diquat - 1/40. dalapon -1/46. TCA 1/120, and aminotriazole «1/150. It should be emphasized that these
figures refer to agricultural rates of application: hazards from the con­
centrates might be different.
Perch ( Perea fluviaiilis) and roach ( Rutilus m tilus) are unlikely
to be affected by MCPA, 2,4-D, or 2,4,5-T (B a n d t 1957) at rates of
application used for aquatic weed control, although a commercial mix­
ture of 2,4-D and 2,4,5-T was more hazardous. In later trials B andt
et al. (1962) found threshold values for toxicity to perch and roach of
MCPA of 200 to 215 mg./litre, of 2,4-D of 75 mg./litre, of 2,4,5-T of
55 to 60 mg./litre, and of 2,4-D + 2,4,5-T mixtures of 5 to 12 mg./litre.
These results show rather greater toxicities of 2,4-D and 2,4£-T com­
pared to MCPA than were shown in H o ld en s (1964) calculations
(see above). The much enhanced toxicity of the 2,4-D + 2,4i3-T mix­
tures may have been an effect of the particular formulations used, or*

* Aldrin - 1.2,3,4,10,10-hexach]oro-l,4,4a,5,8,Sa-hexahydro-exo-l,4-«ido-5,8dimcthanonaphthr.lcnc; PCP - pentachlorophcnol; paraquat - l,l'-diaethyl-4,4'bipyridylium dichloride; simazine - 2-chloro-4,6-bisethylamino-l,3,5-tKizmc; di­
quat - 9,10-dihydro-8a,10a-diazonaiaphenantlircne dibromide; dalapon - sodium '
2,2-dichIoropropionate; TCA - trichloroacetic acid; and aminotriazole > 3-amino1,2,4-triazolc.

DOW 43l>204

Toxicity and hazards of auxin herbicides

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o
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rc
o
cn

J. M. W ay

could demonstrate a synergistic effect of one compound on the other.
D avis and H audcastle (1959) established median tolerance limits
over a 24 hour period (TLm=<) for bluegill sunfish ( Lepomis macrochirvs) to a number of herbicides. Values obtained when the com• pounds were added to relatively pure water were 2,4-D »39 p.p.m.,
MCPA - 20 p.p.m., 2,4-DB - 20 p.p.m., and 2,3,6-TBA -1,800 p.p.m.
C ope (1966) noted delays in spawning of bluegill sunfish of up to two
weeks after treatment oi water with the propylene glycol butyl ether
ester of 2,4-D at five and 10 p.p.m. However, no other effects were
noted on reproduction or on survival of fry. In pond experiments, death
of some fish as a result of 2,4-D treatment led to increased size in the
•* survivors, probably as a result of the greater food supply available
to the individual fish. In further trials with bluegill sunfish, H ughes
and D avis (1963) and D avis and H uches (1963j" reported on effects
of different formulations of 2,4-D and other auxins. Their tests showed
2,4-D and 2,4,5-T esters to have TLm5 4 ranging from 1.8 to 10 p.p.m.
depending on the ester used. Dimethylamine salts of 2,4-D and 2,4,5-T
had TLm» 4 of 162 to 542 p.p.in. and 144 p.p.m., respectively, com­
pared to the alkyl amine salt of MCPA of 163.5 p.p.m. and of 2,4-D
acid of 8.0 p.p.m. This work (which is referred to in C ope 1966, see
above) shows the wide differences in toxicity that can occur In differ­
ent formulations and the care which must therefore be taken in assess­
ing the toxicity of an individual product before recommending it for
use as an aquatic herbicide.
j
In addition to work on fish, W alxeh (1962) has reported effects,
on a variety of bottom-feeding fish food organisms following applica-j
tion of 2,4-D to plastic enclosures at 1.0 to 4.0 p.p.m. L hoste (1959) !
has reported that ester formulations of 2,4-D or mixtures of 2,4-D and
2.4.5-T affected crustaceans, aquatic insects, and molluscs in the range
of 0.1 to 3.3 p.p.m. In their investigations into the effect of the use
of 2.4-D at rates from 40 to 100 pounds/acre in Tennessee Valley
Authority reservoirs, S m it h and I so m (1967) found no measurable
toxic effect on benthic fauna or significant changes in mean numbers
of burrowing mayflies (Hexagenia) before or after treatment There
were some deaths of caged fish but no dead or distressed free fish
were found. Observations of free living fish indicated that they ap­
peared to move out of the treated area, whilst analysis of a number of
fish for 2.4-D residues gave no measurable results at the limit (0.14
p.n.m.) of detection. However, analysis for residues in mussels (mainly
EUiptio crassidcns) indicated that they concentrated the chemical from
the surrounding water although no mortality was recorded. Analysis
of mud samples showed that significant amounts of 2,4-D (up to 58.8
p.p.m. butoxycthanol ester) were present in isolated sediment samples
up to 10 months after treatment. These fairly high levels in the en­
vironment of a sedentary animal such as the mussel might explain the
residues found in these animals. R a w ls (1965) has reviewed literature

562S
- -

-Hgg.1» 1

on the
and ir..
variety
ccnci-r.

virgini.
rth.ui.-l
less sir.
ethanol
alarm \
for wii
11.02 P
fool ilc
local t
practice
n it­
rates of
icitics (•
the dos.
cstuaricgive rivmover! !
ccptible

Ilaz.:
Runo an
Whilst v
chronic t
oisonin
lowevfi
hcrbiciili
compotu;
for wild!,
compoun
signiiicai:
not to s.i
in hedge
isolated i
over laig
scientific
gradually
margins,
cultural 1
The p

r

0002 554
-V

o

lpound on the other.
Jian tolerance limits
ish ( Lepornis macroined when the cornre 2.4-D « 39 p.p.m.,
3-TBA - 1,8C0 p.Pjm.
;sunfish of up to two
ie glycol hutyl ether
0 other effects were
d experiments, death
increased size in the
>od supplv available
gill sunfish, H u g h e s

1 reported on effects
Their tests showed
rom 1.8 to 10 n.p.m.
of 2,4-D and 2,4.5-T
, respectively, comp.p.m. and of 2,4-D
0 in C o p e 1966, see
can occur in differ^ fc
ken in assess2C
0 i.-iiending it for
has reported effects
; following apolicanm. L h o s t e (1939)!
xtures of 2,4-D and
olluscs in the rangel
e effect of the use!
1 Tennessee Valley
md no measurable
>in mean numbers
r treatment. There
distressed free fish
ated that they ap­
sis of a number of
at the limit (0.14
in mussels (mainly
the chemical from
recorded. Analysis
2,4-D (up to 5S.8
sediment samples
i levels in the enmight explain the
eviewed literature

53

on the toxicity1of herbicides to estuarine fauna, principally Crustacea
and mollusca, and described experiments from his own laboratory. A
variety of formulations of 2.4-D affected several species of animals at
concentrations of one to 10 p.p.m. Larvae of oysters ( Crassostrea
virginica) were killed over a period of time by one p.p.m. of butoxyctlianol ester and dimethyl salt of 2,4-D, and though oyster eggs were
less susceptible, their development could be affected by 2.4-D butoxycthanol ester at 10 p.p.m. These effects might give some cause for
alarm when rates of application of 20 to 30 pounas/acre are required
for weed control in estuaries. Doses of this sort would give 7.34 to
11.02 p.p.m. if evenly distributed throughout one acre of water one
foot deep. Because of tidal fluxes and uneven distribution only very
local concentrations at toxic or near toxic amounts may occur in
practice.
The results of these various investigations suggest that at bcrbicidal
rates of application of auxins the hazards from acute or chronic tox­
icides to aquatic organisms are low. Nevertheless in some instances
the dose rates required for effective herbicidal action, for example in
estuaries or where the chemical is likely to be rapidly dispersed, may
give rise to local and perhaps short term concentrations not far re­
moved from those required for toxic effects on some organisms at sus­
ceptible stages of their life history.

Hazards to wildlife from auxin herbicides have been reviewed by

R udd and G enelly (1956), Sptjxcer (1957), and M e ix a n b y (1967).

Whilst with any material having biological activity a risk of acute or
chronic toxicity is always present, authenticated incidents of widescale
poisoning of wild animals by these herbicides have not been reported.
However, there is the further hazard to all forms of wildlife from
herbicides of altering the habitat. Whilst herbicida] applications of the
compounds to agricultural land undoubtedly modify the habitat there
for wildlife, it is unlikely that individual instances of spray drift of the
compounds at sublethai doses, although undesirable, nave any really
significant effect on habitat of adjoining non-cultivatcd land. This is
not to say that some food plants for animals are not regularly killed
in hedgerows and other field margins from spray drift, nor that in
isolated instances of gross mishandling more severe effects do not occur
over larger areas. It is probably true, although there is little or no
scientific evidence, that the continued use of herbicides in the U.K. is
gradually leading to the impoverishment of the flora of the field
margins, and consequently to the gradual impoverishment of agri­
cultural land as a whole as a habitat for wildlife.
The principal problem from the use of auxin herbicides in relation

436206

Toxicity and hazards of auxin herbicides

O
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CO
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ro

o

54

-a

J. M . W a t

to wildlife, is not toxicological but ecological in terms of scale and
intensity of use, especially on non-agricultural and industrial land.
Thus the total destruction of a small area of vegetation on one occa­
sion might be less serious for the wildlife of a region than the selective
destruction of vegetation over a wide area at regular intervals. The
hazard trom the use of herbicides lies in the tact that tney can now
be used tb affect vegetation over very wide areas m a short period or
tim erthurelim inating 'rtscrvoirs' or wiidine and wndkie habitat tli'af
would have gtlrerwlssTarvlved:-------"

underesli
neclar-pi
hazards r
been stud
decrease«
in muni t
populatic
been asx<
host plan
Toxicity t
on the foi
is low an
direct ha:
relation l-

Summary
Toxicities and hazards to man, domestic and laboratory animals,
wildlife, bees, and other insects, soil animals and fish from MCPA,
MCPB, 2,4-D, 2,4-DB, 2,4,5-T, mecoprop, and 2,3,6-TBA are discussed.
In man only one authenticated instance of death by poisoning has been
noted in West European and North American literature. Authenticated
case histories ot sublethal effects are also very rare. However, com­
plaints ot transient dizziness, sickness, and other symptoms are made
rom tima to tllBé by workers engaged in Held applications, especially
under coriditlCSi ivhcrc the spray is inhaled éXCesslVüly. Tllé possibility
of"ihüH ¿jL‘L|uliing uitIi ' dnxK bt these compounds in food milk, or
water appears to be very low.
Toxicity tests on a range of laboratory and domestic animals with
a wide range of formulations of the compounds tend to show greater
differences between formulations than between compounds. AmomgsT
tne domestic animals studied dogs appear to be more suscepuble than
the others? Acutely imilti Ü0üés"5f~thé compounds to farm animals and
fowls are shown to be greater than the ammais could normally ingest
from treated vegetation or water tohowing an agricultural application.'
Hazards to stock trom tne concentrated materials are empnasized, but
the ottensive and unpalatable nature ot the undiluted chemicals would
probably preclude any normal animal trom ingesting a toxic dose. TKe
dancer or chronic toxicity is álS6 Shown TC'be very 10\V and continuous
high dosing over periods ot months rather than weeks are required
to-Tiroduce severe symptoms ot poisoning, in many animals a high
proportion of the active chemical is voided in the urine within 24 to
48 hours ot ingestion, secondary hazards to ammais as a result of"
changes in the chemical constituents ot rood plants may occur, for
insTgneS~t5xicitv~hgS"ljLieii associated with temporary increases ill thtr
nitrate contents of some plants after spraying. Current recommendationrfgTstock to be excluded trom treated pasture for ¿ penod~after
treatment should be observed.
4 7,1
Hazards to wildlile exist from the use of herbicides but are also
associated with other changes in agricultural practices, ihc effects
of hci biddes miiiabUUl are serious lor wiidlite and should~not~bo

a c te ris tirs
O th er th a t,

in excess <

Sont di
doniesthj!:
insectes, la
MCT1J; 2.:
un seul ce.
bibliograp:
même, les
moins, des
temps à a;
exposent à
symptômes
l'homme dtrès limité'*
Les épr
boratoirc i •'
rences sont
qu'en ce rp
tiques, le t
trovoqucr
es volaille
ingérer à p.
à la suite •
risques po’.
agressive • :

I

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5630
J 3 0 0 2 S 5 'c
. J1WW1

8 0 Z 9 e t ’M O a

Toxicity and hazards cf auxin herbicides

55

underestimated. Deaths of bees have occurred following spraying of
»¿ctar-producing plants wlicn in flower. It is possible tiiat simitar
Hazards cxisrro'r~oth'ei,'iieLtai-ij.ediiiU Imdcls. Vv’Here othtf lim;tU have
been studied, increases m population have been noted more often than
decreases. With some~5phftt5 this has been associated with rilducllun
in numbers of predators~No effects have been noted in studies of
populations ot soil artnropods. increased activity by nematbtlcs has
been associated'~with the favourable conditions for invasion of ihe
host plant created by auxin induced teratomlS and uQjliT efieels.
Toxicity~td~fish rind~ofher aquatic organisms has been shown to depend
on the formulation of the compound used. In general, toxicity to fish
is low and herbicidal applications to water no not present a severe
direct hazard. The dangers of secondary effects must be assessed m
relation to other methods of control and the environmental Chat?
actenstics- of ..the water body to be trpatpd <tnmP agnafjc organisms
other thanfish mav be directly affected at dose rates not very much
in excess of those recommended for herbicidal use in water.
’
t
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Résumé *
Sont discutés les toxicités et les dangers, pour l’homme, les animaux
domestiques et de laboratoire, la vie sauvage, les abeilles et autres
insectes, la faune du sol et les poissons, des composés suivants: MCPA;
MCPB; 2.4.D.; 2.4.DB; 2.4.5.T; mecoprop et 2.3.6.TBA. Chez l’homme,
un seul cas authentifié de mort par intoxication a été relevé dan; la
bibliographie de l’Europe occidentale et de l’Amérique du Nord. De
meme, les cas authentiques d’eifets subléthaux sont très rares. Néan­
moins, des sujets affectés à l’cpandage de ces produits se plaignent, de
temps à autre, particulièrement lorsque les conditions de dispersion
exposent à une inhalation excessive, de vertiges passagers et d’autres
symptômes et se sentent malades. L’éventualité d'absorption par
l’homme de doses toxiques avec les aliments, le lait et l’eau semble
très limitée.
Les épreuves de toxicité sur une grande variété d’animaux de la­
boratoire et d’animaux domestiques tendent à montrer que les diffé­
rences sont beaucoup plus grandes en ce qui concerne les formulations
qu’en ce qui concerne les matières actives. Parmi les animaux domes­
tiques, le chien parait être le plus sensible. Les doses susceptibles de
irovoquer des effets de toxicité aigüe chez les animaux de ferme et
es volailles sont supérieures à celles que ces animaux pourraient
ingérer à partir des plantes traitées ou de l’eau éventuellement polluée
à la suite des applications en Agriculture. L’accent est mis sur les
risques pour le bétail des formulations concentrées, mais la nature
agressive et le mauvais goût de ces formulations rendent très impro-

Î

* Traduit .par R. Tauhaut.

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bable l’ingestion d’une dose toxique par un animal normal. Les risques
de toxicité chronique sont également très faibles, car des doses élevées
doivent être répétées, non seulement pendant des semaines mais
pendant des mois, pour pouvoir provoquer des symptômes graves
d’empoisonnement. Chez beaucoup d’animaux, une forte proportion des
matières actives est excrétée dans l’urine dans les 24 à 48 heures qui
suivent l’ingestion. Des risques secondaires pour les animaux peuvent
résulter de modifications dans les constituants chimiques des végétaux
consommés; par exemple, des effets toxiques ont été associés à des
augmentations temporaires des taux de nitrates de certaines plantes
consécutives aux traitements. Il convient de respecter les recommanda­
tions relatives à l’exclusion du bétail des pâturages traités pendant une
certaine période après le traitement.
Des dangers pour la vie sauvage peuvent résulter de l’usage des
herbicides, mais ils peuvent également être causés par d’autres varia­
tions dans les pratiques agricoles. Les effets des herbicides sur l'habitat
sont graves pour la vie sauvage et ne doivent pas être sous-estimés.
Des morts se sont produites chez les abeilles à la suite de traitements
de plantes mellifères à l’époque de la floraison. Il est possible que des
risques analogues existent pour les insectes qui se nourrissent du nectar
des fleurs. Les études relatives à d’autres insectes ont révélé des aug­
mentations de populations plus fréquemment que des diminutions.
Dans le cas de quelques especes de pucerons, cet accroissement était
associé à une réduction du nombre des prédateurs. Aucun effet n’a
été observé dans les études sur les populations d’arthropudes du sol.
L’accroissement d’activité des nématodes était lié aux conditions favo­
rables pour l’invasion des plantes hôtes créées par les tératomes et
autres effets dûs aux auxines. La toxicité pour les poissons et les autres
organismes aquatiques s’est révélée dépendre du type de formulation
utilisée pour l’épandage de la matière active. En général, la toxicité
pour les poissons est basse et les applications d’herbicides à l’eau ne
présentent pas de risques directs graves. Les risques d’effets se­
condaires doivent être évalués en liaison avec d’autres méthodes de
lutte et les caractères de l’environnement des eaux à traiter. Quelques
organismes aquatiques autres que les poissons peuvent être directement
affectés à des doses ne dépassant pas beaucoup celles recommandées
pour l’emploi des herbicides dans les eaux.

eines
nordar.
letlulc
Zeit K

andere
auf de:

Spray-:

dieser
reiche::
To.\
mit ci:
neigen
zwischi
deren .
toxisch,
als sic
Wasser
Die Ci
hervort
verdüm

der Au.
nischcn
liehe 1
lieh, un
wird ei;
Stundet
Tieren .
von Na
zität bc
gewisse,
die Fcr
einige ?
k

Eine
Ilerbizit
in der
Standor
Tod vor.
Bilanzen
für Nek!
wurde i
Bei Blat
der Bau!
von Pop
durch N
Befall d.

Zusammenfassung *
Toxizität und Gefährdung bei Mensch, Haus- und Laboratoriums­
tieren, Wild, Bienen und anderen Insekten. Bodentieren und Fischen
durch MCPA, MCPB, 2,4-D, 2,4-DB, 2,4,5-T, Mecoprop und 2,3,6-TBA

* Übersetzt von F. Bau.

00026ar n

5632

.

t ..»«;ALi<&

• - fcv« *

Les risTit-s
oses clcvecs
mines mais
imcs graves
aporlion des
lieures qui
m x peuvent
les végétaux
ociés à des
ines plantes
commanda>cndant une
l’usage des
mtres variasur l’habitat
ous-estimes.
traitements
blc que des
it du nectar
■lé des aug3im!" 'tions.

cm

était

jn effet n’a
«les du sol.
'.’tiens favoératornes et
?t les autres
formulation
, la toxicité
; à l’eau ne
d’effets se*
éthodes de
Quelques
directement
Dmmandées

jratoriumsnl P ¡«eben
2,3,6-TBA

DOW436210

Toxicity and haznrds of auxin hcrbicidcs

57

werden diskutiert. Beim Menschen wurde nur ein verbürgtes Beispiel
eines Todesfalles infolge Vergiftung in der westeuropäischen und
nordamerikanischen Literatur berichtet Verbürgte Fälle von sublethalen Effekten sind auch sehr selten. Jedoch werden von Zeit zu
Zeit Klagen über vorübergehendes Schwindelgcfühl, Übelkeit und
andere Symptome von Arbeitern vorgebracht, die in der Anwendung
auf dem Felde tätig sind, besonders unter Bedingungen übermässiger
Spray-Inhalation. Die Möglichkeit für den Menschen, toxische Dosen
dieser Verbindungen in Lebensmitteln, Milch oder Wasser zu er­
reichen, scheint sehr gering zu sein.
Torizitätstestc bei einer Heilte von Laboratoriums- und Haustieren
mit einem weiten Bereich von Formulierungen der Verbindungen
neigen dazu, grössere Differenzen innerhalb der Formulierungen als
zwischen den Verbindungen zu zeigen. Von den untersuchten Haus­
tieren scheinen Hunde empfindlicher zu sein als andere. Die akuttoxischen Dosen der Verbindungen für Vieh und Geflügel waren höher
als sie die Tiere normalerweise aus behandelten Pflanzen oder aus
Wasser nach Anwendung in der Landwirtschaft aufnehmen können.
Die Gefährdung des Viehbestandes durch konzentriertes Material wird
hervorgehoben, aber die agressive und widerwärtige Natur der un­
verdünnten Chemikalien würde wahrscheinlich jedes normale Tier von
der Aufnahme einer toxischen Dosis abhalten. Die Gefahr der chro­
nischen Toxizität erwies sich auch als sehr gering und eine kontinuier­
liche hohe Dosierung eher über Monate als über Wochen ist erforder­
lich, um schwere Vergiftungssymptome zu erzeugen. Bei vielen Tieren
wird ein hoher Anteil der aktiven chemischen Steile im Hörn 24-43
Stunden nach der Aufnahme entleert Sekundäre Gefährdung bei
Tieren als Ergebnis von Veränderungen der chemischen Bestandteile
von Nahrungspflanzen kann Vorkommen; zum Beispiel war die Toxi­
zität begleitet von einem vorübergehenden Anstieg des Nitratgehaltes
gewisser Pflanzen nach dem Besprühen. Laufende Empfehlungen für
die Eamhaltung des Viehbestandes von der behandelten Weide für
einige Zeit nach der Behandlung sollten befolgt werden.
Eine Gefährdung für Wild besteht durch die Anwendung von
Herbiziden; sie ist jedoch ebenso verbunden mit anderen Änderungen
in der Landwirtschaftspraxis. Die Effekte der Herbizide auf dem
Standort beim Wild sind ernst und sollten nicht unterschätzt werden.
Tod von Bienen erfolgte nach dem Besprühen von Nektar-erzengcnden
Pflanzen in der Blüte. Es ist möglich, dass eine ähnliche Gefährdung
für Ncktar-frcsscnde Insekten bestellt. Beim Studium anderer Insekten
wurde ein Populationsanstieg häufiger als eine -abnahme festgestellt.
Bei Blattläusen war(dics verbunden mit der Herabsetzung der Anzahl
der Haubinsekten. Keine Effekte wurden festgestellt in Untersuchungen
von Populationen von Bodenarthropoden. Eine gesteigerte Aktivität
durch Nematoden war verbunden mit günstigen Bedingungen für den
Befall der Wirtspflanze, erzeugt durch Auxin-induzierte Teratome und

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andere Effekte. Die Toxizität für Fische und andere Wasscrorganismcn
zeigte «me Abhängigkeit von der Formulierung der angewandten
Vcrbindang. Im allgemeinen ist die Toxizität für bische gering und die
Herbizifanwendung zum Wasser stellt keine schwere direkte Gefähr­
dung dar. Die Gefahren sekundärer Effekte sind in Beziehung zu
setzen zd anderen Kontrollmethoden und zu den Umwelteigenschaftcn
der zu behandelnden Wasserfläche. Ausser den Fischen können gewisse
Wasserarganismen direkt beeinflusst werden bei Dosisbeträgen, die
nicht sehr weit über den zur Herbizidamvendung im Wasser empfoh­
lenen liegen.

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References
(

J

J. B.: Effects of spraying 2,4-D amine on coccinellid larvae. Can. J. Zool.
38, 2S5 (I960).
----- , and M. E. Drew: Crain aphids in New Brunswick. Ill Aphid populations
in habicide treated oat fields. Can. J. Zool. 43, 789 (1965).
Alabaster, J. S.: Toxicity of weedkillers, algicidcs and fungicides to trout Proc.
4th Brit. Weed Control Conf., p. 84 (1958).
Aldhous. J. R.: 2,4-D residues in water following aerial spraying in a Scottish
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A ntoine, O.: Les antiparasitaires et les anhnaux domestiques, risques pour lt*s
poissons, les abeilles et la vie sauvage. Parasitica (Cembloux) 22, 107 (1966).
Avscs, L. J.: Herbicide behaviour in llie soil. In: The physiology and biochemistry
cf herbicides (Ed., L. J. Audus). London-New York: Academic Press (1964).
Bache, C A., D. J. L ise, D. G. W acner, and R. C. W arner : Elimination of 2methyi-4-chlorophenoxyacetic acid and 4-(2-methyl-4-chlorophcnoxybutryic)
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Balayaknis, P. C., M. S. Smith , and R. L. W ain : Studies on plant growthregulating substances. XIX. Stability of 2,3,6-trichlorobenzoic add in wheat
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B
, B . J.: Uber die Giftwirkung von Herbiziden auf Fische. Z. Fischerei 6,
121 (1957).
----- , D. N ehrinc, and M. Schlüter: Schadwirkung und Nutzen von Herbiziden
in der Fischerei Tagungsbericht 2. Inter. Arbeitstagung der Arbeitsgeme­
inschaft. Toxicologie von Pfianzensch. Mitt. Berlin 54, 45 (1962).
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culture. In: Chemicals and the land, p. 135. Proc. Symp. Yorkshire Agr. Soc.,
Askham Bryan, York, England (1965).
Bauer, FL: Studien über Nebenwirkungen von Pflanzenschutzmitteln auf Fische
und Fischniihrtierc. Mitt. biol. Bundesanstalt Land- u. Forstwirtsch., BerlinDahlem 105, 5 (1961).
B eevers, L., and R. H. H acemak : Effects of 2,4-D application on nitrate metabo­
lism in higher plants. Plant Physiol. 37, suppl. XXIII (1962).
Berc, R. T„ and L. W. McElroy: Effect of 2,4-D on the nitrate content of
forage crops and weeds. Can. J. Agr. Sd. 33, 35-1 (1953).
B jorklcnd, N.-E., and K. E rne : Toxicological studies of phenoxyacetic herbicides
in animals. Acta Vet. Scand. 7, 364 (19C6).
*
Bollejo W. B.: Interactions between pesticides and soil microorganisms. Ann.
Rev. Microbiol. 15, 69 (1961).
Bradley, W. B., H. F. E ppson, and O. A. B eatm: Livestock poisoning by oat hay
and other plants containing nitrates. Wyoming Agr. Expt Sta. Bull No. 241,

i:.

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Adams,

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H a v iv , N.

IH u a m , It
S« n-m.*

\ liiir.

f a i r of
Khwamh. t
a m id .,.'
I- NC. K :

snima!'
—

T e n ii

Si and.

* '" r . S. I.
I l.jru: f •i*Sn:, S.
NE. W
------- l l c t l i

44 (10

I H lW IV , S i
2.4-1) .
1 m i. C . H

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(KMO).

la lln li

56 34
°002GC*

aijgi ,iw i

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1. J. Zoul.
jpulationx
out. Proc.
i Scottish
pour lev
T (1906).
ch er';' ,ry

1c

).

ion \,. 2ybutryic )
groxvt’uiu wheat
965).
ichcrel 6,
erbiziden

ritsgemein agriigr. Soc.,
if Fische
Berlinmetabontent of
nbicidcs
as. Ann.
oat hay
N’o. 241,

59

E., and Y. A. Nisiuoka; Pesticides in water. Pesticides Monitoring J. 1,
38 (1967).
Buck, W. B., W. Dinns, L. J ames, and M. C. W illiams: Results of feeding of
licrbicidc-treated plants to calves and sheep. J. Amer. Vet Med. Assoc. 13S,
320 (1961).
Byrdy, St.: Untersuchungen über die Wirkung des 2,4-D-Praparates "Pielik” auf
Bienen. Tagungsbericht 2. Inter. Arbeitstagung der Arbeitsgemeinschaft.
Toxicologic von Pilanzcnsch. Milt Berlin 54, 15 (1962).
Case, A. A.: Some aspects of nitrate intoxication in livestock. ]. Amer. Vet Med.
Assoc. 130, 323 (1957).
C lause, D. E., J. E. Youkc, R. L. Younger, L. M. H unt, and J. K. Mc L aran:
The fate of 2,4-dichlorophenoxyacetic acid in sheep. }. Agr. Food Chem.
12, 43 (1964).
Cope, O. B.: Some responses of fresh-water fish to herbicides. Proc. ISth S. Weed
Control Conf. p. 439 (1965).
----- Contamination of the freshwater ecosystem by pesticides. In: Pesticides in
the environment and their effect on wildlife. J. Applied Ecol. 3 (suppL), 33
(1966).
D add, C. V.: Weed control in cereals: A review. Proc. 6th Brit. Weed Control
Conf., p. 123 (1962).
D alcaahd-Mikeelsen, S. V., and E. P oulsen: Toxicology of herbicides. Phar­
macol. Rev. 14, 225 (1962).
----- , F. Rasmussen, and 1. M. Simonsen: Om toksiciteten af ukrudtbekaempelsesmidlct 2-mctyl-4-klorfenoxyacetat for kvaeg. Nord. Vctcrinaimed. 11, 469
(1959).
D axteson, W. B., J. L. D oughty, and J. L. Bolton : Nitrate poisoning of live­
stock. Can. J. Comp. Med. Vet. Sd. 5, 303 (1941).
D avis, B. N. K.: The immediate and long-term effects of the herbicide MCPA on
soil arthropods. Bull. Entomol. Research 56, 357 (1965).
Davis, J. T., and W. S. H ardcastle: Biological assay of herbicides for fish toxicity.
Weeds 7, 397 (1959).
----- , and J. S. Huchrs: Further observations oa the toxicity of commensal herbi­
cides to blucgill sunfish. Proc. 16th S. Weed Control Conf., p. 337 (1963).
D obson, N.: Chemical sprays and poultry. Agriculture (London) 61, 415 (1954).
D uccan, R. E., and ]. R. W eatherwax: Dietary intake of pesticide chemicals.
Science 157, 1006 (1967).
D unachie, J. F., and W. W. F letcher : Effect of some herbicides on the hatching
rate of hen’s eggs. Nature 215,1406 (1967).
E dwards, C. J„ and W. E. Ripper : Droplet size, rates of application and the
avoidance of spray drift. Proc. 1st Brit. Weed Control Conf., p. 348 (1953).
E rne, K.: Distribution and elimination of chlorinated phenoxyacctic acids in
animals. Acta Vet. Scand. 7, 240 (1966 a).
——— Toxicological studies of phenoxyacetic herbicides in animals. Acta Vet
Scand. 7, 364 (1966 b).
F aust, S. D.: Pollution of the water environment by organic pesticides. Clin.
Pharmacol. Therap. 5, 677 (1964).
F ektic, S. N.: Livestock poisoning from herbicide treated vegetation. Proc. 6th
NE. Weed Control Conf., p. 13 (1952).
----- Herbicidal poisoning of livestock. Proc. 7th NE. Weed Control ConL, Suppl.
44 (1953).
F etisov, M. E.: Problems of occupational hygiene in work with herbicides of the
2,4-D group. Clgiena i Sanit. 31, 28 (1966).
F ish, G. R.: Some effects of the destruction of aquatic weeds in Lake Rotoiti,
New Zealand. Weed Research G. 350 (1966).
F rank, P. A., and B. H. Gricsby: Effects of herbicidal sprays on nitrate accumu­
lation ip certain weed species. Weeds 5, 206 (1957).
B row n,

¡anismen
wandten

Geiälirhung zu
!schaflen
:gewisse
gen, die
empfoh-

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Toxicity and hazards of auxin herbicides

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A. C.: Balance of pesticides: The benefits and risks. Froc. 2nd Brit In­
secticide Fungicide Conf, p. S (1963).
Fheiberc, S. R., and H. E. Clare: Effects of 2,4-dichlorophenoxyacetic acid upon
the nitrogen metabolism and water relations of soybean plants grown at
different nitrogen levels. Botan. Gaz. 113, 322 (1932).
F beweich, E. J., E. A. G euak , D. P. B a u , L. H. Schmidt, and H. E. Skipper :
Quantitative comparison of toxicity of anticancer agents in mouse, rat,
hamster, dog, monkey and man. CanceT Chemotherapy Rept. 50, 219 (1966).
Gilbert, C. S, H. F. E ppson, W. B. Bradley, and O. A. Beath : Nitrate ac­
cumulation in cultivated plants and weeds. Wyoming Univ. Agr. Expt Sta.
BuU. No. 277 (1948).
G lynne J okes, G. D , and J. U. C onnell : Studies of the toxicity to worker
honeybees (Apis mellifera L.) of certain chemicals used in plant protection.
Ann. Applied Biol. 41, 271 (1954).
C oldstein, H. E., and J. F. L onc : Observations on cattle, sheep and swine ex­
posed to 2,4-D, 2,4,5-T and Dalapon herbicides. Proc. 13th S. Weed Control
Conf, p. 5 (1960).
Gricsby, B. H , and C. D. Ball : Some effects of herbiddal sprays on the hydro­
cyanic acid content of leaves of wild black cherry (Prur.ut terotina Ehr.).
Proc. 6th NE. Weed Control Conf., p. 327 (1952).
G utenmann, W. H , and D. J. L ise: Conversion of 4-(2,4-DB) herbicide to
2,4-D by blue gills. N.Y. Fish Game J. 12, 103 (1965).
——, D. D. Hardee, R. F. H olland, and D. J. Lise: Disappearance of 4-(2,4dichlorophenoxybutyric) acid herbicide in the dairy cow. J. Dairy Sei. 46,
931 (1363 a).
— ----- — — Residue studies with 2,4-dichlorophenoxyacetic acid herbi­
cide in the dairy cow and in a natural and artificial rumen. J. Dairy ScL 46,
1287 (1963 b).
H aracsimova, L.: Einfluss der in der Tschechoslowakei im Pflanzenschutz
gebräuchlichen chemischen Mittel auf die Honigbiene (Apis mellifera L.).
Tagungsbericht 2. Inter. Arbeitstagung der Arbeitsgemeinschaft. Toxicologie
von Pflanzensch. Mitt Berlin 54,35 ( 1962).
H olden, A. V.: The possible effects on fish of chemicals used in agriculture. J.
Inst. Sewage Purif. 4, 361 (1964).
H uches, J. S, and J. T. D avis: Variations in toxicity to bluegill sunfish of phenoxy
herbicides. Weeds 11, 50 (1963).
I shu, S, and C. H irano: Growth responses of larvae of the ricestem borer to
rice plants treated with 2,4-D. EntomoL Expt Appl. 6, 257 (1963).
J ohaksek, C.: Bee poisoning. A hazard of applying agricultural chemicals. Wash.
State Coll. Agr. Expt. Sta. Cire. No 356 (1959).
St. J oun, L. E , D. C. Wacner, and D. J. Lise: Fate of atrazine, Enron, Silver
and 2,4,5-T in the dairy cow. J. Dairy Sei. 47, 1267 (1964).
Kephart, L. W.: Moderator of session. Proc. 2nd N. Central Weed Control Conf,
p. 73 (1945).
Kepunczr, M. L , and W. B. D eichmann: Acute toxicity of combinations of
pesticides. Toxicol. Applied Pharmacol. 10, 586 (1967).
Kiianna, S , and S. C. F anc : Metabolism of C14-labellcd 2,4-dichlorophenoxyacetic add in rats. J. Agr. Food Chcm. 14, 500 ( 1966).
Kikc , C. C.: Translocation of C14-2,4-D and CI4-amitrole or their metabolites to
nectar in plants. Research Rept 17th N. Central Weed Control Conf, p.
105 (1960 a).
Effects of feeding herbicides to honey bees. Research Rept 17th N. Central
Weed Control Conf, p. 105 (1960 b).
Lhoste, J.: Les repercussions de l’emploi des désherbants chimiques sur la faune
aquatique C r. Réun. Tech. d’Athènes de l'UICN 4, 253 ( 1959).
F razer,

4

L ux, D.
:
L ynn , C. E ,
leaves s-

2.4J5-T.

M artin , J. '
Rl.ickwt
M axwell , 1'
broad I.
M ki.lanrv , :
Collins '

----- . it. A.
E ntom c'
Monarca, C
diclorofNielsen, K..
diclilori
niatcri.d

Palmfi«, J.

Vct. Mi
------, and R.
eidos or.

P almoh-Jon !
Zealand

------ Effect •
i hi* \ y

ih c iic fl

Rawlx, C.

C hcsapt
RonrnTs, R.
Poultry
Robinson , A
ris ), ..a;
growth*.
Rowi:, V. K.
and 2,-1
associati
Reno, R. l.„
wildlife.
S avage,

A: .*
13, 9 ( :

S m it h , C . K
of 2,4-D

(1967).-

S pringer , P.
Pesticidi
S tahlkii. L.
levels ii,
S ih a c ii , S ,
preparai
Sinirixi.ANn.
culture
tlie n iv :

3 ( i?»no

5636

0002662

Toxicity and hazards of auxin herbicides

tmai:
Mt,

19G3).
ite ac’t. Six.
A-orker
action.
ne cx-onUol
lydro-

Ehr.).
ide to
-(2.4■ i. 40,
berbi-

i. V

ichutz
L.).

□logic

rc. J.
cnoxy
cr to
•Vash,
jilvex

lonf.,
ns of
ioxyes to
•. Pntral
tune

o

ex

D. J.: How now brown cow? Farm Research 32, IS (I960).
Lynn, G. E., and K. C. Barrons: The hydrocyanic (HCN) content of wild cherry
leaves sprayed with a brush killer containing low volatile esters of 2,4-0 and
2,4,5-T. Proc. Oth NE. Weed Control Conf., p. 331 (1952).
Martin, J. T.: Insecticide and fungicide handbook for crop protection. Oxford:
Blackwell Scientific Publications (1963).
Maxwell, R. C., and R. F. Harwood: Increased reproduction of pea aphids on
broad beans treated with 2,4-D. Ann. Entomol. Soc. Amer. 53, 199 (I960).
Mellanby, K.: Pesticides and pollution. New Naturalist No. 50. London-Clasgow:
Collins (1967).
.----- , R. A. F rench, and J. R iches: Herbicide spray and frit fly attack on oats.
EntomoL Expt. Appl. 2, 319 (1959).
Monarca, G., and G. Di Vrro: Sull’ intossicazione acuta da diserbante (addo 2,4diclorofenossiacetico). Folia Med. (Naples) 44, 4S0 (1961).
N ielsen, K., B. Kaempe , and J. J ensen-H olm : Fatal poisoning in man by 2,4dichlorophcnoxyacetic add (2,4-D): Determination of the agent in forensic
materials. Acta Pharmacol. Toxicol. 22, 224 (1965).
P almer, J. S.: Chronic toxicity of 2,4-D alkanolamine salts to cattle. J. Amer.
Vet. Med. Assoc. 143, 398 (1963).
----- , and R. D. Rabeleff: The toxicologic effects of certain fungicides and berbitides on sheep and cattle. Ann. N.Y. Acad. Sci. I ll, 729 (1964).
Palmer -Jones, T.: Effect on honey-bees of some chemical weedkillers. New
Zealand J. Agr. Research 3, 485 (1960).
----- Effect on honey-bees of 2,4-D. New Zealand J. Agr. Research 7, 339 (1964).
P hillips, W. E. J.: Metabolism of carotene and vitamin A following ingestion of
the herbicide MCPA. Can. J. Animal Sci. 44, 29 (1964).
R awls, C. K.: Field tests of herbicide toxicity to certain estuarine animals.
Chesapeake Sti. 6, 150 (1965).
Roberts, R. E., and B. J. Rogers: The effect of 2,4,5-T brush spray on turkeys.
Poultry Sd. 36, 703 (1957).
Robinson, A. G.: Note on fecundity of the pea aphid Acyrthosiphen p h u m (Har­
ris), caged on plants of broad bean Vicin inbo L., treated with various plant
growth-regulators. Can. Entomologist 91, 527 (1959).
Rowe, V. K., and T. A. H ymas: Summary of toxicological information on 2,4-D
and 2,4,5-T type herbicides and an evaluation of the hazards to livestock
associated with their use. Amer. J. Vet. Research 15, 622 (1954).
Rudd, R. L., and R. E. G enelly : Pesticides: Their use and toxicity in relation to
wildlife. Calif. Fish Game, Bull. No. 7 (1956).
Savace, A: Nitrate poisoning from sugar beet tops. Can. J. Comp, Med. Vet Sd.
13, 9 (1949).
Sm ith , G. E., and B. G. I som : Investigation of effects of large-scale applications
of 2,4-D on aquatic fauna and water quality. Pesticides Monitoring J. 1, 16
(1967).
Sprjncer, P. F.; Effects of herbiddes and fungicides on wildlife. N. Carolina
Pestidde Manual, p. 88 (1957).
Stabler, L. M„ and E. I. W kttehead: The effect of 2,4-D on potassium nitrate
levels in leaves of sugar beets. Stience 112, 749 (1950).
Strach, S., and M. B oiiosiewicz: Badania nad toksycznoscia chwastobofczego
preparatu 'Pielik* dla twin. Med. Wetcrynar. (Poland) 20, 662 (1964).
Strickland, A. H.: Some estimates of insecticide and fungidde usage in agri­
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the environment and tlitir effects on wildlife. J. Applied Ecol. 3 (Suppl),
3 (I960).
Suno, J. M„ M. J. W right, and J. Simon : Nitrates in weeds cause abortion in
cattle. Proc. ]7th N. Central Weed Control Conf., p. 31 (1960).

436214

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C. R., and W. C. Shaw : The effect of 2,4-dichlorophenoxyacetic acid on
die hydrocyanic add and nitrate content of sudan grass. Agron. J. 46, 418
(1854).
Vallet, C.: Les intoxications par les herbiddes r^cents. Concours Med. S7, 4633
(1865).
W aulec, B.: Bina och hormonderivaten. Vaxtskyddsnotiser, Stockholm 14, 45
(1850).
W a l k s , C. R.: Toxicological effects of herbicides on the fish environment Proc.
8 th Air Water Pollution Abatement Conf., p. 17 (1962).
Way, J. M., N. W. Moons, J. F. Newman, and M. Owens: Some ecological
effects of the use of paraquat for the control of weeds in small lakes. In
preparation (1968).
W ebstes, J. M.: Some effects of 2,4-dichIorophenoxyacetic add herbicides on
neaatode-infested cereals. Plant Pathol. 16, 23 (1967).
W htieseao, E. I., J. Kersten, and D. J acobsen: The effect of 2,4-D spray on the
nitrate content of sugar beet and mustard plants. Proc. S. Dakota Acad. Sd.
35,106 (1956).
W illard, C. J.: Indirect effects of herbiddes. Proc 7th N. Central Weed Con­
trol Conf., p. 110 (1950).
W illiams, M. C., and E. H. C ronin : Effect of silvex and 2,4,5-T on alkaloid
content of tall larkspur. Weeds 11, 317 (1963).
W illiams, S.: Pestiddc residues in total diet samples. J. Assoc. Oflic. Agr. Chem­
ists 47, 815 (1964).
W oodford, E. K.: Weed control in arable crops. Proc. 7th Brit Weed Control
Cost, p. 944 (1964).
----- , and S. A. E vans (Eds.): Weed control handbook, 4th ed. Oxford: BlackweB Sdentific Publications (1965).
Swanson,

to
C71 .

I. In!rr.
II. Drs.
a) C
b) S.
c ) I)

J) n
111. 0|X'i.
!V.
\
.

a ) A;

b) I*c ) S!

d) K

e) C!

f) D;
VI. C rac:
VII. Discu
VIII. Eluti'
IX. T j t o
Summary . .
lU'sumé . . .

Z'n.iinmcnf.i
References .

The pr

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ami varioi;

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0002664

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5639

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/ / 'a

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/

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/ / y - t - ' ■■■

c:

' " ,/f / y
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y ~ -W v .\

r / . /). i 'j o t £
/¿ ¿ n fU K u *
\{-/H

7 //’

f/y /jo

70-0152. Zink, P. (Institul fucr Gcrichtlichc Medi/in und
•O
; Kriminalislik, Univcrsitact lirh-agfii-Nucrnbcrg, UnivmitactsCn
i ttrassc 22, 8520 lirlangcn, Germany). Idcntidcation of various
cr.
hcrbicidal chlorinated phcno^y-carboxylic acids by means
CD
■ mas* spectrography. Arch. Toxiknl 25(1): 1-4; IW J. (.5
O
I references) (German)
i
Two cases o f poisoning described in the literature
I (Gcldmachcr-vnn Mallinckrodt and l.aulcnhach. this journal.
; 21: 261; l% 6 ) served as the stimulus to assess the possibilities
! o f identifying chlorinated phcr.oxycaibuxylie acids by mass
. spccuography since the presence o f very small quantities o f
the substances made analysis difficult. Comparative \pcvin*grams were prepared, using the Cll 4 (AtlasAYcrke MA I J n uv.
spcetiom ctci, for MCI*A (2-mcjjiy M-clil»unt>licnux\ jcctrc — "" ^ r v ^ '
- a c i d ) , M il'll (yaHi/rM^4-ehimiiO nieihylplivini\y) bulviie
_
iCt-u' *

of

ncjd,^liC£P (n^hq-(^Quti-2d]LCiliyhieuoxy^:pippionjc^

8cid)f 2,4T)/(2,4-dicltloruphcnoxyacciic_acid), 2,4-DP (nip ha-.
(;M-dT^TTo!riipIicnoxy)propioj]ic acid) and 2,4,5-T (2,4,5tridildrophcnoxyacclic.acid). The highest peak values arc
taGulaTclThi mass units and on the basis o f 1007i. The number
o f carbon atoms in each compound was another differentiating
characteristic and this could he calculated for all the com ­
pounds from the molecular peak groups, which were high for
th e mass spectra o f all the substances. In the two cases o f
intoxication cited above, liver and urine were (he specimens
analyzed. A crystalline substance was obtained from the urine
extract (acidic medium w ith ether and subsequent vacuum
sublimation in tem perature gradients after Schmidt's method)
w hich precipitated four insignificantly separated fractions in
the sublimation lube on the sublimation layer. The fractions
analyzed into a mixture of 2,4-D, MCI’A and M-'PU, a little
2,4 ,5-T and traces c f 2,4-DP; each fraction showed the same
m ixture relationships. The liver extract (after Valov) yielded a
greasy residue. Mass spcclrograpiiically this proved to be
contam inated with a chlorine-free compound. Small am ounts
o f 2,4-li were identified by the molecular peak group at 220
w ith the typical 2-diluro distribution as well as peak groups o f
. the main fractional part at 162; other chlorinated carbonic
ccids were not found. Tims, the analysis showed the com­
pound to be pure 2,4,1).

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.OoCi'.ILlv^CS,

The only salvation for the bceheep
in.; industry i:; for each and everv beeireencr to been hammering home,
especially to nor city fathers, die in>
iiOriar.ce of live honeybee and the deva. j

7

• ' •

• >v k*‘wi. —

«•* 4

*■

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iinicr cor.tr

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They fed herbicides at concent rations
v' (), id. 100 and 1,000 parts per rr.il.i.r:i (ppm) in a 60-per cent solution of
rue rose syrup to newly emerged honey#i* I
< 1 «V >w levtls are much higher
U.lCCi li.’ V. O lila O» weed
normally
.mm
N -% 1I*Ci*
'«*■ U.' I «;ij<o were:
A «èw
1CSÎS ir. crons.
Nor.-t vaxIc at all concentrations. Si;J • ^*w r h r , 2 , 4 - D . r/cioram . 2.4. •5:X
•; w**.• i OlW*
>1 »..* A . , '2-cn Io roothanei mospnor.ie acid,
■. J •'» 7 "»'»i
1 i//a

i

' .«v;
••r"i
l «I W

toxic at

i.i. P a r . cpiii.,

100 ano

. . t I

'

4SI i

OCe.S O f

demie ol encephalitis and. i ashed :
ni air.. v'iji.it ci^ïi.'iinnlcs» .in api*. !e
and u *s | fMiiiii^ ICiiiiA r, ^ .. s v*ne ea
i thought to myseif as 1 watched
plane ioavc the »round that possi
many of these expensive protjr.
have been sold on fear, and icar c
rather than on their actual need.

g «• J 'g '

Ug A

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wg '■¿J .

;«i■■v.■»i»vs:
.ThAw:;
ij

/vis. y
tested die
'»l<bees
'i\ »*•*ides
C■rv conditions.
O

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44
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C • *•«4*•• V»*Vf
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n fi

W COOPi-KATlVlS studios with ihe
* A r i z o n a Agricultural
Ex *ner invert:
4*
Nation, Tucson, p l a n t physiologist
iKovaru I.. Mo 'ton and C\ilor.'ioio Sists
•T
s cn.’i O. Mo
i J Robert •l'i.
4

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k * V «•» 4

iiCvvn * 0 •. \ Z. C** 4»>t vI v• V>1 I I . ,

I * ■^

i ,0 0 0
.»iS.>ii'v, OSÌV ì / v,

hexafluarate, cacodylic acid, and cr
thali.
Moderately toxic at 10 ppm: P
ciuat, MA A, MS MA, OS ivi A, and i
*»
¡iv
t ■»
U«il . UIVi

4. I

Arn[nc__sa]ts c i _2.4 - 0 and 2,4,:
cster of_2,4,5-T, ami potassintTL-siàli
¿ieioram apnlicd in water did not
creasc bee mortai iiy. li ut an esjer
2.4,S - T .and an oster of sjjvcx anp
in a diesel oil carrier causcd”hi»h n
tality on the first day after treairm
Ah becs sprayed with paraeuar, ca
dylic acid, and Ivi SM A in water c.
within 12 uays.
While the
ili L L ilw ^ 'v d u
^I
4 1AÎkii
;y.
I
• 4« 4 4* W M 4 . V
proved toxic to honeybees in
i. i l l e s e
periments are hazardous at rates
cuired tor weed control
GLÜAOirVGS IN ot:e C U L T I

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results

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Peak

70%

at

are

the
based

a spiked

area was

used

and
in

run

<1
<1

‘

<1

091640

million

sample

<1
2

and
on

4 part

the

unspiked
the

per

sample

calculations.

4/13/70

r

0G05736
*

5643

# ‘DOW 979350

The

1
r

The

ASSAY

analytical method

dioxins

not work with

bioassayed

these

in

the

rabbit ear

(<

1 p p m of TCDBD)

2,4-D

2,3,7,8-tetrachlorodibenzo-p2,4-D

compounds

test
See

and

and SILVEX

found

attached

or

silvex.

for

chloracne

However,

we

response

them negative.
letter.

J

C00573?

979351

have

does

for

IN

DOW

DIOXINS

3

RIDER AMINE

2,4,5-T

Company

Contains

2,4,5-T/gallon

lb

Triethylamine

salt

of

2 , 4 , 5 - T ............

7.-..

57%

(■: /.- / ; i'U*-"-

\

5645

CG0S733

979352

Diamond Alkali
4

DOW

CROP

/ -

T H E

1
MIDLAND
A p r i l 13,

E.

C H £ M I C A L

C O M P A N Y

1970

DOW 9 7 9 3 5 3

G.
Ag.

D O W

Lynn

Products

9008 Building
RESULTS OF C H LO RA CN E STUDIES C O ND UC TE D O N A S S O R T E D 2,4-D,
2 , 4 , 5-T, S I L V E X S A M P L E S A N D T H E I R E S T E R S , O R A N G E A N D
D I AM ON D' S C R O P R I D E R A M I N E 4T-2.
A s p e r o u r t e l e p h o n e c o n v e r s a t i o n o n A p r i l 1, 1 9 7 0 h e r e i s
a s u m m a r y of results of all chloracne studies re c e n t l y c o m ­
p l e t e d o n a s s o r t e d 2,4-D, 2 , 4 , 5-T, S i l v e x S a m p l e s a n d t h e i r
Esters,

Orange

Chloracne
T-Number

and Diamond's

26t)l
2609

Name
2, 4,5-T
2, 4,5-T
2,4,5-T .
2, 4,5-T
2 , 4 , 5-T
2,4,5-T
2, 4,5-T
2,4,5-T

2611

2,4,5-T

2578
2586
2587
2588
2589
2590

2612

2,4,5-T

2602

2,4-D
2,4-D

2603
2604
2605
2606
2613

2,4-D
2,4-D
2,4-D

Crop Rider Amine

Chloracne
Response
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative

Reference Number
120449
120349
120369
120419
120459
120479
120070
Butyl Ester 60/40
PPI-93-3
Iso Octyl Ester

Negative

PPI-93-5
D o w a n o l E s t e r ■(PiB)
PPI-93-6
L o t # 130667 '.
Lot # 90530
Lot # 90540
Lot # 90670
Lot #
Lot #

90970
94847

‘

2669

2.4- d

Butyl Ester 60/40
ASI
Iso Octyl Ester

2670

2,4-D

AS-7
2/5/70
Iso Octyl Ester

2.4- D

4T-2.

DE

20 A

2/6/70

Negative

Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative

5646

CÖ05733

5847

.oil (-S e e î.. SO) 'i)2ôVi«5. C h a n g e a in e h e sn . c o m p a . o i m o la s
d e p e n d ia g ©b 'c Sjîc î I oil y e a ? In v/îaâda o n g a r b ee£ s w e r e . procès:

oO^ 75SSS9 V)>j
C 004E 62

\

H8 — P I L A M ?

MAT,

9Q143G T o s k k y a a d h a z a r d G to m a n , d o m o s ik a n im a ls , &
w ild life fr o m Gome c o m m o n ly u s e d a u n in h e r b ic id e s . W c
J o h n M ic h a e l ( P lo n k s W o o d E x p . S t a ., 'H u n tin g d o n , E n g l
R e s id u e R e i \ 1969, 2 6 , 3 7 -6 2 (E n g ).
A u x in -t y p e h e r b ic id e s
are r e v ie w e d , w ith s o m e p o s s ib ly n e w in te r p r e ta tio n s . R e p o
o f t o x ic it y o f th e I.in m a n m a in ly refer t o a c c id e n ta l p o is o n in g
c h ild r e n . I t s e e m s p r o b a b le t h a t to x ic h a z a r d s fro m r e sid u e s
2 in fo o d s a r e v e r y s m a ll. A c u t e a n d c h r o n ic to x ic itie s r e p o r t
in a n im a ls h a v e b een b a se d o n a m t s , o f I w e ll in e x c e s s o f t
l a t s . lik e ly t o b e e a te n a s p a s tu r a g e or o th e r fo o d .
H o w ev c
it is p o s s ib le t h a t a p p lic a tio n o f 1 t o n itr a te -a c c u m u la tin g p la t
in c r e a se s t h e h a z a r d o f n itr a t e p o iso n in g in h e r b iv o r o u s a n im a
A p p lic a tio n s o f l t o p la n ts in flo w er c a n c a u s e a rea l h a z a r d t o be
( A p i s - m e i l i f e r a ) a n d p o s s ib ly o th e r n e c ta r -fe e d in g in s e c t s .
F
s o il a n im a ls a n d A sh es t h e h a z a r d s o f I a r e lo w , b u t v a r y w i
th e fo r m u la tio n a p p lie d . O n ly 1 a u t h e n t ic a t e d f a t a lit y in m
from t h e a c tio n o f X h a s so fa r b e e n r e p o r te d .' 10 2 r e fe r e n c e s.
9Q 144t HefBteSfiGS u n 3 m i l b io lo g y . G o r z c la k , Andrz*
S y h v a n 1 9 6 9 , 1 1 3 ( 3 ), 4 3 - 8 ( P o l) .
A r e v ie w in d ic a te s t h a t a n
o f h e r b ic id e s u s e d in w e e d c o n tr o l, in c lu d in g tr ia z in e s , c ld o r in a t
f a tt y a c id s a n d p h e n o x y a c e tic a c id s , a n d c h lo r a te s , c a u s e d
tu r b a n c e s in s o il m ic r o o r g a n ism s, e s p . in fo r e s ts w h ic h a rc leo
d istu r b e d b y h u m a n a c t iv it ie s . A lth o u g h h e r b ic id e s w e r e a l
i. d b ito r y t o s o il o r g a n ism s u n d e r c o n d itio n s o f c u l t i v a t i o n , i t w
c o n c lu d e d t h a t t h e e ffe c ts w e r e n e g lig ib le a n d c o m p le te ly d
a p p ea r e d d u r in g t h e y e a r fo llo w in g a p p l i c a t i o n . ^ ^ j t K le n h a
9 0 1 4 5 u B in d in g o f s a lt s a n d e s t e r s o f 2 ,4 - D , 4 -c h lo r o m e th v ÎD h e n o x v a c e tic a cid a n d 2 .4 .5 -T in soil and natural s o r b e n t

J

s1

;jO .

<6

levels administered, did not result in signs of cholinesterase inhibition. Caroaryl
administered daily at 300 rcg/kg to the guinea pig from day 11 to 20 produced a
maternal mortality;of 3<# and fetal mortality of 17.5-2, but no terata were observe-.:.
After the fetuses were cleared and stained, examination of the skeleton disclosed ;.
bone defects appearing mostly in the cervical vertebrae. When carbary1 adminis­
tration was repeated as a single treatment on selected days between day 11 and 20,
terata were found only in seme of the litters treated on days 12 and 16 of gestat­
ion. The teratogenicity of DM20 in the hamster at 1 mg/100 g body weight-' was con­
firmed. Thiram was teratogenic when suspended'' in CMC at 250 mg/kg; v;hen it was
dissolved in DMS0. the teratogenicity was additive or possibly more than additive.1:.'
When DM30 was used as a solvent for disulfiram, the-high levels of disulfiram
were more teratogenic than v/as DMS0.alone. Disulfiram as a CMC. suspension vxao not
teratogenic. Ref: Health .Aspects of .Pesticides, Vol. ‘3, Ho. 5, Kay, 1970.
2. A.5-T CAKCELUTIOMS - U.S.A
v;^vfi|drv/Tv;r':-'': ’In’Jfiid-April the Pesticide Regulation Division of U2DA suspended the use
& all formulations of 2,A,5-T for uses in lakes, ponds or on ditch banks and '
suspended liquid foiralations for use .around the home, recreation areas, and' -y
.similar sites.
;
'■.-V '•
On Kay 1 cancellation of all granular .formulations for use around the home, recreation areas, and similar sites, and all uses of the chemical on food crops
intended for human consumption v/as announced. Cancellation or suspension v/as
. based on new research information which show 2,A,5-1 to'cause abnormal rtovolopr.er.t inunborn mice. The Curgeon General of the Department of Health, Education
and ’Welfare says that exposure to this herbicide may present an imminent hazard
to women of child bearing age. These actions do not eliminate registered use ,f
2,A,5-T for control of weeds and brush on range, pasture and forests or on rightof-ways ana other non-agrlcultural land. ■ ’
Ho cancellation of uses have bec-nr nr.-junced for 2,A-D or 2,A ,5-TP(silvex) which
are used in many lawn formulation: . Kef: Chemicals Pesticides Program, Vol. C,
Ho. 2, Va y 16, 1970.
• ddt ma y i?rrB-.y£?J5 with p a ii ; m edicatioh . acco ?'.diho to
• hCr»ALE, 3 HEALTH bULLrTlII.
The discovery was made oy scientists of the Karolinsk Institute of Gv/ejon
who found that the action of antipyrine,- a fever-pain medication, v/as interfered
with by pesticides. According to the researchers, the amount of antipyrines In
the blood decreased by half in about seven hours in these who were- exposed to
: DiTT and other pesticides, v/hile the normal half-life of the anti-pain men Leal ¡.on
v/as about thirteen hours. It is believed that insecticides stimulate product:-..a :
o-f enzymes in the liver which metasclize the drug more quickly. He:’: mnvironmena
Jan./reb. 1970.
•

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K'- </&? - M
i< n §
lipidi’iirioIngy. Prcvciitiiin and Treatment
J hy i>rgjno|>hiisplitirus insecticides and in reverse
if acetylcholinesterase. Atropine protects the
tin against aeetylcliolme. but u d im not reverse
on o f acet> Icltohnestcrasc. Animals injected with
dotes o f organnphosplionis insecticides liave
•T tieatment with PAM in combination with
mba ami lliraki 1
Wilfotd <1*170) and an
c i ,VrH j»,Y: 1.17; 1*1711) support the use of
injunction with atropine as an antidote against
tale insecticide poisoning.
u J. S.: Lee. II. II.: Kim, II. Y .:Il.n g . W. P.: Lee.
of internal Medicine. College of Medicitic. Pusan
Pusan. Korea). Oinical obsenraiions on parathion
in
Tavluni A'antnw H a k k n «• C h a p a
■'V70. 117 rcfcrenccsMKorean)
ohservatnins were made in 52 cases of acute
osicalKHt studied in the Department of Internal
san University Hospital from Januaiy I
in
H ie totlnwing icsults were obtained. 1 lie causes
tng were: I ) suicidal attempt IMF.': 1. 2| mtiixicajving 15**7 )■and .1) ime case of intoxication due
ig and one due to ingestion of contaminated
ales were affected three times nsire often than
ersus 25*7 ). Tlie peak incidence of poisoning in
patients was between the ages of 211 and 5*>. In
onai prevalence, intoxication was mo si common
r.especially in July and August. Doses»f (»in 21
e were required for full atiopini/aiioii ( I 'i to x
Jusesof 10 to 5K_5 mg lor clinical tccovcry (X lo
■pine and 2-PA.M were applied Minulianeinisly in
except one recovered clinically. Therapeutically,
•ry fiom parathion intoxicaiiiHi was achieved in
>1 pml 1.1 cases (.W 7) died. lAuilmr abstract

52 cases.

detl. licaliiH'nl consist o l of i.v atropine (50 lo 200
mg/anuiul) ami loxogoniii-Mcick (1500 to 17*0 mg/aiiiinall.
This was jceoinpaiih'd hy symptomatic treatinenl. lo
eouiiteraet great lluiu loss, iiiilusuin ol glucose and
electrolytes were given to a total <>l 54 l/aninul. To cnunieiaxt
diatrliea. tiK'dicalnm hy nasal intubation was applied. Other
drugs were given to sustain tlie blood system and die liver
Willi this treatment 12 animals ( I I cows and one hulli
survived. Milk pioductNm drastically declined. Analysis of the
hiam of smite
die dead annuals sitowed choliiicslctase
activny to he at die lowei Itiiui of detectability. In the urine
ol' two animals. /Mittrophetiid. a tnetahohte ol I*. (i05. was
ohscived. Tlie detailed symptoms of tlie illness, duration ol
tieatment and ouieome in each of the 17 altcctcd aniinals i*
tabulated. four annuals leeovered wuhoiii i remittent \
general discussant ol the iiatuieot orgarntplHtsphoriis uiioxiva
l«m and of the telalhiu between the antieliolincsieiase aetivilv
of tlicse compounds and.'or then breakdown piodocts and die
pallmgemc potccssand its symptoms is ptescnied.

K
/ i.
A' w i"t. •/
714)709. Pinsent. I* J. N’.. I niK. J. (». (Dept, of Veterinary
Medicine. U. ol Hristul. Ij i .: ford llmise. I.anglord. ttiislol.
l-ngland). A case of possible 2,4- 1) and 2. 4JS-T poisoning in
the burse. IV/. R er> m i •17(S ): 247. 117ll.
TIk* |Mtssihiluy that die ingcslMin of nettles and herbage
tlui liad been tteated or contaminated with a mixture of 2.4-1)
and 2.4,5-T was irsponsihlc tor tlie illness and death ol several
horses is considered. A mixture coni amine tlw above lierbicnles was used to destroy nvtlles around tlw cdgcol a pasture
ctmtaiiutig six liorscs. Tlie wind earned die quay mlo die
gru/.ing arc« contaminating tlw water trough and a eousideiable quantity ol tlw licihagc. |-tmi of tlie Inn sc-. weie le-t-wed
from the pasture alici 24 hr. The two mhci Imises icniamcd
foi 12 days. They were both brouglil in when one ot them
became ill. l-.arlv symptoms included depressmn. disiiitcre-i m
drinking water. salivatHm. smacking of lips anti lautidice ol the
mucous memhianes. Alutieiilaiy stasis devebnted with some
abdominal i Iis I c iis h u i and pain. Ixtcpt loi small aiimuuls ol
daik leees adherme to die innciuis mcintsiane die tectum was
empty. Ntt leees weie passed flic symptoms also mcliiilcd
m uscular tremors, patchy sweating, teguigitaiing
foul-smelling stomach eoitteuls ami tlie ma-nuiy to swallow.
Water which was taken in altet die jiimul developed a dmran hack out of the mouth, liodv temperature was normal but
the pulse increased to m i mm I lie aiiiuul was destroyed alici
10 days of illness. Kcd ami wlute cells ws>re within the miimal
range. SCOT values lud increased lo I '? mu-ml. while blood
urea increased to 500 mg' l<M ml. lliere were 100 mg of
proiviii/|00 ml ol urine. 1lie second iHtise developed smul.u
symptoms and died wiihm 2-1 lu alter rer.iov-al tiom die
pasture. Autopsy lindiugs included extensive lesions m die
esophagus and stomach with dcgvnctaiive changes in tlie liver
and kidnevs. Histological examination revealed a proliferation
ol squamous epillieliuni o| the esophagus and stomasli wuh
some inlcispciscd uieeiaied aicav I here was w e ie laity
change in the livei and sonic evnleiiev* of gloineiular ami renal
lubuljr damage. Ivvo udiet horses beeau.. ill hut survived. At
this wilting, the exact cause ol die liluess and deaths is
iimleteriuiued

j. I-..: Mass. H. (Klunk lilt 1 leigcnurlslullc uud
inkhcitcn. Frciv Umvcrsiiaci. Koetngswcg («5 . I
rmaiiy). E 605 putsuning in a herd of Berlin
rztl. I 'm * hull 2S (J ). I2S-.1) . I<l7tl. (Hi
.*rman)
lent o f poisiHiiiig foiluwmg (lie use of !-. '>05
a skin spray lo control mange in a group of l‘>
J two hulls is dcscidicd. The toxicant as a 0 .25'*
spiavcd in Mich a way lhal the heads of the
mangels ol foodiacks were ‘xposed. The dosage
4 ing paialhHin/kg body weight. Of ihv 21
were affccied. in tlie two hulls, symptoms
.n the day of expoMire. One hull alter 10 days of
•vered: the other died after 4 days o f treatment,
at was last 1» develop symptoms did so on the
vposure day. The rest nt the cattle displayed
•otsninng vaiioiisly on pnslcxpoMiic djvs I. 2. 5 .
le symptoms ohscived were- massive sahvaiiou.
tea. muscle tremois. gioaiung. noisy outaakness ol the hindqiiarleis and paitial
hJ retusal of feed, Body lempcutuie and pulse
om u l taiigv. 01 the 17 allcctcd animals, live

196

C O O le S i
.

5650

5651

^

^

*7C O C O / ,

A h r \r v

',7©-®344. Gaiston, A. 17. (Dept. of Biology, Yale U., New o
Mavcn, Conn.. 05520). PLbüato, jpeospHe aiiid p d ife s. BmSdence
ro
oo
20(7): 405-10; 1970. (17 references)
o
Contemporary man is confronted with many problems
concerning social unrest, pollution and overpopulation. Some
o f the chemicals that man depends so much on to produce
f
high levels o f agricultural products are .having deleterious
effects on man and his environment. Chlorinated hydro­
carbons like DDT, dieldrin and aldrin, once considered very
useful, have now become menaces because of their persistence
in the biosphere and their potential poisoning o f various kinds
o f creatures. Present and future pesticides must be more
thoroughly tested and then chosen and used with greater
selectivity. Some o f the more recently introduced herbicides
H ve been shown to be potential herbicida! analogs o f DDT.
Üne o f the suspected chemicals is 4-amino-3,5,6-trichlorok
picolinic acid (picloram or Torfon). Under optimum
conditions • in some soils, 20 t o . 50% o f applied picloram T
disappeared after 467 days (Youngson et qL , 1967). On other
soils low in moisture and poor in inorganic matter and air,
only 3.3% o f the applied picloram disappeared in a similar
period; It takes between 10,000 and 100,000 parts of
exogenous carbon to oxidize one part o f Tordon (Tschirley,
1969). The dangers o f further use o f this herbicide are evident.
Another chemical, 2,4-D, can also become a menace under £
certain-conditions by causing a massive increase in the nitrate r
content o f pasture plants as to sicken animals eating these
phnts (Stabler and Whitehead, 1950) and must be used very
carefully. Pesticides fabricated around heavy metals, such as
!ead and mercury, or other elements such as arsenic (trivalent)
Jo not become completely detoxified once applied. The
cresence o f rising Q u an tities of lead nnH m^rpnrv ¡n tkn

G et

V»

environment lias caused growing concern in public health.
S c ie n c e m u s t not be misapplied. Thoughtless and
. indiscriminate use of- chemicals such as 2,4-D, picloram,
(»8 ¿^jKH^and arsenical cacodylic acid by industry, the military
. “and other groups and organizations has and may continue to
^ * cause ecological havoc. T he legal structure o f our government
- ' as well as - t h e : influence o f international committees and
. commissions offer many ways to promote rational use and
limited control o f our environment and must be employed
before it is too late.

7 M 3 4 5 . Anonymous. Agriculture will continue to approve
2,4,5-T regnstetnon. Chem. Eng. News 48(16): 21; 1970.
Despite a first round o f Senate hearings, Agriculture will
continue to register uses for 2,4,5-T, with restrictions, if any,
depending on the results o f tests and research currently under
way. Ned Bayley, Director o f Science and Education for
Agriculture, holds that there is insufficient evidence to
suspend or cancel registration. In reference to the study by
Bionetics Research Laboratories showing that 2,4,5-T induced
birth defects in laboratory animals, it was stated that
“preliminary” government studies indicated that the defects
were due to contaminant dioxins rather‘than to 2,4,5-T itself.
. -, — -- -------------------------- ----------

i

0G 0486J.

o rc k o n

7ÎMD24S—9

5653

\ /

Toxicology and Pharmacology

BCM had been formed. Similar experiments with thiophanate
yielded BCM. The findings indicated that the fungitoxicity of
methyl thiophanate and thiophanate may come from their
transformation into the more potent fungicides, BCM and the
ethyl analog of BCM. respectively.

embryos were sacrificed between the 20 th and 22 nd days ofO
development. Eggs of the three species used usually hatch after ro
about 24 days of incubation. 2,4-D was very toxic to therO
developing embryos. Of the 320 pheasant eggs, 343 red-legged "'si
partridge eggs and 201 grey partridge eggs, 399, 148 and 148 X 1
embryos, respectively, died prior to the 19th day of incuba-Xj
tion. Most of the surviving chicks were either partially or
completely paralyzed. The embryos from the treated eggs were
smaller than the normal control embryos of the same age.
They exhibited a series of anomalies including lordosis of the
neck, fusion of certain cervical vertebrae, atrophy of the leg
muscles and clenched digits of the hind limbs. Feathers of the
ventral regions were depigmented and certain pterylae were
absent. The chicks were so deformed that they could not
break out of the eggshell at hatching. 2,4-D also had
pronounced effects on sexual differentiation during the
development of the embryos of all three species. The test
compound seriously affected the fertility of the treated birds.

71-1433. Bahr, T. B.; Ball, R. C. (Inst, of Water Res., Michigan
State U., East Lansing, Mich. 48823). Action of DOT on
evoked and spontaneous activity from the rainbow trout
lateral line nerve. Comp. Biochem. PhysioL A 38(2): 279-84;
1971. (8 references)
The possibility that DDT-induced tremors and convul­
sions in rainbow trout (Salmo gairdneri) could arise from
postsynaptic events initiated by intense afferent activity
impinging on the central neurons was investigated by monitor­
ing the frequency of spontaneous discharge and the patterns of
evoked discharge from the lateral line nerve before and after
the onset of tremoring. The trout were anesthetized with 100
mg MS 222/1 and permanently immobilized by severing the
spinal cord at the foramen magnum. The fish were placed on
their sides in a water-filled chamber (13 C) and their gills were
irrigated. Bipolar silver wire electrodes were looped under the
distal end of the centrally cut lateral line nerve midway along
the trunk and water was excluded from the recording site.
Neural activity was monophasicaily displayed on a Tektronix
502A cathode ray oscilloscope after amplification (Grass P-8).
The DDT dissolved in ethanol was administered in the water
(13 ppm for 6 hr prior to preparation) or directly into the
blood by cannula (0.01, 0.1, 0 2 , 0.4, 0.5, ID and 2D mg
DDT dissolved in ethanol and suspended in Courtland’s saline).
Responses were evoked by directing drops of water to the
surface of the water over the innervated portion of the lateral
line. The frequency of spontaneous neural discharge, as
determined by electrically counting the compound action
potentials on a decade scale and from photographs of
oscilloscope displays, revealed no apparent differences between
treated and untreated fish with either method of DDT
administration. Since obvious tremoring in the absence of
lateral line involvement was observed, it is believed that
afferent activity from the lateral line nerve does not play an
important role in nor provide a sensitive index for analyzing
the neurotoxic action of DDT but that the central nervous
system is the primary target for DDT in fish.

71-1435. Bratkowski, T. A. (The U. of Wisconsin, Madison,
Wis. 33706). Isolation and properties of a DDT-sensitive
adenosine triphosphatase complex from rat bain. Dissertation
Abstr. Intern. 31(9): 5149B-50B; 1971.
Copies of the thesis are available from University
Micronims, order No. 70-22,637.
71-1436. Roelofs, R. D. (Oregon State U., Corvallis. Oreg.
97331). Effects of dieldrin on the intrinsic rate of increase of.
the guppy, Poecilia reticulata Peters. Dissertation Abstr.
Intern. 31(9): 5319B-20B; 1971.
Copies of the thesis are available from University
Microfilms, order No. 71 -6785.
71-1437. Kapoor, I. P. (U. of Illinois at Urbana-Champaign,
Urbana, 111. 61801). Comparative metabolism of DDT,
methoxychlor and methiochlor in mammals, insects and in a
model ecosystem. Dissertation Abstr. Intern. 31(9):
5408B-9B; 1971.
Copies of the thesis are available from University
Microfilms, order No. 71-5145.
71-1438. Kuhn, P. J. (U. of Massachusetts, Amherst, Mass.
01002). Microbial interaction with diazinon, an organophosphate pesticide. Dissertation Abstr. Intern. 31(9): 551 IB;
1971.
Copies of the thesis are available from University
Microfilms, order No. 71-6388.

71-1434. Lutz-Ostertag, Y.; Lutz, H. (Laboratoire de Biologie
Animate, Faculte des Sciences, Complcxe Scientifique des
Cezaux, B.P. No. 43, 63-Aubiere, Puy-de-Dome, France).
Deleterious effect of the herbicide, 2,4-D, on the embryonic
development and fecundity of winged game. Compt. Rend.;
Ser. D 271(25): 2418-21; 1970. (French)
The effect of 2,4-dichlorophenoxyacetic acid (2,4-D)
was studied on the development of the embryos of the
pheasant (Phasianus colcfticus), red-legged partridge (Alectons
mfa) and grey partridge (Perdrix perdrix). Nonincubatcd eggs
and eggs incubated for 3tt days were placed in artificial nests
and sprayed with a 2,4-D solution which was applied at a rate
of 1 and 2 1/ha. The treated eggs were aired and turned twice
daily. Control eggs were sprayed with pure water. The

71-1439. Benz, F. W. (The U. of Iowa. Iowa City, la. 52240).
Stereo- and bio-chemical evaluation of a series of oxime
reactivatois of diethylphosphorylacetylcholiiicsterasc. Disser­
tation Abstr. Intent. 31(9): 5526B-7B; 1971.
Copies of the thesis are available from University
Microfilms, order No. 71-3712.
392

G0 C 1G37
et Q rr «

O W 256993 •

71-1433-9

3-1 - A

----------u-u—

CHDfliCAL BIOLOGY RESEARCH
charge 199-0000909

SUBMI TTCO OY

177-9007000

G. E. Lynn

NB T 2 3 .1 4 -1 1 -1 4 ,
NB T 2 5 .1 4 -97-5

oow c h e m i c a l u s . A

K NUMICR

DATS

8 /2 4 /7 1

2372, 20054. 7797

T H E E F F E C T O F 2 , 4 - D I C H L O R O P H E N O X Y A C E T I C A C I D (2,4-D) A N D E S T E R S O F 2 , 4 - D
.ON R A T E M B R Y O N A L , , P E T A L A N D N E O N A T A L G R O W T H A N D D E V E L O P M E N T
P . j. G e h r i n g 0 2 ^
B .A .S c h w e t z , G . L . S p a r s c h u

m
reported

BY

checked by:

p.

j.

G e h r i n g _______________

IN F O R M A T IV E SUWKMKV WITH C O N C L U S I O N ! B A S E D ON TH E S A M P L E R E C E I V E D . A D D I T IO N A L I NF OR MA TIO N IN C LU D I N G TH E E F F E C T
r e p e a t e d e x p o s u r e MAY r e R E Q U I R E D AS S P E C I F I C u s e s a n d f o r m u l a t i o n s a r c d e v e l o p e d o r i f p r o c e s s c h a n c e s o c c u r

This s t u d y e v a l u a t e d the e f fe ct s o f 2,4-D, the p r o p y l e n e g l yc ol b u t y l
A
e t h e r e s t e r o f 2 , 4 - D (PGBE) a n d t h e i s o o c t y l e s t e r o f 2 , 4 - D (10) o n
fetal de v e l o p m e n t and neonatal growth and survival.
D o s e l e v e l s o f 2 , 4 - D p:
u p t o a m a x i m u m t o l e r a t e d d o s e o f 8 7 .5 m g / k g / d a y o r m o l a r e q u i v a l e n t s
C
of P G B E or 10 w e r e administered to pr eg na nt Sprague-Dawley rats on day
t h r o u g h 15 o f g e s t a t i o n .
Fetuses were delivered by Cesarean section
to
on d a y 20 o f g e s t a t i o n and w e r e e x a m i n e d grossly, m e a s u r e d and weig he d.
F o ll ow in g routine preparations, the soft tissues and skeletons we re
examined.
S i g n s o f e m b r y o t o x i c i t y a n d f e t o t o x i c i t y , s u c h as d e c r e a s e d
fetal bo d y weight, subcutaneous edema, dela ye d ossification of bone,
'SJ_ *
lumbar ribs and w a v y ribs w e r e ob se rv ed at h i gh dose levels; considered
overall, the responses were dose related.
Teratogenic responses, however,
were not seen at any dose level.
2,4-D di d not affect fertility, gestation,
viability or lactation.
PGBE and 10 had no effect on fertility and
gestation indices, bu t highest dose levels decreased viability and
lactation indices.
Neonatal growth and de ve lo pm en t we re not altered
by treatment du r i n g pregnancy.

DISTRIBUTION
J. E. Johnton*
H. H. Mclatyro
C. E. Kiwwnol
L. K. Frovol
0. D. McCollistor ^2)
F. 0. Amo

B. E. Bwytft
H. L. Gordon, MD*
E. H. Bioir *
A. J . Schwort, MD *
S . M. MocCuichoen

0 . Kilion, M0 *
B. Horvath
H. Edwards
R. J . Sbavar
V. Bo Robinson*

B. Hotdor, M0 *
L Silvarstoin
L. Pitchforth
C. A. Coring *

cri (5) *

G. E .

Lynn*

C. S . "W illiam s* (4)
♦C om plete r e p o r t

QFSTR1CTFD FOR USE OF DOW EMPLOYEES ONLY

5655
0001838

5656

J

i-.2 ^

Japa.-.-ose J c a r n a l o f Ir.o-j= t r i a l Ha a 1th
V o l . 12, :;urvhar 3 i O c t c b s r , 137C)
jr, sgctiv.:;!

THE FATE CF 2. -I, 5-TEICHLCHCPHZNOXYACETiC ACID IN MAX
A:<:>.i;a M.ATSUWfL'P.A*

2,^,5-M '; = = 7 s ; ^
f-'.v

15 C 1

•r;

recovered from the urine. T h ese facts suggest th a t
analogically in th e Case of m a n , o rally a d m in istered
2. 4, 5 -T w ill be excreted in th e urin e. T h e a u th o r
exam ined w h e th e r or not in gested 2. 4, 5 -T w as
■excreted in the u rin e unchanged and in v e stig a te d
th e fate of it from the p h arm acodynam ic point of

T h e effectiveness c{ eh lsrir.ated phensxy acetic
acids s s p U n : grow th : : ; u h ' . 5 : i h is a e -s recognised
for i s u r i i c r i f
In e a rlie r re p o rts’. - I ) , 2.
d ie h lo ra p h e n ssy ie ttic acid (2. 4 -0 } '.vis re tire d to
h r r.cn-toxic :o snim nls ir.d mar.. 2 u : la te r. th e
tonicity of 2, 4 - 0 and 2. 4. o -tric h lo ro u h rr.o x y ire tie
acid (2. 4. 3 -T ) for ex p erim en tal an im als h r» b te n
rev ealed by various p a p e rs)—*).
A lthough trc.T.rr.dcus a s o s r .ts c i 2. 4-D . 2, i. 5T and th e related com pounds h av e brer. —anufaettirrd m d used, clinical rep o rts or poisoning 3 r : rare.
Niels-*:;*) reported a ease of suicide w ith ingestion
of d icth y larr.in i sait of 2, 4 -D . He observ ed o.O g
o f 2. 4-D in the corpse a: the victim , corresponding
to 30 mg/}:g. S ta b u ry i) re p o rte d an o th e r case. H e
ad m inistered to a p atie n t, suffering from d issem in ated
ceccidicid n y re s is. 3.5 Z of socium sals of 2. 4 - 0
through intravenous infusion.
T h e p a tie n t w as
troubled w ith tw iehir.gs of th e musoies anc fell into
stu p o r, b u t recovered.
A couple c f y ears ag o , th e a u th o r found such
sym ptom s and signs as n a u se a , vom itin g , -veigh:
loss, h e a d a rh c , fatigue and skin rash am ong th e
■workers of the test o p eratio n m ixing an am m onium sulfam ate d eriv ativ e, 2. 4, 3 -T and tw o o th e r kinds
of chem icals. T ho u g h th e epidem iological study
re v ealed 2, 4, 5 -T to be noxious, th e au th o r failed
to suppo rt the nncir.g because th e fate of 2 ,4 , 5 -T
in th e hum an body w as obscure in those days.
L’nsub stitu tcd o r p-ehlorophenoxyacetic acids are
excreted in urine by m a n , dog and ra b b i: in an
in tact form because -of th e e th e r b o n d a g e S -ti).
Clark'-'-), using C'-M abellcd 2 , 4 -D . ctm c.-.atrated
th a t S 5 ;i of orally ad m in iste re d 2. 4 -D was excreted
unchanged by sheep w ith in 72 ho u rs. Leigh'-)) cc
al. rep o rted the fate o f 2. 4. 3 -T in dairy cows.
C u t of 4 ;4 mg fed, 420.7 m g of th e 2. 4. 3 -T w as

t EjjMitn««? o:
Hf-illS, Hj'»salsa
o! Mriiria«, Sapors.
”! \£ r t ; T e i far «aMl cat ; sn A u j . 5, 13TD.

33:z u z i a m x i :

I V,

S u b jects
A v o lu n teer (2$-year-old male} w as ingested w ith
130 m ; (2 .2 m g ;k g ) of 2 .’ 4, 3 - T a fte r b re a k fa st.
Blood sam p les w ere c o lle c te d .il l-to 2-hour in te rv a ls
d u rin g th e first 6 hours and less freq u en tly th e r e ­
a fte r. •
T w o volu n teers (27- ar.d 23-year-old m ales} w ere
given ICO mg of 2 ; 4 , 5 -T o ra lly . U rin e sa m p les
w ere collected a : 2- to 5-ho u r in te rv a ls for 72 hours.
C ollections of 24-haur urine w ere o btained from
21 w orkers of a chem ical m an u re factory.
M e th o d s
1)

J SA 20 1

D eterm in atio n of 2. 4, o -T
D eterm in atio n of 2. 4, 5 -T w as carried out by
X ielsett’s m ethod w ith m odifications as follow s.
T w e n ty m illilite rs of d ilu ted urin e (tw o - o r th r e e ­
fold) o r d ilu ted blood plasm a (th re e - o r fo u rfo ld )
w as tak en into a 50 m l-esn trifu g in g tu b e w ith a
glass sto p p er.
A fte r a d d in g 1 g of c ry s ta llin e
ta rta ric ac id , 4 g of anhydrous X a jS O t, 2 -ml cf
chloroform ar.d 10 sr.f of eth y l e th e r, th e tu be w as
vigorously shaken for 5 os is tires and c e n trifu g ed fo r
3 m in u tes at 1.200 G.
T h e o rganic p h ase w as
decan ted and extractio n of the resid u e w as p e rfo rm e d
t-vicc m ore w ith 3 n l of eth er. T h e co m bined
o rg an ic phase w as ex tracted tw ice w ith 15 m f and
3 m f of 1/20 M p h o sp h ate buffer ( p i l 7 .4 ). A fte r
b eing w ashed w ith n aif th e volum e of c h lo ro fo rm ,
th e com bined phosphate buffer e x tra s: w as re n d e re d
stro n g ly acid wish 1.5 mf cf SN -HeSO s to bo e x ­
tra c te d tw ice w ith chloroform . T h e c h lo ro fo rm
solution w as e x tra c te d w ith 20 m l of b o ra te buffer
. (pH 10.5). T h e aqueous solution w as e x tra c te d

(2 0 )

585 ‘0003537

>*7

■vith-2 2 .3 .-)/ of c h lu m io rn a fte r i: had been made
strongly acid w ith 1.5 si/ of SM -llsSC t. T o r t - r .i v :
the tu rb id ity , the o.-cante e x tra c t w as f.ltrn trd
through anhydrous N ataO i.
O.'.c m illilite r or tv. a (ur —o re. :n the case of
l ow concent.-atinns) si :kv chic.-a form sclutian was
poured into a sm all flask to make it ev ap o rate
cautiously ju st to cryr.css. T w o m illiliters c: cr.rom otropie a rid in cor.-. 1I;3C< solution was poured
into the ftask, w hich was p r r p a rtd by dissolving
0.10 g of th e sodium salt of c h ro n o tro p ic acid (1.3d ih y d rex y n ap h th airn e-fl.aa.su l.'o r.la arid') :r. ICO mf
of co n cen trate d sulfuric a rid . T e n m illilite rs (o r
more according to the csr.rer.tratio n s) of Sr.Ci: solu­
tion p rep ared hv dissolving 25 g at atanr.ous ch lo rid e.
SnCIa-2!IaO. in 500 mf of 2N -H :S O i w as added to
the flask a fte r it h ad been h ead ed a: 143ab2*C for
ten m in u tes. C oncentration v.-is e stim ated a t 573

3 S

■iso

7in lmu if:

zap.
2)

Identificatio n of ex tracted 2, 4. 3 -T
2. 4, S -T in the urine w as identified by the color
reaction w ith ^chronotropic acid ar.d by the apectrophotom etric m easu rem en t of chloroform ex tracts.
G as-ehrojnalograohie identification w as accom plished
a fte r m eth y l e ste r of 2 .4 . S -T w ith boron trifluoride
being m ad c'.S .
E x p erim e n ta l conditions of gasch ro m ato g rap h ic analy sis w ere show n in th e feet
n ote of F ig . 2.
3) E stim a tio n s of 2. 4. S -T in th e surroundings of
a chem ical m an u re factory
2, 4, 5 - T in th e surroundings w as sam p led w ith
a high volum e a ir sa m p ler (G e im a n . T y p e A ). 2.
4, S -T a t the b re a th in g point of the w orkers w as
sam p led w ith a personal a ir sa m p le r w ith a glass
i b e r n ite r.
A fte r sam p lin g . .'¡Iter p ap ers w ere
rem oved in to a continuous e x tra c to r, c o n tain in g
a p p ro x im ately ICO m ( of chloroform . 2. 4. 5 -T ,
e x tracted La chloroform w as estim a te d w ith th e
m ethod m entio n ed above.

F ig . I.

A bsorption spectrum of 2. 4. 5 -T e x tra c te d
from th e urine when reacted w ith chrom o­
tropic acid at 143 to 14S*C for 10 m in u tes.

F .esuK s
D e te rm in a tio n o f 2. 4. 5 -T in th e u rin e
F o r a p re lim in a ry ex p erim en t. SCO and ¿00 ug of
2, 4. S -T h ad been added to 130 m / o f th e urin e of
an' a d u lt ar.d th e urin e w as an aly sed to o b ta in th e
recovery ra te s as h ig h as 95.6. 92.1?-» resp ectiv ely .
2) Id en tificatio n of extracted 2 . 4. 5 -T
A p ecu liar w in e-purple color developed w hen th e
e x tra c t w as h e a te d w ith eh ram otroplc acid in the
presence of concentrated sulfuric acid. T h u a b so rp ­
tion sp ectru m o f th e color is p resen ted in F ig. 1,
show ing th e m axim um absorption ra te a t 57S zap.
S p cetro p h o to m c tric m e asu rem en t of chloroform
'.solution, one step before the le s t of the p to ccu u rc,

et>l a ln l il l

1)

F ig . 2.

G as-chrom atogram c f 2 . 4, S -T
o b tain ed from the u rin e.

yielded the c h a racteristic absorption c u rv e o f]2 . .1.
3 -T . T h e resu it of g as-chrom atographie a n a ly sis is
re p resen ted in r i g . 2. on w hich the sa m p le coincided
w ith p u re chem icals its reten tio n tim e of a b o u t 3
m inutes. It is cle a r from these o b serv atio n s th a t
2. 4, S -T was excreted in th e urine in an in tact
form .
3) D istrib u tio n ar.d elim in atio n of 2, 4 , 5 -T
As w ill "be seen in r i g . 3 . the p lis m a eoncer.tra-

C 21 )

5658

0003538

443
line w ith a g rad ien t of —0.505, w hich indicate th a t
the absorption is also o f the ârst o rder.
T h e excretion o i 2. 4, 5 -T in th e urine a fte r a
single oral a d m in istratio n w as rep resen ted in F ig.
4 and F ig . 5. A fairly la rg e am eu n t of 2. 4. 5 -T
w as recovered from the initial sam p le but only about
45?« of orig in al dose w as found in th e urir.e col­
lected during the first 24 hours a fte r a d m in istratio n .
T h irty -six hours a fte r tre a tm e n t, 6 0 Ji had been

o
o
?**•
S ^Jiet U
ail«.>sif »»list M'<(*
:>S3( êi ¿ 4.¿-T 4jui)stt;f4
F ig . 3.

ip.

C o n centrations o f 2. 4, 5 -T in th e
blood p lasm a.

tion of 2. 4. 5 -T rose ra p id ly d u rin g th e first 3
fcours and th e peak p lasm a level (21.1 og/m f) w as
a tta in e d a t 4th h o u r a fte r a d m in istratio n . T h e
sem i-log arith m ic ear.centratior.-tim e curve -.vas lin e a r
a fte r 4 h o u rs’ post t r e a t m c a i . j n i i ç ^ t h i i ’â 'n rs i o rd er
e lim in a tio n -ra le w ith a g ra d ie n t of —0.C53 o btained
w ith the m ethod of least s q u a re s .. Zergstim e p lasm a
level (2S.0 fg /.T ./) w as o b ta in e d by ex tra p o la tin g
the lin e a r p a rt of the plot.
*■
T h e differences b etw een th e observed co n cen tra­
tions (C ) and the resp ectiv e ones o b tain ed from
ex trap o la tio n (C Q ) w ere a b le to be plotted on a

lue»*«»/ Ou)

j *

r

Tla» tMus)
S » S J « c t t* i7 - T « « r .o ld ;« ! « . X i r * « l ( * t S 3 t ; .
1 0 0 c ( * f 1 ,4 .4 -T i t e U i i u r f i

F ig . 5.

E x cretion of 2, 4, 5 -T in th e u rine.

recovered and a t th e end of 72 h o u rs m o re th a n
20*« w as recovered.
4) E stim atio n s of 2, 4, 5 -T in th e #u rin e as a
p ractical trial
T a b le 1 d em o n strates th a t a re la tiv e ly sm a ll
am o u n t of 2, 4. 5 -T w as found in th e u rine of
w orkers. In some of th e m , it w as im possible to
; d etec t it w ith th is m ethod. T h e co n cen trations of
j 2, 4, 5 -T in th e w orking a re a w e re listed in T a b le
“ 2 and T a b le 3. None b u t th e v a lu e o b ta in e d in th e
to*
* w eighing box of 2, 4, 5 -T exceeded th e rero m j m ended value by A .C .C .I.H ., w hich is 10 m g/m -.

to

*■

29 *Tr»t*e l4 » » ; # . i a i r
J C G i< i f

F ig . 4.

S9W*

4 « J ;iA ;a if;i£ « {¿ U ? .

E xcretion of 2 , 4, 5 -T in the u rin e.

D isc u s sio n
T w o m a jo r b an d s of absorption by th e pher.oxyacetie acids a re found in u ltra v io le t region!?). T h e
one band occurring in th e region of 223-230 m u is
p robably due to th e carbonyl group of th e sid e
ch ain ,, w h ile th e o th e r occurring around 2S0 m/f is
due to th e ben te n rin g . N ielsen?) a n aly zed th e
concentration of 2, 4-13 w ithin th e ra n g e of 230-33C
n / t . In th e m ethod, w hich relic s upon th e ab serp -

( 22 )

I

5659
c

1

0003539

449
2 .1 .3 ■T in the urine of w orkers of a
chem ical m anure fue :ory.

T able 1.

D ate

A pr.

N am e

9

A ge

A . Mo*
Y. I .
T . Ai.

23

M ale

2. C50

:o
so

M ale

1.4C0

1.5
—

M ale

1.370

—

40

1. C20

1.3

1. c:o

1.2

S .H .

25

F em ale
F em ale

T .T .
A.

29

M ale

370

0 .5

23

M ale

2. C00

s. s.

£0
60

M ale

2.220

Y. I.

M ale

2.400

3 .5
—
—

’ S .H .

25

F em ale

1.250

—

T .N .

33

F em ale

1,750

2 .7

A pr. l o

A pr. 15

s.x .

21

M ale

1, 400

3 .6

T . X i.

40

F em ale

750

Y. 1.

60

M ale

S. S.

£0
41

M ale

1, 570
3. 020

1.0*
—

M ale

1.470

2.2

53
33

F em ale
F em ale

450
250

3.5*
—

T . K i.

50
40

M ale
F em ale

S30
ISO

Y . 1.

SO

M ale

1.130

—
l.S *
■—

3 .N .
T . X o.
A p r. 22

Y . O.

s. s.

1

U rine
2.4. 5-T
volum e (m g/day)
(m l/d a y )

Sex

—

m illig ram s p e r lite r.
T ab le 2.

C oncentrations of 2. 4. 5-T in the
surroundings of a chem ical m anure
factory.

S am pling pointa

C oncentrations*

W eighing box
In le t of m ix e r

15.4
1 .3 7

O u tlet of m ix er

0 .8 2

C enter of v.-ork-rsom

0 .5 2

*

m illig ram s p e r cubic m eter.
C oncentrations of 2, 4 , 5-T a t the
b re a th in g location of w orkers.

T a b le 3.

W orkm en

C oncentrations*
0.21
0.31

A
B
C
D

-

■s m illig ram s p er eubie m eter.

0 .6 7
0.33

tion 'in the u ltra v io le t region, it is ra th e r u n sa tis­
factory to m easure the concentrations low er th an
] mg/lOO rr.l because of the low abso rp tio n ra te .
U sing the color reaction w ith c h ro n o tro p ic acid, I
can m easure concentrations low er th a n th at a c ­
curately.
,
On th e o th e r h a n d , in d u strially m an u factu red 2.
4. S -T seem s to be inevitably c o n tam in ated w ith
2 . 4. S -trichlorophcnoi, the p recursor of 2. 4, 5 -T .
o r o th e r chlorophenol d eriv ativ es. A s long as th e
m easu rem en t is carried out in the u ltra v io le t region,
a t 223 m t h e contam inants possessing the ber.cen
rin g can not be excluded. C h ro n o tro p ic acid en­
hances n o t only sen sitiv ity but also specificity.
T h e dev elo p m en t of the color d ep en d s upon a
reactio n b etw een 2, 4, 5 -T ’ and ehrom otropic aeid
in the concentrated sulfuric acid. I t is said th a t •
th e h e a tin g of 2. 4, 5 -T in the presence of concen­
tra te d su lfu ric acid resu lts in the fo rm atio n ' of for­
m ald eh y d e w hich th en reaets w ith th e ehrom otropic
aeidlO . T h e reactio n , highly sen sitiv e to a sm a ll
am o u n t o f phcr.oxyacetic acid, is re la tiv e ly non­
specific. A n u m b er of o th e r com pounds such as
acetic acid, fa ts and w axes m ay re a c t and g iv e a
sim ila r color. B ut in th is stu d y ^ sam p le s free from
2 . 4, 5 -T did not develop the c h a ra c te ristic p u rp le
color. S o m etim es w hen the sam p les w ere h e a te d
above 153*C, they developed a m b e r color, w hich
w as b leached by a stannous chloride solution. S ince
th e w ine-purple color of ehrom otropic acid h ad th e
m axim um absorption at 5^5 m p , th e co n cen tratio n
w as estim a te d in this region.
In p re lim in a ry ex p erim en ts, th e reco v ery of 2. 4 ,
5 - T , added to the u rin e of an a d u lt, w as satisfa c to ry
and th a t of 2. 4. 5 -T , added to th e ho m o g en ate of
th e o rg an s of ra ts, w as shown clsew herclS ).
T h e id e n tity o( 2, 4, 5 -T , e x tra c te d from th e
u rin e , w as judged by the follow ing c r ite r ia : a
c h a ra c te ristic color reaction w ith eh rom otropic acid
re p re se n tin g the absorption m ax im u m a t 575 m^r,
coincidence of the absorption m ax im u m w ith pure
2 . 4, 5 -T in th e u ltrav io let ra n g e a t 233 m ^ , and
coincidence of a reten tio n tim e in g a s-ch ro m ato ­
g ra p h ic analysis. T h e acetic acid o f th e side chain
w as d e m o n strated by th e positive color reac tio n
w ith ehrom otropic acid. T h e b e n re n e rin g w as
in v estig ated spcctro p h o to m ctrically in th e u ltra v io le t
reg io n . N o t only th e intactr.css of e th e r bondage
b u t also the intactn css of the chlorine o f th e side
chain w ere confirmed by g as-ch ro m ato g ram s. I t can
b e concluded from these o b serv atio n s th a t in g ested
2 , 4, 5 -T w as e x e rtte d in the u rin e u n ch an g ed .
O rally ad m in istered 2, 4. 5 -T w as re a d ily absorbed
a s in dicated by the o bservation th a t th e p eak p la sm a

C-

CO
’ ¿71
CJ

(2 3 )

5660
0003540

«so
concentration w as atta in e d w ithin a few hours a fte r
a single ad m in istratio n .
L in earity of the sem ilogarithm ic eo n rrm ra tio n -tim e curve in its te rm in a l
course indicates th at 2. 4. 5 -T w as elim in ated from
the blood plasm a a: the firs: o rd e r elim ination rate.
On the o th e r h an d , n o n -lin ear p a rt of the plot re ­
presents th e absorption ot 2. 4, S-T from th e
digestive tr ie t.
T h e absorption ra te could be
estim ated in d irre t'y by p lo ttin g th e differences b e t­
w een th e observed concentrations and respective
ones obtained from c.M rapoiation on scm i-lcg arith m ie
scale a g ain st tim e , which stan d s for the absorption
Tate'*).
D istribution and elim in atio n of 2, 4, 5 -T have
rarely been in v estig ated . Only Kurt-E.*ne'>3) re p o rted
th a t the plasm a concentration tim e curves as w ell
as th e plasm a h alf-life v alu es of 2. 4, 5 -T w ere
sim ilar to those o b ta in e d w ith 2. 4-D in 'e x p e ri­
m ental anim als. A ccordingly, it is of in te re st to
com pare the fate of 2. 4, 5 -T in m an w ith th a t of
2, 4-D in experim en tal an im als. D istribution volum e
can be estim a te d from zero-tim e plasm a level and
th e dosage given a t 7.9?£ of th e body w eig h t, a p ­
proxim ately as larg e as the to tal blood volum e. It
i s said th a t 2, 4 -D could reg u larly be d etected in
blood cells in concentrations ra n g in g 10 to 207# of
the corresponding plasm as. E ased on these findings,
tw o th ird s of the orally a d m in istered 2. 4. 5 -T
-would be d istrib u ted in the blood in e a rlie r sta g e s
o f elim ination.
C larklD described in his p a p e r th a t the rad io ­
activity of lab elled 2, 4-D rose rap id ly in the blood
plasm a of sheep during th e first h alf-h o u r, reach ed
th e peal: a fte r l 1/« hours and d im inished rap id ly
th e re a ftc r. A bout 15f» of th e o rig in al dose w as
excreted in the u rin e in the first 1*/« hours. E ig h t
and a h a lf hours a fte r tre a tm e n t. 50?» h ad b een
recovered in th e urine and by th e end of 23 hours,
over SO;« of th e dose h ad been ex creted . In co n trast
to these o bserv atio n s o f C lark , th e p eak p lasm a
lev el of 2, 4, 5 -T w as a tta in e d 4 hours a fte r a d ­
m in istratio n and th e plasm a lev el declined g ra d u a lly ,
in d icatin g the p la sm a h alf-life as 11 hours. W ith in
24 hours, only 45*» of the o rig in a l dose w as found
in the u rin e o f v o lu n teers. Low elim in atio n ra te ,
th e re fo re , w ould m ake 2. 4. 5 -T m ore toxic in m an
th an in e x p e rim e n ta l a n im als.
I t is m ig h ty fav o rab le for p rev en tio n and tr e a t­
m en t of 2, 4. 5 -T in toxication th a t th e a g e n t is
excreted in the urine unchanged. T h e in tak e of 2,
4, 5 -T could be e stim a te d by urin aly sis. As a
p ractical tr ia l, th e u rin aly sis of th e w orkers w as
perform ed to find out th a t a co m p arativ ely sm all
am ount of 2, 4, 5 -T w as recovered. In som e of

th e m , the ic e n t could not be d e te c te d w ith the
m ethod m entioned above. I t is to b e accounted
for by the low en v ironm ental 2. 4, 5 -T concentra­
tions, w hich w ere due to sev eral kinds o f im prove­
m e n t of operations. F u rth e rm o re , w o rk ers had b e n t
provided w ith such protectors as gloves, m asks,
hoods and p ro tectiv e suits and th ey took a b a th at
the end of duty. In the case of a w ell-controlled,
o p eratio n , the u rin ary exereations w ere occasionally
low er th an 1 m g /d a y . T h u s, it w ould be in ad eq u a te
to d etec t 2, 4, 5 -T w ith the m ethod m en tio n ed , if
th e chronic toxicity of 2, 4, 5 -T could no t b e ruled
o u t in such levels.
,

Summary
T o clarity the fate of 2, 4, 5 -T in th e h u m an
body, v olunteers took 100 to 150 mg of 2, 4 . 5 -T
orally . Blood and urine sam ples w ere co llected a n d
analyzed to find out th a t
1) orally ad m in istered 2, 4. 5 - T w as re a d ily ab­
sorbed and elim in ated g ra d u a lly from th e blood
p lasm a, show ing a first ord er elim in a tio n ra te .
2) m ore th an 807» of orally a d m in iste re d 2, 4. 5 -T
w as excreted in th e urine in a n in tact form w ith in
72 hours, and
»
3) elim in atio n from the blood p lasm a and ex eretion
in th e urine by m an w ere slow , a s co m p ared w ith
those in e x p erim en tal an im als.
A ccording to th e m ethod e la b o ra te d , 2. 4 , 5 - T in
th e u rin e of w orkers of a ch em ical m anure factory
w as analyzed.
*

Acknowledgement
T h e au th o r w ishes his a p p reciatio n to D r. U ed a
and M r. T a ru m i for th e advice an d e n c o u rag em en t
they h a v e m ade d u rin g the course of th is stu d y .

P. C.

References

1) M arth,
and Mitchell. J. W .: 2. 4-Diehioro*
phenoxyacetie Acid as a Differential H erbicide. S o t.
Gaz., ICS: 224—232, 1944.
2) H ildebrand. E. M u W ar on W eeds. Science. 1C3:
■¡55—453, 1945.
3) Bucher. N . L. U.: ESects o f 2. 4-D:ehloropher.o:cracetic Acid on Experimental A nim als, Pzoc. Soe. E sp er.
Biol. &. M ed.. 63: 2C4-2C5. 1945.
4) H ill, E. V . and Carlisle. H .: T oxicity of 2. 4 Dichlorophenoxyacetic Acid for Experim ental A nim als,
J. ln dnsl. H ys- & Toxicol.. 29: S3—93. 1917.
5) Drill; V . A. and Hiretcka, T .: T oxicity o f 2. 4 DiehloropHer.oxysceiic Acid and 2. 4, 5-T riehlorophepoxyscetic Acid. A rch. Indusr. Hyg. Occupational M ed..
7: Cl—57, 1933.
5) Oiorklund. N . E. ami X urt-Erne: Toxieologicat
Studies of Phenoxyacetie Herbicides in A nim als. A cts
vet. stan d .. 7; 391-393. 13£o.

5661

0003541

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451
7) Nicl>en. K., Ka:mpe. 3 . and Jrnsen-llnlm . J.t rata l
Poironinx in Man by 2. 4-[>iehlorop’n enoxyaee:ie Acid
(3. 4-D): Determination of the A srn t in Forensic
Material.«. A e n pharmacol. et toxicol.. 22: 324—221.
_ 1SG3.
3) Scabury. J. H.: Toxicity of 2. 4-Dieh'orophenozyjeetie Acid for Men and D o :. Arch. Environ. Health,
7: 203-2C3. 1953.
3) Levey, S. and Lewis. H. 3 .: T he Metabolism of
Phenoxyacetie Acid, ita H nm oio;ues. and some Mono*
chiorophcnoxyaccte Acids. New Example* of ¿-Oxida­
tion. J. Biol. Chsm., ISa: 213-221. 1947.
10) W illiams. R. T .t Detoxication Mechanisms, 2nd
- Ed.. 220. Chapman ¿t Kail. London. 1933.
11) Clark. D. E ., Y our.:. J. E .. Y ounser, R. 1—. Kur.t.
L. M. and McLaran. J. K.t T he Fate of 2. 4-Diehlorophenoxyacetic Acid in Sheep, J. A sric. rood Chest.,
12: 4 3 -4 3 . IE « .
12) Leizh. E .. V fa jn e r. D. G. and Lisk. D. J.t ra te of
Atrazine, Kuron, Siivcx and 2. 4. 5 -T in the DairyCow, J . Dairy Science. 47: 1257—1270. 1554.
13) Banduraki, R. S.t Spectrophotometrie Method for
Determ ination of 2 ,4-Dichlorophenoxyace::e Acid.
Bot. Caa.. 1C3: <45—449, 1946-47.
14) LeTournesu, D. and K roz. PL: T h e Use of Chrostotropic Acid for the quantitative Determination of
2. 4-DlchIorophenoxyacetic Acid. Plant Physiol., 27:
622-623. 1932.
15) Mataumura, A . and A be. S.t Studies on the T oxi­
city of 2. 4. S-Trieh!orophenoxyaeetie Acid. Report I.
On the Determination of 2. 4. 5-T in Tissue Hopenates,
Jap. J . Indust. Health, 11: 103. 1559.
*
36) Metcalfe. L. D. and Schmitz. A . A .: T h e Rapid
Preparation of Patty Acid Esters for Cza Chroma to. jrmphie Analysis. Anal. C hest.. 23: 353—354. 1931.
,17) N 'aka;aki. M .t D r.-; T ransfer in 3io(o;iaal Systems.
1st E d , 153. NANKODO. T okyo. 1 9 3 .
13) X url-Erne: Distribution and Elimination of C h l^
rlnated Phenoxyacetie Acids in A nim als. A cta v et.
scan d , 7: 240—355. 1356.

2.4.5 - M ; a s 7 s )

(2.4.5-T)

on W i f j i r t i i □ a - l ' D . cJvfcEai-j
fc.2-5rL.
K'j'tifajaiifitaLfc.

'.t'.V 'J W 'i'V 2. 4 .5 -T O fiJK 'L 'V tlC Si N'iciscn hr>
Z i'J i& Z L 'i L .

F i z m f 5 2 . 4 . 5 -T

iiAFJrli 150mg O 2.4.5-7
a n s i •>•

i ? = -i- 1-

-f*.
4 s?sq-c i \ s a

21.1 «/m f ic;iL . £12;A ri:ii£T L i:. i i ^ i - o O '

Siti;R + i 9 f t ! i S L i : r ; r i l f y : 0 S ' 3 0 3 f i s '? i a i i L i : t S
i!i. 2 . 4 . 5 - T t i t h o a r t . - i i l - -C f/V iii .

E502fc1f 3

1

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< « C.

1) SSaT&*i*-C* o * h» —^ SiE oU V n
i : - / K - a 2 .ic s 2 .L i:.
3) iA SiijO 7P3

C'

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<o2Sii5ri-.*.lt 233 tn/r 1C*, 9 R iJo u O -riii—S L
*-A?fl -rh/57i -*CK2i LilSSiTc. Ri*2?W«iiV

8 ft-C* 9 C S ijiO -= :h .i-a L i:.
SA J5?2iSiC 100mg o 2,4,5-T S rE c a -^ L .
T’^nnicdp/zoTI?.!?.L7 -^ ¡-5 )2 S iii:2 . 4, 5-T crJS
¿LLi:- S24i!£2;:-ir.n-C-G-?-iVoii45;i, -usitVj'CJSre
723y,‘ul-cso;.'LlL-V-ir,!ci 1 i ; ■f) ieJJ’iul
o i 1'X.P3.it.F,<Op2S.;ci;-ov''Ci25'lS-j'C2.4. s - T o ^ r t f i

A 2L *t£:£T o t m > < o - Z £ i z i L*C. * % fiS tJ2 rrZ «
fE3i?f0243-rrj]l? ..-o i« 'C S ‘7=2.4,5 -T 4 rS lS L i:. f?
3i%S5ij*:pO 2.4.5-T O S (S li 0.62 mg/m1—>15.4 mg/
mJ -C5,9. S0^ LfcI521dS*jSS:a!!t 3. Smg'day -C£>

-o/z- if:fi^ aO :T ;* -C -tiD 0 7 jL S lci -,-C2.4.5-T

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5663

120330

Che.-n.'caf Abstracts

Vol. 7 2 . 1 9 7 0

N |i

240

r---- -------

us«-l i t 4 kg Ti.i prtrr:< -ri'.n:e, did not d c rt-i.»:
N O )', NH»,
and l*;Oj Icv»N i:i the »¡1. I t s tin .u la if 1 th e activ ity uf the soil
mi- i i .H.im an^l inerra-.-d the co n ten t of soil nuti---:««. A
30 ‘■‘•'."r incr. i«,: in N content » a s n->t-.-l in the pr«--»--.» ..f the
htrhi-iile. On T ro ia n -tre a te d castar b*-ins the
1 <>f >■•••!«
was l«H% hig h er th an with the c o n tr ol. 1 he con»- nt of N' v d P
in plants cu ltiv ated in the presence of T rcllan » a s hi<l>»-r than
in the control.
A. Rad*-cki
120331s U se of herbicides oo turnip plantings. Puzin.n'te, I.
(U SSR ). K h im . Set. Khaz. 1970, 3(2). 131-2 (R u -0 - Dacrhal
4 -8 , diphenam iil 3 -6 , trifluralin 2 -6 , and .un-hen 2 -3 kg. "ha w-re
to t e d in 3-year field expis. against weed i in turnips planted in
snd-podzols (pH 321-6.3). T he effect of th e -: fcerb-'cides on
turnip yield w«re compared w ith a double hand wc>d'ng as the
control. T h e yield was always less th a n in the control. The
m ax. yield decrease (30 and 29 % , re -p .) was with 4 k j dacrhal
a n d -5 kg a m ib rn /h a . W ith 3 kg dipht-nutiid and 4 k-g triflu ralin /
ha a m in. yield decrease (10 ami 11% , resp.) was obtained.
D iphenam id was also most effective, even at 3 k g /h a , against
Raphanus raphar.iUrum or S te lti'u i media, killing 73.3% when
before thinning and 32% before h a rv e st. D acthal in d trifluralin
were less effective, and low levels of am iben had no effect; a t 3
k g /h a am iben was to e'e to tu rn ip s.
J . Maleic
12033 ’ t Preharvestir.* chem ical drying of fodder lupine in
the w estern U kraine. C hernobai, X . K h .; Proskura, I. P .;
M efn ich u k . P . D .; B ugaiskaya, Z. Y a . (U SSR). K h im . Set.
Khaz. 1970, 3(2), 133-3 (R uss). P reh arv est defoliants and
desiccants for fodder lupine were te ste d in 3-year field expts.
T he most effective chems. (dose in k g /h a given) were: X H .SC X
(2. 3, 10, 25), X aSC N (2. 5, 10, 23), dinitro~a-:rcsol (1, 2, 3,
10), N aO H '3 . 10. 23. 50), Reglone (0.5. 1.0, 2.5. 5.0), and
Gramovor.e 0.5, 1 .0 ,2 .3 .5 .0 ). S praying -if X aSCN or XH.SCN"
10-20, diniiro-o-oresol 2 .5 -5 .0 . and Cramox-jne or Reglone
1.5-2.5 kg. h a accelerated seed m atu ratio n by 3-17 days, in­
creased seed germ inating by 9 -1 3 % , a n d increased seed crude
protein by 0 .4 -l.6 % .
J . M alek
120333u R esults of testing b iennial weed control in w heat
with new herbicides based on s-triazin es, substituted ureas, and
fluorenol. C hiapparini, L .; Soldini, G . B. (Sez. P ato l., O sserv.
M aiattie P ian te, M ilan, Ita ly ). K alis. M at. Pianle 1969, Xo.
80-81, 209—43 (Ita l). Excellent weed control with min. phyto­
toxicity w ere obtained w ith te rb u trin [2-((er.'-butylamino)-4(ethylam ino)-6-m ethylthio-s-triazine] applied preemergence a t
1J kg of activ e ingredient,1h a , m etoxuron [Af-(3-chloro-4m ethoxyphenyl)-iV ',iV '-dim ethylureaJ, neburon [X -b u ty l-Y '(3,4-dichIorophenyl)-.V-m ethylurea], a n d prepn. 3633 (X-benzothiazolyl-.V,2V'-dimethylurea) postem ergence a t, resp., 3.2 o r
4.3, 3.0 o r 3 .6 , and 1.4 kg activ e in g re d ie n t/h a .
»/
Felix Saunders
• X r - 120334V Toxic effect of som e h erb icid es on fish an d aquatic
/'i n v e r te b r a te s . B elyavskaya, L . I . ; K onstantinov, A . S .;
K onstantinova, X . S . (U SSR ). Vidovoi Soslav, Ekol. Prod.
Cidrobiontoo Volgograd. Vodokhran. 1 9 6 9 ,9 3 -8 (R uss). E d ited
by K onstantinov, A . S . Izd . S a ra to v . U n iv .: S arato v , U SSR.
F o r th e prepn. of huge w ater-reservoir beds, woods an d shrubs
a re often destroyed w ith herbicides e .g ., th e Bu ester o f 2,4-D ,
the Bu ester of 2,4.5-T , Foredex 75, a n d Fortex (aq. emulsions
of 2 ,4 -0 a n d 2,4,5-T , resp .). A fter 20-30 m in of co n tac t of
these em ulsions a t 60-120 m g/1., crayfish (Simeeephalus vetzdus)
died; on c o n ta c t w ith a q . em ulsions contg. 10 mg/1., th e y
survived for 5 d ay s. Of all herbicides tested on invertebrates,
which serve as food for fish, 2 ,4 ,5 -T w as th e most toxic, an d it
lcilled them all a t 2 mg/1. T hese herbicides a t 4 and 6 mg/1.
were toxic to 7 ty p es of fish w hen added to Volga w ater a t
14-16* for 1 d a y . Solar oil was also toxic to th e fish and th e
fish feed. A t th e ir present level o f use 6 k g /h a of shrubland,
the concn. of herbicides ia w ater in th e studied reservoirs will
soon reach fa ta l levels to th e fish a n d th e ir food. However, if
th e herbicide tre a tm e n t were lim ited to 0 .0 1 -0 .1 % of th e to ta l
bed-area of th e reservoirs, th e resulting concns. in th e w ater will
be harm less to the fish an d th e ir bio-food.
S . K . R am an
12033Sw Com bined effect of trifiu ralm and M SM A [mono­
sodium m eth an earso n atej o n Jo h n so n g rass control in cotton.
K leifeld, Y . (Volcani In s t. A gr. R es., B e t D agan, Israel). Weed
l ’ 7 ° ' ,l 8 ( l) . 16-13 (E n g ). P rep lan tin g application of
trifluralin reduced th e no. of m ono-X a m ethanearsonate (M SM A )
applications needed for th e control of Johnsongrass {Sorghum
htuepense). T h e com bined effect enabled weed control to be
com pleted before co tto n {Gossypium h in u iu m ) began to bloom ,
a n d hence w ith o u t causing dam age to th e crop.
R CK Z
120336x M etabolism of ehlorbrom uron-“ C la corn and
cucum ber. X ashed, R . B .; K a tz , S . E .; (luicki, R ichard 1).
(Coll of Agr. an d E n viron. Sci., R u tg ers U niv., Xew Brunswick,
M -J.). Weed Sei. 1970, IS U ), 122-5 (E n g ). T h e m etabolic
che
ro o t*aPplied, “ C -carbonyl-labeled, 3-(3-chloro-4-broinoL , : . yl;V-.m. " ho'> " l - ^ h y l u r e a (chlorbrom uron) was investicucum ber
ma?‘ ? ?
4nd ^ ePtib le
•if*trlv Thm
,a **-«*. v ar M ark eter) m z tune-course
*u
r
'« c u b o liie found in corn shoots an d roots
was th e uonphytotoxic S - a - c h t o r o - g - b r o i n u p h e n ^ .l - m l ^ “

u rea. Evidence of binding was found in both shoots and r<»its
of corn. Xo evidence was found for the metabolism of rhlorbromurop. in cucum ber; how ever, some binding occum--! in b->th
sh-jots and routs.
_
RCVCZ
1293J7y Effect of the ratio of soil to water on the if .,
n
of linuroc and itriz in e . G r »ver, Rajba.as; H ance, R . J . (C m .
-Agr. R es. S tn ., R eg in a, Sa-»k.). S o il Sci. 1970, 109(2), 136-3
(E ng). Begbroke soil was p rep d . b y air-drying and pn-.ing
through a 10 meah sieve. T h e soil contained 1.93% C, 15.6%
clay, 18.4% silt, and 66.0% san d . Its water holding capacity
was a b o u t 24 % . T he pH of a 1:1 (soil : w ater) suspension was
7.1. Five herbicide concns. were used in the range of 5-25
pp:n lin-arm and 0.5 -5 ppm atrazir.e for the 1:10 fsnihw ater)
ra tio ; 5-25 ppm linuron for the 1:1 ratio; and 15- «3 ppm
Iinuron and 5-20 ppm atrazin e for th e 4:1 ratio . The linuron
soins, were m ade in 0.1 S f C aC l, an d th e atrazine soins, contained
no CaClj. A dsorption isotherm s were tinear in each ca.-c and
comparison was made using th e Freundlich relation with values
of k reported. T here was a 5-fold increase in th e adsorption of
linuron a t the 1:10 ratio (ft 12J ) compared to the 4:1 ratio
<k 2.7); adsorption in th e 1:1 m ix t. showed k 4.4. For atrazine
th e difference between slu rry conditions (1:10) and the 4:1
ra tio was approx. 3-fold, th e k values being 0.8 and 2.1, resp.
I t was concluded th a t adsorption o f herbicides under field condi­
tions, w here th e soil aggregates are relatively large, may not he
as high as those estd. u n d er slu rry conditions. There m ay be
v ariations in th e exten t of adsorption of a herbicide within 1 field
if aggregate size d istrib u tio n varies.
Peter Coad
120336Z P henothiazines as p lant growth regal a r t s . Farbenfabriken 3-zyer A.-G. F r. D em ande 2,901,531 (Cl. A Oln. C
07d), 26 Sep 1969, C er. Apfil. 08 Feb 1968; 7 pp. Pheaothiazines (I) <R, R* - halogen, alkyl, alkoxy, C Fj, alkylcarbonyl,

r*O Û 3 - r’* w
T ’

ANRIP

w'

o

2

^

A represents alkylene, R l, R* — alk y l or form together wjthr.the
X atom a 5- o r 6-m embered rin g ; n — 0 , l , or 2) and th eir salts,
are used as p lan t grow th reg u la n ts. Compns. w ith v a r i o u ^ ^
concns. of I can a c t as grow th stim u lan ts, growth inhibitors,
herbicides. T he com pns. generally contain (>.1-95% I and i r t ^
applied a t th e ra te of 0.001-100 k g /h a . I may be applied t o r “
dicotyledons (cotton, b eets, c arro ts, beans, potatoes, coffeejJO
m ustard, w ater cress, b ed straw , camom ile, and galingosa)
monocotyledons (maize, rice, o a ts, barley, w heat, millet, sugar
cane, c a t's ta il, spear grass, ra y grass, and panic grass).
G . P . X atus
120339a Im idazoles a s h erb icid es. D raber, W ilfried; Falbe,
Jurgen F .; Buechel, K arl H .; K o rte , Friedrich W . A. G . K .
(Shell Oil C o .) U .S . 3,501,286 (C l. 71-92: A O ln), 17 M ar 1970,
A ppl. 29 J u n 1964; 4 p p . Im idazoles (I) where R is H , C i-w
alkyl, cyanoethyl, or allyl, a n d Y is H , Ci_» alkyl, C X , halogen,

R
o r XOt were activ e a s general preem ergent herbicides a t 10
lb./acre, while th ey selectively controlled broadleaf weeds a t
2 lb /a c re . W ettable pow der, d u s t, an d emulsifiable cone,
com pns. are given. A d u st con tain ed 4,5-dibromoimidazole 4,
M g stearate 1, kaolin 58, a n d gypsum 37 p arts by w t. In field
tests I(Y - B r, R - H ) gave 9 9 % control of broadleaf weeds a t
2 lb /a c re , b u t only tem p o rary b u rn in g of grasses. G erm ination
o f weed seeds was p revented b y incorporating 2 -8 lb I (Y « B r,
R m H ) o r I (Y “ I, R *■ H )/a c re in th e soil.
L . Tetzloff
120340u D ialkylam inoalkyiene sulfide herbicides. B ordenca,
C arl (SCM C o rp .) G e r. O ffen. 1,929,390 (Cl. A Oln), 19 Feb
1970, US A ppl. 27 J u n 1968; 24 p p . d- Dialkylam inoalkyiene
sulfides R SX X R 'R * (I), optio n ally as stable salts thereof, are
used; R — linear o r b ran ch ed C«_u alkyl, preferably octyl an d
decyl branched or n o t, X ■» lower alkylene, preferably ethylene,
R 1 and R’ - sam e o r d ifferent low er alkyl, preferable E t. d-D iethylam inocthyl n-octyl sulfide. 3-diethylainm oethyl 3 ,7 -dim ethyloctyl sulfide, a n d d-dicthylam ir.oethyl 4,6,3-trim ethylnonyl sulfide, optionally a s hydrohaiides. are claimed. I w ere O
tested as ore- an d post-em ergence herbicides.
CJ
......M anfred K naack
^
120341V H erbicidal C ym etrin [2-m ethylthio-4,6-bis(ethylam ino)-»-tnazine| an d benzyl alkylthlocarbam ate m ixtures.
K im ura, Ichiro; S ugiyam a, H iro n ari; K ado, M asaru (K um iai
Chemical In d u stry C o ., L td .) G e r. Offen. 1,943,983 (C l. A
O ln), 12 M ar 1970, J a p a n . A ppl. 28 Aug 1968-07 Ja n 1969;
24 p p . A m ix t. of C y m etrin
(I) a n d thiocarbam ates (II) II
(R - R‘ - E t. X - 4-C1) (b ,.„
127-31*) 7% . 1 1.5% . Xa ligninsulfonate 2 % , woo«l ashes 5 % , a n d bentonite 34.5% by w t.
were ground an d m ixed to hom ogeneity, kneaded w ith w ater,
and granulated to granules of 14-32 m esh. T he In rbiculal mi <t.

-SEATTLE SALES OFFICE
Bellevue* Washington
April 23, 1980
J. R. Beachell
K. D. Bohlander
A. J. Herrera
R. F. Kincaid
J. C. Mitenbuler
W. H. Walker
P. H. Williams

Zt Z9 ¿ 0 NN
* B fc 0 6 T M O a •

DOW C H E M IC A L U .S.A.

cc:V C. TfóM&k
L. E. Warren, Davis
M. L. Smith
D. M. Frederick, 9008 Bldg.
SUBJECT:

ESTERON 245/ESTERON BRUSH KILLER/KURON

As you will note on the attached specimen labels for subject products,
the latest revisions, dated January 1980, clarify the Environmental
Protection Agency's most recent rulings on use of these products.
As the brush begins to leaf out in farmer's fence rows, this would be
a good time to remind your distributor salesmen— and the dealers you
are calling on— that growers may purchase and use these products
legally.
Of course, additional specimen labels are available for distribution
as you determine appropriate in your sales territory.

District Sales Manager
Agricultural Products

pmg
Attach.
5 6 6 5

0001741
A N O PER A TIN G UN IT O F THE D O W CH EM ICA L C O M PA N Y

5666

334
rnKOM.47S3

no tes

MN07 2 6 4 9

Simultaneous gas chromatographic determination of 2,4*0 and dicamba ¡r
human blood and urine

t

The compounds 2 ,4 -dichlorophcnoxyarctic acid (2 ,4 -D) and 2-methoxy-i o
dichlorobcnzoic acid (dicamba) are herbicides commonly used to control a variety Q
of broadleaf plants. An acute poisoning case was recently investigated bv the Hawaii
Community Study on Pesticides in which the victim intentionally ingested a forma,
lation containing 2 ,4 -D and dicamba. The occurrence of this attempted suicide created
a need for a rapid and sensitive method for detecting these compounds in small
o
amounts of human blood and urine.
.H
Several methods for detecting 2 ,4 -D residues in a variety of substrates wer* C
ZD
considered for the analysis of 2 ,4 -D and dicamba in human blood and urine'-*
While generally sensitive, these methods were neither rapid nor applicable to the CO
CO
relatively small samples available.
The Hawaii Community Study on Pesticides uses the method cf B evexue elal*
to determine pentachlorophenol (PCP) residues in human blood. :n an evaluation
done by this laboratory*, the procedure was shown to be rapid, accurate, sensitive
and reproducible. Because 2 ,4 -D and dicamba display chemical characteristics
similar to those of PCP, these compounds are also detected by the procedure.
This note describes the application of the method of B evence et al* to the
simultaneous determination of 2 ,4 -D and dicamba in human blood and urine.
Experimental
Solvents and reagents. The following solvents and reagents were used: benzene,
hexane, isooctane (Mallinckrodt Nanograde Reagents), used as received; 0.1 .V H.SO,.
benzene extracted; 2 ,4-dichlorophenoxyacetic acid; 2 -methoxy-3 ,6-dichlorobenzoic
acid; N-methyl-N'-nitro-N-nitrosoguanidine, supplied by Aldrich Chemical Co.,
Milwaukee, Wise.

The diazomethane solution* was prepared as follows: hexane (25 ml) and 2 ml
of 2 0 % aqueous NaOH were combined in a 50 -ml erlenmeyer flask and 0.5 g of
N-methyl-N’-nitro-N-nitrosoguanidine was added in small increments. When the
reaction was complete, the hexane layer was decanted into a small glass bottle
equipped with a Teflon-lined screw cap. The diazomethane solution was kept in
a freezer for up to one week. Because diazomethane is a skin irritant and a carcinogen,
its preparation and use was confined to a well-ventilated hood.
Standard solutions. Stock solutions of 2 ,4 -D and dicamba in benzene were
prepared, each containing 1 mg of compound per ml of solvent. In separate 10 0 -mJ
volumetric flasks 1 .0 ml of each stock solution was treated with 0 .5 ml of diazomethane
solution and allowed to stand for 1 5 min. The solutions were then diluted to too ml
with isooctane, thus giving solutions which contained 10 //g ml of each compound
as its methyl ester. From these solutions, working standards w;ere prepared in appro­
priate concentrations.
Apparatus. Gas chromatographic determinations were made on a MicroTck
MT 220 gas chromatograph equipped with a *H electron capture detector and a
0 .32 cm I.D. x 1 S0 cm borosilicate glass column packed with a mixture of 4 *,
J . Chroma ! . , g . , jo (1970) 3 3 4 -33 7

5667

\

8.«

•*fJB . . *• •l ■

SOTES

.

335

SE-30 and 6 ° 0 QI:-i on Chromosorb W (AW-DMCS), high-performance grade.

So- 100 mesh.

The instrument conditions were: inlet temperature 220 a, detector temperature
20S0, column temperature 190 s; nitrogen flow rate S5 ml/min.
The 25 -ml evaporative concentrator tube was Kontes No. 570050-2526 , the
evaporative concentrator column Kontes No. 569251 -0324 .
Extraction. One milliliter of blood serum or 5 ml of urine were combined with
20 ml of 0.1 X H-SO4 and 12 ml of benzene in a 125 -ml glass-stoppered erlenmever
flask equipped with a i-in. Teflon-coated magnetic stirring bar and mixed for 20 min
at 50° on a heated stirplate. After cooling, the contents of the flask were transferred
to a 35-ml centrifuge tube and centrifuged for 15 min to effect phase separation.
The benzene (top) layer was transferred to a 25 -ml evaporative concentrator tube,
and the aqueous phase was re-extracted three times with 5 -ml portions of benzene.
.Yith each wash the mixture was shaken vigorously for 1 min, and the phases were
separated by centrifugation. The benzene extracts were combined in the 25 -ml
evaporative concentrator tube, a boiling chip was added, an evaporative concentrator
column was attached, and the combined extract was concentrated to 2 ml in a waterbath at too3.
.1/ethylation. Diazomethane solution (0.2 ml) was added to the concentrated
extract, and the mixture was allowed to stand for 15 min. Excess diazomethane
»as then removed by gently bubbling dry nitrogen through the extract until the
yellow color disappeared, and the volume was adjusted to 10.0 ml with isooctane.
Gas chromatography. The methylated extract was diluted as necessary, and
- to S /¿I were injected into the gas chromatograph. Following each extract injection,
an amount of standard which gave a response of similar peak height (± 10 %) was
injected.
Results and discussion
The 2 ,4-D and dicamba levels in the poisoning case samples are given inTable I.
The results of a recovery study to determine the applicability of the procedure to
the analysis of human blood and urine for 2 ,4-D and dicamba residues are given
•n Table II. The samples were fortified, prior to extraction, with 2 ,4-D and dicamba
acids at levels which compared generally with the wide range of concentrations
found in the poisoning case samples.
In all cases responses to 2 ,4 -D and dicamba (as their methyl esters) were
fluantitated by comparison with standard responses in terms of peak height. By
comparing responses with [>eak heights differing by 10 % or less, quantitation problems
associated with lack of linearity and changing sensitivity, sometimes encountered
w'th electron capture detectors, were minimized.
The limits of detectability in 1 ml of blood serum were 0.03 p.p.m. for dicamba
0.05 p.p.m. for 2 ,4 -D. In 5 ml of urine the limits of detectability were 0.01 and
° °2 p.p.m. for dicamba and 2 ,4 -D, respectively-.
Dnder the conditions given, dicamba and 2 ,4-D methyl esters had retention
hmes of 1.2 and 1.9 min, respectively; I*CP methyl ether had a retention time of
2,2 min. Because the three compounds are well separated on the gas chromatographic
r,,Iumn. the procedure is useful for detecting anv or all of the comixmiuls in the same

'ample.

Lo

LO

TA IH.K I
J . i - 1 » A N II IIICAMIIA l.l - V I - l s IN l'O lK U N IN fi CASI'. SAMI'I.ICS

i
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. -------

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7 |o
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Mi.|<)
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l.|0 0
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2 1 50
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21.00

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_. ----------- _. .

S8G S09 M O Q

117

J 90
t -5 1

7 7 <l
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.1 SS
15'
2.1)0
I.5 S
• 71
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S a m h iiu g

.VOTES

337

TABLE II

Sample*

Fortijication level [p. p. 1111

Found (p. p . I I I . )

Dicnmba

2 .4 -D

Duamba

65.0
13 °
1.3°
0.13
O.OI3
0.000

6.S5
137
*3-7
»•37
0.137
O.OÓS

I’rine

Blood serum

_

—

65.0
Ó.50
0.Ó5
0.005
0.000

0.S3
6S.5
0.S5
0.6S5
0.066

< 0.01c
6S.S
12.5
1.15
0.1 r
0.01
< 0.0!«

<

< 0.03'
ót.o
5 91
035
0.07
< 0.03'

<

recovery

J. 4 -D b

Dicantha

0.02c
779
*311.6
‘ 35
0.15
0.07

too
96
66
¡>5

0 05'
636
6>-0
5.90
0.00
O.On

94
9‘
«5
to-s

2.4-D

_

77

—

“ 4
9Ó
»5
9S
109
103

—

"

93
tot
37
MS

* Each sample a single analysis.
s Detected as their methyl esters* Limit of detectability.

No interfering responses were encountered in any of the samples analyzed;
however, the use of the diazomethane solution in amounts greater than those indicated
in the procedure can lead to interferences due to impurities in the reagent.
Using this procedure, an experienced analyst can process approximately
sixteen samples per day.
The Hawaii Community Studies Pesticides Project is supported by Contract
Xo. 86-6 5 -7 9 with the Division of Community Studies. Office of Pesticides and
Product Safety, Food and Drug Administration, Public Health Service, Department
°f Health, Education, and Welfare, Chamblee, Ga., U. S. A.
Hawaii Community Studies on Pesticides,
Pacific Biomedical Research Center,
t diversity of Hawaii,
Honolulu, Hawaii qhSjJ (C. S. A.)

J e r k y B. R i v e r s
W ill ia m L. Y a c g e r . J r .
H o w a r d W. K i. e mm ICR

* b. E. C l a r k , I'. C- W k io iit an o I.. M. H u n t . J. Agr. Funit Ch. ■ " . 15 ( m » ; i 171.
1 b. J . Lisk . \V. H. ( »u t m n m a n n , C. A. B a c h e . K. Ci W a r n e r vno I). ( 1. W.A<..\hK. J . Dairy
4*> do'M ) M.I5t o G. C r o siiy a n d J . H. B o w e r s , H ull, liu v irn ti C n n la iu iu . Tun. /.. I fin'>u| 104.
A. R e v e n u e , .M. L. K mf. knon . I.. J . C a sa k k tt a n d \V. L. Y.w c k k . J k . y . C hrom a/"*., j * (uji.-S)

{J B. RIVKks,

rrMilts.

C* anm kk a n d J . I ' r k a l . |HTs«»nai c o m m u n ic a tio n .

^«reived April 1 4 th, 19 7 0
/. I

unit.

. JO IH17111 I H - . U 7

DOW 605986

RECOVERY OF 2.4-D AND OICAMUA ADDED TO HUMAN BLOOD SEKUm' a XD URINE

TV

I

BACKGROUND
The herbicide 2,4-dichlorophenoxyacetic
acid (2.4-D) was developed in the mid­
forties and was the forerunner of a group of
phenoxy herbicides that have Jbeen instru­
mental in the control of broadleaved weeds
in food crops and undesirable brush species
on industrial rights-of-way. In addition these
products have contributed immeasurably to
beef production by controlling weed and/or
brush on pasture and rangeland, resulting
in increased grass production and corre­
sponding increase in carrying capacity for
livestock.
Besides 2.4-D , 2.4.5-trichlorophenoxyacetic acid (2 ,4 ,5 -T ). 2-(2 .4 ,5 -tric h lo ro phenoxy) propionic acid (silvex or 2,4,5-TP),
and 2-methyl-4-chlorophenoxy acetic acid
(MCPA) are major phenoxy products of
similar chemical structure, as shown be­
low. but with unique characteristics of
their own with respect to species controlled
and crop selectivity.
CHRONOLOGY OF EVENTS
Over the years there has been consider­
able improvement in phenoxy herbicides
and their use. Development of new formula­
tions, performance information, crop safety,
timing of application, spray equipment,
toxicology, use hazards, and environmental
implications have contributed to both better
product and specific directions for use.
These herbicides are not protected by
patents, so seven commercial companies
were manufacturing one or more of the four
phenoxies (2,4-D, 2,4,5-T. silvex. MCPA)
at the time when the USDA announcement
to abolish the no residue status was issued
on April 13. 1966. Basic manufacturers in­
cluded: Diamond. Dow. Hercules. Monsanto,
Rhodia (then Chipman), Thompson, and

A. 2.4-dichiorophenoxyacetic acid
B. 2,4.5- trichlorophenoxyacetic acid
C. 2-{2.4.5-trichlorophenoxy)-propionic acid
D. 2-methyl-4-chlorophenoxyacetic acid

The status of 2,4-D ,
2 ,4 ,5 -T , Silvex and
MCPA Herbicides
By C. S. Williams
R & D Herbicide Tech. Specialist.
The Dow Chemical Company,
Midland. Michigan.

12

Thompson-Hayward. These compatii:
joined to form the Industry Task Forcée
Phenoxy Herbicide Tolerances (ITFPHÏ;
The chronology of major events associate
with this has been as follows:
April 13, 1966
USDA announcement to abolish no res
due status—industry must comply by oir
taining tolerances for residues in all treats
food and feed products and byproducts fe
December 31, 1970.
August 23, 1966
Industry Task Force on Phenoxy Herbidd
Tolerances was formed to handle 2,4-C
2,4,5-T. MCPA and silvex.
|
•Jt

December, 1967
|
Submitted petitions to FDA for tolerance
of 2.4-D, 2,4,5-T, silvex and MCPA cove
ing all food crop uses listed at that time;
the USDA Summary of Registered Agric£
tural Chemical Uses. Extension of registn
tion was requested for uses of these herb
cides in pasture and rangeland.
|

f

April, 1968
5
Industry Task Force advised of inadf
quacies. in the petitions plus requiremeri
for information on all metabolites of £
herbicides that might occur as residues)
food.
4
September, 1968
'j
Review of literature on metabolism sii
mitted to resolve metabolite question. |
During 1968 programs were establish^
to determine on which crops additions
work would be undertaken and what sped?
projects would be done by each comps
in the Task Force.
October 29, 1968
Industry Task Force requested extensif
for continued use of 2,4-D, 2.4.5-T, silv
and MCPA on pastures and rangeland. §
tension was granted until January 1.191

January 31, 1969
Use of 2.4-D, 2.4,5-T and silvex on aqu
sites extended to January 1. 1970.
Crop samples for residue analyses
collected during summer and fall of 19$
Protocols for milk and meat studies w*
established during 1969. It was decided^
analyses of animal tissues would include»
phenol corresponding to each phenoxyc
pound. Dow handled dairy cattle feeding!
milk analysis for all four phenoxies
began feeding February. 1970.
USDA was scheduled to handle feeding
phenoxies to beef cattle and sheep durfc
winter. 1969-1970.
J

r

[

'.October 29, 1969
■The Office of Science and Technology
lied statement on teratogenic hazard of
4.5- T based on work by Bionetics Re­
arch Laboratory.
December 22, 1969
Phenoxy registration for rangeland use
¿fended until January 1, 1971, except for
4.5- T. Dow undertook feeding of 2,4.5-T
beef animals.
December 3 1 , 1969
Petition for tolerances of 2,4.5-T in food
oops was withdrawn by Industry Task Force
! tolerances could not be established by
January 1, 1970. the deadline set by the
Office of Science and Technology.
r January 19, 1970
[Registration of 2,4-D . silvex and MCPA
'¿tended by USDA for use on food crops
jntil January 1, 1971.

S' •

\ March 4, 19 7 0
! Registration of 2.4,5-T was also extended
until January 1. 1971 for use on apples,
blueberries, grains, pastures, rangeland.
! and sugarcane.
.April 15, 1970
Results of additional work on teratogenic
properties of 2.4,5-T prompted suspension
by USDA of 2.4,5-T for aquatic and home
-uses.
May 1 ,1 9 7 0
Cancellation by USDA of 2.4,5-T for use
onfood crops. Uses on pasture, forests and
industrial areas not affected.
May 28, 197 0
[ Dow appealed the cancellation of 2.4.5-T
for use on rice Hercules and Amchem also
appealed cancellation for rice usage. Each
company could appeal only crops listed on
their labels for 2.4,5-T products. The appeal
istobe reviewed by an Advisory Committee
appointed by the National Academy of
Sciences.
June 16. 1 9 7 0
USDA began the beef feeding studies
scheduled to have been done the previous
winter.
November 2 4 , 1970
' Crop residue work completed. Milk anal­
ysis completed. Meat analyses underway.
Completed data will be submitted as amend­
ment to petitions prior to December 31.
1970.
' To date, no official notice has been
received concerning appointment of Na­
tional Academy of Science Advisory Com­

m ittee to review the
cancellation of rice.*

appeal

on the

REGISTRATION STATUS
Tolerances have been established for
2 .4 - D in applies, barley, grapefruit, lemons,
oats, oranges, pears, rye and wheat. There
is also a tolerance for the sodium salt of
2 .4 - D in asparagus.
Data originally submitted with the peti­
tions in December. 1967 is expected to be
sufficient for 2.4rD in blueberries, cranber­
ries. grapes and raspberries; for silvex in
apples, pears and prunes; and for MCPA in
peas. Data on residues in grass includes
that from previous work and from 1970
residue samples additionally analyzed by
Dow for support with respect to pasture
and rangeland usage.
The Industry Task Force supported work
for determining residues as shown in Table 1.
For virtually all of this work, new analytical
methods had to be developed that would
permit analyses down to 0.1 ppm phenoxy
acid in crops and to 0.05 ppm acid or cor­
responding phenol in the animal tissues and
milk.
For present registered uses residue anal­
yses indicate < 0.2 ppm for all phenoxy
acids in all crops at time of harvest.
The phenoxies were fed at levels of 30.
100 and 300 ppm for two weeks and 1000
ppm for three weeks in the total diet of
dairy cows. Milk was collected and analyzed
for residues. Grazing restrictions compatible
with levels of phenoxies in milk as related
to levels in forage remain to be determined.
There was no evidence of accumulation of
phenoxies in the cream.
At time of this writing, December 1,1970.
analyses are being run on samples of mus­
cle. kidney, liver and fat of beef animals.
Data are expected to be available prior to
December 31. 1970.
Amendments to the petitions for toler­
ances for 2,4-D, silvex and MCPA will be
submitted to the appropriate agency in
charge, including uses in pasture and rangeland. The 2.4,5-T petition will be reactivated
—deadline for this compliance will be ac­
complished prior to December 31, 1970.
TOXICOLOGY
Negligible residue tolerances can be
obtained based on information from 90 day
toxicology studies in two species of mam­
mals. However, tolerances at higher (per­
missible) residue levels require two-year
feeding studies on rats and dogs, plus
fertility and reproduction studies on rats.
At the time the phenoxy herbicides were
* See update i t end of text ( t ).

13

✓

5674

developed these long term feeding studies
were not necessary, since these com­
pounds were registered on a "no-residue"
basis.
FDA has conducted tw o-year feeding
studies on 2,4-D including reproduction
and fertility. Dow has conducted two-year
feeding studies on silvex but not reproduc­
tion or fertility studies.
Ninety-day feeding studies have-been run
on rats and dogs for 2,4-D, 2,4,5-T, silvex
and MCPA. The no-ill effect levels are shown
in Table 2.
Based on single oral doses in rats. 2,4-D,
2.4.5-T, silvex and MCPA are classed as
"slightly toxic" with LDso values ranging
from three hundred to seven hundred mg/kg
body weight.
2 ,3 ,7 , 8 TETRACHLORODIBENZO-P-DIOXIN
The word teratology has recently become
much more familiar. It was tied to 2,4,5-T
when studies by Bionetics Research Labora­
tory implied that 2.4,5-T was teratogenic
(producing malformed fetuses) in mice and
rats. Subsequent studies have shown that
a potential toxic contaminant, 2,3.7.8tetrachlorodibenzo-p-dioxin, is responsible
for the findings attributed to 2,4,5-T. The
sample of 2,4,5-T employed in the Bionetics
study contained 27 ppm 2,3.7,8-tetrachlorodibenzo-p-dioxin.
Additional studies have shown that oral
administration of 2.4.5-T containing < 1
ppm 2 , 3 . 7 ,8 - tetrachlorodibenzo - p - dioxin

produced no teratogenic effects on rats;
rabbits or mice. The obvious concern is toi
produce 2,4,5-T without the contaminant*
The 2 ,3 .7 , 8 -tetrachlorodibenzo- p-dioxiiL
can be formed in the manufacture of the?
precursor 2,4,5-trichlorophenol. The condk
tions required for its formation are high)
temperatures and basic conditions. This cant
occur in the alkaline hydrolysis of 1,2,4,5)|
tetrachlorobenzene to the trichlorophenol|
No detectable dioxins have been observed]
in 2,4-D. This is due to the fact that th£
precursor 2,4-dichlorophenol is made by)
direct chlorination of the phenol and notbyj
alkaline hydrolysis of 1,2.4-trichlorobeivi
zene.
?
To date analytical methods have been;
developed and validated for a method sen-.,
sitivity of 0.5 ppm for 2,3.7, 8-tetrachloro;;
dibenzo-p-dioxiri in 2,4.5-T acid. With proper;
manufacturing controls there is no problem.’
in producing 2,4,5-T with no 2,3,7,8-tetra-'.
chloro-p-dioxin as indicated by these ana-’
lytical methods.
THE FUTURE
By December 31, 1970 the Industry Task'
Force on Phenoxy Herbicide Tolerances will'
have furnished to FDA the supplemental”
residue data necessary for the continued;
evaluation of the petitions to establish negli­
gible residue tolerances for 2,4-D, 2,4,5-T,
silvex and MCPA on the appropriate foodcrops and meat and milk tissues. Registra­
tions (USDA) are expected to remain in
force.

jTflBLtZiHisuhi from.90-day *UMrotfiwinn;stodi5W:wnoi5^heno*nie8ieiaes mvitfr* ^d « isL -„«

gate

ii&0k

[Data provided to date indicate no hazard
no significant residues in food crops,
¡toxicology data support the use claims. The
er manufacturing of 2.4.5-T will allethe problems associated with 2,3.
JjS-tetrachlorodibenzo-p-dioxin.
¡¿The USDA has recently stated that probiting the use of phenoxy herbicides
uld cost the U. S. farmers an additional
I million to maintain current agricultural
duction. In addition, farmers and their
nilies would have to work 20 million more
to control the weeds without these
Mcides. For this extra labor, the farmers
ild obtain no additional income",
veral hundred thousands of dollars
been expended over the past several
»to prove the safety of phenoxy herbi; to man and his environment. From a
ntific base the phenoxy herbicides can
tribute economically, efficiently, and
sly in the future for the control of broadI weeds and brush on food crops, pasB,rangeland, and non-cropland areas as
[have for over 20 years.

tion on D ecem b er 2 2 , 1 9 7 0 a n d EPA advised
o n D ecem ber 3 1 . 1 9 7 0 a petition num ber h a d
been reserved b u t th e y (EPA) w ish ed to k n o w
w h ic h specific 2 .4 .5 - T form ulations w ere to
b e covered b y the petitio n a n d for ind u stry to
id e n tify th e ingredients (active a n d
co ntain ed in these products.

inert)

(3 ) The EPA has also advised th a t a specific
tolerance fo r phenoxies in grass is necessary;
a tolerance in grass h a d n o t been requested b y
IT F P H T as it d id n o t appear necessary. In
com pliance w ith th e EPA req u est e level o f
3 0 0 p p m fo r a ll phenoxies in grass was re ­
quested on Jan uary 6. 1 9 7 1 b y ITFPHT.
(4 ) On J an u ary 11, 1 9 7 1 D o w w as in fo rm ed
th a t the advisory co m m ittee from th e N a tio n a l
A c a d e m y o f Science h a d been a p p o in ted on
N ovem ber 2 .

1 9 7 0 to re v ie w the fo o d crop

cancellation o f 2 ,4 . 5 - T.
A t this w riting. D o w a n d Hercules h a d each
been given an h o u r on February 1. 197 1 to
present in fo rm atio n relative to the safety o f
2 .4 .5 - T as used on fo o d crops.

ICAffT CHRONOLOGICAL EVENTS

•PREPARATION OF MANUSCRIPT
s.of January 2 3 . 1 9 7 1 several sig n ifican t

rhave occurred.
fy/The fTFPHT su b m itted am en d m en ts for
D.silvex an d M C P A p etitio n s o n D ecem ber

5676

i 1970. The E n v ir o n m e n ta l P r o te c tio n
(EPA) ackn ow led g ed filin g o f these
nents Decem ber 3 0 . 1970.
\Jhe fTFPHT also refiled th e 2 .4 .5 - T p e ti­

15

5677

316

Reprinted Iront the Archives o l Environmental Health
March 1971. Volume 22
Copyright 1971. American Medical Association

IS
PESTICIDE
PUBLICATION

I

4 S 2

A H e a lth S u r v e y o f W o r k e r s
i n a 2 ,4 - D a n d 2 ,4 ,5 - T P l a n t
W ith Special A ttention to Chloracne,
P o rp h y ria C utanea T ard a, and
Psychologic P aram eters
A la n P. Poland, M D ; D onald S m ith , M D ;
G erald M e tte r. A tla n ta : a n d P a u l P ossick, M D , Cincinnati

f rim iu iir n v n iii rTOKCIlOT ■

Chamóle«, Gtorfia
o

CO

©

OO

Ol

V*l

or
-vT

0
01

A study of 73 maie employées in a 2,4,5-T fac­
tory was made. Chloracne was found in 13 (18% )
workers. Severity of chloracne correlated signifi­
cantly with the presence of hyperpigmentation,
hirsutism, eye Irritation, and a high score on the
manic scale of the Minnesota Multiphasic Per­
sonality Inventory. Chloracne was not, however,
correlated significantly with job location within the
plant, duration of employment, or coproporphyrin
excretion. Although 11 subjects with uroporphy-

rinuria and at least three with overt porphyria cu­
tanea tarda had been found in a study of the same
plant six years ago, no clinical porphyria could
be currently documented and only one worker had
persistent uroporphyrinuria. Evidence of toxicity
in other organ systems was markedly less than
that reportad In previous studies and could not be
shown to differ from normal populations in most
instances.

, T h E herbicides 2,4-D (2,4-dichlorophenoxyacetic acid) and 2,4,5-T (2,4,5-trichlorophenoxyacetic acid) are widely used
throughout the world. Several distinct medi­
cal problems have been described in workers
involved in the production of these com­
pounds. These problems can arbitrarily be
subdivided into ( 1 ) chloracne and mucous
membrane irritation, (2 ) hepatotoxicity,

neuromuscular symptoms, psychologic alter­
ations, and other systemic symptoms, and
(3) porphyria cutanea tarda (PCT).
Chloracne and Mucous Membrane Irrita­
tion.—Chloracne (pema disease, perchloronaphthalene disease) has been described
as an extremely refractory acne seen in
workers involved in the production of sever­
al chlorinated aromatic compounds.1:1 Kimmig and Schulz4*0 described this dermatolog­
ic condition in workers in a 2,4,5-T factory.
The condition is characterized by inclusion
cysts, comedones, and pustules, with eventu­
al scarring, and originates in the temple-zyg­
omatic area with spread to the pinna, nape
of the neck, back, upper chest, and inguinal
area. Many of these patients also had ble­
pharoconjunctivitis. = The investigators
showed that purified 2,4,5-trichlorophenol
(TCP) was not acnegenic, but that 2,3,6,7tetrachlorodibenzodioxin (TCDD) isolated
from the crude TCP reaction mixture was
an extremely potent acnegenic agent when
applied to rabbits’ ears . 4 The TCDD is oon-

Submitted for publication April 27, 1970: accepted
M ay 27.
From the Division of Pesticide Chemistry and
Toxicology and the Division of Community Studies,
Hureau o l Foods and Pesticides, and Office of
Product Safety. Food and Drue Administration. US
Department of Health, Education, and Welfare.
Atlanta (Drs. Poland and Smith and Mr. M etier),
and Medical Services Branch, Occupational Injury
and Disease Control, Bureau of Occupational Safety
and Health, Environmental Control Administration,
Cincinnati <Dr. Possick). All correspondence direct­
ed to Dr. Poland should he forwarded c/o Rockefel­
ler University Hospital. Rockefeller University, 6Gth
and York Avenue. New York 10021.
Reprint requests to Division of Pesticide Chemis­
try and Toxicoloiry, Food and Drug Administration,
Public Health Service. 4400 BufTord Hwy, Chamblee. (>a :<0341.

Arch Environ Health— VoI 22. March 1971

00024:

5678

317

sidercd an important, but not necessarily medication histories and the detection of any
the only, acncgenic compound in 2,4,5-T neurologic or dermatologic signs and symptoms.
Physical examinations were performed hy three
production.
dilTcrent
physicians; each physician's findings
Hepatotoxicity, Neuromuscular Problems, were considered
final and were not confirmed
Psychologic Alterations, and Other Systemic by
a
second
physician.
Symptoms.—In addition to chloracnc, The Minnesota Mulliphasic Personality In­
studies of workers in 2,4,5-T plants have ventory
(MMPI)1® was administered to all
frequently noted other signs and symptoms employees in a quiet atmosphere at work su­
suggestive of systemic toxicity. These in­ pervised by management The only exceptions
clude anorexia and weight loss, abdominal were a few plant administrators who completed
pains and postprandial flatulence, headaches, the inventory at home. The individual tests
weakness in the legs, and hepatic dysfunc- were scored by computer, and the mean and
tion.'M0 The following psychological altera­ standard deviations were computed for each
compared with normative data .111 In
tions were also noted: lack of vigor, drive, scale and the
frequencies of high and low twoand libido; easy fatigability; emotional in­ addition,
point
scores
were
determined.
stability; and diminished ability to leam.
Each
employee,
after fasting for at least
Porphyria Cutanea Tarda.—Porphyria three hours, was given
a bottle of a beverage
cutanea tarda is an acquired porphyria, gen­ containing 75 gm carbohydrate (Glucola) to
erally occurring in men after age 40, in drink. Two hours after ingestion, a blood sam­
which significant liver dysfunction and usu­ ple was obtained and the serum glucose and
ally a history of alcoholic abuse are found. other analyses were performed in an automated
The syndrome consists of vesiculobullous le­ clinical laboratory. In persons reporting after
sions on areas of the body exposed to light, 135 minutes, the two-hour serum glucose was
invalid and not included in the
hirsutism, excessive mechanical fragility of considered
statistical
analysis.
In addition to the glucose
the skin, hyperpigmentation, and excretion determination, serum
oxaloacetic
of red urine containing an increased amount transaminase (SGOT), glutamic
lactic
dehydrogenase
of uroporphyrin or coproporphyrin, or both, (LDH), alkaline phosphatase, cholesterol, bili­
but not a large increase in urinary {-amino­ rubin, albumin, total protein, blood urea nitro­
levulinic acid fALA) or porphobilinogen gen (BUN), and hemoglobin values, and an
(PBG ) .11*12 Frequently these patients have hematocrit reading, red blood cell count, white
hyperferremia and hemosiderosis, and phlebot­ blood cell count (WBC), serum iron value, and
omy often reverses the clinical picture and iron-binding capacity were disclosed for each
employee.
decreases porphyrin excretion .1* 14
A routine urine specimen was collected from
In 1964, Bleiberg et al 13 reported studies each
volunteer, the pH was adjusted to 7.0, and
of 29 workers in a 2,4-D and 2,4,5-T factory. the specimen
was frozen until porphyrin analy­
Many of the workers had chloracne and 11 sis.
An
attempt
was made to shield the urine
had abnormal excretion of urinary uropor­ from ultraviolet light
but was not entirely satis­
phyrins. Many of the workers with and factory in the industrial setting. In addition,
without uroporphyrinuria had hirsutism, hy­ each urine sample was analyzed for ALA and
perpigmentation, and increased skin fragili­ PBG by the method of Marver et al17 and for
ty. Liver dysfunction was noted in two hos­ uroporphyrin and coproporphyrin by the
method of Schlenkler et al.1® The porphyrin
pitalized patients.
As a follow-up of the observations of Blei­ and porphyrin precursor levels were expressed
berg et al15 of a toxic PCT in these factory per gram of creatinine.
analysis, employees were sub­
workers, we restudied all the employees in For statistical
into several dilTcrent groups according
the same plant in February 1969 with par­ divided
location in the plant, exposure to chemicals,
ticular emphasis on PCT, chloracne, hepato­ to
and
educational level. All quantitative results
toxicity, and neuropsychiatric symptoms.
were reported os niean ± one standard devia­
tion. percent of prevalence, or a correlation
Methods
coefficient between two variables. Significance
was determined by Student's t-test or x- test:
All volunteers submitted to u medical history results reported os nonsignificant are those
and physical examination; s|>ecial emphasis was with the significance level (probalTility value)
placed on occupational, smoking, drinking, and of greater than 0.05. Where distribution of

2 iS i,8 e M o a

H E A LTH S U R V E Y IN 2.4-1) A N D 2,4,5-T P LA N T— POLAND E T A L

Arch Environ Health— Voi 22. March 1971

5679

0002495

318

H E A L T H S V IiV E Y IN 2.4-1) AN D 2.4 â -T P LA N T—PO LAN I) E T AL

lesions and the scarring was generally in­
creased. No erythematous or edematous le­
sions were found, as previously reported by
some investigators studying intense or mas­
sive exposures.’1
“Active acne” was defined as the presence
Results
of cysts, comedones or pustules (but not
scarring). Each of the three lesions was
Descriptive Data.—Seventy-eight persons graded according to the severity (grade 0 to
were studied in the plant. Five women em­ 4) in each location <five possible areas: face.Q
ployed in the office were excluded from the neck, back, chest, and other). The severityQ
analysis. The remaining 73 employees, all grades for each lesion were summed over all^£
men, included one part-time worker and five locations. Finally, these severity su m s^
four persons who had not worked in the were totaled to produce an overall active GC
plant for one to six months. The group of 73 acne score (maximal possible score: grade 4 CC
persons was subdivided into four basic occu­ in all five locations • 2 0 for each of three hU
pational subgroups: ( 1 1 administrators and lesions » 60). This arbitrary rating allowed çj -j
technical help, 2 0 men including engineers, us to deal with the degree of active acne in a <1
business office workers, laboratory techni­ statistical form. Scarring was scored sepa­ CO
cians, and janitorial help, all housed in a rately by the same system (grade 0 to 4
building separated from the production area multiplied by five possible areas - maximal
and, thus, less exposed to production chemi­ score of 2 0 ).
cals; ( 2 ) supervisors, 1 1 foremen spending Twenty-five persons had an active acne
most of their time in the production area; score of 0 (no lesions), 35 had a score of 1
(3) on-line personnel, 28 persons working in to 8 (minimal), and 13 employees had a
the production area; and (4) maintenance, score of 9 or greater (moderate to severe).
14 men with various occupations (plumbers, Scarring was also a highly skewed parame­
electricians, etc); some were confined to the ter: 37 employees had a score of 0; 29 had a
maintenance shop and others were frequent­ score of 1 to 8 ; and 7 had a score greater
than 9. As seen in Table 3, with increasing
ly in the production area.
As seen in Table 1 , the average age of the active acne scores, the prevalence of scar­
18 Negro and 55 white employees was ring, hyperpigmentation, hirsutism, and com­
39.3 ±11.1 years and the average years of plaints of eye irritation was greater.
schooling, 11.9 ± 2.6 years. The average du­ Another way of expressing the same data
ration of employment was 8.3 ± 7.6 years is to convert the skewed active acne scores
(Table 2). Of 64 workers questioned, 22 to a more normal distribution by logarith­
missed more than one day from work due to mic transformation (see Methods) and com­
illness in the past year. The average number pare these transformed scores with other
of days lost for 63 employees was 2.8 days parameters by correlation coefficients. Scar­
per year. (This excludes one person hospi­ ring, also a skewed parameter, was similarly
talized for almost two months with a uri­ transformed.
nary tract infection.)
Active acne was correlated with the de­
Acne.—A specific dermatologic history gree of scarring (r - 0.80), the presence of
with special emphasis on acne and porphy­ hyperpigmentation (r - 0.38) and hirsutism
ria was taken from all the employees and all (r ■ 0.44), and complaints about eye irrita­
were examint'd by one of us (P.P.).
tion (r - 0.39). All the above correlations
The minimal acne lesion consisted of co­ have a significance level of P < 0.001. The
medones and inclusion cysts in the teniple- active acne scores were not significantly cor­
zygomatic area and the pinna of the ear. In related with the duration of employment nor
more severe cases, cysts and comedones were with the excretion of coproporphyrins per
accompanied by pustules on the face and gram of creatinine.
ear, nape of the neck and back, and in the
The occurrence of acne was not signif­
worst cases, the chest and inguinal area. icantly greater in Negro employees than in
With anatomic spread, the severity of the white workers (*= lest). Although all six
resu lts on a given con tin u o u s variable was not
n o rm al logarithm ic tran sfo rm atio n s were m ade
before in f o r m in g /-tests o r calculating co rrela­
tion coefficients. M ean s and sta n d a rd deviations
a re alw ays rejiorted as untran sfo rm ed values.

Arch Environ Health— Vol 22, March 1971

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H E A LTH SU R V E Y ¡N 2.4-1) A N I) 2.4.5-T P LA N T—POLAND E T AL

319

Mucous Membrane Irritation.—Seven em­
ployees complained of itching of the eyes,
14 of frequent tearing, five of "bloodshot
No. of
Years of
Group
Schooling*
Employees
Age •
eyes," and seven of sties. On physical exami­
nation, however, only three (4.1%) had ap­
A dm in istra to rs,
lab technicians#
preciable conjunctival injection. Twentyja n ito rs
20
3 4 .0 ± 12.2 13.8 ± 2.3
three (31.5%) employees had hyperemia of
S upervisors
12.7 ± 3.0
46.8 ± 7.4
11
the nasal mucosa and eight n0.9% ) had
On-line personnel
28
40.1 ± 10.0 10.6 ± 1.7
M aintenance
39.1 ± 10.5 10.6 ± 1.8
14
inflammation of the buccal mucosa.
Total plan t
39 .3 i 11.1 11.9 ± 2.6
73
Hyperpigmentation and Hirsutism.—Hy­
. * Mean ± 1 SO.
perpigmentation, Usually most prominent on
the face, consisted of a grayish or brownish o
tone to the complexion. Hirsutism was most o
Table 2.— Duration of Employment in Plant
noticeable in the area between the outer
No. o f YearsNo. o f Employees
edge of the eyebrow and the temple hair £
0-4
33
margin. Both characteristics were recorded
10
4-8
only
as being present or absent. As seen in
1
9-12
Table 3, 16 subjects had facial hypertricho­
29
> 13
sis and 30 had hyperpigmentation. Correla­
• Mean ± 1 SO — 8.3 ± 7.6 years.
tion between hirsutism and increased hy­
perpigmentation was significant (r - 0.25,
individuals who stated that they had had P < 0 .0 5 ). As previously mentioned, hirsut­
teen-age acne and who had worked in the ism and hyperpigmentation are significantly
plant for more than 14 months do presently correlated with the severity of active acne,
have active acne, the prevalence of acne in but neither is significantly correlated with
this group ( 10 0 % ) was not statistically coproporphyrin excretion.
higher than the prevalence of acne in a
Urinary Excretion of Porphyrins and Por­
group who had also worked in the plant for phyrin Precursors.—The average values of
more than 14 months but had no history of ALA, PBG, and coproporphyrin found in
teen-age acne (62% [ys « 3.40; 1 degree of routine urine specimens of 72 workers in the
freedom, 0.05 < P < 0.10] ).
plant are shown in Table 4 along with the
The 73 male employees were subdivided normal values from the literature .19 The ex­
into groups with respect to present work cretions of ALA, PBG, or coproporphyrin
location in the plant: TCP formation, 2,4- were not abnormally elevated. One employ­
dichlorophenol (DCP) formation, phenoxy- ee, hospitalized several years ago with se­
acetic acid production, esterification, formu­ vere PCT, still has mild uroporphyrinuria
lation and storage tanks, maintenance, (107/ig/gm of creatinine). However, no overt
supervisory, laboratory help, and administra­ clinical cases of porphyria were found dur­
tion. Often one man might work in several ing this study. Only four of the 1 1 work­
locations (eg, supervisors might work in ers with uroporphyrinuria originally de­
both T C P and DCP production and in scribed by Bleiberg et alls were still working
phenoxyacetic acid production and esteri­ in the plant. Several of the seven employees
fication). In these cases, each area was com­ with uroporphyrinuria who had left the
pared (xs test) to see if the severity of the plant had been retested by Dr. Bleiberg
active acne of the employees might be worse before they left and had no elevation in
in certain locations in the plant. There were urinary porphyrin excretion.
no statistically significant differences, per­ Several pertinent observations can be
haps because of the small size of each group made about these urinary porphyrin values.
and the mobility of the workers. However, The excretion of ALA and PBG, although
as migi?t be expected, the maintenance men not abnormally elevated, nonetheless tended
tended' to have the most acne (having to to be relatively increased in the 15 mainte­
clean and repair vats and pipelines ) and the nance men versus the other 56 workers. The
administrative jjeople (housed in a separate coproporphyrin excretion per gram of crea­
building) tended to have the least acne.
tinine, although again within normal limits,
Table 1.— Distribution, Age. and Educational
Level of Employees According to Job Category

384574

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H E A LTH S W iV E Y /.V 2.4-1) AMI) 2.4 A-T P L A N T —PO LAMI) E T AL

was significantly elevated in the mainte­ includes mean values). Two subjects had
nance men versus the same 56 workers alkaline phosphatase values slightly above
148.3 £ 14.4 vs 36.7 * 19.6, P < 0.025 ). Per­ the normal laboratory range (18.7 and 19.0
sons with higher values for coproporphyrin King-Armstrong units/100 cc, respectively
excretion ( ^ 50 mg/gm of creatinine, N - [normal range, 7 to 18 King-Armstrong
15.) did not have any greater duration of units / 1 0 0 ccj); the other liver function tests
employment in the plant than those with were normal. Two employees had SGOT
lower values (^- test). There was a signif­ levels elevated above the normal range (48
icant positive correlation between ALA ex­ and 121 Karmen units/ml, respectively I nor­
cretion and coproporphyrin excretion fr - mal range, 7 to 45 Karmen units/m l | ', but
0.42, P < 0.001 ) and between ALA excre­ otherwise had normal liver function tests.
tion and age ( r - 0.42, P < 0.001 ). There The employee with an SGOT value of 48
was a significant negative correlation be­ Karmen units/ml was receiving lincomycin^f
tween ALA excretion and serum bilirubin injections at two- to four-week intervals/"}
The SGOT value of 121 Karmen u n its/m S
concentration <r - -0.26, P < 0.05).
Cardiovascular Findings.—Pulse rate and was not confirmed by a second testing, and%
blood pressure recordings were taken. The the subject had no other evidence of liver
average pulse was 85.1 ± 10.2 beats per min­ malfunction. Six persons had palpable liveiCO
ute; systolic blood pressure, 126.6 ± 12 .8 edges; five of these six findings were obOD
mm Hg; and diastolic blood pressure, 75.4 served by one of the three physicians. T w (A
± 7.8 mm Hg. Only four persons had a of these six subjects were heavy beer drinlf^T
diastolic pressure greater than 90 mm Hg. ers (at least four cans of beer per day), an&Jf
Cardiac evaluation was unremarkable ex­ one had emphysematous lung changes wifiif
cept for histories of myocardial infarction in lowered diaphragm. Only one of the six
subjects with a palpable liver had an abnor­
three employees.
Pulmonary Findings.—Twenty of 73 em­ mal liver function test, an alkaline phospha­
ployees (27.4%) gave a history of a chronic tase level of 19 King-Armstrong units/100 cc.
cough, emphysema, or fairly consistent rais­ Gastrointestinal Results.—Several studies
ing of early morning sputum. Of these 20 of 2,4,5-T workers have mentioned high
workers, 16 (80%) currently smoke ciga­ prevalences of anorexia and weight loss,
rettes and the other four ( 2 0 % ) had smoked postprandial flatulence, nausea and vomit­
considerably in the past. In the entire plant, ing, abdominal pain, and documented cases
42 employees (57.5%) currently smoke ciga­ of gastritis and ulcers.•'■ ,i•'!■,', At the time of
rettes and another 15 (20.6%) gave a histo­ the examination 2 2 workers in this plant
ry of smoking in the p ast The group of (30%) were intermittently experiencing at
current smokers averages 25.6 pack-years.
least one of the following symptoms: nausea,
On physical examination 18 workers were vomiting, diarrhea, abdominal pains, or
judged to have some impairment of maximal blood in the stool. Two persons in this
diaphragmatic excursion. Fourteen of these group and four other persons had a history
currently smoke and three had smoked in of “ulcers” (6 of 73 persons or 8.2%). We
the post Eight of these 18 workers gave a found no results from other industrial set­
history of chronic cough or frequent early tings with which to compare our data.
morning sputum and one gave a history of Neurologic Findings.—Several authors
asthma.
have noted lower extremity weakness and
Although the incidence of chronic pul­ fatigue in many of the patients with chlormonary disease in the plant seems to be acne in 2,4,5-T factories. 1'7 Only seven of 73
appreciable, it cannot be attributed to any (9.6%) employees in our study complained
industrially related cause, as suggested by of lower extremity fatigue or difficulty in
the report of Bauer because 78% of the climbing stairs. Five of these workers gave a
employees now smoke or have smoked ciga­ history of arthritis in the lower extremities
rettes previously.
or heart disease with dyspnea; thus, only
Hepatic Function.-—All serum total biliru­ two (2.7%) experienced leg fatigue totally
bin values and albumin concentrations were unexplained by other concomitant disease.
within the normal laboratory range (Table 5 Lower extremity weakness was not detected
Arch Environ Health— Voi 22. March 1971

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H E A LTH S U R V E Y IX 2.4.1) AXI ) 2.4JÎ-T P L A N T —POLAND E T AL

321

5
7
6

1971

4

H e a lt h — V o ! 2 2 . M a r r h

8

E n v ir o n

3

A rrh

DOW

Distribution oI Certain Signs, Symptoms, and Test Scores
in the neurologic examina­ Table 3.—Among
Three Categories ot Active Acne Severity
tion of any subject.
Active Acne Score
Eight workers (11%)
complained of nonspecific
1-8
0
>9
headaches. Ten workers had No. of em ployees
decreased auditory acuity H irsutism
3 /2 5 (1 2 % )
4 /3 5 (1 1 % )
9 /1 3 (6 9 % )
1 5 /3 4 (4 4 % )
(inability to hear a watch H yperpigm entation 5 /2 5 (20% )
1 0 /1 2 (8 3 % )
1/2 5 (4.0% )
2 2 /3 5 (63% )
1 3 /1 3 (100% )
ticking 1 cm from the ear); Scarring
irrita tio n
1 /2 5 (4.0% )
8 /3 5 (2 3% )
6 /1 3 (4 6 % )
four of these had tympanic Eye(h isto
ry of tearing,
membrane disease. No dis­
itching , sties,
bloodshot)
crimination was made be­
score of Mama
55 .4 ± 7.7
58.8 ± 10.2
64.9 ; 10.5»
tween nerve or bone conduc­ Mean
tion defects. A decrease in scale o f MMPI
p e rio n s w ith acne ic o r e t o f > 9 had sig n ifica n tly higher scores on
olfactory discrimination was th e• The
Mania scale on the M M PI (P < 0.05).
observed in only one em­
ployee and was most likely
due to nasal obstruction. Two persons had a was higher than that of the 52 individuals
decreased sense of proprioception (one with whose results were < 140 mg/100 ml (39.3
history of chronic lumbar arthralgia secon­ years; range, 21 to 56). One of the six was
dary to traumatic injury to the back). In the only known diabetic in the plant; he had
three persons, Achilles tendon reflexes were a 19-year work history in the plant and a
absent without other neurologic deficits (one two-year history of diabetes which is cur­
had a two-year history of diabetes mellitus). rently being treated with tolbutamide. The
Tremors of the hands were observed in three average period of employment in this plant
employees. We found no abnormalities of for the six individuals was 9.2 years, with
cranial nerves other than those noted above: the range being 1 to 19 years; three had
no clonus, no alterations in deep tendon minima] chloracne and one had severe chlorreflexes of musculi biceps or triceps brachii acne. None of the six had a family history of
or quadriceps femoris, and no alteration in diabetes; one was obese. The prevalence of
pain sensation.
10.3% hyperglycemic results in this factory
Results of Other Laboratory Determina­ is consistent with a 14% prevalence figure of
tions.—Only one employee had an elevated two-hour postglucose hyperglycemia in a
BUN value, as shown in Table 5 (29 random sample of a normal population in
mg/100 ml; normal range, 6 to 24 mg/100 Bedford, England.st
ml), and none had an elevated uric acid
Only two of 72 employees had a hemo­
level (normal range, 3.0 to 7.9 mg/100 ml). globin value of less than 13 gm/100 ml of
The serum cholesterol (normal range, 133 to blood, and 16 employees ( 2 2 % ) had a se­
294 mg/100 ml) was elevated in seven of 71 rum iron determination of less than
persons. There was an unexplainable eleva­ 75/ig/l00 ml. The mean serum iron value
tion in the serum LDH (normal range, 50 to was 97 ± 32/ig/100 ml and only two employ­
160 Wacker units/m l) in 20 of 70 employees ees had a value of 160/ig/100 ml or greater.
(28.6%); this may have been due to centri­ The average value for saturation of the
fuging clinical chemistry blood specimens iron-binding capacity was 27.4% ± 9.0%,
more than two hours after sampling.
with 27 persons (37%) having a value of
The two-hour serum glucose levels, as de­ less than 25%. Seven persons had a WBC of
termined by the automated ferricyanide less than 5,000 cells/cu mm, but it was 4,000
method for glucose determination, were cells/cu mm or less, in only two employees.
judged satisfactory with 58 employees (see None of the seven had an absolute granulo­
Methods). Six employees (10.4%) had a cytopenia (< 1,500 neutrophils/cu mm ) - 2
two-hour serum glucose value of ^ 140 and only one worker had a lymphopenia
mg/100 /tnl2" after a 75-gm oral carbohy­ (< 1,000 lymphocytcs/cu mm) Since the
drate load (145, 148, 152, 171, 174, and 176 normal hematologic values used here are the
mg per 100 ml). The average age of these 95% confidence limits of a study of a nor­
six individuals (50.6 years; range, 39 to 62) mal population ,23 single cases of granulocv-

C 0 0 2 4 9 9

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H E A LTH SU R V E Y IN 2.■ /-/> AND 2J.5-T P L A N T —POLAND E T AI.
Table 4 .— Urinary Porphyrin Excretion

PBG
C oproporphyrin
U roporphyrin

Plant Values,
pg /grn o f C reatinine*
1.021 i 406
444 ± 2 3 7
39.3 ± 19.0
Tr«cet

N orm al Values'*
1.900 ± 60 0 tig /g m creatinine

U pper Lim it of Norm al,
¿ig/gm of Creatinine
3.100

700 ± 4 0 0 pg/grn creatinine

1.500

0*175 ¿¿g/titer
0-15 „ ( / l it e r

• Mean ± SD, N » 72.
t M easurable in only one person; value. 107Mg/gm of creatinine.
♦ Values derived fro m norm al by assum ing a 70*kg man excretes creatinine at ^
urine.

175J
is :

1.5 g m /d a y and 1,500 m l of

topenia and lymphopenia would not be un­ defensiveness in a person (an unwillingness
expected in a population of 73. The red cell to admit that he differs in any respect from
morphology and platelet count, determined what he considers normal); this score in­
by scan of the peripheral blood smear, were creases with educational level. The mean K
scores for both groups in this plant are
normal in all subjects.
M M PI Results.—The M M PI scores of normal for their educational levels and do
production workers (including on-line per­ not significantly differ from each other.
sonnel, supervisors, and maintenance men The only significant differences in means
[N - 52] ) and the administrative staff (in­ on all scales between administrative staff
cluding laboratory technicians [N » 17]) and production workers were on the L and
were analyzed separately because of the dif­ Hypochondriasis scales, with the production
ference between the two groups in environ­ workers presenting a more hypochondriacal
mental exposure and educational level. Four picture than the administrative staff. When
janitors, currently working in the adminis­ scores of both groups of employees were
tration building, were eliminated from the compared with the normal values given for
analysis of the administrative staff because the M M PI test scales (N, approximately
of a different educational background and 300; mean, 50; standard deviation, 10), ad­
previous exposure as production workers.
ministrative staff and production workers
M M PI results are based on an analysis of varied significantly from normal on a total
scores of all subjects on 13 separate scales. of six and nine scales, respectively (Table
Ten of these scales are designated by com­ 6 ). The normal values for the MMPI, how­
mon clinical terms and are thus somewhat ever, were based on scores of a small popu­
self-explanatory (Table 6 ). Detailed de­ lation of Minnesota males who took the test
scriptions of each scale are found in A n just before World War IL,ti Since many
M M P I H a n d b o o k . ™ The remaining three obvious demographic differences exist be­
scales, the L, F, and K scales, measure the tween the plant population and the “normal
validity of the test results as an accurate population,” it was thought that a more
profile of the subject A high L score indi­ meaningful definition of the plant popula­
cates that the subject answered most ques­ tion would be obtained by searching for
tions according to socially desirable stan­ personality patterns appearing in high fre­
dards rather than according to individual quencies within the plant. A high-point code
preferences. More highly educated subjects system was used which grouped subjects
tend to have lower L scores; this is consis­
to the two scales in which they
tent with our finding of significantly lower L according
scored
the
The following high-point
scores in the administrative staff. A high F codes had highest
the
greatest
frequency (F): ad­
score may be obtained if a subject answers ministrative staff, Kypomanic-hystcricnl,
fin a prankish way, if he has a poor ability to
23%;
production
workers,
hypomanic-psychoread or comprehend the items, or if he is
severely mentally disturbed. Since only one pathic, f - 12%. It is apparent from these
subject oiit of 69 had a high F score (F > small frequencies that no one personality
76), the test seems to have been both under­ pattern tended to dominate eithes- adminis­
stood and taken seriously by the workers. A trative or productive group.
high K score indicates a high degree of Correlation coefficients were calculated
Arrh Environ Health— Vol 22. March 1971

¿ ¿ S k seA o a

Test
ALA

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H E A LTH SU R V E Y I S 2.4-1) AHI ) 2.4 ¿ -T P L A N T —POLAND E T AL

Arch Environ Health— Voi 22. March 1971

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Table 5.— Clinical Laboratory Data
for several M M PI scales
with other parameters, such
Mean
SO
N*
as porphyrin excretion, BUN (m g /1 0 0 mTest
3.9
14.5
72
l)
chloracne, job history, and Uric »cm (m g /1 0 0 ml)
5.4
72
1.0
several biochemical tests. A Cholesterol (m g /1 0 0 m l)
44
237
71
32
581
95
significant correlation was Two-hour serum glucose (m g /1 0 0 m l)
phosphatase (King-Arm *
found between the score of Alkaline
71
2.5
11.2
strong units)
severity of active acne and LDH
46
70
146t
(W acker un its)
the hypomanie scale (r —
71
13
(K arm en un its)
22
0.33, P < 0.01 ). Further­ SGOT
0.1 6
71
. 0.44
B ilirubin (m g /1 0 0 m l)
more, the mean manic scale A lbum in (g m /1 0 0 m l)
71
4.6
0.3
score for the group with the H em oglobin (g m /1 0 0 m l)
72
14.9
1.1
most severe chloracne was Serum iron (/jg /1 0 0 m l)
97
32
70
significantly higher than the Total iron-omOing capacity
44
69
348
O ig /1 0 0 ml)
means of the two groups S aturation
69
27.4
9.0
of iro n -b in d in g capacity (% )
with less severe acne < P < WBC (ce lls/cu m m )
72
2.1 90
7,160
0.05 [ Table 31).
• V ariation in N secondary to sam pling d iffic u ltie s : tor N m 73. mean age
+ SO « 39.3 * 11.1: 25 % of population are Negroes, 75% are w hite.
Comment
~ t For N « 58, m ean age ± SO « 39 .3 1 10.9; 29% of population are N e­
groes. 71% are w hite.
Acne.—Fourty-eight of 73 t Elevated value is p robab ly due to poor sam ple handling.
( 6 6 % ) employees in our
study had some degree of acne, but only 13 the presence of chloracne than with uropor(18%) had moderate to severe lesions. Most phyrinuria, and ( 2 ) the findings of other
previous investigations of acne occurring in studies of 2,4,5-T factories' - ’1 in which
workers in 2,4,5-T plants have selectively workers were noted to have chloracne and
presented clinical and pathologic findings in hyperpigmentation but no mention was
those severely affected. In contrast, the pres­ made of skin fragility, vesicular eruptions,
ent study surveyed the prevalence of acne in or red urine.
As stated in the introduction, Kimmig
the entire employee population and found
the acne absent or minimal in 82% of the and Schulz,tt have demonstrated the potent
acnegenic properties of TCDD, an unwant­
workers.
The severity of active acne- correlated ed side product in the synthesis of 2,4,5-T.
with the presence of scarring, hyperpigmen­ There is a tendency in the plant we studied
tation, hirsutism and complaints about eye for the maintenance men, who have the
irritation (Table 3). Dugois et al"’ reported most direct exposure to all products of syn­
a 50% incidence of conjunctivitis in 2,4,5-T thesis, to have the most severe acne and for
workers with acne. Although we had many the administrative personnel, who have the
complaints about eye irritation, only three least exposure, to have the least acne. How­
workers were observed to have conjunctivi­ ever, an association of a higher prevalence
tis. However, 23 ( 32%) of the employees of acne with any particular location in the
had hyperemia of the nasal mucosa and plant could not be demonstrated, eg, in the
eight ( 1 1 % ) had hyperemia of the buccal T C P synthetic area where one would expect
TCDD concentration in the environment to
mucosa.
Our findings that the presence of hyper­ be the highest. The plant has taken steps to
pigmentation and hirsutism correlate quite decrease the contamination of the TCP end
significantly with the severity of acne and product by dioxin, and the concentration of
not a t all with coproporphyrin excretion the contaminant in the TC P has dropped
suggests that these two signs, which are from 10 to 25 ppm to 1 ppm (see the
usually thought of as part of the PCT syn­ following subsections on chemistry and the
drome, rhay be more closely related to chlor- plant). Al the time of the study the effect of
acnc in 2,4,5-'!' plants. Other studies support the safety measure to decrease contamina­
this view, such as ( 1 ) the findings of Blei- tion on the severity or prevalence of chlor­
berg ct al’* that the presence of hirsutism and acne in the factory could not lie evaluated
hyperpigmentation correlated better with since ( 1 ) there were no previous prevalence

C00°r-501

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324

H E A L TH S UDVEY I X 2.4-1) ANI ) 2.4JÌ-T P L A N T — POLAND ET AL

the TCDD to be present in
10 to 25 ppm. About six
months before our survey,
Scale
Mean
Mean
SO
P*
P*
SO
the company installed a de­
L
52.5
8.2
46.1
NS
5.8
NS
vice
which removed most of
Ft
7.4 < 0 .0 0 2 5
54.4
54.6
8.8
NS
the
TCDD.
The TC P thus
K
8.9 < 0 .0 2 5
55.4
53.2
6.3 < 0 .0 2 5
produced
contained
less than
55.5
9.8 < 0 .0 0 0 2 5 48 .8
8.5
NS
H ypoch ond riasis:
1 ppm TCDD.
D epression
11.2
56.7
<
0.0001
11.5
59.5
<0.01
The TC P reacts with
H ysteria
59 .0
8.8 < 0.0001
58.9
6.7 < 0.0 01
m onochloroacetic acid
Psychodeviance
59.8
< 0.0001
55.5
12.1
12.1 < 0 .0 2 5
(MCA) to form 2,4,5-T.
M a sculinity
57.4
7.6 < 0.0 001
60.7
6.1 < 0 .0 0 0 2 5
The latter is esterified with
Paranoia
9.6
52.9
8.3 < 0 .0 5
52 .6
NS
Psychasthenia
10.4
8.9
52.1
NS
50.8
NS
.various alcohols or allowed
S chizophrenia
9.8
NS
51.2
NS
51.2
10.0
to react with dimethyla- .
Mania
12.4 < 0 .0 1
60.0
9.0 < 0.0 001
56.8
mine, and then formulated
Social introversion
8.4
51.4
NS
50.1
9.8
NS
and packaged. Some 2,4,5-T
Q
Y ear* o f schooling
10.9 * 2.1
P
15.2 ± 1.5
and
2,4-D
is
sold
as
the
un0 .0 0 1 1
esterified add in solid form.
C opropo rph yrin
38 .8 * 16.0
37 .9 * 24.7
NS!
The starting materials for
excretion, j<g/gm of
cre atinine
2,4-D are phenol and chlo­
No. 01 persons w ith
3 6 /5 2
10 /17
N S|
rine (gas) which react in an
som e degree o f acne
exothermic
process to form
Aetive acne score
6.2 ± 9.6
2.5 ± 3.2
NS)
DCP.
Subsequent
reactions
• S ignificance values by S tu d e n t's t-te st. com parin g the above p o pulatio ns
with MCA to the phenoxyto th e n o rm al population as de fin e d in th e te xt o f th e results.
t The tw o p la n t groups are significa ntly d iffe re n t (P < 0 .0 0 5 ). T his would
acetic acid are similar to
be expected on an education basis,
2,4,5-T. In 2,4-D produc­
t The tw o pla n t groups are sig n ifica n tly d iffe re n t (P < 0.0 1).
t S ignificance by S tu d e n t's t-te s t.
tion there is no formation
| S ignificance by X1 te s t.
of the TCDD.
figures on chloracne in the plant with which
T h e Plant .—The plant employs several
to compare and ( 2 1 the effect of such a measures in an attempt to improve indus­
measure on the severity or prevalence of a trial hygiene. Clean work dothes are issued
chronic disease such as chloracne may take daily, and separate lockers for street and
longer than six months to become evident
work dothes are provided. All production
Individuals seemed to vary greatly in workers said they showered daily before
their susceptibility to acquire chloracne. leaving the plant. The DCP and T C P are
This observation is consistent with (1) the made in one building and the 2,4-D and
lack of correlation between the duration of 2,4,5-T (acids) and their esters in another.
employment in the plant and the severity or Attempts have been made to improve venti­
presence of chloracne and ( 2 ) the tendency lation, and recently the company has been
toward an increased susceptibility to chlor­ successful in removing most of the TCDD
acne in persons with history of teen-age before the TCP leaves the area in which it is
acne.
synthesized. A dermatologist employed by
Chemistry .—Although the structures of the company visits the plant at least weekly,
2,4-D and 2,4,5-T (Figure) differ only by largely to treat the chloracne.
one chlorine, the commercial syntheses of Almost all operations are carried out in
the two compounds are dissimilar. The closed processes (eg, tank car to pipeline to
starting product for 2,4,5-T is 1,2,4,5-tet- reaction vessel to next pipeline to next reac­
rachlorobenzenc which reacts with metha­ tion vessel). There is of course some leak­
nol and aqueous sodium hydroxide to form age, and frequently men working in mainte­
2,4,5-tnchlorophenate, 2,4,5-trichloroanisole, nance must clean the inside of the reaction
and small amounts of several unwanted side tanks or open pipelines.
products, eg, TODD. The reaction product Porphyria Cutanea Tarda.-—A large out­
is steam-stripped to remove the anisole. At break of PCT in Turkey in 1956 due to the
this point, sampling of various hatches shows accidental ingestion of hexuchlorobenzene- 1
Table 6.— Scale Scores on the MMPI
52 P roduction W orkers

17 A d m in istra tio n W orkers

384579

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325

1.2 .4.5 tetra
chlorobenzene

2 .4.5 trichluro
phenate sodium

2 .4.5 trtchloro
anisole

Arch Environ Health— Voi 22, March 1971

DOW 384580

Porphyria cutanea tarda (ie, uroporphyplus the high prevalence of PCT described
by Bloiberg et al 13 in this 2,4-D and rinuria, skin fragility, and vesicular eruptions
2,4,5-T plant in 1964 support a direct toxic on the skin) does appear to be a symptometiology of many cases of PCT. The actual complex distinct from that of chloracne (ie,
etiologic agent of PCT in this plant is un­ cysts, pustules, comedones, and scarring of
known. We have administered reagent grade the skin); both were found in workers in
DCP to rats and observed an increase in the this plant in which 2,4-D and 2,4,5-T are
urinary excretion of coproporphyrin (un­ manufactured. The independence of these
published data). A single oral dose of DCP syndromes is supported by ( 1 ) the lack of
caused a threefold increase in the activity of correlation between the severity or even
hepatic ALA synthetase, the rate-limiting en­ presence of chloracne and uroporphyrinuria
zyme in porphyrin biosynthesis. Rimington in the previous study ,13 and ( 2 ) the persis­
and Ziegler- 0 reported a series of structurally tence of chloracne in the factory six years
similar chlorinated benzene compounds which later, despite the disappearance of the uro­
caused experimental porphyria in rats. The porphyrinuria and skin fragility. Even if the
TCDD, because it is a potent hepatotoxin etiologic agent is the same, the syndromes
and acnegen, was not examined for its ability apparently can occur independently and cer­
to cause porphyrinuria in experimental ani­ tainly have different clinical courses.
mals.
The statistically higher coproporphyrin
In the report of Bleiberg et al ' 5 in 1964 of excretion in the maintenance men suggests
this same plant, 1 1 employees had uropor- that they have a higher toxic exposure; they
phyrinuria and several had hirsutism, hy- also have a tendency towards more severe
perpigmentation, or skin fragility, or all acne. These men repair the inside of reac­
three. In the three case histories presented, tion vessels and leaking pipelines, and al­
two patients had vesicular eruptions on ex­ though their exposure is sporadic, it is ap­
posed areas of the body, two had hepatic parently more intense.
dysfunction, and the tissue obtained from
Recently, much attention has been direct­
the two reported liver biopsies fluoresced ed towards the finding of hemosiderosis and
intensely with ultraviolet irradiation. These hyperferremia in patients with PCT, often
three case histories unquestionably fulfilled in those with an alcoholic history .30-32 Al­
the criteria for the diagnosis of PCT.
though studies of iron metabolism were not
Our reexamination of the same plant six reported in the patients with hexachloroyears later revealed no clinical PCT; only benzene intoxication, chelating agents did
one employee had mild persistent uropor- appear to reverse the clinical symptoms and
phyrinuria. The reason for the decrease in porphyrin excretion .33 In our study, despite
severity and prevalence of PCT in this plant the absence of uroporphyrinuria in the
is unknown. Possible reasons include ( 1 ) workers at the present time, a tendency
increased encouragement of personal safety towards hyperferremia might be expected.
habits of workers, and ( 2 ) decrease in the On the contrary, we found an unexplainable
contamination of PCT by the hepatotoxin, trend towards hypoferremia and a lower
TCDD, and perhaps simultaneously by por- iron-binding saturation, with little demon­
phyriagenic chemicals.
strable anemia.

2 .3 .6 . 7 ■tetraclilorn
<1iben/odio»in

0002503
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H EALTH SU R V E Y IN 2,4-D A N D 2.45-T P LA N T— POLAND E T AL

DOW 384581

Hepatotoxicity.—T f’DD, which has been of these findings in this population would
incriminated as a potent acne genic agent, is only be conjectural.
MM PI.—The M M PI results are impor­
also very hepatotoxic. A single oral dose of
0.05 to 0.1 mg/kg to rabbits resulted in tant in that they offer an objective profile of
death in one to two weeks. Autopsy revealed the workers in one herbicide synthesizing
extensive necrosis and enlargement of the plant. Conclusions about personality changes
livers.' Similarly, dermal application of the secondary to chronic exposure in a plant
tarry residues in 2,4,5-T synthesis caused in which 2,4-D and 2,4,5-T are manufac­
gross hepatic lesions and rapid death in tured can only be tenuous, however, be­
guinea pigs."1 Clinically, several investiga­ cause we have no suitable group with which
tors have looked for evidence of hepatotoxic- to compare these results. The comparison of
ity in 2,4,5-T-plant workers affected with M M PI results of these factory workers with
chloracne. Bauer et alT noted that two of nine the .normal population of pre-World War II.
cases had slightly delayed sulfobromo- white subjects from Minnesota was inescap­
phthalein excretion and three had abnormal able since the scoring system for the test is
liver biopsies. In the report of Bleiberg et based upon this "norm.,,1,, Nonetheless, we
al*-' of patients with chloracne and porphy­ consider these groups incomparable. The
ria, two subjects had hepatic dysfunction only evidence to suggest any environmental
and two had abnormal liver biopsies. Dugois effect of this herbicide plant on personality
and co-workers*11* have noted hyperlipemic is the significant correlation between severi­
and hypercholesterolemic sera in their sub­ ty of chloracne fa known toxic effect) and
jects. Hofmann and Meneghini- 1 found no the score on the hypomanic scale. This is an
hepatic damage in six patients with chlor­ interesting finding and somewhat contradic­
acne. Of the liver tests performed in this tory to the apathy and psychomotor retarda­
study (bilirubin, albumin, SGOT, and alka­ tion reported by Bauer et al .7
line phosphatase), only four values were
M any previous investigations of both
elevated. Three of these four elevations were acute and chronic poisoning in 2,4,5-T
extremely mild, and the fourth was accom­ plants have reported the symptoms of ano­
panied by no other abnormal liver test val­ rexia with weight loss, fatigue, debility, and
ues. Of the six patients with palpable livers, asthenia .4-7 However, only one author has
only one had a mildly elevated liver func­ described in detail personality changes
tion test Although TCDD and other chemi­ thought to be associated with chemical expo­
cals produced in 2,4,5-T synthesis may be sure. Bauer et al 7 studied nine workers with
hepatotoxic in humans, the prevalence of severe chloracne which still persisted five
demonstrable chemical liver dysfunction in years after termination of their exposure in
this plant is minimal.
a 2,4,5-T plant and described a syndrome of
Systemic Toxicity.—As noted in the in­ general weakness, fatigue, apathy, and de­
troduction, a variety of other signs, symp­ creased libido alternating with fits of anger
toms, and laboratory findings have been not­ and irritability. Rorschach tests showed a
ed in various studies of 2,4,5-T workers with weakened emotional reaction, a lack of con­
acne. We found no unexplainable significant centration, slowed thought processes, and
increases in abnormal function of cardiovas­ perseveration. This whole syndrome was
cular, pulmonary, intermediary metabolic, designated by the term “psychovegetative
or hematologic systems in comparison with disorder.”
normal populations. Although the preva­ The inconsistency of our finding of a sig­
lence of gastrointestinal complaints seems nificant correlation between hypomania and
high f30%) and although physical examina­ chloracne and the previous description of
tion revealed occasional unexplainable neu­ lethargy, dulled emotional response, and
rologic deficits (decreased hearing, six of 73 apathy 7 remains to be adequately explained.
subjects; diminished proprioception, one of Possible reasons include a basic difference in
73; absent ankle jerks, two of 73), we could methodologies and a difference in testing
find no figures of comparable studies with period with relation to the timeTrf exposure.
which to compare; hence, a judgment on the Additionally, the severity and chrouicity of
normality or abnormality of the prevalence the organic disease in subjects studied by
Arch Environ Health— Vol 22, March 1971

H E A L T H S U R V E Y ¡N 2,4-D A N D 2.4 S -T P L A N T —POLAND E T AL

Bauer et al7 may have induced personality
changes which masked any manic effects
found with less severe disease.

References
1. Schwartz L, Tulipan L, Birmingham U: Occu­
pational Dictate* of the Skin. Philadelphia, Lea &
Febiger Publishers, 1967, pp 336-345.
2. Hofmann HT: New experience with highly
toxic chlorinated hydrocarbons. Naunvn Schmiedeberg Arch Pharm Exp Path 232:228-230. 1957.
3. Olivier NE: Chloracne. Arch Derm 99:127, 1969.
4. Kimmig J , Schulz KH: Occupational chloracne
caused by aromatic cyclic ethers. Dermatalogica
115:540-546, 1957.
5. Kimmig J , 'Schulz KH: Chlorinated aromatic
cyclic ethers as the cause of chloracne. Naturuiistentchaften 44:337-338. 1957.
6. Schulz KH: Clinical and experimental studies
on the etiology of chloracne. Arch Klin Exp Derm
206:589-596, 1957.
7. Bauer H, Schulz KH, Spiegelberg U: Occupa­
tional intoxications in manufacturing chlorphenol
compounds. Arch Gewerbepath 18:538-555. 1961.
8. Uugois P, Colomb L: Chloric acne: Case re­
ports arising from the preparation of 2,4.5-trichlorophenol. J Med Lyon 38:899-903, 1957.
9. Uugois P. Colomb L: Acne chlorique au 2,4,5trichlorophenol. Lyon Med 88:446-447, 1956.
10. Uugois P, Maréchal J, Colomb L: Chloric
acne caused by 2,4.5-trichlorophenol. Arch Mai Prof
19.-626-627, 1958.
11. Brunsting L: Observations on porphyria cuta­
nea tarda. Arch Derm Syph 70:551-564, 1954.
12. Goldberg A. Rimington C: Ditease* of Prophyrin Metabolitm. Springfield, III, Charles C Thomas
Publisher, p 110.
13. Epstein JH . Itedeker AG: Porphyria cutanea
tarda symptomatica (PCT-S). Arch Derm 92:286290. 1965.
14. I<undvnll O, Weinfeld A: Studies of the clinirnl and metabolic effects of phlebotomy treatment

porphyria

cutanea

tarda. Acta

Med

Scand

iokx

15. Bleiberg J . Wallen M. Biodkin K. et al:
Industrially acquired porphyria. Arch Derm 89:793797 1964.
16. Oahlstrom S, Welch G: An M M PI Handbook.
Minneapolis, University of Minnesota Press, 1960.
17. Marver HS, Tachudy UP, Perlroth MG, et al:
The determination of aminoketones in biological
fluids. Anal Biochem 14:53-60,1966.
18. Schlenkler FS. Uavis C L Kilchell CL: Uri­
nary total, aqueous, and ether soluble porphyrins.
Techn Bull Regitt Med Ttchn 33:57-66, 1963.
19. Haeger B: Urinary {-aminolevulinic acid and
porphobilinogen in different types of porphyria.
Lancet 2:606-608. 1958.
20. Klim t CK. Proul T E , Bradley KF. et al:
Standardization of the oral glucose tolerance test:
Report of the committee on statistics of the Ameri­
can Uiabetic Association, June 14, 1968. Diabetes
18:299-310, 1969.
21: Sharp C L Butterfield W JH. Keen H: Giabetes survey in Bedford 1962. Proc R oy Soc M ed
87:193-202, 1964.
22. Osgood EE, Brownlee IE, Osgood MW, el al:
T otal differential and absolute leukocyte counts and
sedimentation rates: Determined for healthy per­
sons 19 years of age and over. Arch Intern Med
64:105-120, 1939.
23. Hofmann MF. Meneghini CL: Apropos of folliculoais caused by chlorinated hydrocarbons (chlo­
rine acne). G Hal Derm 103:427-450, 1962.
24. Cam C: Cutaneous porphyrin related to intox­
ication. Dirim 34:11-15, 1959.
25. Ockner HK, Schmid R: Acquired porphyria in
man and rat due to hexachlorobenzene intoxication.
Nature 189:499, 1961.
26. Cam C, Nigogosyan G: Acquired toxic por­
phyria cutanea tarda due to hexachlorobenzene.
JA M A 183:88-91. 1963.
27. Schmid R: Acquired porphyria. JA M A 183:133134, 1963.
28. Schmid K: Cutaneous porphyria in Turkey.
N ew Eng J Med 263-397-398, 1960.
29. Rimington C. Ziegler G: Experimental por­
phyria in rats induced by chlorinated benzenes.
Biochem Pharmacol 12:1387-1397,1963.
30. Lam ent NM. Hathom M, Joubert SM: Por­
phyria in the African: A study of 100 cases. Quart J
M ed 30:373-392. 1961.
31. Strickland GT J r Porphyria cutanea tarda in
association with hemosiderosis of the liver Iron
absorption studies and evaluation of therapy by
phlebotomy. Amer J Gattroent 50:202-207, 1968.
32. Felsher BF, Redeker AG: Acquired porphyria
cutanea tsrda. primary refractory anemia, and he­
patic aiderosis. Arch Intern Med il8:163-167, 1966.
33. Peters HA, Johnson SAM. Cam S, et al:
Hexarhlorobenzene-indured porphyria: Effect of
chelation on the disease: Porphyrin and metal
metabolism. Amer J Med Sci 251:314-322, 1966.

gflW 384582

Thomns Ashby, MU, Augustin Gombart, MU,
Jerome Schulz, MU, and Arthur UcPalma, MU,
provided clinicni assistance. Patricia Hickman per­
formed the ALA, PBG. and porphyrin determina­
tions. Jan et Gating, MS, Community Study on
Pesticides, Washington State Gepartm ent of Health,
assisted in preparing a subset of our data for com­
puter analysis. Frank Clark, PhU, Assistant Professor
of Psychiatry (Psychology) at Emory University,
assisted in interpretation of M M PI results.
Jacob Bleiberg, MU, gave permission for entry of
the factory studied and provided written records of
past medical histories for comparison on some
workers.

in

iiu .io i.m u

327

A tv h E nviron H e a lth — V a l 22, M arch IS T I
Printed end Published in the United States ot America

0002505
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K 'e z i l i K .

• 210

NATURE VOL. 231 MAY 28 1971

3 , t Y— i - J — /

msw WOKLD

PSMisessii ire M l-!
9 “9 ^ '

.

k : '■ ¿fud

i f?7'

f i

by our Washington Correspondent

In October 1969. the President’s science PSAC panel apparently completed its a kow-tow to the military is an awlc. fS5
adviser. Dr Lee Dubridge, set up a report and confirmed that the Bionetics ward adornment of what purports to
panel to study the widely used results could not be dismissed, that the be an "outstanding case study of Ol
herbicide 2,4,5-T and its suspected Pentagon's long attachment to 2,4,5-T government regulation of pesticide", cn
teratogenic properties. The panel duly was broken. On April 15 last year the since if partisan evidence from the
reviewed the available evidence but,-as Surgeon General announced that 2,4,5-T Defense Department can be quoted CO
is customary with reports compiled would be suspended for use over water, without challenge how is the reader to
for the President's Science Advisory around the home and on food crops, be sure that similar courtesies have
Committee, its findings were not made enabling the Defense Department to not been extended to the manufac­
public. The report of the PSAC panel declare at the same time that the mili­ turers of a . pesticide and their
would probably be of little more than tary use of the herbicide.would fall in customers?
Another anxiety raised by the PSAC
historical interest had it not recently line with domestic use.
If the purpose of the PSAC panel on study lies in its treatment of the
been discovered and issued this month
as a model of what such a report should 2,4.5-T was simply to strengthen economic consequences of 2.4,5-T. The
be. T h is review represents an out­ Dubridge's hand by providing an weighing of economic benefits against
standing case study of government authoritative review of the then avail­ the possible health hazards of a pesti­
regulation of pesticide." states a press able evidence, the panel members seem cide is of course a crucial question for
release issued by the Office of Science to have performed a thorough and regulatory authorities and one that -an
and Technology. In a preface to the careful task. But their report, which "outstanding case study” might weil be
report Edward E. David, who succeeded does not seem to have changed signi­ expected to address. But although the
Dubridge as science adviser and chair­ ficantly since they completed it, is being panel attempts to estimate the costs of
man of PSAC last August, says that the presented by the Office of Science and substituting alternative herbicides to
report is "an excellent case study” 'and Technology as an outstanding case 2,4,5-T for a variety of uses, the report „
that the principles it brings out "will be study of government regulation, which contains no suggestions as to how these £
useful in dealing with other pesticides, for all intents and purposes covers the should be weighed against the toxico- |>
L
and indeed with chemicals of all kinds.” evidence on 2,4,5-T up until March logical risks.
This failure to grapple with aa
How far does the review live up to the 1971, the date of David’s preface.
Judged in this light, the PSAC report important issue is in a way not sur-?;
claims made for it?
Some people who have seen the has several curious features. One is prising-since the necessary expertise inireport call it excellent, others say it the inclusion of a section on the import­ social science is lacking. None of the£contains glaring and serious omissions. ance of 2,4,5-T as a defoliating agent. members of the PSAC panel seems to F.
The verdict may vary according to the This is an issue which might properly be an economist, there are only three t
perspective of the beholder and the be regarded as beyond the competence social scientists on PSAC and the£
date and purpose for which the review . of a scientific review panel; if it- were Office of Science and Technology half
was compiled. Members of the PSAC to be considered, an impartial presen­ yet to appoint its first social scientist
panel say it was written so long ago— tation of evidence might be expected. staff member. This composition off
about March or April 1970—that they The PSAC report is not impartial—it course reflects the long-standing dosnf
can remember little about it. One quotes at length, though admittedly inance of physical scientists on PSACf.
Even when on home territory. th:i
member believes that, to begin with, the without endorsement, a series of anec­
only real data available were the study dotes told by an Admiral W. E. Lem os PSAC panel report does not seem tcf.
conducted, by the Bionetics Corporation indicating that defoliation and crop have covered all the ground. "For wha?
which first showed that commercial destruction by herbicides are militarily it does say, it is a reasonably go«’
preparations of 2,4,5-T could produce effective. A staff member of the OST report, but there are two importac:
malformations in the offspring of has said that this was the only sort of , recommendations that should haw
evidence available. In fact there exist been made but were not." sav
rats.
Although the PSAC panel was told several studies made in or for the Matthew S. Meseison. professor oi
to concentrate on the domestic use of, Department of Defense that challenge biology at Harvard University f
the herbicide, this member recalls there the effectiveness of defoliation and ‘ Meseison believes the report shouL
was the feeling that evaluation of the crop destruction programmes, includ­ have been more circumspect in it*)
military use of 2,4.5-T as a defoliant ing, for example, a survey compiled by consideration of the dose-respon<£
in Vietnam was the real impetus for the Rand Corporation in 1967 sug­ relationship between 2,4,5-T and thi
undertaking the study. Dubridge, in gesting that only S per cent of enemy terata it induces, and that more auej
the first public reference to the soldiers depended on locally grown don should have been devoted to tj.
possibility that dioxin, the highly letfc?
Bionetics study in October 1969, said crops.
Why did the PSAC panel fail to cite contaminant ot ¿.O -T . preparation
that the Defense Department in its
defoliation operations over Vietnam these reports or to present the evidence may accumulate ..in body tissues, if
"will restrict the use of 2,4,5-T to areas objectively? "The reason PSAC did it essence, the PSAC panel report ss:-{'
remote from the population”, but for was a sheer tribute to the political that there exists a minimum doss:|
another six months he was unable to power wielded by the Joint Chiefs of below which the herbicide will c|
stop either the Defense Department or Staff,” says one observer. "It was not cause damage, so that animals
the Department of Agriculture from the habit of PSAC to buck the Joint humans may safely be exposed to cc.exposing the populations under their Chiefs of Staff, at least not under ccntralions of 2,4.5-T that are small,
respective controi to 2,4,5-T. It was Dubridge.” Whatever the reason, the by a safety factor of 10 to 100 th-j
not until April 1970, shortly after the abandonment of scientific objectivity in this minimum or threshold, -dosa-i

~_5£9I

3?

NATURE VOL. 231 MAY 28 1971

' 0 0 N2 I 5 5 0 3 5

211

Mcscison agrees that the idea of a plctcd about August 1968, and it was dangers to this country because of new
threshold is plausible, though unproved, not until 14 months later, after exten­ developments in Soviet weaponry . . .
but he believes the PSAC panel, report sive prodding by individuals inside and In reasonably good humour I say to my
should have pointed out the crucial outside the government, that the exis­ colleagues and the taxpayer, ’Here we
nature of this assumption, and that in tence of the report was first made go again’”. Within a few days of
Senator Symington's observation, John
its recommendations for further re* known by Dubridge.
search it should explicitly have cited The PSAC panel report does not S. Foster, the director of defence re­
the need for establishing a dose* deem it worth describing this extra­ search and engineering in the Depart­
response curve for the teratogenicity of ordinary chain of events in any detail. ment of Defense, launched his
2,4.5-T. The consequences of the Another recommendation of general springtime offensive against Congress on
response being linear would be pro­ importance—now embodied in an behalf of the S7,880 million the
administration bill before the Congress Pentagon is requesting for research and
found.
Meselson also considers the report —is that a legal mechanism should be development in the coming financial
should have mentioned the possibility established for restricting a pesticide year. The two prongs of his attack
that the dioxin eontaminant of 2,4,5-T temporarily on the basis of new infor­ were that the Soviet Union is now
may accumulate, in the body fat, as mation that indicates it may be spending the equivalent of 53,000 mil­
might be expected from its chemical hazardous. Of the two mechanisms lion a year more than the United
structure. Edward J. Burger, an OST available at present, cancellation is too States in these areas and that, as a
staff member of the panel, says this mild—the manufacturer can continue result, “the Soviet Union could assume
issue was discussed in panel meetings to sell the substance pending an appeal technological superiority in military re­
but not in the report since it was just —and suspension is too drastic, since a search and development in the latter.,
/fl r
one among several unproved possibOi* compound can only be suspended if half of this decade“.
J J
ties. But the possibility that an shown to be an imminent hazard, and
" 7 f ' extraordinarily toxic contaminant of a once suspended is unlikely ever to
/
j
widely used herbicide (the LD„ of return to the market.
dioxin is 0.0006 parts per million for A third recommendation is-a level­
&
--female guinea-pigs) may be sufficiently headed warning on the interpretation
stable in the environment and soluble of teratogenesis. Most chemicals might
in fat and liver to enter food chains . be shown to be teratogenic in animals,
and the human diet does seem worth the PSAC report says, and it is not posmentioning in what is claimed to be a • sible to extrapolate the results of
model report. Moreover, the PSAC animal experiments directly to man.
panel report offers no scientific justifica­ “To restrict or ban usage of chemicals John & Foster, director of defence research
tion for its recommendation that the on the basis of demonstration of terato­ and 'engineering. (From The Washinglevel of dioxin in 2,4,5-T should be genicity at dose levels which far exceed Ionian.) (By John Lewis, ©1971 Washinglimited to not more than OJ ppm and actual or expected exposures is un­
toman Magazine.)
should be reduced to 0.1 ppm. This is reasonable and could well deny the
good news for manufacturers because, usage of chemicals whose benefits far The Senate Armed Services com­
mittee, before which Foster delivered
as the PSAC panel report points out, outweigh the risks.”
present technology allows 2,4,5-T to be The President’s Science Advisory this testimony, may well have been im­
manufactured with less than 1 ppm of' Committee won its spun as a. body of pressed not only by the bad news but
dioxin.
physical scientists, concerned almost also by Foster’s magnificent for­
But the smallest quantity of lethal exclusively with defence matters, whose bearance in not demanding an extra
impurity a manufacturer is capable of particular forte was the ability to take 53,000 million for himself. Last week, in
admitting into his product is not neces­ on issues such as strategic weapons that an unprecedented open hearing, the ad
sarily the quantity guaranteed to be cut across different parts of the govern­ hoc subcommittee on research and de­
harmless to man and beast. A very ment. In some ways PSAC is less velopment of the Senate Armed Services
rough calculation indicates that even important than it was since Congress committee had the chance of hearing
the 0.1 ppm limit urged by the PSAC is beginning to take an interest in the Foster's claims dissected and dismissed
panel report raises questions. Animals questibning and analysis of defence by the Federation of American Scien­
are quite often found to contain in their issues which was formerly done by tists. Pentagon statements about Soviet
bodies concentrations of DDT of the PSAC or not at all. The panel report research, the federation said in a 50order of 10 ppm ; if the pesticide were on 2,4,5-T suggests that PSAC, in try­ page report entitled is there a R e ­
not DDT but a solution of 2,4,5-T ing to extend its sphere of authority to search Gap?, have amounted to a
containing 0.1 ppm of dioxin, the the civilian issues more usually covered “classical numbers game featuring
animal would conain 10“ B pans' of by the National Academy of Sciences, selective disclosure, questionable as­
dioxin, which is only a thousand times has yet to acquire the tact and urge for sumptions, exaggeratedly precise esti­
l/'*
less than the LDN for guinea-pigs completeness with which the academy mates, misleading language and
alarming non-sequitur conclusions”.
(2,4,5-T does not accumulate, but seeks to hallmark its public reports.
The federation, a small but ener­
'dioxin may). The PSAC panel report
getic lobbying group, devotes most ofcj
includes no calculations of this kind, OEFENCE SPENDING
its energy to issues of arms control and£D
much less any discussion of their
disarmament
Its' membership, c o m O
significance.
Faster
posed almost entirely of scientists, in-CO
Of wider interest than the issue of FAS Flays
by our Washington Correspondent eludes several former members of th < ^
2,4,5-T, the PSAC panel recommends
- that new information on a pesticide “Many new occurrences inevitably President’s Science Advisory Com</J
“should be disseminated promptly to come with spring,” Senator Stuart mittee. The analysis of the DefcnscQO
individuals and .organizations that are Symington told the Senate in March Department's new claims about Soviet
legitimately concerned”. The original this year, “some pleasant—warmer military research spending was put to­
experiments on 2,4,5-T by the Bionetics weather, Japanese cherry . blossoms; gether by a federation committee which
Corporation, under contract to the some unpleasant proposed higher do- included Pmfaunr UMvin I

5692

5893

P* 7 J w “

THE

DOW

CHEMICAL

COMPANY

April 27, 1971

J.
C.
H.
C.
V.

E. Johnson, Corporate Research & Development, 2020 Bldg.
W. Hinman, corporate Research & Development, 2020 Bldg.
L. Gordon, Corporate Medical, 2030 Bldg.
G. Kramer, Corporate Medical, 2030 Bldg.
K. Rowe, Toxicology, 1803 Bldg.

DOV026820

MIDLAND

RESULTS OF THE PILOT STUDY ON THE ABSORPTION AND EXCRETION OF
2,4-D and 2,4,5-T BY HUMAN SUBJECTS
A report of the results of the indicated study is enclosed.
Considering the quality of the subjects, as well as the in­
vestigators, the data were quite good.
I feel that this study
should be expanded to include 3 more subjects for each compound.
Recommendations for future work are given at the end of the
report.

P. J. Gehring
Toxicology
Building 1803
slk

0000497

S69S

UL

File :

K-2372,p T 2 3 .1 4 -1 1 -1 7 (2,4-D )
K-4568,'7T23.1 4 - 2 3 - 2 3 ( 2 , 4 , 5-T)
D O W 026S2Ì

RESULTS OF A PILOT STUDY ON THE ABSORPTION AND EXCRETION OF
2,4—DICHLOROPHENOXY ACETIC ACID (2,4-D) AND 2,4,5-TRICHLOROPHENOXY ACETIC ACID (2,4,5-T) FOLLOWING A SINGLE ORAL DOSE
TO HUMAN SUBJECTS

P. J. Gehring
Toxicology
H. L. Gordon
Corporate Medical

April 27, 1971

5698
0000438

OBJECTIVE

026822

The objectives of the work reported herein were two-fold: (1)
to determine the concentrations of 2,4-D and 2,4,5-T in blood as
a function of time following oral administration, and (2) to
determine the amount of these agents excreted in the urine
during the first 48 hours following administration.
METHODS
Two human male volunteers weighing 76.8 and 81.0 kg ingested
5 mg/kg 2,4-D (Sample AGR 30653C) and 2,4,5-T (Sample AGR 86187),
respectively. For ingestion, the samples, which were analytical
standards prepared by The Dow Chemical Company, were mixed in
milk.
Blood samples for compound analysis, 10 ml, were collected
immediately prior to ingestion and at 2, 7, 12, 24 and 48 hours
following ingestion. Samples of the urine, collected 0 to 23,
23 to 24, 24 to 47 and 47 to 48. hours following administration,
were used for compound analysis. Analyses for 2,4-D and 2,4,5-T
(R. Papenfuss, AL28-109, 1971) were performed as described by
Marquardt, 1970 (Dow Report ML-AM-70-95).
RESULTS AND DISCUSSION
The concentrations of 2,4-D and 2,4,5-T in the blood at various
times following administration are given in Table 1. Before
doing a kinetic analysis of the data, the concentration in plasma
was calculated (see footnote to Table 1). This transformation
was needed to derive a volume of distribution from the kinetic
analysis.
In previous studies conducted in this laboratory, as well as
in other laboratories (Courtney, 1970,* Erne, 1966a), the dis­
appearance of 2,4-D and 2,4,5-T from the plasma of .rats, pigs,
calves and chickens followed first order rate kinetics. There­
fore, the kinetic model describing the concentration of these
agents in plasma as a function of time was assumed to be that
given by the equation
Apkf
cs =

-k t
-k t
(e e - e f )

Vd (*f-ke>

5.697
0000499

where
kf = the apparent first order absorptionconstant (hr )
ke = the apparent first order disappearanceconstant (hr
V3 = the apparent volume of distribution (1/kg)
AQ* = the dose administered (mg/kg)
t = time (hrs)
Cs = drug concentration (^gm/ml)

)

DOW 026823

- 2 -

The values of kf, ke and V3 which provide the curves of best fit
for the experimental data were determined using a computer pro­
gram recently developed by G. E. Blau (NCM-10526, 1970). The
concentrations of 2,4-D and 2,4,5-T found in plasma together
with the curves of best fit are illustrated in Figures 1 and 2,
respectively.
The rate constants (ke ) for the elimination of 2,4-D and 2,4,5-T
were 0.035 + 0.001 and 0.045 + 0.004 hr“ ^-. These values correspond
to a half-life (t^) of 19.8 and 15.4 hours, respectively.
In
other species (rat, pig, calf, chicken) the t% ranges from 3 to
12 hours (Erne 1966a).
The volumes of distribution (V<j) for 2,4-D and 2,4,5-T were
0.114 + 0.001 and 0.066 _+ 0.002 1/kg. The plasma volume of man
is 0.04 1/kg and the extracellular volume is 0.18 1/kg. There­
fore, these data suggest that the distribution of these agents
is extracellular. Since these agents are bound to plasma protein
(Erne, 1966b), a volume of distribution less than 0.18 1/kg is
expected. In previous studies using rats, the volume of distri­
bution of 2,4-D was found to be 0.11 1/kg.
The excretion of 2,4-D and 2,4,5-T in the urine is depicted in
Tables 2 and 3, respectively. Within 48 hrs, 94.4% of the dose
of 2,4,5-T was excreted in the urine of the individual receiving
this agent. Only 72.4% of the dose was excreted in- the urine of
the subject given 2,4-D. This difference is very likely an in­
dividual difference rather than a difference in the excretion of
the compounds.
In a recent study using rats, it was found that
as much as 22% of the 2,4-D excreted may be excreted via the
feces. Therefore, the frequency of defecation may influence the
amount excreted in the urine as well as the rate of clearance
from the body.

0000500

5898

D

-3 -

o

In conclusion, the following suggestions are made for future
studies.

o
fo
cn

00

1. Additional blood samples should be obtained at 32, 56 and
72 hours following the administration of the compounds.

r\o

2. The analyses for 2,4-D and 2,4,5-T should be conducted on
plasma rather than on whole blood.
3. The plasma protein binding of 2,4-D and 2,4,5-T should be
determined.
4. Urine should be collected from 48 to 72 and 72 to 96 hours
following administration.
5. Feces should be collected from 0 to 24, 24 to 48 and 48 to
72 hours following administration.

REFERENCES
Courtney, K. D. 2,4,5-T in the rat: Excretion patter, serum
levels, placental transport, and metabolism. Pesticide
Symposia, Inter-American Conferences on Toxicology &
Occupational Medicine, University of Miami School of
Medicine, 1970.
Erne, K. 1966A. Distribution and elimination of chlorinated
phenoxyacetic acid in animals. Acta vet Scand. 7:240-256.
Erne, K. 1966b. Studies on the animal metabolism of phenoxy­
acetic herbicides. Acta vet Scand. 7:261-271.

0000501

Table 1
CONCENTRATION OF 2,4-D AND 2,4,5-T IN THE BLOOD OF MAN
AS A FUNCTION OF TIME FOLLOWING THE ADMINISTRATION OF 5 MG/KG
Time (hrs.)
post-administration

Uqm/ml blood

0

0 .0

2

2,4-D
uqm/ml plasma1

uqm/ml blood

5-T
uqm/ml plasma:

0 .0

0 .0

0 .0

21.0

35.3

25.0

42.0

7

20.0

33.6

33.0

55.5

12

18.0

30.3

30.0

50. 5

24

15.0

25.2

19.0

32.0

48

2.1

\ 3.5

3.9

6.5

1 The ratio of the concentration of 2,4-D between plasma and red cells at equilibration is
approximately 1:10 (Erne, 1966). Since the hematocrit of both subjects was 45%, the concentration
of 2,4-D and 2,4,5-T in plasma was estimated using the following equation:
0.55y + 0.45 (l/10y) = C
where y is the concentration in plasma and C is the concentration in blood.

cn
^3

o
o

o
O
O
CD

cn
O

ro

228920 M O a

Table 2

URINARY EXCRETION OP 2,4-D BY A MAN GIVEN 384 MG, 5 MG/KG

Duration of
collection, Hr
post-administration

Volume of
urine voided
ml.

Volume of urine
following dilution
ml.

2,4-D
concentration
uqm/ml

0 to 23

1320

15001

79.0

118.5

30.9

30.9

23 to 24

56

56

98.0

5.5

1.4

32.3

24 to 47

1400

15001

99.0

148.5

38.7

71.0

47 to 48

180

180

29.0

5.2

1.4

72.4

2,4-D Excreted
Total,mq
Cumulât:
%

1 Sufficient water was added to the undiluted urine sample to give the volume indicated.

o
o
o

QI
O

o

CM

o
CO

928920 Moa

DOV

’6827

Ta bl e 3

U1
1

URINARY EXCRETION OF 2,4,5-T BY A MAN GIVEN 405 MG, 5 MG/KG

Volume of
urine voided
ml.

Volume of urine
following dilution
ml.

concentration
uqm/ml

0 to 23

1680

20001

105.0

210.0

51.8

51.8

23 to 24

103

103

97.0

10.0

2.5

54.3

24 to 47

2340

2340

68.0

159.1

39.2

93.5

47 to 48

204

204

17.0

3.5

0.9

94.4

to

Duration of
collection, Hr
>ost-administration

4 , 5-T Excreted
%
Total, mq
Cumulative

1 Sufficient water was added to the undiluted urine sample to give the volume indicated.

o

o

cn
cb
to

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o

OT

o

S

O ai
CO O

en

is

5705

1

r

M

T 3 6 . 25-66681-14
0

6

9

79 7

3 £>, Iai

A PROBE STUDY OF THE ACUTE ORAL TOXICITY OF 2,3,7,8TETRACHLORODIBENZO-£-DIOXIN IN THE CANINE
Norris

This study was conducted to define a range of toxicity from
a single oral dose of 2,3,7,8 -tetrachlorodihenzo-£7 dioxin
(TCBD) in the canine.

In addition, clinical and pathological

evaluations were conducted in an effort to further define
the toxic properties of TCBD.

This study is intended to he

a probe study and the data must be interpreted in this con­
text.
EXPERIMENTAL METHODS

(

2, 3, 7,8-Tetrachlorodibenzo-]>-dioxin (TCBD), reference number
was
340-2-54B, /administered to unconditioned pound dogs. The
study was conducted in two parts; first^male dogs,

2 /treat-

ment level, were given 3 mg/kg or 300 ug/kg TCBD and 2 months
later female dogs, 2/treatment level, were given 100 or 30
l^g/kg TCBD.
The TCBD was suspended in an 8:2 corn oil-acetone solution
and administered as a single oral dose in gelatin capsules.
The dogs ranged in weight from 8.0 to 17.5 kg and were given
from 8-17 ml of the TCBD corn oil-acetone mixture.
In the male dogs^ evaluations of blood urea nitrogen (BUN),
serum alkaline phosphatase (AP) and serum glutamic pyruvic^ ^ q ^

OOGd.'ÎÆÆ

DOW 749678

By: G. L. Sparschu, J. E. Bourne, P. J. Gehring and J. M
OBJECTIVES

i

-2 transaminase (SGPT) were made on test days 1, 8 , and 15.
Hematology studies consisting of packed cell volume, hemo­
globin, total erythrocyte count and total leukocyte cotint
and differential were conducted on test day 18 for the two
male dogs given 300 ug/kg of TCBD and on test day 64 for
all the female dogs.
Body weights were taken on all dogs prior to dosing and
weekly thereafter.

Complete gross and histopathology were

conducted on all dogs.
RESULTS AND DISCUSSION - MALE DOGS
The clinical signs for all four dogs, reg.ardless of dose,
were essentially the same.

The clinical signs consisted of

anorexia, dehydration, depression, emaciation, and gastroin­
testinal hemorrhage.

The two dogs given 3 mg/kg of TCBD

died after 9 or 15 days on test.

Those dogs given 300 ug/kg

of TCBD were killed after 18 or 22 days on test due to their
extreme cachexic and moribund condition.
The clinical chemistry results are given in Table 1.

All

dogs showed slight to moderate elevations in SGPT and dog
70-3171 had a terminal elevation of BUN.

r „ AKl|

0004345

Dog 70-3170

a.

had a decreased PCV and both male dogs had/(leukocytosis.
Table 3 contains the gross and histopathology results.

The

749680

The hematology results are given in Table 2.

digestive tract appears to be particularily sensitive to the
affects of TCBD resulting in focal areas of inflammation,
necrosis, hemorrhage and ulceration.

The major organ

affected was the liver; focal areas of necrosis, usually
central lobular with only a minimal number of hepatocytes
involved.

Another treatment related observation is the

vasculitis that is most prominent in the omental and mesen­
teric vessels.
culitis.

An adjacent steatitis accompanied the vas-.

The profound anorexia that the TCBD produced

resulting in extreme emaciation and dehydration no doubt
was a major contributing factor in the death or moribund
t

condition of the male dogs

receiving- 3$ mg or 300 jjtg/kgtTC'& ® •

RESULTS AND DISCUSSION - FEMAT.F. nnfls
The first several days after treatment the dogs appeared
clinically normal.

By eleven days they were dehydrated,

anorexic, depressed, and had intestinal hemorrhage.

Dog

71-424 (30 pgAg) appeared near death and had a generalized
partial alopecia.

Dog 71-425 (100 iig/kg) had minimal shedding

of hair and both dogs given 100 yg/kg TCBD had unsteady

.

5708.

CG04G4G

gaits.

At fourteen days the dogs still had evidence of

intestinal hemorrhage; the condition of dog 71-424 (30 ug/kg)
\A/

DOW 749681

was unchanged.

Dog 71-425 (100 ug/kg) was not showing extensive

alopecia and dog 71-423 (30 jjg/ k g ) was starting to shed hair.
At 18 days and thereafter, there was no evidence of intes­
tinal hemorrhage and all four dogs appeared more alert
suggesting partial recovery.
The dogs treated with 100 gg/kg TCBD continued to lose body
weight the first 30 days after which they began to gain weight;
by 64 days they were approaching their pre-test body weight
(Figure 1).

The dogs given 30 ug/kg TCBD also lost body

•

weight, but after 15 days they began to gain back their
weight.
The hematology results, Table 2, show a decreased PCV, total
erythrocyte count, and hemoglobin with a leukocytosis.

The

decrease in erythrocitic values is probably a reflection of
the intestinal hemorrhage clinically observed.

Terminal

hematoxylin and eosin stained sections of bone marrow were
unremarkable; there was no evidence of bone marrow depression.
The pathology results are given in Table 4.

Various degrees

of alopecia were seen in all dogs, with the 100 u g A g treated

5709
GG04347

-5-

Microscopically the skin

lesions consisted of a folliculitis with epidermal inflam­
matory cells and focal ulceration.

There was no marked .

hyperkeratosis or keratotic cyst formation to relate the
skin lesions to chloracne.
Dog 71-426 (100 pg/kg) had' scattered inflammatory cells in
the hepatic sinusoids and dog 71-425 (100 ugAg) had a mild
chronic colitis.

(

\

0QW 749682

dogs showing the most hair loss.

SUMMARY
Two unconditioned male pound dogs/group were treated with
3 mg/kg or 300 ug/kg of 2, 3,7,8 -tetrachlorodibenzo-]3-dioxin
(TCBD).

The TCBO was administered as a single oral dose

suspended in an 8:2 c o m oil-acetone solution.

All male

dogs became anorexic, depressed, emaciated and had gastro­
intestinal hemorrhage.

The dogs receiving 3 mg/kg died on

test days 9 and 15 and those receiving 300 jjg/kg were killed
on days 18 and 22 because of their moribund condition.
Lesions noted in the digestive tract included ulceration,
inflammation, necrosis and hemorrhage.

All dogs had minimal

to moderate amounts of hepatic necrosis.

There was a vascu­

litis present, especially in the omental and mesenteric
vessels, with an adjacent steatitis.

The profound anorexia

and dehydration were important contributory factors as to
the cause of death or moribund condition of the male dogs
treated with 3 mg/kg or 300 ng/kg of TCBD.
Two unconditioned female pound dogs/group were treated with
100 jjg/kg or 30 ug/kg of TCBD.

Several days after treatment

they became anorexic, dehydrated, depressed, lost weight und
had intestinal hemorrhage.

Approximately eighteen days

after treatment the dogs became more alert, began eating and

; 5711

CG04343

0DW 749683

-6 -

intestinal hemorrhage stopped suggesting the beginning of
recovery.

The dogs receiving 100 ug/kg started to show

weight gains 30 days after treatment and those receiving
30 ug/kg showed weight gains after 15 days.

All female dogs

t.896i£* AlSP^c

-7-

had various degrees of alopecia with the 100 \jg/kg level
being the most severe.

Histopathologically, the skin lesions

consisted of inflammatory cells in the epidermis and a folli­
culitis.

The female dogs were killed 64 days after treat­

ment and at this time one dog at the 100 u3 /kg dose had a
mild chronic colitis and the other dog at this dose zjc had
scattered inflammatory cells in the hepatic sinusoids.
It is concluded that single oral doses of 300 ug/kg and above
of 2,3,7,8 -tetrachlorodibenzo-£-dioxin are lethal in the dog.
Single oral doses of 100 ug/^g and 30 ug/kg of 2,3,7,8tetrachlorodibenzo-ja-dioxin are severely toxic to the dog,
but recovery does occur.

5712

0G04G30

TABLE 1
STUDY: Single Dose Oral Probe
TEST MATERIAL:
2,3,7,8-Tetrachlorodibenzo-£-dioxin
TEST ANIMAL: Unconditioned Pound Dog - Male

CLINICAL CHEMISTRY. BODY WEIGHT AND MORTALITY DATA
Dose and
Animal
Number
3 mg/kg

Day
of
BUN (mq%)
Test. ...1
8 15

AP
(K.A. Units)
8 15
1

SGPT
(Karmen Units/ml)
1
8
15

Body Weight
Loss (kq)
8
18
22

Test Day
of Death

70-3167

11

25

—

10.9

32

—

56

92

—

2 .1

—

—

9

70-3168

18

18

—

10.9

6

—

68.7

53

—

0 .6

—

—

15

70-3170

14

21

15

8

7

5

58

32

60

1.3

2 .8

3.4

22 *

70-3171

15

17

90

11

15

20

53

56

100

1 .2

4.2

—

18 *

300 \jg/kg

♦Killed because of moribund condition.

C G 0435Ì

or
CO

S896H MÖ.0

'A • '

TABLE 2

STUDY: Single Dose Oral Probe
TEST MATERIAL: 2,3,7,8 -Tetrachlorodibenzo-£-dioxin
TEST ANIMAL: Unconditioned Pound Dog
HEMATOLOGY SUMMARY
Dose and
Animal
Number
300 ug/kg

DiiPferential Couni
PCV

Sex

Test
Day

%

70-3170

M

18

70-3171

M

71-425
71-426

x 10 ®

Hemoglobin
q /1 0 0 cc

WBC
x 10 s

Neut
Seq.
B/J

39

5.9

14.2

27.1

70

18

54

7.3

19.8

31.2

F

64

32

4.9

1 1 .6

F

64

33

5. 3

71-423

F

64

35

71-424

F

64

30

RBC

(%) ,______

Lymph

Mono

Eo3in

Base

10

16

4

6

0

79

51

9

7

0

0

2 2 .1

33

0

15

10

42

0

11.9

24.2

51

3

21

11

14

0

5.9

1 2 .6

24.8

63

1

22

7

7

0

5.1

1 0 .8

20.9

53

3

31

6

7

0

loo u g A g

30 p g A g

CJ1
<1

n 9 9 B W
0004332

4MDQ

TABLE 3
STUDY: Single Dose Oral Probe
TEST MATERIAL: 2,3,7,8-Tetrachlorodibenzo-|)-dioxin
TEST ANIMAL: Unconditioned Pound Dog - Male

GROSS AND HISTOPATHOLOGY SUMMARY
im al

-3167

Dose
3 mg/kg

Gross Pathology
Died« cachexic; buccal ulcers; liver—
congested with lobular accentuation;
kidneys— swollen; omentum— white lobulated mass; adrenals— enlarged; gastric
ulcers; GI tract empty and congested.

3168

3 m g/kg

Died« cachexic« Dirofilaria, liver— pale
and granular; perirenal edema and renal
swelling; mesenteric lymph node hemor­
rhagic; omentum— lobulation of adipose
tissue; GI tract— edema, congestion and
focal hemorrhage.

riber

OI
< s

h*

Ol

C G 04353

His t o p a t h o l o g y

Thrombus in renal arcuate artery; splenic
lymphoid depletion and focal hemorrhage;
diffuse pulmonary congestion; Vasculitis
of vessel in coronary groove; liver—
j
central lobular congestion with diffuse
focal hepatocyte necrosis and leükocyte
infiltration; enterocolitis; omental stea-!
titis; a nonspecifiq vasculitis mainly in-*
volving the arteries in the omentum and
I
mesentery; focal necrosis and leukocyte
j
infiltration of adrenal gland adjacent to
necrotizing vasculitis; mesenteric lymph
node— lymphoid depletion and perilymphadenitis adjacent to the vasculitis of the
surrounding adipose tissue.
Chronic prostatitis; acute inflammation of
adrenal with periadrenal hemorrhage;
chronic nephritis; lung— vasculitis with
Dirofilaria: mesenteric lymph node— lymph­
oid depletion, hemorrhage, edema and vas­
cular necrosis; spleen— lymphoid depletion;
liver— central lobular necrosis and fatty
metamorphosis; omentum— steatitis; acute
enteritis with hemorrhage and nécrosis.
----------------------- — —

CONTINUATION OF TABLE 3
•

[mal
nber
-3170

Dose
300 p g A g

Histopatholoqy
Gross Patholoqv
Killed, cachexie; moribund; buccal ulcers; Acute prostatitis; liver— central lobular
necrosis; acute myosititis of adductor
intestinal hemorrhage; liver— mottled.
group; enteritis with ulceration and
necrosis; omentum— steatitis; chronic
colitis.

-3171

300 v g / k g

Killed, cachexie; moribund; Dirofilaria;
urinary bladder— serosal hemorrhages;
gall bladder— edematous and distended;
omentum— lobulation of adipose tissue; GI
tract— edematous with focal serosal and
mucosal hemorrhages.

en
-vJ

GG04354

Spleen— lymphoid depletion; lurtg;— vascu­
litis with Dirofilaria; acute esophaqitis.
focal myocarditis, renal abscessations,
liver— fatty metamorphosis; omentum— stea­
titis and vasculitis; serosal ¿utface of
abdominal viscera— chronic hemorrhage with
fibroblast proliferation.

i

8 8 9 6 p £

M O Q

TABLE 4
STUDY; Single Dose Oral Probe
TEST MATERIAL: 2,3,7,8-Tetrachlorodibenzo-p-dioxin
TEST ANIMAL: Unconditioned Pound Dog - Female

GROSS AND HISTOPATHOLOGY SUMMARY
limal
imber
-425

Dose
io o vigAg

Gross Patholoqv
Skin— symmetrical bilateral alopecia of
abdomen, sides, and legs with erythema­
tous papule^ and focal scab formation in
areas of alopecia.

Histopatholoqv
Chronic colitis-minimal; chronic lymphad­
enitis of mesenteric lymph node; skin—
focal ulceration with acute and chronic
folliculitis.

-426

lo o p g A g

Skin— symmetrical bilateral alopecia of
abdomen, sides and legs with erythematous
papulen* and focal scab formation in areas
of alopecia; focal ulcerations on hard
and soft palates; renal capsule thickened.

Focal ulceration, necrosis and hemorrhage
in palatine tissues; chronic interstitial
nephritis with focal parasitic granuloma;
liver— acute and chronic inflammatory
cells in sinusoids; skin— focal Ulceration
with acute and chronic folliculitis.

-423

30 (jgA g

Skin— ventral alopecia with minimal pus­
tular scab formation; Dirofilaria, intes­
tinal tapeworms.

Focal chronic interstitial nephritis;
focal dermatitis with folliculitis-minima].

-424

30

OT

minimal alopecia of front legs with
vg/kg Skin—
slight erythema and papule formation.

0004355

Dermatitis with focal ulceratiort-minimal.

Xi r r m - m

m n T a n

18

# 71-426 (100 Ug/kg)
17
16
15
14
13
12

(100 p g A g )

# 7 1 -4 2 5
# 7 1 -4 2 3

( 30

# 7 1 -4 2 4

( 30 ggAg)

\ £ / k g )

11
10
9
8
7

6

DAYS ON TEST

noor * f /AGO

5719
y

/
/

Reprinted from BioScien

r

The Public Health Implications of Widespread
Use of the Phenoxy Herbicides and Picloram
Julius £. Johnson
Martin and Duggan, 1968;Comeliussen, J
1969).
*V
Air and water are potential sources
of exposure of m m to these herbicides.
Manigoid and Schulze (1969) have re­
ported on pesticide residues in selected
streams in the West such as the Missouri,
Yellowstone, Colorado, Arkansas, Rio
Grande, Snake, and othen. Phenoxy
herbicides were detected as shown in
Table 3. Of the 320 samples taken,
fractional parts per billion concentra­
Tolerance» proposed to FDA
tions were reported in 78 samples. The
measurement
of picloram residues in
2,4-D
2,4,5-T
MCPA
Siivex
Pidorem
water has been reported following direct
X
X
spraying o f a static pond in Texas
X
X
(Hoffman et al., 1969) and a watershed
X
X
X
X
experiment was conducted in North
X*
G ra in s ftic m
X
X
X
Carolina using 2,4-D, 2,4,5-T, and picF is * S * t(j
X*
X
•ioram (Sheets and Lutz, 1969). The
X
results of these studies are summarized
X
P otato**
S u g a rc a n e
in Table 4. It is noted that picloram
X
persisted longer than 2,4-D or 2,4,5-T.
Hey
X*
Parage Gramme*
Air
samples collected in spring and
X*
summer from two wheat-growing areas
Meat
X
in the State of Washington were ana­
fo titb a tolerances except those marked*)
Milk
(Ail negligible
lyzed for phenoxy herbicides by Bamesberger a n d A d a m s (1966). The results
,,3te to the health and safety ddes in foods as confirmed by the F D A
are summarized in Figure 1. Phenoxy
may t r i ‘r n e data presented will empha- market basket survey of pesticides in herbicides are widely used for early
of ju^' -ctirar toxicity, and chemistry.
___ composites of 12 categories of foodpostemergence control of weeds in these
°l ”
I uriiJ comment
cu,?“‘ on exposure to stuffs. Of the 1548 samples analyzed, crops. There were detectable levels of
Fit51, *
1 f and
. There is very little only 36 positive identifications were 2,4-D and 2 ,4 ,5 -T in the air up to 0.06
®zecirst,
„fid airfood,
toa« to ** Phen°*y herbi- made. All but one was less than 0.2 Mg/m3. M a n will inhale about 30 m 3 of
lOStfte ° ffh food. The principal uses ppm. 2,4,5-T was found in three of the
air per day. A t a level of 0.06 ng/m3,
e*P
»ogfr
cides ttir°ftTrbicideS ® *rowing fo°d surveyed samples (Duggan et ai., 1967; this would a m o u n t to an exposure of
of these “’ooginaliy registered on a

/ / O r*

t
This paper is concerned primarily
Table 1 shows a condensed version of
2,4-D, 2,4,5-T, siivex, and
tolerances
proposed for 2,4-D, 2,4,5-T,
^¿rJoram-the phenoxies
because V
of
m__
H MCPA, siivex, and picloram. Those
spread use; picloram, 2,4,5-T, and
marked with an asterisk are for toler­
D more specifically because of their
ances at a permissible level for 2,4-D in
^ an military defoliants. The purpose
rice and flax seed and for picloram in
discussion is to explore possible
forage grasses and meat. The remainder
£ f* f j l i c health consequences of wide- are negligible residue tolerances.
use. This presentation will be
Table 2 demonstrates the infre­
g Ç * yrj/iei primarily to information as it
quency of residues of phenoxy herbi-

,

crops * ert„cis. A change “ po,icy now
no-«»dul! f e registrations be supported
^quires
residue tolerance. An
hV a negilgJv force has formally reindustry
to establish these tolerquested FD^ uest for 2.4,5-T has been
ances. (The * qitJ|dfawn without preju-

nporaniy fjJing,)
' ,e to futur*

The author

■Msident *"4 Director of

” .1mmoany.

i residues 1965-68
Number of Semples with Residues of 1548 S am p le « A n a ly z e d
Total

PPM
2,4-D
MCPA
2,4,5-T
Siivex

0.001 - <0.1
23
2
2
2

o .l - < 0.2

4
1
i
0

Kesearch, Qow C hem ic al C om pany,
it

V

i c t - f T c * .- -

I Public
Presented
»(Implications
/tie Sym posi
m
Health
ofuWidgB
*xaibu

of Herbie,a# (Chairman: Dr. Arthur r*.1* u**

ABmlo om
cnctn
institute of Btolopcn
aig ton.
indent. 26 Auput l 9?Q tn c ts '

Duggan. B. E., H. C. Barry, and L. V. Johnson, 1967. Parr*
Martin , ft. J„ end R. E. Duggan. 1968. A»*—
Corneiiuswn, 9. E. 1969. A—

0 -4
O
1

o

0

27
4
3
2

5720

8o

5727

■-U-.

Q & 'A lC A L BIOLOGY RESEARCH
SCdMlTTCO

/

BV

D O W C H E M I C A L U .S.A.

NB T36.25-66681-22
K NUMBER

DATE

CHARGE

.1

FILE

01269-000-026
10/25/71
K-66681
EYE IRRITATION STUDY CONDUCTED ON:
2,3,7, 8 -TETRACHLORODIBENZ0-p-DIOXIN
REPORTED BY

J. M. Norris

checked b y :

P. J . ~Gehring

C t)

IN F O R M A T IV E SUMMARY W ITH C O N C LU S IO iWs
N S BASCO
SED ON
C
T H E S A M P L E R E C E IV E D . A D D IT IO N A L IN F O R M A T IO N INCLC) SING T H E E F F E C T S
R E P E A T E D E X P O S U R E MAY B E R E Q U IR E D AS S P E C IF IC USES A N D F O R M U L A T IO N S A R E D E V E L O P E D OR IF P R O C ESS C H A N G ES O C C U R

A sample of...2, 3,7/8-tetrachlorodibenzo-p-dioxin, identified by #‘340-2-54B,
was submitted to Chemical Biology Research "for evaluation of eye irritation
properties.
Twenty-four hours prior to use, the eyes of an albino rabbit were examined
and established as being without defects or irritation. The test procedure
involved instillation of 2 mg of the test compound into the conjunctival
sac of the left eye. The right eye was used as an untreated control.
The treated eye was examined for conjunctival inflammation, iritis and
corneal injury immediately after instillation, at 1, 24 and 48 hours and
13, 22 and 27 days post-instillation. The behavior of the animal was
observed for indication of pain or discomfort at the time of instillation. *

r»T • n T

The compound caused discomfort on instillation and very slight irritation f
of the conjunctival membranes which persisted for 24 hours. Delayed
(
conjunctival chemosis causing the eye lids to be more than half closed
was observed on day 13 and by day 22 the condition was severe. On day 27
the chemosis had subsided, however, the eye lid rim was thickened and
encrusted.

0004353
DISTRIBUTION
J. E. Jehnton
H. H. M clniyr*
C. E. Kiminel
L . K. F r . v . l
D. D. M cCol!■ •••' (2)

B. E. Burger*
H. L . Gordon, MD
E. H. B lair
A. J . Schwan, MO
S . M. MacCutcheon

D . K ilia n , MU
B. H o rv a th

H . Edward*
R. J . Shavor
V. B. Robinien

B. INolder, MU
L . S ilv o n to in
L . Pftchforth
C. A. Goring

CHI (9)

e*"

ri*

0 (¿

s\

M

8\

• D O W t 478694
BACKGROUND
The herbicide 2.4-dichlorophenoxyacetic
acid (2.4-D) was developed in the midforties and was the forerunner of a group of
phenoxy herbicides that have been instru­
mental in the control of broadleaved weeds
in food crops and undesirable brush species
on industrial rights-of-way. In addition these
products have contributed immeasurably to
beef production by controlling weed and/or
brush on pasture and rangeland, resulting
in increased grass production and corre­
sponding increase in carrying capacity for
livestock.
Besides 2.4-D . 2.4.5-trichlorophenoxyacetic acid (2 .4 .5 -T ). 2 -(2 .4 ,5 -tric h lo ro phenoxy) propionic acid (silvex or2.4.5-TP).
and 2-methyl-4-chlorophenoxy acetic acid
(MCPA) are major phenoxy products of
similar chemical structure, as shown be­
low. but with unique characteristics of
their own with respect to species controlled
and crop selectivity.

CHRONOLOGY OF EVENTS

ci

Cl

Over the years there has been consider­
able improvement in phenoxy herbicides
and their use. Development of new formula­
tions. performance information, crop safety,
timing of application, spray equipment,
toxicology, use hazards, and environmental
implications have contributed to both better
product and specific directions for use.
These herbicides are not protected by
patents, so seven commercial companies
were manufacturing one or more of the four
phenoxies (2.4-D. 2.4.5-T, silvex. MCPA)
at the time when the USDA announcement
to abolish the no residue status was issued
on April 13. 1966. Basic manufacturers in­
cluded: Diamond. Dow. Hercules. Monsanto.
Rhodia (then Chipman). Thompson, and

12.021

Thompson-Hayward. These com:
joined to form the Industry Task Fc
Phenoxy Herbicide Tolerances (IT>
The chronology of major events assc
with this has been as follows:

April 13 , 1966
USDA announcement to abolish nc
due status—industry must comply t
taining tolerances for residues in all t
food and feed products and byprodu
December 31. 1970.

August 23, 1966
Industry Task Force on Phenoxy Her
Tolerances was formed to handle .
2.4.5-T. MCPA and silvex.

December. 1967
Submitted petitions to FDA for toler
of 2.4-D, 2.4,5-T, silvex and MCPA
ing all food crop uses listed at that t
the USDA Summary of Registered Ac
tural Chemical Uses. Extension of rec
tion was requested for uses of these
cides in pasture and rangeland.

April. 1968
Industry Task Force advised of i
quacies. in the petitions plus require
for information on all metabolites
herbicides that might occur as resid
food.

September. 1968
Review of literature on metabolism
mitted to resolve metabolite question
During 1968 programs were estab
to determine on which crops add;
work would be undertaken and what sp
projects would be done by each cor
in the Task Force.

October 29. 1968
A . 2.4-dichlorophenoxyacetic acid
B. 2 .4 .5 - trichlorophenox y acetic acid
C. 2-{2.4.5-tr<ch/orophenoxy)-propionic acid
D. 2-methyl-4-ch/orophenoxyacetic acid

Industry Task Force requested exte
for continued use of 2.4-D, 2.4.5-T, £
and MCPA on pastures and rangelan
tension was granted until January 1.

January 3 1. 1969

The status of 2,4-D ,
2 ,4 ,5 -T , Silvex and
MCPA Herbicides
By C. S. W illiam s
R & 0 Herbicide Tech. Specialist.
The Dow Chem ical Company.
M idland. M ichigan.

_ .«

\ a

Use of 2.4-D. 2.4.5-T and silvex on ac
sites extended to January 1. 1970.
£ ro p samples for residue analyses
collected during summer and fall of
Protocols for milk and meat studies
established during 1969. It was decide
analyses of animal tissues would indue
phenol corresponding to each phenoxy
pound. Dow handled dairy cattle feedir
milk analysis for all four phenoxies
began feeding February, 1970.
USDA was scheduled to handle feec
phenoxies to beef cattle and sheep c
winter. 1969-1970.

n n n > * i4 a m

I
I

L
TABLE I
The EHact «1 R a iiita a t Variety end Crop R oution on W ilt Control and Pirform anct
IM S (P itt Walt« and Vanca Counties)
fle Remise

■

Remise

Veite
«/Acre

WRt<
VMstf

w ar
M u

Valse
•/Acre

1 Mrsase By
Retatioa

as

Cater 8 4

It

1»

M

120

AC. B it

74

«41

a

IMS

»1

l# ti|k t S-B

»

734

a

IM I

B7

Cater 111

n

in

it

M4

1M

<W ilt M a x indicate! extent of root damage 0 - no damage. 100 « maximum disease development

TABLE S
Tho EHoet of R oaiitant Variety and Chemical Soil Traatmam w ith TELONE C an
WIR Control and Parformanca (P in . Walt« and Vanca Counties)
Re Tostasti«

ta sta sti«

«H it'
latex

Wee
(/Acre

Cater 2S4

17

a c . a it

74

le s iti« e-ts
Cater 111

Variety

WRt1
tatet

Was
1/Acts

« tacrsase ly
fe u ifitie e

171

It

1227

a t

147

tt

Ml

ta

a

1M

a

107

to

71

111

n

744

1M

1W ilt Index indicates extent of root damage. 0 » no damage. 1 0 0 - maximum disease development.

]
TABU 10
Tka Effect of Resistant Variety, Crop Rotation and Chemical Soil Treatment w ith
TELONE C on W ilt Control and Performance (P itt Wake and Vance Counties)
W ilt M as1
Variety

te Re Retati«

Wae t/Acra

Retati« A
Fatefatiw

Rs Retati«

'R etati« 1
f «sayni«

tarresta

Cater 2M

17

II

17*

ito

3M

A C .812

74

47

147

1RM

M2

tastata G-I t

33

11

734

1212

471

Cater 311

n

•1

IH

IH

3M

' W ilt Index indicates extent of root damage 0 - no da>‘H > 1 0 0 - maximum disease development.

TABU 11
The Effect of Crop Rotation & Chemicsi Soil Treotment w ith TELONE C en Control of Root Knot
and Stack Shank in Two Resistant Varieties (Columbus, Edgecombe and Jones Counties)
Variety-Cater B k

Variety -S fatata G-H

IIM Cresi

Seat
lu t
M u1

Hack
Stete
latex'

a VW«

Rest
Kata
latex'

Back
Skate
latex'

a Vatas

1244

la ta ! fin is Fame

New

M

a

1137

1

1

Cara

Maas

71

B

IM2

1

1

12M

Can«

Meat

41 f

il

iia

1

1

ia i

leytean

Meat

41

il

11M

1

1

U SI

Fossa

He«

31

14

I1H

1

1

1223

Street Famen

Meat

11

34

11-2'

1

1

UM

Atem« al tal Retati«

Ne«

M

24

iia

1

7

1271

U1

3

7

IB I

1H7

2

t

UM

Tatarts

Nan

11

4*

Tettate

TetaaeC

M

24

' Root Knot and Black Shank Index indicates extent of root damage 0 - no damage. 100 - maximum
riisrias* develnfm icnt

• 'V

alone or continuous tobacco, plus TELONE
C. These results suggest that a complete
control program involving several practices
is needed where resistant varieties (regard­
less of level) that are susceptible to the
southern root-knot nematode are used. They
also suggest that the use of a variety with
high resistance to wilt and root knot might
permit the omission of crop rotation or the
use. of a multi-purpose chemical soil treat­
ment from the control program. Results are
presented in Table 10.
A second series of integrated disease con­
trol tests was established in 1968 at three
locations. These tests were located in Co­
lumbus. Edgecombe and Jones counties in
fields where root-knot nematodes and black
shank were present at low to moderate
levels. Sixteen treatments involving seven
two-year cropping systems were compared
with continuous tobacco culture. Alternate
crops included in the different cropping sys­
tems were corn, cotton, small grain fescue,
soybeans, sweet potatoes and peanuts. Two
disease resistant varieties were used within
all cropping systems including Coker 254
(high resistance to black shank, bacterial
wilt and root knot) and Speight G-36 (high
resistance to black shank and bacterial wilt
but susceptible to root knot). Two soil treat­
ments (one using TELONE C soil fumigant
and one using no soil treatment) were used
in plots where continuous tobacco culture
was practiced. No chemical soil treatment
was used in rotated plots. Results from
these tests provided a comparison of sev­
eral alternate crops a'nd TELONE C in re­
ducing root knot and black shank and their
effects on performance.
Disease development was low in the
Coker 254 variety at all locations. Rotation
with such crops as corn, cotton, peanuts,
small grain fescue, soybeans and sweet
potatoes failed to provide additional con­
trol or increase in performance of this
variety. (Table 11).
The Speight G-36 variety was damaged
severely by both root knot and black shank
in most testis where tobacco was planted
continuously. Black shank root damage was
reduced by all cropping systems and root
knot by all with exception of corn and sweet
potatoes as compared with continuous
tobacco culture. (Table 11).
The use of TELONE C in plots where
tobacco was planted continuously gave
equal disease control and a slightly higher
value increase as compared with most crop­
ping systems in both the Coker 254 and
Speight G-36 varieties. (Table 11).

0007109

5731

1
October 29, 1969
The Office of Science and Technology
issued statement on teratogenic hazard of
2.4.5- T based on work by Bionetics Re­
search Laboratory.

December 22, 1969
Phenoxy registration for rangeland use
extended until January 1, 1971. except for
2.4.5- T. Dow undertook feeding of 2.4.5-T
to beef animals.

December 3 1 , 1969
Petition for tolerances of 2.4.5-T in food
crops was withdrawn by Industry Task Force
since tolerances could not be established by
January 1. 1970. the deadline set by the
Office of Science and Technology.

January 19 , 19 70
Registration of 2.4-D. si Ivex and MCPA
extended by USDA for use on food crops
until January 1. 1971.

March 4. 19 7 0
Registration of 2.4.5-T was also extended
until January 1. 1971 for use on apples,
blueberries, grains, pastures, rangeland,
rice and sugarcane.

April 15 , 19 7 0
Results of additional work on teratogenic
properties of 2.4.5-T prompted suspension
by USDA of 2.4.5-T for aquatic and home
uses.

*

May 1. 19 7 0
Cancellation by USDA of 2.4.5-T for use
on food crops. Uses on pasture, forests and
industrial areas not affected.

May 28, 19 70
Dow appealed the cancellation of 2.4.5-T
for use on rice Hercules and Amchem also
appealed cancellation for rice usage. Each
company could appeal only crops listed on
their labels for 2.4.5-T products. The appeal
is to be reviewed by an Advisory Committee
appointed by the National Academy of
Sciences.

June 16, 19 7 0
USDA began .the beef feeding studies
scheduled to have been done the previous
winter.

November 24, 19 70
Crop residue work completed. Milk anal­
ysis completed. Meat analyses underway.
Completed data will be submitted as amend­
ment to petitions prior to December 31.
1970.
To date, no official notice has been
received concerning appointment of Na­
tional Academy of Science Advisory Com­

m ittee to review the appeal
cancellation of rice.*

on the

REGISTRATION STATUS
Tolerances have been established for
2 .4 - D in applies, barley, grapefruit, lemons,
oats, oranges, pears, rye and wheat. There
is also a tolerance for the sodium salt of
2.4- D in asparagus.
Data originally submitted with the peti­
tions in December. 1967 is expected to be
sufficient for 2.4-D in blueberries, cranber­
ries, grapes and raspberries; for silvex in
apples, pears and prunes; and for MCPA in
peas. Data on residues in grass includes
that from previous work and from 1970
residue samples additionally analyzed by
Dow for support with respect to pasture
and rangeland usage.
The Industry Task Force supported work
for determining residues as shown in Table 1.
For virtually ail of this work, new analytical
methods had to be developed that would
permit analyses down to 0.1 ppm phenoxy
acid in crops and to 0.05 ppm acid or cor­
responding phenol in the animal tissues and
milk.
For present registered uses residue anal­
yses indicate < 0 .2 ppm for all phenoxy
acids in all crops at time of harvest.
The phenoxies were fed at levels of 30.
100 and 300 ppm for two weeks and 1000
ppm for three weeks in the total diet of
dairy cows. Milk was collected and analyzed
for residues. Grazing restrictions compatible
with levels of phenoxies in milk as related
to levels in forage remain to be determined.
There was no evidence of accumulation of
phenoxies in the cream.
At time of this writing. December 1.1970.
analyses are being run on samples of mus­
cle. kidney, liver and fat of beef animals.
Data are expected to be available prior to
December 31. 1970.
Amendments to the petitions for toler­
ances for 2.4-D, silvex and MCPA will be
submitted to the appropriate agency in
charge, including uses in pasture and rangeland. The 2.4.5-T petition will be reactivated
—deadline for this compliance will be ac­
complished prior to December 31, 1970.

TOXICOLOGY

*

Negligible residue tolerances can be
obtained based on information from 90 day
toxicology studies in two species of mam­
mals. However, tolerances at higher (per­
missible) residue levels require two-year
feeding studies on rats and dogs, plus
fertility and reproduction studies on rats.
At the time the phenoxy herbicides were
*Sm upd*M w *nd o* t u t (| V

s

5732
0 0 0 /1 1 0

TABLE t. Cray* and tistM S m nplri lor mid«« «u/yjii { t ) ttn iir th i fTTPHT prsgnm.
0 c
MCPA c r

2.4-0

2.4.5-T

Corn

+

+

Rico

+

+

+

+

F lu

-

-

-

+

Tolerances

—

-

+

Small Grains
(Barley, Oats. Ryo. Wheat)

Silva*

/

cc

Granted

Sorghum

+

- Sugarcam

+

+

+

-

Milk

+

+

+

+

Moat

+

+

+

+

• ........"■

developed these long term feeding studies
were not necessary, since these com­
pounds were registered on a "no-residue"
basis.
FDA has conducted tw o-year feeding
studies on 2.A-D including reproduction
and fertility. Dow has conducted two-year
feeding studies on silvex but not reproduc­
tion or fertility studies.
Ninety-day feeding studies have been run
on rats and dogs for 2.4-D. 2.4.5-T. silvex
and MCPA. The no-ill effect levels are shown
in Table 2.
Based on single oral doses in rats. 2.4-D.
2.4.5-T. silvex and MCPA are classed as
"slightly toxic" with LDso values ranging
from three hundred to seven hundred mg/kg
body weight.

2.3,7,8-

*

TETRACHLORODIBENZO-P-DIOXIN
The word teratology has recently become
much more familiar. It was tied to 2.4.5-T
when studies by Bionetics Research Labora­
tory implied that 2.4.5-T was teratogenic
(producing malformed fetuses) in mice and
rats. Subsequent studies have shown that
a potential toxic contaminant. 2.3.7.8tetrachlorodibenzo-p-dioxin. is responsible
for the findings attributed to 2.4.5-T. The
sample of 2.4.5-T employed in the Bionetics
study contained 27 ppm 2.3.7.8-tetrachlorodibenzo-p-dioxin.
Additional studies have shown that oral
administration of 2.4.5-T containing < 1
ppm 2 .3 .7 .8 - tetrachlorodibenzo - p - dioxin

produced no teratogenic effects on r.
rabbits or mice. The obvious concern is
produce 2.4.5-T without the contamin.
The 2.3.7.8-tetrachiorodibenzo-p-dic
can be formed in the manufacture of
precursor 2.4.5-trichlorophenol. The cor
tions required for its formation are h
temperatures and basic conditions. This <
occur in the alkaline hydrolysis of 1.2.*
tetrachlorobenzene to the trichloropher
No detectable dioxins have been obser
in 2.4-D. This is due to the fact that
precursor 2.4-dichlorophenol is made
direct chlorination of the phenol and not
alkaline hydrolysis of 1,2.4-trichlorob
zene.
To date analytical methods have be
developed and validated for a method s*
sitivity of 0.5 ppm for 2.3.7.8-tetrachlc
dibenzo-p-dioxin in 2.4.5-T acid. With pro:
manufacturing controls there is no probi
in producing 2.4.5-T with no 2.3.7.8-tet
chloro-p-dioxin as indicated by these a;
lytical methods.
THE FUTURE
By December 31. 1970 the Industry T i
Force on Phenoxy Herbicide Tolerances \
have furnished to FDA the supplemer
residue data necessary for the contini
evaluation of the petitions to establish ne<
gible residue tolerances for 2.4-D. 2.4.5
silvex and MCPA on the appropriate fc
crops and meat and milk tissues. Regist
tions (USDA) are expected t<^rerpain

force'

o o o v m

573

’t

,

TABLE 2. R nottx frw i IB -A iy n k a c o tii fa rtin g i t r t i n 4 n tim a p h tn o ir k tfb ic M tt ¡a ra ti aad Baps.

Approximate No-Ill Effect Levels (Mg/Kg/Oay)

i
2.4-D

2.4.5-T

Silvax

»CPA

Bits

30

30

HO

18

Dags

10

5

0-10
.TTCJn

Data provided to date indicate no hazard
and no significant residues in food crops.
Toxicology data support the use claims. The
proper manufacturing of 2.4.5-T will alle­
viate the problems associated with 2.3.
7.8-tetrachlorodibenzo-p-dioxin.
The USOA has recently stated that pro­
hibiting the use of phenoxy herbicides
“would cost the U. S. farmers an additional
$290 million to maintain current agricultural
production. In addition, farmers and their
families would have to work 20 million more
hours to control the weeds without these
herbicides. For this extra labor, the farmers
would obtain no additional income".
Several hundred thousands of dollars
havH been expended over the past several
years to prove the safety of phenoxy herbi­
cides ;o man and his environment. From a
scientific base the phenoxy herbicides can
contribute economically, efficiently, and
safely in the future for the control of broad­
leaved weeds and brush on food crops, pas­
ture. rangeland, and non-cropland areas as
they have for over 20 years.

tio n o n D ecem b er 2 2 , 1 9 7 0 a n d EPA advised
on D ecem b er 3 1 . 1 9 7 0 a p e titio n num ber h a d
been reserved b u t th e y (E P A ) w is h e d to k n o w
w h ic h sp ecific 2 .4 .5 -T fo rm u latio n s w ere to
b e covered b y th e p e titio n a n d fo r in d u stry to
id e n tify

th e

in g red ien ts

(a c tiv e

a n d • in e rt)

c o n ta in e d in th ese products.
(3 ) The EPA has also ad vised th a t a sp ecific
to lerance fo r phen o xies in grass is necessary;
a to lerance in grass h a d n o t been requested b y
IT F P H T as i t d id n o t ap pear necessary. In
co m p lian ce w ith th e EPA re q u e s t a le v e l o f
3 0 0 p p m fo r a ll p h en o xies in grass w as re ­
q u ested on J a n u a ry 6 . 1 9 7 1 b y fTFPHT.
(4 ) O n J a n u a ry 11. 1 9 7 1 D o w w as in fo rm ed
th a t th e ad viso ry c o m m ittee fro m th e N a tio n a l
A cad em y o f S cien ce h a d been ap p o in ted on
N ovem ber 2 . 1 9 7 0 to re v ie w th e fo o d crop
c an cellatio n o f 2 .4 .5 -T .
A t th is w ritin g . D o w a n d H ercules h a d each
b een g iven a n h o u r on F ebruary 1. 1 9 7 1 to
p resen t in fo rm a tio n rela tiv e to th e s a fe ty o f
2 .4 .5 - T as u sed o n fo o d crops.

i SIGNIFICANT CHRONOLOGICAL EVENTS
SINCEPREPARATIONOFMANUSCRIPT
A s o f Jan u ary 2 3 . 1 9 7 1 several s ig n ific a n t
events have occurred.
(1 ) The fT F P H T s u b m itte d am en d m en ts fo r
2 .4 -D . silvex a n d M C P A p e titio n s o n D ecem ber
22.

1970.

5734

T he E n v iro n m e n ta l P ro te c tio n

A gen cy (EPA) ackn o w led g ed filin g o f these
am en d m en ts D ecem b er 3 0 . 1 9 7 0 .
(2 ) The IT F P H T also re file d th e 2 .4 .5 -T p e ti­

0007112

u w u

] *
Ib
\4 -

Sx

5735

8X

Acta pharmacol. et toxicol. 1971,29, 81-86.

O

O'*

From the Department o f M icrobiology, Dental Faculty,
University of Oslo, Norway

^1
*o

The Herbicide'2,4-DichIorophenoxyacetic~Add2
I :/Effects on' L Cells1
By
Jan Kolberg, Kristen H dgdam l, Jon Jonsen and Olav TJeltreit

543831

(Received M ay 19, 1970)

Abstract: The effect o f 2,4-dichlorophenoxyacetic acid (2,4-D ) on L 929 cells in
monolayer cultures has been studied. I t was found th a t^ A D in the range of-50
¿o-500-ug/m i had --a-dosa-dependeat. inhibitory- effect.on cell-growth. With~350
and 500 pg/m l-com plete.inhibition-of growth. occurred.after.about 24 hrs- s t.in ­
tu b a tio n . On removal o f 2,4-D a rapid resumption o f cell m ultiplication took
place. This occurred even after exposure to 500 pg 2,4-D /m l fo r 12 days. In the
presence o f 250 to 500 pg/m l, «ecuoies, evhn.ii siaineU "W ltlrlipid* »oluble-dyes,
appeared- in-the-cytoplasm . On prolonged incubation w ith the herbicide, these
vacuoles disappeared.

.

’

K iy -w o rd s : Herbicide - L cells.

2.4- dichlorophenoxyacetic acid (2,4-D) belongs to the phenoxy group of
herbicides which at low concentrations induces growth responses similar to
the plant hormone auxin. At higher concentrations of these herbicides an
excessive, uncontrolled growth leading to the death of the plant is observed.
This is believed to be based on an abnormal metabolism of RNA ( H a n s o n &
S l if e

i

1969).

2.4- D is probably used to a greater extent than any other herbicide, and
descriptions of its toxicity and hazards to man, domestic animals and wildlife
have been summarized by W a y (1969). The acute toxicity following oral
administration appears to be moderate. The LDS0 values found for a number
of experimental animals range between 300 and 800 mg 2,4-D/kg body
weight. In a recent, yet unpublished, study commissioned by the National
Cancer Institute (U. S. A.) this herbicide was labelled as being a potentially
teratogenic compound needing further study (Science 1969,166, 977 news
and comment).
Since 2,4-D might be a teratogenic agent, studies to elucidate the mecha­
nism of action on mammalian cells is of importance. For this purpose, cell
cultures provide a suitable tool. The only study in this field has recently been
(

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5736

JAN K O L S E R G ET AL.

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made by Li &. Jo r d a n (1969) w h o found a transient growth inhibition in
suspension cultures of L cells exposed to 5 and 10 pg/ml of an ester of 2,4-D.
The present paper describes the effect of different concentrations of 2,4-D
Na-K salt on the growth and morphology of L 929 cells in monolayer cul­
tures.
Materials and Methods
Cell culture techniques.
Monolayers of mouse fibroblasts, strain L 929 (S a n f o r d et al. 1948) were. used. The
cultures were grown in Eagle’s Minimum Essential Medium supplemented with 10 %
calf serum, streptomycin and penicillin. Plastic petri dishes, 60 X 15 mm (Falcon
Plastic Inc.) were seeded with 0.4 X 10** cells suspended in S ml medium and incubated
at 37* it* a humidified atmosphere of COs in air. After incubation for 24 hrs, the
medium was replaced by test media containing 2,4-D. The media were renewed every
third day.
In reversal experiments the 2,4-D containing medium was removed and, before ad­
ding the control medium, the cell layers were washed twice with 2 ml of the latter
medium.
For growth measurements, the cultures were trypsinized and counted in a Biirker
haemocytometer. Each point on the growth curves represents the mean of the counts
from 2 cultures.
Cytochemical analysis of lipids were carried out with Oil Red 0 in 100 % isopropanol
or Sudan Black B in 70 % ethanol and counterstaining with Hams haematoxylin and
neutral red respectively. The cells were not fixed before staining.
Cultures cultivated in Sykes and Moore chambers, volume 0.7 ml (Sykes A M o o r e
1960) were photographed at 2 frames/min. by means of a Reichert inverted phase-con­
trast microscope with a 16 mm Beaulieu camera loaded with Kodachrome II A film.
To reverse the effect of 2,4-D, the chambers were perfused with 23 mi control medium.
H erbicide.

A stock solution containing 25 mg 2,4-D/ml was prepared by dissolving the acid
(Eastman Organic Chemicals) in aqueous NaOH-KOH (0.1 N : 0.1 N) and adjusting
the pH to 7.3 with HO. The solution was sterilized by filtration through a 0.22 p
Millipore^nembrane filter.
Results

On~exposure-of L-929 cellsin monolayer cultures to-2,4-D in concentra­
tions from SO to-500 pg/ml,a-dose dependent inhibitionof growth was-found
(fig. 1). Complete inhibition-was found^after 24- hrs in the-presence of-350
and-500-fig-2,4-D/ml. Cytopathogenic changes such as rounding up and
detachment of the cells were not seen, however, in the treated cultures.
When 2,4-D was removed from the cultures after incubation in the
presence of 500 pg/ml for 3 or 12 days, inhibition of growth ceased (fig. 2).
On removal of 2,4-D after exposure for 3 days there was a rapid increase in
the number of cells concomitant with a disappearance of the vacuoles,
whereas after 12 days there was a lag period of about 24 hrs before cell
*•

0007409
5737

V

2,4-D AND L CELLS

83

inhibition in
es- <f2,4-D.
ntions of 2,4-D
Tionolayer cul-

and, before admi of the latter
ted in a Burker
m of the counts

DOW 1 543833

> were used. The
cnted with 10 %
IS mm (Falcon
m and incubated
for 24 hrs, the
•e renewed every

\\

-l

2

3 .4

TIME (OATS)

S

Fig. 1. The effect of various concentrations of 2,4-D on growth of L 929 cells.

0 % isopropanol
tematoxylin and
irx E S &. M o o r e

-rted phase-conf"«ne II A film,
rol medium.
oiving the acid
) and adjusting
rough a 0.22 ¡i

in concentsath was-found
sence of-350
ding up and
dtures.
ation in thè
ased (fig. 2).
d increase in
he vacuoles,
s before cell

Fig. 2. Resumption o f cell growth after removal o f 2,4-D.
■ -------- ■ untreated cultures.
□ -------- □ cultures containing 500 pg/ml.
• ---------« cultures from which 2,4-D has been removed after
3 or 12 days of treatment.

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multiplication sta
prolonged incuba
tion to this compc
In the presenc.
which stained wit
after about 3 hrs
21 hrs. On furthc
for 4-5 days the
vacuoles were ob
incubation. No a
with 50 and 150
The observatic
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2,4-D/ml no cell
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The cultures wer
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pound. No obvic
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A concentrati
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hrs, and with 3
cells was found
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tions which mit
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a synchronizing
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0007411

5739

2,4-D AND L CELLS

85

I

Discussion

~
fast microscope.
30 hr*.

A concentration dependent inhibition of growth was found, when monolayers of L cells were exposed to 2,4-D. The inhibition was evident after 24
hrs, and with 350 and 500 jig 2,4-D/ml no net increase in the numbers of
cells was found during the next 5 days of incubation. Some growth occurred,
however, during the first 24 hrs in the presence of these two high concentra­
tions which might indicate that 2,4-D was selectively toxic to a particular
stage in the cell cycle. As this inhibitory effect of 2,4-D was readily reversible,
a synchronizing effect would be expected if such a mechanism was operating.
When cultures treated with 500 pg 2,4-D/ml for 33 hrs were transferred to
control medium and examined with time-lapse cinemicrography, no burst of
synchronized cell division was seen. This makes the above explanation un­
likely.
The rapid resumption of cell growth on removal of 2,4-D after treatment
for 3 and 12 days seems to exclude a state of unbalanced growth. This condi­
tion usually results in cell death on prolonged exposure to the inhibitor
(Mueller 1969).

During a period of 21 days, the L cells exhibited an adaption to growth in
the presence of 500 pg/ml. This may be due to a selection of resistent cells
and/or a prolongation of the generation time.

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DOW 1 543835

multiplication started. This experiment also shows that the cultures on
prolonged incubation in the presence of 500 ng 2,4-D/ml exhibited an adap­
tion to this compound and some growth occurred.
In the presence of 350 and 500 ng/ml, vacuoles of uniform size (fig. 3),
which stained with Oil Red O and Sudan Black B, appeared in the cytoplasm
after about 3 hrs of incubation, and their number increased during the next
21 hrs. On further incubation, the vacuoles disappeared, and after treatment
for 4-5 days the cells were free from lipid-containing particles. At 250 ng/ml,
vacuoles were observed after 24 hrs, but they disappeared within 2-3 days of
incubation. No accumulation of vacuoles was found in the cultures treated
with 50 and 150 pg/ml.
The observations made in the previous experiments were confirmed by
time-lapse cinemicrography. During 33 hrs in the presence of 500 ng
2,4-D/ml no cell division was seen, and vacuoles were formed. When 2,4-D
was removed a rapid resumption of cell growth took place, and the vacuoles
disappeared within 22 hrs without being extruded intact into the medium.
The cultures were followed for 32 hrs after removal of 2,4-D and there was
no indication of a synchronizing effect from the treatment with this com­
pound. No obvious effects of 2,4-D on pinocytosis and the undulating activity
of the cell membranes were found.

►

*

t

<w

86

It has previously been shown that changes in the extracellular environment
cause accumulation of lipid-containing particles in cultured mammalian cells.
These changes include a decrease in the extracellular pH, the addition to the
medium of phenol or aliphatic polyalcohols, the replacement of horse serum
by rabbit or human serum (M ackenzie et al. 1967), and the addition of an
unsaturated fatty acid such as oleate, linoleate and linolenate (Moskowitz
1967). These vacuoles, like those induced by 2,4-D, stained with lipid soluble
dyes and exhibited little or no tendency to fuse. However, these investigators
found no growth inhibition with lipogenic factors in the medium, except for
some growth retardation caused by the polyalcohols (M ackenzie et al. 1968).
On continued incubation with 2,4-D, the vacuoles disappeared. Moskowrrz (1967) observed a similar phenomenon in oleate-treated L cells. He
suggested that the particles-might have been extruded intact into the medium
by a secretory mechanism or, alternatively, that an intracytoplasmic hydro­
lysis might account for the breakdown and removal of the lipid particles. In
favour of the hydrolytic mechanism, he demonstrated an increased lipase
activity in steatotic cells. Whether a similar mechanism operates in our cells
has not been investigated.
We found a transient lipid accumulation in 2,4-D treated cells and a
release of growth after changing to the control medium. These findings sug­
gest that the occurrence of lipid vacuoles in cell cultures is not necessarily
indicative of degeneration.

DOW 1 543836

.

JAN KOLBERG ET AL.

REFERENCES
Hanson, J. B. St F. W. Slife: Role of RNA metabolism in the -action of auxin-herbicides.
Residue Rev. 1969, 25, 59-67.
Li, M. F. ¿t C Jordan: Use of spinner culture cells to detect water pollution. J. Fish.
Res. Bd. Canada 1969, 26, 1378-1382.
Mackenzie, C G., J. B. Mackenzie St O. K. Reiss: In: L ip id metabolism in tissue culture
ceils. Ed.: G. H. Rothblat <k D. Kritchevsky. The Wistar Symposium Monograph
No 6. Wistar Inst Press, Philadelphia, Pennsylvania 1967, pp. 63-81.
Mackenzie, J. B., C. G. Mackenzie St O. K. Reiss: Regulation of cell lipid metabolism
and accumulation VII. Increase by glycerol of the polar lipid and triglyceride
content of cultured ceils. Proe. Soc. E xp tl. B io l. M ed. 1968, 128, 42-46.
Moskowitz, J' S.: In: L ip id metabolism in tissue culture cells. Ed.: G. H. Rothblat St
D. Kritc .-sky. The Wistar Symposium Monograph No. 6. Wistar Inst Press,
Philadelphia, Pennsylvania 1967, pp. 49-59.
Mueller, G. C : Biochemical events in the animal cell cycle. Fed. Proc. 1969, 28,17801789.
Sanford, K. 1C, W. R. Earle St G. D. Likely: The growth in v itro of single isolated
tissue cells. J. N a t. Cancer Inst. 1948, 9,229-246.
Sykes, J. A. St E. B. Moore: A Simple tissue chamber. Texas Rep. B io l. M ed. 1960,18,
288-297.
Way, J. M.: Toxicity and hazards to man, domestic animals, and wildlife from some
commonly used auxin herbicides. Residue Rev. 1969,26,37-62.

V

000741a
f
1

J
*

5741

83

5742

83

THE

DOW

CHEMICAL

COMPANY

M IDLAND
F e b r u a r y 19, 1971

€
DOW 049272

H . L . G o rd o n , M. D.
C . W. H in m an
J . E . Jo h n so n
C . G . K r a m e r , M. D.
V . K . R ow e -

E n c lo s e d is a s u g g e s te d p r o to c o l fo r the c o n su m p tio n of 2 ,4 - D and •
2 ,4 , 5 -T by J . E . Jo h n so n and C . W. H in m a n . T h e p r o to c o l fo r the
p ro p o s e d s tu d y " A b s o rp tio n and E x c re tio n of 2 ,4 - D an d 2 ,4 , 5 -T "
to be c o n d u c te d in In d ia is a ls o in c lu d e d . T he l a t t e r p r o to c o l in c lu d e s
b a c k g ro u n d to x ic o lo g ic a l in fo rm a tio n , m e ta b o lis m d a ta , an d s a m p le
s p e c if ic a tio n s . A ll of the in fo rm a tio n sh o u ld be s tu d ie d p r i o r to c o n ­
d u c tin g th is s tu d y .
S in ce I h a v e th e s a m p le s of 2 ,4 - D and 2 , 4 , 5 - T , th e s tu d y c a n be
in itia te d w h e n e v e r f e a s ib le . I s u g g e s t th a t th e c h o s e n g u in e a p ig s
and w itn e s s e s c o m e to 1803 B u ild in g w h e re th e m a t e r i a l s w ill be
w e ig h e d o u t a n d c o n s u m e d .

r-. j . u e n r in g , D .V .M . , Ph-. D .
C h e m ic a l B io lo g y R e s e a r c h
1803 B uilding
6 -1 0 8 9

,

A tta c h m e n t
CONFIDENTIAL - SUBJECT TO INJUNCTION
D .C , E D . M l. 4 -4 -7 8 ; D O W /E P A AGREEMENT 9 -7 9

0000795
5743

(K
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oo

PROTOCOL:

HUMAN TOLERANCE OP 2,4-D AND 2,4, 5-Tv..',;

OBJECTIVE:

To establish that the doses of 2,4-D and 2,4,5-T
recommended for use in the study entitled "Absorp­
tion and Excretion of 2,4-D and 2,4,5-T In Man"
are safe.
v
J. E. Johnson- Director of Corporate Research and
Development

C. W. Hinman- Director of Chemical Biology Research
COMPOUNDS :

f

Samples of 2,4-D (Sample AGR30653C) and 2,4,5-T‘ (Sample AGR86187).
These samples sure analytical standards. The same materials are
being used in the study "Absorption and Excretion of 2,4-D and
2.4.5- T In Man".

DOW 049273

SUBJECT:

vÿ

PROCEDURE :
A copy of the proposed study "Absorption and Excretion of 2,4-D
and 2,4,5-T In Man" is attached. In the study proposed here,
J. E. Johnson is to consume 81 mg of 2,4,5-T (1 mg/kg) and
C. W. Hinman 79 mg 2,4-D (1 mg/kg). One week following this
treatment, J. E. Johnson is to consume 405 mg (5 mg/kg) of
2.4.5- T and C. W. Hinman 395 mg (5 mg/kg) of 2,4-D. For ad­
ministration, the compounds are to be mixed in a glass of milk.
The milk is to be consumed in the presence of witnesses and a
sworn affidavit obtained.
MEDICAL SURVEILLANCE:
.The information provided in the accompanying protocol "Absorp­
tion and Excretion of 2,4-D and 2,4,5-T In Mam" should be
studied by Doctors Harold Gordon and Charles Kramer.
<<

Prior to administration of the agents, it is to be ascertained
whether there are any medical reasons for not using the chosen
subjects. For a minimum of 24 hours following administration,
a physician should be readily available. Physical examination
prior to and following administration of the agents will be per­
formed at the discretion of the physician involved.
ANALYTICAL WORK:
It is suggested that a 10 ml sample of blood be obtained at
2, 7, 12, 24 and 48 hours following administration of the 5 mg/k
dose. These samples should be analyzed for 2,4-D and 2,4,5-T.
It is requested that all urine voided during the first and second
24 hour periods following administration be collected. A sample of
these composite samples is to be analyzed for the agent in question.
574§000706

I

ABSORPTION AND EXCRETION OF 2,4-D AND OF 2,4,5-T IN MAN

SUMMARY OF TOXICOLOGY LITERATURE
There are a number of reports in the literature in which the acute
and chronic oral toxicity of 2,4-D and 2,4,5-T were determined
Cor a variety of animal species.
In 1947, Hill and Carlisle
reported the acute oral toxicity of 2,4-D in mice, rats, rabbits;
guinea pigs and monkeys.
Of these species the monkey was most
sensitive showing no adverse effects at 214 mg/kg dosages but
definite signs of toxicity at 428 mg/kg.
The toxic signs ob­
served were nausea and vomiting, stiffness of the legs with some
muscular incoordination, lethargy, hanging of the head and ptosis
ol c.ht? eyelids.
Ln 1948, Bicrn and N'orthen reported the acute oral lethal dose
of 2,4-!': for chickens to be between 380-765 mg/kg.
In 1953,
Drill, and Fiiratzka reported on studies with dogs indicating the
acute oral LL\, value for 2,4-D to be about 100 mg/kg.
When
2, j o r ’.f; mg/kgfduy of 2,4-D was administered 5 times/week for
90 days, no adverse effects were noted; however, 20 mg/kg caused
the death of j of 4 dogs on days 18, 25, and 49 of the test.
The toxic signs which w e r e 'observed varied from mild ataxia and
svi.ffrt.-ss of the hind logs to a definite myotonia, weakness, dif­
ficulty ir. swa 1 Lowi:k ;, anorexia and occasional bleeding from the
g ur s .
r: 1: 4, ¡-nvo.and Hymns reported the acute oral toxicity of 2,4-D
to rats, mice, i u l u -m plus nr.d chickens.
The acute oral LD^
•m f:es .¡¡id the;r c,r; confidence limits were as follows:
rats,
■:•
ke • - >2-4v ': - :i.i ?e, >f.8 mg/kg (312-434); guinea pigs,
•
::u k ■
■> i; and chicks, 54 L mg/kg (358-817).
The
•o x .ci ty ;:.cu':t.d v i :epeated oral doses of 2,4-D in rats and

5746
0000797

DOVP049274

The extensive use of 2,4-dichlorophenoxyacetic acid (2,4-D) and
2 , 4 , 5 - trichîorophenoxyacetic acid (2,4,5-T) as selective herbi­
cides ir. many areas of the world has increased the possibility
of exposure of animals and ma n to these materials.
This has
created the need for creater knowledge and understanding of their
fate after ingestion by man, as well as other animals.
Such
understanding is of great value in using data obtained from
animal experimentation to assess safety for man.

chicks was also reported.
Rats fed 2,4-D five times a week for
4 weeks at levels of 3, 10, and 30 mg/kg/day showed no adverse
effects of the treatment, whereas rats receiving levels of
100 mg/kg showed varying degrees of gastrointestinal irritation,
slight cloudy swelling in the liver, and depressed growth rate.
Animals receiving 300 mg/kg failed rapidly and died with severe
gastrointestinal irritation being the principal effect observed.
In L963, Palmer described the effects observed in cattle treated
with varying amounts of 2,4-D five times/week for varying periods
of time.
A steer receiving 112 daily oral doses of 50 mg/kg
showed no adverse effects.
Another steer receiving 100 mg/kg/day
developed a digestive disorder characterized by extreme tympany
after 86 treatments.
Another steer given 200 mg/kg/day developed
muscular weakness in the hindquarters and a staggering gait after
34 days.
A steer given 250 mg/kg/day developed moderate tympany
and meiena which was first noticed after 15 days on treatment.
A limited number of reports have appeared in the literature
dealing with unusual exposure of humans to 2,4-D.
In 1959,
Goldstein, Jones and Brown reported 3 cases of exposures to an
unspecified ester of 2,4-D.
Severe sensory and motor symptoms
necessitated hospitalization in each case.
The disorders began
some hours after the arms and/or legs of the patients had b e ­
come wetted with unknown amounts of the 2,4-D preparation being
used to kill weeds.
The symptoms progressed for a period of
days until pain, paraesthesia and paralysis were severe.
D is­
ability was protracted and recovery was incomplete even after a
lapse of years.
Electromyographic examination supported the
*. diagnosis- of. -peripheral n e u r o p a t h y » . In 1962«. Todd, described a
patient with peripheral neuritis lasting almost two years
which was presumably caused by the same agent.
In 1963, Berkley
and Magee reported the development of a neuropathy in a farmer
following exposure to the dimethylamine salt of 2,4-D.
In 1'h 5, Neilsen, Kaempe and Jensen-Ilolm reported the only known
fatal case or j ,4-D poisoning.
The suicidal patient had in■:ested approximately )'-'0 mq/ka o f 2,4-D b u t the actual lethal
Jo so could not
estanl ished.
I:i 1 *70, Berwick >_oporr.ori o:i a farmer who accidentally inrod about -D . 1. of a concentrated weedkiller formulation
c o - ’...lining
.
. . 4 - b i sooctv lester , 4 9 % S - e t h y l d i p r o p y l t h i o carb.in.ar.'- (ig.’ar , •)' .--.erosone, 0.5'i epichlorohydrin and 5%

0000703

( b . z f i t ’O M o a

- 2 -

■
f.

■- . a«»-»
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-3-

DOW 049276

emulsifiers. The amount of 2,4-D ingested was calculated to
be 110 mg/kg as the free acid. In addition, 230 mg/kg of Sethyldipropylthiocarbamate, and 2.3 mg/kg of epichlorohydrin
must have been ingested. The patient was hospitalized and his
progress through the acute stages and subsequent recovery were
closely monitored. Symptoms associated with intoxication in­
cluded nausea and acute gastritis, breathing difficulties,
,
skeletal muscle injury and weakness. The levels of IDH, SOOT,
SGPT, aldolase and creatine phosphokinase in the serum were
elevated. No symptoms of peripheral neuropathy were detected.
The patient was discharged from the hospital after 2 weeks.
Laboratory studies one month after ingeàtion of. the material
showed normal values for the serum enzyme levels, the complete '
blood cell count and the urinalysis.

The most meaningful data relative to establishing, the safety
of the doses of 2,4-D suggested foruse in the present study were
reported by Seabury in 1963. He reported on the administration
of 2,4-D as a treatment for two terminal patients with dis­
seminated coccidioidomycosis. One patient received 12.712 g
of the sodium salt of 2,4-D during a period of 34 days without ,
observable toxicity. The drug was administered both intra­
muscularly and intravenously. On the 34th day, 2 gm were given
intravenously without detectable untoward effects. On the
36th day, a dose of 3.600 g of sodium 2,4-D was given intravenously
over a period of 2 hours. Untoward effects associated with the
administration of the latter dose were stupor, hyporeflexia,
urinary incontinence and muscular weakness. Within 48 hours
after treatment the patient had returned to his prereaction
status and no further evidence of neurologic or muscular change
appeared in the subsequent two weeks of his life.
Another study in which thè dose of 2,4-D was accurately quantitaed was that of Assouly (1951). He ingested 500 mg/day for
3 weeks without experiencing adverse effects.
The survey of the li tara tiare revealed only limited information
on the toxicity of 2,4,5-T to animals. No cases of unusual
human exposures have been reported. In 1953, Drill and Hiratzka
reported the acute oral LD^ of 2,4,5-T in dogs to be in the
range of 100 mg/kg or higher. No marked toxic effects were noted
but there was some wieght loss, anorexia, ataxia and slight to
moderate stiffness in the hind legs in dogs receiving higher
doses. Dogs receiving 2, 5 or 10 mg/kg of 2,4,5-T five days a

0003739

In 1954, Rowe and Hymas reported the acute oral toxicity of
2,4,5-T in rats, mice,guinea pigs and chickens. The acute
oral ID values and the 95% confidence limits were 500 mg/kg
(391-640), 389 m g A g (245-619), 381 m g A g (307-472) and 310
m g A g (211-456), respectively. The toxic signs at the higher
levels of treatment were anorexia, weight lose, depression,
rough hair coat and muscular weakness.

Recently, a S’tudy of the effects of 2,4,5-T consumption for
90 days has been conducted using male and female rats. (Study
conducted during 1970 by The Dow Chemical Company, Midland,
Michigan and as yet unpublished.) In this study, 2,4,5-T was
included in the diet such that daily doses of 100, 30, 10 and
3 mgAg/day were given to groups of male and female rats, 10
animals/group. Parameters evluated were appearance, behavior,
growth, food intake, hematology, urinalysis during the 12th
week of the experiment, terminal determination of blood urea
nitrogen (BON), serum alkaline phosphatase and SGPT, terminal
organ weights and gross pathological and histopathological exa­
mination of the tissues. There was 100% survival in all groups.
At the 100 mgAg/day level the following signs of toxicity were
observed: depressed body weight, depressed food intake, a
two-fold increase in serum alkaline phosphatase, a 20% increase
--in SGPT (observed: only in males), inconsistent paleness and
accentuated lobular patterns in the liver of some rats, and
cloudy swelling of the hepatocytes. At the 30 mgAg/day level,
the only changes associated with treatment were increased liver
and kidney weights in males and a slight elevation of serum
alkaline phosphatase and SGPT in the females. No changes were
detected in rats given 10 or 3 mgAg/day.
Studies designed to reveal whether 2,4,5-T is teratogenic have
been conducted by The Dow Chemical Company, Zionsville,
Indiana (In Press.) In these studies, rats were given 1, 3,
6, 12 or 24 mgAg/day 2,4,5-T as a 0.25% suspension in
METHOCEL" by oral gavage on days 6 through 15 of gestation and
rabbits were given 10, 20 or 40 m g A g / d a y orally in gelatin
capsules on days 6 through 18 of gestation. At the doses indi­
cated, no untoward effects were detected in either the mothers
or the fetuses.

5749

0000200

Do w O-1927?

week for 90 days showed no adverse effects. The daily admini­
stration of 20 m g A g produced death in 4 dogs after 11, 49,
59 and 75 days of treatment. In the dogs that died, toxic signs
were weakness, stiffness of hind legs, difficulty in swallowing
food and bleeding from the gums.

-5-

PHARMACOLOGY OF 2,4-D AND 2,4,5-T
Absorption and Distribution

DOW 0 4 9 2 7 8

Orally administered, 2,4-D or 2,4,5-T is rapidly absorbed from
the stomach and distributed to all tissues.
The compounds have
been detected in blood and organs as early as 1 hour after
dosing, with peak levels occurring between 4 and 8 hours after
administration in several mammalian species (Khanna and Fang,
1966; Shafik et al, 1971; Erne, 1966; Courtney, 1970).
The
greatest concentrations occur in plasma, liver, kidney and
lung.
Metabolism and Excretion

2,4-D and 2,4,5-T have been shown to b e eliminated completely
and intact in urine of cattle (Lisk et al, 1963; Bache et al,
1964; St. John et al, 1964) and sheep (Clark et al, 1964).
Courtney (1970) reported that greater than 90% of an administered
dose of 2,4,5-T to rats was recovered unchanged within 72 hours
after treatment.
She suggested that 2, 4 , 5-T was apparently not
metabolized to any extent in the rat.
However, Shafik et al
(1971) found that treatment of rats wi t h 50 mg/kg 2,4,5-T re­
sulted in the excretion of at least three metabolites in
addition to the parent compound.
In the same study, rats
dosed with 2,4-D excreted the m ajor portion of the administered
herbicide in the urine.
No metabolites of 2,4-D were detected.
Khanna and Fane (1966) administered l-C**-2,4-D to rats and
found no C1' in expired air during a 3-day period following
dosing.
In the latter study counter-current separation of urine
and tissue extracts revealed a very small quantity of an un­
identified m e t a b o l i t e ( s ) (0.25% of total radioactivity in urine).

I

The excretion rate of 2,4-D and 2,4,5-T appears to b e somewhat
dependent upon the dose administered.
High dose levels (>250
m q / k q ) are excreted at a slower rate than low dose levels
(Khurinn and Fang, 1966; Shafik, et al, 1971).
The plasma
ha if-Life values are about 3 hours for rats, 8 hours for calves
and chickens and 12 hours for pigs (Erne, 1966).
The major
excretory route is via the kidneys in all m a m m a l i a n species
studies.
Following administration o f a tracer dose of C1+-2,4-D
to sheep, 96';' of the activity was excreted in the urine and 1.4%
Lr. the feces in 72 hours (Clark et al, 1964).
Only low levels
of 2,4-D were found in feces of rats and pigs and in bile; of
pigs g Lven 2,4-D orally (Erne, 1966).
Renal excretion of

5750
0000801

phenoxyacetic acids has been reported to be the predominant
mode of excretion in cattle and sheep (Lisk et al* 1963j
Bâche et al, 1964 and St. John et al, 1964). Shaflk et a)L
(1971) found that in rats, orally administered 2 , 4 -0 and 2 , 4 , 5-T
were almost completely excreted in the urine within 2 days.
Khanna and Fang (1966) reported 94 to 99% of 2,4-D is excreted
in urine and feces of rats within 48 hours following the admini­
stration' of 1-10 mg 2,4-D per rat. Of this, 93 to 96% was ex­
creted in the first 24 hours; almost all was excreted in the
urine.

m o n

-6 -

EVALUATION OF THE SAFETY OF THE PROPOSED STUDY ON MAN
Acute and chronic toxicological studies on various animal species
(absorption, distribution and excretion) indicate that there is
no significant difference in either the toxicity or metabolism
of 2,4-D and 2,4,5-T. In the most susceptible animal, the dog,
the LDm for a single dose of either 2,4-D or 2,4,5-T is at
least 100 mg/kg. In this specie, a dose of 10 mg/kg/day of
either compound is without adverse effect when given 5 times a
week for 3 months. Data from various studies on other species show
all of them to be less sensitive than the dog.
Reports of accidental ingestion and therapeutic administration
of 2,4-D to man strongly suggest that man is not uniquely sensi­
tive to 2,4-D in comparison to other species. Based on the
similarity in biological effects of 2,4-D and 2,4,5-T in animals*,
it seems reasonable to expect 2,4,5-T to be similar to 2,4-D in
.its effect on. man.
Thus, based on those experiences in which the ingestion of 2,4-D
by man was quantitated arid on the biological similarity of 2,4-D
and 2,4,5-T in animals, a single oral dose of 5 mg/kg of either
2.4- D or 2,4,5-T is believed to be safe for man.
PRE-EXPERIMENTAL SAFETY CHECK

Although the available data suggest that a dose of 5 mg/kg 2,42,4,5-T will not cause any untoward effects in man, it
would be prudent to establish the subjective tolerance of this
dose before conducting the experiment. Therefore, it is recom­
mended that two test subjects per material be given 1 mg/kg
2.4- d and 2,4,5-T. When it is established that this dose does
not produce untoward effects, the dose should be sequentially in­
creased t o 3 mg kg and then to 5 mg/kg. If the experimentalists
believe tr.nc selected clinical studies such as those proposed
D and

5751 0 0 0 0 8 0 2

-7 -

EXPERIMENTAL DESIGN
1.

Subjects: Five male volunteers between the ages of 20 and
40 years will be used for each compound. Prior to exposure,
the individual will be examined and judged to be of normal
health and nutritional status and should not be on medica­
tion. Clinical parameters to be assessed at this time and
during the experiment are indicated below.

2.

Samples of 2,4-D (Sample AGR30653C) and 2,4,5-T (Sample
AGR86187). The samples of these material will be analytical
standards supplied by Dow. Specifications fb r these samples
are included.
Administration: 2,4-D and 2,4,5-T will be given in a single
dose of 5 mg/kg in a gelatin capsule. The test material is
to be given one-half hour after a standardized light breakfast. Further meals for the first 24 hours should also be
standardized.

4.

Sampling Schedule
a. Blood: Blood samples should be obtained as indicated
below.
2,4-D or 2,4,5-T Time
Analysis
0 (Pretreatment)
20 ml
2 Hours post-treatment
10
H
10
5
II
10
8
II
10
12
II
10
16
•1
20
24
II
20
48
II
20
168

Clinical
Chemistry
30 ml
-

20
20
20

Thu blood samp Les for analysis of 2,4-D and 2,4,5-T are
to be mixed w i t h 2 mg potassium oxalate per ml of blood
and cciitr.fuued. Plasma is to be collected for compound
analysis. Duplicate analysis should be made if possible.

5752

0 0 0 0 3 C3

082610 M O Q

(Paragraph 6 of Experimental Design) are desirable, they should,
of course, conduct them. The test subjects used for this study
should not be used in the absorption and excretion experiment.

> \

-8-

The blood samples for clinical chemistry should be col­
lected without anticoagulant. The clinical chemistry
determinations will be made on serum.
b.

Urine: A pretreatment 24-hour urine sample is to be
collected as a control. Following treatment, all urine
is to be collected. During the first 24 hours, the
volume of the urine voided and the time following treat­
ment at which it was voided should be recorded. A 50 ml
aliquot of each urine sample is to be taken for compound
analysis. Clinical chemical evaluations are to be con­
ducted on another aliquot. After the first 24 hours, a
composite urine sample for each subsequent 24-hour period
is to be collected and an aliquot, 100-200 ml, retained
for compound analysis.

c.

Feces: All feces voided on each of t h e 7 days following
treatment should be collected. The time collected and
the weight of each sample is to be determined. A repre­
sentative aliquot of the feces, 100 to 200 gm, is to be
retained for analysis.

(All samples - plasma, urine, feces - are to be refrigerated
as soon as possible after collection and prior to analysis.
The analytical procedures to be used will be submitted with
the protocol.)
Analysis: The procedures for the analysis of 2,4-D and 2,4,5T are included. Prior to attempting analysis of specimens
obtained ..from .test subjects, known amounts of 2,4-D and 2,4,5T should be added to plasma, feces and urine and'the percent
recovery should be established. Analysis of samples obtained
from tlie test subjects should not be conducted until a re­
covery of greater than 90% has been established. This is
necessary to provide a basis for the evaluation of the vali­
dity of the analytical methods in the hands of the experimen­
talist.
The evaluation of results from the first test subject for
each compound si.ould be completed before using subsequent
test subjects to confirm that the dose was appropriate and
the sampling times were appropriate. Adjustments can be
¡-ado as tie-.ano-d necessary.

5753

000030,1

in addition to the clinical evaluation prior to treatment,
it. should be repeated 12, 48 and 168 hours following the
••¿ministration of the test compound.
Expected beta:
Examples of the character of the data to
be obtained from these experiments are shown in the attached
hypothetic ii. figures.
Once the data have been obtained,
v.e ha-’c a computer program which will allow us to determine
tr.e r-ite oj absorption, the rate of disappearance and the
vciume of distribution for each experimental subject.
These
■alu'.-s will Then be subjected to a statistical evaluation
to determine the mean and standard deviation.

5754

0000805

vd

If currently feasible, the clinical tests should include the
following:
electrocardiogram, electroencephalogram, electromyogram, urinalysis (color, transparency, specific gravity,
pi: and concentrations of protein, glucose, acetone, m yo­
globin, biood, bile, as well as microscopic examination of
the sediment , SGPT, SGOT, serum LDH and component Isozymes,
serum aldolase and serum creatine phosphokinase.
Renal
function is to be evaluated by determining the clearance of
.'’Ah and creatinine.
Hematological evaluation is to include
■. complete blood count and hemoglobin determination.

0

Physical and Clinical Evaluation of Subjects:
In addition
to obtaining a history, a thorough physical, examination,
includi;:-: a neurological evaluation, will be conducted on
each subject.
Any evidence of abnormal nutrition, gastro­
intestinal, urinary, cardiac, muscular, or neurological
conditions will exempt the individual from use in this study.

7

6.

Fin aa o n

-9-

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5755

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c o m p a n y

MIOLAKO MtCl'tOAN
ANALYTICAL LABORATORIES
REPORT

19G9

(

CHARCE

PR08LEM _5602322.

AL kumse* ___ .7r23-2_____
TOUS KUMP CR___

.12 G l-02 6,

FULL DESCRIPTION OF ANALYTICAL STANDARD 2,4-D
_______(2 ,4 -DICHLOROPHENQXYACETIC ACID)________

OEJCR'PTlO:».

Analytical standard 2,4-D, AGR 30653C, was found to be 99.7%
pure. Identity was confirmed by data obtained by elemental analysis,
mass spectrometry, and nuclear magnetic resonance spectrometry.
1. Specifications for analytical standard 2,4-D: (Batches not
meeting this specification will be reworked until they do or will be dis­
carded).

2,4-D
Volatile Impurities (100°C)
Impurities determined by Differential
Scanning Calorimetry

£826M}iV\Oà

oate__October 13,

% Minimum % Maximum
99.0
100.0
0.0
0.5
0.0

2. Identity confirmed by:
(a) Elemental Analysis (+0.3%):
Carbon
43.2
Hydrogen
2.45
Chlorine
31.8
• - (b) Mass Spectrometry
(c) Proton Nuclear Magnetic Resonance Spectrometry

1.0

43.8
3.05
32.4

3. Purity of the analytical standard will be determined as follows:
(a) The purity of the analytical standard will be calculated
as follows:
% 2,4-D = 100.0 - A B
Where A - % volatile impurities
B = % impurities determined by differential
scanning calorimetry
(b) Determine volatile impurities by the following procedure:

ine. 1

nm 23

ocr

1-1

A!':••• o v ' ; o ;::

AR 823

sieves

R. Hummel
PmO.sE M E* __3543____ BLOC.1- .574_______

5757

000CSG3

Alj l-3o<i

-2 -

Weigh one gram of the standard into a dish and place the
dish in a 100°C oven for 30 minutes. Cool the dish in a desiccator and
reweigh. Calculate the percent volatile impurities from the loss in
weight.
(c)
Determine impurities in the dried analytical standard
by analysis of the melting curve obtained with a Perkin-Elmer Differential
Scanning Calorimeter:
(1) Accurately weigh three mg of the dried analytical
standard into a sample pan and obtain its melting curve by heating in the
calorimeter through a range of 405 to 420°K using a chart range of
2 m e a l./s e c ., a heating rate of 0. 625°C/min. and a chart speed of one
inch per minute.
(2) Calculate the mole percent impurities in the sample
from the melting curve following the directions given in Anal. Chem. 41,
330 (1969).
4.
The identity of the analytical standard will be confirmed by
the following procedures:
a. Elemental Analysis:
(1) Determine carbon and hydrogen by "Rapid Multiple
Microdetermination of Carbon and Hydrogen", Mikrochimica Acta 1964,
631, or equivalent.
(2) Determine chlorine by method MLS. 66.1 (semi­
micro oxygen flask combustion, potentiometric titration) or equivalent.
b. Mass Spectrometry:
The mass spectrum obtained with a suitable spectro­
meter should show:
(1) A parent ion at mass 220 with isotope peaks
indicating the presence of two chlorine atoms.
(2) A fragment ion at mass 175 (loss of -COOH).
(3) Fragment ions at mass 162 and 161 (loss of -CHCOOH
and -CH2COOII).
• (4) A fragment ion at mass 145 (loss of -OCHaCOOH).
c. Proton Nuclear Magnetic Resonance Spectrometry:
The 60 MHz proton spectrum obtained by scanning
a 20^ solution of the analytical standard in hexadeuterated acetone with
tctramethylsilane as internal standard should show:
(1) A singlet at -4.82 ppm (methylene protons in -OCH2-).
(2) A doublet at -7.04 ppm, J = 8.8 Hz, a doublet,
J = 8. 8 Hz, of doublets, J = 2.5 Hz, at -7.26 ppm and a doublet at -7.42
ppm, J =2. 5 Hz. (Chemical shifts and coupling constants characteristic
of a 2 ,4-disubstiluted phenoxy ring).
(3) A singlet at -9.15 ppm due to an exchangable proton,
(carboxylic acid).
(4) Integrals of the ratio: 2 mclhoxy protons: 3 aromatic
pro tons: one acid proton.
*

5758

0000.809

98'36l?CW\oa

O c t o b e r 13, 19UU

O c to be r 13, 1969

-3-

AL 7-932

5.
Synthesis, purity and identity of the 2,4-D standard now in
use (July 17, 1969). Purity and identity established by methods in sections
3 and 4 respectively.
a. Synthesis and purification of analytical standard 2,4-D,
AGR 30653 C:
Technical grade 2 ,4-dichlorophenoxyacetic acid was
purified by dissolving in sodium hydroxide solution, precipitating with
acid, wasliing with water and drying.
b. Tests for purity of analytical standard 2,4-D, AGR 30653C:
2,4-D
99.7%
Volatile Impurities
0.20%
Impurities determined by Differential
Scanning Calorimetry
0.06%
c. Tests for identity of analytical standard 2,4-D, AGR 30653C:
(1) Elemental Analysis:
■(a) Carbon
43.8%
(b) Hydrogen
2. 9%
(c) Chlorine
, 31.8%
(2) Mass Spectrometry:
The mass spectrum showed:
(a) A parent ion at mass 220 with isotope peaks
indicating the presence of two chlorine atoms.
(b) A fragment ion at mass 175.
(c) Fragment ions at mass 162 and 161.
(d) A fragment ion at mass 145.
(3) Proton Nuclear Magnetic Resonance Spectrometry:
The proton spectrum showed:
(a) A singlet at -4.82 ppm (methylene protons).
(b) A doublet at -7.04 ppm, J = 8. 8 Hz, a doublet,
J = 8. 8 Hz, of doublets, J = 2.5 Hz, at -7.26 ppm and a doublet at - 7.42
ppni,' J = 2; 5 Hz-,- “(aromatic protons).
(c) A singlet at -9.15 ppm due to an exchangable
proton (acid proton).
1
(d) Integrals of the ratio: 2 methylene protons:
3 aromatic protons: 1 acid proton.

k

5759 0000310

o
O
.i i
o
CO !
SO-*
CO ‘

AL 7-932

▲Wj ±

References

R R 17. R Vll M O O

Proof of Structure:
Chemical-Physics Research Lab. combined report IR 203894,
MS 205431, NMR 205275 by S. T. King, L. Shadoff and T. E. Evans.
Elemental Analysis:
Analytical Laboratories AL 9-150, J. Simpson, C. Mendoza.
Preparation of Analytical Standard:
L. White, Process and Formulations Research Lab.
Additional Analytical Work on Sample AGR 30653C

-

It was necessary to dry the 2,4-D standard in order to obtain a
meaningful purity result with the differential scanning calorimeter.
Without drying a result of 99.0 mole percent was obtained. Karl Fispher
determination of water in the standard showed that the drying loss was
due to water (0.19% water, AL 9-645, R. A. Dutcher). To confirm the
suitability of the calorimetric purity determination 2,6-D was added to
a portion of the standard and satisfactorily delected using the differential
scanning calorimeter.
A 0.4% unknown found on gas chromatograpliic analysis after
méthylation with diazomethanc is thought to be a result of the diazomethane
reaction as it is found in about the same amount in all phenoxyacids
methylated in this way (AL 5-666, J. Russell). This unknown was not
found after esterification with methanol-sulfuric acid.
ajg
i

0 0 0 i\VQo
5760

SH

y**'py •«
KJ £ \J JL

i

By Or. Julius E. Johnson
Vice-President and Director of Research
The Dow Chemical Company

safttv in the Development
of Herbicides
The consideration of this broad topic can
well be divided into three parts—past, pres­
ent and future.
The portion entitled "past" will deal pri­
marily with 2,4,5-T and other phenoxy
herbicides. The chapter on the "present" will
give you a portrait of the events currently
involved in establishing the safety of a new
pesticide. The concluding remarks on the
"future" will present some of my opinions
on what is needed to encourage and sustain
a healthy weed control enterprise for the
future.
Past
A controversy surrounding 2,4,5-T
emerged on October 29,1969. The results of
a National Cancer Institute study on tera­
tology conducted by the Bionetics Labora­
tory were made known through an an­
nouncement by the President's Science
Advisor, Dr. Lee Dubridge. Immediately
thereafter I was personally involved in re­
porting to the Government the possibility
that the sample used in the Bionetics test
was contaminated. The suspected contaminant was 2.3.7.8-tetrachlorodibenzo-p-dioxin. inese suspicions were later confirmed
'byAnalysis of the Bionetics sample. Efforts
were immediately instituted to test the ef­
fect of commercial 2,4,5-T containing less
than 1 part per million of the contaminant.
Tests were conducted in Dow laboratories

after thorough discussions with government
scientists concerning the methods to be
used. Sprague-Dawley rats were the rec­
ommended test animals. Repeated daily oral
administration of 2,4,5-T by stomach tube
to the pregnant rats was the recommended
route of administration. The first series of
dosages ranged up to 24 milligrams per kilo­
gram body weight, the second series to 100
milligrams per kilogram. None of these
dosages produced the symptoms of tera­
tology claimed in the Bionetics report. The
tetrachlorodibenzo-p-dioxin, on the other
hand, was toxic to embryos at a dosage level
of 0.125 micrograms per kilogram per day;
0.03 micrograms per kilogram was b e lo w
th e n o -effect level. Only by combining 2,4,5-T
at a dose of 50 milligrams per kilogram with
the tetrachlorodibenzo-p-dioxin in a third ex­
periment. was it possible to produce cleft
palate in a few of the test rats. (Figure 1).
This is as close as we were able to come
confirming the Bionetics work in rats. Later
tests with 2,4-D and silvex also failed to
produce terata at elevated dosages.
It is noteworthy that the tests which were
conducted by Bionetics Laboratories and
by the National Institute of Environmental
Health Sciences that were interpreted as
showing 2,4,5-T to be teratogenic were ad­
ministered by subcutaneous injections of
2,4.5-T dissolved in dimethylsulfoxide. The
doses were up to 113 m iilig ^ f^ g ^ k ilo DOWN TO EARTH. VoL 27. No. 1. Summer 1971

1

FIGURE 1 —Teratology Study in Rats: 2.3,7,8-tetrachlorodibenzo-p-dioxin
Added to 50 MG pure 2,4,5-T/KG Body W t/Day.

FIGURE 2 -A c u te Oral Toxicity, Single Oral Dose.

LQi*XI flig/Vg SedyWeigh

Treatment

No. of
Litters

No. of
Fetusos

Litters
with
Cloft
Palato

Control (Vehicle)
50 mo Pure 2.4.5-T/kn
+0.0Ì MB TCOBD
+0.03 MB TCDB0
+0.06 MB TCDBO
+0.125 MB TCDBD
+0.5
MB TCOBD
+ ÌJ0
MB TCOBD

17
17
15
17
17
15
14
15

194
172
150
173
155
134
76
46

0
0
0
0
1
0
4
5

Littore
with
Intstnl.
Hemorr.
0
0
0
0
8
11
13
8

2.4-0
2.4.5-T
Silvex
Pidoram
DDT
Parathion
23.7,8-Tetrachlorodibenzo-p-dioxin

-Bat

Gointa r

375.0-666.0*
5000
500.0
8.2000
1500-8000*
1.7-30.0*

lOOOi

8517
• 3000:
400:
93-31!

0022-0.045*

OOOOf

so:

*Varies according tovehicle or sex

gram body weight in the Bionetics test',
There was ample justification for cor
by government officials over the results
and up to 100 milligrams per kilogram in the
covered with the original sample use.
NIEHS te sts2.
Bionetics. This sample, however, (colle
These procedures are not at all relevant
in
1964) was not typical of carefully
to actual exposures and it is questionable
duced 2.4,5-T. 2.3.7.8-tetrachlorodibe
whether the demonstration of teratogenicity
by these methods is meaningful. Golberg3 p-dioxin is an extremely toxic subst:
(Figure 2). This is good and sufficient re:
has recently pointed out several complica­
to maintain the tetrachlorodibenzo-p-di
tions worthy of citation —
concentration as low as.possible. With
"In checking on potential teratogenicity of
analytical methods and production ttrace chemical contaminants in food, the
niques available in 1965 a one part per
height of absurdity is achieved by the
lion level was a realistic specification,
combination of a maximum tolerated dose,
has since been reduced to 0.5 parts
a parenteral route of administration, and
million.
the application of zero tolerance on the
A few comments on the characteristic
basis of the results. In this area we know
2.3,7.8-tetrachlorodibenzo-p-dioxin wili
full well that any one of a host of adverse in­
of interest. This compound can be forr
fluences on the mother is reflected in fetal
during the manufacture of 2,4.5-trichlc
deaths, resorptions and/or abnormalities.
phenol where two moles condense to fc
Teratogenic effects are elicited by: trans­
a
dioxin (Figure 3).
port of mice by air on days 12 and 13 of
The following information is known at
pregnancy (Brown. Johnston & Niswanthe stability and properties of the 2.3.'
der. 1970): fasting for 24 hr. or less at a
tetrachlorodibenzo-p-dioxin:
critical stage of gestation (Kalter & War(a) in solution it is rapidly degraded by
kany, 1959) which may be brought about
traviolet light at wavelengths that
inadvertently or unsuspectingly by in­ >, f
pear in the spectrum of the s
ducing som nolence, lethargy, muscle
(Figure 4).
weakness or ataxia; a diet of raisins for
(b) The tetrachlorodibenzo-p-dioxin i
one day (Peters & Strassburg, 1969): se­
one-fifth the solubility of DDT in we
vere limitation of movement or avoidance
and one-sixteen hundredth the sc
behavior (Rosenzweig & Blaustein, 1970):
bility of DDT in benzene (Figure
and many other nonspecific factors prob­
Thus, there is probably less tender
ably acting through a stress mechanism,
to concentrate in fat. but the quest:
as well as hyper- or hypothermia and
is really academic because the quar
endocrine influence (Kalter & Warkany,
ties of tetrachlorodibenzo-p-dioxin
1959). Even subcutaneous sodium chlo­
the environment are exceedingly sm
ride is teratogenic in mice (Nishimur^ &
Concern has.been expressed that thecc
Miyamoto, 1969).
bustion of 2,4,5-T (even though the pare
Here, above all. is a situation th at de­
material contained no tetrachlorodibenzo
mands the utmost care in selecting doses
dioxin) would cause dioxin formation,
that do not render the mother sufficiently
laboratory experim ent w as conduct:
ill to produce even transient inappetence.
wherein agent orange (a defoliant contai
The use of a maximum tolerated dose over­
ing equal quantities of the n-butyl esters
looks the possibility of non-specific toxic
2,4-D and 2.4,5-T) was applied to 18.5 c
stress ...
—uj n o
2

DOWN TO EARTH. Voi. 27. No. 1. Summer 1971

57bo

Iter paper at rate of 24 lb. per acre (Fig¡6). This is equal to approximately 10 lb.
2.4,5-T acid equivalent per acre. The
lerwas burned and combustion products
/eight ."S
his air drawn through an absorber packed
Guinee F iji
glass beads cooled with liquid nitro1.000.0 «
.
The
combustion products plus the ash
380J)f
ias solvent extracted and analyzed by gas
S50ÜÎJ
3.000-0*
natography. The burning temperatures
dOOHi
countered are shown in Figure 7. There
9.3- 310*3
bsjio 2.3.7.8-tetrachlorodibenzo-p-dioxin
ILOOOS*
letectea in tne combusion products at
A Similar experiment
(«as tried using woo'd as a substrate but
were substances in the combustion
products which interfered with the analy. Speculative claims are widespread that
2,4.5-T residues on vegetation might be
[«inverted to 2.3.7.8-tetrachlorodibenzo-pDn for concej »ioxin if the dead foliage is burned. All availKableevidence to date indicates this conver
he results dj!
Ision does not occur. The alleged precursoi
mple used?
[is in dilute form oh the substrate, the re a c ­
ver. (collect!
tion is bimolecular—the molecules must be
carefully pi
[freed close together to react.
hlorodibenzt
Concern has also been expressed that
ic substani
[traces of preformed tetrachlorodibenzo-pficient reasorfl Idioxin contaminating 2.4,5-T sprayed into
enzo-p-dioxmS Itheenvironment would accumulate. The evible. With tjiff
I (fence to date is skimpy but nonetheless
uction teci
[worth mentioning. Cu -labeled 2,3,7.8-tetrap«
er mi@ dilorodibenzo-p-dioxin dispersed on clay
fication. Thij loam containing 3 percent organic matter
1.5 parts pej
gave off a very small amount of C,4C0 j in
[two weeks when incubated at 24°C. (75°F.).
acteristics:
[This evidence suggests that degradation
oxin will
occurs slowly but it will require further obn be former | sen/ations to quantify the rate.
1,5-trichlorcS Kearney4 has reported that the 2.3,7,8arise to foi
| tetrachlorodibenzo-p-dioxin is quite immocnown aboi | bile in soil. Even in sandy soil there is little
| tendency to leach into the soil profile. Al­
the 2.3.7.8J
though persistent in soil the dioxin is not
readilytaken up and transported to the aerial
graded by ul| portion of the plant.
;ths that ap3 | Silvex is also made with 2,4,5-trichloroof th e sun? ! phenol as a starting material. A total of 8
lots of silvex have been analyzed, obtained
-dioxin has| from our 1967, 1968 and 1969 production.
)DT in waterj No detectable amounts of 2.3,7,8-tetrah the solu-i chlorodibenzo-p-dioxin have been found
(Figure 5 j| using a method having a sensitivity of 0.1
as tendency ppm. Before leaving the subject of con­
he question taminating chlorodibenzo-p-dioxins it is
the quantj£ noteworthy that no detectable dibenzo-p-p-dioxin irra dioxins (including the 2.3,7.8-tetrachlorolingly smallj
or the 2.7-dichloro-) have been found in
lat the com!"
2.4- D samples analyzed. The sensitivity of
■th
arenf!
adii. ,zo-p3 die analytical method used was 1 ppm.
rmation. As The formation of 2.7-dichlorodibenzo-pconducted) dioxin does not occur in the manufacture of
nt contain^ 2.4- dichlorophenol since it is made by di­
yl esters of| rect chlorination of phenol and not by alka­
to 18.5 cmj line hydrolysis of a trichlorobenzene. Re-

F1GURE 3 -F o rm a tio n of Z3,7,8-tetrachlorodibenzo-p-dioxin. •

-

FIGURE
-. Solubility at 25"C
Solvont
,.

,

•

.

: '

;

TCDBD

’ Water
- Benzene

'

DDT

ml

g/100

FIGURE 6 —Burning Agent Orange Applied to F ilter Paper

FIGURE 7 —Combustion of Agent Orange on F ilte r Peper.

C
Papor
S o rtici
Body of
Flame
From of
Filma ~

boo- boo

\

1200 ;

300- « :

550-65

700-75C

m.

r

URE-8—Safety Evaluation For New Pesticide—..Minimum Registration Requirements.

'

'-

*1950

'

•I960*
Acuta,
- Lib. Animals
90 day. rat
■90 diy, dog
year, rat:
P :*
1 year, dog
‘

TAB0USM
(ALYTÎCÀl:
•-•T.",-'--

•

ology;

esticide only

1970

Acuta,
Lab. Animals
90 day, rat
90 day, dog
. 2 yarn, rat
2 yiar, dog
Reproduction. 3 gen. rat
Teratogenesis. rodent
Fish, shellfish, etc.
Birds
'
Animal
(min.)
Rodent and/or dog
¿ T y
-.
'
Plant
cwp*,:1 PPM* . Food craps 0.1 ppm** Food craps 0X1-0.05 ppm**
~ Meat 0.1-ppm.'
Meat 0.1 ppm
Milk 0.1 ppm'
Milk 0X05-0.05 ppm
ENVIRONMENTAL
. - V .'. • ' - , \
Stability
Movement
u;;
Spectrum
-i:/'. vr’
Accumulation

V..
V * - - • ■’ -■•-•Lib. Ammali “ .
30-90 day. rat

** Pesticide plus toxic metabolitels)

action conditions do not favor bimolecular
decision by j.
condensation to form a chlorodibenzo-pfunctions.
dioxin. Moreover. 2,7-dichlorodibenzo-pIn brief, Ste
dioxin is more difficult to form than the ■ Stage 2 idem
2,3.7 .8 - tetrachlorodibenzo - p - dioxin even : by limitations
when reaction conditions are favorable.
' can be identit
I would conclude these remarks by stating
commitment
the firmly held opinion that commercially
money coverii
produced 2,4,5-T containing less than one residues, m
part per million of 2,3.7.8-tetrachlorodistudies. Stag
benzo-p-dioxin does not present a hazard to research outs
health. It is unfortunate that tests on one im­ ' essary confirm
pure sample coupled with emotional concern and (b) the re
over the use of 2.4,5-T as a defoliant in Stage 5 is th
Vietnam have created unwarranted public
The costs c
fear concerning the appropriate use of this ; 9. A half milli
valuable herbicide.
1 the end of St?
• 2 may have .
10). In this c
Present
out
on the ba
Let me now turn to the present and de­
scribe some of the experiments primarily : work with ac
relating to safety as they occur in the de­ lion dollars. ;
velopment of a new pesticide. For the pur­ _ by the end c
poses of this discussion we could substitute years of time
the word herbicide for pesticide because production pr
most of the safety considerations involve tation. The pr
handling hazards, residues, non-targel expensive los
organisms and environmental considera­ .Stage 4 (Figi
tions. I will briefly describe the stages tion limitatio
through which a candidate progresses and Another exam
then present some of the safety and cost ■ ure 13, wher
considerations in each respective stage ■vested at the
In a study reported by Von Rumker, Guest ; nated. Incide
and Upholt*. it was claimed, in 1969, in . vested at 8 % i
a survey of 13 companies that one com-' Imposition of pit
pound out of 5.000 tested emerged as a r plus interest,
marketable product. Testing required ap­ ¿ million dollar:
proximately five years prior to marketing hand a winnir
and a cost of approximately four million e still substant:
dollars. This reported cost did not include ¡-'cash position
process development or pilot plant studies *15) compared
I would estimate today that one new pestK (Figure 16) ha
cide emerges per 10.000 tested. The time { invested at 8
from discovery to market ranges from eight ¡¿interest are si
A winner is
to ten years and the cost is ten million
dollars or more. This cost includes all re­ i market lasting
search. development and technical service; i sales level of
process development, and pilot plant costs and a return
It includes losers as well as winners. Tm .tax. A good
other research-intensive companies re­ remember the
cently surveyed have privately given com­ the losers. The
a mixture of
parable estimates to us.
The challenge is to identify and eliminate ■some "real life
the losers early. Conversely, the expensive Project A wa
way out is to have the second best hang 5600.000. Pro
in a poker game. You may be interested« 000, Project
spending $4,0
comparing the minimum toxicological
ing $1.5millio:
quirements for registration in 1950. 1
50.5 million, r
and 1970 respectively (Figure 8 ). In the
ceeding discussion. I will refer to Stage I: tinuing and he
2. 3. 4 and 5 as steps in the process
The time to
development of a new pesticide. Promoti
and hopefully
2. The present
to each higher stage represents a formali;

I
IGURE 10 —Sunk Costs 7- Stage II. > : v y

'•!

- ■

0

IX r
Cjimlcthrt Costs, M$ 1
2X
J •'

3X

Stages..

I

t * *-i ■* .« 1*'l

, •. I

i. M I •. »i‘I ,i ■ I■ a 1.1 ’ I

.
-7

' -f '

. »- -

é•t- ' . " ‘

5765

bimolectila [decision by personnel in many interrelated

adib^zo*! Ifunctions.
adit
,0?j | In brief. Stage 1 is the exploratory stage,
n tharvrtra tstage 2 identifies the key questions where­
dioxin-eve! by limitations of a new discovery hopefully
yorable;^ lean be identified early. Stage 3 involves the
s by statin] Icommitment of extensive time and lots of
jmmerciail [money covering efficacy, toxicity, process,
s than-or [residues, m etabolism , and ecological
:rachloro98 [studies. Stage 4 Involves (a) cooperative
a hazards? [research outside of Dow to obtain the nec; on oriein lessary confirmation of efficacy and safety
nal coricer [and (b) the registration of the new product.
defoliant™ Stage 5 is the initial sale.
ited pulili? I' The costs of Stage 1 are shown in Figure
use of tms |9. A half million dollars may be involved by
I the end of Stage 1. The end point of Stage
2 may have cost a million dollars (Figure
10). In this case the product was flunked
out
on the basis of residue and metabolism
rnt and de
s primarily work with accumulated costs of one mil­
• in the de lion dollars. $1.900.000 could be invested
or th e p'urj by the end of Stage 3 and consume four
years of time (Figure 11). In this case the
i substitiiti
|
production process was the deciding limile becaus
3ns involy|i i tation. The product cost too much. A very
non-targefl Iexpensive loser is one which flunks out in
considers !Stage 4 (Figure 12). In this case registrathe stages] I tion limitations were the limiting factor.
ret
an] Another example of a loser is shown in Fig­
y and cosi ure 13. where 2.1 million dollars were intive staged [ vested at the time the project was terminker, Guesfj I nated. Incidentally, that same money in­
n 1969, :f^ vested at 8 % interest would result in a cash
one corn! position of plus 3.4 million dollars, principal
erged a s ;^ plus interest, as opposed to a loss of 2.1
quired a p | million dollars (Figure 14). On the other
marketing hand a winning product for which there is
our million? still substantial hope involves a negative
tot include! cash position of 4.6 million dollars (Figure
rnt studies^ 15) compared to a plus 6.1 million dollars
new pesti^ (Figure 16) had that money been alternately
I. The time invested at 8 % where both principal and
from eight; interest are shown above the line.
A winner is defined as a project with a
ten million^
ides all rej market lasting at least 9 years at an annual
:al service^ sales level of 10 -20 million dollars per year
)lant costsl and a return on investment of 40% before
oners. Tw§? tax. A good investment all by itself, but
oanies regl remember that the winners must support
given conri| the losers. The total agricultural business is
Si *3 a mixture of winners and losers—and for
d eliminate! some "real life" examples note (Figure 17).
i expensive! Project A was terminated after spending
best handf $600,000. Project B after spending $1,000.terested iris 000, Project C. an expensive one, after
>lo<
re-i spending $4,000,000. Project D after spend­
950. 1960| ing $1.5 million, and Project E after spending
In the sue« $0.5 million. Projects F, G and H are con­
tinuing and hopefully will result in winners.
o Stage
The time to kill a loser is in early Stage 1.
process og
Promotion! and hopefully no later than the end of Stage
formalized! 1 The present costs as they relate to safety

É.'

Y

FIGURE 1 4 - Cumulative Cash Position <rf,a " L o s e ^ . B .

«' •

r/gjfe
... Millions ct Doll«« |- v’.;

f P^'act

|

S Î4 111

'. v : ; U*a*d. ,~;T«nninitid > .1

42.1 *

;* •'

■■-_jl r I" n ---r ' r->. r

t-

--T

1360 ‘61 .■62 :;*63 .'64r:651.,6 6 -‘«7 , ’68 ’SO ’70 .

SURE 1 6 — Cumulative Cash Position Of A " W in n e r " - B.
________ Cumulati»« Cash Position Of A “ Winner“

'WUtoKOf Hatten

*6.1il

'4

443SI

'

;

~T I ■■' l' :,

I _J_

L.

1__ I

l

1___ I__________

. ;^ r 7 >1360-61J82.-Ê3 '34 ‘65 -66 ‘87 '68 '63 1370

GURE 1 7 -E xp e n ditu re Pattern, Eight Typical Agricultural R & D Projects

1360

1362

1364

1368

1366

fPBn-iFgT a XS - m |

| PROJECT BIT S-II.OB
4.
T C
PROJECT
CV 1 - 44.0 Bf I • /-V.
'Ü
.1 :
PROJECT p Y z - l i i S l

I•

■¿i'L:
.. 'ì ■

-------------

j I .. .

, m' '

lasSl -^.;L

PROJECT f ' ■I ts. 463 M I
~ f 1 • PROJECTC“'

government c
fhade with goc
on fact.
Another sug
4220.000
[be developed f c
,2300 'te*22.000%
laboratories. T
U
- f p u ---'ratories, u n iv e r
•laboratories
ar
TOXICOLOGY
Greater public
METABOLISM
iopedfortheres
ANALYTICAL
stories
if cert
ECOLOGY
^qualifying boa
¡^adequacy of pi
and ecological considerations in Resear ttment and hous
and Development are shown in Figure II ^Improve and m
Early toxicological work co sts abouft ‘of the data use
$22.000 through Stage 2. Other safety costs! ¿A ssum ing th
(metabolism, analytical and effect on er ifjed, then on
/certified labore
vironment) increase toxicology expenses
Stage 3 totalling $220.000. Further ex­ ETor the suppo
panded work continued through Stage j|for the suppo
^further sugges
resu lts in an accum ulated expense
$692.000. But the most important consider! feof- a pesticide
ation is tim e. The time value of money and I patent expires
the effective time of life o f a patent a^ ^certified data
^either from the
often underrated.
¿from a govern
jjjective would I
Future
Let's compare .the toxicology requin |£lier so that
^competitive a
ments in 1950, 1960 and 1970 with the
¡one who has
tential needs that m ig h t become requ
ments between '71 and '80 as presented! ¡development.
¿With a prov
Figure 19. These would include addition
^tHe
only type
testing for carcinogenesis, primate toxic
¿ible
for suppe
ogy, human toxicology and mutagenea
Perhaps human metabolism will also be~ ¡requirement tr
Required to pa
quired. plus intensified information
environmental effects. You will recall! possible to lc
'Tolerance petit
$692,000 figure for the cost of toxicol
to encourage t
metabolism, analytical and ecology tests
in 1970. The added requirements c™ ^ata at this p
jections. Toda\
involve another $400,000-$500.000,
lator nor the p
cost is not as important as the added ti
¿they want th
Once 5 million dollars has been invest^
ijcious. Morec
a three year delay could cost an added!]
ipected to he
million dollars in terms of interest exper*
luct, do not ha
and losses of earnings while the proji
ion informat ic
is held up. Developing pesticides is tr
^(tension spe
a high risk business. Changes and imprr
ie validity c
ments will have to be made in the way
loreover. the
manage our destiny.
[registration at
I would like to submit some suggestiq
under pressun
for consideration. In the first place, 41
'Trbids h im fr
government should do s better job of
jpn-; available
fining te s t requirem ents, yet the ii
:itioner. The
methods or protocols need flexibility^
iblic
distrus
individual judgment, a n d provision for''"
remot
out in •
erate change. Change - in require
iWe
should
r
should not be capricious but should
of the ex
from advances in science and advance^
listration sc
new knowledge. Any change, howr
^ ile can be ac
should involve understanding and pai
®^sion. This w
pation by industry, not by sudden units
FIGURE 18-P ro je c t Technical Manpower
For Safety R & D ...1 9 7Q
Agricultural Chemicals

1370

:- II

-i- j ,

T » 16.7 M

iji.iijHjmiim.'.i

JGURE 19—Safety Evaluation For New Pesticide—Miniinuni Registration Requirements,
:^
v ; ? a s t P rese« and Possible Future

:: .> V -1350
- ■ 1360 -• —? '1370 r' 1371-'8Qffl Vr
Acuì»,.;
V: Acuta,;
•Aaite',K
.
tub. Animals
f=-l- ¿F>; - Ub Auinnb
lull. Animals'
'LatLAnimals
’30
day, rat
-30 Äiy.nit'^iÄiiSB.dey; rat •_ ' S;<-.30-30 day. n t 90 day, dog
'SOday. dog - =-r '30 day, dog
2 year, rat
' ’*■'2.year, rat- ■
.2 year, ret
2 year, dog y ; !. ; 1 year, dog • •
...1.:year, dog
'•Raproduclion, y'.s'. 'Reproduction. {JXICQLOGY
3 gon. rat . y -/.
■■^ü5^JCx,<3 gaiCrat -¿ßg.
•• -Taiatogenasis! ’ :Taratogan«sis.
rodent
• rodant
Fish, shellfish, ate.
Birds

Fish, shollfish. ate.

Birds
•y(carcinogenesis)
Primate
“ Human viv?
; Muttganasis ■
V-.'
Animal (min.) -r g r Rodent and/ordog Rodentand/ordog
V l e n t . . Plant
ietabousm .
Human
••
Food
crops.
031^■<v’'.vFood«apsiÿi ' : . Foodwop*.vi'Vÿ-'Food crops; 031-' • .V035 ppm** . ; : ..
ppm*<^#Kii0J)S;ppm**•**#.Meat 0.1 ppm
MALYnCAL . ;
" \ Moataippm .f^MÔ«t0.1ppm
iw;
Milt 0.1 ppm <^Millt0.005-aQS ppm .Mint 0.005-0.05ppm
‘ENVIRONMENTAL;;;■
,^5Stability **Movement
coiocY
" r v_,v.
¡ «»ut
t y ^.v
- .
j
:
.. Blo»am
Spactnm ■
" Accumulation
s.
'■. ~i. V-;
~ -A
.-‘^4.
\ ‘'-iV,
. ~l^atieideplus t o x i c m a t aNb:- o.:..'l.'f-‘
i t é t « ) T-■
v 2 yr, hamster...

lESLOaj

(government decree. Changes should be
[•made with good and sufficient reason based
[on fact.
Another suggestion is that a mechanism
bedeveloped for certification of toxicological
laboratories. This means industrial labo­
EEEHSEE" ratories, university laboratories, commercial
laboratories and government laboratories.
[Greater public confidence could be develj-opedforthe results issuing from such labora­
tories if certification required th a t a
¡¡qualifying board periodically rule on the
[¡adequacy of personnel, procedures, equipions in Research ; ment and housing. The objective would be to
w n in Figure.noJ improve and make more uniform the quality
rk c o s ts abo?
of the data used for support of products.
)ther safety cosul
Assuming that laboratories could be certi­
id effect on J ffi fied, then only toxicological data from
>logy e x p e n s e s ^ certified laboratories should be admissible
)00 . Further exT for the support of product registration or
:hrough Stage^
for the support of residue tolerance, it is
ted ex p en se-o
further suggested that the second supplier
portant consider-! of a pesticide after the original petitioners
ue of money anSi patent expires, be permitted to purchase
of a patent arej certified data. This could be purchased
either from the original supplier or perhaps
from a government agency. The main ob­
jective would be to burden the second sup­
¡oology require
plier so that he does not have an unfair
970 -ith the
competitive advantage compared to the
bet
e require
one who has borne the original cost of
) as presented.^ development.
iclude additional^
With a provision of certified data being
primate toxicoj-, the only type of toxicological data admis­
id mutagenesis^ sible for support of tolerance, and with the
1 will also be 1
requirement that the second supplier be
information <
required to pay his way. then it would be
u will recall _
possible to lay the registration and the
st of toxicology^ tolerance petitions open for inspection and
ecology testing ? to encourage publication. Openness of the
lirements could' data at this point would relieve many ob­
-$500,000, but; jections. Today neither the qualified investi­
the added time,; gator nor the public have access to the facts
been invested,^
if they want them, hence the public is sus­
)st an added 12 t picious. Moreover, the experiment stations,
nterest expense
expected to help support the use of a prod­
lile the projects uct. do not have access to certain registra­
nicides is trulyj tion information. Experiment stations and
es and improve^?
extension specialists need confidence in
2 in the way we;5
the validity of the back-up information.
S i Moreover, the public official charged with
ne suggestions^*
registration and tolerance proceedings is
irst place, theg
under pressure because the present policy
;tter job of de^i] forbids him from making certain informa­
yet the te st
tion available w ithout co n sen t of th e
1 flexibility for
petitioner. The whole situation promotes
•/¡sin- *or delibpublic distrust because supporting facts
i\
rements
are not out in the open.
t should derive,
We should more aggressively extend the
id advances in
use of the experimental labels prior to full
ige. however,
registration so that typical, though limited,
ig and particisale can be achieved under qualified super­
dden unilateral
vision. This would permit the development

of meaningful use experience with limited
exposure of the total population. The ob­
jective would be to discover unexpected
phenomena difficult or impossible to ex­
p o se under -laboratory or limited field test
conditions.
One of the weakest links in the chain is
in diagnosis of pest problems and applica­
tion of pesticides. I do not wish to discredit
those competent professionals who do a
good job. nevertheless, in the country at
large the process of diagnosis and applica­
tion involves people, usually unskilled,
poorly paid, and nonprofessional. We have
many elegant tools but poor craftsmen. To
overcome this difficulty I would suggest
that we classify pesticides in two categories
—fo r professional use o n ly and fo r n o n -p ro ­
fes s io n a l use. Those for professonal use
could be applied by licensed professionals
only—skilled in diagnosis and application.
Moreover, since there is a scarcity of this
kind of skill, a training of a new kind of pro­
fessional should be developed by our agri­
cultural schools, th is will take time, but
it's needed and the professional licenses
should be granted only to those qualified.
The grower would then pay for results and
the whole process of pest control, includ­
ing weed control, should be less poundage
oriented and more result oriented. This
will require some drastic rethinking on the
part of industry on how they can make their
contribution and reap their share of the
reward. I am convinced that the American
consumer can well afford to pay a higher
percentage of the shelf price of food or
fiber for pest control if the job is profes­
sionally done with well designed products.
In other words, there is economic room to
maneuver if everyone is compelled to com­
pete by equitable regulations.
As a final note—I would strongly advocate
that we worry less about conflict of interest
and see to it that government, industry and
university personnel communicate more
effectively.
REFERENCES
1. National Institute of Environmental Health
Sciences. Research Triangle Park. North Caro­
lina. Summary Teratogenic Study NIEHS. Type­
script draft of record of hearing on 2.4.5-T
before the Subcommittee on Energy. Natural
Resources and the Environment of the Senate
Committee on Commerce. Philip A Hart, Chair­
man. April 15.1970. pp. 225-232.
2. Science. 168 (864-866) 1970.
3. Brief of the USDA. In The United States Court
of Appeals For The District of Columbia Circuit
(No. 24.434). November 2. 1970.
4. Kearney. Phillip C. Report Presented before a
joint meeting on Pesticides. United Kingdom.
Canada and United States. Sponsored by the
Council on Environmental Quality and Presi­
dent's Cabinet Committee on the Environmental
Working Group on Pesticides. Washington.
D.C., November 5. 1970.
5. Bioscience. 2 0 (1004-1007) 1970.

5768

DOWN TO EARTH. Vol. 27. No. 1. Summer 19

0
R i v k i n ,R a d l e r ,D u n n e & B a y h
E A B PLAZA
UNIONDALE, NEW YORK 11556-OIH
(516) 3 5 7 - 3 0 0 0

r

TELEX: 6 « 4 5 -0 7 < 4 • T E L E C O PIE R : ( 5 1 6 ) 3 5 7 - 3 3 3 3 • CABLE! AT LAW
3 0 NORTH LASALLE STREET

1 5 7 5 EYE STREE T, N.W.

2 0 4 0 CENTURY PARK EAST

6 0 5 THIRD AVENUE

CHICAGO, ILLINOIS 6 0 6 0 2 - 2 5 0 7

WASHINGTON, O.C. 2 0 0 0 S - I I 0 5

LOS A NG ELES, CA 9 0 0 6 7 - 2 5 9 2

NEW YORK, N.Y. 1 0 0 2 2 - 7 5 1 3

(3 1 2 ) 7 6 2 - 5 6 8 0

(2 0 2 ) 2 6 9 - 8 6 6 0

(2 1 3 ) 2 0 1 - 0 5 1 0

(2 1 2 ) < 4 1 8 - 5 2 0 0

August 10, 1989

W R I T E R 'S O I R E C T D IA L

Mary E. Alexander, Esq.
Cartwright, Slobcdin, Bokelman,
Borovsky, Wartnick, Moore & Harris, Inc
101 California Street, Suite 2600
San Francisco, California 9411

RECEIVED
AUG a 4 ^989

CARTWRIGHT ET AL

Re: Davis v. The Dow Chemical Company
Dear Mary:
In Dow's continuing effort to comply with the Court's
directions, Dow has searched its files for records of shipments or
sales between 1954 and 1973 of products containing 2,4,5-T to the
United States Forest Service. Dow has recently discovered certain
records which may be responsive to the Court's most recent order
and encloses them without conceding their relevance or
admissibility at trial both in terms of product identification and
time frame.
Please feel free to contact me if you have any questions or
comments.
Very truly yours,
Rivkin, Radler, Dunne & Bayh

JHK:lc
Enc.

T H E P A R T N E R S H IP IN C L U O E S O N E O R M O R E P R O F E S S IO N A L C O R P O R A T IO N S

5770

. M*|"4Í¡ C U S T O M E R ORDER N*rs1,ew,,CMt* *•*-«4 m o o u c t
. *AX

372 ORDER 603 KPLTQ .

f l Í,¿-----------j7 5 6 0g 72
M l M I M I I I I

¡Us 0 A

I I I « I I I I • »

II

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TOXICOLOGY
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20351

Some Effects of 2,4-D and
2,4,5-T on Ehrlich Ascites
Tumor Cells In Vivo and In Vitro
Ernest M. Walker, Jr., PIlD., Richard H. Gadsden, Pli.D.,
Loretta M. Atkins, B.S., and Glen R. Gale, Pli.D.

q

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2,4-D and 2,4,5-T shows appreciable inhibitory effects on the in vivo develop*
ment of the Ehrlich ascites tumor in mice.

INTRODUCTION
he application o f 2,4-D, 2,4,5-T, and similar herbi­
cides to plants impregnates plant cells w ith a persis­
ten t, spurious auxin which prevents the fluctuations o f
physiological auxins required for norm al grow th and
differentiation. Hanson and Slife1 have made detailed
descriptions o f the chemical and m orphological changes
in plant bodies and-cells following exposure to 2,4-D and
related chlorophenoxycarboxylic acid herbicides. Such
changes include: little or no ro o t elongation, abnorm al
growth in the basal stem , abnorm al leaf developm ent,
and a num ber o f other disruptions th a t may lead to
alteration or death o f the plant. The fact that the effects
o f 2,4-D, 2,4,5-T, and similar herbicides on plants
apparently depend upon an auxin-like action led to the
assumption th at these agents would n o t be expected to
alter or damage mammalian cells. Certain developm ents,
which center largely about the potential toxicity and
teratogenic activities o f these herbicides in animals, par­
ticularly 2,4,5-T, have led to serious reconsideration o f
this assum ption.
T he Bionetics Research L aboratory o f L itton Indus­
tries, under NTH contract, tested various pesticides and
related com pound: f-” *<'rntogeii:e activity.2 2,4,5-T. ad­
ministered in honey or DMSO, proved to be teratogenic
in mice by causing increased num bers o f abnorm al
fetuses and a statistically higher proportion o f litters
affected. The chief anom oilcs observed in affected off­
spring were cleft palates and cystic kidneys. Similar re­
sults were seen in Spraguc-Dawley rats given various oral

T

The following abbreviations are uved: 2.4-D. 2,4-dicliloropncncxyjcctic acid; 2,4,5-T, 2.4,5-trichloroplicno\>acetic acid;
MEM. Eaele’s minimum essential medium with flanks* balanced
salt solution; DMSO. dimethyl sulfoxide; TPCV, total packed
cell volume of Ehrlich uscucs tumor: DNA, deoxyribonucleic
acid: RNA, ribonucleic acid; i.p., intraperituncally.

doses o f 2,4,5-T on days 10-15 o f gestation. T he highest
dosage o f 46.4 mg/kg/day resulted in 60% fetal mortality and a high incidence o f abnorm alities in the survivors. These investigators reported th a t 2,4-D also produced an increase in th e proportion o f abnorm al litters
in mice.

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An anonym ous rep o rt sum m arizing some studies o f
VI
the Nationai Cancer Institute covers, in part, the p oten­
tial teratogenic activities o f 53 com pounds, w ith the
m ost conclusive results being fo u n d for 2,4-D and
2,4,5-T.3 Doses o f 113 m g/kg o f a b u ty l ester o f 2,4,5-T,
adm inistered from days 6-14 o f g estatio n in mice pro­
duced a total o f 54% m alform ations in the mice born
from treated m others, as com pared to 1% in those born
from control m others. M alformed m ice were seen in the
litters o f all six treated m others. A dose o f 9 8 mg/kg o f
the butyl ester o f 2,4-D, given during the same interval
o f gestation, produced an increased incidence o f malfor­
m ations in the litters o f fo u r o f the six treated m others.
Several entries concerning the an ti-tu m o r activities o f
2.4- D (NSC 2925) and 2,4,5-T (NSC 4 3 0 ) are listed in
the Negative Results o f the Cancer Chemotherapy
Screening Data o f the United States Public H ealth Ser­
vice. Entry No. 22683 reports th a t tw ice daily injections
o f 250 mg/kg o f 2,4-D for seven days caused 43% inhibi­
tion o f Sarcoma-180 in Swiss mice. In ad d itio n , th e same
entry reports that II daily injections o f 75 m g/kg 2,4-D
caused 63% and 42% inhibition in tw o d ifferen t experi­
m ents using A denocarcinom a 755 in CBFy mice. How­
ever, the entry reports th a t daily injections o f 250 mg/kg
2.4- D had no effect on L-t 210 leukem ia in BDFy mice
(mice were treated daily for 30 days o r until death). cC ^ *7 T
E ntry No. 37090 reports th at twice daily injections o f £)'*• 4
S8 mg/kg o f 2,4.5-T for seven days caused 60% and 43%
inhibition o f Sarcoma-180 in Swiss m ice in t w o separate
experim ents. The same entry rep o rts that 2,4,5-T had 0 0 0 2 4 8 1

TABLE 1. Effect of 2,4-D on Development of the Ehrlich Ascites Tum or In Vivo
Dose
mx/kglday0

tXtttnhcr o f
injections’*

Average weight
change (gm) T/Cc

45
65
75
77

6
6
6
5

-0.4/l.S
-1.7/1.6
0.2/3.2
-0.1/5.3

Mortality
C
T

Average TPCVd
TIC. ml

r*
/«
inhibition

0/S
0/10
0/9
0/9

1.061/1.549
0.924/1.744
0.728/1.572
0.983/2.000

32
47
54

0/8
1/3
2/9
2/9

51

S.D.C
TIC

P

0.330/0.355
0.431/0.320
0.460/0.26S
0.393/0.330

<0.05
<0.0005
<0.0005
<0.0005

¿Dimethyl sulfoxide was the vehicle
^Injected intraperitoneally, once per day
cTrcated/Control
^Total packed cell volume
cStandard deviation of the treated/control groups

Dose
mg/kg/da}'a

Number o f
injections0

A verage weight
change (gm) T/C0

Mortality
C
T

Average TPCV4
TIC, ml

%
inhibition

62
78
SO
85

6
6
6
5

1.7/3.7
-3.3/1.6
-1.5/5.S
-0.3/3.2

0/8
1/8
0/10 4/10
0/9
3/9
0/9
3/9

1.235/1.772
0.485/1.744
0.728/2.000
0.580/1.572

30
73
64
59

S.D.e
TIC

P

0.341/0.250
0.321/0.320
0.266/0.330
0.286/0.205

<0.005
<0.0005
<0.0005
<0.0005

DOW 37578

TABLE 2. E ffect o f 2 ,4 ,S-T on D evelopm ent o f th e Ehrlich A scites T u m o r In V ivo

¿Dimethyl sulfoxide was the vehicle
hjnjcctcd intrapcritoneally, once per day
•Trealed/Control
dToul packed cell volume
cStandard deviation o f treated/control

little effect on Adenocarcinoma 755 in CBFi mice (11
daily injections of 44 mg/kg) or L-1210 leukemia in
BDFi mice (mice were treated daily for 30 days or until
death). Consideration of the foregoing studies of 2,4-1)
and 2,4,5-T, especially the implications of their teiatogenic potentials, prompted us to investigate the effects
of 2.4-D and 2,4,5-T upon Ehrlich ascites tumor cells,
including certain in vivo and in vitro parameters.
MATERIALS AND METHODS
2.4-D and 2,4,5-1" were provided in a highly puuficd
form (99.0%) by the Pesticides Research Laboratory.
Perrine, Florida. Thymidine-methyl-311, uridinco-Ml.
L-leucinc-i4C, and sodium Gormate-tJC were obtained
from New England Nuclear Corporation. Ascites tumor
cells were maintained in BALB/c mice (Flow Research
Animals, Inc.).
The investigation included the following parameters
which have been described in earlier reports as indicated:
rates of synthesis of DNA. RNA, and protein4-5; de
novo synthesis of RNA purines0-7 ; development of the
Ehrlich ascites tumor in vivo.8 In addition, the survival
times of mice bearing ascites tumor cells and treated

with each herbicide were assessed.
DMSO was used as the solvent system for all parame­
ters in the investigation of 2,4-D and 2,4,5-T except the
survival experiments in which 20'.”- DMSO in 0.9% saline
was used. The volume of DMSO solution injected was
0.1 ml per 40 grams body weight of each control or
treated mouse. In the in vitro experiments, the final con­
centration of DMSO was l%(v/v), and the same concen­
tration of DMSO was contained in all control vessels.
RESULTS AND DISCUSSION
Effects o f 2,4-D and 2,4.5-T On Development o f the
Ehrlich Ascites Tumor In I 'ivo
The inhibitory effects of 2,4-D and 2,4,5-T on de­
velopment of the Ehrlich ascites tumor in vivo arc shown
in Tables 1 and 2, respectively. Both compounds were
effective inhibitors of tumor development, with 2,4,5-T
appearing to be slightly more effective than 2.4-D atf ) 7 7 8
equivalent dose levels. Increasing the dosage of 2,4,5-T
from six injections of 62 mg/kg/day to six injections of
S0-S5 mg/kg/day doubled the extent of inhibition of
tumor development. Similar increases in dosage of 2.4-D

0002482

TABLE 3. Effects of 2.4-D and 2.4,S-T Upon the Average Survival
Times of Mice Inoculated with Ehrlich Ascites Tumor Cells
Number o f death:
during Treatment
Control
Treated

Dosage
m s/kg/daya

Number o f
Injectionsb

2,4-D

75

5

0
8

2.4.5-T

70

5

0
S

Compound

Ave. Time o f
Survival In Days
Treated
Control

% o f Control
Survival
Time

S.D. in Days
Treatedc
Control

0
8

18.4
14.6

126

2.7
3.9

<0.025

t
8

20.6
14.6

141

6.8
3.9

< 0 .0 2 5

P ■

a207S DMSO in 0.97» saline was the vehicle
bn ie first i.p. injections of 2.4-D or 2,4.5-T in the above vehicle were given 24 hours after inoculation of
each mouse with approximately 6.xl0b Ehrlich ascites tumor celts. Mice received one injection per day for five days.
cStandard deviation of the treated/control groups in days.

DOW
inhibited to about the same extent 48 hours after injec­
tion, indicating that the inhibitory effects may be
exerted on the common pathway of purine synthesis
(prior to synthesis of inosinic acid).
Effects o f 2,4-D and 2,4.5-T on the In Vitro Incorpora­
tion o f Radioactive Precursors into DNA, RNA, and Pro­
tein o f Ehrlich Ascites Cells
The effects of 10-* M 2,4-D on the in vitro incorpora­
tion of radioactive precursors into DNA, RNA, and pro­
tein are shown in Table 6. Incubation of the cells for
periods of an hour or less with 10-* M 2,4-D caused a
small and probably insignificant increase in the incor­
poration of thymidine-methyl-3H into DNA. Similar
periods of incubation produced no notable effects on
tiie incorporation of uridine-5-3H into RNA or on the
incorporation of L-lcucine-i4C into protein. Incubation
of the cells with KM M 2,4-D for periods of two hours
resulted in slight stimulation, producing average in­
creases of 17%, 37%, and 22% in the rates of incorpora­
tion of radioactive precursors into DNA, RNA, and pro­
tein, respectively.
The effects of KM M 2.4,5-T on the in vitro incor­
poration of radioactive precursors into DNA, RNA, and
protein arc shown in Table 7. Incubation of ascites cells
witii the compound for intervals of an hour or less pro­
duced slight increases in die incorporations of
thymidine- 3H into DNA and uridinc-5-3H into RNA.
Increasing the incubation period to two hours produced
no further increase in the incorporation of isotopic pre­
cursor into DNA and resulted in the loss of the stimula­
tory effects of RNA synthesis seen after shorter incuba­
tion periods. Incubation of ascites ceils with KM M
2.4,5-T for an hour or longer resulted in stimulation of
the incorporation of L-leucine into protein. Increases of
23% and 28%. were obtained after one and two-hour
incubation periods, respectively.
In summary, 2,4-D and 2,4,5-T showed appreciable
inhibitory effects on the in vivo development of thc^ q' 0 2 4 S 3

375782

did not result in such marked increases in the degree of
inhibition as was observed with 2,4,5-T.
Effects o f 2,4-D and 2,4,5-T on A verage Survival Tunes
o f Mice Bearing Ehrlich Ascites Tumor Cells
The average survival times of mice previously inocu­
lated with Ehrlich ascites tumor cells and treated with
each herbicide arc shown in Table 3. Both compounds
produced moderate but significant increases in the
average survival times of tumor-bearing mice. 2,4,5-T
caused a slightly'greater increase in the average survival
time (41 %) than 2,4-D (26%), but the 2,4,5-T group had
a higher standard deviation for survival of mice in the
group (±6.8 days) than seen in the case of the 2,4-D
group (±2.7 days).
Effects o f 2,4-D and 2,4,5-T on RNA Purine Synthesis
In Vivo
Data presented in Table 4 demonstrate the effects of
2,4-D on de novo RNA purine synthesis in vivo. The
effects of 2,4-D on RNA guanine synthesis were not
consistent. Dosages of 75 mg/kg and 100 mg/kg ap­
peared to produce slight stimulatory effects upon RNA
synthesis when injected i.p. 24 hours prior to the injec­
tion of sodium forinate-i4C. Averages of 16% increase in
RNA guanine and S% increase in RNA adenine were
found when 100 rnnlk?. 2.4-D was injected i.p. 48 hours
prior to tiie injection of isotopically labeled foimatc.
Table 5 shows the effects of 2,4,5-T on dc novo
purine synthesis in vivo. 2.4,5-T appeared to produce a
slight stimulatory effect upon RNA adenine synthesis
when injected i.p. 24 hours before the injection of
sodium formate-i4c by the same route. The effects on
RNA guanine synthesis 24 hours after injection were not
consistent. However, 2,4,5-T, 48 hours after i.p. injec­
tion of 100 mg/kg, produced an average of 28% inhibi­
tion of RNA adenine synthesis and an average of 30%
inhibition of RNA guanine synthesis. Thus, the rates of
synthesis of RNA adenine-i4C and guaninc-i4C were

TA B LE 4. E ffect o f 2 ,4-P on RNA Purine S y n th esis
in Ehrlich A scite' T u m o r Cells In Vivo3
CPMimg Da++ RSA
Guanine
Adenine

% Change
Guanine
Adenine

2.4-n
mg/kg

I/nurs after
hifectianh

0
25
50
60
75
100

24
24
24
24
24
24

4608
3645
3552
4S76
5087
4036

2558
2725
2739
3464
2804

0
100

48
43

3336
4712

1354
2246

—

0
100

48
48

33S7
3074

2369
2221

—

+13
+ 9

1.50
1.42
1.30
1.74
1.47
1.44

+21

1.80
2.10

- 6

1.43
1.3S

3064
-17

-21
-23
+ 6
+ 10
-12

-n
•u

—

+41
—

- 9

Guanine
Adenine

a2.4-D in DMSO. at the doses indicated was injected i.p. to groups o f 3-5 mice per group.
^Represents interval between i.p. injections of 2,4-D and l'ormate-l4 C. Animals were
sacrificed three hours after the i.p. injection of formale-l3 C. Cells were removed and
processed as described in reference 7.

CO

cn
<r
CO

Hours after
injection*

% Change
Guanine
Adenine

0
25
50
60
75
100

24
24
24
24
24
24

3657
3419
4563
3646
3363
4212

1256
1147

—

m,,

- 7
+25
0
+ 6
+ 15

- 9
+ 6
+ 5
+ 12
+15

0
25
50
60
75
i00

24
24
24
24
24
24

4377
3597
4019
5370
4160
2992

2593
3448
2777
3813
2636
2795

—

—

-18
• 8
+23
- 5
-32

+33
+ 7
+47
+ 2

0
100

1S54
1067

—

—

43

3336
2207

0
100

'48
48

3387

2369

—

2513

2038

•

2,4.5-T
mg/kg

1336
1277
1401
1443

-34

-26

+ 8
-»2

—
-14

Guanine
Adenine
2.91
2.98
3.42
oc
ri

CTM/mg Da ++ RHA
Guanine
Adenine

00

TA B LE 5. E ffect o f 2 ,4 ,5-T on RNA Purine S y n th e sis in
E hrlich A scites T u m o r Cells In Vivo3

2.76
2.91
1.69
1.04
1.45
1.41
158
1.07
1.80
2.07
1.43
1.23

a2,4.5-T in DMSO, at the doses indicated was injected i.p. to groups o f 3-5 mice per group.
^Represents intercal between i.p. injections of 2.4.5-T and forinate-l’ C. Aninuls were
sacrificed three hours after the i.p. injection of form ale-l'C . Cells were rem ised and
processed as described in reference 7.

5780
0002434

INDUSTRIAL MEDICINE, VOL. 41. NO. 1. JANUARY 1972

21-

T A B L E 6. E ffects u f 2,4-D o n th e In V itro
In c o rp o ra tio n o f R adioactive P recu rso rs into
D N A , R N A , and P ro tein in E hrlich A scites Cells®
Synthesis
of
DNA

RNA

Protein

Sampleb

Hours o f
Ineuhationc

Average
CPMd

Control
2.4-D
Control
2.4-D
Control
2,4-D

0e
0
1
I
2
2

69138
70207
62294
65667
48211
56266

Control
2,4-D
Control
2,4-D
Control
2,4-D

0
0
1

75788
69457
50149
49799
31856
43528

' Control
2.4-D
Contrcl
2,4-D
Control
2,4-D

0
0
1

1
2
2

-

1
2
2

7644
7424
7250
7111
5567
6771

Average 7
o f Control

TABLE 7 . E ffects o f 2,4,5-T on th e In V itro
In c o rp o ra tio n o f R adioactive Precursors into
D NA , R N A , an d P ro tein in E hrlich A scites Cells®
Synthesis
of
DNA

102
105
117
RNA
92
99
137
97
98
122

aThc cells were incubated with the appropriate radioisotopes for
periods o f 20 minutes, at the end of which the reactions vere
terminated by the addition o f an equal volume of 10% TCA
(trichloroacetic acid). Radioactive precursors and final activities
were: for DNA. thymidine-mcthyl-^H (10 pCi/ml); for RNA,
u r id in c - 3 ]( (]() pCi/ml): and for protein, L-leucinc-l®C.
uniformly labeled (2 uCi/ml).
^Concentration of 2,4-D, 10~* M in incubation medium.
•Time interval between addition of 2,4-D and addition of
appropriate radioisotope to ceils.
^Average o f two similar experiments in which duplicate tubes
were included for each sample (average o f four tubes).
*CcUs were added to incubation tubes containing 2.4-D and
radioisotope.

Protein

Sampleb

Hours o f
Jneuhationc

Average
CPMJ

Control
2,4,5-T
Control
2,4.5-T
Control
2.4,5-T

0«
0
1
1
2
2

37425
39599
28877
33568
2449S
26944

Control
2,4,5-T
Control
2.4,5-T
Control
2,4,5-T

0
0
1
1
2
2

39846
44453
34 953
33714
29962
28765

Control
2.4,5-T
Cunt rol
2.4.5-T
Control 2
2.4,5-T

0
0
1
1
2
2

135SS
12149
74 K8
9181
5379
6875

Average %
o f Control

o
J

106
116
110

CO

112

cn

96

CO

96
89
123
128

¡■The cells were incubated with the appropriate radioisotopes for
periods of 20 minutes, at the end of which the reactions were
terminated by the addition of an equal volume of 1IV,7 TCA
(trichloinucctic acid ). Radioactive precursors ami final .n ioiiies
were: for DNA. thymidinc-mcthyl-?II (10 uCifml): (or RNA,
u tid in c -^ H (10 uCI/ml): and for protein, I.-leucine-I^C,
uniformly labeled (2 uCi/ml).
^Concentration of 2.4.5-T. 1(H M in incubation medium.
cTime interval between addition of 2.4,5-T and addition of
appropriate radioisotope to cells.
•^Average of two similar experiments in which duplicate tubes
were included for each sample (average of tour tubes).
••'Cells were added to incubation lubes containing 2.4,5-T and
radioisotope.

y

5781
0002485

Ehrlich ascites tumor in mice and caused mild to
moderate effects on selected parameters of nucleic acid
and protein syntheses as assessed by the preceding in
vivo and in vitro studies. However, the extents of these
effects may not be adequate to explain the relatively
marked and significant degrees of inhibition of tumor
development in vivo. These findings, therefore, tend to
indicate a need for determining the effects of 2,4-D and
2,4,5-T on a number of additional biochemical and phar­
macological parameters of cellular metabolism in
mammalian cells.

4. Gale, G.R. and Hynes, J.B.: Effects of Certain Arylhydroxamic Acids on Deoxyribonucleic Acid Synthesis by
Ehrlich Ascites Tumor Cells /« Vitro, J. AJed. Chem. 11:
191-194, 1968.
5. Gale, G.R., Simpson, J.G., and Smith, A.B.: Studies of the
Mode of Action of N-isopropyl-(2 mcthyihydrazinc)-ptoluamide. Career Res. 27: 1186-1 191. 1967.
6. Gale. G.R., Ostrander. W.F... and Atkins, L.M.: Effects of
Alanosine on Purine and Pyrimidine Synthesis, Biochem.
Phcrmccoi. 17: 1S23-1S32,1968.
7. Gale, G.R. and Schmidt. G.B.: Mode of Action o f Alanosine,
Biochem. Pharmacol. 17: 363-368, 1968.
8. Gale, G.R., Smith, A.B., and Walker, E.M., Jr.: Selective
Inhibition by Sorbylhydroxamic Acid of Deoxyribonucleic
Acid Synthesis in Ehrlich Ascites Tumor Cells, Cancer Res.
30: 24-29, 1970.

REFERENCES
1. Hanson, J.B. and Slife, F.W.: Illinois Res. 3: la . 1961.
Quoted from Oberbcck, J. Van, Survey in Mechanisms of
Herbicide Action, in: The Physiology end Biochemistry o f
Herbicides, edited by L.J. Audus, New York: Academic
Press, 1964, p. 3S9.
2. A Report of the Secretary's Commission on Pesticides and
Their Relationship to Environmental Health, U.S. Govern­
ment Printing Office: 0-371-074, 1969, pp. 664-677.
3. Anonymous. Thalidomide Effect from Defoliants, Set Res.
4: No. 23,11-12, 1969.

Tills investigation was supported by the following: Trust Fund
LOU, Department of Biochemistry; S.C. Community Pesticide
Study (FDA contract no. 5 SOI RR 5420-09); S tate Appropri­
ated Research Funds (College of Graduate Studies); and N11I
Research Giant GM-1395S.

•

•

•

© 1 9 7 2 M.P.I., Inc.

DOVi/ 375785

Ernest M. Walker. Jr., Pli.D., Richard H. Gadsden, Ph.D., Loretta M. Atkins, B.S., and Glen R. Gale, Ph.D., Department o f Biochemistry,
Department o f Pharmacology, and Veterans Administration Hospital, Medical University o f South Carolina, Charleston, S .C 29401.

Reprinted from

INDUSTRIAL MEDICINE A N D S U R G E R Y

VoJ. 41, No. 1 —Ft. Lauderdale, Fla. 33304
Printed in U.S.A.

5782
0002486

«Ì7

5783
S (o a .

f

Im a j

... .. Ajt.ia .• I..., ,i .. . , , ....-

-~n

-2 3 .‘• 'a u rc s s y c h ia e n c Fm ditiqx in P .B .5 . Expo*«d P.at*«r.ts by
J iic iin iy J c r o s s , M.D., U n iv e rs ity o f M ichigan tte c ie a l
C e n te r. Ann A rbor, Ml; R obert K. N ixon, M .o ., K tnry Ford
H c s p ita l, D e tr o it, Mt; M arvin 0 . A nderson. M.O., Henry Ford
H o s p ita l, D e tr o it, Ml.
F o r ty -fo u r M ichigan f a r » a e n b e rs underw ent e x te n s iv e c l i n i ­
c a l in v e s c i e j t i c n fo r p o s s ib le h e a lth e f f e c t s seco n d ary to
s i g n i f i c a n t P . 3 .a . e x p o su re . I n d u c e d in th e stu d y were
p s y c h ia tr ic - p s y c h o lo n ic e v a lu a tio n s and n e u ro lo g ic t e s t i n g
ir.c lu d ir.e s ta n d a rd e le c tro m y o g ra p h ic and n erv e c o n d u c tio n
d e t e n t i n a t i o n s . C om plaints c f d e p re s s io n , m e n o rv -c o n c e n tra tio n d e f e c ts and p a r e s th e s ia s w ere f r e q u e n t, r e s u l t s i n d i ­
c a te th e p re se n c e o f a r e a c t iv e r a t h e r th a n an o rg a n ic
d e p re ss io n m th e s e p a e ie r .ts . h 'e u r o lc g ic a liy , a s i g n i f i ­
c a n t in c id e n c e o f p e r ip h e r a l n e u ro p a th ie s , c h ie f ly s e n s o ry ,
v e re found by n e rv e c o n d u ctio n s t u d i e s . There was no
c o r r e l a t i o n betw een th e s e fin d in g s and t i s s u e le v e ls o f
P .3 .3 . I t i s co n clu d ed t h a t th e r e a r e n e u ro lo g ic changes
w hich nay be due t o t i s s u e t o x i c i t y by P .B .B . The d e p re s ­
s io n e n co u n tered i n th e s e p a t i e n t s was n o t o f an
endogenous o rg a n ic c h a r a c te r .

A s t r i k i n g n o to r d is tu r b a n c e , " sp in n in g syndromer d ev elo p ed
w ith a h ig h freq u e n c y i n th e weaning n ic e whose d o ss re c e iv e d
o r a l 3 , 4 ,1 ’ , 4 ’ te tra c h lo ro b ip h e n y l(4 - C 3 ) d u rin g th e g e s t a t i o n .
The syndrome i s p erm anent and i s c h a r a c te r is e d by s w if t
c i r c l i n g »ovestent (SO to 150 t u m s / n i n ) , r e s t l e s s n e s s and
h y p e rk in e s ia . The s p in n e r s (24) o f v a rie d ages a lo n g w ith
age-rtaechsd c o n tr o ls were s u b je c te d f o r h is to p a th o lo g ic ,
h i s to f lu o r e s c e n t and e le c tr o n m ic ro sc o p ic s t u d i e s . The s e a t
r e l i a b l e h is to p a th o lo g ic m arker f o r th e s p in n e r s i s th e
p re se n c e o f c y l i n d r i c a l CIS p en in su las(C C ?s) in th e s p in a l
n erv e and c r a n i a l n e rv e r o o t s . The CC?s c o n s is t o f e i t h e r
CMS-type m y e lin a te d f i b e r s o r a s t r o g l i a l b u n d le s and a re
e n c lo se d ir. e basem ent membrane. A d is tu rb a n c e in s y n a p to o e n e s is by 4-C3 i s th o u g h t a t t r i b u t a b l e to th e developm ent
o f th e CC?s. I n te r f e r r e n e e w ith sy n a p to o e n e sis nay have
a ls o o ccu red in th e d o p a n n erg ie sy stem . T h is lik e lih o o d i s
su g g e ste d by e le c tr o n m ic ro sc o p ic and h i s to f lu o r e s c e n t
c atec h o la m in e s t u d i e s on th e n u c le u s accumbens and th e n ig ro
s t r i a t a l pathw ay and a ls o by th e re s p o n se s on a d m in is tr a tio n
o f d o p am in erg ic a g e n ts , d o p am in erg ic r e c e p t o r s tim u la n ts
and b lo c k e r s .
-2 5 LCSG-TZSM TOXICOLOGIC STUDIES OF
3,7,3-TETRACHLQRQDIBENZO-p-OIOXIN (TCDD) IN LABORATORY ANIMALS by R .J . JCociba,
D .V .M .,P h .D ., D.C. K eyes, B .S ., J .E . B ey e r, B .S ., R.M. C arrc o n , B .S . and P . J . C eh rin g , D.V.M., P h .D ., Dow C hem ical
USA, M idland, M ichigan.
R ats w ere g iv e n d i e t s su p p ly in g 0 .1 , 0 .9 1 , and 0 .0 0 1 Ug
TCDD/kg/uay, e q u iv a le n t to 2200, 210 and 22 ppc o f TCDD f o r
2 y e a r s . In g e s tio n o f 0 .1 u g /k g /d a y cau sed m u ltip le in d i ­
c a tio n s o f t o x i c i t y , in c lu d in g in c re a s e d m o r t a l i t y , de­
c re a s e d w e ig h t g a in . In c re a s e d e x c r e t io n o f p ro p b y rin s and
m orpnologic changes i n m u ltip le o rg a n s y ste m s , e s p e c i a l l y
th e l i v e r . T h is h ig h dose cau sed on in c re a s e d in c id e n c e of
h v p a c o c e llu la r carcin o m as and squamous c e l l carcin o m as of
th e lu n g , h a rd p a i a t e / n a s a l t u r b i n a t e s o r to n g u e , w hereas a
d e c re a s e d in c id e n c e was n o te d i n th e p i t u i t a r y , u te r u s ,
aam aary g la n d , p a n c re a s sod a d r e n a l . R ats g iv e n 0 .0 1 ug /
k g /day had a l e s s e r d e g re e o f t o x i c i t y , lim ite d p r im a r ily
to in c re a s e d u r in a r y e x c r e t io n o f p o rp h y rin s p lu s some
l i v e r and lu n g l e s i o n s . In g e s tio n o f 0 .0 0 1 u g /k g /d a y
caused none o f th e a d v e rse e f f e c t s see n w ith h ig h e r dose
l e v e l s . Thus, c o n tin u o u s d o ses o f TCDD s u f f i c i e n t to
c au se s e v e re t o x i c i t y in -r e u s e d th e In c id e n c e o f seme ty p e s
o f tu m o rs, w h ile d e c r e a s in g o c h e r ty p e s . No in c r e a s e in
tum ors o c c u rre d in r a t s g iv en s u f t i e i e a c TCDD to induce
s l i g h t o r no t o x i c i t y .
'•
*

•-2 6 -

LC::G TEAM EFFECTS OF TOCO MO HCDO IN MICE ANO PATS by
?. A. Holr.es, j . H. Rust, W. R. Richter ana A. M. Shefner.
I IT Research In stitu te ana University of Chicago, Chicago.
Tetrachlorodibenzo-p-dioxin (TCEO) and a mixture of
isomers of hexachloroaibenzo-o-oicxin (HCSO) were given by
gavane in corn-oil and acetone to r.ice ana ra ts ar.e skinpainted in acetone on mice fo r 2 years. HOOD in femaie
ra ts slig h tly increased hepatic tumors but nerp lastic
nodules were increased strik in g ly . HCED treated ra ts of
botn sexes had a marxea m ultifocal pulmonary a d erc ratcsis.
In male mice. HC3D and TCC3 by gavage increased hepatic
tumors. In female mice both TCE3 and HCEE markedly increas­
ed e p ith e lia l tumors.
In mice whose backs were painted with a solution of 7CC3
or HC33. skin tumors increased in numoers and in aggression.
EM3A pretreatm ent had no Influence in e itn e r case. This
study suggests th a t TCEO and HCD3 are weak carcinogens civei
o ra lly ana are complete carcinogens when applied to th e '
skin.
These studies were carried out under Contract NCI-CP12338 with the National Cancer In s titu te and sub-contract
74-22-106002 with Tracor J itc o , Inc. (Contract N01-C?43350 with NCI).
-2 7 STUDIES WITH CHLORINATED DIBENZO-P-DIOXLNS. POLYGROMINATED BIPHENYLS AND POLYCHLORINATED BIPHENYLS
IN A TWO-STAGE SYSTEM OF MOUSE SKIN TUMOR IGENESIS:
POTENT ANTI-CARCINOGENIC EFFECTS." David L. ie rrv .P h .D ..
Thomas ). Slags. Ph.D;, Oak Ridge N at'l L ab.. Oak R:cae. T N ..
JohnDiGiovanni. Ph.D., and Mont R. Juchau. Ph.D.. L'rjv. of
Washington. Seattle. Wa.

DOW37317Z

n r" -.^t.’.ilaw.h .»x-n1*. »

-2 4 SEUF9PATHCLDGY CF "SPINNING SYNDROMS" INDUCED BY PRENATAL
: ntc:;: cat : on with a ? cb in mice by s . m. Chou, md. ,p h D ., t
Mi i k e . MO.• W.M. P ayne, MO., and G .J . O a v is, PhD., West Va
Cniv Med C e n te r, M organtown, w .v a ..

nr III ...... .

Au.

A

Topical pretreatment with 2 .3 .7 .3-teaachlorodibeszo-p-dioxin
(TCDD) or Aroelor 1254 (PCS1 inhibited the skin tumor-imeating
ability of the polycyclic aromatic hydrocarbons (?AH1 7 .12-dimethylbensfalantbracene (DMBA1 and benzo(a)pyrcne ( SaPl in female
CD-I mice. TCDD inhibited skin tumor-lnicanon by DM5A in a
time dependent relationship. TCDD and Aroelor 1254 were very
weak skin tumor initiator] at doses of 2 ug and 100 ug respectively,
topically followed by 32 weeks of promotion by 12-0-tesradecanovlphorboI-13-acetate (TPa ). The potency of the anti-carcinogenic
effects resulting from pretreatment with TCDD and PC3 correlate
with the ability to induce epidermal monooxygenase enzymes that
convert the PAH into reactive electrophilic intermediates and with
the marked reduction in amounts of DMBA bound covaier.tlv in vivo
to epidermal DNA and RNA but not protein. ’ Rescarcn scunsorcu
joinrlv by NCI Grant CA20Q76 and Div.of Biol, a Environ. R e s..
u.S.DOE under contract W-7405-ecg-2b witn the U.C.C.
-2 8 REPRODUCTIVE EFFECTS OF HAIOGENATED AROMATIC HVOROCARSONS ON NONHUMAN PRIMATES by J.R . A llen, O.A, B a rso tti,
l.K . lambrecht and J.P . Van M iller, Univ. of Wisconsin,
Madison, UI.

^

Polychlorinated and nolvbrominated biphenyl mixtures and
'2.3,7,8-tetrachlorodibento-o-dioxin have been fed a t low
levels to female rhesus monkeys over an extended Period.
Their general e ffe c ts on reproduction are n u ite sim ilar
with modifications in serum e strad io l and progesterone
being associated with changes in the menstrual cycle.
D iffic u ltie s with conception, early abortions ana b irth
of undersized infants were observed. Slow growth, tnymic
involution, and increased su s c e p tib ility *.o infection
were present in the postnatal Period. Altered learning
and benavioral patterns of the Infants were also noted.

0002357
^___

5785

iviutagenic Effects of TCD D
on Bacterial System s .
By S . Hussain, L. Ehrenfcerg, G. Lofrolh and T. Gct'vall, Radiobiology Department,
Wallenberg Laboratory, University o( Stockholm , Sw eden

I
TCDD, which occurs as a contaminant in the herbi-ide 2,4,5-T and possibly in other
compounds derived from 2,4,5-trichlorcphcnol, is among the most toxic substances
known. 2,4.5-T is widely used ns a defoliant. The authors of this report claim that TCDD
is rnut2 genic. Their experiments indicate that the muugcnic effect of TCDD is caused
by intercalation with DMA.
A^
2,3,7, 8-tetrachlorodibcnzo-p-dioxia
(TCDD) occurs as an impurity in 2.4.5trichlorophcnoxyacctic acid (2,4,5-T)
and possibly also in other compounds
derived from 2,4.5-trichlorophcnoi (I).
TC D D was early implicated as the cause
o f chloraene in workers subjected to
occupational exposure in 2.4.5-Y plants
(2, 3). TCDD ccntaminatio.i as high as
30 ppm in 2,4.5-T has been reported
(4), but 2,4,5-trichlorophenoi that is
currently produced is reported to con­
tain less th-n 3 ppm (1). In addition,
TCDD is formed when vegetation
treated w'ith 2,4,5-T is burned (5).
TCDD seems to have an exception­
ally high toxicity. An LDeo (a dose leth­
al to SO % o f tha population) o f 0.6
/ig /k g has been reported for female
guinea pigs (6). A single oral dose of
10 /rg/kg in rabbits is lethal and 1 uSJ
kg causes serious liver damage and
chloraene (I), whereas rats seem to be
less sensitive in view of the fact that
ten consecutive oral doses o f 8 n g;k g
each, given daily during pregnancy,
caused death only in one out o f eight
treated animais (7). In the same study
on rats (7), foetal toxicity w as observed
at a dose level o f ten consecutive doses
o f 0.125 f t g /k g each.
y

s

c

/

© '-t
vn

i*4 S i
V I ^

2. Reversion to histidine prototrophy
1. Reversion to streptomycin inde­
in Salmonella typhim urium strains de­
pendency in EschericiJc colt Sd-4. A
ficient in uv excision repair. A treat­
test procedure which lias been described
ment procedure similar to that used for
previously was followed for the most
E. coli was followed except mat cniy
part (9, 10). The required amount of
0.1 ml DM SO was added to 0.9 ml of
TCDD (11), dissolved in 0.2 ml dime­
the bacterial suspension. For the deter­
thyl sulphoxide (DMSO), was added to
mination of survival, the treated bac­
the bacteria in lag phase susoended in
teria were plated on nu'.rien' agar. The
0.S ml of a 0.1-.M sc-dittm phosphate
minimal medium described by Hart­
and potassium phosphate buffer o f pH
man (12) and supplement according to
7.0 containing 4.0 g;l sodium chloride
Ames (13) was used for the determina­
and 0.2 g/1 magnesium sulphate. The
tion of mutants. The mutant coicnies
treatment was carried out at 57°C for
were counted_after four days.
one hour and was stepped by diluting
. i) S. typ h im u riu m strain T A 1520.
ten times with buffer solution at 4°C .
This strain has a base substitution in
The results are summarized in Table
one codon for the first enryme o f the
1 and show that there is a high muta­
histidine biosynthesis. A one hour treat­
tion frequency at a TCDD concentra­
ment with TCDD concentration ci 1
tion o f about 2 /ig/m i and at a survival
and 10 u g /m l resulted in a «yrvival o f
which excludes a prc.srcu-'iai sc lectio.;
JO and less than 1 per cent. resp. N o \
o f spontaneously occurring mutants.
mutations above the spontaneous level
Parallel tests with acridine jotar^tj
were found in this ranee.
showed that the mutation frequency
ii) $. typ h im u riu m strain T A 1532.
could be raised to 50 per 1C4 at a concentration o f ¿<3—70 «y. nil and a sur- v This strain is assumed to nave a base
J * pair in excess o f the aminotransferawvivai o f about 50 per cent.
— IT
locus. The effects cf-ncridine mu.-tari
•ll?N • \wV*
O
rfTCR-ITOl^uiisolved in both DM SO
TCDD
\ Mutation Number o t
and water, were investigated parallel
concentration Survival (;caucn:y muianu
to the tests with TC D D dissolved in
»C/ml
U
x I0~' ' counted
DMSO (Fig. 1.). N o increased ir.uta22!
7
(100)
tion frequency was opserved at concen0
3
6
100
tiations o f TC D D and acridine nu'turd
0.5
90 \
which caused a survival in the range cl' .-O r
I#
■
3
A»
50— 100 per cent. i.c. at concentration
90 ]
!•
10 v
34
is ;
less than 2 —3 //g .m l. A t higher con­ ' S
ro
46.
11 ..2 5 6
centrations and lower survival w c mu­ .¿a»
I
(201
4
3
tation frequency increased, reach.i'-:
10s— ID* per 10' surviving bacteria for i to
Table 1. Fieveriisn to '.»•eptomyci.i indeTCDD around one per cent survival.
penuency in £. co.'/ J«M treated with
Acridine mustard, dissolved both m
1CDD.
DMSO and water, gave sbphily lower
*) D u u liu to «ampie* ci'irtn*) treatment. A

5*

Studies or! genetic effects of T C D D
have not been published except for a
preliminary r.ntc on 'Aline date -from
the present report t.s). The re........ for
the initiation of the experiments des­
cribed here is that the three rir.i; -tin c­
ture of TCDD resembles that of acri­
dines and oilier compounds w his.lt' in­
duce mutations through intercalation

fw

5786

p r e n c i a» number
of mutants per I0 1
surviving cells,
versus survival in
S. typhununum TA
1532.
° treatment with
TCDD in DMSO
• treatment with
•cridir.e mustard
In DMSO
• treatment with
. acridine mustard
In water

10 •

19 ■ v
100

50

10
5
SURVIVAL. PER CENT

05

(Hill
). Kimnua and K . II S-Su1/’. D erm alolofieis 11?. pp. J 4, — S-t', <|-/? 7 |.
It. Iliu.-r. K . II. S.he'.t a n j U. Spicselberg. A r e l.ie li.r ( it-* i V - i i * m ul
G e * tr l\ m i c e re la. pp. 5)v—??? (tSS!).

K. Dtar.c Courtney, O W. G.i>!:r. M. D.
Horan. II. L. fa ir. K. It. II-.!-.-» and I.
Mitchell. Scien ce li.J. pp. :M— it-6 ( tv701.
N. P. Ouu-lloi, C . Saint-Ruf. P. U.^ol
and M. Mur.gane. Complet R end u s d es
s è m e s d e l ’A e eJ em .e des Sciences Scries
D. 273. pp. 7)3—711 iw :n .
Sature 231. pp. 2 H —211 (1971).
C. L. Sparschau. H. L. Dunn and V. K.
Rom:. Food end Cosmetics Toxicology
9, 'p p . <95— 112 (1971 >.
S. Hussain. L. Ehrenberg. C . Lo.'roth and
T. Gcjsall in Fen-n/snor (report by an
et pert committee c i the 5>»ed.»h National
Poisons and Pesticides Dîard. Stockholm,
.1971).
L 7 unôazky and L. Ehrenberg. Mutation
Research S, f-p. 224—lès (I9Ù4I.
C. Ahnstrôm. L. Ehrenberg. S. Hussain
and A. T. Natarajan. Mutation Research
10. pp. 247—2? J 1147)).
A sample of TCDD c i 'ri per cent purity
obtained from FDA. v.'ashi.-.aten D. C.
P. E. Har.man tn Gcnette Studies uith
Bacteria (Carnegie Institution c i Washington. Washington. D. C - Publication 612.
19*67 pp. 35—6!.
B. N. Atpa-s in Chemical M utèrent. VoL
1» A Holbcndcr. Ed. (Pie.tum Press. New
York. 1471) pp. 267—2 s2IL Markovich and R. Laiartet. Advances
in Blolovcci and Medical Physics 6, pp.
7S—94 (195S).
B. Hcinemnn in C h cn o cn l .\fu m em s Vol.
L, A . Holber.der. Ed. tP:--num Press,
New York. 1971) pp. 2:5—266.
N. P. Buu-Hc-L Do-Phuoc Mien. G. SaintRuf and J. Servoin-Sideir.e, Comptes
Rendus de séances de rAcademie de
Séances Séries D, 272, pp. 1447— 1450 '
(1371).
17. G. R. Higginbotham. A. Huang. D. Fireseoe. J . Verreli. J. Rees and A. D.
Cur.pbell, Sature 220, pp. 702—3 (I96à).
R W. Boader and H. 3. Bauer, Industrial
Medicine end Surgery 20, pp. 2S6—2>0
(1321).
19. J. G. Vos. J . H. K oerun. H. L. ve.-i drr
üaas, M. C. ten Houver de Urat-w and
JL H. de Vos. Food end Cosmetics Tosucdogy 8. pp. 625—633 (1970).
20 .& Katsuki (Chairman) Fukuoka Acta
Medico 60. pp. 4)3—553 (19691.
.Tais investigation has keen supported by
pants from the Swedish Atcmtc Research
Council and the Hirrta-ll.-uius huunduion. The authors are gratetul to Dr U.
X Ames, University s( California. Berki y for supplying the S . r>ptnmurium '
o ain s. to Dr D. Tirevtone. Fo>;d and
Drug Administration, Washington. D. C.
far supplying the TCDD sample, a n j to
Dr II. 3. Creech. Institute uf Cancer
Research, Philadelphia for supplying the
ICR-170.
Received November 1, 1971.

3. Prophage induction. £. coli K-39 TCDD demonstrated here fevidently a
cells were harvested in log phase, consequence of intercalation) and the
washed, diluted to about J0 > cclls/ml efficient induction of microsomal hy­
and treated for 0.5 hour under condi­ droxylases (16) suggests lines of inves­
c
tions described above: 0.1 ml DMSO tigation which could expiain the extra­
was added to 0.9 ml of the cell suspen­ ordinarily high toxicity of this com­
C
sion. Following the ten-fold dilution pound.
Besides TCDD, other chlorinated
with buffer solution at the end of the
treatment, the bacteria were once more dibenzo-p-dioxins, originating from
U3C
washed with buffer solution and then chlorinated phenols, may become en­
suspended in I ml nutrient broth and vironmental pollutants (17). Occupa­
V \c
incubated for 2 hours at 37SC Plating tional exposure to pemachlorophenol
was done with £. coli K-19 as indicator of unknown purity has been reported
on an appropriate mqciun (14). The to cause chloracne-Iikc symptoms (IS).
LPC
plaques were counted after iS hours. Some of the widely used commercial
The applied DMSO inhibited the re­ formulations of polychlorinated bi­
lease of phage in comparison with the phenyls (PCB's) contain impurities of
control test without DMSO. whereas chlorinated dioenzofurans (19) having a
TCDD at a non-toxic concentration of three ring structure resembling that of
S.
0-5 pg/ml partly reversed this effect, dibenzo-p-dioxin. Although no ana­ I
producing an increase in the number of lyses with respect to impurities have
plaques from 1.9 X10 5 per ml in the been reported it is noteworthy that a
DMSO control to 5.3 X 10 ' per ml in widesptead in.OAicaiU.i ih e to inges­
the case of TCDD (Table 3).
tion of a formulation of polychlorin­
Treatment of 5. typhimurium TA ated biphenyls was associated with
1530 with TCDD did not show any chloracne-like symptoms (20 ).
mutagenic effect, whereas high muta­
21
tion frequencies were observed in the
strain TA 1533 which is known torevert by frameshift mutation. This in­ Table 2
dicates that an acridir.c-iike behaviour
• Bacteria
causes the genetic effects of TCDD.
surviving
TC D D
< The prophage induction was diffi­ concentration treatment, Plaques
No./m l
No./m!
cult to detect, but there seems to be 0/ pg/m l
x 10-»
x I0-»
certain activation of rluce in the pres­
ence of TCDD and DMSO which is not 0 (w ithout 4.4
7J
higher than the control value without
.
D M SO )
DMSO. Although it is difficult to 0 (w ith
IJ9
4.2
disentangle the effect of DMSO from
0002480
that of TCDD. the data couij be in­ C 5 DM SO ) 4.1
5.4
terpreted in terms of TCDD’s weak
T
a-Î3b'«(/*)2. treated
Prophaijo activation*) in £. co/>
1.8
2J>
prophage-inducing actum, in view of 1I J
K
»¡th 1COD.
p H q h
2.6
2o2
the similar action of acridines (15).
*) Ihe term "prepharys induction" is pDr-f O i
25
1.7
1.6
poiely avoided here.
The strong mutagenic action of
Q)

DO
on

5738

00
co

»

!> Sri

I: Vnjmr
l HI.

'unt-ilN i»f
in*tiv»ly. A't’ir
■•<•1 n f »tilTrvrnt
lr.7i ()(«•/<tryng
l . r .lr. <-t nl:
fllivli'tio

:o.

nnd Hliiimil.

r r 0.1 : M rc h a-

IMisint' «*( Oli"”
H ea lth l.V.1 1.1h*m H A .

c t nl:
o( Inlm ralnry
■rmm'iil 17:72l>nlinn ln \i> itirn
arm aeol
ctTrct.i o f nerni-l Ip m iio Up o n

la Inoculala. C
'.inn V I.: jlii»tf nil- iiy iiliim i-

inai.

form uli U-livilo
oi^pniiisin. Gig
i(.

miiiiiolic

• T h cr 127:25»c t nl: Aflrpn-

A reh Intern
•led nroloW p-

!CJ- I74:U-15>.
,led nrrlnlflph 21:354-337,
■ TW .

c t nl:
Sensitive new
: inni (inn nml

Anal Chem
enninp «-nsi• drnj'one lest
in a ir. A nal

s prodi tel ion
-litflips iisinf:
■..338, 15)70.
od o f d elcr>kc: A p p lin i' lieti/.ril nml
ler It: Nnsnl
pxpprimrnlfi:

. «moke nip
i. 1371.

i

A Uevinv Including Chlorinated Dilii-nzodioxins :ind Chlorinated Dilien/.oruians

i

Ileiiu .e />. h'imlirm igli. M l ) , C h m n h lrr, (la

Various chlorinated technical compounds,
namely, 2,4.5-trichlorophcnol, 2,4,5-trichlorophcnoxyocctic acid (2,4,5-T), and European ch lo ri­
nated biphenyls (Phcnoclor DP6 and Clophcn
A60). have been lound to be contam inated w ith
trace amounts o f chlorinated dibenzoturans o r
chlorinated dibenzodioxins. Toxic tat which pro­
duces hydropericardium in chickens also contains
chlorinated dibenzodioxins. These and other tech­
nical chlorinated compounds such as the tech­
nical pentachlorophcnol have been im plicated in
causing chloracnc, liver disease, teratogenicity,
x-discasc in cattle, and chick edema. The litera­
ture on the toxicity o( the chlorinated technical
compounds is reviewed. It is mentioned that
2,4.5-T and the chlorinated biphenyls also induce
porphyria. W hether the various disease entities
arc caused by the contaminants, com binations
o l the chemical and its contaminants o r by the
chemicals themselves needs further evaluation.

Chloracnc

This disease is descrilx’d by the formation
of comedones with or without cysts and
pustules. The follicular orifices are filled
with sebaceous and keratinous material.
Melanosis and a secondary inflammatory
reaction may exist. Chloracnc was descrilx-d
for the first time by Herxheimer' in 180?)
who thoupht it was produced hy free chlo­
rine. Waucr5 in 1918 and Tcleky 1 in 1927
suppcslcd the term ‘‘pernakrankheit.’’ They
felt that this skin disease, was produced by
certain chlorinated hydrocarbons. Jones and
Aldcn' were amonp the first to rcjx>rt a case
of chloracnc in a 26-year-old male Negro
who worked for three years distillinp chlori­
nated biphenyls. This patient also com­
plained of lassitude, loss of appetite, and
loss of libido.
7
Chloracnc is one of the most frequent '
R l'.(T .N T L Y several chlorinated com­
pounds, such as (he jxilychlorinated biphe­ forms of occupational dermatitis, and many
nyl and 2,4,5-trichIorophcnoxyncctic acid eases have been reported in llic United
(2,4.5-T), have received a great deal of at­ Slates as well as in Europe, particularly
tention. Folychlorinatcd biphenyls (PCB) Germany and England.
Tcleky 5 reported in 1949 that ho j>orson.nlare incrcasinply found in our environment
and 2,4,5,-T lias been used extensively as ly had seen at least "150 to 280 workers” in
an herbicide. Occasionally these and oilier four different factories with chloracnc. He
chlorinated technical compounds have caused stated that liver disease occurs independent­
ly from chloracnc, and usually manifests
diseases tliat bear similarity to each other.
Some of these chemicals have been found itself after an exposure time of four to six
to lxi contaminated with chlorinated diben- months, but may occur in as short a time as
zofurans or chlorinated dibenzodioxins. The seven weeks. Occasionally loss of appetite,
various diseases produced by one or more of nausea, and edema of the face and hands
the different technical compounds arc dis­ arc the first symptoms. Abdominal pain and
cussed in this paixir. Attention is focused on vomiting follow, and then jaundice develops.
the various contaminants and their possible At autopsy, acute yellow atrophy of the
relationship to different diseases in a variety liver is found. He cites Flinn and Jarvik"
of species. The need for further well-coordi­ who reported eases of acute yellow atrophy
nated epidcmiolopical and experimental ani­ with fatal outcome following exposure to
Halowaxes. Halowaxes, which are chlorinat­
mal studies is pointed out.
ed
naphthalenes, as well as the FCB (AroS iihm iltcfl for pnblim lion Oct 8, li>71; nrcejitrtl
clors)
were increasingly user! after 1950 to
M n trh 3. 1372.
From the Chnnthlec Toximlof-v Ijibom tory, F.nvi- insulate cables. TelckyB cites 'm any other
roninenliil Protection Ai:enry, Clinnihlee. (In.
ease reports (Drinker et nl.7 Jones,* GreenIteprint request* to Chnnthlro Tnxirolocv lolm m lory, Knvironmenlnl Protection Apenry, 4770 lln- burg ct nl ,9 Cotter , 10 Mclicchtchio and
Robertson ,11 and Collier19). Von Oeltinford Hwy, Chnmblcc, Qn fJtXMl (D r. KiitibrntiRli).

7 7. Z Z L Z I A O Q

•''ll 1*1

Toxicity of Chlorinated Hydrocarbons and
Related Compounds

Arrh Environ Health— Val 25, Aug 1972

5788002;}

73

r.!i;

t o x ic it y

n r iiY i> i:n cA iiii(h \'S

i <i m n n n tir .it

POW37 3323

the r.ahliil ear. (The rabbit. ear
i:i n;vfl
ns n KTriTiiiii); Inst In determine whether n
compound is likely In prorhir»* chloracnc.
’Ilie chemical, diu’inlved ill jirnpylcno. glyeol,
is painted nil the rabbit ear. If Ilie. leal is
positive, erythema occurs williin Iwn dnys
nnd in a few weeks hyperkernJosis develnps.) 'IIin raliliil skin nfTers ns an experimenial method that will indicate Iho ability
of Kiihslnnees to produce arneifnrm derm.nlilis in man .31 I’ure 2,4,5-triehlorophenol did
not affect the rabbit car, and neither did
1.2.4.5- lctmchIorobcnzcne, the storting ma­
terial in the manufacture of 2,4,5-lrichlorophcnol. However, technical 2,4,5-lrichIorophcnol which was used in the production of
2.4.5- T did produce hyperkeratosis of the
rabbit car. It was concluded that by-prod­
•THftK.M
twndihwiwIioytfl
ucts which had resulted during alkaline hy­
Chemical structure of a chlorinated dibenzodioxin drolysis of 1,2,4 1.'i-tclrachlorol>cnzcnc were
and a chlorinated dibenzofuran.
the causative agents.
Kimmig and Schulz3" isolated several con­
descrilies the occurrence of taminants
and found that some, of them,
chlornaic in workers cxjxwed to these chem­ namely tctrnchlorodilxmzofumn
at
icals. He cites several references which indi­ a concentration of 0.0.l5% ns well(Figure)
ns
tetracate that dermatitis is tlic result of the local chlorodibenzodioxin (Figure) at a concentra­
efTect or direct contact with the chemical, tion of 0.005%, produced hyperkeratosis in
while the toxic hepatitis results from absorp­ the rabbit car. A hcpntoloxic cfTcct was
tion.
also observed with these compounds. They
Chloracnc has been rcjxjrlcd in workers also found that pure pcntachlorophcnol did
exjposed to chloronaphlhalenes, chlorobi- not produce hyperkeratosis of the rabbit car.
phenyls, chlorodiphcnyloxides, certain pe­ Chloracnc was observed in workers of a
troleum products, and solid chlorophcnols .14 West German plant who produced tech­
..More recently Meigs ct al,B and Hofman nical pcntachlorophcnol from hcxachloronnd Meneghini10 rcjtorlcd 14 nnd 13 cases of benzene .33 The technical material also in­
chlorncne, respectively. Exjtosurc to chlori­ duced hyperkeratosis in the rabbit car. It is
nated biphenyls was established in most possible that technical pcntachlorophcnol
of tliesc cases. Birmingham 17 observed contains toxic impurities, particularly since
chloracnc in 15 employees wlx> had painted- pcntachlorophcnol is produced by alkaline
flat sections of glass with enamel and then hydrolysis from hcxachlorobcnzcnc. Dibcnbaked it. The enamel had Aroclor (a PCB) zodioxin and dibenzofumn themselves do not
incorporated in it. Plcwig1* produced chlor- cause liver necrosis or hyperkeratosis when
acne experimentally on the upper back of applied to the rabbit ear; however, they
eight male adults hy applying Hnlownx become highly toxic when they contain three
1014, pentachloronaphthnlene, and hcxachlo- or more chlorine atoms .30 Some of the workmnaphthnlcne.
ers with chloracnc also develop eye irritnBauer et nP° rej>ortcd chloracnc in work- lion, hepatotoxicity, intolerance to alcohol,
ers Hint handled technical 2,4,5-lrichIoro- neuromuscular symptoms, porphyria cutaphenol. In some of these workers neuromus- nea tarda, nnd |Psychologic alterations .3-1 Accular,weakness, j»sychopnlhologicnl changes, cording to Braun,•’* chloracnc scents to roblcpharoconjunclivitis, nnd liver involve- suit from direct contact with the chemicals
menl also occurred. Kimmig nnd Schulz 30 producing i t Crow ,30 on the other hand,
found that pure 2,4,5-lrichIorophcnnl did points out that chloracnc can also bo pronot produce hyperkeratosis when applied to duerd by systemic absorption.
Arch Environ Health— Vo! 25, Aug 1972

0002

X-disCMSC

linphlhulcni*!;, bill also with petroleum prod­
ucts such ns crank «rase nil, 'Jim toxic prod­
ucts were excreted in the milk and produced
x-disense in the (•-•lives drinking the milk.
'Jliey cited other authors who were able to
produce the disease with a complex wood
preservative and a lubricant.

Ar ch Environ Health

DOV/373324

Thin disease in raillc was described by
(liaison7" in l!M7. A few years later evi­
dence was published showing (.bat highly
chlorinated naphthalenes cruised the dis(’allle wilb x-diseasc show a rapid
Ilell -’13 tested the ability of various cumdivline in vilaiuin A plasma levels. Syinptoms of poisoning include excessive lacrima- pounds of the chlorinated naphthalene
lion, diarrhea, polyuria, marked salivalinn, group to produce x-diseasc. lie discovered
and discharge from (lie nostrils. A chronic that dichioriailed nnd IrichlorinaU-d naph­
couch. ]Mior apiKilile, nnd numerous red thalenes did not produce (he disease while
maculae in the huccal mucosa develop, anil lelrnchloronnphthnlcnc liad an cfTect, and
hyperkeratosis of the skin follows. Micro­ Uie higher chlorinated nnphllialetie, pen Liscopic examination of autopsy material chloronaphthnlcne, hcxachloronaphllialono,
shows central lobular degeneration of liver hepLichlomnaphlhalene, nnd odachloronnphcells with bile duct proliferation and dila­ thalcnc, caused severe disease. Octachlorotion of the glands in the wall of the gall­ nnphlhnlcnc was less toxic Uian hcxachlorobladder. Cystic dilation of the collecting naphthalcnc and hcptachloronaphthalcnc.
tubules of the renal cortex, with a moderate
degree of fibrosis nnd degenerated cells in the
Chick Edema
pancreas, was also observed.1®In sheep, ne­
crosis nnd cirrhosis of the liver, damage to
In 1957,n,-1G a disease occurred in a large
the nephrons, and squamous metaplasia of number of chickens which, a t first glance,
the endometrium were the principle findings seemed to represent an epidemic. It was
after ingestion of feed containing highly soon discovered that the residues of certain
chlorinated naph^haIcncs.-,,' Huber and dislillcd animal fats produced the condition
Link™ fed hexachloronaphlhalenes to young when they were added to the chicken
swine and produced degenerative lesions of d ie t 30.37 The disease was called chick edema
the liver and kidneys and hyperplasia of the because it manifests itself with hydroperi­
vaginal epithelium with keratin formation. cardium and ascites in chickens. Ducks and
A depression of the vitamin A plasma level turkeys experience a reduction in growth.
was also observed.
Allen and Lalich3* produced hydroperiDrinker and co-workers7 found that the cardiuin and ascites in chickens when
liver of rats was affected when the ani­ enough toxic fat was given to kill most of
mals were fed high doses of tclrachloro- the birds in five weeks. ("Toxic fat” is a
naphihalcnc,' pentachloronaphlhalene, and term used for fat found in chicken feed that
hcxachloronnphtlialonc or chlorinated bi­ induces chick edema.) When the concentra­
phenyls. When Schocttlc et al 31 fed hexa- tion of toxic fat was reduced nnd fed to the
chloronaphtlvilcnc to rats, they observed mild chickens for 150 days, hydropcrcardium and
to moderate fatty degeneration of the liver ascites developed less frequently, but testicu­
with ccntrilobulnr vncuolalion of hepatic lar hypoplasia became apparent Simpson el
cells. Degenerative changes were observed in al3B described proliferation and hypertrophy
the kidneys and the skin showed hyperkera­ of the vascular endothelium and passible
necrosis of hepatic and bile duct tissue in
tosis.
It can be concluded from these reports chickens and turkeys. When monkeys (M a­
that highly chlorinated naphthalenes pro­ caco mulatto) were fed toxic fat, alopecia,
duce liver changes in several species. In subcutaneous edema, decreased total serum
cows, x-disease of the skin, together with a protein (with a reversal in the albumin-glol»drop in the vitamin A plasma level, is one of ulin ratio), and reduced hcmnto|miesis nnd
the leading manifestations of the disease. It sjK*rmatogen«,sis dcvolojied. Gastric ulcers
is possible that Uic vitamin A deficiency occurred in fifi% of the animals. Focal areas
represents a manifestation of concomitant of necrosis nnd degeneration were observed
in the liver; and the bile duct epithelium
liver disease.
Sikes et al 35 found that x-diseasc could be was found to be affected when it was exam­
produced in cattle with highly chlorinated ined under the electron microscope. Dilnta-

9

— Vo! 25, Aufi 1972

5791
2M 2375

Tnblo 1.— Dlscnso Rcsultinfj From Expoiuro
P ath o lo g ical rin d in g *
of Iho (.Ivor

Chlorjtcno
C ertain ptMtnl'Mim p ro d u cts
C hliuotM iililhtilniPs
(m ainly p rid a ,
Itrx a, am i hoptn)
ChlurnhiphonyJs
C lilfirotltphniyloiltlcs
Cltlo’ophnm ils
(l«*cfmicaf 2. 4. 5-lrichlorophcnol,
prnlaehlorophftnol)
T echnical 2. 4, 5-T

O irlh O efects and
Lothal F actor

CMIor!natr*i| n a p h th a le n e s C ertain lcclmir.nl 2,4 , 5-T
C hlorinated hlphonyls
Plldflod m aterial
T echnical 2.4, 5 *T ow l
from toxic fat*
o th o r chlorophonols
Polychlorinated
biphenyls

In C attle
Highly ch h irm alrd
n a p h th a le n e s
P etro leu m p ro d u c ts

* ''T oxic f a t" is a term iisnd lor ta t found in chicken feed th a t in d u c e s chick n d em a.

7332

(ion of the heart and hypertrophy of the that the Aroclors can produce chick edema.
cardiac muscle were: also present :40
The Aroclors (FOB) arc used primarily as
Toxic fat is not the only product callable dielectric fluids for capacitors and trans­
of producing the chick edema syndrome. formers, as industrial fluids for hydraulic,
Chlorinated biphenyl products, used ns a for gas turbine and for vacuum pumps, and
plasticizer in a paint, have caused hydropor- as heat transfer fluids. They arc also used as
icardium and ascites in chickens ,41 and two plasticizers and are widely distributed in the O
of seven Bengalese finches fed PCB dc- environm ent 40 Aroclor is a trade name un- Q
vclojicd hydropcricardium .42 The seven der which these compounds arc marketed in ^
finches of a total of 56 birds died during the the United Slates. They can be found in 5 5
course of the experim ent A mixture of pen- synthetic resins, synthetic and natural rub­
Inchlornnnphthnlcnc and hcxnchlornnaphtha* bers, cellulose resins, paint varnish, wax,
lone, when fed to chickens, resulted in chick asphalt, and in allyl starch. They have been
edema.4"* The toxic fat which contained the employed for dust prevention, moisture
chick edema factor was studied intensively. proofing, scaling, impregnation, and vapor CJ"t
Flick et nl 44 sejianited a purified crystalline suppression to prolong the residual life of
concentrate which produced chick edema and insecticides. They increase the toxicity of
decreased the haldwihilily of injected e.ggs. dicldrin and DD T in •insects.00 Depending
Embryonic deformities were also produced. on the am ount of chlorine they contain they
Flick et al 40 wore not able to produce testic­ arc assigned certain numbers. Aroclor 1260
ular hypoplasia in cockerels when they fed for instance, contains 60% chlorine, while
low doses of toxic fat ( 0 .6 % and 1 .0 % ) to Aroclor 1242 has an approximate chlorine
the Cockerels for 12 weeks, as had been re­ content of 42%. Miller ,01 in 1944, tested a
ported by Allen and Lnlich.3*
commercial chlorinated biphenyl with 42%
In 1967, Cantrell et al4# reported that chlorine and observed liver damage in rab­
1,2,3,7,8,9-hexachlorodibcnzo-p-dioxin was bits, guinea pigs and rats. Skin changes
one of the toxic compounds producing chick were observed in the animals who received
edema. Tom ita et al 47 had shown earlier sulxmlancous injections and application of
that the healing of pcntnchlorophcnol pro­ the material to the skin. The skin lesions
duced octachlorodilK'n/o-p-dioxin. Higgin- produced by subcutaneous injection were
Ixtllvim et a l 10 showed llint (he chick edema histologically similar to those of chloracnc
factor represented chlorinated dilionzo-p-di- in man. Direct application to the skin pro­
oxins. The two principal comixnmds iso­ duced inconsistent lesions compatible with
lated were 2,3,7-trichlorodilx'nzn-p-dioxin low-grade irritation. Von Oeft¡ngon,34,,',300■:" ,7,
and 2,3,7,8,-tetmch lorod ilxinzo-p-d ioxin. Tire discusses some of the earlier work on the
authors suggested, as a possible source of toxicity of (he Aroclors in his lxrak T h e
contamination, fats and fatly acids contain­ llalogcnated Hydrocarbons, Toxicity a n d
ing commercial chlorophcnols. When crude Potential Dangers. Nishizumi52 olserved
fats and tallows arc heated to produce fatly changes in the livers of mice and monkeys
acids chlorophcnol residues might be con­ after fcerling them chlorinated biphenyls.
verted to a chick edema factor.
Tire author undertook this work because of
It was mentioned
earlier in this article an outbreak of poisoning that involved at
/ /
Arch Environ Health— Vol 25, Aug 1972

5792

Tnblo 2.— Toxic Contam inants Found in Somo
Technical C om pounds*

. ¡* to Technical Compounds

2f4t 5*irlchlorophcnol

C hick Criem*
luxlr, fat«
C hlorinated IWphonyl*
M filiim of prnlnchloro*
nnpldhnlcnfl
a n d ItcxAchloro*
nA phthateno

2. 4, 5-T

ÎT etrnchlorndihnn/nlurnn
ITctrnchlorodJIjcnrodloxfn
fT etrachlorodlbcnzofuran
IT ctrachlorodihcnzodloxin

11. 2. 3. 7, 8.9 hexachlorodlbenzo-p-dlosln
12, 3, 7,8 tctrachtorodlbcnzo-p-dloxln

Toxic (At

1 2, 3,7 trlchlorodihenzo-p'dioxln

E uropean chlorlnnlcd
biphenyls (P hcnoclor
DPG And Clophen A 60)

fT ctrnchlorodibcnzofuran
<P cn tach lo ro d ib cn zo fu ran
1H o x sch lo ro n sp h lh alen e

* Listed In Tablo 1.

PO W 373326

least GtX) people in W estern Japan who had by Emerson ct al,T found a teratogenic effect
ingested rice bran oil that had been contnm- of technical 2,4,5-T in rate. Apparently, the
inalcd with clilorobiphenyls, n Japanese technical 2,4,5-T was contaminated with
r e » (Kancchlor 400). Tho clilorobiphenyls 27 ppm of 2,3,7,8-tclrachlorodibcnzo-p-dioxin.
leaked into Hie rice bran oil through pin­ In the study published by Courtney ct al ,66 it
holes in a piixj used for heat exchange in the was staled that the technical 2,4,5-T which
manufacturing process. An increase in pig­ was found to be teratogenic in two strains of
mentation and abnormal grayish dark- mice and one strain of rats was con­
bnnvn pigment deposits in the skin of still- taminated with 30 ppm 2,3,7,8-lclmchIoroIxini and newborn infants was observed.™ dibenzodioxin. Emerson ct al6T lasted 2,4,5Stillhirllis were unrelated to FCB ixiisoning T that only contained 1 ppm 2^,7,8-le.trnof the mother. Adults showed chlomcnc, urt- chlorodibcnzo-p-dioxin at comjinmblc dosage
orexia, fatigue, edema of the eyelids, cheese- levels and did not elicit a teratogenic ctTcct
like discharge from the mcilximian glands, This finding was substantiated by Sparschu
and dark-brownish pigmented nails .64
ct al6n who studied teratogenic ctTcct of 2,Vos and Koemnn 66 studied the toxicity of 3,7,8-tctrnclilorodibcnzo-/Mlioxin in the rat.
several polychlorinated biphenyls in chick­ A slight ctTcct was observed a t a dose of
ens, quail and rats. The authors compared 0.125/ig/kg/day when the material was giv­
the toxicity of two European commercial en by gavage from day 6 through day 15 of
polychlorinated biphenyls (Phcnoclor D P 6 gestation. At the level of 0.5pg/kg/day, the
and Clophen AGO) and one (Aroclor 1260) effect was quite pronounced. Courtney and
which was produced in the United States. Moorcr,° were able to produce cleft palatas
The European compounds showed higher and kidney malformations in three strains of
toxicity and produced ccntrilobular liver ne­ mice with high doses ( 1 0 0 m g/kg subcuta­
crosis. Chick edema was a common finding neously daily from day 6 to 15 of preg­
with European compounds and rare with nancy) of analytical grade 2,4,5,-T that
Aroclor 12G0. All three compounds pro­ contained less than 0.05 ppm 2,3,7,8-tclraduced porphyria. Chemical analysis of these chlorod ibenzo-p-d ioxin.
three compounds revealed tctrachlorodiKcplingcr et alni reported a decreased
ben/.ofuran, pcntachlorodibcnzofuran, and survival of pups in rat reproduction studies
hcxnchloronaphlhaicnc as a contaminant in when fed 100 ppm of Aroclor 1242 and 1254,
the European samples but not in the sample and poor hatchnbilily of eggs from chickens
from the United Stales .66 Since- hydroperi­ fed 10 or 100 ppm of Aroclor 1242 or 100
cardium occurred occasionally in chicks fed ppm of Arochlor 1254. It has been said*-*
Aroclor 1260, the authors felt that this was that a purified crystalline concentrate from
indicative of small quantities of a toxic fac­ toxic fat led to the development of embryonic
tor in this preparation.
deformities in chickens.
Comment
Recently, Bionctics Research Laboratory,
In somo instances, the same chemical comBethesda, Md (unpublished data), ns cited ixiunds arc responsible for the occurrence of
Teratogenasis

/ ' '
A rch Environ Ilcnltli— Vnl 25, A ug 1072

5793

0002377

1:10

t

T O X IC IT Y or IlYD U O C A JtnO N S— KIM IlllO IJC Il

«•lilur.-icuo in pcuplr, liver disease in several contamination of the technical chemicals
specirs, x-disease in cattle, chick edema, mid with various highly toxic substances, well
teratologic or oilier effects on 11«: felas. NoL coordinated epidemiological studies, and ad­
nil of Ihp disenso entities hnvo thus fnr been ditional ex|>erimental studies in nnimals are
produced by nil of the comi«>nnds (Table needed to clarify these problems.
1). Some of (hr compounds in <|tiostinn hove
Chlornene has lieen produced ox|»crimcnllieen shown to l>p rontnminntrd with chlori­ nlly in men by Shelley nnd Kligman (Arch
nated dilienzo-p-dioxins or chlorinated di- D e r m 75:689, 1957). The authors cite addi­
lieiizofiirans ('I'nhln 2). The decree of con­ tional references on experimental dermal ex­
tamination serins to vary with different posure of animals nnd men to chlorinated
comjiounds. Some com|>anies manufacturing naphthalenes.
these compounds have instituted clean-up
procedures which remove the crea test part
References
of the contaminants. Improved hygiene
amone Hie workers nnd improved ventila­ 1. Herxheiincr K: tiller Clomkne. M tlnch M ed
tion of the working area have, in many W tcbr 40:278. 1899.
2. W aiter H: Gcwcrlilirlio Krkrnnkungen durrh
instances, reduced the incidence of chlor- g clilo
aene. Whether all disease entities dcscrilicd 1018. rlo KoliIcnwmwcrNlnlTu. Xbl (lew Ily a G: UX),
H. T ricky Ig Din Pemnkrnnklieil {O dors- c).
are caused by the chlorinated dihonzodiox- Klin
u
Wtr.hr r.:847-K4H-, K07-!X)I, 1!«7.
ins or the chlorinated dilienzofurans is not 4. .Innas
iJW, Alrlrn IIW : An nenciform dcrmnlcrO
yet known. Some of these chemicals arc posiM. Areh Drrm Syph ¡0:1022-10.14, lil.’IG.
Teleky Jg liber Ncurcre l-’orschnnKwiirl linden
$
highly toxic, as illustrated by the fact that unit5. I'orsi
nuf deni Gcliiot dor Gcwcrl>okrnnkC
O
two applications of 10/<g of 2,.3,G,7,-(clmchIor- heiten. Klinluinccn
W tehr 27:240-2.17. 1049.
odibenzo-p-dioxin applied to human skin"2 6. Klinn KM, .Inrvik N14: Actions of rertnin rhlnr- <E
naphthnlcncs of the liver. Proe Hoc K ip lliol
CO
produced chloracnc-likc symptoms. Single inntrd
.11:118-120, 19.1«.
CO
oral doses of 20/ig to 50/ig/kg/body weight M7.ed Drinker
CK, W nrrcn M F, P en n ell GA: The
ro
problem of pnssihlo systemic cfleets from cerlnin
resulted in fatal liver necrosis in rabbits."2 rtilorinntcd
hydromrixm*. J Induttr lly g Toxic
There is no readily available information 19:282-111, 1917.
on the. metabolism of Urcsc comixninds 8. Jones A l': T lie etiology of note with *|iet-inl
to nrne of nrrutHilionnl origin. J In d u ttr
in animals and their breakdown in the referrnee
H ya Toxic 21:2:10-112, 1941.
environment.'12 It is probable that they are 9. Grrcnhtirg U Mayer* M il, Moss-Smith A: Tlic
stable. Crosby et al"' found that 2,3,7,8-tcl- systemic effect resulting from exposure to certain
Hydrocarbons. J In d u ttr Ily g Toxic
mchlorodilicnzo-p-dioxin and its homo­ rtilorinntcd
21:29-18. 1918
logues decomposed rapidly in alcohol solu- 10. Culler l i t : Pcnlnrhlorinnlcd nnphllinlcncs in
J A M A 125:271-274. 1944.
tionnmclcr artificial light and natural light industry.
11. M rljerhlrhic NGH. Rnlicrtsnn D : O ilorinnted
However, photodccomposition was negligi­ naphthalene
firit M ed J ldi!II-«92, 1942.
ble in aqueous susjicnsions and on wet or 12. Collier jioisoning.
K: Poisoning by chlorinated naphtha­
Ijw c r t 244:72-74, 1911.
dry soil. It is unknown whether the disease lenes.
11. Von Octtingen WK: T h e Haiogenaled Hydroentities discussed arc the result of one or sev­ enrhant.
Toxicity and Potential Danger, publication
eral contaminants or the combined effect of 414. Public Health Service, 1911.
14. Seliwarlx L, Tuli|win I* Mirmingh.-im D-J: Oetin: chemical compound nnd its contaminant. eupnlinnnl
D iteatet of the Skin, cd 1. Philadelphia,
Another unsolved problem is the observa­ last and l-'cbiger
l ’uhlishcr*. 1917, pp 11«-145.
15. Meigs W.l, Alliom .1.1. K artin MI.: Chlornene
tion that some of these comixninds such ns from
and unusual cx|x?suro to arorlor. J A M A
technical 2,4,5-T induce porphyria.22 The 154:1417-1418,
195-1.
chlorinated biphenyls also cause ¡jorphy- 1«. Hofiunnn M I', Mcnegliini C L A prosixwito
follimlnsi da idmrnrhuri rlom snslituili (A rne
ria.22 Whether porphyria is the result dclle
clorirn). (! Uni Derm 101:427-410, 1902.
of the chemicals themselves or of the con­ 17. Mimiinglinm 1).I: Ocruimlinnat dermatology:
taminants, or both, is not known. Crow1'-" Current prohiema. Skin 1:1H-42i, PM2.
Plcwig G: /.nr Kinctik der Coincdonenliildiing
suggests that the porphyrin produced by liei18.Chlornene
(tlnlow axam e). Arch Klin K xp Derm
technical 2,4 ,5-T may have been caused by 218:228-241, 1970.
■
contamination of the chemical with crude 19. It.-iuer H. Si-hut/. K H, Spiegellierg U : n eru flii-iie Vergifltingcn liei dcr Iferslelliing vou Clilnrchlorobenzenes, which were used to produce phenol-Vcrliindungrn.
the chlorophcnols from which the 2,4,/i-T brhyg 1H:i:18-111,PKlt. Arch Gerteerbepath Geneerwas made. Hotter QWnrcncss of the jxjssiblc 20. Kimniig J , Srhulx K II: Meruflirlio Akne (Nog

o.i..
A ll..
21
re-:|r
tlnv»-

2:1 4
22

lion
20 2>
21

h e al'

2.4.1
24
nteii.

21.

1970
20
tic . <

27
lion
Imvi:

whe:

25
(ion
chi'.

■29

Oilo
Amr

10
e h lo

4:27

31
ICxp
•nd
12

men
r a il!

prod

:r

p a ir
vine
197

3--

la rv

131:

¡V

lion

• V
diw
pre!

112

3

effc

d

S ci

pro

Din

1

cn<:.

uni-

11-1
4nii<
kes
19.
4
per
by.

4

nm
av.

/

K\

Arch Environ llrnlllt— Vol 21, Aug ¡072

5794

oofeora

»
«I

r n s i c r r v n r n v n n n c A H n u N s -K iM hitniir.ii
]
!

wvll

j

il ml*
s nr«
A r r /i

rultliil cx-

uitpd

M rd

¡lurch
0:100.

A it im i* « K M .

ini c r­
im in i
rnnk■hlorllio l
The
rlnin
Vaxic

1

It.

The
rlnin

oxic

a in

nterl
12.
Ihn-

drnlion
Ochin,
me
AM

silo
me

vy-

■ ing
•rm

•niI
l

nf

in liln l

/ v k ll'w 'llll

I i ì m I i M I ) , N | h *m i *i *i * I I I ! , r i
a k in

In

in iii| t t iiiin l'i

I llltll|r ili!i|.

r l i i i Itr llil

III

l n * x m - l i I m m i n t i l i l I i i i I i *i i i **i .
n i : 'U n *

ir |n iili* il

In

m i x I l l M I I l f |>t’ l 1I n . „ m l
P ooh Set 7 M -4 2 4 C i l ) , lp . 7' 1.
ii

4 1 . | - * li r k 111*', l - ' i i r - i l i H i i * I ) , M i i i l i m * .1 J S l i n t i i - i n f
l i l t * r l i i i ' k i v l t - t i n i i l i i H ' i i f i r ; J | . I ' r i ' I H i m l i i H l m i l l I ■I •«11■rr -

ir n l
r llr r lii n f
n i* r v ii( iill! iii*
i* liir k
I* |||* I||||
f i„ * |/ ir
Imvi* i*itn*i«*il iirni'lnrni ilrriinililiH. //nlim lr A/ri/ Nnrg
I'lnin l i l r i i l r . Poult Sri * 1 4 : 1 2 1 4 * 1 2 2 2 . I f X if i,
2:1 I. l!MI.
11*.!. Iliiinlrr KW. Iliiitrr ll.l: !niln*ili*inl iiil<iKÌi*n*
43. M irk 1)1**, 0*1 >rll R fl, Hnris VC: Sli>iilii**i nf llm
liitn ilu r In |M*iiliii'liliini|>lii*nnl. Im i M c d Sur/t
I’liirk rilrllln lliw nar; Ism e -lrm i, lnw-lrvi*l firilin g
Ip.M.
of chirk rd rin n fnrlnr. P oult S ri 40:180-191, 11X17.
211. t'nliind A l1. Sinilti J), M r llr r lì, e l ni: A
40. C niilrrll .IS, Wi-lil» NO, Mnliia A.I: S rn rrh for
hr.-illli aiti-vi-v a l wm krr* in n 2.4-1 ) pillili nini
rliii-k riln iin fnrlnr. Clu-m K m t N r on 43:10. f!X!7.
2.4.3-T |iliinl. A rd i Kneirun llr a lth 22:.’llli-:)27, 1971.
4 7 . T nm il/i M , Uriln S, Nnrlamln M : I lil x * n / . r l - p 21. llrnim W : K liniarhr llrnlnirliliinitcn / u r Knl- dinxiil ilrrivnlivra: X X V If. S ynlhm ia nf |M>lyliitli|rlialiliiing ilrr O ilo n irn r. H n uln rzt 111:1211*1211. 1959.
l»rn/.i)-/i-iliiixma. Yiilmr.nkti Xmmhi 79:180-192, 1939.
25. Cimv K I): Clnrnriir. Urti J Derni 8*1:799.000,
48. I liccinlmllintn fill, 1 funne A. Kirralnno I), e l
11170.
nl: Clicinirnt mill Inxiiailnctml cvnlunlinna nf ¡«mint­
2lì. Olnfann )’: lfv|>crkcnilnain (x-diacnac) nf cnl* ed nnd Hynllictic rhlorn derivntiven of dil/cnxo-/>Ile. Ctirnrll V rl 37:279-291, 11M7.
dioxin. N ature 220:702-707, 190A
27. 1Innari W, Oli»fsnn p , M rKult*r K : T lie isnln49. RiarlirniiL’h RW , Rieelie 1’, I'rnknll D ll, c l nl:
lion nml id rntilirnlion of Ilio rnuaiilivo ngcnt of
I’olyrhlnrinntrd liiplirnvls in this global econyntctn.
bovine hy|icrkcm loaia (x-diarn.ar) froni n pmecnacd
N ature 220:1098-1102, 1908.
w lirnt rnncT nlm lr. Cornell V r t 43:94-101, 1055.
60. I-iriw nslrin K l’, Schulr. K R , Kuhrcm nnn T W ,
28. Sikra I). llridgca M li: K xiw im entnl producc l nl: Rinlngiml inlcm rlion liclw ren plnalirizrrn nml
lion of liv|irrkrntlnnin (x-diaenar) nf m ille willi n
innrrlirirlra. J Krnn K ntnm 02:701-703, 1909.
rlilorinnlnl napltlhnlrnc. Scirncr 1 10:330-507, 111.72.
61. M illrr .IW: Pnllm lngim l rltnngra in nniinnla
29. llroek \VK. Jo n ra KW. M nrV irnr 11, e t ni:
ex|Kwcd to n coinm ereini rhlnrinnled liiphenyl. P ub­
d ilo rin n lril nn|tlillinlrnr inloxinitinn in aheep.
lic H ealth Itrp 09:1083-1093, 1944.
A m ar J V r t U n 18:G25.(2Ì0. 19.77.
52. N islii/um i M: U g h t nnd c lcrlm n mii-roaropr
.10. Hula*r W15, I jn k I l i ’: T nxic pileria of hcxnnludy nf Hilom biphrnyl poianning. A rch Knuiroa
Hilonninplillinlpne on aurine. T nxic A p p i Pharmacal
H ealth 21:020-032, 1970.
4:2.77-21 >2.1902.
63. T nki I, Miannngn S, Amngnae Y : R rim rla of
n i. Schedile CK. Rclicr KK, M o irill C C ,e t ni:
Iho atudv of "Y uaho." Fukuolta A cta M cdica
l*!\|x'rim rnIni production of hy))rrkcrn Inaia in mia 00:471-474, 1909.
nml hnm slcra. A m rr J V r t U n 10:18*1-188, 1977.
34. Okum um M , K nlnuki S: Clinim l nliacnm lion
¡12. Sikra I), W iae JC . llridgca M H : T h e cx|ierion "Y uaho." Pukunha A cta M cdica 00:440-440. 1!X!9.
m m ln l proiliii*lion of x-diacnae (hypcrkrrntoaia) in
53. Voa JG , Kocmnn -TH: O im phm ljvo toxicolog­
m ille svilii rliliirinnlcd nnplillinlrnea nml pclrolcnni
ic aludy with polyrhlorinnled hiphenyla in rh irk en a
pnalurla. J A m rr V rt M ril A m ie 121:337-344, 1972. w ilh H|M>rinl refen-nre lo porphyrin, cdrm n forinnXI. llrll W ll: llrln liv c Inxirilv nf llir rhloritinn, liver nrrm aia nml liaauo reniduca. T oxic A p p t
n n lrd nnplitlinlrnm in rx |«rrinim lnlly producivi ImPharmacol 17:030.008. 1970.
v in r liv|M'rkernlnnin (x-diacnac). V cl Mal 48:177-1*10,
30. Voa JG . Kocm nn J H , Vnn D r r M nna I l f , c t
111.7*1.
nl: Tdrnlifimlinn nnd Inxirnloyirnl cvnlunlinn of
.*14. Sniigrr V I , S rn lt I , lln m ily A, e t ni: A lim rnrh lo rin n lrd ililM*nznfiinni nnd chlorinnlrd nnphlhn(nrv Inxriiiiii in rliirkcna. J A m r r V c t Mnl A tto c
lenc in Iwn rtm n n rrrin l |M ilyrhinrinntcd hiplicnyla.
1.77:172-171!. 19.78.
Food Cnxmrt T oxic 8:023-1221, 1970.
,*17. A nnnym om : T lir rhirk cd cn in fnclor. N o tri57. Kmerann J I „ 'llioinfMon D J , R lrrhing R .I, c t
tinn tir o 20:2.8.30, 4908.
nl: T cm lngcnic nludiea of 2.4.3-lricliloroplim 70. S i-lunilllr SC. lidw im la H M , M orria DA: A
o x y n rrlic nriil in the m l nnd rnlil>it Faatl C o tm rl
d ian rd rr nf rh irk rn a jimlinlily d u e lo n (oxic feedT oxic 9:395-404, 1971.
prcliininnrv iT|mrL J A m r r V r l A f r i A n n e
38. C ourtney K D , G nylor D W , Hngnn M D , c t nl:
1:12:210-210, 19.78.
T crnlogrnic cvnliuition of 2,4„7-T. Science 108:80437. lù lg n r SA, lln n d DS, M rliu s P , e t ni: T he
80«, 1970.
cfTn*t nf n loxir nulislnncc in fnt on jioullry. Potili
59. Sim rarhu GT, D unn KT, R ow e V K : S tu d y of
S ri 37:1200-1201, 1978.
Irm ln g rn irity at 2.3,7,8,-lclm rhlnrm liiicnzo-p-dinxin.
78. A llrn .111, I ¿dirli .1.1: Reaponae of rhirkenn lo
Fatal Caxmrt T a xir 9:407-412, 1971.
pm longcd (ordine of m u lo loxic fot. P roc Snc K xp
<!0. C ourtney K D . M oore .IA: T em lology alm lica
Hinl M rtl 109:48-71, 1902.
w ith 2.4,3-lrirhlnm phrm ixynretic ncid nml 2,3,7,8-tct39. * Siin|isnn CK. l ’rilchnrd W ll, H nrm a R H : An rnrlilom«lilK*n/.<*-/>-<lioxin. T oxic A p p l Pharm acol 20:
390-407, 1971.
m illilitri inaia in rliirkcna a n d lu rk ry a m tiscil liy nn
u n id rn lilird dicln ry fnclor. J A m r r V e t M r d A n oc
01. K cplingrr M l - K nnrlirr OH, Cnlnndrn JC :
134:410-411!, 19.79.
Toxicologic almlica w ilh polyrhlorinnled hiphenyla.
40. Alien .IR , Cnmlena I-A: IJ g h t nml electron
Roml licfnrr th r Irn lh nnnunl m eeting of (he Society
micniwnpii* olmrrvnlinna in M ara erti m ulatta mnnof Tox irologv, W nahinglnn, DC, 1971.
krv* fixl tnxic fnL A m r r J V r l U n 28:1313-1320,
02. Si Inil/. Kl f : K liniarho und rx |ierim m (e lle Iln 1907.
Irraiirliungrn /.nr A lnlngir d r r C hlnracne. A rch K lin
41. M rf'm ic H I , Xnvnge JK . 0*1 M I RI,* H ydroK xp Derm 21X1:589.500, 1957.
|M*rirnnliiun nnd narilra in rliirk a ferì n chlorinntcd
(21. Alirlaon 1*1 f: Pollulinn liy organic rhcm icnla.
hydm cnrlm n. Ptndl Sci 41:293-299, 1902.
Science 170:497, 1070.
42. P rra ll I, .IrfTrrica 11.1. M nnre N W : Pnlvrlilori04. Croaliv D G , W ong AS, l’lim m rr J f l , e t «1:
rnili-d liip h n iy la in wild lórda in llriln in nini llirir
Ph(ilndiaaiiii|x>ailiiin o r rld n rin n lrd dihrnzn-p-dioxnvinn Inxirilv. Knuirnn P o llili 1:3-20, 1970.
ina. Science 173:7-18.749, 1971.
43. l'iH lrlkirw ir/. W J, 1loilrlior RV , Cnllrnluirli
03. Crow K l): O iln m m e . T r e n t S i J o h n l l n t p
KW, c l ni: Suino pliyamlogicnl rea|iuitacn of New
D erm Sue 60:79-99, 1970.

Arch Environ Health— Voi 25, Am : 1972

DOW373328

k n c l.

■ki*

‘i '. l .

m **i | m i i i *m*

ni'nf-

lurTr-

1*1

I I llu m in i* I lim i l i r l lllll ¡
II* m * l | l l i l l i ' * r t l l «
Ath*'v. / i . j (»i n f i i / t i l i . . i . i o M i ; , i : « , v .

1.11

ooo:2379
579;

5796

Z%A

il •

10/6/72

SUMMARY

TERATOLOGY

T he n o -e f f e c t d o s e l e v e l of 2/A-D a n d 2 A 5 - T
IN A VARIETY OF SPECIES IS IN THE RANGE OF
25 - 50 m g / kg /d a y t h r o u g h o u t o r g a n o g e n e s i s .
c

{

ZZtpnn

2/3,7,8-t e t r a ~ a nd h e x a c h l o r o d i b e n z o -p -d i o x i n
ARE HIGHLY TOXIC TO THE DEVELOPING EMBRYO AND
FETUS OF THE RAT; 1/2/3^-TETRA^ , 2 , 7 - D I ^
AND OCTACHLORODIBENZO-P-DIOXIN ARE NOT.

TERATOLOGY - 2 > D
AUTHORS

SPECIES

FORM OF
2,4-n

Co l l i n s & W i l l i a m s ,
1971 (C.6.8)

Ha m s t e r

Acid

Or a l

200,60,100

Kh e r a & Mc K i n l e y ,
1972 (c.6.6)

Ra t

Ac i d ,
Sa l t

Or a l

2 50100,150

Ra t

ESTEá

Ora l

12.5050,75,87

-

Sc h w e t z E l AL»

1971
) =

(PP 8F0670)

No -e f f e c t d o s e

Ac i Dj

l e v e l for e m b r y o and

DOSE LEVELS
ROUTE

m g /kg /d a y .

FETAL TOXICITY.
5797
0002855

TERATOLOGY -2,4,5-T
FORM OF
SPECIES g A H

RQÍÍIE

(C. 6 .8} *

Ha m s t e r

Ora l

Co u r t n e y ex al..»
1970 ( c . 6 . 9 ) ♦

Ra t Ì
Mo use j

Co u r t n e y & Mo o r e ,

Mouse
. Rat

AUTHORS.

DOSE LEVELS
- mg/ kg/ d a y

Co l l i n s & W i l l i a m s ,

1971

(C.6.11)*
(C.6.12)t

Kh e r a & Mc K i n l e y ,

1972

(C.6.6) «

ÍÍSUBERT & DlLLMAN,

1972

(C.6.13) »

1971 ( c . e . i 4 ) *

Po l l ,

Ra t

Em e r s o n EI AL* a *

1971

(C.6.3) *

Mouse
Mouse

(C.6.4) #

(C.6.2) ft

^

pt>M 2,3,7,8-TCDD

Acid
Aci d

sc
sc

Ac i d , ^
Sa l t , l
Esters j

Oral

Ora l

Mouse

Acid

Ora l

Ra b b i t
Rat

Acid
Acid

Or a l
Or a l

Ra t

Acid

Or a l .

Sheep -

Acid
E ster

Ora l

J , 100,125,150
10,22j@,80,250

Ora l

135,60,90,130

-

B inns S Ba l l s ,

1971

20 j©,80,100

ä

Acid
Bu t y l
Ester

Sp a r s c h u EI AL.,

1971

Acid

AA t ~ \ r \

1971

5798
)

=

No -e f f e c t d o s e

le v e l for

♦See PP 1F1102, May 1973

EMBRYO AND FETAL

TY.

0002S56

TERATOLOGY - DIBENZO-p-DIOXINS

C£
rc

AUTHORS
Sp a r s c h u El AL»
1971 (C. 6 . 5 ) Jf

-DIOXIN
2,3,7,8-Te t r a ~

SPECIES ROUTE
Ra t

Oral

DOSE LEVELS *■

^G/KG/DAY

(gTp,0,125,0,5,:
8

Kh ERA & RUDDICK,
1971 ( C . 6 . 7)*

2,3,7,8-Te t r a ^

Ra t

Or a l

Cffj2l),0,25,0.5,:
2 ,4 ,8 ,1 6

1,2,3,9-Te t r a ^

Ra t

O ral

50^100,200,400,

2,7-Di

Ra t

Ora l

250,500,10004^

2,7-Di ^
Hexa ^
Octa

Ra t
Ra t
Ra t

Or a l
Oral
Oral

Co u r t n e y &
Mo o r e , 1971

2,3, 7, 8-Te t r a d

Ra t
Mouse

SC
SC

NEU3ERT &
D i l l o n , 1972

2,3,7,8-Te t r a -^

Mouse

Ora l

Sc h w e i z e i a l >
1972 (c.7.5)*
-

(C .6 . 1 1 ) «
(C .6 .1 2 )*

0,3,3,4,5,9

(C.6.13)«

(§ 3 )

•See PP 1P1102, May 1973

000285 ?

5799

8«?

5800

i------- " IN S

TR.*'
C
C r ' ^

~"ET OR
TISE
— NTTAL

Summary

of

Teratology

compiled
e

Dow

by

Chemical

Marguerite

Company,

October

Teratogenic
number

of

studies

its

Studies

have

derivatives

L.

conducted

in ra t s

with

(Schwetz

McKinley,

1972),

in

hamsters

(Collins

and

in s h e e p

(Binns

and

Johnson,

1970).

level

of

In

study

conducted

the

propylene
given
and
on

glycol

orally

87.5
days

mg
6

by

of

was

were

fetuses

daily

for

half

body

weight,

bone,

lumbar

with

her

on

development

In

conducted

(khera,

1972)

2,4-D

rats

at

doses

50,

100,

or

and

the

from

150

animals

and

its

to

mg/kg/day
wore

300
on

killed

and

would

day

dose

(87.5

day

on

dose

day

levels
fetal

ossification

of

effects

not

not

through

were

seriously

inter­

was

at

noted

50 mg/kg/day.

Drug

Directorate

butoxyethanol,

orally
with

0

were

decreased

isooctyl,

of

each

considered

and

22

75,

(The

Food

day

50,

taken

Canada

6

25,

were

were

at

days

esters

Fetuses

noted

mg/kg/day,

its

caused

effect

given

and

this

were

were

on

2,4-D

highest

No

found.*

embryotoxicity

These

they

a no­

than

was

delayed

butyl,

derivatives
25

higher

a

case,

weight

included

ribs.

the

was

highest

smear

survival.

by

1971)

12.5,

body

the

and

effects

Williams,

gestation.

given

because

Khera

and

of

rats.

of

edema,

wavy

minor

dimcthylamine

20

1971,

isooctyl

The

fetotoxicity

or

and

of

a vaginal

mothers

25 mg/kg/day

and

day

of

teratogenic

kg

pregnant

in

and

doses

al.

1971)

and

levels

pregnancy,

ribs,

study

per

because

seen

of

at

and

In each

(Schwetz,
(PGBE)

2,4-D

et

50 m g / k g / d a y

gestation.

subcutaneous

the

Dow

rats

the

Signs

in

considered

in

to

ether

of

section

noted

fere

15

chosen

pregnancy.)

by

equivalent

toxicity

sperm

25

pregnant

acid

Caesarian

which
of

to

least

butyl

through

mg/kg/day)
signs

at

Michigan

1972

and

effect

2,4-D

Leng

Midland,

18,

been

with

to

most
15

of

pregnant
tests

at

gestationf

pregnancy.

( Spcrma-

5801

0002802

*See also attached tabular summary of teratology studies with
2 , 4 - D , 2 , 4 , 5 - T a n d v a r i o u s d i b e n z o - p - d i o x i n s ( S c h w e t z 10/6/72.)

-2-

DOW

tozoa in the vagina on the morning fo llo w in g ov ern ig h t mating
was considered day 1 of g e s t a t i o n . ) They r e p o r t e d t h a t e f f e c t s
noted a t 50 mg/kg/day were not s i g n i f i c a n t l y d i f f e r e n t from
those in u n t r e a te d anim als. Doses of 100 or 150 mg/kg/day
o f 2 , 4-D induced f e t a l deaths and caused an in cre as ed in­
cidence of s k e l e t a l anomalies. Normally o c c u rrin g anomalies
included wavy r i b s , e x t r a r i b s , delayed o s s i f i c a t i o n of s k u l l
bones and s e v e r a l changes i n the sternum. They a ls o observed
a low incidence of anomalies a t the high dose l e v e l s which
were not p r e s e n t i n c o n t r o l anim als. These included fused
r i b s and bent or d i s t o r t e d l e g bones. Some animals were
allowed to d e l i v e r normally and the weight g ain and v i a b i l i t
o f the o f f s p r i n g were w ith in normal l i m i t s .
c

c

In the study conducted by the U.S. Food and Drug A d m in is tra tio n
( C o l l i n s , 1971), 2 , 4-D from t h r e e d i f f e r e n t m anufacturers was q
given by o r a l i n t u b a t i o n to hamsters a t doses from 20 to 100
mg/kg/day on days 6 through 10 o f g e s t a t i o n and the animals
were s a c r i f i c e d on day 14 of g e s t a t i o n . The incid en ce of
f e t a l anomalies was low and c o n s i s t e d of delayed head o s s i f i c a ­
t i o n or e a r abnorm ality in only two f e t u s e s from t h r e e groups
given 100 mg/kg/day, and fused r i b s i n a t o t a l of nine f e t u s e s
from t r e a t e d groups compared to t h r e e in the c o n t r o l group.
The au th o rs concluded t h a t the % a b n o r m a li t i e s , even a t 60 and
100 mg/kg/day^ were not s i g n i f i c a n t l y d i f f e r e n t from the c o n t r o l
percentage.
In the study conducted by the U.S. Department of A g ric u ltu re
(Binns, 1970), pregnant ewes were given 2g of 2 , 4-D mixed
with a l f a l f a meal each day f o r 30, 60, or 90 days a f t e r breed­
ing. (This i s e q u i v a l e n t to approximately 40 mg/kg/day in
sheep). No s ig n s of c o n g e n it a l malformations in the lambs,
or c l i n i c a l poisoning or h i s t o p a t h o l o g i c a l l e s i o n s in the
dams were noted.

. 5802
0002S53

-3 -

Schwetz, B. A., G. L. Sparschu, and P. J. Gehring (1971).
The Effect of 2,4-Dichlorophenoxyacetic Acid (2,4-D) and
Esters of 2,4-D on Rat Embryonal, Foetal, and Neonatal
Growth and Development. Food Cosmetics Toxicology 9_,
801-817.
(C.5.3)
Khera, K. S., and W. P. McKinley (1972). Pre- and Postnatal
Studies on 2,4,5-Trichlorophenoxyacetic Acid, 2,4-Dichloro­
phenoxyacetic Acid and Their Derivatives in Rats. Toxicology
and Applied Pharmacology 22_, 14-28. (C.5.4)
Collins, T.F.X., and C. H. Williams (1971). Teratogenic
Studies with 2,4,5-T and 2,4-D in the Hamster. Bull.
Environ. Contam. & Toxicology 6_(6), 559-567.
(C.5.5)
Binns, W., and A. E. Johnson (1970). Chronic and Tera­
togenic Effects of 2,4-D (2,4-Dichlorophenoxyacetic Acid)
and Atrazine (2-chloro-4-ethylamino-6-isopropylamino-striazine) to Sheep. Proc. N. Cent. Weed Contr. Conf. 25,
100.
(C.5.6)

Addendum, August 1973.
Also attached are excerpts from two papers presented at the
Eighth Inter American Conference on Toxicology and Occupational
Medicine in Miami, July 1973.
(a)

Clegg, D. J. and K. S. Khera. The Teratogenicity of
Pesticides, Their Metabolites and Contaminants.
(Phenoxy Herbicides p. 270-273)

(b)

Durham, W. F. and C. H. Williams. Mutagenic, Teratogenic
and Carcinogenic Properties of Pesticides.
(2,4,5-T
and 2,4-D p. 316-317)

58030 0 0

2S54

l'

■

t

Eil'ccl of Telraelilorodibenzo'p-Dloxin on
Growth Rale and ihe Synthesis
of
•»
Lipids ami Proteins in Rais
Ly II. M. Cunningham ami I). T. W illiams
li esecrili ¡.abiirnturìff, fo tti! am i DrUf: litrccturnrr
T im m y 's l ’asturr, O ltan a

,*■ ».

D O V /3 7 3 3 1 5

Chick edema disease was first observed in 1957
when thousands of broilers were lost (1) but it was
not until 1966 that chlorodibenzo-p-dioxins were found
to be the causative agent (2). One of the most toxic
dioxins was 2,3,7,8-tetrachlorodibenzo-p-dioxin (3).
The most prominent toxic effects in chickens are
hydropericardium and hydroperitoneum (4,5,6) while
in mammals the liver appears to be the main point of
attack showing enlargement and fatty infiltration (6,
7). In one report, enlarged livers in rats were not
accompanied by an increase in liver fat (8). The
present experiments were conducted to determine if
dioxins affect the ability of the liver to synthesize
lipids and protein, and the dosage and time required
to produce maximum effects.
MATERIALS AND METHODS

A

i

In each experiment 12 to 16 weanling male Wistar
rats were allotted at random into two to four treat­
ment groups. 2,3,7,8-tetrachlorodibenzo-p-dioxin
(99% pure, courtesy of the Dow Chemical Company) was
dissolved in corn oil and given by oral intubation
at levels of 0 to 10 pg/kg of body weight. Both
treated and control rats received corn oil at a level
of 10 ml/kg. One to seven days later each rat received
an intraperitoneal injection of 1.0 pc of 1-14C-Lleucine and 10.0 pc of 3n-Sodium acetate'. -Exactly 1 hr
later the rat was killed by a blow on the head, the
liver was removed and 1-g samples were homogenized with
4.0 ml of 0.9% NaCl. The lipids were extracted by a
procedure similar to that of Folch et al. (9) and the
radioactivity was counted with a liquid scintillation
counter (10) . In some experiments the lipids were
partitioned by thin-layer chromatography (10). .The
proteins were separated from the aqueous phase of the
Folch extraction by centrifuging, washing twice with
5% trichloroacetic acid and twice with 2:1 methanol:
ether (v/v). They were dried under nitrogen and 20 mg
aliquots were solubilized in scintillation vials with
1 ml of Soluene (Packard Instrument Company) at 55°C
in a shaking water bath. In some experiments lipids
were arso extracted from adipose tissue and proteins
isolated from heart and skeletal muscle.

ì

45

Hullrlin of KurilMtitiM'til il Goiiluiiiiii.itioii Ä sin.
Voi. 7, Nu. I IV.«!, yi >|nih|’«r
i\ i*w \oii li«
t •1

1,1■ • »n

5805
00023G6

RESULTS

DOW373316

Experiment 1. A comparison was made between levels
of 0, 1.0 and 10.0 yg of dioxin per kg of body weight
on the incorporation of labelled acetate and leucine
into liver lipids and pro"sins. The dioxin signifi­
cantly reduced body weight gain (P<0.05) and the
radioactivity of the free fatty acids in the liver
(P<0.01) along with reductions in other liver lipid
fractions (Table 1). The specific activity of liver
protein, on the other hand, increased (P<0.05) when
dioxin was given. The 10.0 ug/kg dose level appeared
to have a greater effect on most measurements than
the 1.0 ug/kg level but the differences between them
were not large.
TABLE 1
Effects of Dioxin on Liver Lipid and Protein Synthesis
3 days after dosage
O
o
H

Number of rats
4
Initial weight, g
49.5
Weight gain per day, g
3.1
Liver lipids
(DPM 3H/mg liver)
total lipids
23.66
triglycerides
3.47
free fatty acids
0.31
diglycerides
13.95
phospholipids
5.91
Liver protein
(DPM 14c/mg protein)234

O•

O

Oral dioxin dosage (yg/kg)
S.E.
4
51.2
0.4

4
51.0
0.2

2.1*

15.46
2.09
0.13
10.68
2.58

11.46
2.17
0.08
6.63
2.66

4.25
0.53
0.05**
2.67
1.26

330

330

28

*P < 0.05
**P < 0.01

I
\

Experiment 2. The 10.0 ug level of dioxin was again
compared with a zero control over a 3-day period.
It again had a similar effect on growth rate and the
incorporation of labelled acetate, and leucine into
lipids and proteins but with more rats per group
more of the differences were significant at the 1%
level of probability (Table 2). The 14c-leucine
incorporation by the heart was not affected by dioxin
Aut there was a significant reduction (P<0.05) in
the incorporation of 3n-acctate by epididymal adipose
tissue.
46

000236?
5806

TABLE 2
Effects of Dioxin on Lipid and Protein Synthesis in
Liver Compared with that in Heart and Adipose Tissue
3 days after Dosage
Oral dioxin dosage (uq/kg)
0
10.0___S.E.____
8
76.2
0.36

0.35**

6.92
1.31
0.07
1.62
1.98

1.86**
0.39*
0.07
0.95
0.64**

71.7

37.4*

223

6.9**

33

7.0

DOW373317

8
Number of rats
76.1
Initial weight, g
2.25
Weight gain per day, g
Liver lipids
(DPM 3H/mg liver)
15.62
total lipids
2.69
triglycerides
0.08
free fatty acids
4.02
diglycerides
5.55
phospholipids
Epididymal adipose tissue
215.8
(DPM 3H/mg lipid)
Liver protein
166
(DPM l4C/mg protein)
Heart protein
38
(DPM l4C/mg protein)

*P < ’.05
**P < 0.01
Experiment 3. The 10.0 vg level of dioxin was used
to determine the number of days after dosing at which
it would have the greatest effect on liver lipid
synthesis (Table 3). There was a slight reduction in
^H-acetate uptake by liver lipids (DPM/g of liver) at
.2 days and a significant decline (P<0.05) at 7 days.
TABLE 3
Effect of Dioxin on Liver Lipid Synthesis at Varying
Intervals After Dosage
Days after qivinq 10 uq dioxinAq
2
3
7
Controls 1
Number of rats
Initial weight, g
Weight gain per day, g
Total liver lipids
(DPM/mg ¿liver)
*P < 0.05

4
61.5
3.4

4
64.0
2.5

4
65.7
1.9

' 4
62.0
2.8

16.38

16.79

13.78

14.35

4
'63.5
3.1
9.00*

47

00023G8

Experiment 4. Graded levels of dioxin were used in
this experiment to determine the smallest dosage that
would have a noticeable effect on protein or fat
synthesis 7 days later (Table 4). There was a pro­
gressive increase (P<0.05) in liver weight as the
level of dioxin intake increased with a maximum
increase of 21% at the 10 ug/kg level and a minimum
of 9% at 0.1 pg/kg. There also was a trend towards
reduced 3n-acetate incorporation into liver and adi­
pose tissue lipids with dioxin treatment but with
only 4 rats per treatment the differences did not
reach statistical significance. Part of the reduction
in the incorporation of 3H-acetate per g of liver
could be accounted for by an increase in the size of
the liver.

Effects of Varying Levels of Dioxin on Lipid and Protein
Synthesis 7 days after dosage
Oral dioxin dosage (ucr/kg)
10.0
S.E.
1.0
0.1
0
Number of rats
Initial weight, g
Weight gain per day, g
Liver weight, g
Liver lipids, total
(DPM 3H/mg liver)
Epididymal adipose tissue
(DPM 3H/mg lipid)
Liver protein
(DPM 14c/mg protein)

4
63.2
4.6
4.3

4
64.2
5.0
* 4.7

4
69.2
5.1
4.8

4
65.7
5.1
5.2

0.43
0.21*

14.93

11.34

9.12

9.51

1.88

322.3

125.2

56.3

138.0

86.0

186

168

153

143

17.0

D o w 3 ? 3 3 i8

TABLE 4

Experiment 5. This experiment compared protein and fat
synthesis at 3 and 7 days after dioxin dosage (Table 5).
The dioxins had little effect on rate of body weight gain
but liver weights were noticeably increased 3 days after
treatment (P<0.05) and slightly increased after 7 days.
The increase in liver weight was accompanied by a
large increase in liver lipids which also was more
apparent 3 days after dioxin treatment than after 7 days.
The total incorporation of ^H-acetate into liver lipids
(DPM x 103/liver) was unaffected by dioxin but the
increase in unlabelled liver lipids diluted the labelled
acetate so that the specific activity of the liver
lipids (DPN/mg lipids) was significantly (P<0.05)
Reduced.
*
48

Û0023G9
-

5808

!%J ' •

»..fS’
* • ••. »/ • * V*

Adipose tissue in the flank region of rats
incorporated considerably less ^H-acetate into lipids
than epididymal tissue in earlier experiments but
dioxin appeared to have a greater ..’.nhibitory effect
on incorporation at 7 days than at 3 days after treat­
ment, although neither of the differences reached
statistical significance.

■

•¿i T

The protein concentration in the liver (mg/g)
was slightly reduced 3 days after dioxin was given,
apparently due to the larg^ dilution of the liver
with lipids. At 7 days the protein concentration
in the liver was unaffected by dioxin. The incor­
poration of l4C-leucine into liver protein was
significantly (P<0.05) increased 3 days after dioxin
treatment and only slightly increased after 7 days.
The specific activity of skeletal muscle was not
affected by dioxin treatment.

O
£
CO
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DISCUSSION AND CONCLUSIONS

• ;••• : .

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Most of the animal experiments reported to date
in the literature have used "toxic fats" or fractions
of these fats containing mixtures of different chlorodibenzo-p-dioxins. The tetrachlorodibenzo-p-dioxin
used in the present experiments was 99% pure and the
highest dosage used (10 yg/kg) was close to lethal for
when this amount was given orally to rats each day in
a preliminary experiment they all died within 2 to 4
, days. The lowest level in a single dosage that caused
an increase in the liver weights of rats was 0.1 yg/kg.
The dioxin had no effect on the rate of incorporation
of 3H-acetate into liver lipids but since large quan­
tities of lipids accumulate in the liver it would
appear that in some way the chemical restricts the
transport of lipids out of the liver. This storage
reached a maximum at about 3 days after dioxin was
given and was accompanied by a significant increase in
the incorporation of 14c-leucine into liver proteins.
Seven days after dioxin was given the incorporation of
14c-leucine into liver proteins varied considerably
between experiments being slightly higher than controls
in Experiment 5 and slightly lower in Experiment 4.
Weight gain per day showed an opposite trend being
lower than controls in Experiment 5 and slightly higher
in Experiment 4 but none of these differences was
statistically significant. It is possible that thé
increased synthesis of liver proteins at 3-days was the
result of an induction of liver enzymes (11) in response
to the dioxin and that the time required for it to
decline aghin varied slightly between experiments.

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TABLE 5
v\

Effect of Dioxin on Lipid and Protein Synthesis 3 and 7 days after Dosage
3 days

qt

no

7 days

Dioxin oral dosage (ug/kg)

0

10

S.E.

0

10

S.E.

Number of rats
Initial weight, gl
Weight gain per day, g
Liver weight, g
Liver lipids
mg/g liver
mg/liver
DPM 3H/mg liver
DPM 3H/mg lipid
DPM 3h x 103/liver
Adipose tissue, flank
DPM 3H/mg lipid
Liver protein
mg/gm liver
mg/liver
DPM Hc/mg protein
DPM 14c x 103/liver
Skeletal mifScle
DPMl4c/mg protein

4
63.0
5.2
3.78

4
63.0
4.9
4.80

0.7
0.4
0.20*

4
64.0
5.1
4.03

4
65.0
4.5
4.65

1.4
0.3
0.30

60
229
15.3
258
58

132
640
12.1
98
58

25
138
4.5
42*
22

56
226
21.7
394
86

92
428
17.5
190
82

19
28**
. 3.2
'62*
9

1.61

1.53

1.33

2.30

1.35

0.53

277
1046
135
141

241
1157
156
178

14
66
6*
7*

252
1015
144
14 5

252
1169
152
177

13
83
8
11

42

42

2

40

39

2

*rats on 3-day treatment were dosed 4 days after initial weights taken and were then
killed on the same day as 7-day rats.
*P < 0.05
**P < 0.01

0002371

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Acknowledgments

^ i H i r i< f lè w l > f i i M ' i > r i i l i * r ' i k i

We are grateful to Mr. R.C. O ’Brien who assisted
in the chemical analyses.
References
ANONYMOUS, Am. Hatchery News, 33, 17 (1957).
CANTRELL, J.S., WEBB, N.C. and MABIS, A.J., Chem.
Eng. News 45, 10 (1967).
3. HIGGINBOTHAM, G.R., HUANG, A., FIRESTONE, D.,
VERRETT, J., RESS, J. and CAMPBELL, A.D. Nature,
220, 702-703 (1968).
4. YARTZOFF, A., FIRESTONE, D., BANES, D., HORWITZ, W.,
FRIEDMAN, L., and NESHEIM, S. J. Am. Oil Chem.
Soc. 38, 60-62 (1961).
5. SANGER, V.L., SCOTT, L. , HAMDY, A., GALE, C., and
POUDEN, W.D. J. Am. Vet. Med. Assoc. 133, 172176 (1958).
6. FLICK, D.F., DOUGLASS, C.D. and GALLO, L. Poultry
Sci. 42, 855-862 (1963).
7. FRIEDMAN, L., FIRESTONE, D., HORWITZ, W . , BANES, D.,
ANSTEAD, M. and SHUE, G. J.A.O.A.C., 42, 129-141
(1959) .
8. CAMPBELL, T.C. and FRIEDMAN, L. Proc. Soc. Expt.
Biol. Med. 121, 1283-1287 (1966).
9. FOLCH, J., LEES, M. and SLOANE-STANLEY, G.H. J.
Biol. Chem. 226, 497-509 (1957).
10. CUNNINGHAM, H.M. and LEAT, W.M.F. Can. J. Biochem.
' 47, 1013-1020 (1969).
11. CONNEY, A.H. Pharmacol. Rev. 19, 317-366 (1967).

DOW373321

1.
2.

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18,140

W es sci .

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M.

Transi. MID
ICIIER ZZITJ’
JG
no.70
p.

Ko, 9506
February

12, (1973)

Dioxins-insidious poisons in our
environnent,
Author:
Hans Paul Bosshardt
Translator: P. Fisher
Date: May, 1973
Requested by:
Translation no.:

M. Irons, 2040 Bldg.
73-3-8

r e c e iv e d

J!!N 2 5 1373:
registration

0000520

8 511-373
73 3-8

'

-

Translated by:
Phil Fisher

C
0

Neue Zürcher Zeitung

Number 70/Noon/Tebrvaïy 12, 1973

DOWÜ32372

-

Dioxins-Insidicus Poisons in Our Environment.
Origin, properties and
behavior of polychlorinated dibenzo-p-dioxins
By Hans Paul Bosshardt*

In the large scale production of chlorinated phenols
as side products, very toxic polychlorinated dibenzop-dioxins are formed which are damaging to the fetus.
These impurities in their environmental behavior are
similar to chlorinated hydrocarbons: therefore one
has to count on accumulations within biological
material and consequently on irreparable damages.
In the U.S.A. millions of chickens fall victim to
these toxins.

In order to keep our environment and our food and nutritional sources
free of these dangerous toxins, according to a decree from the Research
Institute of Wadenswil, the especially dangerous tetrachlorodibenzo-pdioxin must not be detectable in plant protection agents and weed d e s ­
troying agents.

It is to be hoped that the Confederated Office of Health

will establish similar specifications for other polychlorinated phenols
and all compounds produced from them which are broadly used outside of
agriculture in industry and technology, for example in wood protection
or that their further application will be forbidden on the basis of the
toxic r;:hr.tp:*.ccc lav.».

0000S21

Reliable analytical proof of polychlorinated dibenzb-p-dioxins is
extremely difficult and requires the use of modern devices
chromatography coupled with masspectrometry)

(gas .

cie se iV A o a

2

which cannot yet be

introduced into the municipal and Cantonal laboratories probably for
financial reasons.

In order to protect human and animal life and

health, and especially in order to prevent possible birth defects,
legal-industrial and technical steps are urgently required.

On a worldwide basis, residues of chlorinated hydrocarbons which are
used as insecticides

(DDT etc.), fungicides

insulation materials

(PCB) or as solvents can be detected in biological

material.

(HCB), lubricants and

By way of preventative measures for protecting possible

irreparable environmental damages, the use of such substances in many
countries, also in Switzerland, has been limited or completely forbidden. In addition to these rather well known impurities, with progressive
development in analysis technology additional chemical environmental
problem substances are beginning to become detectable.

With respect

to their amount they are indeed less, ecologically however'they can be
of at least equal significance.

By way of an example of such analytically,

very difficultly detectable environmental toxin we will describe here
polychlorinated dibenzo-p-dioxins in somewhat greater detail which have
already caused serious toxic catastrophes although they are present in
only relatively minute amounts.

0000622
5815

to
CO

Impurities of Chlorinated Phenols
In the technical ma nu fa ct ur in g of chlorinated phenols, by wa y of
impurities due to the co ndensation of two molecules, polychlo ri na ted
dibenzo- p- di oxi ns form.

This co ndensation reaction occurs at temperature

over ro ug hl y 200°C almost qu an t i t a t i v e l y and in an explosive manner;
few factory installations have a l re ad y fallen v i ct im to this.

a

Under 180°<

in te chnically flawless installations these side products are formed only
to a m i nu te extent.

Therefore, it was possible for the rese ar ch agen cy

of Wa de n s w i l to establish for weed killers produced from t r i c hl or op he nol
ba se d on 2, 4, 5- T a degree of p u ri ty of less than 0.05 p p m of 2,3,6,7t e tr ac hl or od ibe nz o- p- di oxi n w i t h r e sp ec t to 2,4,5-T acid;
a similar spec if ic at io n was esta bl is he d

in Ge rm an y

(0.1 ppm).

Toxifi ca ti on Ca tastrophes in Chicken Farms
Due to the da ng er ou s toxicological pr op er ti es of te tr ac hl orodibenzo-pdioxin, s t ri ct p u ri ty c r it er ia have to be maintained.
toxicity

The acute oral

(LD^ q ) of 2 , 3 , 6 ,7-tetrachlorodibenzo-p-dioxin is:

• ^ d 50

gAg

(1 pg' = 0.001 mg)
Rats, male
female
Guinea pigs

r

22

45

0.6

000C623
5816

uo£,w ;

DOW

The

for the lower and for the higher chlorinated d i b e nz o- p- di oxi ns

is somewhat more favorable:

2, B-dichlorodibenzo-p-dioxin rats orally
"> 2,4,8-trichlorodibenzo-p-dioxin

5000 mg /k g
5000 mg /k g

Hexachlorodibenzo-p-dioxins

1 mg/kg

Oc tachlorodibenzo-p-dioxin

1000 mg/kg

The acute toxification phenomena with chlorinated di be nz o- p- di oxi ns is
shown in the picture of chloracne: brea ki ng out of the skin, d i f f i c u l t
to heal.

.

Especially dangerous are the p o ly ch lo ri na ted dibenzo - p-dioxins due to
their high dama gi ng effect on the embryo

(teratcgeneity): even 0.125

-7

yg/kg of 2,3,6,7-tetrachlor odi be nz o- p- dio xi n cause ph ot ot ox ic damages
in rats if they are administered da i l y from the 6th to the 15th d a y of
pregnancy.

Al so in mice such t r ea tm en t w i t h 1 u g / k g leads to cleft

pallets.

The exact evaluation of these and a whole series of further

v.<_

pertinent investigations is in d e t a i l quite difficult,

still no doubt

exists as concerns the fact th at te tr ac hl or od ibe nz o- p- di oxi n is high ly
teratogenic.

The same is also, true for the isomeric hexach lo ro di oxi ns

wh ic h when administered to rats in 10 da il y doses of 1 y g / k g

caused the

number of fetuses to drop by one half.

Octach lo ro di oxi n, on the other

hand, does not seem to be teratogenic.

Data co nc er ni ng ex perimental

investigations on chronic toxicity of chlorinated d i b e nz o- p- di oxi ns is
locking.

0000624
5817

1

The U.S. Food and Drug Admini st ra ti on has available a copius backlog
of experience concerning the effect of these substances on poultry.

DOW03237G

5

In the years 1957 to 1969 a disease broke out repeatedly in various
States in the U.S.A. on chicken farms and many millions of animals
fell victim to it.

Exhaustive investigations were able to trace this

disease back to the chicken feed whose fatty components were made'impure
with polychlorinated dibenzo-p-dioxins, namely the isomeric hexac,hlorodibenzo-p-dioxins.

In addition to liver and kidney damages, the autopsy

in the animals concerned showed a hydropericardium, that is to say an
accumulation of liquid in the heart cavity.
"chick-edema-disease".

This disease was named

Such toxification catastrophes in chicken farms

are a warning to be taken seriously because dioxin contaminations do not
necessarily have to remain restricted to animal feeds.

Also, in addition

to the various possibilities of ingesting the toxin through the mouth
(orally), probably many possibilities exist that it can also reach the
body through the skin (dermally) .

As an example of this v/e should point

out the toxification of infants due to pentachlorophenol; pentachlorophenol was used in an American drycleaning establishment for treating
diapers and sheets and thus led to the death of two newborn infants.

Large Scale Technical Production
Th e qu estion as to whence and in wh at w a y chlo ri na te d di oxins reached
ch ic ke n feed has no t been decisively cl ea re d up because several possibility
exist for this.

Chlorinated phenols and their d e ri va ti ve s arc consumed

in count) ess area;: of application;

by way of examples wo will me n t i o n here

•O00CS2S
5818

the use of pentachlorophenol in wood pr ot ec ti on and of trichlorophenol

> O W U3237V

6

for p r o d u c i n g the weed de st ro yi ng agent 2,4,5-T or the bactericide
he xachlorophene.

Although no pr od uc ti on figures are known, it is easy

to e s t i m a t e that the world pr o d u c t i o n of chlorinated phenols reaches
v e r y large dimensions.

The p r o d u c t i o n alone of 2,4-D and 2,4,5-T in­

cr ea se d in the United States fr o m ro u g h l y 15 mi llion kg in the year
1958 to 40 m i l l i o n kg in the y e ar 1966, and for the d e s t r u c t i o n of the
V i e t n a m e s e jungle in the years 1962 to 1970 some 22 m i l l i o n kg of 2,4,5-T
w e r e al so u s ed in addition to an essent ia ll y larger a m o u n t of 2,4-D.
The annu al pr od uc ti on of sodium pentachlor op hen ol at e in 1969 alone in
J a p a n a m o u nt ed to over 16,000 tons.

The us e of p e n t ac hl or op hen ol as

d e f o l i a n t on Amer ic an cotton fields was me nt io ne d as a possib il it y of
c o n t a m i n a t i o n of chicken feed.

The m o s t probable source of contamination

is c o n s i d e r e d to be however the tr ea tm en t of cowhides w i th p e n t a c h l o r o ­
phenol.

This fat adhering to animal tissues is mi xe d into chicken feed,

p r o v e d to be v e r y toxic and showed a h i gh content es pe ci al ly of hexachlorodibe nz o- p- di oxi ns .

Since technical pentachlorophenol, in addition to

some 8% tetrac hl or op hen ol and 0.1% trichlorophenol, contains larger
am o u n t s of p o l y ch lo ri na ted dibenzo-p-dioxins, these co mp ou nd s went
d i r e c t l y into the fat on the one ha n d or were able, on the ot he r hand,
to be fr e s h l y formed in addition from the chlorinated phenols during
the m e l t i n g of the fat

In a d d i t i o n to these acute oral or d e r m a l toxications w i t h chlorinated

can scar ce ly bo :;K'a:.urcd by do ct or s and patient

5819

DOWÜ32378

7

Only the damages can be measured'
The chemical and physical properties of chlorinated dioxins ma ke such
ch r o n i c damages very probable.

Polychlorinated dibenz o- p- di oxi ns are

stable and prin ci pa ll y fat-soluble compounds.

Therefore, in the

envi ro nm en t they behave according to the p a tt er n of chlorinated hy d r o ­
carbons.

Te tr achlorodibenzo-p-dioxin was de co mp os ed using ul tr a - v i o l e t

o r sunlight, wh er ea s octachlorodioxin is stable un de r these conditions.
As i d e from this reaction, until today no d e gr ad at io n m e ch an is m is k n o w n
7 >:
w h i c h can ma k e these dangerous compounds di sa pp ea r fast enough.
2,2,6,1tetr ac hl or od ibe nz o- p- di oxi n is heat-stable up to 700°C and only decomposes
co mp le te ly at 800°C.

If paper wh ic h was treated w i t h sodium pentac hl or o-

ph en o l a t e is burnt, the concentration of octa ch lo ro di oxi n even increases.
T e t r a c hl or od iox in is extremely pe rs is te nt in va ri ou s soils and over the
course of 160 days is only unessentially degraded. ' Du e to the low w a t e r
so lu bi li ty

(0.2 ppb) one cannot anticipate its be in g washed out into the

grou nd water, however, absorption into plants —
—

co ul d be detected.

ev en to a minu te degr ee

The absorption of larger amounts, however, is
i

improbable du e to the extremely di ff ic ul t so lu bi li ty even in apolar
solvents

(best solvent:

o-dichlorobenzene,

0.14%).

These properties favor a broad di stribution in the 'environment so that
d i o x i n contaminations,

in addition to the re la ti ve ly larger contaminations

w i t h respect to amounts with chlorinated hy dr oc ar bo ns in biological
material arc very difficult to detect.

Since di oxins do not all have

the ¡M...... Li clerical effect, the possibili Ly exists that this ur.:..» a sural; la
contamination coo]cyirnlly is as significant as the me as ur ab le one.

In

*<

0000627
5820

damage:

no th in g is me as ur ab le and d a ma ge s can appear however at.

bi o l o g i c a l foci.

The contamination on l y becomes me as ur ab le w h e n the

DOVW3237Í)

this sense di ox in -c o n t a m i n a t i o n is a pr im e example of environmental

da m a g e s are irreversible.
f

Pr eventative M e a s u r e s

—

•

In order to pr ev en t such damages, in the n e ar future 'the technical
p r o d u c t i o n of ch lorinated phenols and all subsequent pr od uc ts m a n u ­
fa ctured from t h em and formulations m u s t be improved in such a wa y th at
n o m o r e po ly ch lo ri na ted d i b e n z o- p- di oxi ns are formed.

Should this no t

be possible, then the use of these pr ep ar at io ns should be d r as ti ca ll y
limited or co mp le te ly forbidden.

In a n e w Co nf ed er at ed Toxic Substances

Law, wh ic h is in force n o w fo r 1 year, legal bases are prov id ed which
m a k e it poss ib le to rapidly control this pr o b l e m area.

The manu fa ct ur er s

of chlorinated phenols will probably l e nd a hand in b r i n gi ng about a
hy gi en ic al ly satisf ac to ry control and d o everything to m a k e their p r o d u c ­
ti on units inert w i th in a useful a m ou nt of time.

It is un de rs to od that such pr oblems c a n n o t be solved in Sv/itzerland alone
Inte rn at io na l c o op er at io n is indi sp en sa bl e and f o rt un at el y has already
b e en exte ns iv el y realized.

It w o u l d be qu it e p r o p e r ho wever for our

c o u n t r y in such ma tt er s to lead the w a y and work ou t p r op os al s for
p o s s i b l e solutions of problems of pr ev en ti on and r e s t o r a t i o n . o f chemical
environmental c o n t a m i n a t i o n s .
(A

l i t : : ..ture i iv.'ck can l._-

availab le upon request.)

5 8 2 !0^00S28

U

5822

J

AN ANALYTICAL ME7HC.

DL

CTiMC DIOXIN*

Robert T-r:uc; hmnn
‘ M a tth e w M
H a r v a r d :;;; ¿varsity

Cambrj d g e , M a s 5a chusetts

02138

3

&
o
u
13
•0
O

2,3,7,8-Tetrachlcrodibenzo-p-ciio:<ir. (TCDD) is an extraordinarily’
toxic substance that is produced as an unwanted side product in
the industrial synthesis of 2,4,5-trichlorophenol, an intermediate
in the manufacture of the herbicide 2,4,5-trichlorophenoxyncetic
acid (2,4,5-T).

1

2

Because of its chemical stability *nd its

'

lipophilic nature, the possibility exists that TCDD released into
A direct test

o o

the environment could accumulate in food chains.

of the possibility of biologically significant accumulation in

? <)

/

animal tissues requires an analytical method able to detect TCDD

9 ? 9

at leveis well below those knevn to be toxic.

The lowest value

known for the lethal dose cf TCDD is that observed in the guinea
pig, for which the single cral dose L D rn is SCO parts oer trillion
(ppt) body weight.

3

jU

*

Allowing for sublethal toxic effects and pro­

viding for a conservative margin of safety, it seems desirable
to have an analytical sensitivity of at least one ppt.

For a on<-

gram sample this moans the method must have a sensitivity cf abwiil
10

-1 2* grams or one Dicogram (pg).

The most common method for analysing chlorinated organic compounds
in tissue samples is gas-liquid chromatography (glc) v;ith an
electron capture detector.

Its limit of detection for TCDD, 10

-9

*Paper presented April 2, 1973 at the Conference on DAbunzodioxins
and Dibenzofurans hold by the national Institute of Environmental
Health Sciences, Research Triangle Park, North Carolina.
To be
published in Envircnv.cntal Health Fersooctiv--.~.
~ .. .

5823
000CSS2

ft

9

-2 gram, is inadequate.

This method is also susceptible to intci-

ference from other compounds and so is not very specific.

D o

Mass spectrometry offers better possibilities. It is highly
sensitive and in the high resolution mode of operation it is
highly specific. We have previously described a time averaged
4
mass spectroscopic method with an adequate limit of detection.
However, full sensitivity could not be realized in most sample
types because of interference from DDE (a major degradation
product of DDT) and polychlorinated biphenyls (PCB's). In this
paper we describe a cleanup procedure that overcomes this
difficulty.

w
o o
8 9
9 1 0

Homogenized samples are saponified in alcoholic potassium
hydroxide and extracted with hexane. The extract is shaken with
sulfuric acid and chromatographed on alumina. Elution with
carbon tetra.chlorid e-hexane removes most of the DDE and PCB's.
Chlorinated dioxins are then eluted with dichloromethane-hexane.
The TCDD containing fraction is further purified by preparative
gas-liquid chromatography and analyzed by mass spectroscopy, using
a multi-channel analyser (CAT) to average successive scans.
We also report the levels of TCDD found in a limited number of
samples of fish and crustaceans from locations in South Vietnam
near areas heavily exposed to 2,4,5-T.

5824
0 0 00 5 G 3

-3-

EXPERIMENTAL SECTION

A. ' Reagents and Apparatus.
1. Hexane: Pesticide grade (Fisher Scientific).
2. Dichloromethane: Reagent grade (Eastman).
3. Carbon tetrachloride: Reagent grade (Merck).
4. 95-97% Sulfuric acid: Reagent grade (Dupont).
5. Sodium carbonate (powdered): Reagent grade (Mallinckrodt).
6. Ethanol: Pesticide grade (Matheson, Coleman and Bell).

/666<y<70/M 0(C.

7. Activated alumina: Fisher No. A-540, activated at
130°C for 24 hours.
,
8. Gas chromatograph: Bendix Model 2200 equipped with
thermal conductivity detector.
9. Gas chromatographic column: 5% SE-30 on 60/80
Chromosorb W, 2 m x 2 mm (id) stainless steel. .
10. Trap for preparative gas chromatography: 150 mm x
1.5 mm (id) glass tube packed with 30 mm of glass wool.
11. Mass spectrometer: Associated Electrical Industries
MS-9 double focusing mass spectrometer.
12. A Varian 1024 time averaging computer (CAT), inter4
faced with the MS-9 as described earlier.
B. Cleanup Procedure for the Analysis of TCDD in Tissue Samples.
1. Weigh the sample and homogenize it with 1.0-1.2 parts
EtOH.

5825

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' * 1-

-b

VV

¿

» ">~-^,
-------fT

/'/ F

^

'f

x,r

Y I b t> b-8 O O M P < £

u

1v t /
'+

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o o o c o.r.
/
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5826

-4 -

2. Transfer the homogenate to a round bottomed (RD)
flask equipped with a reflux condenser (Teflon tape
should be* used on the ground glass joint). Spike
37 '
the sample with approximately 1000 ppt
Cl TCDD,
add 2 parts 40% aq. KOH, and reflux for 2 hrs.
3. Let the solution partially cool and add 1 part hexane.
4. Transfer the solution to a separatory funnel, separate
phases and extract with three more identical portions
of hexane. Collect the hexane in the original RB
flask.
5. Transfer the hexane to the separatory funnel, rinse
the RB flask twice with a few ml of EtOH and then
twice with a few ml hexane, refluxing solvent each
time, and extract the hexane with 1 part 1.0 N NaOH.

7. Extract the hexane with 1 part water and add several
grams of Na2C03 to the hexane.
8. Filter the hexane through a column of Na2C03 (100 mm
x 10 mm id for 300 ml hexane). Prewash Na2C03 with
several ml of hexane.

D o W o o P 9 999.

6. Extract the hexane four times (or until acid phase
is colorless) with 2 parts 95-97% H^SO^. Break up
emulsions with a few drops of saturated Na^CO^
solution.

9. Concentrate the hexane (Snyder column) to 3-4 ml.
10. Chromatograph the hexane residue on a column of
activated A1203 (50 mm in a 5 mm disposable pipette).
Do not prewash column. Elute with 12 ml of 20%
CClg in hexane, then 1 ml of hexane, and finally
4 ml of 20% CH2C12 in hexane.
11. Concentrate the 20% CH2C12 fraction carefully to__
about 50 y u lt add 100-200 ,«1 benzene and concentrate

582 7q 0009

-5-

agaJn to 2 0 ^ 1 .

12. Add a few/«g of m-tcrphenyl in benzene to the residue
and preparatively chromatograph. The retention time
of m-terphenyl relative to that of TCDD should be
determined beforehand
and used to make certain
that the TCDD collection is carried out at the
right retention time.
13. Elute the glc trap containing TCDD with 60 /fl
followed by 10 /tl of benzene. Measure the total
amount of eluant collected and calculate the fraction
size in /<1 for the planned number of fractions
(typically ten).

£*(, fc 6<S

o Q /H

14. Prepare the fractions for TCDD analysis in the sample
4
tubes described previously. Add a known amount of
TCDD to three or more fractions for quantitation of
any TCDD observed. The amount of TCDD added per
fraction for quantitation should be approximately
3-4 times the amount expected to be present.
15. Analyze the fractions with the MS-9/CAT. Typical
conditions are: source 220°C, resolution 10,0G0
(based on a 10% valley between peaks), trap current
1.0 mA (rhenium filament), electron multiplier 700,
ionizing voltage 70 eV, time averaging at four scans
per second.
16. Measure peak heights at m/e 321.894. Compute the
-12
quantity of TCDD (grams x 10
) present in the
fractions to which TCDD has not been added from the
ratio of their mean peak heights to the mean peak
heights found with added TCDD.
17. Repeat steps 14-16, but add ^7C1 TCDD and measure
peak heights at m/e 327.885 in order to compute the
amount of 37 Cl TCDD recovered. Calculate the recovery

5828

00003

.

through the complete cleanup procedure bared on the
37
amount of
Cl TCDD added to the sample at the
beginning of the cleanup.
18. Correct the quantity of TCDD computed in step 15 by
the recovery factor obtained in step 16 to give the
final result.
C. Sample Collection.

D
o
w
O O

P9? 9 V

Freshly caught fish and crustaceans were collected in South Vietnam
in August and September 1970 from local fishermen. The samples
were homogenized with a meat grinder, placed in' acetone-rinsed
glass bottles with aluminum foil lined caps, and immediately
frozen in dry ice. Later on the same day, samples were placed in
a Linde LR-35 liquid nitrogen refrigerator where they remained
until analysis. Water blanks were present in the liquid nitrogen
refrigerator throughout the storage period and were analyzed with
the samples. Fresh Cape Cod butterfish (Poronotus -tricanthus,
'family Stromateidae) were obtained from a local market, homogenized,
and kept at -20°C until analysis. Domestic beef livers were ob­
tained and treated similarly.

RESULTS
A. Methodology.
37
The mass spectra of natural and
Cl TCDD are shown in Fig. 1.
The most intense signal for natural TCDD occurs at m/e 321.894
(nominal m/e 322), corresponding to the isotopic isomer with one
atom of
Cl and three atoms of
Cl. The natural abundances of
the Cl isotopes are 75.53 and 24.47 percent, respectively.- The...

'

5829

0 0 00908

-737

observed spectrum for the synthetic
Cl TCDD corresponds tc an
isotopic purity of 95.5 percent 37ci, the same as the value
claimed by the manufacturer of the NaCl used in the synthesis of
the labeled TCDD. The synthetic 37C1 TCDD contributes only 0.042
percent as much to the peak at m/e 322 as to its most intense
signal at m/e 328. The contribution at m/e 320 is even lower, by
37
a factor of nearly 100. This allows an excess of .Cl TCDD to be
added to each sample before cleanup without interfering with
37
analysis of natural TCDD at m/e 322 and 320. The addition of
Cl
TCDD provides a carrier and makes possible tfie calculation of
absolute recoveries.

♦

D o

An alumina chromatography step has been developed which, when
combined with the cleanup steps described previously, makes
possible the measurement of picogram quantities of TCDD in samples
initially containing more than a million-fold excess of DpE and
PCB's. Fig. 2 shows the effectiveness of this procedure.

w
o o

P ? *9 jr

The calculation of TCDD levels described in steps 14-16 of the
experimental section assumes a linear relationship between peak
height and amount of.TCDD present in any given sample. Fig. 3
demonstrates that the response is indeed linear over the full
range of TCDD amounts introduced into the MS-9 in the course of
the analyses reported here.
The reproducibility and overall recovery of the complete analytical
procedure is illustrated in Table 1. A sample of beef liver was
homogenized and divided into three portions each of which was then
37
spiked with 20 ppt TCDD and 1000 ppt
Cl TCDD. The three samples
were independently put through the cleanup procedure up to the
glc step. Each sample was then split into three portions before
preparative glc and mass spectrometric analysis, giving rise to a
total of nine separate values for the recovery of both TCDD and
37C1 TCDD. The average recovery was 3 4 + 7 percent for TCDD and

5830

00 0 0 9 0 9

8
37

2 7 + 5 percent for
Cl TCDD. When the slight background signal
at m/e 322 in an unspiked sample of the same liver is taken into
account, the calculated recoveries from the spiked samples become
even more nearly equal. Experiments performed separately with
each individual cleanup step established that the step with lowest
recovery is preparative gas-liquid chromatography.

B.
TCDD
■ ■ !■ ■ ■Observed
■ ■ ...
■ I I ■ Levels.
■ ■»

✓

Signals at m/e 320 and 322 were conspicuously present in each of
the fish and crustacean samples from Vietnam. The calculated
levels of TCDD, summarized in Table 3, range from 18 ppt to 814 ppt,
based on total wet body weight.
No peak was observed at m/e 320 or 322 with Cape Cod butterfish.
The background signal corresponded to a level of ^ 3 ppt of TCDD.
No peaks were observed in water blank samples present in the liquid
nitrogen refrigerator throughout the sample collection and storage
period.

9 fc 6 6 3 o o A i o ( L

We conclude from these and other controls that the present analyti
cal method provides the sensitivity and reproducibility required
for biologically meaningful analyses of animal tissue samples.
The method makes possible investigations of such samples at levels
approximately ten thousand times lower than those reported here­
tofore.^

Confirmation that peaks observed at m/e 320 and 322 are in fact
produced by TCDD is routinely provided by the criteria outlined
in part A of Table 2. All three of these criteria are- met by the
mass spectra from each of the Vietnamese samples.

5831 OOOIOCO

-9 The additional confirmatory procedures listed in part B of Table

2 were carried out on a sample of Vietnamese fish.

This sample,

carp from the Dong Nai River, exhibited a mean TCDD level of 540
ppt.

The mass spectrum in the regi on m/ e 322 is shown in Fig. 4.

The compound observed in this fish behaved identically to TCDD
in each of the three additional confirmatory tests. We consider
it extraordinarily unlikely that this compound is anything other
than a tetrachlorodibenzo-p-dioxin. In contrast to the signifi­
cant amounts of 2,3,7,8-tetrachlorodibenzorp-dioxin known to have
been disseminated as a contaminant of 2,4,5-T,^ we know of no
mechanism by which other isomers of TCDD might have been introduced
into thè Vietnamese environment.

w o o 8") 9 9 7

The locations from which the Vietnamese samples were obtained are
designated in Fig. 5. The letters correspond to those in Table 3.
Areas heavily treated with 2,4,5-T before its use was ordered dis­
continued in April 1970 are shown as stipled. The number of samples
is not adequate to permit reliable conclusions concerning the dif­
ferences between various locations and species, although this cer­
tainly should be a subject of future studies.

3 o

We have ruled out the possibility that the- TCDD was formed during
the saponification step of the cleanup procedure. It is known that
chlorophenols and chlorophenoxyphenols can form dioxins vinder alka­
line conditions at high temperature.^ A sample of Dong Hai catfish
was worked up omitting the saponification step. The sample was
digested in concentrated sulfuric acid and carried through the rest
of the usual cleanup. The TCDD level, 304 ppt, was somewhat lower
than the 522 ppt observed after the complete cleanup but the dif­
ference was not significant.

oaoioot

C O ''-

I

-1 0 -

Con sidering the limited number of samples we have analyzed and the
fact that they were collected two and a half years ago, it does not
seem appropriate to attempt any detailed evaluation of the possible
toxicological significance of our results. Such discussion is made
even more difficult by the complexity and incompleteness of the
existing toxicological data. However, in order to provide perspec­
tive for such discussion, a tabulation of some of the principal
toxicity data on TCDD is presented in Table 4. .It may be noted that
guinea pigs consuming their weight of food contaminated with TCDD
at <1 level of 600 ppt would have ingested a quantity correspondirnj
to the lethal dose. In contrast, a far greater quantity of TCDD
is required to reach the LD5Q cited for rats. The table shows that
teratogenesis in the rat occurs at doses substantially lower than
those required to kill.

Do W c o P
9P

Feeding studies in monkeys show that dioxin poisoning is cumulative.6
Various levels of a toxic fat known to contain chlorodio^ins were
incorporated into'the daily diet of Macaca mulatta monkeys. As
pointed out by the investigators, the mean survival time depended
inversely on the daily dose. A plot of their data (Fig. 6) conforms
rather well to the relation (mean survival time) ■ X/(daily dose) +
K', where X and X' are constants corresponding, respectively, to
the accumulated lethal dose and to the lag time between the accumu­
lation of this dose and the time of death. No departure from this
relation is seen even at the lowest level of toxic fat tested, where
the mean survival time was 445 days. The importance of this result
is that repeated intake of quantities of TCDD individually equal
to only a small percentage of the single oral dose LD^g may over
time cause serious poisoning. Unfortunately, the LD^g for TCDD in
these primates cannot be computed since all of the animals died (5/5)
even at the lowest dose level and the concentration of TCDD in the
toxic fat employed has not been definitely established.

0001002

1

-1 1 In South Vietnam itself we have little information regarding «ho
possible occurrence of toxic effects of TCDD in humans nor has
it been established whether or not measurable levels of TCDD
have accumulated in exposed populations. Certainly, it should be
pointed out that while we were in South Vietnam in 1970, the medi­
cal member of our group, Dr. John Constable, Professor of Surgery
at Harvard Medical School, did not encounter evidence of any
severe and widespread unusual illness in visiting Can Gio and
several other villages or in discussions with officials of the
South Vietnamese Ministry of Health. However, it was felt that
certain indications in birth statistics ought to be investigated
further for possible connections with herbicide exposure.^ It
is of obvious interest to.survey appropriately.chosen populations
in South Vietnam more closely, especially if TCDD residues should
be found in human tissue samples.
Vi

o

Finally, turning from questions of environmental toxicol&gy to the
biological mechanisms of action, we note that TCDD seems to be
particularly toxic to proliferating tissues, as suggested by its
effects on spermatogenesis and hematopoiesis and its apparent
toxicity to the intestinal epithelium and the thymus.
These
indications are consistent with the effects of a mitotic poison,
such as TCDD is known to be in the African blood lilly*0 and
possibly in Drosophila melanogaster.
We are led by these obser­
vations to speculate that TCDD may be able catalytically to dis­
rupt microtubules, the subcellular elements of which spindle
fibers are constructed and which are ubiquitous in their structural
roles in cell extension and cell movement.

5834

o
o

«0

-o
•«

.0
«0

CO

000100

<

12SUMMARY
]. A procedure has been developed for the reliable detection of
-TCDD in animal tissues down to levels approaching one ppt.

It

makes use of chemical cleanup, preparative gas-liquid ch romato­
graphy and analysis b y time averaged high resolution, mass sp ec­
troscopy.

2. A limited number of fish and crustacean samples.was collected
in South Vietnam in 1970 near areas heavily exposed to the herbi­
cide 2,4,5-T. TCDO was detected in these samples at levels
ranging from 18 to 814'ppt. TCDD was not detected in a sample
of Cape Cod butterfish used as a control.
DO W

3. These results suggest that TCDD may have accumulated to biolo­
gically significant levels in food chains in some areas 'of South
Vietnam exposed to herbicide spraying.

o

O

•*o
O
O
o
0

5835
0001004

-1 3 -

•ACKNOWLEDGEMENTS

I

He thank Professor Bui Thi Lang, Mr. Robert Cook, Professor Arthur
Westing, and Or. John Constable for aiding in tho collection of
samples, and Professor Lang for helping to identify the specimens
collected. We also thank Kenneth Gross and Lesley Newton for
their expert assistance in the laboratory.

Q o p 6 Q O f M O <£

-This research was initiated by the Herbicide Assessment Commission .
of the American Association for the Advancement of Science. The
work has been supported by funds from the AAAS, the Ford Foundation,
and the National Institute of Environmental Health Sciences
(NIH grant no. 1 ROI ES0C851-01 TOX).

•

5836

000 1•*\Jn 'Jn,0rr

REFERENCES

1. "Report on 2,4,5-T," A Report of the Panel on Herbicides of
the President’s Science Advisory Committee, Executive Office
of the President, Office of Science and Technology, March 1971.
2. "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,
91st Congress, April 7 and 15, 1970.
3. Sparschu, G.L., P.L. Dunn and V.K. Rowe, "Study of the Terato­
genicity of .2,3,7,3-Tetrachlorodibenzo-£-dioxin in the Rat,”
1971. Fd. Cosmet. Toxicol.
405.

€. Allen, J.R. and L.A. Carstens, "Light and Electron Microscopic
Observations in Macaca Mulatta Monkeys Fed Toxic Fat," 1567.
Am. J. Vet. Res. 28: 1513^
7. Neubert, D. and I. Dillman, "Embryotoxic Effects in Mice Treated
with 2,4,5-T and Tetrachlorodibenzo-£-dioxin," 1972.
Naunyn-Schmledbergs Arch. Pharmacol. 272: 243.

8. Verret, J. in "Effects of 2,4,5-T on Man and the Environment,"

Co 0 0 6 0 0

5. Woolson, E.A., W.I. Reichel and A.L. Young, "Dioxin Residues
in Lakeland Sand and Eagle Samples," in press. Adv. in Chem.
119.
“
/

n* o <£

4. Baughman, R. and M. Meselson, "An Improved 'Analysis for 2,3,7,8Tetrachlorodibenzo-£-dioxin," in press. Adv. in Chem. 119.

Hearings before the Subcommittee on Energy, Natural Resources,
and the Environment of the Committee on Commerce, United States
Senate, 91st Congress, April 7 and 15, 1970.

9. Poland, A. and E. Glover, "2,3,7,8-Tetrachlorodibenzo-£-dioxin:
A potent Inducer of ¿-Aminolevulinic acid synthetase, .1973.
Science 173: 243.
10. Jackson, W.T., "Regulation of Mitosis: III. Cytological Effects
of 2,4,5-T and of Dioxin Contaminants in 2,4,5-T Formulations,"
1972. J. Cell Sci. 10: 15.
11. Herbicide Assessment Commission, "Background Material Relevant
'to Presentations at the 1970 Annual Meeting of the AAAS" and
"Preliminary Report of the Herbicide Assessment Commission,"
both reprinted in Congressional Record IIS(32): S3226-3233,.• 3 March 1972.

5837

0001006

-1 5 -

12. Ruu-Hoi, N.P., Phairf Huu-Chanh, G. Sesquel, M.C. Azum-Gclaile
and G. Saint-Ruf, "Organs as Targets of Dioxin Intoxication,"
1972. Naturwissenschaften 59: 174.
13. Davring, L. and M. Sunner, "Cytogenetic Effects of 2,4,5Trichlorophenoxyacetic Acid on Oogenesis and Early Embryogenesis
in Drosophila Melanogaster," 1971. Hereditas 68: 115.
14. Porter, M.L. and J.A. Burke, "Separation of Three Chlorodibenzo£-dioxins from Some Polychlorinated Biphenyls by Chromatography
on an Aluminum Oxide Column," 1971. J. Assoc. Off. Anal. Chem.
54: 1426.
15. Woolson, E.A., R.F. Thomas and P.D.J. Ensor, "Survey of Polychlorodibenzo-£-dioxin Content in Selected Pesticides," 1972.
J. Agr. Food Chem. 20: 351.

D o W o o nr o o a 3

16. Crosby, D.G., A.S. Wong, J.R. Plimmer and E.A. Woolson,
"Photodecomposition of Chlorinated Dibenzo-£-dioxins," 1971.
Science 173: 748.
■
/

0001007

5838

Figure 2.

Mass spectra showing reduction of DDE and PCB levels
in fish residue by means of alumina chromatography.
Following the sulfuric acid cleanup step, the residue in
hexane is added to a column of activated alumina. The
trace in part A of the figure is from the material eluted
by 20% CH-Cl., in hexane after the column was first
eluted with 20% CC1. in hexane. The trace in part B
was obtained from a4similar 20% CH.Cl. in hexane elu­
tion after the column was first eluted with 1 %
CH-Cl- in hexane. Elution with 1% CH-Cl, in hexane
was reported to be effective in reducing * the amount
of PCB residues (Porter and Burke, 1971). Elution with
20% CC1. is clearly even more effective and was routinely
used in obtaining the results reported here.

Figure 3.

Linearity of response for TCDD in the presence of beef
liver residue. The TCDD values are the amounts intro­
duced into individual runs on the MS-9.

Figure 4.

TCDD signals observed in fish samples. A. Vietnamese
carp plus 60 pg TCDD. B. Vietnamese carp. C. Cape Cod
butterfish. The wet weight of fish represented in run A
and run B is 0.18 g. In C it is 0.16 g.

Figure 5.

Map showing sampling sites in relation to rivers and
principal sprayed areas. Sites A and B are located on
the Dong Nai River, site C is on the Sai Gon River,
and site D is on the coast at Can Gio. Sprayed areas
are depicted only within the region bounded by the
dashed lines ( __).

Figure 6. Mean survival time of monkeys fed toxic fat (Allen and
Carstens, 1967) plotted against the reciprocal of the
percent of toxic fat present in the diet.

0001008

5839

O OQ b O O

Mass spectra of TCDD (A.) and.-^Cl labelled TCDD (B.).
The isotopic purity of the J/Cl is 95.5%. The
asterisk denotes an impurity. The multiplicity of
lines associated with each major molecular species
results from the presence of various isotopes of
Cl and C.

h

Figure 1.

0(£

FIGURE LEGENDS

5840

V

J

e address presented a t the 2 5 th a n n u a l m e e tin g o f the S outhern W eed
S o c ie ty a t Dallas. Texas. Jan. 18. 1972)

n e w s p a p e rs re p o rte d th a t th e re had been |
an in c re a s e in h um an birth d e fe c ts in some
p a rts of th e co u n try . T h e s u g g e s tio n was
m a d e th a t th is in crease w a s s o m e h o w re­
la te d to th e u se by th e U n ite d S ta te s Armed
F o rces of d e fo lia n t ch em icals.
T h e s e re p o rts of p o ssib le teratogenic
e ffe c ts in V ie tn a m m ig h t h ave gone un­
n o tic e d had th e y n o t been fo llo w e d in Octo­
ber. 1 9 6 9 . by th e a n n o u n c e m e n t of Dr. Lee
A. D u B rid g e , th e n th e P re s id e n t's Science
A d v is o r, th a t use of 2 .4 ,5 -T in th e United

Teratological
potential of
2,4,5-T

By James G. Wilson. Ph.D.
Children's Hospital Research Foundation and
Departments of Pediatrics and Anatomy
University of Cincinnati College of Medicine

w a s a c o n s e q u e n c e of th e re le a s e a few
d a y s e a rlie r o f th e resu lts o f a s tu d y of a

of p a g e s of rese
and s u rv e y resL

n u m b e r o f p e s tic id e s a n d in d u s tria l
c h e m ic a ls by B io n e tic s L ab o ra to rie s which
in d ic a te d th a t m ice tre a te d d u rin g early
p re g n a n c y w ith large d oses of 2 ,4 .5 - T gave

p a rtic u la rly duri:
N e v e rth e le s s , in
p o rt* w a s subm i
h aus. w h o h ad.

b irth t o m a lfo rm e d offsp ring .
D r. D u B rid g e 's a n n o u n c e m e n t, together
w ith th e V ie tn a m re p o rts th a t h u m an birth
d e fe c ts h ad p o s s ib ly res u lte d fro m exposure

a p p o in te d Adm :
re p o rt rep resen ;
m e n t re a c h e d bm itte e e x c e p t fo
w a s D r. T h e o d o r
n o t a q u a lifie d
asked t o a p p e n r
and e x c e p tio n s

o f p re g n a n t w o m e n to th is h e rb ic id e , had
w id e s p re a d re p ercu ssio n s. D u rin g th e fol­
lo w in g m o n th s a v e rita b le e p id e m ic of high
level d is c u s s io n s o ccu rred in th e fo rm of:
g o v e rn m e n t-s p o n s o re d p an els o f experts:
c o m m is s io n s s e t up by s c ie n tific societies;
c o n fe re n c e s o f s c ie n tis ts fro m universities,
c h e m ic a l in d u s trie s and th e governm ent:
and e v e n a s u b c o m m itte e o f th e U .S . Senate.
T h e s e s u n d ry g ro u p s d is c u s s e d th e matter
a t le n g th b u t w e re unable to reso lve the
m a in is s u e o f w h e th e r 2 ,4 ,5 -T . as currently
p ro d u c e d an d u sed , re p re s e n te d a real risk

o p e ra te d

spec:

lecse
u n til de
■ ta k e n b y th e Er
a rtic le s appears
a lm o s t e x c lu s i'
e x c e p tio n s o f *
N o m o re th a n ;

J a n u a ry , 1 9 7 1 , a t w h ic h tim e it w a s inherited
by th e n e w ly fo rm e d E n v iro n m e n ta l Protec­

to th e c o n c lu s k

tio n A g e n c y (E P A ). D u rin g th e p re v io u s year

a n is ts a n d bioc
in th e m a in rep
By all a p p e a r

rem ain ed

re s tric tio n s

had

b een

placed

on th e use of 2 ,4 .5 - T , both fo r m ilita ry pur­
p o s e s in V ie tn a m and fo r s e v e ra l ty p e s of

o f m o re p u b lic c o n c e rn th a n a n y o th e r p e s ti­

u se in th e U n ite d S ta te s th a t, it w a s th o ug h t,
m ig h t re s u lt in e x p o s u re o f p re g n a n t w om en. .
M a n u fa c tu re rs of 2 .4 ,5 - T as w e ll as agri­
c u ltu ra l in te re s ts th a t used it w e re entitled
u n d e r la w to a s k fo r s c ie n tific re v ie w of
a n d /o r p u b lic h e a rin g s on th e b ases for
th e re s tric tiv e a c tio n s . T w o m an u fa c tu re rs.

c id e e x c e p t th e in s e c tic id e D D T . S in c e e a rly
in th e V ie tn a m e s e w a r , 2 .4 ,5 - T , to g e th e r

T h e D o w C h e m ic a l C o m p a n y an d Hercules
In c o r p o r a t e d , h a d a lr e a d y r e q u e s t e d a

w ith s e v e ra l o th e r h e rb ic id e s , w a s e x te n ­
s iv e ly used as a d e fo lia n t to re d u ce th e

s c ie n tific re v ie w , seve ra l m o n th s
E P A o ffic ia lly c a m e in to being.

before

lik e lih o o d o f a m b u s h in a reas w h e re d e n s e

O n e o f E P A 's e a rlie s t fu n c tio n s w a s to

u n d e rg ro w th in th e fo re s ts an d s w a m p la n d s

a c tiv a te in J a n u a ry , 1 9 7 1 . a S c ie n tific Ad­

o f V ie tn a m fa v o re d g u e rilla w a rfa re . D u rin g

v is o ry C o m m itte e w h ic h w a s c h a rg e d with

J u n e an d J u ly o f 1 9 5 9 , S o u th V ie tn a m e s e

re s p o n s ib ility

'*• TO EARTH. Voi. 28. No. 4. Spring 1973

A lm o s t im m er
to t w o s c ie n tif
and S cience) ir
re g u la tio n s un.

u n a n s w e re d in

q u e s tio n

g o v e rn m e n t

h e rb ic id e 2 .4 , 5 - T h a s received m o re

C o m m itte

S ta te s w a s s o o n to be re s tric te d by certain
g o v e rn m e n t ag en cies. The announcem ent

The

n o to r ie ty an d p ro b a b ly h a s been th e c a u s e

The

m o n th s in w h ic
all in fo rm a tio n t t
d ence an d to pr
a fo rm id a b le job

fo r h u m a n p reg n a n c y .

The

dence
on
th e
(c a p a c ity t o caus
and to re c o m m e
th a t w o u ld s e e r
s c ie n tific d a ta . A
s e le c te d fro m a I
by th e N a tio n a l
reg ard ed b y th e .
priate q u a lific a tic

to

re v ie w

all a v a ila b le evi­

5841

th e e ig h t to x ic

b eh in d th e s e c
h o p e d t o d e s tr
p o rt a n d arous
s c ie n tis ts and
had an o p p o rti
je c tiv e w a y . n
s id e re d a c tio n
ta k e n t o d a te .
M r. R u c k e ls h a i

*Copies of the Repor
available by w riting :
Mira. Betty Bazille
Room 3125
Environmental Prot
South A griculture t
12th Street and Inc
W ashington. 0. C.

■-¡a

th a t t h e r e h a d b e e n !
b irth d e fe c ts in sorriep
T h e s u g g e s tio n wasf^

dence o n
th e
te r a to g e n ic p o t e n t ia l
(capacity to c a u s e b irth d e fects), o f 2 ,4 , 5 - T
and to re c o m m e n d a n y c o u rs e s of a c tio n

" A p u b lic h earin g w ill be h e ld in th e fa ll to
o b ta in a d d itio n a l fa c ts on 2 .4 , 5 - T w h ile th e
c a n c e lla tio n o rd e r c o n tin u e s o n th e use of

>e w a s s o m e h o w r£ lj

that w o u ld s e e m to be in d ic a te d by th e
scientific d a ta . A n in e -m a n c o m m itte e w a s

th e

selected fro m a list of s c ie n tis ts s u b m itte d
by th e N a tio n a l A c a d e m y o f S c ie n c e , and
regarded by th e A c a d e m y as h a vin g a p p ro ­
priate q u a lific a tio n s .

fu rth e r q u o te d as s a yin g th a t " w h ile th e
A d v is o ry C o m m itte e R e p o rt s ig n ific a n tly
ad d s to an u n d e rs ta n d in g o f 2 .4 .5 - T , q u e s ­

U n ite d S ta te s A rm e d i
m ic a ls .
D ossible te r a to g e n ic !
ig h t h a v e g o n e u n | |
ie n fo llo w e d in O c to £ |,
•u n c e m e n t o f D r. L e e j|
P re s id e n t’s S c ie n c e t
I . 4 . 5 - T in th e U n it e d !
r e s tr ic te d by c e rta in ^
T h e a n n o u n c e m e n t^
•f th e re le a s e a fe w ^

The C o m m itte e w a s a llo w e d o n ly fo u r
months in w h ic h to c o lle c t an d e v a lu a te

h e rb ic id e

on fo o d

h u m a n c o n s u m p tio n . . . "

c ro p s

g ro w n fo r

R u c k e ls h a u s w a s

tio n s rem ain as to th e b a la n c e b e tw e e n
b e n e fits and risks fro m u se o f th e h e rb i­
cid e." T h e n e w s release n o te d th a t th e C o m ­

all in fo rm a tio n th a t had a n y s c ie n tific c re ­
dence and to p re p a re its re p o rt. T h is w a s
a fo rm id able jo b in v ie w o f th e th o u s a n d s
of pages o f res e a rc h d a ta , e x p e rt o p in io n ,

m itte e w a s n o t c h a rg e d w ith a s s e s s in g
b e n e fit-ris k fa c to rs and th a t a p u b lic h earin g
w o u ld be d e s ira b le to re s o lv e th is issu e. N o
p u b lic h earin g has y e t b een s c h e d u le d .

u lts o f a s tu d y o f a~L

and su rvey re s u lts th a t h ad a c c u m u la te d ,

es

particularly d u rin g th e p re v io u s tw o years.

; L a b o ra to rie s w h i c h |a

N evertheless, in M a y , 1 9 7 1 , a 7 5 -p a g e re ­

If it w a s th e in te n t of th e p e rs o n s w h o
leaked th e re p o rt to d is c re d it it b e fo re its
le g itim a te re le a s e , th is o b je c tiv e w a s o nly

re s te d

port* w a s s u b m itte d to W illia m D. R u c k e ls haus. w h o h a d , a fe w m o n th s e a rlie r, b een

p a rtia lly ach ieved . In th e fir s t p la c e th e
m a g a zin e re p o rts w e re s o fla g r a n tly b iased

appointed A d m in is tr a to r o f th e E P A

th a t e v e n re a d e rs u n fa m ilia r w ith th e s u b ­
je c t co u ld h a rd ly m is s th e s e le c tiv ity used

and

in d u s tr ia l^

d u rin g

earty^fl

o s e s o f 2,4,5-T gavejfl
pring.

The

>u n cem en t. t o g e t h e r ^
rts t h a t h u m a n b irth 3

report re p re s e n te d a c o n c e n s u s of a g re e ­
ment reach ed by all o f th e n in e -m a n C o m ­

s u ite d fr o m e xp o su re” !
th is h e rb ic id e , had.
on? D u rin g th e fo lbl
id e m ic o f high
rre a in th e fo rm of:

mittee e x c e p t fo r o n e m em b er. T h e d is s e n te r
was Dr. T h e o d o re S te rlin g , th e o n ly m e m b e r
not a q u alified b io -m e d ic a l s c ie n tis t, w h o
asked to a p p e n d a le n g th y lis t o f o b je c tio n s
and e x c e p tio n s to th e m ain re p o rt.

p a n e ls of e x p e rts ; ■
s c ie n tific s o c ie tie s ;
ts fr o m u n iv e rs itie s ,
id t h e g o v e rn m e n t; !
;e o f th e U .S . S e n a te ,

A lm o s t im m e d ia te ly th e re p o rt w a s leaked
to tw o s c ie n tific n e w s m a g a zin e s (Nature
and Science) in s p ite o f th e fa c t th a t th e
regulations u n d e r w h ic h th e C o m m itte e
operated s p e c ific a lly p ro h ib ite d a n y re­

is c u s s e d th e m a tte r

fo rtu n a te c o n s e q u e n c e s m a y be th a t the
re lia b ility o f s c ie n tific jo u rn a lis m w ill rem ain

2 .4 .5 - T , as c u rre n tly

lease u n til d e fin itiv e a c tio n h ad been
taken by th e EPA . H ig h ly c ritic a l m a g a zin e
articles a p p e a re d d u rin g th e s u m m e r b ased

^ re s e n te d a re a l ris k

alm ost e x c lu s iv e ly o n th e o b je c tio n s and

th o u g h le tte rs w e re w r itte n t o th e m a g a zin e
p rin c ip a lly in v o lv e d , o b je c tin g t o th e o v e rt

a b le t o re s o lv e th e

exceptions o f th e o n e d is s e n tin g m e m b e r.
le d

u n a n s w e re d

in d e s c rib in g th e c o n te n ts o f th e re p o rt.
T h e care fu l e v a lu a tio n o f d a ta an d ju s tifi­
c a tio n of c o n c lu s io n s p re s e n te d b y th e A d ­
v is o ry C o m m itte e w e re n e ith e r q u o te d n or
d is c u s s e d . O n th e o th e r h a n d , m o s t o f th e
o b je c tio n s an d c ritic is m s o f th e o n e d is s e n t­
ing c o m m itte e m e m b e r w e re re p o rte d in as
fa v o ra b le a lig h t as th e w r ite r c o u ld m a n a g e ,
d e s p ite th e fa c t th a t th e s e o b je c tio n s w e re
based o n little m o re th a n p erso n al o p in io n s
an d s u b je c tiv e re a c tio n s . O n e of s e v e ra l un­

in d o u b t in th e m in d s o f m a n y re a d e rs . A l­

in

No m o re th a n p assin g re fe re n c e w a s m ad e

b ias and u n fa irn e s s of th e a rtic le s on th e

tim e it w a s in h e rite d

to th e c o n c lu s io n s a n d re c o m m e n d a tio n s o f

2 .4 . 5 - T

v iro n m e n ta l P ro te c -

the e ig h t to x ic o lo g is ts , te r a to lo g is ts , b o t­

o n ly le tte r y e t to be p u b lis h e d w a s th a t

ng th e p re v io u s y e a r
s h a d been p la c e d
o th f o r m ilita ry p u r­
e r s e v e ra l ty p e s of
tn a t. i t w a s th o u g h t,

anists and b io c h e m is ts w h o h ad c o n c u rre d
in th e m ain rep o rt.
By all a p p e a ra n c e s th e p e rs o n o r p e rs o n s
behind th e s e d is to rte d m a g a z in e a c c o u n ts
hoped to d e s tro y th e c re d ib ility o f th e R e­

s ig n ed by th e e n tire m e m b e rs h ip o f th e
C o u n c il erf th e S o c ie ty o f T o x ic o lo g y . T h e
p ro te s t o f a p anel s o h ig h ly q u a lifie d to
p a s s ju d g m e n t co u ld h a r d ly be ig n o re d ,

o f p re g n a n t w o m e n .

port and a ro u s e p re ju d ic e in th e m in d s of
s cien tists an d th e p u b lic b e fo re th e E P A
had an o p p o rtu n ity t o e x a m in e it in a n y o b ­

d e la y e d s e v e ra l m o n th s b e fo re
p rin te d . (Science, N o v. 5 . 1 9 7 1 ).

jective w a y , m u c h le s s ta k e c a re fu lly c o n ­
sidered a c tio n . T h e o n ly a c tio n E P A has
taken to d a te , h a s b e e n a n e w s re le a s e by
Mr. R u c k e ls h a u s o n A u g u s t 9 , 1 9 7 1 , s ta tin g .

re a lly te ra to g e n ic ? T h e a n s w e r is ves. b u t

-T as w e ll as a g rised it w e re e n title d
s c ie n tific re v ie w o f
on th e b a s e s fo r
Tw o m a n u fa c tu re rs ,
tp an y a n d H e rc u le s
e?
re q u e s te d a
rai ,.io n th s b e fo re
being.
: fu n c tio n s w a s t o
’1 . a S c ie n tific A d w a s c h a rg e d w ith
a ll a v a ila b le e v i­

‘ Copies of the Report of the 2.4.5-T Advisory Committee are
available by w riting:
Mrs. Betty Bazille
Room 3125
Environmental Protection Agency
South A griculture Building
12th Street and Independence Avenue. SW .
Washington. D. C. 20460

A d v is o ry

C o m m itte e

R e p o rt,

th e

n e v e rth e le s s , th e le tte r o f th e C o u n c il w a s
it

w as

B u t w h a t a b o u t th e m a in is s u e — is 2 , 4 , 5 - T
s o are h u n d re d s o f o th e r c o m m o n ly used
d ru g s , p la n t p ro d u c ts an d e n v iro n m e n ta l
c h e m ic a ls (T a b le 1). T h e s e c o m p o u n d s w e re
g iv e n t o p re g n a n t la b o ra to ry a n im a ls in
d o s e s u s u a lly - g re a tly in e x c e s s o f th a t
e q u iv a le n t t o th e h u m a n th e ra p e u tic d o s e , in
th e c a s e o f d ru g s , or o f th e lik e ly e x p o s u re
level, in th e c as e o f e n v iro n m e n ta l c h e m ic a ls .
T h is lis t o f c h e m ic a ls n o w k n o w n t o be
te r a to g e n ic in ra ts , m ic e o r ra b b its is s o
DOWN TO EARTH. Vol. 28. No. «

I
»

&

.exten sive t h a t it is q u ite n a tu ra l To ask if
n o t all c h e m ic a ls m ig h t be d am aging to
e m b ry o n ic a n im a ls u n d er th e rig h t condi­
tio n s . A s a m a tte r o f fa c t, in vestig ato rs in
th e fie ld o f te r a to lo g y te n d to assu m e that
th is is in d eed th e c ase. O ne of th e widely

^SSelicylates (e.g, aspirin, o il of wintergreon)

a c c e p te d p rin c ip le s in th e fie ld is called
K a rn o fs k i's L aw , w h ic h s ta te s , "th a t any

^•C ertain A lkalo id s (e .g , caffeine, nicotine, colchicine)

d ru g a d m in is te re d a t th e p ro p er dosage, at
th e p ro p e r s ta g e o f d e v e lo p m e n t, to em­
b ry o s of th e p ro p e r s p e c ie s , w ill be effective
in ca u s in g d is tu rb a n c e s in e m b ry o n ic de­
v e lo p m e n t."

Tranquilizers (e .g , meprobamate, chlorpromazine, reserpine)

- ;

I 7^-'

Antihistamines (e .g , budizine, meclizine, cyclizine)
Antibiotics (e .g , chlorampbenacol, streptonigrin, penicillin)

A lth o u g h K a rn o fs k i's Law s p e c ifie s only
d ru g s , it u n d o u b te d ly h o ld s tru e fo r many
o th e r, if n o t all. ty p e s of ch em icals. All
p e s tic id e s w o u ld c e rta in ly b e s u s p e c t be­

Hypoglycémies (e -g , carfautemide, tolbutamide)
Corticoids (e .g , triamcinolone, cortisone)

c a u s e th e y , like a n tib io tic s , sulfonam ides,
a n titu m o r c o m p o u n d s an d im m u no -su p p ress iv e d ru g s , w e re d e s ig n e d a n d developed
e ith e r to kill livin g o rg a n is m s o r to sup­

Alkylating agents (e .g , busulfan, chlorambucil, cyclophosphamide, TEM)
Antimalarials (e .g , chloroquine, quinacrine, pyrimethamine)

'

-

ir --

Anesthetics (e .g , halothane, urethan. nitrous oxide, pentobarfaital)

p ress th e ir m e ta b o lis m and g ro w th . Three
y e a rs ag o a s u rv e y of th e lite ra tu re (Table

AntimetaboHtes ( e ^ , folic acid, purine and pyrimidine analogues)

2 ) reve a le d th a t s e v e ra l p e s tic id e s had at
th a t tim e a lre a d y b e e n re p o rte d to b e terato ­
g e n ic in m a m m a ls . T o d a y th e list w o u ld be
a t le a s t tw ic e as long. T h u s it is probably
s a fe to a s s u m e th a t m o s t p e s tic id e s given
in h ig h d o s a g e to p re g n a n t m a m m a ls would,
if th e y did n o t kill th e m o th e r fir s t, interfere
w ith th e g ro w th a n d /o r m e ta b o lis m of the
e m b ry o s . N o b io lo g ic a l s y s te m is k n o w n to
g ro w fa s te r th a n th e m a m m a lia n embryo
d u rin g its e a rly d iffe re n tia tio n and organ
fo rm a tio n .
W h y th e n , if m o s t p e s tic id e s are likely to

' v Sohmnts (e .g , benzene, dimethylsutfoxide, propylene glycol)

,¡1
ili

Pesticides (e .g , 2,4,5-T, carfaaryl, captan, folpet)
Industrial effluents (e .g , some compounds of Hg, Pb, As, Li, Cd)

.

Ili.
: Il

i
;

Miscellaneous (e .g , trypan blue. Tryparanol, Diamox, etc.)

be te r a to g e n ic a t h ig h d o s e s , did so much
e x c ite m e n t fo llo w th e re p o rt in 1 9 6 9 th a t 2,
4 ,5 - T at h ig h d o s e s c a u sed m a lfo rm atio n s
in m ice? T h e a n s w e r is n o t e n tire ly clear,
b u t it p ro b a b ly re la te s to all o f th e fo llo w ing
. i

Capian and Folpet .;

;

! £ > : . v ^ ; ;Carbaryl (Sevin) ■ .-d

j p ■"

- rabbits and mice

mice and dogs

c o n d itio n s : 1) th e h ig h level o f e m o tio n that
s u rro u n d e d all a s p e c ts o f th e V ie tn a m War, I
2 ) th e u n s u b s ta n tia te d re p o rt th a t hum an I
b irth d e fe c ts h a d re s u lte d fr o m d e fo lia tio n I
in V ie tn a m , fo llo w e d w ith in a fe w w e e k s by 1

Dieldrin. Chlordane and Keptone

-m ic e -

" Diquat and Paraquat

v.;':•-/ '•

rats

• ;

; ' Various organomercury compounds

mice

th e re p o rt o f th e te r a to g e n ic e ffe c ts o f 2 ,4 .
5 - T in m ic e . 3 ) th e w id e s p re a d p u b lic con­
c e rn a b o u t m a n y a s p e c ts o f e n v iro n m e n ta l
c h a n g e a n d th e ir e ffe c ts on " e c o lo g y ", 4)
o v e r-z e a lo u s a n d s o m e tim e s irresp o n sib le 1
e ffo r ts b y s o m e a g e n c ie s an d in d iv id u a ls to |
in flu e n c e p u b lic o p in io n fo r p e rs o n a l gain. |
o r fo r s in c e re b u t ill-fo u n d e d re a s o n s .

>

k
j :-

L

2 .4 3 -T * and 2.4-D

rats and mica

** ..'.Teratogenic effects may have been caused by an im purity.

i'

■'

,,■..

;ki ..-i

16

DOWN TO EARTH. Vd. 28. No. 4. Spring 1973

|

N o w I w o u ld lik e to s p e a k d ire c tly to the f
s u b je c t o f th e te r a to g e n ic p o te n tia l o f 2 ,4 . f
5 -T . T h e A d v is o ry C o m m itte e a g re e d in a |
ra tio o f 8:1 t h a t a c c e p ta b le d a ta on th e e m - |
b ry o to x ic ity o f 2 . 4 . 5 - T w e re a v a ila b le fo r 6 |
m a m m a lia n s p e c ie s ; n a m e ly , m o u s e , rat. |

5843

I

'1
hamster, ra b b it, s h e e p , and rh esu s m o n k e y .

¿ ig /k g

of T C D D

None of th e s e s h o w e d ad v e rs e e ffe c ts a t a
dosage of 4 0 m g /k g / d a y d u rin g th e p erio d

young

w ith

on

d ays 6 - 1 5

p ro d u c e d

so m e e m b ry o to x ic sig n s. No

evidence o f d o m in a n t le th a l m u ta tio n s w a s

of organogenesis. T h e m o u s e w a s th e m o s t
sensitive of th e s p e c ie s s tu d ie d , s h o w in g
a low level o f te r a to g e n ic ity , m a in ly c le ft
palate, a t 1 0 0 m g /k g /d a y g iv e n th ro u g h o u t
organogenesis. H a m s te r and ra t re q u ire d

s een

in

s u rv iv in g

m ales

a fte r

an y dose.

higher d o sag e to o b ta in c o m p a ra b le e ffe c ts .

p o s itio n . p e rs is te n t in so ils and s lo w ly
d e g ra d e d in s o ils to p o lar m e ta b o lite s .

K e a rn e y a n d a s s o c ia te s at th e U .S . D e ­
p a rtm e n t o f A g ric u ltu re h ave re p o rte d th a t
T C D D is im m o b ile in so ils, n o t read ily ta k e n
up

by

p la n ts ,

s u b je c t

to

p h o to d e c o m ­

It was th o u g h t lik e ly th a t all s p ecies c o u ld
have been c a u s e d to s h o w e m b ry o to x ic ity
ff. 2 .4 .5 -T d o s a g e h a d been raised h ig h

" S u b s e q u e n t s tu d ie s reve a le d th a t e n v i­
ro n m e n ta l c o n ta m in a tio n by T C D D is e x ­

enough. F o r e x a m p le , p re g n a n t ra ts g iv e n a

tre m e ly

single dose o f 2 0 0 m g /k g o n d a y 9 , th e m o s t
susceptible tim e fo r te ra to g e n e s is in th is
species, c a u s e d m o d e s t in c re a s e s in th e

lo g ical s a m p le s ."

percentage o f m a lfo rm e d s u riv io rs as w e ll as
in percentage o f in tra u te rin e d e a th .
The d io x in c o n t a m in a n t 2 . 3 , 7 . 8 - t e t r a chlorodibenzo-paradioxin (T C D D ) h a s a ls o
been show n to h a v e a lo w te r a to g e n ic p o ­
tential at d o s e s in e x c e s s o f 0 .0 0 1 m g /k g .
This dosage level is v irtu a lly im p o s s ib le to
achieve w ith c u rre n tly p ro d u c e d 2 ,4 .5 - T ,
which rarely e x c e e d s 0 .5 p pm and u s u a lly
contains m u c h less.
Careful s c ru tin y o f th e re p o rts of a d v e rs e
effects on h u m a n re p ro d u c tio n a fte r u s e of
2.4.5- T as a d e fo lia n t in th re e s e p a ra te
locations, n a m e ly , V ie tn a m : G lo b e . A riz o n a ;
and Sw ed ish L a p la n d , y ie ld e d n o a c c e p ta b le
evidence th a t th e a lle g e d e ffe c ts w e r e re ­
lated to e x p o s u re t o 2 .4 .5 -T . It w a s c o n ­
cluded th a t, as c u rre n tly p ro d u c e d a n d as
applied a c c o rd in g t o th e re g u la tio n s in fo rc e
prior to A p ril. 1 9 7 0 , 2 ,4 , 5 - T re p re s e n te d n o
hazard to h u m a n re p ro d u c tio n .
This w a s th e s itu a tio n as of M a y 7 . 1 9 7 1 ,
when th e A d v is o ry C o m m itte e s u b m itte d
its report. H a s a n y th in g h a p p e n e d in th e
meantime t h a t w o u ld m a k e it n e c e s s a ry o r
desirable t o c h a n g e th is c o n clu sio n ?
New d a ta fr o m

G e rm a n y (N e u b e r t an d

Dillman) re p o rte d t h a t o n e s tra in o f m ic e
showed s ig n ific a n t in c re a s e in. c le ft p a la te
incidence a t a d o s e o f 4 5 m g /k g o f p u re
2.4.5- T o n d a y s 6 th ro u g h 1 5 o f g e s ta tio n .
An increase in c le ft p a la te w a s a ls o n o te d
after tre a tin g

such

m ic e

w ith

doses

of

TCDD e x c e e d in g 1 f i g / k g . M o r e s i g n i f i ­
cantly. p o t e n t i a t i o n o f t h e t e r a t o g e n i c
effects oif 2 , 4 , 5 - T by T C D D w e re o b ta in e d .

but only w h e n th re s h o ld o r h ig h e r doses o f
bath substances w ere use d T h is d e m o n ­
strated th a t a T C D D c o n te n t o f < 1 p p m w a s
unlikely t o c o n trib u te t o th e e m b ry o to x ic
effect of 2 .4 , 5 - T . e v e n in th is h ig h ly s e n ­
sitive s tra in o f m o u s e .
Canadian w o r k e r s (K h e ra

an d

R u d d ic k )

have s h o w n t h a t W is t a r r a t s g iv e n 0 . 5

s m a ll an d

W o o ls o n

an d

n o t d e te c ta b le in b io ­

a s s o c ia te s

fro m

several

g o v e rn m e n ta l ag e n c ie s fo u n d n o d io xin
re s id u e s in e a g le s a m p le s fro m v a rio u s g e o ­
g ra p h ic a l re g io n s of th e U .S . o r in soil
s a m p le s a fte r a p p lic a tio n o f 2 .4 ,5 - T in to ta l
a m o u n ts of 9 1 2 Ib /a c re o v e r th e s p an of
7 years.
T h e s e a n d o th e r d a ta h ave b eco m e a v a il­
a b le s in c e th e A d v is o ry C o m m itte e s u b ­
m itte d its re p o rt. T h e n e w in fo rm a tio n in
large m e a s u re re p re s e n ts re fin e m e n ts and
m o d e ra te e x te n s io n s of in fo rm a tio n th a t
w a s a lre a d y a v a ila b le , or co u ld re a s o n a b ly
be d e d u c e d fro m d a ta e x a m in e d by th e C o m ­
m itte e . In a n y e v e n t, th e n e w d a ta d o n o t
in d ic a te t h a t a sin g le o n e of th e re c o m m e n ­
d a tio n s m a d e in th e o rig in a l w o u ld need to
be c h a n g e d ff th e re p o rt w e re r e -w ritte n
to d a y .
T h is is p ro b a b ly re fle c te d in a n e w s item
in th e O ttaw a J o u rn a l of D ec. 6 , 1 9 7 1 . to th e
e ffe c t th a t th e C a n a d ia n G o v e rn m e n t has
b e g u n t o re la x th e re s tric tio n s p la c e d on
th e use o f 2 ,4 ,5 -T .
" S ta rtin g n e x t y e a r,

It w a s s t a t e d
m a n u fa c tu re rs

th a t
who

p ro d u c e 2 .4 , 5 - T w ith a d io x in c o n te n t of
less th a n 0 .5 p pm w ill be ab le to sell th e ir
p ro d u c ts w ith o u t th e w a r n in g s — n o w
re q u ire d on all 2 ,4 . 5 - T lab els." A n in te r­
e s tin g c o m m e n ta ry on th e s itu a tio n in th e
U n ite d S ta te s is c o n ta in e d in th e sam e
a rtic le . "T h e c o n tro v e rs y a b o u t th e a p p li­
c a b ility o f a n im a l te s ts t o h u m a n s s till ra g e s ,
p a rtic u la rly in th e U .S . w h e re th e h e rb i­
cid e 's u se h a s p o litic a l o v e rto n e s ." In fa c t,
th e a rtic le m a k e s c le a r th a t th e C a n a d ia n
G o v e rn m e n t is re a d y to a d o p t e s s e n tia lly
all o f th e re c o m m e n d a tio n s s e t fo rth in th e
R e p o rt o f th e 2 ,4 , 5 - T A d v is o ry C o m m itte e .
U n o ffic ia l re p o rts in d ic a te th a t re s tric tio n s
on th e u se o f 2 .4 , 5 - T m a y als o be relaxed
in 1 9 7 2 in S w e d e n and Fin lan d .
T h u s th e old a d a g e a b o u t "a p ro p h e t w ith ­
o u t h o n o r in h is o w n c o u n try -" s e e m s to
h a v e b e e n o n c e m o re e x e m p lifie d in th e
R e p o rt of th e 2 ,4 . 5 - T A d v is o ry C o m m itte e .-^
DOWN TO EARTH. Vd. 28. No. 4. Spring 1973

17

TCDD
residue
disappears

A

to x ic c o n ta m in a n t th a t o n c e a p p e a re d

in th e p e s tic id e . 2 .4 .5 - T , w a s n o t d e te c te d
in re c e n t s tu d ie s o f soil sam p le s and bald
e a g le tis s u e and so d o e s n o t a p p e a r t o be a
resid u al th re a t t o w ild life .
The
c o n ta m in a n t.
2 . 3 , 7 . 8 -te tra c h lo ro d ib e n z o -p -d io x in (T C D D ), can be fo rm e d

2 ,4 ,5 -T . A n a ly s is fo r T C D D residu es bega:
in 1 9 7 0 .
A t o ta l o f 9 4 7 p o u n d s o f a c tiv e 2 .4 ,5 -T pe
ac re w a s a p p lie d o v e r a 3 -y e a r period to th
L akelan d san d. T h is is a m as s iv e dosew he
c o m p a re d t o n o rm a l a p p lic a tio n rates fc

c h lo rin a te d

bru sh c o n tro l on g razin g land of 2 pounc
per a c re , o r 6 p o u n d s in a 3 -y e a r spar

p hen o ls if te m p e ra tu re s ab o ve safe lim its
are u sed (A G R . R E S .. O c to b e r 1 9 7 1 , p. 8).

U s in g a n a ly s is te c h n iq u e s c a p a b le of de
te c tin g le s s th a n 1 ppm o f T C D D , th e sc

P rio r to 1 9 6 9 , tr a c e s of T C D D w e re fo u n d in
s o m e s a m p le s o f th e h e rb ic id e 2 . 4 , 5 -T .

e n tis ts fo u n d

W h ile re g u la tio n s an d in d u s try q u a lity
c o n tro l h ave b een e s ta b lis h e d to e lim in a te
th e th re a t o f T C D D c o n ta m in a tio n in p e s ti­

N o T C D D re s id u e s w e re fo u n d a t th e lowlim it o f d e te c tio n —0 .0 5 ppm in e a g le tissu

c id e s , c h e m is t E d w in A . W o o ls o n an d te c h ­
nician P e te r D. J . E nso r of A R S in v e s tig a te d

fro m 1 9 c a rc a s s e s c o lle c te d in 1 5 states ;

d u rin g

m a n u fa c tu re

of

so m e

th e p o s s ib ility th a t T C D D res id u e s fro m o ld .
e x tre m e ly h e a v y 2 . 4 , 5 - T a p p lic a tio n s m ig h t
s till pose a th r e a t to w ild life .

no re s id u e s

in 3 -fo o t co:

sam p le s .

B ald e a g le tis s u e e x tra c ts w e re obtaine
w id e ly s e p a ra te d as A la s k a . M a in e . Florid
and M is s o u ri. E agles w e re used as repr;
s e n ta tiv e s o f th e to p o f a fo o d ch ain .

life R esearch C e n te r, L au rel. M d ., and A lv in
L Y o u n g , fo rm e rly a p ro je c t s c ie n tis t w ith

T h e s c ie n tis ts s u g g e s t s e v e ra l reasot
w h y no T C D D w a s d e te c te d in e ith e r tt
so il o r e a g le tis s u e sam p le s . F o r exampi
d is s ip a tio n fro m th e soil m a y h a v e re s u lt
fro m m ic ro b ia l d e g ra d a tio n , photodecor
p o s itio n , v o la tility , a n d /o r w in d e ro sio n ,

th e U .S . A ir F o rc e . Eglin A ir Force B ase, Fla.

th e c a s e o f th e e a g le tis s u e s a m p le s , t:

T h e y a n a ly ze d soil s a m p le s fro m e x p e ri­
m e n ta l p lo ts o f Lakeland san d in F lo rid a

re s u lts s u g g e s t th a t T C D D re s id u e s frc
p ast p e s tic id e a p p lic a tio n s w e r e n o t ava

t h a t had re c e iv e d m a s s iv e d o s e s o f 2 ,4 . 5 - T
by aerial a p p lic a tio n d u rin g an 8 -y e a r perio d

ab le t o e n te r th e fo o d chain.
T h e s e re s u lts , c o m b in e d w ith th e to le ra n

T h e re s e a rc h w a s d o n e in c o o p e ra tio n
w ith W illia m L R eich el. c h e m is t w ith th e
U .S . D e p a rtm e n t o f In te rio r. P a tu x e n t W ild ­

fro m 1 9 6 2 to 1 9 7 0 . a p erio d d u rin g w h ic h

lim it o f 0.1

T C D D tra c e s o f up to 4 0 p a rts p e r m illio n

fro m h e a lth h a za rd s d u e to T C D D contarr

(p p m )

n a tio n .

had

b een

d e te c te d

in c o m m e rc ia l *

p pm

TCDD

a s s u re s freed c

* From October. 1972 issue of Agricultural Research.

5845
18

DOWN TO EARTH. Voi. 28. No. 4. Spring 1973

Amendment to PP 8F0670 - 2,4-D - June 1973
C.7.0

Summary of Toxicology Studies with 2,4-D in Livestock,
June 1973.

.■--■

As discussed in Section C of .the petition submitted by the
Industry Task Force on Phenoxy Herbicide Tolerances in
December 1967, no effect was produced by repeated oral doses
of 2,4-D salt and ester formulations at 50 mg/kg in cattle

100 mg/kg in sheep, even when administered five days per

week for as long as two years.

Levels of 250, 500 and 1000

DOW

and

mg/kg caused anorexia and resulted in death after several
repeated doses.

The following tables summarize studies by

USDA with various phenoxy derivatives in livestock.

(Complete

reports of the USDA studies can be found in Sections C.l,

C
f

C.2 and C.3 of the December 1970 amendment and in Section
C.7.1 and C . l . 2 of this June 1973 amendment.)

These and other

studies with 2,4-D in livestock are also summarized in
Section C.0.2 of this June 1973 amendment.
The effects of exaggerated rates of intake were studied by
USDA and by Erne in Sweden (C.7.2 and C.7.3).

In the USDA

study, administration of the 2-ethylhexyl ester of 2,4-D as
an emulsifiable concentrate formulation or as technical
material at 250 mg/kg/day for 9 to 56 days caused ill effects
in all seven animals after 3 to 14 doses and death in one
calf after 14 doses and in two sheep biopsied after 17 and 29
doses.

A significant increase in plasma magnesium:calcium

ratio was noted in the animals that died.

These animals also

had increased blood urea nitrogen (BUN) levels, and patho­
logical examination at necropsy showed kidney damage and
swollen, blood-engorged thyroids from this high dose rate.
In the Swedish study, various effects were observed in pigs
given 2,4-D triethanolamine salt or 2,4-D butyl ester at
doses of 50 to 300 mg/kg/day for 5 to 103 days

(C.7.3).

.

Lesions

5847

00028G2
■

r -

7

-

2-

were observed mainly in the digestive tract and the respiratory
and excretory organs.

Persistently high 2,4-D plasma levels

(200 - 400 yg/ml) were found in animals which developed
definite signs of poisoning indicating that their threshold
level had been exceeded. On the other hand, 2,4-D plasma
levels declined to about 10 yg/ml within 24 hours in
animals tolerating repeated administration of 50 mg/kg/day.
The 2,4-D amine was also administered at 500 ppm in the diet
of pigs

(equivalent to

20 mg/kg/day)

for up to

12 months.

Growth rate was irregular and depressed and locomotory dis­
turbances developed.

This exaggerated dietary level also

caused difficulties in pregnancy and parturition in the one
sow studied, and the piglets were underdeveloped.

Surviving

piglets fed 500 ppm 2,4-D in the total diet for 35 weeks
grew more slowly than controls and also exhibited locomotory
disturbances.

The 2,4-D plasma half life was highly variable

in the pigs in these studies compared to the normal value
of 12-2 hours obtained after single oral doses of 50 or
100 mg/kg (C.6.1).
In the USDA studies, 2,4-D salt and ester formulations were
administered by drench or in gelatin capsules five days
per week for prolonged periods.

Although not representative

of ingestion of declining residues of 2,4-D in treated forage,
they show that spraying pasture and rangeland with 2,4-D
for control of weeds and brush presents no hazard to livestock.
As discussed in Section D.5.0 of this June 1973 amendment,
residues of 2,4-D are not expected to exceed 300 ppm in
grass immediately after spraying at 2 Ib/A.

(Brushy areas

requiring higher rates of application generally contain
little or no grass and afford poor grazing for livestock.)
The residues in grass decrease rapidly to less than 10 ppm
within a few weeks after treatment, depending chiefly on

5848
00 028G3

geographic location.

This residue amounts tn * level of

about 1 mg/kg/day in livestock ingesting 3% <<r their body
weight daily on a dry weight basis.
The lack of hazard to livestock from residues in grass was
also demonstrated in feeding studies conducted by USDA and
The Dow Chemical Company.
levels up to

Phenoxy herbicides were fed at

2000 ppm in the total diet of c a t (_ie and sheep

for four weeks prior to slaughter and sampling of tissues
for residue analyses.

Based on measured feed intake, in-

(

dividual animals ingested 2,4-D at 55 to 62 m-j kg/day
Similar levels of 2,4,5-T and silvex in the diot caused
anorexia in some animals.
(Section D.6.4 of
amendment is a complete report of this study
sheep and of a similar study at up to
cows.

11,13 June 1973
1 cattle and

5 0 9 1 3 4

continuously for four weeks without apparent iii effect.

1000 ppm in dairy

It also shows the residues found in milk, cream,

muscle, fat, liver and kidney of animals during ingestion
and following withdrawal from the herbicides . \

0 0 0 2 8 G '1

'is

5850

A m e n d m e n t to PP 8F0670 - 2 ,4-D - June 1973

C.6.0

Summary of Metabolic Studies with 2,4-D in Animals,
June 1973.

A review of available information on metabolism of 2,4-D
and other phenoxy herbicides in animals was submitted as
part of a supplement to PP 8F0670 in September 1968.

c

Work

at Cornell indicated that phenoxy herbicides were rapidly
excreted unchanged in the urine of ruminants (Ref. 42, 43,
48, 49, 51, 52).

O

Work at Oregon State University in rats

showed that maximum residues occurred in tissues 6 to 8

on
o
co
to

hours after a 1 mg dose and 8 to 17 hours after an 80 mg
dose with highest levels in kidney (Ref. 50).
metabolite were detected in liver.

Traces of a

Total residues decreased

rapidly to less than 0.02 ppm in all tissues except the
stomach within 16 hours after a 1 mg dose.

Elimination was

also rapid following subcutaneous injection of 2,4-D, its
butyl and isooctyl esters at 100 mg/kg in mice by Bionetics
Laboratories

(Ref. 53).

The butyl ester was hydrolyzed more

rapidly than the isooctyl ester.

Only 5 to 10% of the

2,4-D remained in the carcass after 1 day and no 2,4-dichlorophenol was detected in extracts of whole mice.
Erne in Sweden has studied the distribution and elimination
of 2,4-D in rats, calves, pigs and chickens (C.6.1). Single
oral doses of 50 to 200 mg/kg were readily absorbed and
peak plasma concentrations were attained within 2 hours after
dosing in chickens and-within 4 to 7 hours in mammalian species.
A dose of 100 mg/kg gave peak plasma levels of about 100 to
200 ug/ml in the species studied.

The peak was attained

more slowly after administration of the butyl ester in calves
and was much lower after administration of the ester to rats.
Plasma levels declined and urinary levels increased when rats

5851
0002358

-

2-

and pigs were given repeated doses of 50 mg/kg/day, except
in two pigs which exhibited signs of poisoning at this dose
level.

A similar decline in peak plasma level was noted in

chickens given 2,4-D amine at 300 mg/kg/day.
Erne also studied tissue levels of 2,4-D in pigs maintained

O
C

on a diet containing 500 ppm of the herbicide and in rats,

-<

chicks and chickens given 1000 ppm in their drinking water
for 2 months to 2 years.

However, the levels found are not

representative of normal tissue distribution because the
threshold dose was exceeded as evidenced by anorexia and
reduced weight gain.

Only low levels of 2,4-D and no intact

Cl

o
cp

to
CO

ester were found in tissues of pigs given the butyl ester at
50 mg/kg/day for 1 month.

In all cases, the lowest levels

were found in fat and muscle with higher levels in liver and
kidney.

Similar results were .found in feeding studies con­

ducted by USDA in calves and sheep (Section D.8.0, June 1973)
Erne also demonstrated that 2,4-D is bound to plasma protein
and that a portion is excreted in the urine in conjugated
form '(C.6.2).

In his method of analysis for 2,4-D in biologic

material, the samples were extracted with an organic solvent
under acid conditions, followed by solvent partition, thin
layer chromatography, or photometric estimation of the color
produced with chromotropic acid (C.6.3).

However, some

conjugates may require alkaline extraction and hydrolysis (D.8.11)
Recent work at Dow in pregnant rats has demonstrated that 2,4-D
and 2,4,5-T undergo placental transfer both into and out of
the fetuses (C.6.4).

Placental and fetal levels were

proportional to maternal serum levels in this study and in
another with, labeled 2,4-D in mice (C.6.5).

The radioactivity

showed a slight tendency for accumulation in the visceral yolk
sac, passed to the fetus and was rapidly eliminated

(within

24 hours) from all tissues.

5852
0002859

-3-

In related work with 2,4,5-T, it was shown that uptake,
distribution and elimination depend on the species and on
the dose level(see PP 1F1102, Section C.8.0).

A single oral

dose o.f 5 mg/kg was excreted rapidly unchanged in the urine
of rats and man, but was retained longer and was partially
metabolized in dogs.

The distribution, metabolism and

O

o

excretion of 2,4,5-T in rats was much the same following
doses of 5 or 50 mg/kg but was markedly different following
doses of 100 or 200 mg/kg.

Thus detoxification processes

may be quite different at high doses such as those which
caused effects in teratology studies with 2,4-D and 2,4,5-T
(Section C.5.0 of this June 1973 amendment).
A gas chromatographic method has been reported recently
for the determination of trace quantities of 2,4-D and
2,4,5-T and their respective phenolic moieties in urine
(C.6.6).

In rats given 2,4-D at 10”5 to 10~^ of the LD5Q

(375 mg/kg), the major portion of the administered herbicide
was excreted as the unchanged compound and no metabolized
residues were detected.

Detectable residues of 2,4-D were

r

present in urine within 24 hours after administration of
3.75 yg/kg.

Urine samples from occupationally exposed

people were found to contain 0.2 to 1.0 ppm 2,4-D by this
method.

5853
0002860

cc
ÎO
CO

%V

A m e n d m e n t to PP 8F0670 - 2,4-D - June 1973

SECTION C.

FULL REPORTS OF INVESTIGATIONS MADE WITH RESPECT
TO THE SAFETY OF THE PESTICIDE CHEMICAL

C.0.2

Summary of Toxicology Studies with 2,4-D, June 1973

The following tables and abstract summarize the data available
on acute/ subacute and chronic toxicity of 2,4-dichlorophenoxyacetic acid (2,4-D) in a variety of species.

A more

descriptive summary was presented in Section C of PP 8F0670
Tolerances in December 1967.

It was repeated in an amendment

submitted in December 1970, along with several reports on
toxicity studies conducted by USDA with 2,4-D in cattle,
sheep and chickens

(Sections C.0, C.l, C.2, and C.3).

An

amendment submitted in September 1971 included a summary of
two-year feeding studies in rats and dogs and of a repro­
duction study in rats conducted by FDA (Section C.4).

A

published report of the long term studies is included in
this June 1973 amendment (Section C.4.1).
Teratology studies were initiated in 1970 as requested in
PR Notice 70-8 and a report of a Dow study with several
2,4-D esters was included in the September 1971 amendment
(Section C.5.2).

Section C.5.0 of this June 1973 amendment

summarizes four teratology studies conducted with 2,4-D
in rats, hamsters and sheep, followed by published reports
of the studies by Dow~(C.5.3), Canada Food and Drug Directorate
(C.5.4), the Food and Drug Administration (C.5.5) and the
U. S. Department of Agriculture (C.5.6).

In each case, no

effect was produced by administration of 25 to 50 mg/kg/day
during organogenesis.

0002840

Qiirnnn

submitted by the Industry Task Force on Phenoxy Herbicide

A m e n d m e n t to PP 8F0670 - 2,4-D - J u n e 1973

SECTION C.

FULL REPORTS OF INVESTIGATIONS MADE WITH RESPECT
TO THE SAFETY OF THE PESTICIDE CHEMICAL

C.0.2

Summary of Toxicology Studies with 2,4-D/ June 1973
<D

Dow 5094 07

The following tables and abstract summarize the data availabl
on acute, subacute and chronic toxicity of 2 ,4-dichlorophenoxyacetic acid (2,4-D) in a variety of species.

A more

descriptive summary was presented in Section C of PP 8F0670
submitted by the Industry Task Force on Phenoxy Herbicide
Tolerances in December 1967.

It was repeated in an amendment

submitted in December 1970, along with several reports on
toxicity studies conducted by USDA with 2,4-D in cattle,
sheep and chickens

(Sections C.0, C.l, C.2, and C.3).

An

amendment submitted in September 1971 included a summary of
two-year feeding studies in rats and dogs and of a repro­
duction study in rats conducted by FDA (Section C . 4 ) .

A

published report of the long term studies is included in
this June 1973 amendment (Section C.4.1).
Teratology studies were initiated in 1970 as requested in
PR Notice 70-8 and a report of a Dow study with several
2,4-D esters was included in the September 1971 amendment
(Section C.5.2).

Section C.5.0 of this June 1973 amendment

summarizes four teratology studies conducted with 2,4-D
in rats, hamsters and sheep, followed by published reports
of the studies by Dow (C.5.3), Canada Food and Drug Directorate
(C.5.4), the Food and Drug Administration (C.5.5) and the
U. S. Department of Agriculture (C.5.6).

In each case, no

effect was produced by administration of 25 to 50 mg/kg/day
during organogenesis.

00028*1

-

2-

A review of information on metabolism of 2,4-D and other
phenoxy herbicides was submitted in September 1968 and the
portion on animals was repeated in Section D.6.1 in December
1970.

Section C.6.0 of this June 1973 amendment summarizes

a number of additional studies with 2,4-D in animals, including
rats, pigs, calves and chickens

(Section C.6.1 and C.6.2),

pregnant rats (C.6.4), and pregnant mice (C.6.5).

These

show that 2,4-T is readily absorbed into the bloodstream and
unchanged 2,4-D.

Placental transfer of 2,4-D occurs both into

and out of fetuses.
Residue studies have been conducted in dairy cows, beef
calves and sheep which showed that levels of at least 1000 ppm
2,4-D in the total diet were required to cause detectable
residues (0.05 ppm) in milk, muscle or fat, while low
levels were found in liver and kidney at 300 ppm in the
diet (Section D.6.4 of this June 1973 amendment).

Low levels

of 2,4-dichlorophenol were also found in liver and kidney of
ruminants fed 2,4-D, partly in conjugated or "bound"
form which require alkaline hydrolysis prior to quantitation
(Section D . 8 .11).
Several methods of analysis have been used recently for 2,4-D
and dichlorophenol in biological materials.

These include

spectrophotometric measurement of the color produced with
chromotropic acid (Section C.6.3), cleavage of the ether
link with pyridine hydrochloride and gas chromatographic
determination of the liberated 2,4-dichlorophenol (Ref. 45,
September 1968), and gas chromatography as methyl esters
(Sections D.7.3, December 1970; D.8.9 and D.8.10, September
1971; D.8.12, June 1973) or ethyl esters (Section C.6.6,
June 1 9 7 3 H
This amendment to PP 8F0670 also contains a number of

5857

additional reports on toxicology studies with 2,4-D salts

0002S42

POF’finn 4a

is excreted rapidly in the urine relatively quantitatively as

-3-

and esters in livestock.

Section C.7.0 includes summary

tables for studies conducted by USDA (Sections C.2 and C.3,
December 1970; C.7.1 and C.7.2, June 1973) and a discussion
of an extensive study in Sweden (C-7.3). Some animals
tolerated repeated daily doses of 50 or 100 mg/kg by capsule
or drench without ill effect while others showed symptoms
of anorexia resulting in reduced weight gain.

Similarly

no effect was noted in calves and sheep maintained for 4 weeks
on a diet containing 2000 ppm 2,4-D, equivalent to ingestion
of 55 to 62 mg/kg/day (Section D.6.4).

However# pigs given

r

500 ppm 2,4-D amine in the diet for 12 months did not gain

^

as well as controls and developed locomotory disturbances

c

(C.7.3).

c
c

Toxicity studies with 2,4-D in birds are summarized in
Section C.8.0.

Repeated oral doses of 2,4-D amine or ester

by pipette caused reduced weight gain at levels of 100 or
250 mg/kg/day (C.7.0).

No effect was noted at dietary levels

of 500 and 2000 ppm for 7 days in cockerels (C.8.2), of
up to 1000 ppm for 3 weeks in chicks

(C.8.3), and of 50 or

150 .ppm for 28 to 48 weeks in laying hens (C.8.4).

Con­

tinuous ingestion of 500 ppm in feed or 1000 ppm in drinking
water for up to 5 months caused renal hypertrophy in chicks
and decreased egg production in laying hens
C.8.5).

(C.7.3 and

Spraying of chicken and pheasant eggs with 2,4-D

at normal and exaggerated rates had no effect on hatchability
in two studies in the U. S. and Canada,

(C.8.6 and C.8.7),

but was reported to be very harmful in a French study (C.8.8).
However, no data were provided on hatchability of untreated
chicken and pheasant eggs under the inadequately described
conditions used in the latter study.

0002S<J3

c

Cl

Formulation

Organism

Dose

Effect

R e fe re nc e

Triethanolamine

Swine

50 m g / k g

N o ef fe ct

(a)

Triethanolamine

Sw in e

500 m g / k g

Lethal

(a)

Bu ty l e s t e r

Sw in e

100 m g / k g

N o ef f e c t

(a)

Triethanolamine

Ch i c k e n

300 m g / k g

No effect

(a)

Butoxyethanol eater

Phytoplankton

1 ppm

l6 H de c r e a s e in
C O g fi x a t i o n

(b)

Dimethylamine

Phytoplankton

1 p p m (U h r s)

N o effect on
C O 2 fi x a t i o n

(b)

Ethylhexyl ester

Phytoplankton

1 p p m (•» hrs)

•♦9% de c r e a s e in
C02 fixation

(b)

Propylene glycol
butyl ether ester

Phytoplankton

1 p p m (U hrs)

d e cr ea se in
C O g fi x a t i o n

(b)

Butyl ester

Rats

620 m g / k g

l d 50

(c)

Unspecified

Trout

3 mg/l*

L e t h a l 2h hrs

(d)

Unspecified

Trout

2 .2 m g / l *

L e t h a l U 8 hrs

(d)

Unspecified

Man

689 m g / k g

Lethal

(d)

Is o p r o p y l es te r

R a ts

700 m g /k g

l d 50

(e)

Pr op yl en e glycol
b u t y l et he r es te r

Bluegill

3 ppm

U 8 hr T L n

(f)

r

»

.

* mg/l is approximately the same as ppm.

Olf'GO Q

MOCI

ACUTE TOXICITY OF 2,li-D

0 0 0 2 8 4 4

- - - ' — ----------------- — ------------------------------------------»

1

Fo rm ul at io n

Reference

* 0 5 PP«n

*♦ 8 hr T L n

(g>

I s oo ct yl e s te r

Bluegill

9 pp m

1*8 hr T L a

(g)

Bu ty l e^ster

B l ue gi ll

1 ppm

1*8 h r T L m

(g)

I s op ro py l es te r

Bl u e g i l l

1 ppm

1*8 h r T L n

(g)

Dimethyl&mine

Bl u e g i l l

166 p p m

1*8 hr T L m

(g)

Alkanolamine

Chicks

38 0- 76 5 m g / k g

tr\

0

(h)

R a ts

700 m g / k g

U J jO

’ (h)

Is op ro py l e s t e r

Chicks

ll*20 m g / k g

^50

(h)

Isop ro py l e s t e r

G u i n e a pigs

55 0 m g / k g

L D jO

(h)

Bu ty l e s t e r

R a ts

620 m g / k g

^50

(h)

Bu ty l e s t e r

G u i n e a pigs

81*8 m g / k g

l d 50

(h)

Butyl es te r

Chicks

2000 m g / k g

ED50

(h)

P r op yl en e g l y c o l
b u t y l e t h e r es te r

R a ts

570 mg/kg

O
tr\

(h)

Acid

Dogs

100 m g / k g

l d 5o

(h)

Acid

Chicks

5^1 m g / k g

l d 5o

(h)

Unspecified
am in e

M a l l a r d du ck s

2000 m g / k g

l d 5o

(i)

A c id

Ph ea sa nt s

U 72 m g / k g

l d 50

(i)

Acid

M u le d e er

1*00-800 m g / k g

l d 50

(i)

'

T “ ~T' ■*’ A ~ f-* i \

ACUTE TOXICITY OF 2,U-D (Cont.)

Bluegill

9

>r..
Cl

Ef f e c t

9

o
o
fo
CD

Dose

A l ka no l& mi ne

Is o p r o p y l es te r

OX
GO
CT>
o

Organism

ACUTE TOXICITY OF 2,U-D
Literature Cited

n

Bjorkland, Nels., and Erne, K. — 1966 (See C.7.4)
Toxicological Studies of Phenoxyacetic Herbicide in Animals
Acta. Vet. Scand. 7: 36^-390.

Edson, E. F., Sanderson, D. N., and Nookes, D. N. - 196U
Acute Toxicity Data for Pesticides.
World Review of Pest Control U(l) Spring 1965»
McKee, I. E., and Wolf, H. W. - 1971
Water Quality Criteria - Publication 3-A, April 1971.
State of California, Water Resources Control Board.

yt rnnc

aa

Butler, P. A. - 1965
Effects of Herbicides on Estuarine Fauna.
Southern Weed Cont. Conf. Proc. 18: 567.

Hayes, Wayland J. J. - 1963
Clinical Handbook on Economic Poisons.
U. S. Dept. Health, Education and Welfare.
Hughes, J. S., and Davis, J. T. - 1966
Toxicity of Pesticides to Bluegill Sunfish Tested During 1961-1966.
Report to Louisiana Wildlife and Fisheries Commission,
Monroe, Louisiana.
Lawrence, J. N. - 196U
Aquatic Herbicide Data.
USDA, ARS Agricultural Handbook 231.
Rowe, V. K., and Hymas, T. A. - 195U (See petition, 12/67)
Summary of Toxicological Information on 2,U-D and 2,U,5-T
Type Herbicides and An Evaluation of the Hazards to Livestock
Associated with Their Use. Am. J. Vet. Res. 19: 622-629.
Tucker, R. K., and JCrabtree, D. G. - 1970
Handbook of Toxicity of Pesticides to Wildlife. Resource
Publication No. 8U. Bureau of Sport Fisheries and Wildlife,
U. S. Dept, of Interior.

00028 *

I

I

J

Fo r m u l a t i o n

Organism

Dose

f

cn
CD

cn

Triethanolamine

Sw in e

50 / m g / k g / d a y

Butyl es te r

Sw in e

50 / m g / k g / d a y

T r i e th anolamlne

Sw in e

300 p p m in feed

Triethanolamine

Rats

1 0 0 0 p p m in w a t e r

Triethanolamine

C h ic ke n

1 0 0 0 p p m in w a t e r

No t sp e c i f i e d

Dog

500 p p m in feed

N o t s p ec if ie d

Rat

1250 p p m I n feed

N o t spec if ie d

Rat

... 500 p p m in feed

to

o
o
o
ro

00
*

*See summary which follows.

Effect

Reference

8-10 doses

Minor transient
effects.

(a)

None

(a)

1 month

Some locomotory disturbance, depressed
growth rate, no gross
pathology

(a)

10 mos.

Depressed growth rate,
no gross pathology

(a)

Dally from
Egg size normal,
hatching
production reduced 30
through first
2 mos. of egg
production.

(a)

2 years

None

(b)

2 years

No effects on growth,
survival hematology
or tumor incidence.

(b)

No effects in repro­
duction studies.

(b)

5 doses

2 years

r* T

t

r> <-i

CHRONIC TOXICITY OF 2,U-D

Duration

*

'

Fo r m u l a t i o n

Organism

Ethylhexyl ester

Cattle

250/m g/ kg /d ay

lU days

S i c k in 3 days,
s u rv iv e A r e c o v e r
f r o m 9 doses.
lU do se s lethal.

(c)

Ethylhexyl ester

Sheep

250/o g / k g / d a y

1 7 days

S i c k in 3 days 1 7 do se s lethal.

(c)

N o n e to m i n o r ef fe ct s

(c)

E t h y l h e x y l es te r

Sheep &
Cattle

Dose

100/ m g / k g / d a y

Duration

10 days

Effect

Reference

n
§
H
O
8

X

H
2 , U - D in w a t e r

H u m a n taste
th re sh ol d

0 - 01m g / l *

n

B a d taste

(d)

a

0

►u
Alkanolamine

ai
go

en

oo
O
O»
O
r.i
DO

Sheep

100/ m g / k g / d a y

**8l days

No effect

(e)

Cattle

50/ m g / k g / d a y

112 days

N o ef f e c t

(e)

Pr o p y l e n e g l y c o l
butai ether ester

Sheep

100/ m g / k g / d a y

**01 days

N o effect

(e)

Alkanolamine

Ch ic ke n

100/ m g / k g / d a y

10 days

N o effect o n weight gain

(f)

Pr o p y l e n e g l y c o l
b u t a i e t h e r es te r

Ch i c k e n

50 mg / k g / d a y

10 days

N o effect on weight gain

(f)

Pr o p y l e n e gl y c o l
b u t a i e t h e r e s te r

Ca tt le

100m g / k g / d a y

10 days

N o ef fe ct

(f)

Acid

Mule d e e r

00 & 2l»0mg / k g / d a y

30 days

M i n o r symptoms no w e i g h t loss

(g)

-

OO

m c o s M o a

1

ti

Alkanolamine

«•ng/l is a p pr ox im at el y the same as ppm.

tr

0
0
a

rr
t

Chronic Toxicity of 2,4-D

5 m 0 9 * O d

Literature Cited
(a)

BJorkland, Nels., and Erne, K. « 1966 (See C.7.4)
Toxicological Studies of Phenoxyacetic Herbicide in Animala
Acta. Vet. Scand. 7: 36U-390.

(b)

House, W. B. et al - 1967
Assessment of Ecological Effects of Extensive or Repeated Use
of Herbicides. Final report on Midwest Research Institute
Project 3103-B under Dept, of Army Contract DAHC 15-68-C-Q119.

(c)

Hunt, L.M., Gilbert, B.N., and Palmer, J. S. - 1970
Effects of a Herbicide, 2-ethylhexyl Ester of 2,U-D on Magnesium:
Calcium Ratios and Blood Urea Nitrogen Levels in Sheep and Cattle.
Bull. Environ. Contamination and Toxical 5: 5^-60.

(d) McKee, I. E., and Wolf, H. W. - 1971
Water Quality Criteria - Publication 3-A - April 1971.
State of California, Water Resources Control Board.
(e)

Palmer, J. S., and Radeleff, R. D. - 196U
The Toxicologic Effects of Certain Fungicides and Herbicides
on Sheep and Cattle.
Ann. N.Y. Acad. Sci. Ill: 729-36.

(f)

Palmer, J. S., and Radeleff, R. D. - 1969
(See C.7.0)
The Toxicity of Some Organic Herbicides to Cattle, Sheep and
Chickens. Production Research Report No. 106 - ARS - USDA.

(g) Tucker, R. K., and Crabtree, D. G. - 1970
Handbook of Toxicity of Pesticides to Wildlife. Resource Publi­
cation No. 8U. Bureau of Sport Fisheries and Wildlife.
U.S. Dept, of Interior.

5

8

6

4

0002849

il

\

!

p DOW 090038

J

uV JAH ICC ClXCCLAfJD AND KCVIfJ P. Ci:C A
'’'•
•»*\r \ 1

¿Is, yon isuy rpt races ckm yon pay for

o \ l - SO«
nns-.siijy

u f c::c!!)u.:ti:.
t o x ic to luim.m hcmirs mil
!o Hie .•nv'!''!iiiion? is Mic p ro d u c ts o f side reactions

w liiv.li o c c u r Itinnu H:'\;:',.in ¡i.icHux.' m’ .«urictiirur:il .tml industrial ch e m i­
cals. Twi- .x’cenllv p ia d ic i/c d ox.inip'eK ire '..V7..s-ietr:«clilor'nlihi;n/op ilin x m
:< i)l) .»r . ¡ i i i i i i i a u«!iuv 'u is o n o u s chem ical found a:» a
c u n ia m iiu n r in Hie »..July used lcrhicide 2 4.5-T : and the chlorinated
d i K m / o f , n n s . fou.fd is con ram in am s m so m e I’oreipn-inaile p n lychlpri:ill led iMniie n v f »^»«' ^
•■»■■‘■Minds, lio lh ch em ic a ls are far m ore t o x ic
than the c o m p o u n d s they c o n ta m in a te and in tact are imomt the m ost
. t o x i c ch e m ic a ls k . a i v b . Pints w hile m e n t i o n has been focu sed o n the
to M c ily i:»'a persistent p esticide, .¡«purities w hich mav :n fact he m ore
t o x ic have ¿one u n n o tic ed tor mar.', years.
Xertcultural chem tca.s. w id e ly dispersed m the en viron m en t, usually
con tain vary tilt:'.timutr.rs o f im purities. R ec en tly , workers in the l :.S.
D ep artm ent ot' Xdncuiture n . S I M i anal'./.ed a van elv o f .lyrieultural
ch em ic als tor p ossible co n ta m in a tio n w ith d io x in s. The group ot' p esti­
cides w h ic h w ere a n alysed were ail derived, like 2.4..5-P from a i ip ’MP o f
ch em ic als k n o w n as c h lor op h en ois. <h ie o f these chem icals, p entaeliioroplien ol <pt p i turned >>ut to be o f particular interest, because it con tain ed
significant q u a n tit ie s o f ih c d io x in s. O f further interest is the fact that it
' s b een used as j replacem ent for highly t o x ic mercury c o m p o u n d s for
siim e c o n t r o l in the m anu factu re o f paper pulp Ironically. I’< |» m paper
mill w a ste w a te r is turm n” out t o be ab out a s hazardous to atfiialic life as
are th e m ercu ry c o m p o u n d s it is replacing
and m ore t*< l’ ili.m mereury
is n eed ed t o cou n teract the d un e.
- PCI* has b een used extensively ind for i variety o f purposes o ilier than
lor slim e c o n t r o l ■■ver Hie sears Xs a i . n e i c i d e it has been used m the
processing o f oils. leathers, paints clues, and 'c x lite s and has K e n
in corporated in to hair sh a m p o o s anphcui "ii • i*< !> • .v.»ut provides
p ro tec tio n against erm ites and w ood-hi rime insects is -sell is m old s.'
I*' P in.iy also c o m e directly <nto con tact .vuh I'.-nd
¡1 has been used as
a w oiui ¡’le se ix a liv e m crates used !> r . iw len.a.itui'.ii n .il e r u lv as a
luneikitle in Iv e i ■>iis raj m the c.r.k.-' . , .rij : vat ' • d .Miiauiers In
i
ii..ue 4' os.« o o ii (s, 1112^1 s ■•( |u ¡* \ i ■ p! ■ i] ,^.ai • 11 it I m .-ic : hail
oiU'-ti.d! *'l tills 1« I.ii w.js . cit as * '» il :*■ s»'i'. at o e s
i ,:us. the possilMlus oi Muin.in ex p o su re i::.l 0 ee \:r.
:i:.d . oiitair.i;i.iti«iii with pi I*

ami ils toxic im purities in great.
In ihe i: u> \ slm ly. 12'* .aniplcs o i
I " ilil lerent p estititles were analyzed liv­
ing ;ias chrom atography in u len lity the
presence o f die varnm s dioxins.4 Pil'fcreni tliuxins can be tlisiininiisiictl from
each .n h e r by th e num ber n i chlnrincs
w inch th ey co n tain , th ere are eifiiit p o ­
sitions w here chlorine could he a tta c h e d
lo th e basic dih cn /o -d ioxin stru c tu re , as
shown in Figure t. This m akes a variety
o f dioxins possible ranging from (lie
m ono form lo n e chlorine ato m ) to Ihc
oriu form ieight chlorine atom sI. Then,
depending on th e n rienlalion o f these
chlorines in space about the m olecule,
various isom ers m ay also he form ed
(■il in all are possible for I his particular
chem ical stru ctu re. Although the d io x in
found in 2 .4 .5 -f is the letra form (fo u r
chlorines are attach ed lo ihe basic d i­
oxin stru c tu re at Ihe 2..1.7. and x posi­
tions! and this form i l i ' l i n i has re ­
ceived m uch a tte n tio n , this is n o t (he
only toxic form , as we'll see later.
In Ihe i i s i i \ investigation. 2.< o l 4 2
sam ples o f 2 .4 .5 -1' were co n tam in ated
CD
w ith dioxin as well as 4 o f (i sam ples oi
CD
in c h lo ro p h c n o l. all 5 sam ples ol letra00
ch lo ro p h en o l. and ID o f II sam ples m
LO
l*riv th e d io x in s containing 4.i> .i. aim
S chlorines were found.
Ih e co n tam in atio n of r<T by dm xir.
was co n ltrm ed in a later study in w in d :
the investigators looked at l'( I* alone
Hoih dioxin and an o ih er co n tam in an t
d ih e n /o tu ra n . w ere loiinil.. In all. i '
O
parrs oer tnillioti.ip.ptni ..| llie lie\a->l.
o
•m il were t.iu lid . l a s ppm ol Ihe hepo
la-ilioxill. and I f4 ppm .It th e . u t a •!.
■vm. Ilcta- m d hepl.it h l.ir.lib en /. . 1 .
o
tails were ..leiuitie.1 I’nt the 111.011:1 .
n-•
ts -U' a . ‘I >1.-le i M ii u e . I * I he I’j s i . -I: ..
CD

I II ' ■! I.l'fll/.ll II III s IIs. I s||. >wu

•

I gin • I i While ’le* et e viinplc' I - •l
in litis slii.lv .vele pi.Hli.s.-d r e t i
I o .o ilu- j iili.ir. pointed ml th at •• i

f

1
samples from a 1970 batch in another
study xfac contained ¿toxins - 0.5 to
37 ppm of the hexa-dioxin and t o to
I3S ppm of the bcptatiioxin. The pres­
ence of the dioxins has also been con­
firmed by studies in other parts of the
world - a recent study by Swedish in­
vestigators revealed the presence of SO
ppm of octa-dioxin in a sample of Per.5
: r-

. Form ation o f D ioxins and Fusaas
H ie dioxins found in contaminated a n , JJles of l,4 i- T and in the chiorophenols
arc formed during the manufacturing
process (Figure 11. During me process a
reaction between two molecules of trichlorophenol may occur resulting in the
formation of tetra-dioxin (Trunk* The
formation of TCDD during the manu­
facture of trichlorDphenol and 2.4 ,5-T
has been documented by a number of
chemical companies both in the U25.
and Europe.
The same sort of conditions (high
temperature, high pressure, and alkalini­
ty ) arc necessary for the production of
P C P when hcxacfalorobenzene is used as
the starting chemical (sec reaction 2.
Figure I ). Under these conditions both
/ the dioxins -and the furans may be
formed.5
Alternately, the cfalorophenuLs may
be produced by the direct condensation
if chlorine and phenol (see reaction 3,
Figure I ). When too much heal is ap­
plied. especially during the manufacture
o f tetra- and pentachlorophcnol, the
more highly chlorinated dioxins can be
formed.
It has also been proposed that the
dioxins may be formed after the manu­
facture of P C P , for example when wood
is treated with this substance under
highly alkaline conditions. Wood may
also be pressure-impregnated with pcp ,
with the combination of high pressure
and alkalinity leading to the formation
o f dioxins.
Once in the environment there is the
chance that p c p and 2,4,5-T (which
may have chlorophenol impurities) can
become exposed to very high tempera­
tures. This might occur when brush
sprayed with either ol these chemicals ts
burned or when wood products impreg­
nated with PCP arc incinerated after dis­
posal. Dioxins might in this way be re­
leased into the environment. (One way
o f producing dioxins, for which there is
a Canadian patent, is to heat 5 grams a gram is equivalent to 0.03S ounce o f pcp at 300.degrees for twelve hours.
This vieids 1.5 grams of the ucta-dioxin r

fact that thrt a*r extreme!' toxu sub­
stances For example, tin- iIikto I 7(’t>l>
-needed to kill 50 percent ot an experi­
mental group of guinea pip. is ().<- microgram nine microgram e- one-milluinth of a gram I per kilogram of body
weight." In an experiment in which rab­
bits were fed 1 Ci>l>, a Jose of 10 micrograms per kilogram killed* all the ani­
mals. while at a lower dosage of I mi­
crogram per kilogram, liver damagr and
cUoracnc (a skin diseawi resulted.
PCP on the other hand, is toxic to ex­
perimental animats in the milligram per
kilogram range." (A milligram ts onrthousandth of a gram.)
One of the higher chionnaled dioxins
fthc hex»-dioxw> found in pcp has abc
hecn shown to hr toxic. This informa­
tion was gathered after the accidental
poisoning of millions of commcrcialfy
raised chickens in the I'-S. in l u5~. in
some flocks, therr was as high as 50
percent mortality. Affected chickens
had droopy and ruffled feathers as wel!
as difficulty m breathing Autopsies
showed fluid accumulation in the mem­
brane surrounding the heart ' hvdroperi­
cardium). beneath tht skin <subrutaneous edema l. and in tltr abdominal cavi­
ty, and ii*cr and kidnry damage. Un:i:
the substancr responsible lo* :lu- ep>demir was later idcnttfied. _it was callet
the "duck edema factor.*'1*
The cause of ‘.hr discasi was at first
traced to par. ot the birds' diet - to tin
fats added to commercial poulry feed
Later, a toxic fraction was isolated Iron:
these fats and found to contain cm:»
tine. It was suggested at fust that the
substance could hr a chlorinated naptiwlcnc.13 The chlorinated nap'.lialenc:.
have been known to cause a condition
called hyperkeratosis in cattle, f Hyper­
keratosis involves thickening and hard­
ening of the skin end proliferation o)
crib of the mucous membranes i How­
ever. this substance was ruled nut when
samples of letmchlorunapthaiem and
hexachloronapthaieac failed to producc
tbe symptom of edema disease seen in
chickens. Finally, in 1906, x-ray crystallngraphers at Proctor and Gambit- de­
termined that the toxi; substance was
hexacfaiurdihenzo-p-dinxir."* This teaconfirmed when a chemist at Proctor
and Gamble synthesized the hcxa-di.-xtii
end showed that it prnduerd chick eiirBn disease.
During tfar search lor :ht- nientitt ot
the unknown chemical substance whicti
had caused the disease sampler ot u.xi.
lat were tested in a variety
iahoratt>*\
ammris. Adverse eftect: we*t- roiuid it.
munkrvs. guinea pip. tiop rat-, and

Ptf1

Toxicity
neern about the reieasr of the dioxins
-*to the environment stems from the
36

Became thr toxic lacto; c.-uid fir
recovered front Uw tissw ->! chid enled thr substance ttu cucral Food and
Drug Administration u ;.-. i ruled in

l i ( I that eoRimercu! fats bad to hr
screened lor the -chick etlema factor hr
fore being placed .us thr market. Thr
chick edrma (actor hid atv> hem found
in fat samples which wen lo hr used in
food tin human hemp.1*
After identifying thr factor as a di­
oxin. the sourer ol the dioxin was still a
puzzle One suggestitm was that thr di­
oxin fin contaminated samples of
2.4.5-T or P i n was originally sprayrd
onto crops (such as enrni from which
vegetable oil was later processed. Anoth­
er suggestion' was that since heat is
needrd during thr preparation ol thr O
fats, tfar dioxins could have hern
formed as condensation products ot
p( P or other cfalorophenob which
might have been present in thr onpnal
CO
vegetable material.

CO

o
o

CO

The chlorodilienzoiurans. which as wr
mentioned earlier were found as con­
taminants in » re samples along with thr
dinxins are ais<> toxic Researchers have
noted a possible relationship between
tin- toxicity ol ;hr Pi h:. ilfar potvefaiorraa'ed biplicp-’L-. i and their level ol eontammatior with tetra- and pentachlonlibenzoturai. in a recent study, three
samples o! pi b-PlM.-nocior i produced in
Francri. Clophen (produced in (¡ermanyt and Arocior t produced by Monsan­
to ir thr l -S I - were fed to chickens
lo: od days, after which thr surviving
animals wife killed and autopsaed. The
animals receiving Phenoctor and Ck»
phen all died during thr course of th r
experiment; then symptoms included
ruffled leathers, loss of feathers, and
kiss ot (macular coordination. awl thry *
wen- found to have hydrupcncardium,
alulomuial and subcutaneous edema,
and liver necrosis (death ol liver crilsi.
On thr other hand, only 15 percent of
thr group receiving Arocior died during
the experiment, and only a few (tfarer
of twenty) developed hydrnpericardium: Five Japanese quail and eight
white rats were also fed Phenodnr. The
quail all died within 13 days; necrosis ol
tin- livet was revealed upon autopsy All
eight rat: died wuhtii
days and
showed' enlarged Inert, iwith local ne­
crose.) and disappearance of part ol the
structure of thr spleen."'
A subsequent investigation turned ut>
tin- tact that Phenocior awl Oopfaer
b.<;n contained tetra- and pentacfalord*henzoturan The authors at’.ribu'rd Ibr
greater toxicity o; ;im r cr-mpound
th r iactot aw! -aiggr'.’rd that tin *u-ar.
could *n , lo’nird wtien c*wi» p h r
dis'.ibeu
O ccupation! Hazard
Tin cfalortna'.rd pvdr-i-arbvins have king

(mmanw. d« it. *•» V
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K-i-n a k n o w n h a /a i d In w o rk e rs. Ilic
chcm icals w h ic h w c have m e n tio n e d m >
I j r m its. c h lo rin a te d n a p th a le n c s.
th lo r o p h e n o ls i cause a c o n d itio n k n o w n
as chloracm .-. o n e o l th e m ost fre q u e n tly
in c u rrin g to im s o l o c c u p a tio n a l d e rm a litis I uit lam in j l ion o f I In- sk in I.1* ) lie
iliH-jM- in n o l cau sed by c h lo rin e a lo n e ,
h u l hy c h lo rin e m c o tn h in a lio n w ith
c e rta in o rg a n ic eultstancc-s. m i c I i a s p h e ­
nol.
In laic I ’M n . several i j m -n o l c
racne were rc-poMed ill a (•e rin a n ci. ...tcal p lant in v o lv ed ill th e p ro d u c tio n o f
i-i I*, usiiip lie x a c lilo io h e n /e iie in th e
pro cess S o m e o f th e s y m p to m s , w hich
th e j lt c c l c d m en b e g an to sllow n e a iiy
live m o n th s a lte r th e s ta rt >>t i -i l* p ro ­
d u c tio n . in c lu d e d severe a c n e , in v .rn e
>ases w ith u ile c tio iis . h ro w u sk in pigilic n ta lio ri. a n d neuralgic i nerve I pain in
th e low er e W rc m ilie s. It-w .is rc-cogm /cd
th a t c ilh e r I’l l' ilsc-ll o r an in le ru ie d ia iy
p i o d n d in its p ro d u c tio n c o u ld have
lieeu tlie- c a u s jtiv e .i|!ent
in a d d ih o n .
com e ot th e m en llad a lso been e x p o se d
to le tia c h K -ro h e n /o l a n d t in h lo r o p h c nol 1 '
\ lalei re p o rt ae.m i tro rn t ••'iiu.iiiv .
in I*'- s chi-w ed th a t :t.e .<* iilal:u:iatilin . l.li 'ii 't'h e t.o ! pit -d tic 11*ill in d ee d ..ill
.aasc . In .|_c lie In iliic .jc .- -I w. •: se : w li-- | - : . | . i ; . d
I n . 10
I.--I .u..i

PCP » (1‘oxm <ontjrnifijritS

2 .4 .5 -I d e v e lo p e d c i.lo l.ic n e . U 'lU ) was
•.solateli Iro m th e lu n c h e d p ro d u c t jn d
w as lo u n d t o Ih; a p o te n t a c n e -p ro d u c ­
ing atteri I in ra b b its . I r i - a t i d te tra c h lo rodilH -ii/.oluran w e re also te s te d o n e x ­
p e rim e n ta l a n im a ls a n d fo u n d In be a c ­
tiv e a c u e -p io d u c in t! atteri Is
In ta c t.
I ( t il l p ro v e d to be su c h a slro n tt at-’e n t
l a **.415 to O.tlfll p e rc e n t s o lu tio n was
n e ed e d In p ro d u c e a c n e o n th e ra b b it
e a r l d ia l ill a la te r a tte m p i lo d e v elo p a
s ta n d a id i/e d p ro c e d u re lo r i|iia n tily iu t!
th e e tle c ls ol c h lo ra c u e producili)! s u b ­
sta n c e s I r 'l i l i w as chosell as th e lesi
s u b s ta n c e ." 1
< lllo ra en e h a s also a p p e a le d j s a
p ro b lem m l-.S . w o rk e rs. In I'M*# an
acc id e n t ill w in ch iiiteiliie d ia rv . Ilciuicals w ere le le a se d al a 2 .4 .5 - 1 plant
I M o n san to t led to I I ■' cases o l c h lo taciie In a late r in c id e n t hi th e I .Y . th is
lim e at H ow t heiiuc.il in l'»r.4. n n n
was singled o u t a s th e cau sativ e aeen l
a . t i l l l a d l e o u lb ie .il. Ill o ld e r to in ­
crease the- p ro d u c tio n rale <>i J .f .5 - 1 .
llocc had .h a u tte d l- m-.i . I iou .o n d ilio n s le s itllni)* in a n h i . tea se in l< l ’ l>
c o lllelll '*

p ro d u c e b u lli d e le c ts beeau w illi a
stu d y carried o u l h y llio n e iic s Ke scare li
la b e lH-lweett l'» its aliti I -*»•-'< u n d e r
e o u lra e t lo th è N a tio n a l I aiicei In stitu te : 2 .4 .4 -1 w as aiuoli)! ihe 4 * pestieides w liicli w e re slu d iv d II vvas lo u n d
th a l pre)!ii.inl lin c e , tre a te d w illi 2.4 5-1
al a Ic-vcl ol I I .* n iiliig ra m s per k ilo e ia m
o l b o d y wei)!ht per day p ro d iic c d a
g rc alcr p c rc c n la p e o t a b n o rm a l litte is ol
• il|s|iriri|! as w ell as ari ilici case in th è
peicenla|!c- ->l a b n o iiii.il Iclli't-s per li! ter. I Ile- iii .iio i ly p e >d abuoim .ibli-.-s
intlc-tl w ere d e l l p a la le am i sy slic kid*
lice . .Ahiiotm allllc-e. as well as ilii 'cV'C-s
Iti te la i n io iia lily . veere al'-* piotili- e d in
la te al v.uioiis dosc-s Al all Jusi-s ili Ibe­
la i. h e n io rth a p e ol ilu- ¡; islio iiile -liii.il
tr a ile o| th è le tu s veere o h e e tc -.l I ln-s-.re su lis. Iioccever. c o n iti n-d be ilvttrtrli le
linke .1 1- .4.>• 1, h k•!**>!c* ¡1 teas ll.M
er ed lll.it (lu* N jinplc II>1 il iin rli,L Nl'ItlV
;o p ¡'»Il «l(
to rn a m e li
I t i ) l> * ’
r. .li tu .1 li. •V.CVl'f
II tl.f Njfi.r
Iti il 1« HI *
c ir. . . *' •- *•li 1(| ili'! Vat n
Idi .1if 1
'
whenI !..l l-t l’fl'L’Jl.lM
« !I*.
li. ile 1ri .1 *;;ulv ni i.l.- le« 1*
i!

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li Mi ‘ 'I l In- ill-11 1'i . •Il I I .1 * *1 In ; i i . u . l

• *1

• *11 I1.Hi' *u l ' i . i '•

m in ' .nut in m u ' s t u m • -I 1 .1 I •> I’ •1
p ii>ilHifil i ll'll p a la le III .ill llili'i* "I'.iilii
ni unii- lull u n i in tlif M l. f nl nc y .il'
lu iii'ljlilii'i iii'ii; seen ill b o th I In- m in '
•uni tin* u t v *

. ou1.1 .ii i liluul.iie

Il I•I •

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11' , | i'il

mi

llllfc

M i .i i i i -

L iiv innim i'iitul (.'tm ia m iiu iim i
llu- ilinxiiiN. 2 .4 .5 -7 . jm l f i r iim i c u ­
ter ilic I'liv iim iu ii'iii lim n .i n u m b c i ul
sm itccs. O n e so u rc e n l M iilm ni-fi r is
dii' i'll Iiia i l u l p a p er p u lp la c to n e s
which use I'l l’ i.im p o u n iK in s liJ il ol
nii'ii'iiry a s slim c -c m ilio llin g a g e n ts.
M any e x p e rim e n ts have sh o w n d ia l
I'l l', as n o il a s iiic io u ry , is to x ic to t'lsii
in m u' e x p e rim e n t o n e -h all o f dio
guppies k 'sli'il at a li'Vol u l
ppm I'll*
were killed in sox on d a y s. T h e p u p p ie s
itiu lil survive a lelh a ! d o se n i S p pm
only a lle r tirsi h av in g licon a c iliiiia lc il
at low er d o se s (th a t is. p laced in w a te r
w ith a low I'lin e o n tra tim i ol I 'l l ' to i a
few d jy s t. T h e a u th o r s o l th is s tu d y
com pared th e to x ic ity o l I ' d ' to th a t nl
m ercu ri' c o m p o u n d s (su c h as p h c n y lm ercuric a c e ta te a n d in e rc u iic c h lo n d e l
and fo u n d d ia l i'l l' is as to x ic in 25
species o f fish a s m e rc u ry is In p u p p ie s,
ra in lx in t r o u t, a n d stic k le b a c k . l u r l h c r m ore, th e a m o u n t o f I'l'l* n e ed e d fo r
e d ic ic n t slim e re m o v a l w as a b o v e th e
lethal ranpe lo r m a n y fish, a n d m o re
I'l'l* th an m e rc u ry is n e e d e d .2 '*
T he a u th o rs n o te d th a t th e c o n c e n trai ion o f I 'l 'l ' n e e d e d t o kill slim e w as
above d ia l w h ic h c o u ld kill a q u a tic in ­
v e rte b rate s (su c h as insect larvae a n d
snails). In fa c t. IV I' h a s been used to 'k ill
sc h isto so m e-(a ty p e o f p a ra site I c arry in g
snails d ia l live in I g y p tia n irrig a tio n
d itch es. L evels o f 10 p p m o f c o p p e rI'ci* are le th a l t o so m e snails, a n d 15 lo
20 ppm w ere used o n th e l-.pyptian
snails. T hese c o n c e iilia lim is . m o re o v e r,
also a ffe c te d th e local lish p o p u la ­
tion.*
While u e k n o w (h a t Ihe d io x in s a re
e x tre m e ly to x ic su b s ta n c e s , a n o th e r im ­
porla!!! q u e st im i is w h e th e r <>r n o t th e y
persist in d ie e n v iro n m e n t. A lth o u g h
Ihe d io x in s a re heal sta b le i i i n n is
stab le up In HOIJ d e g ree s c e n tig ra d e , o r
1.472 degrees T aliren h e il I th e y can he
d e co m p o sed by s u n lig h t'u n d e r e e rla in
c irc iim slu m es. T \p e rim i-iils have sh o w n
lh.it m a lc o h o l solili u n is, um ici n a tu ra i
siinliglil. i r m i
tlccm nposi-s rapidly
I u ni n i Immusì W hen l i UH III m ilh a uoi was pl.iii-d m i sm l Ira lh e r iliau in
he.ikeis m ijo s i'.l lu b i's i. h o u e i e i . so
Hk
till-1fi J I,i .| Wiilklii cv.ip* >1 ali*.
>1» li.iii tu »7 vi i \ * »::iip* iN(.J .ilU 'l !• tur*
l e m il.tV *
1 (u* .itili h . t ' . . | rh i^ ' •1Ull \
y'» l u i . li 1ll.ll Ih» w .1 ».
k l t l u h ..1
II'.Ai ' ••! .» 1». ' 1 1 . tile i . > l i . :.i Ih u 'lt l • ; i v f

IIn v< ;;»» 111: 1. (;• •r: I . Jn*‘:<u j llv t .1'» ( h i
ir.

A III* *' r

is'. " I l l

.:( . . | V . .|li‘ t

. it.h

• Il l l l l l l l l l . l l l" M 11• <•" • •• 1111

1• i* i*

i f i i t i i - i l i » * ii.i<

1 h i-.

. h

ll.lllo ll' o l I I I • I > I I I H . .11 .>1 M in p p m
III h c u /c iic l n e ie apt - l i . i l i.» r . \ . . l*. PV*.
• >| i o ||. ,i vindy '.oil alul a . lay lo.im . Soil
sam ples w ere th e n te s te d j l t c i .’’l. 4 0
fill. |d ll. a n d ¡5 0 d a is . A llc l .¡5u il.i'. s.
54 p c ric n l ol d ie I p pm H l>l> -a m p le
was ic c o ie r c d lo i b o ili ty p e s oi s m l.. »»•
p e rn -ill o | th e Kilt p p m sa m p le was
rc c o ic ic il ill th e sandy so il, a n d " I p eteen l n l d ie K m p p m sam ple w as i c i n i ered hi th e clay sm l * '
I h e p o ss ih ih ly d ia l d ie d io x in s couM
be lo im e d hv b a c te ria l a i l n m in d ie sm l
was also te s te d , b e ea u se il lias been
sh o w n , lo r e x a m p le , d ia l m ic o l Ihe
b re a k d o w n p r o d u c ts o f p ro p a m l (a n
h e rb ic id e ). .t.4 ili-c lilm o a n ilin e . is c o n ­
v e n e d ill th e soil to 5 .5 '4 .4 '-te tr;i.i.'o ih lm b e n /e n e . a su b sla iic e clo sely re ­
la te d In c h e m ic a ls w hich have p ro d u c e d
c an cer hi e x p e rim e n ta l a n im als. D ichloro p h e n o l a n d tric h lm o p h c n o l w ere b o th
in c u b a le d in tw o ty p e s ol soil hi o rd e r
t o d e te rm in e w h e th e r o r n o t a b a ite ria l
c o n d e n s a tio n re a c tio n m ight c o m e r i
th e m lo d ie d io x in s . T h e y w ere iiicub a le d at levels o f It). 100. a n d I .m m
ppm lo r 7 0 d a y s
n o d io x in s , h o w e v ei.
w ere re e o v e ie d a lte r th is tim e p e rio d .
W hile th e I'h lttro p h c n o ls a n d th e ir derival lie s re p re se n t only a sm all Irac tim i
o f th e a g ric u ltu ra l a n d in d u stria l ill- lin ­
eals e n te rin g th e e n v iro n m e n t, il is clear
d ia l Ih c y c a n y w ith llicm p ro b le m s lar
b e y o n d th e ir o w n d i-siru iliv c p ro p e r­
ties. Sin c a lm o st all o rg a n ic ih e m ic a l
re a c tio n s p ro d u c e c o m p o u n d s o th e r
th a n th e o n e sp e c ifica lly so u g h t ( t h e
p o p u la r insect icid c ‘lo x a p h e n e w as u c e n tly fo u n d to be c o m p o s e d o f 4 * d o ­
le ren t I'lim p o iie n ts l. Ih e d is e o ie ry ol
th e se to x ic e o n ta m in a n is m ay be a sig­
nal lh a l a th o ro u g h an aly sis o f widely
used c h em ica ls is long o ver d u e .
I i

NOTES
1. R e v e n u e , A r t h u r , a n d

A

H e rru n

H e rb m d d ,

D is cu ssi on

«it

it s

f'r u p c r tic s ¿ n d Its O c c u r ie n e v as «1 M osid u c m
H u m a n a m i / t i m i d i lissn us,** R e s id u e R e*
v ie w s . J 9 H .1 B 4 . 1 9 0 / .
2 . S y n th e tic O rg a n ic C h e m ic a ls . U .S P r o d u c ­
t io n a n d Sales, w .5 I .in»« L u i m -. iv *,i . h .
J. P lim n ic r . J a r * M .. J t.h u f.V h » * ! ., a n d t U
w in A ,
**r/,»ss 'i i i u f i K 'n i p t n i iigr.-ut»lic a liM ti o f trie* M e n ta »md
r. i r d i j t r r i

(jtticii/K
-odt'udix and (•i>.i't"
Ihhcn.j *A q r a n1d*«
r t c n 'u c j i lV " ! j y » d

P o o d C h e m .. 2 U I i * 9 j , J j -> » .*0

i*W «

4. W. m.e...... » i-|,
i* . »«•»• jio
a n d Pet«'» 1» j |
flilfi'tt.'.i ¿j ili • td (.•dilr
ufl*' !." J. Agi 1
l * i/2
*. J«*!.\»
*. . .»«.<

». Tn »«•..i*..
•I. f 1

a.jU

. . .1 '.

... 1 1 ...

•1

• l» *U '.« l •»*» / .4 s t . t * • . .» • •».. »*,...
«•»•»i,.- ••». . .1 • • . ...........». • •
............. /••la .
...............
* . »i*.' .»*• ». ¿4 .* .• •*
•'*. 1» « . V .
19 / 1, li 1‘.
/
•••»>.»•, tr*'»t‘ .a*.. .i*«*! » *•. *. *.p ■ »1
*51
.i**.|» / i »*••«.»•
H * t», f *:i".
*• , " / *
C n a iio n r t tr n t , J .* |h | .*1 , J**t . 1 .*, r i t u

;

x

R e p o rt un 2 .4 .5 T . •»*/ • •? . 0 4 9

9
|M

f , M l . f „ * ? I I . , “ I *» « » t* .|f|............I

f J* tll ll 'i f M . i M I / 'i f ] l 'i « l l l t i /

M ir i li *.f
N a tu r e .

.» .J ,/.«

lti***fl«.||

‘ •»»finii*' .* .4 .5 > *.» h l-.r i O( i «m i «i
? v , j««n| 1 «, 1 9 / 1 .

12. •f *'l*'it:, I f i i t i f l l /« , bVilii.i«*« | rftr lid i.
f «Hii'ti'*.**, ••«••> K.ul* V . K » t/f* !ii* * *i,
tei 1% ■*U " (' Mdt»f»ltS «if K 'itlit* t*(-.«• •j^l»*-***•! .
S*!<lMir<t lV M tiK ld , fK i|titi'ii.tl< :l “ J in d u c i. 1
t ie n e a n d T o * . . ? i ( 5 | t o n I / ? . f / a y 1 9 ( 9 .
1?. " I ite (tue*» >.<|i*in«i
R t r i r e t , •><<r..<lH, 1 9 0 «

t d i 't i » , "

N u tr ii

I J /»»• i / n t l , A u d i« :*» . ( j.i v i *- I iri!st*»m:. D a
fiani*«,. • V iliijrn M ' f f w i t / , 1 #?*• I ii«.*uuian,
S l a n l e / N v U i m i i i i , * * 'it u d iv ‘< n i th e t.iu c t* t
in«i t a«.tor, i l . is o la tim i u t «1 T o » ic *•
s U n ie ,"
J.
ot
A m . O tl
C h e m is t'
&•
3H CO 0 2 . ►«&. i O O l.

Q
0

$
o
co

o
O»Ai

1 4 . '*^ e a n ti tu« C ln c ^ t dcn»a I mc I » * , ” C h e
cal a n d C n t m e e r m t N e w s , Jan . 3 0 . 1 9 0 /
10.
1 5 . U m igiass, t . j r i l i . , a n d O n n a id
H
“ C 'tlU U td J lifU b i'U S M y fi»r C I i k i * f d e n ta f
to#,** J. Assoc. O f f r e . A g r. C h e m is ts . 4 4
I 9 t » l.
li» . V o s . J . i i . , a n d J .M . Kr*e>tian( “ C o m p .
ture 7 d u ( o l u i |i t . U *u d y w it h P d ly c h lir f iiu
& iD h e n y ts m C lu c k c n s w it h Soc*».Mi M e le rò
t u P u rp h y ii.t. t d o m a l o r » n * ti* m , L i^ c r N e t
sis. a n d Tissue M esidues.** T o * . A p p i. Rhar
l/:tiS O *4 » 6 « . 1 9 7 0 .
1 / . V o i . J.Ca ., J .H . K if« iu a ri. H . l . van
W . ij s , M .C . le u N 't t v v r d e U » <iu e, a n d M .H
V o s . '* td e n lilic a tu » n a n d ì u*ic»>l</<jicai b v .
a tm n u t
in a tv d r ) t b e f i /u f u M n a n d O
rin ate ci N a D tlia ic n c »n T w n C n in itu .'ic ia i, P<
c h iu r in a tc d H io h e n y t s /* F d - C o s m e t. T o e ic
B .0 2 5 -Ó 3 J . 1 9 7 0 .
16 . S c h w a » t/, L o u is . L o u i s T tih p in , D o n
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S k iA . L e a A F c P t t e r . P to ia d e ip n ia , 1 9 5 7 ,
33C .
19 . B a a lc t . L . W ., a n d H .J . B a u e r, “ In d u S t
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and
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by R.L. lobasia,' P.l. Gehrinc/ R.l. Kacika/ aid B.A. Schwatz*

Introduction

Pentachlorophenol is a registered anti­
microbial agent whose principal use is for
the preservation of wood. Typical commer­
cial pentachlorophenol contains a variety of
substances which are considered to be “in­
active” from the aspect of antimicrobial e f­
ficacy. Consequently, pentachlorophenol is
sold as an antimicrobial agent with 95%
active ingredients and 5% “inert” ingredi­
ents. Analysis of acceptable commercial
pentachlorophenol is shown in Table 1.
The “caustic insolubles,” sometimes re­
ferred to as the “nonphenolic or neutral im­
purities,” include chlorinated dibenzo-p-dioxins and chlorinated dibenzofurans (I).
Recently developed analytical technology has
allowed quantitation of hexachlorodibenzop-dioxins and octachlorodibenzo-p-dioxin in
pentachlorophenol. Portrayed in Table 2 are
concentration ranges for these two chlorodibenzo-p-dioxins in samples of currently
available commercial grade pentachlorophe­
nol. Techniques capable of detecting 0.05 ppm
showed no 2,3,7,8-tetrachlorodibenzo-p-dioxin
in any sample of pentachlorophenol examined
by us. The absence of this compound in pen­
tachlorophenol is not surprising, because the
appropriate precursors for its formation are
not present.
'Designed Products Department, The Dow Chemi­
cal Company, Midland, Michigan' 48640.
tChemical Biology Research Laboratory, The Dow
Chemical Company, Midland, Michigan 48640.

Table 1. Commercial pentachlorophenol composition.
Content
%
Pentachlorophenol
Tetrachlorophenol
Trichlorophenol
Higher chlorophenols
Caustic insolubles (maximum)

85-90
4-8
<0.1
2-6
1

Table 2. Concentration ranges of some chlorinated
dioxins in commereiai pentachlorophenol.
.Chlorinated dibenzo-p-dioxin

Concentration
range, ppm

2,3,7,8-Tetrachlorodibenzop-dioxin
Hexachlorodibenzo-p-dioxins
Octachlorodibenzo-p-dioxin

None
9-27
575-2510

Heptachlorodibenzo-p-dioxin and hexa-,
hepta- and octachlorodibenzofurans have
been qualitatively detected in commercial
pentachlorophenol However, the lack of ap­
propriate standards for these materials does
not allow their quantitation.
Severe toxicological responses have been
attributed to certain chlorodibenzo-p-dioxins ( 2 ). For example, the LD50 of 2,3,7,8tetrachlorodibenzo-p-dioxin ranges from 0.6
fig /k g in male guinea pigs to 115 pg/kgr in
rabbits of mixed sexes. A benzene solution
of this agent containing as little as 0.04
/xg/ml produces acne in the rabbit ear bio­
assay. Very high embryotoxicity and the
production of edema in chicks are other
properties of this material. The no-effect

September 1973

¡171

0001357

5871

DOW 119368

Chlorinated Dibenzodioxins and
Pentachlorophenol

2

Table 3. Concentrations of chlorinated dioxin “indi­
cators” in commercial pentachlorophenol utilized in
toxicological evaluations.

Chlorinated dioxin

Concentration,
ppm

Octachlorodibenzo-p-dioxin
Hexachlorodibenzo-p-dioxins

1980
19

A chemically pure pentachlorophenol hav­
ing no detectable concentrations of any
chlorinated dioxins was subjected to the
same toxicological tests. The toxicological
data on this chemically pure pentachloro­
phenol, summarized in Table 5, include neg­
ative responses in both the chick edema
and rabbit ear bioassays. In the 90-day rat
feeding study, the only changes noted were
increased liver weights at 30 or 10 mg/kgday and increased kidney weights at 30 mg/
kg-day. However, in contrast to commercial
pentachlorophenol, gross and histopathological alterations did not accompany these in­
creases in organ weights. Thus, by utilizing
the results of these three tests, it may be
concluded that the presence of the contami­
nants in commercial pentachlorophenol may
be detected by toxicological evaluation.
An analysis of a sample of pentachloro-

Table 4. Toxicological data on sample of commercial
pentachlorophenol.
Study

Result *

Chick edema bioassay
Rabbit ear bioassay
Rat feeding study
Food consumption
Body weight
Hematology
30 m g/kg-day
10 m g/kg-day
3 m gA g-day
Urinalysis
Clinical chemistry
30 m gA g-day
10 m gA g-day
3 m gA g-day
Liver weight
30 m gA g-day
10 m gA g-day
3 m g/kg-day
Kidney weight
30 m g/kg-day
10 m gA g-day
3 m gA g-day
Pathology
30 m gA g-day
10 m gA g-day
3 m g/kg-day

+
+
—
—
+

DOW 119370

commercial pentachlorophenol. A positive re­
sponse was noted in both chick edema and
rabbit ear bioassays. In the 90-day rat feed­
ing study, untoward effects were noted in a
majority of the parameters monitored. Hem­
atological examination revealed a depres­
sion of erythrocytes, hemoglobin, and
packed cell volumes at a dose level of 30
mg/kg-day pentachlorophenol.
Clinical
chemistry alterations included an elevation
of serum alkaline phosphatase at 30, 10, or
3 mg/kg-day and a depression of serum al­
bumin at 30 or 10 mg/kg-day. The weights
of liver and kidneys were increased at 30,
10, or 3 mg/kg-day. Pathologic examination
revealed minimal focal hepatocellular de­
generation and necrosis at 30 mg/kg-day.
Thus, it is evident that commercial penta­
chlorophenol induced untoward effects in
each of the three toxicological tests.

—
—

—
+
+
+
+
+
+
+
+
+
-1- •
— :
— .

* -I- denotes effect; — denotes no effect.

phenol representative of that which we are
capable of producing is shown in Table 6.
The toxicological data on this sample of
pentachlorophenol are summarized in Table
7. Both the chick edema and rabbit ear bio­
assays gave negative responses. In the 90-day
rat feeding study, the only unequivocal
changes were increased liver weights at 30
or 10 mg/kg-day and increased kidney
weights at 30 mgAg-day. There were no
gross or histopathological alterations noted.
To reiterate the toxicological findings on
these three samples of pentachlorophenol, a
comparison is provided in Table 8.
Commercial pentachlorophenol gave posi­
tive responses in both the chick edema and
rabbit ear bioassays; in contrast, the chem­
ically pure pentachlorophenol and the im­
proved pentachlorophenol both gave nega­
tive responses in these bioassays.
In the rat feeding studies, commercial pen­
tachlorophenol was associated with hema173

September 1973

0001359
5

8

7

2

Rodenticide Act. The composition specifica­
tions for this new commercial pentachlorophenol are cited in Table 9.
In conclusion, it is feasible to produce a
pentachlorophenol in commercial quantities
which by comparative evaluations mimics
pure pentachlorophenol in toxicological re­
sponses.

Content
Pentachlorophenol
88-93%
Tetrachlorophenol
12- 7%
Trichlorophenol
<0.1%
Higher chlorophenols
0.1%
Chlorinated dioxins
Octachlorodibenzo-p-dioxin
30 ppm (max.)
Hexachlorodibenzo-p-dioxins
1.0 ppm (max.)

1. Plimmer, J. R., Ruth, J. M., and Woolson, E. A.
Mass spectrometric identification of the heptaand octachlorinated dibenzo-p-dioxins and dibenzofurans in technical pentachlorophenol. J. Agr.
Food Chem. 21: 90 (1973).
2. Schwetz, B. A., et al. Toxicology of chlorinated
dibenzo-p-dioxins. Adv. in Chemistry, in press.
3. The Dow Chemical Company. Antimicrobial
agents product literature, Section IV-7, Dow
Chemical Co., Midland, Mich., 1969.
4. Adams, E. M., et aL The response of rabbit skin
to compounds reported to have caused Acneform
Dermatitis. Ind. Med. Ind. Hyg. Sec. 10: (2)1
(1941).
5. Official Methods of Analysis, 10th ed. Associa­
tion of Official Agriculture Chemists, Washing­
ton, D. C., 1965, Sections 26.087-26.091.

DOW 119372

Table 9. Composition and specifications of improved
pentachlorophenol.

REFERENCES

175

September 1973
° 0 0 î3 Q t

5873

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• DOW275Z82

TOXICOLOGY AND APPLIED PHARMACOLOGY 46. 279-303 (1978)

Results of a Two-Year Chronic Toxicity and Oncogenicity
Study of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin in Rats
X»

ro
o

R. J. K ociba, D. G. K eyes, J. E. Beyer, R. M. C arreon , C. E. W ade,
D. A. D ittenber , R. P. K alnins, L. E. F rauson , C. N. P ark , S. D. Barnard ,
R. A. H ummel, and C. G. H umiston

o

■si
03

Toxicology Research Laboratory, Health and Environmental Research, Dow Chemical,
U S A . Midland, Michigan 48640

Received November l , 1977; accepted February 22,1978

Results of a Two-Year Chronic Toxicity and Oncogenicity Study of 2J.7,8-Tetrachlorodibenzo-p-Dioxin in Rats. Kociba, R. J., Keyes. D. G - Beyer. J. E , Carreon. R. M„ Wade.
C. E„ Dittenber. D. A„ Kalnins, R. P„ Frauson. L. E , Park. C. N.. Barnard. S. D..
Hummel. R. A - and Humiston. C. G. (1978). Toxicol. Appi. Pharmacol. 46.279-303. Rats were
maintained for 2 years on diets supplying 0.1.0.01, and 0.001 pg of 2.3.7,8-tetrachJorodibenzop-dioxin (TCDD)/kg/day. Analysis of these diets indicated 2200, 210, and 22 parts per trillion
(ppt) of TCDD. Ingestion of 0.1 pg/kg/day caused an increased incidence of hepatocellular
carcinomas and squamous cell carcinomas of the lung, hard palate/nasal turbinates, or tongue,
whereas a reduced incidence of tumors of the pituitary, uterus, mammary glands, pancreas, and
adrenal gland was noted. Other indications of toxicity at this dose level included increased
mortality, decreased weight gain, slight depression of erythroid parameters, increased urinary
excretion of porphyrins and ¿-aminolevulinic acid, along with increased serum activities of
alkaline phosphatase, y-glutamy! transferase and glutamic-pyruvic transaminase. Gross and
histopathologic changes were noted in the hepatic, lymphoid, respiratory, and vascular tissues.
The primary hepatic ultrastructural change at this high dose level w u proliferation of the rough
endoplasmic reticulum. Terminal liver and fat samples from rats at this high dose level contained
24.000 and 8100 ppt of TCDD, respectively. Rats given 0.01 pg/kg/day for 2 years had a lesser
degree of toxicity than that seen at the highest dose level. This included increased urinary
excretion of porphyrins in females, liver lesions (including hepatocellular nodules), and lung
lesions (including focal alveolar hyperplasia). Terminal liver and fat samples from rats of this
dose level contained 3100 and 1700 ppt of TCDD, respectively. Ingestion of 0.001 pg of
TCDD/kg/day (-2 2 ppt in the diet) caused no effects considered to be of any toxicologic
significance. At this lower dose level, terminal liver and fat samples each contained 340 ppt
of TCDD. These data indicate that continuous doses of TCDD sufficient to induce severe
toxicity increased the incidence of some types of tumors, while reducing other types. During
the 2-year study in rats, no increase in tumors occurred in those ran receiving sufficient
TCDD to induce slight or no manifestations of toxicity.

.

The compound 23,7,8-tetrachlorodibcnzo-/>-dioxin (TCDD) is a highly toxic impurity
that may be formed under certain conditions during the production of 2,4,5-trichlorophenol. T C D D has been considered one of the causes of chloracne, which has been
279

<»«l-00SX/Tg/04M-0279J02.00/0
Copyright § 1973 by Academic Press. Inc.
AU rights of reproduction in tny form reserved.
Primed m Greet Britain

5b i'o
0001969

I

280

«V
ro
00

KOCIBA ET At.

associated with the industrial production of 2,4,5-trichlorophenol and other products
made from 2.4.5-trichIorophenoI.
Most of the earlier toxicologic studies with T C D D were concerned with the
assessment of its short-term toxicity and teratogenic potential. Results of these earlier
studies have been summarized in a previous publication by Kociba et at. (1976). This
same publication also reported the results of a subchronic study in which rats were
given 1.0, 0.1,0.01,0.001, otOftg ofTCDD/kg 5 days/week for 13 weeks. Doses of 1.0
Mg/kg/day caused multiple toxicologic effects, including mortality and morphologic
changes in liver, thymus, and reproductive organs. A dose level of 0.1 //g/kg/day caused
lesser degrees of toxicity, and rats given 0.01 or 0.001 //g/kg/day had no alterations
considered of any toxicologic significance.
More recently, toxicologic studies of T C D D have also been conducted in the
monkey. McConnell et at. (1978) reported a single oral LD50 in monkeys of <70 ftg
TCDD/kg. These monkeys died with loss of hair or nails, keradnization of Meibomian
glands and hair follicles, and hyperplasia of the epithelium of the renal pelvis, stomach,
and bile duct.
Allen et at. (1977) reported on a subchronic study in which monkeys consumed a diet
containing 500 ppt of T C D D for 9 months. It was calculated that these monkeys
ingested 2 to 3 fig TCDD/kg over the course of the 9-month study. Clinically, these
monkeys showed changes similar to those described by McConnell et at. (1978) as well
as hematologic depression and hemorrhages in various tissues. Hypertrophy, hyper­
plasia, and/or metaplasia were noted in the epithelium of the bile ducts, salivary glands,
bronchi, pancreatic ducts, sebaceous glands, skin, gastric lining, and urinary tract
In regard to long-term or carcinogenic studies conducted in rodents with TCDD,
Innes et at. (1969) reported no increase in tumors in mice given 2,4,5-Trichlorophenoxyacetic acid contaminated with a level of T C D D sufficient to supply 0.27 fig of
TCDD/kg/day. DiGiovanni et at. (1977) conducted a study in which T C D D was
reported to be only a weak tumor initiator in the two-stage system of mouse skin
carcinogenesis with 7,12-dimethylbenz(a]anthracene (DMBA).
Van Miller and Allen (1977) issued a preliminary report on a study of small groups of
male rats fed diets containing T C D D for 65 weeks. All 10 rats of each group receiving
1.0, 0.5, or 0.05 ppm of T C D D in the diet died within 4 weeks, with acute toxic effects,
including severe liver necrosis, bile duct hyperplasia and edema, atrophy of spleen and
thymus, gastrointestinal hemorrhages, and decreased spermatogenesis. Groups of male
rats on diets containing 5000 or 1000 ppt experienced increased mortality, decreased
weight gain, and liver toxicity. Dietary levels of 500, 50, 5, and 1 ppt of T C D D were
also studied. Various neoplasms were found in some rats at ail dose levels of 5 ppt and
higher, with only the lowest dose level of 1 ppt reportedly free of any neoplasms. These
same results on tumorigenesis were included in an updated report by Van Miller et at.
(1977), which included the data generated through the end of their 95-week study.
No tumors were reported in the group given 1 ppt of T C D D or in a total of 50 control
rats.
In view of the need for an evaluation of the chronic toxicity and potential for
carcinogenicity of TCDD, the study reported herein was conducted. In this study,
groups of male and female rats were maintained for 2 years on diets supplying

J

5876
0001970

m o o

281

r s z w

CHRONIC TOXICITY OF TCDD IN RATS

various dose levels of TCDD, and numerous parameters were evaluated in order to
assess the potential chronic toxicity associated with long-term ingestion of the material.

METHODS

A

Experimental design. Male and female Sprague-Dawley rats, Spartan substrain,1 6
to 7 weeks old, were randomly placed (two/cage) into suspended wire-bottomed cages
for this study. Food1 and water were available ad libitum. Croups of 100 rats (50 males,
50 females) were maintained for up to 2 years on diets supplying 0.1, 0.01, or 0.001 pg
of TCDD/kg/day. The diet of the control group of 172 rats (86 males, 86 females)
contained the vehicle.
Test material. The T C D D sample used for this 2-year study was prepared by the
D o w Chemical Company. Purification of the crude T C D D was followed by gas
chromatography and mass spectrometry. The final product had a purity exceeding
99%, as determined by electron-capture gas chromatography. This sample was used to
prepare the premixes and test diets according to the following general procedure:
Approximately 1 mg of T C D D was weighed on a microbalance and dissolved in 40 ml
of reagent-grade acetone. This solution was then added to 1000 g of control feed and
mixed for 30 min to prepare a stock premix. A sufficient sample of the stock premix was
then mixed with control feed to produce a working premix to be used in preparing the
test diets. The stock premix was prepared six times during the course of the study. The
test diets were prepared by diluting the working premix with sufficient control feed to
provide dose levels of T C D D as required by body weight and food consumption
determinations in order to maintain the designated dosages on a microgram per kilo­
gram per day basis. Samples of the working premix and the test diets were analyzed
periodically to ascertain that the dietary levels were being maintained as scheduled. The
concentration of T C D D in the premix samples was determined using electron-capture
gas chromatography and gas chromatography-mass spectrometry. The concentration
of T C D D in the test diets was determined by gas chromatography-mass spectrometry
after extraction and suitable cleanup.
Clinical observations. All rats were palpated on a monthly basis, with a recording of
the number of rats bearing palpable masses. Body weights and food consumption of 20
rats/sex/treatment level and controls were routinely recorded for each week of the first3
months of the study and at approximately monthly intervals thereafter. All remaining
rats were weighed monthly throughout the study.
Blood samples for hematological determinations were collected from eight rats/sex/
group at 3, 12, and 23 months of treatment The total erythrocyte count (RBC), total
and differential leukocyte counts (WBC), thrombocyte and reticulocyte counts, packed
cell volume (PCV), and hemoglobin (Hgb) concentration were determined using
automated3 or manual procedures. Urine samples were collected from seven to eight
rats/sex/group at these same time intervals. Urine specific gravity, pH, and the presence
1Spartan Research Animals, Haslett, Michigan.
1 Purina Laboratory Chow, Ralston-Purina Co.. St. Louis. Missouri.
1 Coulter Counter Model ZB-1. Coulter Electronics. Hialeah, Florida.

5877
0001971

4

282

ro
00 ~

¿4. •'

KOCIBA ET At.

or absence of sugar, protein, ketones, bilirubin, and occult blood were determined45at
each of these times, and urinary urobilinogen was also evaluated at Month 23.
Urinary excretion of creatinine, coproporphyrin, uroporphyrin, and ¿-amino levulinic
acid (¿-ALA) was determined by a consulting laboratory* on samples collected from
four to five rats/sex/group at Months 3-4, 12, and 23.
Serum samples were collected from seven rats/sex/group by orbital puncture at
Month 22 for determination of urea nitrogen (BUN), glutamic pyruvic transaminase
(SGPT), bilirubin (total, direct and indirect), cholesterol, and triglycerides. Automated
procedures were used for these determinations.4 Serum samples were similarly collected
from seven rats/sex/group at Month 23 for determination of alkaline phosphatase (AP)
activity, total protein, albumin and globulin.6
At terminal necropsy, serum samples were collected from all survivors or a maximum
of 10 rats/sex/group for determination of BUN, SGPT, AP, and total bilirubin.6 A
consulting laboratory* also made determinations of serum y-glutamyi transferase
activity (GGT).
All rats dying or culled during the course of the study were subjected to a gross
pathologic examination. Representative portions of the major organs and any gross
lesion suggestive of a significant pathologic process or tumor formation were collected
from each rat and preserved in buffered 10% formalin.
Terminal necropsy examination was conducted at the end of 2 years of treatment
(105th week). All rats were deprived of food overnight prior to killing by decapitation.
The eyes of all rats were examined by gently pressing a glass slide against the cornea
under bright fluorescent illumination. Any observations on the eyes were recorded as
part of the gross necropsy observation records. The eyes for a maximum of five
rats/sex/group were preserved in Zenker’s fixative. Eyes from remaining rats were fixed
in formalin fixative.
The weights of the liver, kidney, brain, heart, thymus, spleen, testes, and
ovaries/uterus were recorded for a maximum of 10 rats/sex/group. A bone marrow
smear was prepared from most rats, and filed for future reference, ifindicated. Portions
of fat, liver, and kidney from a maximum of five rats/sex/group were frozen for possible
T C O D analysis, with subsequent analysis of liver and fat samples from three
females/dose level, using gas chromatography-low-resolution mass spectrometry.
Portions of esophagus, salivary glands, stomach, small intestine, large intestine,
pancreas, liver, kidneys, urinary bladder, prostate, accessory sex glands, epididymis,
testes, ovaries, uterus, brain (cerebrum, cerebellum, brain stem), pituitary gland, spinal
cord, peripheral (sciatic) nerve, trachea, lungs, spleen, thymus, lymph nodes, heart,
aorta, skeletal muscle, mammary tissue (females), adrenal glands, thyroid, parathyroid,
tongue, lower jaw, and skull (including nasal turbinates, ear canal), together with any
additional gross lesions were preserved in formalin fixative.
Histologic examination of tissues was conducted on paraffin-embedded sections of
tissues which were stained with hematoxylin and eosin. All rats from the control and top
dose level, regardless of whether they died during the study or were killed at the

j

4 Ames Bililabstix or Multistix. Ames Co.. Elkhart. Indiana and TS Meter. AO Optical. Buffalo. New
York.
5 Bioscience Laboratories. Van Nuys. California.
‘ Technicon AutoAnalyzer. Technicon Corp.. Rye. New York.

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CHRONIC TOXICITY OF TCDD IN RATS

283

termination, were subjected to histologic examination of an extensive list of tissues,
intended to include a majority of those tissues listed above as those collected at the time
of terminal necropsy. All rats from the two lower dose levels were subjected to histo­
logic examination of those selected tissues identified as possible target organs and all
gross lesions suggestive of tumor formation. The actual number of dssues specimens
from each group examined histologically is on file and available from the authors.
Additional sections of liver from selected females from the terminal necropsy were
stained for lipid content using Oil Red O or Sudan IV stains.
Liver tissue collected at the terminal necropsy was examined using an electron
microscope1 to characterize qualitatively the ultrastructure of hepatocytes from three
females/group. The liver tissue was fixed in 2.5% phosphate-buffered glutaraldehyde
and then postfixed in 1% phosphate-buffered osmium tetroxide, dehydrated through
graded ethanol solutions, washed in propylene oxide, infiltrated with Epon 812, and
embedded in polyethene capsules. Sections of 1 ftm thickness were stained with toluidine
blue and examined using light microscopy. Thin sections were stained with uranyl
acetate and lead citrate prior to examination by electron microscopy.
Statistical evaluation o f data. The significance of differences between control and
test values for hematology, urinary and clinical chemistry parameters, body weights,
organ weights, and organ/body weight ratios was statistically determined by one-way
analyses of variance followed by the Dunnett test (Steel and Torrie, 1960). A
significance level of p < 0.05 was used. Data on mortality, palpable masses, gross
pathology, histopathology, and tumor incidences were analyzed using the Fischer exact
probability test, p < 0.05, one-sided test (Siegel, 1956). Mortality data were also
analyzed using the Mantel-Haenszel Test Repeated measures analyses across time
were not appropriate because of mortality and because the assumptions of the statistical
tests were not valid.
Statistical evaluation of gross pathology data collated for the entire study compared
the data of each of the treatment groups with the data of the control group of that sex.
Statistical evaluation of histopathologic observations and tumor incidences compared
the data of the high-dose group with the data of the control group of that sex. The same
evaluation was conducted on the lower dose levels in instances in which comparable
numbers of tissues were subjected to microscopic examination (apparent target
organs).

o
O
ro
TO 03O'i

RESULTS

Dietary Content o f TCDD
Analyses of feed samples indicated the dosage levels of 0.1, 0.01, and 0.001 p% of
TCDD/kg/day equated with approximately 2193,208, and 22 ppt ofT C D D in the diet.
Six repeated analyses of the feed samples indicated good agreement between the
intended content of T C D D and results of analysis for T C D D content.
Clinical observations
Females given 0.1 pg/kg/day had statistically increased cumulative mortality dining
the latter half of the study, whereas those given 0.01 or 0.001 ¿tg/kg/day had mortality
1 Carl Zeiss. Inc., New York.

5879
0001973

LZ it&a
t)82C

284

KOCIBA E T A L .

rates comparable to that of the controls. In males, there were some isolated instances of
statistical differences between the treated and control groups. However, as the mortality
of only the group of males given 0.01 //g/kg/day was significantly different from control
using the Mantel-Haenszel test, these deviations in the male rats were considered of
questionable toxicologic significance. Mean body weights of males and females given
0.1 //g/kg/day were statistically decreased from control values throughout the major
portion of the study, from Month 6 to the end of the 2-year test period. Mean body
weights of females given 0.01 //g/kg/day were decreased to a lesser degree during this
same time interval. The mean body weights of males given 0.01 or 0.001 //g/kg/day and
females given 0.001 //g/kg/day were sometimes lower than controls during the middle
of the study, but only occasionally were the differences statistically significant. During
the last quarter of the study body weights of these groups were comparable to those of
controls.
There were no consistent deviations in food consumption of males or females at any
dose. The few sporadic cases in which there was a statistical increase or decrease
between the control and treatment groups followed no consistent trend, and were
considered of no toxicologic significance. The first palpable mass was noted at Month 5
in a male of the control group. There were no statistically significantdifferences between
the control and treated groups except during Months 15 and 16, when the males given
0.01 //g/kg/day had an increased incidence of palpable masses. This was considered of
no toxicologic significance because of its isolated occurrence and lack of dose response.
During the last 12 months of the study, females ingesting 0.1 //g/kg/day had a
consistent trend toward a decrease in the number of rats with palpable masses. This
observation was not noted at lower dosage levels in the females.
Hematology
The hematology data collected after 23 months of treatment are listed in Table 1 and
are similar to the patterns observed during the study. In rats given 0.1 //g/kg/day, there
were statistically significant decreases in the P C V and Hgb values for males after 3
months as well as decreases in the Hgb values for males and decreases in PCV, total
RBC, and W B C counts and Hgb values for females after 1 year. At the preterminal
examination, this high dose group again had statistically significant decreases in RB C
and Hgb values (males) and P C V and Hgb values (females); reticulocyte counts also
appeared to be slightly increased. Thrombocyte and W B C differential counts appeared
to be unaffected at alldose levels ofTCDD. Rats given 0.01 or 0.001 //g/kg/day had no
hematologic changes considered related to treatment
Urinalyses
Repeated examination of urinary parameters revealed no consistent alterations that
could be attributed to any of the dose levels of TCDD.
Urinary porphyrins and 5-ALA
Porphyrin data collected after 23 months of treatment are listed in Table 2 and are
representative of the patterns observed during the study. Urinary excretion of copropor­
phyrin was statistically increased in female rats at a dose level of 0.1 //g/kg/day after
each evaluation at 3-4, 12, and 23 months. Coproporphyrin excretion was also

5880
0001974

r

TABLE I
M ean H ematologic Values of M ale R ats (D ay 681) and F emale R ats (D ay 682) on D iets C ontaining TCDD
___________
...__ ___ __ — ■
- —

Dose of
Number
TCDD
of
(MsAgAJay) Sex rats/group
0
0.100
0.010
0.001
0
0.100
0.010
0.001

M
M
M
M
F

F
F
F

8
8
8
8
8
8
8
8

PCV
(%)
46.9 ± 4.7"
43.4 t 4.J
47.6 ± 2.8
47.4 f 1.8
43.6 t 1.4
38.9 t 3.8*
45.1 ± I I
46.9 ± 4.7

2

50

Reticulo­ Thrombo­
WBC dilferential count (%)
RBC
Hgb
cytes
cytes
WBC
(x IO*/mmJ) (g/100 ml)
(%)
(x lOVmm1) (x lOVmm1) Neut Lymph Mono Eosin Buso
7.99 ± 0.64
7.19 ±0.65»
7.65 t 0.46
7.73 ± 0.27
6.84 ± 0.66
6.38 ± 0.90
7.35 ± 0.32
7.56 ± 0.69

15.6 ± 1.6
13.9 ± 1.5»
16.0 ± 0.8
15.7 ±0.6
14.1 ± l.l
12.5 ± 1.2*
15.1 ±0.4
15.7 ± 1.5*

1.0
2.2
1.0
0.5
0.8
1.2
0.5
0.5

1.195 ± 0.350
1.214 ±0.358
1.482 ± 0.342
1.042 ± 0.143
1.046 ± 0.210
1.176 ± 0.358
0.962 F 0.161
0.857 + 0.239

14.9 ± 5.4
12.1 ± 5.2
15.8 ±4.0
18.7 ± 8.0
9.1 ± 1.5
7.1 ±2.1
9.4 ± 2.2
9.5 ± 1.9

27
33
34
30
34
30
36
23

65
62
60
65
60
67
60
72

7
4
5
4
4
2
3
3

1
|
|

1
2
1
1
2

0
0
0
0
0
0
0
0

o
z
n

H
O
n
3
3
o

n

o
o
2
£

“ Mean i SD.
* Statistically significant from control mean using analysis of variance and Dunncil's lesl, p < 0.03.

K>

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la

000197
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or
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oo

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K»
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r

TABLE 2
U rinary Excretion of C reatinine, C oproporphyrin, U roporphyrin, and ¿-A mino Levuunic A cid for M ale and F emai.e R ai s
(D ays 678-680) on D iets C ontaining TCDD

0.100
0.010
0.001

0
0.100
0 .0 10

0.001

M
M
M
M

F
F
F
F

4
5
5
5
5
5
5
5

Total urine
48 hr (ml;

0

8.£
S3

.g

C OO
•3
3 '¿b
<j£

§•>
u 5

51-0 + 21.663.4 ± 40.5
61.4 ± 26.1
41.0 ± 6.4
60.0 ± 34.9
51.2 ± 22.8
54.2 t 20.5
57.2 + 20.0

27.6 ± 3.2
19.6 ± 9.1
24.4 ± 5.7
30.8 ± 3.2
23.3 ± 6.2
18.6 ± 4.3
19.4 ± 2.3
20.8 ± 4.7

18.0 + 3.7
19.6 ± 11.7
16.5 ± 8.0
23.8 ± 4.8
9.8 ± 1.3
17.4 ±4.0*
16.4 ± 4.7»
8.6 ± 2.0

>» OG
& 'S
ao ■a

u
o. U
o 00
u
Im

se

B

0.69 +0.19
1.22 + 1.08
0.64 ± 0.25
0.78 ± 0.19
0.43 ± 0.49
0.98 + 0.41»
0.83 ±0.18
0.42 ± 0.06

G
la
>»

G
c
>* IT
■C
8*^
o 00

JS

4)
G

fr 1(4

&

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t*
D

2%

se

D 5

5.4 ± 2.2
7.3 ± 4.0
5.7 ± 2.1
5.3 ± 1.6
3.8 ± 1.7
5.7 ± 2.3
3.5 ± 1.1
3.0 ± 1.1

O
u
U
60
S

0.200 + 0.092
0.418 + 0.295
0.228 ± 0.056
0.174 ± 0.053
0.157 ±0.050
0.296 ± 0.074*
0.181 ±0.053
0.143 ±0.037

* Mean t SU.
* Slalislically significant from control mean by analysis of variance and Ounnett’s tcsl.p < Ü.0S.

000197?!

cn
GO

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to

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g o S f

■S C ”

¿ iS

<
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00
E

u
(*>
E

0.27 + 0.38 0.010*0.015
0.08 ± 0.02 0.005 + 0.003
0.26 ± 0.42 0.009 ±0.013
0.06 ± 0.01 0.002 ± 0.001
0.07 ± 0.03 0.003 ± 0.001
0.12 ± 0.05 0.006 * 0.002»
0.08 ± 0.03 0.004 i 0.002
0.08 + 0.02 0.004 + 0.001

KOCIBA ET AL.

o 3

Sex

Q ~
O £

*C
>*

Number of
rats/group

C

o>

Ê

statistically increased in female rats at a dose level of 0.01 /¿g/kg/day after 3 and 23
months. Urinary excretion of uroporphyrin was statistically increased in females after
3 and 23 months of receiving 0.1 /¿g/kg/day and after 3 months of receiving 0.01
/ig/kg/day. Urinary excretion of ¿-ALA was statistically increased in females after 3
and 23 months of receiving 0.1 /ig/kg/day. Total urine volume or creatinine excretion
was not affected by any of these dose levels in the females. Males had no alterations
considered treatment-related in any of these parameters at any of the dose levels of
TCDD.

<1"i
'j
'j
j

DOW £75209

287

CHRONIC TOXICITY OF TCDD IN RATS

Clinical Chemistry
For sake of brevity, only the results obtained at terminal necropsy after 2 years of
treatment are presented in Table 3. Analyses of serum samples collected by orbital
TABLE 3
Mean Terminal (2-Y ear) C linical C hemistry Values for Male and F emale Rats
G iven D iets C ontaining TCDD

Dose
TCDD
0/g/kg/day)

Number
BUN
SGPT
of
Sex rats/group (mg/100 ml) (mU/ml)

0
0.100
0.010
0.001
0
0.100
0.010
0.001

M
M
M
M
F
F
F
F

10
5
4
10
10
4
10
10

33 ± 34“
28 ± 11
36 ± 19
20 ± 9
21 ± 9
20 ± 3
17 ± 3
18 ± 5

AP
(mU/ml)

49 ± 17 87 ± 3 0
42 ± 6
105 ± 17
47+10
88 ± 2 6
43+9
86 ± 2 5
39 ± 11 60 ± 2 9
54 + 12* 205 ± 146*
49 + 7
61 ± 2 0
42 ± 5
54 ± 2 8

Total
bilirubin
(mg/100 ml)

y-glutamyl
transferase
(mU/ml)

0.2+0
0.2 ± 0
0.2 + 0
0.2 ± 0
0.2 ± 0
0.2 ± 0
0.3 ± 0.1
0.4 ± 0.2

0+0
1±0
1 ±0
0±0
0±0
14+ 10*
1 ±0
1+0

* Mean ± SD.
‘ Statistically significant from control mean using analysis of variance and the Dunneu’s test.
p < 0.05.

puncture at 22 to 23 months of treatment revealed no alterations considered related to
treatment in regard to BUN, SGPT, total, direct, or indirect bilirubin, cholesterol,
triglycerides, total protein, albumin, and globulin. Serum A P was statistically increased
in females given 0.1 /ig/kg/day. A statistically significant increase in serum triglycerides
noted in males given 0.01 ^g/kg/day was considered of no toxicologic significance
based on the lack of a dose-response relationship. Analyses of serum samples collected
at terminal necropsy after 2 years indicated a statistical increase in SGPT, AP, and
G G T activities for females given 0.1 ^g/kg/day. Females at the lower dose levels and
males at aildose levels were unaffected in these parameters. The B U N and totalbilirubin
values of either sex were unaffected by any levelof treatment with TCDD.

Gross and Microscopic Observations on Tissues
Detailed descriptions of all gross and microscopic observations made on all rats
killed or dying during the course of the 2-year study are on file and available from the
authors. On account of the voluminous nature of the data, the results are summarized
below. Tumor and tumor-like lesions are listed in Tables 4 and 5.

5883
0001977

.* • . ' ' '
n U o G iZ m o o

r

TABLE 4

00

00

T umor Incidence in M ai.i; Rats M aintained on D iets C ontaining TCDDu
Time iniervals during study:
0

0.1

0.01

0.001

0

0.1

0.01

Number ol'rals examined:

65

41

46

38

15

5

4

1

2

0

4

1

1

0
1
4

0
0
0

0
0
0

1
0
0

1
0
0

'

0

0

0

0

0

0

0

1

Total
0.001

0

0.1

0.01

0.001

85

50

50

50

0

6

2

3

0

0
0
0

0
0
0

2
0
0

1
1
4h

0
0
0

0
0
0

0

0

0

0

1

'

0

1

0

0

1

2

0

0

1

1

0

0

0

1

0

0

0

2

0

0

0

0

0

0

0

1

0

0

1
0
0

0
0
0

0
0
1

0
0
0

0
0
0

0
0
0

0
0
0

|
1
2

1
0
0

0
0
0

0
0
1

0

0

1

0

0

0

1

2

0

0

2

0
6

0
4

0
1

0
1

0
0

0
1

1
0

0
10

0
6

0
5

1
1"

1

.

K O C IB A S T AL.

ai
GO
GO

Terminal kill

Dose level in /rg/kg/day:

Rais with luniors/lum or like lesions
Hepaiocellulur hyperplastic
2
nodule(s)
Hepatocellular carcinoma(s)
1
Bile duel adenoma
0
Strulilied squamous cell
0
carcinoma ol' hurd palate
or nasal turbinales
Paravertebral or sub
0
cutaneous malignant
schwannoma
1
Carcinom a of renal tubules
pelvis, or bladder
Adenoma of renal tubules
0
or pelvis
0
Keratinizing squam ous cell
carcinoma o f lung
1
Pulmonary adenoma
I
Pulmonury adenocarcinoma
2
Oligodcndroglioma/astrocylom a o f brain, or glioma
ol'spinal cord
2
Interstitial cell adenoma of
testes
0
Adenoma ol’prostate
Subcutaneous fibroadenoma/
8
libroma/lipoma

©

©
«

Months 13-24

r

0001379

O l

GO

00
or

Subcutaneous librosareoma
Benign mammary neoplasms
Mammary gland adenocarcinoma
Cutaneous papilloma/basal
cell tum or
Squamous cell carcinoma of
integument
Zymbal gland carcinoma
Pituitary adenoma
Pituitary adenocarcinoma
Stralilied squam ous cell
carcinoma ol' longue
Fibrosarcom a o f tongue
Squamous papilloma/polyp
of gastric mucosa
M ucocysladcnocarcinoma of
small intestine
Leiomyosarcoma of cecum
Intraabdominal
schw annom a/sarcom a
Acinar adenoma of pancreas
Acinar adenocarcinoma of
pancreas
Islet adenoma of pancreas
Islet adenocarcinoma of
pancreas
Adenoma ol' adrenal cortex
Pheochrom ocytom a of
adrenal
Inlerfullieular C ecil
adenoma ol'thyroid
Intcrfollicular C e c il
adenocarcinoma of thyroid
Follicular adenoma of
thyroid
Follicular adenocarcinoma
of thyroid

0
0
2

1
1
0

1
0
0

J

0

0

0

0

0

0
19
1
0

0
10
0
3

1
11
0
1

1
0

0
1

0
1

0
0

0

0
0

2
2

4

0

0

6

3

2

I
0
19

0
5
3

0
2
10

2

4

0

2

1

0

0

I1

11

0

0

0

0
0
0

0
0
0

0
0
0

0
0
0

0
0
2

1
1
0

1
0
0

0
0
0

0

0

0

0

0

3

0

0

0

1

0

0

0

0

0

0

0

1

,
5
0
'

0
7
1
0

0
2
0
0

0
0
0
0

0
1
0
0

0
26
3
0

0
13
0
3*

1
II
0
1

1
6
0
1

0
1

0
0

0
0

0
0

0
0

1
0

0

1

0
1

0
1

2

0

0

0

0

1

0

0

2

0
0

0
0

1
0

0
0

0
0

0
0

1
2

0
0

0
0

3
0

3
0

0
0

1
0

4
0

14
1

2h
0

5
0

7
0

1

6

0

1

2

12

3

3

3

0
0
3

1
0
9

0
0
1

0
0
0

0
0
3

2
0
28

0
Sh
4*

0
2
10

0
0
6

0

6

0

0

0

‘s

4

0

0

0

1

0

0

0

3

1

0

0

0

0

0

0

0

0

1

1

0

0

0

1

0

0

0

1

0

0

CHRONIC TOXICITY OF TCDD IN RATS

0
0
0

M oa
i

TABLE 4 — continued
Months 13-24

Time intervals (luring study:

Terminal kill

Total

0

0.1

0.01

0.001

0

0.1

0.01

0.001

0

0.1

0.01

0.001

Number o f rats examined:

65

41

46

38

15

5

4

II

85

50

50

50

5

0

3

2

0

0

0

1

5

0

3

3

1
1

0
0

0
0

0
0

0
0

0
0

0
0

0
0

1
2

0
0

0
0

0
0

0

0

1

0

0

0

0

0

0

0

1

0

0
0

0
0

0
0

1
0

0
0

0
0

0
0

0
0

0
1

0
0

0
0

1
0

Malignant lymphoreticular
neoplasm
Hemangioma o f lymph node
Fibrosarcom a/osleosarcom a
o f musculoskeletal system
Infraorbital malignant
schwannoma
Mediastinal fibrosarcoma
Not available for
pathological examination

* No tumors occurred during Months I through 6. Tumors occurring during Months 7 to 12 included I subcutaneous fibroadenoma (control), I pituitary
adenoma (0.1 ig/kg/day). I pituitary adenocarcinoma (control), I osteosarcoma (control). These four tumors, which were present in the 10 males dying prior
to Month 13, are included in the above total tabulation.
* Statistically diiTerenl from control data when analyzed using the Fischer exact probability test, p < 0.0S. Appropriate tumor data have been combined Tor the
sake of brevity.

0001980

CA
CO
CO
œ

KOCIBA ETAL.

Dose level in pg /k g /d ay :

TABLE 5
T umor Incidence in F emale R ats M aintained on D iets C ontaining TC D D '
Time intervals during study:

Months 13-24

Terminal kill

Total

0

0.1

0.01

0.001

0

0.1

0.01

0.001

0

0.1

0.01

0.001

Num ber o f rats examined:

60

36

34

32

23

4

14

16

86

49

30

50

2

20

8

1

6

3

10

2

8

23*

18*

3

0
0
0

10
2
4

1
0
1

0
0
0

1
0
0

1
0
0

1
0
0

0
1
0

1
0
0

II*
2
4*

2
0
1

0
1
0

0

7

0

0

0

0

0

0

0

7*

0

0

0
0
1

0
0
0

1
0
0

0
0
0

0
1
0

0
0
0

0
0
0

0
0
0

0
1
1

0
0
0

1
0
0

0
0
0

0
0
0
0

1
0
0
0

1
0
0
1

0
1
0
1

0
0
0
3

0
0
0
0

0
0
1
0

0
0
0
0

0
0
0
3

1
0
0
0

1
0
1
1

0
1
0
1

16
2

3
0

7
3

5
1

12
0

2
0

4
0

7
0

28
2

7*
0

II
3

12
1

6
1
1

4
0
0

0
0
0

1
0
1

0
1
0

0
0
0

0
1
0

0
0
0

6
2
1

4
0
0

0
1
0

1
0
1

R ats with tumors/tum or-like lesions:
Hepatocellular hyperplastic
nodules
Hepatocellular carcinoma(s)
Bile duct adenoma
Stratified squam ous cell
carcinoma o f hard palate
or nasal turbinates
Keratinizing squam ous cell
carcinoma o f lung
Pulmonary adenocarcinoma
A strocytom a of cerebrum
Malignant schwannoma of
pelvic canal
N ephroblastom a o f kidney
Adenoma o f renal tubules
Carcinom a o f renal pelvis
Granulosal cell neoplasm
of ovary
Benign tum or o f uterus
Malignant schw annom a of
uterus
A denocarcinom a o f uterus
Fibrom a o f cervix/vagina
Subcutaneous fibrom a/
fibrolipoma

0001081

or
oo
oo
«vi-

CHRONIC TOXICITY OF TCDD IN RATS

Dose level in ftg/kg/day:

K
vo*

MOO

J

*• ' . i V*i ■ ■\ * ■
V p > ,o i ^
v b o -!
^ o a
K»
VO

r

to

TABLE 5— continued
Time intervals during study:

QO
GO

Total

0

0.1

0.01

0.001

0

0.1

0.01

0.001

0

0.1

0.01

0.001

Number o f rats examined:

60

36

34

32

25

4

14

16

86

49

50

50

0
50

0
22

0
23

1
24

0
23

0
2

0
12

0
It

0
73

0
24»

0
36

1
35

5

0

1

3

6

0

5

1

8

0»

4

4

0

1

0

0

0

0

0

0

0

1

0

0

0

0

0

0

0

0

0

1

0

0

0

1

26
4
1

12
2
2

8
0
0

12
0
0

17
2
0

0
0
0

5
1
0

6
0
0

43
6
1

12»
2
2

13
1
0

18
0
0

0
0

0
2

0
0

0
0

1
1

0
0

0
0

0
1

1
1

0
2

0
0

0
1

1

0

0

0

0

0

,

0

1

0

1

0

KOC1BA £ 7" AL.

0001982

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Terminal kill

Dose level in //g/kg/day:

Subcutaneous fibrosarcoma
Benign neoplasm of
mammary gland
C arcinom a of mammary
gland
Stratified squam ous cell
carcinoma o f digit
Cystadenom a o f Zymbal
gland
Pituitary adenoma
Pituitary adenocarcinoma
Stratified squam ous cell
carcinom a o f tongue
Papilloma o f esophagus
Squamous papilloma/polyp
o f gastric mucosa
Leiom yosarcom a/sarcom a
o f small intestine

GO

Months 13-24

1

0

0

0

0

0

0

0

1

0

0

0

0
1

1
0

0
0

0
1

0
2

0
0

0
1

1
2

0
3

1
0

0
1

1
3

1

0

0

0

1

0

0

0

2

0

0

0

7
4

5
3

1
0

2
1

2
3

0
0

1
1

4
1

9
7

5
3

2
1

6
2

9

4

1

1

5

0

1

0

14

4

2

1

3

2

0

1

1

0

0

1

4

2

0

2

0

1

0

0

0

0

0

0

0

1

0

0

1

1

1

0

1

0

0

0

2

1

1

0

1

0

0

0

0

0

0

0

1

0

0

0

0

0

0

0

0

0

0

0

0

1

0

0

* No tumors occurred during Months I through 6. Tumors occurring during Months ? to 12 included two benign tumors oT the uterus (0.1 pg/kg/day) and one
benign neoplasm of the mammary gland (0.01 ¿jg/kg/day). These three tumors, which were present in the 14 females dying prior to Month 13, are included in lire
above total tabulation.
* Statistically different from control data when analyzed using the Fischer exact probability lest, p < 0.05. Appropriate tumor data have been combined for (lie
sake of brevity

CHRONIC TOXICITY OF TCDD IN RATS

Polypoid adenoma o f large
intestine
A cinar adenoma o f pancreas
Islet cell adenoma o f
pancreas
Islet cell adenocarcinoma
o f pancreas
Adenoma o f adrenal cortex
Pheochrom ocytom a o f
adrenal
Intcrfollicular adenoma of
thyroid
Interfollicular adeno­
carcinoma o f thyroid
Follicular adenoma of
thyroid
Malignant lymphoreticular
neoplasm
Hemangioma o f abdominal
muscle
Not available for pathologic
examination

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294

K0C1BA E T A L .

Gross necropsy examination of the rats of the top dose level indicated the grossly
visible target organs to include the liver, vascular system, respiratory system, and
lymphoid organs; the general body condition was also consistently affected.
Microscopic examination of tissues from rats dying during the study or killed after
2 years revealed the following treatment-related affects:
Liver. The liver was the organ most consistently affected, and rats given 0.1 or 0.01
/ig/kg/day had multiple hepatocellular degenerative, inflammatory, and necrotic
changes noted upon light microscopy. These hepatic changes, which were more
extensive in females than in males, were characterized by cytomegaly, distortion of

F ig . 1. Lesion classified morphologically as hepatocellular carcinoma in liver of rat given 0.1 n of
TCDD/kg/day. Note adjacent fibrosis, inflammation, and fatty infiltration on left. H & E stain, x 200.

lobular pattern, and resultant atrophy of hepatic cords, cytoplasmic vacuolization, fatty
metamorphosis, altered tinctorial properties with increased basophilia, hepatic necrosis
and inflammation, multinucleated hepatocytes, and foci or areas of hepatocellular
alterations. They were accompanied by increased aggregates of pigment, bile duct
hyperplasia, and some increase in fibrosis and periportal inflammation. During the latter
phase of the study and at the terminal necropsy the females given 0.1 ¿ig/kg/day also
had hepatocellular proliferative lesions classified morphologically as hepatocellular
carcinomas (Fig. 1) and hyperplastic (neoplastic) nodules. There was no evidence of
metastasis of any liver neoplasms. Female rats given 0.01 ,ug/kg/day also had an
increased incidence of these hepatocellular hyperplastic nodules. Upon examination
using light microscopy, livers of female rats given 0.001 ¿tg/kg/day had a statistical
increase above the background incidence of foci or larger area of slight hepatocellular
alteration (swollen hepatocytes). However, in male rats given 0.001 //g/kg/day, there
was a statistically significant decrease in the number of livers with an area of hepato­
cellular alteration of this type.

0001384

295

As part of the ultrastructural evaluation of hepatocytes, light microscopy of toluidine
blue-stained sections of liver from females given 0.1 /ig/kg/day revealed an increased
number of individual hepatocytes containing large accumulations of lipid droplets.
Ultrastructural evaluation by electron microscopy of liver sections from this high dose

mo a

CHRONIC TOXICITY OF TCDD IN RATS

5,

•W

ro
CD

F ig. 2. Hepatocyte from female rat given 0.1 n of TCDD/kg/day for 2 years. Note disorientation of
RER and focal cytoplasmic vacuolization. Uranyl acetate-lead citrate stain. x3350.

level revealed the most consistent change to be in the rough endoplasmic reticulum
(RER), which appeared to be undergoing proliferation with some distortion and
fragmentation (Fig. 2). Smooth endoplasmic reticulum (SER) and mitochondrial
structures were within the range of variation observed in the control sections. Other
changes noted at this high dose level included focal areas of cytoplasmic vacuolization,
increased lysosomal activity with residual body formation, and an occasional multinucleated hepatocyte (Fig. 3). Upon ultrastructural examination of hepatocytes from

' 5891
0001985

296

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KOCIBA E T AL.

rats of the 0.01-ig/kg/day dose level, the most notable change was limited to a lesser
degree of proliferation and disorientation of the R E R and some proliferation of SER
(Fig. 4). There was some slight increase in the number of individual hepatocytes with
lipid droplet accumulations.

The hepatocytes of female rats given 0.001 ¿/g/kg/day were ultrastructurally within
the limits of variation seen in the controls (Fig. 5). There was no general increase in the
lipid droplet content, but an occasional cell contained increased numbers of lipid
droplets.
Lymphoreticular tissues. Treatment-related effects, noted only in females of the 0.1//g/kg/day dose level, included isolated occurrences of thymic atrophy and/or splenic
atrophy.

5892
0001986

CHRONIC TOXICITY OF TCDD IN RATS

297

Respiratory system. Treatment-related effects were noted in both males and
females at the O.I-/tg/kg/day dose level but were much more extensive in the female
rats and included an increased incidence of focal alveolar hyperplasia (Fig. 6),
aggregates of hematogenous pigment in lung and thoracic lymph nodes, focal

' *

10

\ Cl
iv
■
to t o

J

F ig. 4. Hepatocyte from female rat given 0.01 n of TCDD/kg/day for 2 years. Note proliferation of
SER and disorientation of RER. Uranyl acetate-lead citrate stain, x 2070.

accumulation of alveolar macrophages and cholesterol clefts, pulmonary edema, focal
interstitial inflammation and fibrosis, keratinizing squamous metaplasia, or squamous
cell carcinoma formation (Fig. 7) within the lung. Focal alveolar hyperplasia was also
increased in females given 0.01 /tg/kg/day. The lower dose level of 0.001 /tg/kg/day
had no discernible effect on the tissues of the respiratory system.
Cardiovascular system. Effects probably related to the ingestion of 0.1 /tg/kg/day
included an apparent increase in the incidence of hemorrhage in the brain and possibly

5893
0001987

2 /

spinal cord of females, an increase above the background incidence rate of
mesenteric/thoracic periarteritis with accompanying changes, such as thrombosis and
hematoma formations in both males and females, and an increase above the
background incidence of myocardial degenerative changes (females only). At the 0.01-

5300

DOW

298

kociba e t a l .

Fio. 5. Hepatocyte from female rat given 0.001 ftg of TCDD/kg/day for 2 year*. Morphology within
normal limits of variation seen in controls. Uranyl acetate-lead citrate «»■«■ x4100.

jug/kg/day dose level, probable treatment-related lesions were limited to an increase
above background incidence of periarteritis and thrombosis of testicular or
thoracic/mediastinal vessels of male rats. There were no alterations considered related
to treatment with 0.001 ig/kg/day.
Reproductive system and mammary gland. Female rats given 0.1 ig/kg/day had a
statistically decreased incidence of uterine changes, including endometrial hyperplasia.

5894
0001388

CHRONIC TOXICITY OF TCDD IN RATS

299

cyst formation, and adenomatous polyp formation. This same group of high dose level
female rats also had a significantly decreased incidence of subcutaneous mammary
tumors. These observations correlated well with a decreased incidence of pituitary

TO
Ol
CO

o

F ig. 6. Focal alveolar hyperplasia near terminal bronchiole within lung of rat given 0.1 Mg of
TCDD/kg/day. H & E stain, x 100.

F ig. 7. Lesion within lung of rat given 0.1 Mg of TCDD/kg/day classified morphologically as squamous
cell carcinoma. Note accumulation of keratinized material within lesion. U A E stain, x 100.

5895
0001389

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300

KOCIBA. FT / L.

adenomas noted in this same high dose level of female rats. There were no discernible
effects in female rats given 0.01 or 0.001 ftgt kg/day.

The reproductive organs of male rats appeared to be unaffected by these dose levels,
with similar degenerative, inflammatory, and proliferative lesions in all treated and
control groups.
Endocrine organs. Female but not male rats given 0.1 //g/kg/day had a significantly
decreased incidence of pituitary changes, including hemangiectasis and adenoma
formation. Adrenal changes noted at this high dose level included a decreased incidence
of medullary hyperplastic nodule formation (males and females), a decreased incidence
of pheochromocytoma formation (males), an increased incidence of cortical necrosis
and hemorrhage (females), and an increased incidence of adrenal hematocyst formation
(males). A statistical increase in the incidence of adrenal cortical adenomas noted for
males given 0.1 ftg/kg/day may have been the result of normal biological variation of
the incidence of this tumor, which does occur spontaneously in this strain of rat
(approximate 10% incidence in the control group of female rats used in this study).
The pancreas of male rats given 0.1 ft g/kg/day had a statistical decrease in the
incidence of acinar adenoma formation. A statistically increased incidence of fibrosis of
atrophic pancreatic tissue noted in the group of females given 0.1 ft g/kg/day may or
may not have been associated with the increased incidence of periarteritis noted in this
group. The thyroid glands of the high dose group of male rats appeared to have a low
incidence of various follicular changes that may or may not have been related to
treatment: this included isolated cases of follicular cyst or microcyst formation,
follicular adenoma, or follicular adenocarcinoma formation. The parathyroid gland was
unaffected by treatment, except for a decrease in secondary parathyroid hyperplasia as
a result of the decrease in severity of chronic renal disease of the males given 0.1
//g/kg/day.
Gastrointestinal system. A wide variety of degenerative or inflammatory lesions
occurred in all control and treated groups, with no indications of a direct treatmentrelated effect in the salivary glands, esophagus, stomach, small intestine, or large
intestine. However, the group of male rats given 0.1 //g/kg/day had a statistical increase
above background incidence of stratified squamous cell carcinomas of the tongue,
which were considered to be probably related to treatment. There was also a statistically
significant increase in the incidence of squamous ceil carcinomas of the hard
palate/nasal turbinate region of male and female rats given 0.1 /tg/kg/day. Historically,
squamous cell carcinomas of the tongue and hard palate/turbinates have occurred at a
spontaneous incidence rate of 1 to 3 % in this strain of rat It appears as if treatment
with 0.1 //g/kg/day increased the incidence of this type of neoplasm. A secondary effect
of treatment was noted in the stomach only of the high dose group of males, in which
there was a decrease in the incidence rate of mineralization of the gastric muscularis and
mucosa. This was secondary to the decreased incidence and severity of chronic renal
disease and uremia in this high dose group of male rats.
Nervous system. The only observation considered as probably related to treatment
was the increased incidence of focal hemorrhage in the brain (and possibly spinal cord)
of female rats given 0.1 //g/kg/day. This was described previously in the description of
the cardiovascular system. All groups of rats had the expected spectrum of
degenerative, inflammatory, and proliferative lesions considered spontaneous in origin.

/

5896
0001390

CHRONIC TOXICITY OF TCDD IN RATS

301

Urinary system. There appeared to be a decrease in the severity of the chronic
nephropathy affecting the kidneys of the male rats given 0.1 /ig/kg/day. Other
degenerative, inflammatory, and proliferative changes occurred in the kidneys or
urogenital tract of control or treated groups, with no observations considered related to
treatment.
Musculoskeletal system, eye. and miscellaneous tissues. Various degenerative,
inflammatory, or proliferative lesions occurred in a scattered pattern in all treated and
control groups, with no indication of a treatment-related effect. No toxicologic
significance was attached to the statistical decrease in the incidence rate of
subcutaneous benign tumors noted in males given 0.001 ig/kg/day.

rw tO
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ow

-Î w

Organ Weights
Statistically significant differences in terminal organ weights considered related to
treatment included (1) an increase in liver weight calculated on an absolute basis (males
given 0.1 or 0.01 //g/'kg/day, females given 0.1 ig/kg/day) and on a relative basis of
liver/body ratio (females given 0.1 or 0.01 /ig/kg/day) and (2) a decrease in the absolute
weight of the thymus of females given 0.1 ¿/g/kg/day. Additional changes in organ
weights were considered to be secondary to decreased body weights due to treatment
with 0.1 jUg/kg/day.
TCDD Content o f Tissues
Results of analysis of samples of fat and livercollected at terminal necropsy of female
rats after 2 years of treatment indicated that rats given 0.1 //g/kg/day had an average
T C D D content of 8100 ppt in the fat and 24,000 ppt in the liver. Rats given 0.01
/ig/kg/day had an average T C D D content of 1700 ppt in the fat and 5100 ppt in the
liver. Rats given 0.001 ig/kg/day had an average of 540 ppt of T C D D in the fat and
also in the liver.
DISCUSSION

The findings of this chronic toxicity study on T C D D in rats are an extension of the
studies of shorter duration reported previously from this laboratory (Kociba et al.,
1976). Continuous ingestion of diets containing approximately 2200 ppt of T C D D (0.1
fig of TCDD/kg/day) for 2 years caused multiple toxicologic effects, including
increased mortality, decreased body weight gain, slight depression of certain
hematologic parameters, increased urinary excretion of porphyrins and ¿-ALA,
increased serum activities of AP, GGT, and SGPT, and morphological changes
primarily of the hepatic, lymphoid, respiratory, and vascular tissues of the body. This
high dose level of 0.1 ig/kg/day also caused an increase in the incidence of hepato­
cellular carcinomas of the liver (females only) and squamous cell carcinomas of the
lung, hard palate/nasal turbinates, or tongue. The occurrence of numerous age-related
lesions usually encountered in this strain of rat, including tumors of the pituitary, uterus,
mammary gland, pancreas, and adrenal gland was reduced at the high dose level. Also
reduced was the incidence and severity of chronic renal disease in the aged male rats.
Female rats given this high dose level for 2 years had 24,000 ppt present in the liver.
This compares with 34,600 ppt of T C D D present in the liver of female rats given this

5897
0001991

DOW 275304

302

KOCIBA ETAL.

same dose level for 13 weeks (Kociba et al., 1976) and indicates steady state concen­
trations were achieved during the early phase of this 2-year study.
Ingestion of the intermediate dose level of 0.01 //g/kg/day (-210 ppt in the diet)
caused a lesser degree of toxicity. The primary effects noted at this dose level included
(1) increased urinary excretion of porphyrins (females), (2) liver toxicity, including an
increased incidence of hepatocellular nodules, and (3) increased incidence of focal
alveolar hyperplasia in the lungs. Terminal liver and fat content of TCDD averaged
5100, and 1700 ppt, respectively. This compares with 3700 ppt present in the liver
after 13 weeks of treatment with this dose level (Kociba et al., 1976).
Lifetime ingestion of 0.001 //g/kg/day (-22 ppt in the diet) caused no effects
considered to be of any toxicological significance. Light microscopy of livers from
females of this group indicated a statistical increase above the background incidence of
swollen hepatocytes; conversely, the livers of the males of the group had a decreased
incidence of this observation. When liver tissue was examined using electron
microscopy, the hepatocytes from the females were within the limits of variation seen in
the controls with an occasional hepatocyte containing increased lipid droplets. The
liver and fat each contained 540 ppt of TCDD at the end of the lifetime ingestion of
0.001 //g/kg/day.
If the results of this lifetime study in rats are compared to the preliminary results of
the study in rats by Van Miller et al. (1977), it will be noted that both studies report
neoplastic responses in the lung and liver of rats maintained for extended periods of
time on high doses of TCDD. In the preliminary report by Van Miller et al. (1977),
5000 ppt produced both liver and lung neoplasms, while in this study, 2200 ppt
produced both liver and lung neoplasms.
Thus, there is agreement between the results obtained in both studies at higher dose
levels of 2200 to 5000 ppt of TCDD in the diet. However, at lower dose levels, there are
differences in the two studies, with Van Miller et al. (1977) reporting a diverse spectrum
of neoplasms in rats given as low as 5 ppt of TCDD, based on a zero incidence of
neoplasms in a total of 50 control rats examined in their study. Conversely, in this
study, there was no carcinogenic response in rats given 210 or 22 ppt of TCDD for 2
years. DiGiovanni et al. (1977) reported TCDD to be only a weak tumor initiator in
studies of mouse skin carcinogenesis with DMBA.
In summary, data collected in the study reported herein indicate that doses sufficient
to induce severe toxicity increased the incidence of some types of neoplasms in rats,
while reducing the incidence of other types. No increase in neoplasms occurred in rats
receiving sufficient TCDD during the 2-year study to induce slight or no manifestations
of toxicity.
REFERENCES
A llen, J. R., Barsottl D. A.. Van Miller, J. P., A brahamson, L. J., and Lauch , J. J.
(1977). Morphological change in monkeys consuming a diet containing five hundred parts per
trillion of 2,3,7,8 tetrachlorodibenzo-p-dioxin. Food Cosmet. Toxicol. 15,401-410.
D iG iovannl J., Viaje, A.. Berry, D. L-. Slaga, T. J., and J uchau, M. R. (1977). Tumor
initiating ability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and Arochlor 1254 in the
two-stage system of mouse skin carcinogenesis. Bull. Environ. Contam. Toxicol. 18, 552557.

5898

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CHRONIC TOXICITY OF TCDD IN RATS

303

275303

Innes. J. R. M.. Ulland. B. M.. Valerio. M. G.. P etrlcelli. L.. F ishbein. L.. H art. E. R..
Pallotta. A. J.. Bates. R. R.. F alk. H. L.. G art. J. J.. K lein. M.. Mitchell. I., and
Peters. J. (1969). Bioassay of pesticides and industrial chemicals for tumorigenicity in mice:
A preliminary note. J. Sat. Cancer Inst. 4Z 1101-1114.
Kociba. R. J.. Keeler. P. A.. Park. C. N.. and G ehring. P. J. (1976). 2.3.7.8-Tetrachlorodibenzo-p-dioxin (TCCD): Results of a 13-week oral toxicity study in rats. Toxicol. Appl.
Pharmacol. 35. 553-574.
McC onnell. E. E.. Moore. J. A., and D algard. D. W. (1978). Toxicity of 2,3,7.8-tetrachlorodibenzo-p-dioxin in rhesus monkeys (Macaca mulatto) following a single oral dose.
Toxicol. Appl. Pharmacol. 43. 175-187.,
S iegel. (1956). Son-parametric Statistics for the Behavioral Sciences. McGraw-Hill. New
York.
Steel, R. G.. and T orrie, H. H. (1960). Principles and Procedures of Statistics. McGrawHill. New York.
Van Miller. J. P., and Allen, J. R. (1977). Chronic toxicity of 2.3,7.8-tetrachlorodibenzop-dioxin in rats. Fed. Proc. Fed. Amer. Soc. Exp. Biol. 36, 396.
Van Miller. J. P.. L aL ich. J. J., and A llen, J. R.. (1977). Increased incidence of neoplasms
in rats exposed to low levels of 2.3.7.8-tetrachlorodibenzo-o-dioxin. Chemosphere 9. 537-544.

)

5893

0001993

5900

t r C â é f

! -

(/< r d )

^

—ï»art I
. During

t h e y e a r s .f r o m . 1 9 6 5 t h r o u g h 1 9 6 9 ,

a chemical plant

producing herbicides - pentachlorophenol and 2 , 4 ,5-trichlorophenoxyacetic acid derivatives
chlorina.

- reported a mass outbreak of acne

The number of persons

l a rg es t, e v e r r e c o r d e d .

stricken,

Furthermore,

78 ,

is o n e o f t h e

s o m e o f o u r p a t i e n t s were,

also stricken by a complete intoxication manifested by liver
lesions,

b y a . d e f i c i e n c y in porphyrin,

a n d b y ne rv ou s an d ph ys ic al changes.
ca ti on w a s fatal.

fat and prot ei n me t a b o l i s m
In t w o cases,

the intoxi­

This part of the research wi ll deal primarily

wi th skin symptoms and the internal and neurological changes will
be discussed

i n a- s u b s e q u e n t w o r k

another work

[32].

(23]

and in more detail in

The acne forming dermatoses are caused in mo st casés by
cr ud e o i l d e ri va ti ve s - known as acne oleosa - a n d le ss of te n b y
tar and its derivatives - in these cases
c o nt ra st to th o s e acnes,

it is a c n e picea.

In

acne chlorina is relatively very rare

b e c a u s e i t is o n l y c a u s e d b y a s m al l n u m b e r o f c e r t a i n c h l o r i n a t e d
h y d r o c a r b o n s w h i c h are p r o d u c e d and u s e d on ly o n a l i m i t e d scale.
Acne chlorina also differs from other acne forming dermatoses by
its

localization,

cl in ic al p i c t u r e an d course.

M a s s occurrences o f acne chlorina have be en re co rd ed during
s e v e r a l t i m e p e r i o d s w h i c h a r e s e p a r a t e d b y d e c a d e s , .in a c c o r d
wi th industrial and chemical development and w i t h the gradual
i n t r o d u c t i o n i n t o p r o d u c t i o n o f new- c h l o r i n a t e d - h y d r o c a r b o n s .
The first cases were described by Herxheimer

[18]

in Î899 in

t h e e l e c t r o l y t i c p r o d u c t i o n o f s o d i u m c h l o r i d e a n d s o d i u m hydroxidf r o m brine..

Herxheimer assumed that the illness was caused by the

i n h a l a t i o n o f c h l o r i n e a n d b y i t s e x c r e t i o n t h r o u g h m e m b r a n e s and.
h e therefore n a m e d th e disease acne chlorina;
The. F r e n c h a u t h o r s T h i b i e r g e

[4 3, 44 ]

i.e.,

and Hallopeau

chloracne.
[16] w e r e of.

th e same o p i n i o n as to the incidence of this disease.
more,

Further­

Ha ll op ea u thought that the black coloration of the comedones

.5901
0011890

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— —

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w a s c a u s e d b y s o m e l e a d c o m p o u n d t h a t f o r m e d on- "the c h a m b e r 4 s
lead w a l l s as a r e s u l t of a r e a c t i o n w i t h ch l o r i n e .

Renon

[35]

a l s o c o n n e c t e d c h l o r a c n e -with l u n g

tuberculosis in one of hi s

patients and ascribed both diseases

to gaseous chlorine.

However,

Hirsch

[20]

concluded in that same ye ar that the

cause m u s t be a solid substance w h i c h formed o n the anodes d u r i n g
electrolysis because exposure to chlorine gas alone did n o t ca us e .
the disease.

This finding was supported by Bettmann

[2] w h o o b ­

s e r v e d 2 1 pa tients, w h o w e r e e n g a g e d i n t h e p r o d u c t i o n o f h y d r o - ch lo ri c acid an d who w e r e in c o n t a c t - w i t h c h l o r i n a t e d t a r c o m ­
p o u n d s b u t n o t w i t h c h l o r i n e gas.
Lehmann

[26],

in 1903,

concluded that the actual causes of the

d i s e a s e w e r e c h l o r i n a t e d p r o d u c t s s u c h as h e x a c h l o r o b e n z e n e , p e n t a chlorobenzoic acid and hexachloroethane which
bon anode during electrolysis.

formed on the ca r-

Because similar cases of ac ne

-

chlorina were also observed in the production and processing of pnitrochlorobenzene,

W. L e h m a n n

[27]

concluded that acne ch lorina

was caused primarily by chlorobenzenes.
we re replaced b y magnetite anodes,

As s o o n as c a r b o n a n o d e s

the incidence of acne d u ri ng

-

electrolysis ceased.
B e f o r e W o r l d W a r I, W a h l e

[46]

observed acne chlorina in two

chemists which was probably caused b y a chlorine co mp ou nd - phenylei
dioxide - not identified or known in m o re detail at that time.
T h e n e x t w a v e o f t h e d i s e a s e 's i n c i d e n c e o c c u r r e d a t t h e e n d
of World War Z during the production of chlorinated naphthalenes
or perchloronaphthalenes.

In this p e r i o d th e d i s e a s e w a s g i v e n a n

additional new name - Peraakrankheit

(Wauer

[421).

naphthalenes and diphenyls with a wa xy consistency,
halowaxes,
ness,

Chlorinated
also kn o w n as

have some very valuable properties such as wate rp ro of­

flame resistance and acid resistance.

These properties

earmarked these materials for the production of pr ot ec ti ve gas
masks and other military equipment.
A n e w in cr ea se in th e i n c i d e n c e o f t h e d i s e a s e o c c u r r e d

in

the ni neteen-twenties d u r i n g the us e of c h l o r i n a t e d n a p h t h a l e n e s

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as an insulating'material, in the m i n i n g i n d u s t r y

{Teleicy {423 ) -

A c n e c h l o r i n a a l s o a p p e a r e d in t h e e l e c t r o l y t i c p r o d u c t i o n o f
c h lo ri ne w h en in su la ti ng coat in gs of tar w e r e used.

■

This fact

brought about numerous disagreements and qu es ti on s as to w h e t h e r

•

*

thè disease, in t h o s e p r o d u c t i o n p r o c e s s e s w a s n o t a c t u a l l y a c n e
picea.

On the other hand,

Teleky supported the contention that

'

the tar acne could be caused by chlorinated hydrocarbons c o n t a m ­
i n a t i n g t h e t a r.
The excellent dielectric properties of the chlorinated
naphthalenes

("haftax", " h a l o w a x " , "nibrene")

led to their w i d e

utilization in the production of cables and condensers and ¿ m o t h e r
mass occurrence of the disease was produced in the ni ne te en -t hi rti e
w h e n th e U.S.A.
individuals.

and other countries recorded hundreds of s t ri ck en

Boltzmann

among the family members
condensers.

[22]

also noted the occurrence of a c n e

-

o f w o r k e r s e m p l o y e d i n t h e p r o d u c t i o n of.

These family members handled and washed work clothes'

contaminated with th e chlorinated naphthalenes.
observations,

Based on these

B o l t z m a n n c o r r e c t e d his p r e v i o u s original' c o n c l u s i o n s

and stated that the chloracne was actually tar acne and ju dg ed
the effe ct of halowaxes to be that of an external acnegenic
Ozmsby

[31]

cause. .

observed acne chlorina with the use of a fungicide

u s e d i n t h e i m p r e g n a t i o n o f lumber* (sodium t e t r a c h l o r o - c - p h e n y l phenolate).
others

Jones and Alden

[24],

Schwartz

[39]

and later m a n y

also observed total disorders in their patients and F l i n n

and Jarvik

[8] n o t e d a d e a t h d u e t o y e l l o w l i v e r a t r o p h y .

Mayers and his coworkers

[28]

In

1938,

were unsuccessful in clarifying the

m e c h a n i s m o f o c c u r r e n c e o f d e r m a t i t i d e s and- a c n e c h l o r i n a i n

their

patients with the aid of epicutaneous

nega­

tests - the tests w e r e

.

tive. •
D u r i n g W o r l d W a r ZI,

chlorinated naphthalenes were used

for

i m p r e g n a t i o n of ships'

hulls when it was discovered th at

these

substances could protect ships from damage of m a g n e t i c mine s.

The

the

use of halowaxes
cal-industry.

increased significantly,

especially in-the

el e c t i . -

Am e r i c a n authors gave acne c h l o r i n a an ot he r name,'

0 0 l i 8 9 ^ 903

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"cable rash",
patients

because of

the o c c u p a t i o n

(Good a n d c o w o r k e r s

(cable p r o d u c t i o n )

(14], S c h w a r t z -140]., .Morris

of the

(29]).

At

t h a t t i m e , m a s s i v e o u t b r e a k s o f a c n e a n d s e ve ra l' d e a t h s w e r e .
a
recorded.
After World War'll, the incidence of acne did no t sub­

side because

t h e un d c s i r a b - l e e x p e r i e n c e s w i t h t h e c h l o r i n a t e d

n a p h t h a l e n e s did not p r e v e n t th e i r us e in thé e l e c t r i c a l .industry
f o r s o m e time.
[19]

in 1947.

An interesting observation was recorded b y Hèrzberg
He observed seven cases of acne chlorina in patients

who had ingested chlorinated paraffins in the form of a food
shortening.

T h i s p r o m p t e d H e r z b e r g to. c o n c l u d e t h a t a c n e c h l o r i n a -

mu st occur after the

i n t e r n a l i n g e s t i o n o f c h l o r i n a t e d -h y d r o ­

carbons.

Fuchs

Similarly,

[9]

al so no t e d the af fl ic ti on .o f t h r e e

children and their mothers after they had eaten potatoes fried in
the "paraffin".

Winkler

[48]

r e c o r d e d 10 cases o f d e r m a t i t i s a n d

1 3 c a s e s o f a c n e c h l o r i n a a m o n g 2 3 w o r k e r s i n a c o n d e n s e r factory.,
t h a t u s e d n i b r e n e i n .t h e p r o d u c t i o n s e q u e n c e .

From these cases

Winkler concluded that "chlorinated hydrocarbons cause t h e forma­
tion of reagents that in turn cause an increased secretion f r o m
lubricating glands".

In 1949 G a v r i l o v a

[10} w a r n e d o f t h e p h o t o - ;

s e n s i t i z a t i o n e f f e c t o f p o l y c h l o r o n a p h t h a l e n e s .'

In re ce nt years,

p l a s t i c s h a v e r e p l a c e d c h l o r i n a t e d n a p h t h a l e n è s i n industrial, u s e .
Even today the production of sodium hydroxide m a y be a source
of acne chlorina.
1965.

W e ha v e ob se rv ed m o r e cases be t w e e n 1950 a n d

The ma in causes of acne chlorina in these cases ha ve been

th e combination of chlorine gas w i th the hydrocarbons in the tar
gaskets and fittings on the electrolytic containers

and tubs and .

w i t h the m i ne ra l oils wh ic h w e r e u s e d t o protect t h e e l ec tr od e
contacts.

T h e a u t h o r s n o t e d s e v e r a l c a s e s o f a c n e 'c h l o r i n a a m o n g

l a b o r a t o r y w o r k e r s a f t e r t h e u s e o f n i b r e n e i n 't h e e l e c t r i c a l
in dustry in th e 1950's.
Furthermore,

the acnegenic effects of the m i n e r a l oil ba se d

coolant mixtures used in metal working increase w h e n chlorinated
hy dr oc ar bo ns ar e added.

Therefore*

.

some cases o f acne oleo sa w e r e

observed which exhibited a partial acne chlorina character

(more

0011803

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distinct follicular hyperkeratosis, less inflammation, cyst forma­
tion and atypical localization).
The last wave of the disease's occurrence was recorded in the
1950's. This outbreak was noted among persons engaged in. the
production of chlorinated phenol type insecticides and herbicides.
From 1950 through’ 1951, Baader and Baüer [1] and. Brinkiaann 171'
published information on the occurrence of acne chlorina which
involved the complete internal,, nervous and psychic symptoms among
17 workers engaged in the production of .pentachlorophenol.; Several
years iater, Hergt [17],. Oettel [30] and Hoffmann [21] observed a
total of .80 afflicted workers who were employed in the production
of 2,4,5-trichlorophenol by the alkaline hydrolysis of
1,2,4,5-tetrachlorobenzene. From 1954 through 1956, Kiramig and
Schultz [25] recorded 31 cases of acne chlorina in one Hamburg
(Germany) plant that produced 2,4,5-trichlorophenoxyacetic acid .
and its esters. By very detailed research and tests, these authors
have shown that’acne chlorina and total intoxication with damage tc
the internal organs, mainly to the liver, with psychovegetative dis
orders were not caused by the basic chlorinated hydrocarbons, i.e.,
tetrachlorobenzene, trichlorophenol and trichlorophenoxyacetic• •
acid, but rather were caused by a ballast substance concentrated
in these compounds in small quantities, namely, 2,3,6,7-tetrachlorodibenzodioxine. These authors contributed significantly
with their other experimental works to the question of what sub­
stances cause ache chlorina.and how these substances act.
.In 1964, Bleiberg and coworkers [3] observed 29;cases of acne
chlorina among persons engaged in the production of 2,4,5-tri- .*
chlorophenol and 2,4-dichlorophenol. In eleven of these cases
they also diagnosed symptoms of porphyria cutanea .tarda. In 1957
Bowen and coworkers [4] observed acne.chlorina among workers em­
ployed in the production of DDT from trichlorobenzene and hexa—
chlorobenzene. The fates.of 53 patients Who in 1953 had worked
.’
In thé alkaline hydrolysis of tetrachlorobenzene. to trichlorophenol
verc described only last year by Goldmann [11]. In addition to
acno, Goldmann also diagnosed in his patients bronchitides and' O -

0011894
'5305
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disorders of the liver, kidney, myocardium and nervous system.
The author described the death of a patient -from “necrosis of -the •
pancreas as a special case. Furthermore, he also observed .
dermatitis in a nurse who had had contact only with experimental
animals. Finally, he diagnosed acne chlorina in the fourteen
year old son of an employee whose disease was contracted only
from the household environment - by wearing his father's contaminated scarf and by usingr his father's towel. .
Actual observations

The process called-PCP (pentachlorophenol) which caused
mass damage in Czechoslovakia was, as far as production is con­
cerned, identical to the process from which Kimmig and Schulz
drew their patients for observation. The final products of this
process were sodium pentachlorophenolate, 2,4,5-tricholorphenoxyacetic acid and the latter's sodium salt and butyl ester. The
primary raw material was technical grade trichlorobenzene which
was produced in a neighboring building for.the production of
hexachlorocyclohexane (HCH) and lindane. .The table shows the
entire production scheme (Table I). The chlorination of tri­
chlorobenzene produced tetrachlorobenzene and hexachlorobenzene.
Sodium pentachlorophenolate was formed by the alkaline hydrolysis
of hexachlorobenzene. Sodium trichlorophenolate was produced by
the alkaline hydrolysis of tetrachlorobenzene. The hydrolysis
of tetrachlorobenzene with sodium hydroxide in the presence of .
methanol took, place in an autoclave at 190°C and at 45 atm of
pressure for one hour. After cooling, the methanol was distilled
from the hydrolysate, the hydrolysate was diluted with water to
a 25% concentration and it was then syphoned into a storage tank.
The. condensation of sodium trichlorophenolate with monochlo'roacetic
acid produced sodium trichlorophenoxyacetate. Following cooling,
I
centrifugation and flushing with water, the dried sodium trichlorophcnoxyacetate was picked out by hand and placed in small barrels.
O .
Some of this product was used to produce the butyl ester ofo
• ro .
03.

Cff

5906

P a ^cf

trichlorophenoxyacetic acid.

This production toôk place in .

another building.
The production of both the primary products took place in a
four story building with grated floors (WEMA grates) which was
one reason why the entire_building was contaminated with the high
risk substances.. The building was not sufficiently airtight, the
local exhausts were in most casés improperly installed and .
hermetization and mechanization'were insufficient.' Initially, the
sodium tetrachlorophenolate [sic] was escaping .from the centri­
fuge into the work space in the form, of a mist. A series of
tasks such as the removal of solid substances and the pumping of
liquids were performed by hand. The workers were dressed in
linen work clothes. The thorough cleaning and frequent changing
of the work clothing was impossible to guarantee over the.entire
interval of production. Rubber gloves and respirators were used
at some work sites. The conditions surrounding the production of
the butyl ester of trichlorophenoxyacetic acid were the same as
those described above.
Kimmig and Schulz [25] found that the main source of acnegenic hydrocarbons was that segment of the production process in
-which sodium trichiorophenolate is formed by the alkaline hydro- '
lysis of tetrachlorobenzene in the presence of methanol. A co­
worker of. these authors. Doctor (of Chemistry) Sorge, had de­
duced on theoretical grounds that if this reaction were conducted
at 190 *C and 45 atm of pressure, chlorinated hydrocarbon by-pro­
ducts such as polycblorodibenzo-p-dioxines, polychlorodibenzo-;
furans, polychlorodiphenyl ethers and polychlorodiphenÿl oxides
may be formed in small amounts (Schulz [36]). Sorge also pre­
pared some of these substances by his synthetic process.
Animal tests carried out by the authors

(Schulz a n d Kimmig).

with tetrachlorodibenzofuran and 2,3,6,7-tetrachlorodibenzodioxine

I

. .

ha ve p r o v e d an ex tr ao rd in ar ily h i g h to xi ci ty f o r t h e s e substances.
A si m p l e , s m e a r o f a 0 . 1 %

solution of tri- and te tr ac hl or od ibe nz o­

.5 9 0 ' 7

DOM2I6059

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A j

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damage and death of the test animal within 2 to 3 weeks {Schulz
(36]).. 2,3,6,7-Tetrachlorodibenzodioxine, which also has very
strong acnegenic effects, was proved to be far more toxic. Only
then was Dr. Sorge successful in the identification of this
enormously toxic substance-ih the production process and in the
final product. He proved that this toxic substance is formed only
at certain temperatures and pressures. If during the alkaline
hydrolysis of tetrachlorobenzene to sodium trichlorophenolate
the temperature is reduced below 153*C, the toxic substances do .
not form. However, at this low temperature, the reaction time
is extended from one to five hours and the productivity is ob­
viously lower. Furthermore, it was proved that the condensation
of two molecules of trichlorophenolate to give tetrachlorodibenzodioxine is an exothermic reaction. Several explosions of auto­
claves with injuries and the lethal intoxication of workers were
reported to be the result of this reaction in the German Demo­
cratic Republic (East Germany) (Schul2 [38], Goldmann [11,12]).
.Tetrachlorodibenzodioxine was also identified in the Czechoslo­
vakian production process and in the products as well.
Thé first two cases of acne chlorinà were noted in 1965 in
two technicians who operated the production machinery. It was as­
sumed at that time that these tw o cases were caused by careless
work and an undeveloped production technology under semiproduction
conditions. After one year without a reported case and during
which production was temporarily halted, a mass outbreak of 78
cases was recorded within the following three years. Finally,
after a detailed investigation and study of the entire problem,
the process was terminated and production ceased in 1968. Workers
from all stages of production were stricken. At that time, it
was impossible to determine which production stage was the most
risky because most of the workers were involved in several stages
at various locations in the plant within a relatively short time.
Workers directly involved in production as well- as maintenance per*
sonne1 and workers digging sewage ditches were stricken. Later
a research scientist working to isolate the acnegenic ballast

substances, from the products of the individual production phases
was even stricken, ilis research work vas carried out outside the
production plant in a research institute's laboratory. In addiz/
■
4•
tion to the 78 patients with dangerous'symptoms of acne ;chlor ina.
or porphyria, other workers in this plant had symptoms of acne
which were, however, symptoms of juvenile acne or of a similar
character and could not therefore be counted as an-occupational
disease.
With the exception of two female laboratory technicians> the
patients consisted of males from 18 to 57 years of age (4 persons
below 20, 46 persons from 21 to 30, 11 persons from 31' to 40; .
10 persons from 41-50 -and 7 persons from 51 to 57). Therefore, .
the acne chlorina patients were mostly young people. On the other
hand, more than half the patients with porphyria cutanea tarda were older than 40 years.
Generally, the disease followed a normal course as described
by other authors. In only six patients did the disease begin sud- '
denly as acute solar dermatitis, i.e., by erythrema and edema
*
\
.
.
in regions exposed to solar radiation and continued by acne sym-'
ptoms (Photographs 1 and 2), as observed by Goldmann [12], Grimmer
[15], Braun [6] and before these authors, also by Touraine and co­
workers [45]. Therefore, chlorinated phenols in some cases have
similar photodynamic effects as the chloronaphthalenes described
by Gavrilova [10]. Erythema and edema in these patients were
obvious mainly on their auricles. Sometimes, small blisters in
thick groupings were observed there.
In all the other patients the acne developed slowly. Among
the less severe cases the disease was limited to single or densely •
grouped comedones, conditoned by follicular hyperkeratosis, which
were so small and sometimes so numerous that they appeared to be.
a slate grayish-brown color, like dirty spots on the skin. These
symptoms were noted mainly on the face, with max ilium occurrence
above the cheek bones (Photograph 3). In the more severe cases; .
the comedones gradually developed into small white cysts similar

rs>

5909 «

to nilia, although with -black centers, and into large cysts
ranging from the size of a pea to that of a nut (Photographs 4,

S and 6). As opposed to other acne forming dermatoses, the
described symptoms and manifestations mainly lacked signs of an
infection. Only in the more severe cases and rather in the vici­
nity of large cysts were some reactive inflammations, infections .
and abscesses noted. The emptied cysts and abscesses left behind '
large atrophied scars with raised edges and with bridges of. intact
skin under which a probe could be inserted (Photograph 7). This
basic clinical picture was supplemented by diffused hyperpig­
mentation in the face and by papulopustules on the torso and ex­
tremities. Patients who were found to suffer from a disorder in porphyrin metabolism .had' symptoms of porphyria cutanea tarda in
regions exposed to the sun (hyperpigmentation and hypertrichosis,-'
blisters,’ excoriation and small scars). However, in this group
there were two patients with prophyria but almost without any
acne at all. ■
,
.
-,
* -.
Histological investigation is not much, help in diagnosis..
The symptoms begin with hyperkeratosis at the mouth, of the follicle
After one to three weeks, the follicle changes into a cystic for­
mation filled with a h o m y substance ‘[keratin?] (Photograph 11)
and to a small extent with sebum. However, the epidermal changes
are small. After the rupture of the full follicle, the contents
of the cyst penetrate into the surrounding tissue and a granuloma
of foreign bodies forms around the area (Photograph 12)..
The disease normally starts on the face above the cheek bones.
However,- in 17 patients the disease started in an unconventional
manner by papulopustùlar eruptions on the extremities, primarily
on the lower extremities. This characteristic, resembling.more ’
acne oleosa, remained in some of the patients for the entire dura­
tion of their illness.
The most common localizations of acne chlorina were.the cheeks
temples and'to a lesser extent, the entire face." Eyebrows, eye­
lids, the perioral region and the scalp and hair area remained
r*o
cn

•5910 cn
0011897

POW2 I6068«
Page 12

~

‘

Other predilected areas vere the auricles, thè back, of the neck,
the back and the chest in .a seborrheic localization and the
genitals. The auricles and genitals and especially the scrotum
were stricken by the formation of large cysts (Photographs 8 and 9)
Among the five most seriously ill patients, the disease spread .
all. over the body and had a discouraging.effect that was heightened
•by the. disease's usual persistence, the long term healing and by
medical helplessness (Photographs 10 and 7).‘ The.contents of the
cysts produced a repulsive and rancid odor. For some of the
•patients the disease disrupted their social and family relations
and caused severe depressions.•
Some authors, such as Braun [6], emphasize itching as the
initial symptom, but we noted this symptom in only three cases.
In 20 patients with extensive symptoms,, the occurrence of
pustules, cysts and abscesses was also noted in axillae although
this location was not mentioned by other authors. Because of the.
great number of follicles in the axillae and the similarity of
the axillary skin to that of the genitals and for several other
reasons, this finding appears to be just as valid as localization
on the genitals and the genital area. On the other hand, it is
surprising that acne chlorina does not strike at the hairline or
scalp. In the case of one patient, the ache symptoms disappeared
in the facial areas when he grew a full beard while in other areas,
the symptoms continued without a reduction in intensity. So far.
there is.no explanation for this phenomenon.
Hypertrichosis or hyperpigmentation or both were, determined
in the faces of 19 patients without a laboratory diagnosis of a
deficiency in porphyrin metabolism. Bleiberg [3] made similar ob­
servations. These symptoms receded simultaneously with the healinc
of acne chlorina.
■M bm younger pogoone-ouffared from aeae e^lerina-than"eldeg
persene.* This study group contained SO persons (85%) below 30
•Traitela begTa. «ate i Beginning-od-this-sentance illegible—
♦Many authors believe that younger, persons, have a greater tendency to
develop acne .chlorina than older individuals. ' (Correction by NIH Translation'Unii

I

Page 13

years of age. However, this fact cannot be used as a hard and
fast rule or to support the findings because the production plant
under study employed mostly young workers. The preillness medi­
cal history among the patients did not indicate any significant
predisposing factors and no incidence of serious illnesses, intoxi­
cation or etiolation*. Furthermore, no relation to hair color, eye or complexion color, to the degree of.body hair, to the per­
spiration of skin oils or to hereditary follicular hyperkeratosis ■*
and other skin diseases was noted. Only one patient reported a
more severe prior case of acne juvenilis, 12 patients reported mild
cases during puberty and 14 patients reported only insignificant
acne juvenilis symptoms. 18 patients also had some infrequent Con­
tact with mineral oils either during the appearance of the symptoms
or at some time in the past. ' These patients were mostly involved
in maintenance work as mechanics. However none of them had ever
had an oil acne in the past. Their illnesses following 'contact
with tetrachlorodibenzodioxine were of the same character as the
illnesses of the other patients. Their diseases were not similar .
to acne oleosa either in appearance or in localization.
The latent period from the initial contact with the risk
substance to the appearnace of symptoms was quite varied.. Same
workers became ill after exposure of from several months to several
years and the disease in their cases.was rather mild although the
indications pointed to a massive exposure. In other cases, severe
damage occurred after a very short time of several weeks and after
an apparently small exposure. For example, one of the most serious:
ill patients worked under the semiproduction conditions for only,
two and a half weeks before the outbreak of the disease (acne and
porphyria cutanea tarda}. After that he had no contact with the
risk materials.
It is not clear whether individual predispbsition or the level
of personal and work hygiene is more responsible for the intensity
*Sdàtse»a note:— This may be a dubious sheiso»■ The Caoch "atylismu
fcgensliterates to »eteiliam^r
.
_
♦alcoholism (Correction by NIH Translation Unit) :
O .

Page 14
i

;

■5913
0011902

09IZHOO

of'damage or latency of the disease. Most authors discount
individual predispositon. However it is believed that the
deciding factor must be the degree of actual exposure which is '
undoubtedly dependent on a series of other factors such as work
discipline, personal discipline, adherence to preventive measures,
frequency of changing work clothes and undergarments, the degree
and regularity of hygienic decontamination, work site order and
cleanliness, dust in the environment, etc: •
These patients also had a series of subjective difficulties:
tiredness, weakness in the lower extremities, muscle pains, sleepi­
ness and insomnia, increased perspiration, lack of apetite, head­
aches and other disorders in the mental and sexual spheres. These
complaints were more recurrent and more intense in patients with
the more extensive skin symptoms. In more serious acne cases, a .
significant weight loss was also noted. It is our opinion that all
these difficulties and symptoms are manifestations of a rather
overall intoxication than of only the acne chlorina. .
All the patients were admitted to a dispensary. Regular
examinations of their dermatological, internal and neurological
conditons were arranged. The patients who were still working were
transferred to prevent further contact with any acnegenic factor.
All contact with chlorinated hydrocarbons of any type was prevented
even with those substances whose acengenic effects have not been
thoroughly proved. Furthermore, all contact with, mineral oils and
tar and its products were prevented. The application of mineral
based ointments was discounted for external therapy. The plant's
medical doctors were cautioned against prescription of any internal
medication with an acnegenic effect (iodine and bromium preparation
corticoids and others). The patients were given detailed medical
directions concerning their daily regimen, personal and work hy­
giene and secondary disease prevention and treatment.
All available medications for acne were utilized in the treat­
ment. The expressions of comedones and the pressure relief of
the contents of cysts and abscesses necessitated many periods of

O)

(

y a g e 'x a —
hospitalization and special ambulatory treatment hours attended
by a specially trained nurse. No special results were, observed
as a result of repeated hospitalization. The best results were
noted after the expression of.comedones by the use of a sauna
constructed for the patients by the factory on the author's re­
commendations. It was not possible to test the effects of vitamin
A acid (retinoic acid) which has been praised by some authors in ■.
the medical literature. However, in agreement with the findings
of other authors, it was determined that no local' or internal .•
medication exists which has an effective and permanent healing
effect on acne chlorine. Some of the patients have been under ..
observation for eight years and their disease still persists.
Dr. Schulz's [38] patients have manifested continuous symptoms
for more than ten years. Goldmann [11] has now. been treating one
patient with comedones and cysts on his penis and scrotum for 18 .
years.! In most of the patients discussed by these authors, the .
symptoms and manifestations of the disease progressed, and reached '
a maximum after about one year, even after transfer of the patient
from the high risk work area. . A regression takes place after two
or three years at the earliest. In some-isolated cases, the pro­
gress of a mild course of the disease was observed even after,
five years. However, the regression of the disease in all the
patients was very slow and, it may be said, even lasts a number
of years.
Of the 78 patients treated by the authors, 76 suffered from
acne chlorina. Of these,.11 simultaneously had hepatic lesions
with a deficiency in porphyrin metabolism. •Two patients did not
have acne chlorina. One of these two had porphyria and the second
one died in 2.966 from acute intoxication. Pentachlorophenol was
considered to be the cause of death in that instance. When the
toxicological analysis was carried out at that time, the investigators were unaware of tetrachlorodibenzodioxine and, therefore#. *
it is our contention now that in that case, this highly toxic
substance was primarily responsible for the acute intoxication^
’• ;

ro

5914 , S
0011903

S,

■nagre“-xu

f

Gombos 1131 mentioned one patient with acne chlorina and porphyria
in connection with this production process.

The authors analyzed '55 patients internally and neurologic
cally in detail.' About.half, of these patients had a deficiency in
lipid metabolism and in more than one-third, some minor.biochemi­
cal deviations.and mild hepatic lesions were noted in the first
stages.of the disease. In 17 patients, the authors found symptoms
of central nervous system disorder, the majority with lesions of.
the peripheral neurons of the lower .'extremities' (verified by EMG .
examination). In most of the patients, physical disorders, pri­
marily an acute neurasthenic syndrome, were diagnosed. In addi­
tion to the death due. to acute intoxication (pentachlorophenol?
tetrachlorodiberizodioxine?), three more patients died.' In one
patient, the.porphyria developed'unusually rapidly into an .- ’
arteriosclerosis of the brain with an atypical morphological diag­
nosis and demention. Two patients died from bronchogenic carcinoma
All these patients with their complete symptoms '«ire discussed
in other papers [32,23]. •
Discussion
In agreement with Kixnmig and Schulz, we cite the tetrachlorodibenzodioxine which.is formed during the production process as ..
the main reason for the skin symptoms as well as for all the other
deficiencies mentioned and diagnosed in the authors1- patients.
This toxic compound was confirmed in considerable quantities in.
the final product (Arboricide E50)., in the butyl trichlorophen- oxyacetate and also in the mortar and wall paint of the factory
building. ' Even adjacent rooms such as worker dressing and locker
rooms were contaminated with this compound. Test rabbits placed
into these rooms soon died.
In his monograph, Braun [5] cited the following chlorinated
hydrocarbons as the then known causes of acne chlorina: chloro-. .
benzenes with various degrees, of chlorination, chlorinated phenols
(sodium tetrachlorophenolate, pentachlorophenol), p a ra -nitrochlorobchzene, sodium tetrachloro-o-phenylphenolate, chlorinated .- O

'5915
0011904

»

naphthalenes, various chlorin ated diphenyls,, trichloroxydiphenyl,
chlorinated diphenyl oxides, sodium o-(2-chlorophenyl)phenolate
and othèrs. However, Kimmit and Schulz [25] ascertained that pure
pentachlorophenol and pure 2,4,5-trichlorophenoxyacetic acid
do not have any acnegenic effects. Therefore, they checked the
presumed effects of the other substances that were blamed for
causing acne and found that neither pure nor chlorinated benzenes
'.(from 1 to 8 chlorinated positions) nor chlorinated diphenyl•
•
*
ethers have any acnegenic effects. A definite acnegenic effect
was proved only for the following substances: chlorinated naph­
thalenes (5 and 6 chlorinated positions):in agreement with Shelley
and Klingman [41], higher chlorinated dlbenzofurans (diphenyl oxide:
and 2,3,6,7-tetrachlorodibenzodioxine.
Therefore, it is possible and even probable that some chlor­
inated hydrocarbons that have been previously considered to have
an acnegenic effect do not have this effect and the actual causes .
were other ballast chlorinated hydrocarbons contained in.those
hydrocarbons.

Tetrachlorodiphenylene oxide
(tetrachlorodibenzofuran)

2,3,6,7-tetrachlorodibenzodioxine (tetrachlorodipheny!
dioxide)
In an animal experiment, Kimmig and Schulz found an ex­
ceptionally high toxicity for tetrachlorodibenzodioxine. In a
local application on rabbits* auricles (keratosis test), this comr
pound produced a strong reaction even at a concentration of 0.001%.
Also; a skin application of this substance at the same concentra­
tion produced acne chlorina (Schulz [38]). •Tetrachlorodibenzofuran
was proved to be 10 to 20 times maker. Chlorinated naphthalenes
had a thousand times weaker effect. Generally, acne chlorina is
caused by the direct contact of tetrachlorobenzodioxine with t£3
skin. Absorption through the skin is not ruled out and ah e f f § g p
_

IS )

'• CO-

001190$

t'aytf— xo

through the ingestive or respiration tracts is also conceded.,.
However, in a pure ingestive experiment with a test animal, acne
chlorina did not occur. However, the cases of acne chlorina con­
tracted after the ingestion of chlorinated naphthalenes as food
fat substitutes should not be forgotten (19,9].
Tetrachlorodibenzodioxine used internally has a strong hepatotoxic effect. A dose of 20 to 50 mg per kg of rabbit's body
weight causes death in all the test, animals by yellow liver atrophy.
Because of this enormous toxicity, all the'preventive measures
taken at the high risk processes in East Germany and Czechoslo­
vakia were proved to be completely ineffective. The only de­
pendable solution to that problem was the reduction of the re­
action temperature and pressure during the alkaline hydrolysis
of tetrachlorophenolate to sodium trichlorophenolate. In East
Germany, the decontamination cost of the high risk areas and
buildings was extremely high. One chemical plant attempted to use
for other purposes a contaminated building which* had not been
decontaminated after the building had remained unused for two .
years, but. new cases of acne chlorina were shortly reported.
With normal decontamination procedures new outbreaks of the disease
occurred and therefore, decontamination had to be carried out using
special pressurized chemical protective suits with pressurized
breathing apparatus because gas.masks were not sufficient. Doors,
windows, the wooden trim and other wooden building components
had to be burned; the steel construction materials in the building
had to be sandblasted; the plaster, facade and floors had to be
torn out and cast into cement blocks together with the tubing,
plumbing and other equipment. These blocks were then transported
to the, Atlantic Ocean and sunk. The disposition of those non- ,
flammable materials by burying would have run the danger of con­
taminating the subterranean water supply. Only the most expensive.
components, autoclaves and some valves, were successfully decon­
taminated by repeated washing with special detergents.
The Czechoslovakian plants are still closed and. their fate^a'
still undecided. The patients are still under a continuous o b - O
servetion.
^
001190$

5917

O)

i*

Page 19

.

1.
Surfer, E. VA, Basar. II. I» Industriai
intoiiealMin Una io pcnueblurplicooL lad.
ihul. Sur*. 23. ISj I. ». 233. — 2. BoUiuaun:
Cblcr-Akuc. clan besandere Fera too prò*
Icssiuaellcr ll>«lcrkr<mka>t DiscIl ocd.
• W tch r, 27. 1*131. x 427. — 3. B U ilu rr. ) ,
IC alles, K , B rodaio, I-* In d o tti JaU raciiair*

• ed purpiirr». Ardi. Dora, U , USI. s.
733—797. — 4. Sansa, S. S , Mmimiod, M.
Tu Oliarcene (a llu oanutactoro oi DOT.
. AreS. Ocra». Syph. (Cblcagol, 75. 1SS7. x
743—743. — 5. Brasa, I7d X^tlaratoo. Mo*
, nogupitiM zor Zolische. Oenilsdcraotesca.
t Dead I , Auletidar! L, WUrtt, Lili'loa Cantor;
„1355. ». 73. — 9. Ilrsun, \ i j Kllnlsdie Oso**
"tuchtuatta sur EautsSuos ile? CSlorscae.7
•Vjlautxrri. 10, 1353, i 123- 123.'—'7.*j*tf«fe£
atesa. Od Fcatachlarpbcaol — Verstltaas.
Vortrac. GcwerUaniL Taxi* la lle a a L W.
USO — eie Oauer — 43.— A. JUsa, F. & .'
JanJk. JL Cr Actlaa ai «ertala chlorfnoted t
niptiklniBi sa tbe tirar. Trac Soc. expor. *
S lo t MeiL. 35. 1933,' S. 333-120. — L '
Fucfcx, Tj d i. Orata — 5. — 10. Catrilcr«, V. H sO poioscacze intodcnaatlior ot cSlo*
- «rotunuych aalultao«, vasta.. Cera. V».*
«■"ber.. 1343. 3. 3, x 32. — 12. CaMaasa. %
I f - Sdiworxts aiuta Cblcratoe dardi ZM*
/ dilatpfarnci-Zcnatrsaxrpradakta. Arfcds*
V modula CASA), 7. 1972, X 12—10. — IL*'
^ D sI-Umob, F. J j Schivante okuio Chlor*.
*?«toQ, e Ina MoxioalataxtkaHca dorch 2,-3, *
l'Q, 7*To(recSIordlSeaiedlada. ILiutari,' 2 .\s
^4373.1.-«JllS-JSL- —*13 :'Sm>taVrs:.*‘lW *
: f it t o t i, A*‘ Ifothv.'
Itavi. £i.*.
«.forpiiTtU cutanea tarda « aeso chiorlna "
/a r i virata crkllckfch cltlAroraafdi ulilo* *
? «sdito«. Arac I3Jc. U«tr, SI. 3903. a. 0*0—
Olx — l i . Cuoi, Qu JC, raaafcj’, i l i Itele*
K it a u u (.cablo rash*], Arde *Dora.
Srph. (Chiuso I, 43. 1343, X 2S1-2S7. —
_ 15. Crlnuner. IL: Dcrnfllch bodlusta
Akne dardi dilorlerto aroautlsdia Fobica*
wassontolte. ZhL Arbeiisaicrf, 5, 1955, x.
•'70. — UL llatlepaaa. IL: Acni chleriiitia.
S ce. «ned. 1500. x 201 — eie Brasa S. — .
27. S erie IF- Dlakusslaa. V/crfclrzte-Koo*
(em ù , 8ad Dartonto, 13S5 — eie Baaor
.43. — 13. llonh ciacr, Zs Oher Cbioretoa. *
* Ittach. BctL VAschr, 40,1333, x 273. — 13.
.SZerxharf, J. Js Chlarakae aadt Casus» voa
cUedertua Feritila. Dana. Wxchr, 113,
.,2947. a. 425. — 20. Dirtele n a sUtftraaksax
.la etner Fakxlk sor elcktrolyttschsa CUar*
. en tep ta s. ZBdt. d. 2cairal»lclla L Arbsl*
lcr>U*ahUabmelarlditaBcsa, 7, 1SZ3. x £1
— clL Orsa 5. — 21. tinilassa. IL Tlu
"Vorlr. Werksrslc-Xcsisrcar, Oad Cr-ndidq. ■
3355 — d e Jiaucr £ 3 .-2 2 . UaUxssau. Xs
Qilaraeaa a. FiaaafcraskbsIL Arslu IItf.
117. 1333. 1. x. su ved eas — dC Uraaa S. .
— 23. Jlrdisk, U. Z slu ci:/. ) . Asbix, IC.
. HttliU, Z . I*axJerari. J. al nU Vilduky
tslUatd imasikaca a alino chiodila pii vj*
»ahi hcrbhdd — piip.uvorduo do Usha. —

i. 2 L Jonca, J. VA. Aldui. IL Sa An aconiarm
* dcnuatorcosti. Arch. Dtan. S/ph. (Chini*
. co)« 13. 1233, x. 2L22—liUX — 23. KìbciLC
L Sdisia, K. IL: OeruUldio Akaa (»or. '
Coloratoci durcb chlorlene orooalUdio
zykllidie ÀUier. Dcrsatolostea, 115. 1557,
. x 540—549. — 20. Lohoiasa, X. IL: Experi* .
.menIella SUuilon aber dea ELnlluts tedi- .
. aitch a. fafciealzdi widillscr Cara u. Oao* '
io o si dea Organimi!«. XL Siudlou Otar.
.Chlorakne*. Arch. n r r . 40, 1003. ». 322—
333. — ClL Urani 5 . - 2 7 . Lrbiaaan, W:
.*Otar Chloratoa. Arch. D ora, 77, 1905. ».
205—203. Uditesi 323—344. — 23. Marasx.
7L K . SOrerhers. K. &s Stia condition»
.Temiuse from exposure to certain chiarina* ¿led hydrocarbons. ). ludusir. llyg. Toxicol,'
.20, 1933, x. 244—253. cU. Uraua 5. — 23. .
Harrix, ‘C. L , Zatasshow, L. JL_*- .Cabla •
. rash* — A nolo on s now deenxiar mix*
' lata. J. Amcr. acd . Axx, 121.1913, x. 132—
{ 333. — d L Brami i — 30. DdloL U s JOl-.
r a u dio a . tlcnujialam tcH a Erlshnagaa
•Jialt liochloxlxchca Chlorkohleatsassemol* *
,i lea; ala Bcltras zna Penu-Prohlam. Wetk* AXnio
Koalernax, Bed DarMiclm,19S5,
ctt.*.
• ......................•
. ,
V
. .
■_!
.Baaor 40.'*—* S t,- Oimihy,' Oj^DUkussf y ,.
p oxointto'k drfnku Jon— ?f. — .32.^».
• T o sin o ti. U tokiL S , Spiritati, I I , ]t- }
. risale, L . XiOsBsk/. J, F itto ti, J, IlxircV. *
A . K t»e»i l II, JUis, J- I’uiLosjal ztlravl- •
pwcMoBU pii vfrobA Z.4.S-1! IchlOrltsiaxy.
ocuna teda di a. fratta. U*v, (v lis ta i.—
3X FlottU. IL: Zur Klsdlk net Ccatsdaacn* ■
BOdtiar U«J Chiaraluto (llalow sxauo).
Arch. klla. sxp. Dorai, 239. 1970. X 223—
24L — 3L FLnrir,
LobalLehsndlui.r dcr
Cbloracae (llalowaxacaal aur vuotala A*
Ware. Haitiani, 21. '35170. a. 405—470. — *
35. Hfacrt. i t i Inioxlcatloa praiesslsndl«
par lux re pours de cblorc; a<to chiurlotto
ot lultsrcàlatc putuuaalre. Scia. m iti. 1305,
x.423. — ctt firata 5 . - 3 « . SelutU, IL Zs
JQlo lecite a. cxpcrloieaudlc Usicraschua*
pea sur Atiolofia dcr Cbloracns. Arch.- *
kiln. cxp. P era, 223. 1357. 589—C30. —
37. Setola, IL IL: Zar KUnik s . AUologlo
dcr Chluraltnc. ArboUsuulizbi (ASA), 3.
1933, X. 25-23. — 33. Sshulx, li. IL: Osotoi
sdii col. — 33. SdnrarU, t s DerstutlUx
i n a syathciic resus osti v tu c x Amur. J. ■
paid, ltllh , 20, 1920, S. 53d—592. — <40L
Schararix, t i Aa ohlbrcuk al llalòuiax
acne {.cablo rash*) aaump clckiricanL J.
Amor. ned. Axs, 122, 3313. t. 2S3. — 4JL **
Shxllc/, 57. A , Xllasfea«, A. ì t s The e»po*
rinculai predaevea ul cetw l«t p&tu* and
JicxachlBttwplitalbae». Ardi. Ucna. Syplu
(C hiùgol. 75. 1357. X. 6SX — 42. Tahikor,
Id Dio I'ltnukraakhd: (Chlorshae). Klla.
VAlChr, 1 2 . 1327. x »45-443, iU iU a 097— .
SOL — 4L Tbiblcrra, Cd Acni coBrfdaa.
ptocralisCs dite 3 Htihslallod pralcsstoael*
le de vapours chloriqucs. Seas vn/.d, 1300,
x. 24, ClL Brau &. —

5918.0011907
___

Ì 0 9 0 9 I.ZM0Q

References

.. Pagê~2'CT

UThiWco;. C., Feg.
Miei, ' >tu L‘ze*»e cü^kji. ui*. a».u iierm. ■_
¿Ji-U.
W X1, tm *15—‘523. — cl». ‘ „
B.-jsa 5. — 4S. Tojrutt'o, A, Soleste, B„ J/
—¿alluri, B . A etna: Si o » de ilcnaitltei '
per ir:ihIurae|tUMllae. tlulL Voc, 'detta,'
MjiB„ i l . 1931 s. 3.5-270. — i 3. WaUlsT- “
i\: Vu.-i 2 Felle «tut .CIiÀetfM'. Iiuvt«* .
Ml DIU. Leipzig. 2914. — ctL litt««. S. — .
47. vreuet: tidoeblirhe ftkrenVang curdi
tiitU u u KiAieiwtuürtuili« ll'e n u b u l» .
liciti. ZK. Ccvcrbciiyt, 9, 1911. «. lüO— ■'
201. — U WlniJitf.

Zar CUck I m (Per-

R.".rssUicÄj. 2. lU st. CjssLIVt^ U .
Ü iU z. 415—491. — 43. Hattet.
Sclial«.

;

X . u , S,>U £ « ltu £. De U ent3lriie y«*7jUla»*

jtc* Itti der HunlcUuas «cs CLierjUiesel—*' -

W itisutnagen, Arck. C airnb^PataoL Ca»'**

•4«txb. lly s, 18, 2931, i. S52-5W

• . -V

[10]

Pathogenesis of photodermatides from chlorinated naphthalenes.

[13]

Gombosr et al., Porphyris cutanea tarda and acne chlorina
..occurring during the production of cyclic chlorinated hydro- .
■.carbons. _

[23]

Symptoms of total intoxication in acne chlorina during the
production of herbicides
in final preparation for.printing.

[31]

Discussion on paper by Jones [24]

•

[32]

.

.

»

• .

4 .

Health hazards'to workers in production of sodium 2,4,5-txd; chlorophenoxyacetate-

I.

;

OOH90S

5919

Page 21
' 7

1 * 0

TABLE I .

P roduction Sehea*.

P lane f o r production o f .
HC8 (htxachiorocydohexane)

1,2,3-erichlorobanzaaa and its'
isooers (1,3,5- and 1,2,40)

..

V»

* ■-»> .

■

•. (
>.
..1 • .*«
if*, .v. k v
M

*■ • : t

pastachlaropbanolaca

1 1.
f l f l . *. v
J
• » i * . • y* + Î R«
*•V •• •
••
• . •*

*. • *«••*

0011909

5920 .

»1909IZMOQ

X

i

fl «ras rematad thnt Um «lila and general »ítactirma tr e n ca ñ a d by 2JA7-l*tretítlorodltoeozadlaxln wblch la tonned dunng alkallcw bydrolyso o í inTirlilniiraciiijji.
uua eodiua irtriUargphanoUiA U m rractloo w at perfonncd by a a anaritabla pepead a ra
ai a im p e m a n o t IW C and p m n n of 49 a in . From tfw -sor* o í Séllala and KUaarig •
tt la kamra Uul liria hlgiriy lóale labrtincat la aoc. tem ad whea iba a b a n n iriin n
la earrMd oat at a lo tñ r (amparaten o< 1S3*C KM onJy «rock*re angagod la prodac
Iloa af feerWcldaB wara alaciad bol also raraataaaaca ararfear
«cartón diggta» ti» am aga cnaaaai.
la ala iba dtaanaa basta aa acata aotar ¿enastilo. tn all tbo nratlndra II
by a atoar arapdoa ot cewtdonaa aad thatr dera lopraeat lata eyata. iMflimraanoaa aad
acam as arara tomad eoly la iba leoei ta r a n a n d e * tanatee atractioaa. Tba aba aad
«tan-al auatfestaiioea «am tpoodad lo d a u taponad la tba 11ten tara, la 17
harn ear. acaa eblortaa-ttartad la a án a ta tl «cay by pa'paloparaulam anpU oaa aa
«stnrattMA la 20 panano tria «oleara tia m a d loeaUaatioa la tba axtllaa «trie* r
oot taponad p n n o a tly. Tba diarara doaa a et atlact tba balry p a n al tba abaü i
la oaa patlaat tt duappaana aran Ireot ibera site* la tba laca and aa tba ¡
ba (tara a baard, wbila tba cooditloa pactaod ai « b a r m o a Pailoats wtm aa uapaind
porpbyná atctabollaat tu lc n d (rom typwal byprrpigmanianaa and brpanrieboaO aad
«wriy naa o( iba« froai bolloait aclínica « aractuaici. Hypmncbociaaad byparptg
u aa atas obrarcad olmo la U tablacta triUt acaa vriinoat labeniar» d y n t al
la tba pattod prior ta tba dlaatar aa «goUicant pndtapeatns factara « ta n i
Tba Utancy d tflm d . la tora# Insuacaa ta r a n dármatniogtcal ,
n lo i t d altar « io n asparon, la « b a r lotiaacrt appanm ly tong a a d .:
c ía n » oaly arild tyatptooo. Oatplta th o tba a atb c n a n caa riactd tbat tba lataaaHp ot
Iba dlaaaro dapaada a t a oo dxpoaan tora pradOporiUoa. Ib a potleao bad ranoaa
corapirtno-tMDtlie. sa n ta l aad aotaal aad roara o í rirara bad canridanblr araight lanar.
Trra dlod froai genaml liuorlc rilnw. uro (ro a hmg cancar ta i ao la r tt la a « ppealbla to
« a ta nlOMy whatbar lt «na relatad te tba oerspaltana! asp aran , p w
rtfect o t cblortaatad bydmcarbom prodacad ht tba patttralar p ía « aras a « :
lar. Tba raedle«! aad aaanlpsicsl tira « arar
la i

iitai'YMOV

'

« sanarsi latoxtcarton
llttla « fa ettn . A cartata bai
rad anear, la an paltana « cooda ry
la srrnrdtnca i
4A 0 7 3 , í . J, A JOS—317.

Hidratara
a, E. W, ia a n , « .
btdestroi
das to pnm tilorpbanol. bid.
Mad. Sor». 20. USL a U A — 2. Satlraaaat
Chler-Afeon. n aa baroadara Fona roa proWaebr, 27. 1S0L a. 4 » . — 2. »tartars . J ,
W a n t. K , A iadtla. Id tadasuM Iy acaalo­
ad porpbyná. Arca. D m . *3,' U H a.
739-7*7. - 4. la m a . A A, M aarnri; M.
Fd O r ia m o bt tba «aaafactan al DOT.
Aten. Dana. Sypb. (C blcaiai, 73. 1337. a
749—743. — 1 S n aa. W j O rio n i« « Ma>
! L, AaOaOorf L W trtt, Cdhton Caator
, a 73. — A I m a , « d KlbrircMt leo worn aar t n oob óng dar Chloracoo.
H aotrnt, M, USE a 123—123. — 7. Irlab-

Vanrag. 0 nr* ranm í. Tagg. « Hata« L W.
ISSO — cri. Baoer-— 43. — A Fibra, V. 3 .
Jarrflb D. Ed Aerina « córtala cblartaaiad
aaphm aaaa rat tba livor, Frac. Soci aspra-.
Slot. Mod. 33. 1393. a 123—1281 — A
Fosb« Fd eli. Brrara — A — 1A l
» . Md O p a la s i ara fctodarranlimr « t
n ratraaaycb aatotbra*. »Sara. Darra. Va*
aar, 134A A 3. a 3A — IL Dtlla a t a. L
Fd SeHo m a r afean QUorabaa darcb TH*
ChlorpbanM-Tai eem ingepiudubta. Arbori*
(ASA|. 7. 1372. A 12—1A — 12.
F. IJ Scbtrrrao abata China*
Mararairaoaifeani darcb A X
A 7-Tttfracftlerdlhaaio dlon a . Haotarct, SA
1373. a 143-132. — 13. t r a t t i, B , IO*

SI«'
[JLJiiura».sa'm a* 'mg-ip

«ÀÉrtt
THIS IS A SIN G LE PH O TO CO PY MADE BY THE NATIONAL LIBRARY OP MEP<

o o im i
"F922

I ta lv ec with V. C. hove. a f Sot, " I t nam in g.
:ne fa llo v in * i

.s« c : t : t :

So« haa 20 casea or enloracno. No ¿ Id not t o l l so
another or not th e ir plant «a« town.
They analyted tfta trlchloropnenoxyacotle acid ve
•one then and found 3 - 10 ppn o f 3 ,* .7 .8 - c n c n lo r o dlbenxodlozaae. Thla, ho atatoo, ho la auro la tno
chloracno acont, although tnoro nay bo other l a p u r it ie s . a t any r a te , no la ao eoneomad that tnoy
ara flavin* a aanaxoaont aootlnx to docldo «notnor to
uao tU M flT o earloada of a a to r ia l vo t*Mc then.

>c
: «
Ob . •
: v
4^ *1
I, >. .I*iV4..l;-.
•\
**Ji s•

3.

Bo had d o fln lto ly cauaod onloroeno on rabbit oari
v lth th la a a t o r ia l. When 1 t r lo t to aay that aoao
o f our poopla nave not boon afclo to cauao I t vitr.
other aato ria l« ho juat aeoffod and aald theta vaa
no doubt la tua alnd that ho can t e l l enloracno by
ta la rabbit oar t e a t and that ho naa run about 500
toota on rabbit ea ra .

b.

Sow haa boon ro eo lv ln c eoaplalnta froa th e ir euatonora (vnothor fo m u la to ra or u ltla a to uaore, 1
don't imam). Bo vaa not too elo a r on vnoao a a to r ia l
I t vaa and ho« auch enloracno developed.

3«

Ho had hoard th a t "the ?HS la looking Into th la
problta and haa eontaetod aono o f Sow'a cuatcnort
concam ln* the oocurrar.ee o f enlsracn e."

*

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^if#ir

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«o aeni
whether or not tnoyy v i l l a tto c t thla

*“.»£.

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• •• »£»■
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lit wanted ste a so r t o f era an Eaetlr4*«-*ith a l l the
produaora to tr y to roach tone o p a c ific a tio n le v e l
in order to aliaH nate th la p a rticu la r contaaina
before the PUS pot Into the a c t.

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a l l or tno aeovo m a naaaac over acalr.. Tm only tnln e ^
A gricu ltu ral D ivision pooplo could add «as th a t they had
no hlatory o f chloracno froai any o f th o ir eu atoaors. Thay
aro so ln c to c a ll AaChoa, «no ha a boucht about 1 .2 d l l l o n
pounds o f 2 ,u .o ,t o find out I f they had any tr o u b le . Ruaor
la to chock Thoapaon Coapany te too I f tho res haa boon a fto r
ho*. Thoy alao atatod that I f auch a a a otlag occurs Dr. Jaeobs,
«no la a acobinatloo q u a lity aentrol/prooosa aan, would bo
th o ir roprasontatlvo. aloe« with aoaoono froa tho Modloal Departa o n t.

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5926

5927

DEBORAH A. BARSOTTI, Ph.D.

I.

QUALIFICATIONS
Dr. Barsotti is an Assistant Professor of Tocicology

in the Department of Pharmacology and Toxicology of the Philadelphia
College of Pharmacy and Science in Philadelphia, Pennsylvania.

Her

curriculum vitae is attached.

II.

SUBJECT MATTER
Dr. Barsotti is a toxicologist who has done substantial

personal investigative work with h a l ogenated aromatic hydrocarbons.
This category of chemicals includes various biphenyls and dioxins,
and specifically includes tetrachlorodibenzo-p-dioxin (TCDD).

She

will testify as to the relative chemical stability and lipophilicity
of these chemicals as well as their resistance to degradation, and
will testify that these qualities are what account for the perceived
and documented bioaccumulation of the chemicals in the human system.
Dr. Barsotti will emphasize the difference in results between chronic
and acute exposures to these chemicals, testifying as to the varying
types of responses to acute and chronic exposures.
Dr. Barsotti will discuss her own work as well as review
the literature in these areas, noting differences in routes of

5928

administration, and in the species and genetic strain of the animals
involved.

She will explain these variations and where they permit

extrapolation of results from animals to humans.

In particular, Dr.

Barsotti will also testify as to the similarities in response to
these chemicals by rhesus monkeys and by humans, and will explain
how the similarities permit extrapolation in this instance.
Dr. Barsotti will also discuss reproductive toxicity,
teratology and post-natal toxicity,

including instances where no

gross or clinical signs of maternal toxicity could be detected-

I I I . SCIENTIFIC PRINCIPLES
See General Statement of Scientific Principles and
Assumed Facts attached.

IV.

SUBSTANCE OF OPINIONS
A.

That dioxins are toxic, and in particular TCDD is

exceptionally toxic.
B.
stability,

That dioxins bioaccumulate because of their chemical

l e p o p h i l i c i ty , and resistance to degradation.
C.

That chronic low level exposure to dioxins is

significantly ^toxic.

5929

D.

That TCDD accumulates and persists in the lipid

E.

That TCDD,

stores.
in chronic doses, is toxic, is parti­

cularly toxic to reproductive system,

is teratogenic, and is

'f e t o t o x i c .
F.

There are ample scientific bases, for extrapolat­

ing animal experimental data to man,

in the case of TCDD, for example,

as demonstrated by Dr. Barsotti's work on rhesus monkeys.

V.

GROUNDS FOR EACH OPINION *
1
A.

General
Review of applicable medical and scientific litera­

ture;

education and training; personal research.

The work on the

chronic toxicity of HAHs in rhesus monkeys has culminated in
numerous publications as presented in Dr. Barsotti's curriculum
vitae.

In addition,

she has been invited to speak or present her

work in various atmospheres including community meetings and
scientific symposia.
B.

Specific
1.

With respect to opinion IV A, see generally

references attached.
2.
I

With respect to Opinion IV B, note the following:

In the course of Dr. Barsotti's graduate and post­
graduate studies, she has had the opportunity to conduct research
and evaluate others' work on members of the class of chemicals
called halogenated aromatic hydrocarbons

(HAHs).

She will discuss

the fact that this class of chemicals includes the chlorinated *dibenzo-p-dioxins and the ha logenated biphenyls
and polybrominated biphenyls)
structure,

(polychlorinated

and that HAHs are similar in chemical

in the pattern of toxic responses that they produce and

in the mechanism by which they are believed to act
1979; Poland and Knutson, 1982) .

(Allen £t a l . ,

Although individual HAHs vary in

potency, their chemical stability and l i p o p h i l i c i t y , as well as
their resistance to degradation results in bioaccumulation

(Isensee

and Jones, 1975; Meselson et a_l. , 1978; Marinucci and Bartha, 1982;
Mes et a l . , 1982).
3.

With respect to opinion IV C, note the follow­

ing:
Thesis work p e r formed by Dr. Barsotti at the U n i v e r ­
sity of Wisconsin with polychlorinated biphenyls indicated that
evaluation of the toxic effects of HAHs in the Rhesus monkeys was
valuable to the understanding of the potential hazards of HAHs in
the environment

(i.e. chronic exposure).

indicated, unlike rodent models,

In addition, this work

that there were similarities in

the response of humans and rhesus monkeys to these chemical toxi­
cants

(Bar-sotti, 1980) .

Like man, the rhesus monkey is genetically

diverse and responded to PCB exposure on an individual basis.

/

Dr. Barsotti will contrast the acute toxicity that one
observes in accidental or intentional ingestion of toxic or lethal
doses of chemicals with the effects of chronic or lower levels of
exposure to chemicals such as HAHs.
4.

With respect to Opin i o n IV D, note the following:

Dr. Barsotti will comment on the state of the
literature on the fate of TCDD once in the body, i.e. TCDD metabolism
(Vinopal and Cassida, 1973; Ramsey ert a l . , 1979; Schlatter, 1979) .
Efforts to identify metabolites in tissues and excreta from various
studies have been unsuccessful.

Recent advances in detection

methodology indicate elevated levels of TCDD in Viet n a m veterans
and other exposed persons.

Generally, however,

the data indicate

that if metabolism does occur the rate is slow (Guenthner et al., 1979)
The ramification of this finding is that the body would be incapable
of or slow in ridding itself of TCDD and TCDD would accumulate and
persist in the lipid stores.

In Dr. B a r s o t t i 's work, as well as

others', this appears to be the case with rhesus monkeys and man
(Montagna et a l ., 1979; Van Miller,
5.

1981; McNu l t y et a J . , 1983).

With respect to opinion IV E, note the following:

Not only did the rhesus monk e y appear a good model
for HAH toxicity but established,

as had been in other animal

species, that the reproductive system was extremely sensitive to
PCBs at intake levels below that required for the manifestation of
toxic signs-in the adult

(Barsotti et .al. , 1976) . "In addition to

PCB induced reproductive toxicity, PCBs were found to be fetotoxic

three months in animals receiving TCDD in the diets at levels of
500 parts per trillion

(ppt) and in animals consuming diets c o n t a i n ­

ing 50 ppt TCDD these same symptoms occurred after consuming the
diets for 30 months

(Allen et _al. , 1977; Schantz el: a_l. , 1979) .

This indicates cumulative toxicity associated with ingestion of
TCDD.

Thus, there is evidence for bioaccumulation and associated

chronic toxicity at lower doses than that required for acute t o x i ­
city in rhesus monkeys and p resumably man

(Pazderova et _al, 1981;

Ideo et al., 1982.)
6.

With respect to Opinion IV F, see generall

references attached and V A above.

I

5 9 3 3

D. A. Barsotti, L.J. Abrahamson and J.R. Allen, Bull. Environ.
Contain. Toxicol., 21, 463,

1979-

R.E. Bowman, M.P. Heironimus and D.A. Barsotti, Neurotoxicol,m 2,
251, 1981.

K.D. Courtney and J.A. Moore, Toxicol. Appl. Pharmacol., 20, 396,
1971.

T.M. Guenthner, J.M. Pysh and D.W. Nebert, Pharmacol.,

19 12,

1979-

G. Ideo, G. Bellati, Al Bellobuono, P. Mocarelli, A. Marochhi and
P. Brambilla, Clinica Chimica Acta, 120, 273, 1982.

A.R. Isensee and G.E. Jones, Environ. Sei. Technol., 9, 668, 1975-

E. E. McConnell, J.A. Moore, J.K. Haseman and M.W. Harris, Toxicol.
Appl. Pharmacol., 44, 335, 1978 a.

E.E. McConnell, J.A. Moore, and D.W. Dalgard, Toxicol. Appl.
Pharmacol.,

175, 1978 b.

W.P. McNulty,K.A. Nielsen-Smith, J.O. Lay, D.L. Lippstreu, N.L.
Kangas,^P.A. Lyon and M.L. Gross,4E d . Chem. Toxic., 20, 371,
19 8 2 .
I

'

5934

I. Manara, P. Coccia and T. Croci, Drug Met. Rev., 13, 423, 1982.

A.C. Marinucci and R. Bartha, Bull. Environm. Contain. Toxicol.,
29, 326, 1982.

J. Mes, D.J. Davies and D. Turton, Bull. Environm. Contain.
Toxicol., 28 97, 1982.

M. Meselson, P. O'Keefe and R. Baughman, In: Symposium on the use
of herbicides in forestry, 91, February 21-22, 1978.

M.-Montagna, A. Fornari and S. Facchetti, In: Forensic Toxicology,
78, 1979-

J.R. Olson, M.A. Holscher, and R.A. Neal, Toxicol., Appl.
Pharmacol., 55, 67, 1980.

J. Pazderova, M. Nemcova, J. Pickova, L. Jirasek and E. Lucas,
Arch. Environ. Health, 36, 5, 1981.

A. Poland and E. Glover, Molec. Pharmacol.,

17, 86, 1980.

A. Poland and J.C. Knutson, Ann. Rev. Pharmacol. Toxicol.,22, 517,
1982.

H. Poger, H. Buser, H. Weber, U. Zweifel and C. Schlatter,
Experimentia, 4 484, 1982.
I

s

R. C. Ramsey, J.G. Hefner, R.J. Karbowski, W.H. Braun and P.J.
Gehring, Toxicol. Appl. Pharmacol., 47, A 162, 1979*

S. L. Schantz, D.A. Barsotti and J.R. Allen, Toxicol. Appl.
Pharmacol., 46, 180. 1979-

S.L. Schantz and R.E. Bowman, The Toxicologist, Vo l . 4, Abstract
number 333» March,

1984.

J.P. Van Miller, Doctoral Thesis, University of Wisconsin, 1981.

J.H. Vinopal and J.E. Cassida, Arch. Environ. Contam. Toxicol. 1,
122, 1973-

I

r>
•J

K.J

I

jmm aw

UNITED STATES PATENT OFFICE
2,509,245
PREPARATION OF 2,4,5-TRICHLOROPHENOL
Edward Joseph Nikawltz, Passaic, and William S.
Gump, Upper Montclair. N. J., assignors to The
Glvandan Corporation, a corporation of Npw
Jersey
No Drawing. Application March 20,1947,
Serial No. 736,1X8
5 CUlnu. (CL 260—623)
1
2
This Invention relates to a process for prepar- be varied. The alkali metal hydroxide is used in
log 2,4,5-trlchloro phenol, and more especially amounts equivalent to at least 2 mols of hydroxto a process wherein 1,2,4.5-tetrachloro benzene ide per mol of tetrachloro benzene. 2-3 mols of
is subjected to alkaline hydrolysis in the presence hydroxide per mol of tetrachloro benzene gives
of ethylene- or propylene glycol (propane­ 6 excellent results. Higher amounts of hydroxide
may be employed, but are unnecessary.
diol- 1 ,2 ).
With regard to the amount of glycol which
2.4,5-trichloro phenol has been prepared from
1.2.4.5- tetra:hioro benzene by hydrolyzing the should be employed in our process, we find that
excellent results are obtained when about 750
latter with alkali in the presence of methyl al­
cohol. the process being conducted under con­ 10 grams of the glycol per 216 grams ( 1 mol) of the
siderable pressure, of the order of 600-600 pounds tetrachloro benzene are used. Larger amounts
per square inch. Special pressure equipment is of glycol may be used, but in such cases no ad­
vantageous results follow. Amounts less than
required for conducting such a process. More­
over. appreciable amounts of the methyl ether 450 grams of glycol per 216 grains of tetrachloro
of 2.4,5-trichloro phenol form when methyl al­ 16 benzene are not recommended, as yield and qual­
ity of the desired phenol are adversely affected.
cohol is employed; and the formation of the
The temperature range at which the hydrol­
ether is undesirable as it decreases the'yield of
ysis may be effected is between about 160* C.
the desired free phenol.
Our present invention overcomes the foregoing and 200° C., the preferred range being between
disadvantages and provides a process for making 20 about 170° C. and 180° C. Higher temperatures
2.4.5- trichloro phenol from 1,2.4.5-tetrachloro ’ are obtainable when propylene glycol fs employed
benzene which can be conducted with cheaper
than is the case when ethylene glycol is employed.
and simpler equipment than is required by theT
A special advantage of this process is th at it
prior art process, and which does not result in can be conducted at atmospheric pressure, under
the formation of any appreciable amount of 25 reflux. However, if desired, the contents may
be heated in a closed system, whereby a slight
ether.
In general, our process may be conducted by pressure is built up. amounting however to not
dissolving an alkali metal hydroxide, such as so­ more than 15 to 20 pounds per square inch, and
dium hydroxide potassium hydroxide and lith­ not necessitating the use of any special pressure
ium hydroxide, in ethylene glycol or propylene 30 equipment in the plant.
glycol, or a mixture thereof, at elevated tempera­
The invention is illustrated by the following
tures while stirring the contents. The tetra­ examples without however limiting the same to
chloro benzene is then added and the mixture is them.
heated for a few hours, normally 3-4 hours being
Example I
sufficient. The end point of the reaction can be 35
determined easily by taking a sample of the re­
60 grams of sodium hydroxide flakes (95%
action mixture and diluting it with water. If NaOH) were dissolved in 500 grams ot ethylene
th e sample is water soluble or practically entirely glycol in a 2 liter three-necked flask provided
soluble in the water, the reaction may be con­ with stirrer and an air condenser. The contents
sidered to have been completed. The desired 40 were heated to 150° C.-160* C„ this step requir­
phenol may be Isolated in accordance with known ing about 30 minutes. 144 grams of 1,2.4,5-tetprocedures. For example, the reaction mixture rachloro benzene were rapidly added to the so­
may be cooled after the test as above shows sub­ lution, and the mixture was heated to 170° C.stantial completion of the reaction, and then
180° C. (inside tem perature), and maintained at
acidified with a mineral acid such as hydro­ 45 th at tem perature range for 4 hours. 10 grains of
chloric acid. The precipitated alkali metal chlo­
tetrachloro benzene sublimed in the air con­
ride is filtered off. The filtrate is poured into denser and were recovered. A sample of the re­
w ater, causing the 2.4,5-trichloro phenol to pre­ action mixture gave a clear solution when dis­
cipitate. The phenol is extracted with benzene solved in 10 times its weight of water.
and the benzenS extract is distilled to remove the BO The reaction mixture was allowed to cool;
benzene and yield the phenol. The aqueous dry hydrogen chloride was passed into it until It
layer remaining after the benzene extraction became acid to litmus. The slight excess of hy­
is fractionally distilled- to remove the glycol drogen chloride was neutralized by the addition
employed.
'
of a small amount of sodium bicarbonate. After
The proportions of the ingredients used may H cooling again to about 20° C* the salt was filtered

y

UNITED STATES PATENT OFFICE
2,509.245
PREPARATION OF 2.4.5-TRICHLOROPHENOL
Edward Joseph Nlkawitx, Passaic, and William S.
Gump. Upper Montclair, N. J„ assignors to The
Givaodan Corporation, a corporation of Npw
Jersey
No Drawing. Application March 20,1947,
Serial No. 736,118
5 Claims. (CL 260—623)
1
2
This Invention relates to a process for prepar- be varied. The alkali metal hydroxide Is used in
lug 2,4,5-trlchloro phenol, and more especially amounts equivalent to at least 2 mois of hydroxto a process wherein 1.2.4.5-tetrachloro benzene ide per mol of tetrachloro benzene. 2-3 mois of
is subjected to alkaline hydrolysis in the presence hydroxide per mol of tetrachloro benzene gives
of ethylene- or propylene glycol (propane­ g excellent results. Higher amounts of hydroxide
diol- 1 ,2 ).
may be employed, but are unnecessary.
W ith regard to the amount of glycol which
2,4,5-trlchloro phenol has been prepared from
1.2.4.5- tetra:hioro benzene by hydrolyzing the should be employed in our process, we find that
excellent results are obtained when about 750
latter with alkali in the presence of methyl al­
cohol. the process being conducted under con­ 10 grams of the glycol per 216 grams ( 1 mol) of the
siderable pressure, of the order of 600-800 pounds tetrachloro benzene are used. Larger amounts
per square inch. Special pressure equipment is of glycol may be used, but in such cases no ad­
required for conducting such a process. More­ vantageous results follow. Amounts less than
over, appreciable amounts of the methyl ether 450 grams of glycol per 216 grams of tetrachloro
of 2,4,5-trichloro phenol form when methyl al­ 15 benzene are not recommended, as yield and qual­
ity of the desired phenol are adversely affected.
cohol is employed; and the formation of the
ether Is undesirable as it decreases the'yield of
The tem perature range at which the hydrol­
ysis may be effected is between about 160* C.
the desired free phenol.
Our present invention overcomes the foregoing
and 200* C„ the preferred range being between
disadvantages and provides a process for making 20 about 170* C. and 180° C. Higher temperatures
2 .4 .5 trlchloro phenol from 1,2.4.5-tetrachloro ‘ are obtainable when propylene glycol fs employed
benzene which can be conducted with cheaper
than Is the case when ethylene glycol is employed.
and simpler equipment than Is required by theT
A special advantage of this process is th at it
prior a rt process, and which does not result in
can be conducted at atmospheric pressure, under
the formation of any appreciable amount of 25 reflux. However, If desired, the contents may
ether.
be heated in a closed system, whereby a slight
In general, our process may be conducted by pressure is built up. amounting however to not
dissolving an alkali metal hydroxide, such as so­ more than 15 to 20 pounds per square inch, and
dium hydroxide potassium hydroxide and lith­ not necessitating the use of any special pressure
ium hydroxide. In ethylene glycol or propylene 30 equipment in the plant.
glycol, or a mixture thereof, at elevated tempera­
The invention is illustrated by the following
tures while stirring the contents. The tetra­ examples without however limiting the same to
chloro benzene is then added and the mixture Is them.
heated for a few hours, normally 3-4 hours being
Example I
sufficient. The end point of the reaction can be 35
determined easily by taking a sample of the re­
60 grams of sodium hydroxide flakes (95%
action mixture and diluting it with water. If NaOH) were dissolved in 500 grams of ethylene
the sample is water soluble or practically entirely
glycol in a 2 liter three-necked flask provided
soluble in the water, the reaction may be con­ with stirrer and an air condenser. The contents
sidered to have been completed. The desired <0 were heated to 150* C.-160* C.. this step requir­
phenol may be Isolated in accordance with known ing about 30 minutes. 144 grams of 1,2,4.5-tetprocedures. For example, the reaction mixture
rachloro benzene were rapidly added to the so­
may be cooled after the test as above shows sub­
lution, and the mixture was heated to 170* C.stantial completion of the reaction, and then
180* C. (inside tem perature), and maintained at
acidified with a mineral acid such as hydro­ 45 th at tem perature range for 4 hours. 10 grams of
chloric acid. The precipitated alkali metal chlo­
tetrachloro benzene sublimed in the air con­
ride is filtered off. The filtrate is poured into denser and were recovered. A sample of the re­
water, causing the 2,4,5-trlchloro phenol to pre­ action mixture gave a clear solution when dis­
cipitate. The phenol is extracted with benzene solved in 10 times its weight of water.
and the benzenS extract is distilled to remove the ¿ 0 The reaction mixture was allowed to cool;
benzene and yield the phenol. The aqueous dry hydrogen chloride was passed into it until it
layer remaining after the benzene extraction
became acid to litmus. The slight excess of hy­
is fractionally distilled- to remove the glycol drogen chloride was neutralized by the addition
employed.
'
of a small amount of sodium bicarbonate. After
The proportions of the ingredients used may K cooling again to about 20* C„ the salt was Altered

5938

by suction and the Balt cake was washed with in the range of th at of the atmosphere up to 20
60 cc. Of isopropyl alcohol. 600 cc. of water were pounds per square inch and for a time sufficient
added to the filtrate resulting In a bottom layer to substantially complete the conversion into
of precipitated trlchloro phenol and a top layer 2,4.5- trlchlorophenol.
of dilute ethylene glycol. The entire mixture was 8 2. The process for preparing 2.4,5-trichloroextracted with 400 cc. of benzene, then with 100 phenol, which comprises heating at 160°-200° C.
cc. of benzene and finally with 80 cc. of benzene. In the following proportions: 1 gram molecular
weight of 1,2,4,5-tetrachloro benzene and at least
The combined benzene extracts were shaken
with 200 cc. of water and the water layer was 2 gram molecular weights of an alkali metal hy­
separated and added to the dilute ethylene glycol. 10 droxide in the presence of at least 450 grams of
ethylene glycol, the reaction being conducted
The washed combined benzene extracts were
dried by means of anhydrous sodium sulfate, fil­ under a pressure within the range of th at of the
tered. and distilled. After removal of the ben­ atmosphere up to 20 pounds per square inch
zene, the residue was distilled a t a pressure of 4 and for a time sufficient to substantially com­
mm. of mercury. 106 grams of 2,4,5-trlchloro 18 plete the conversion into 2.4,5-trichloropbenol.
3. The process for preparing 2,4,5-trichlorophenol, boiling a t 101° C.-105* C., and having a
congealing point of 63-3* C. (uncorrected), were phenol, which comprises beating at 160°-200° C.
In the following proportions: l gram molecular
obtained.
The ethylene glycol can be recovered by dis­ weight of 1,2,4,5-tetrachloro benzene and at least
tillation of the aforementioned dilute ethylene SO 2 gram molecular weights of sodium hydroxide
glycol. The water and isopropyl alcohol were re­ In the presence of at least 450 grams of ethylene
moved in a fractionating still at a pressure of 90 glycol, the reaction being conducted tinder a
mm. of mercury, the tem perature being carried pressure within the range of th at of the atmos­
up to 60° C. The ethylene glycol was then dis­ phere up to 20 pounds per square inch and for a
tilled under high vacuum (3 mm.), 232 grams 28 time sufficient to substantially complete the con­
of the glycol boiling a t 80° C. being recovered. version into 2,4,5-trichloropheiiol.
4. The process for preparing 2.4,5-trlchloroIn order to remove practically all of the ethyl­
ene glycol from the small amount of salt re­ phenol, which comprises heating a t 170°-180° C.
maining In the distilling flask, the temperature in the following proportions: 1 gram molecular
was raised so th at some glycol, boiling from 80' 30 weight of 1,2,4,5-tetrachloro benzene and 2-3
gram molecular weights of sodium hydroxide In
C. to 120° C.. was obtained.
the presence of 750 grams of ethylene glycol, the
E x a m p l e II
reaction being conducted under atmospheric pres­
72 grams of 1,2,4,5-tetrachloro benzene were sure and for a time sufficient to substantially
stirred and heated to 190-200° C. with a solution 35 complete the conversion into 2,4,5-trlchloroof 30 grams of sodium hydroxide In 250 grams phenol.
of propylene glycol, the heat treatm ent being
5. The process for preparing 2,4,5-lrichloroconducted for 6 hours. 24 grams of concentrated
phenol, which comprises heating at 170°-180° C.
sulfuric acid (93% HaSO«) were added to the re­ in the following proportions: l gram molecular
action contents after they were cooled to room •10 weight of 1,2,4,5-tetrachloro benzene and 2-3
temperature (about 25° C.>. The entire con­ gram molecular weights of sodium hydroxide In
tents were poured Into 1000 cc. of water. The
the presence of 750 grams of propylene glycol,
solid material was then filtered and washed With the reaction being conducted under atmospheric
500 cc. of water and finally dissolved in 200 cc. pressure and for a time sufficient to substan­
of benzene. The benzene solution was dried with 48 tially complete the conversion into 2.4,5-trichloroanhydrous sodium sulfate and then filtered.
phenoL
After removal of the benzene by distillation,
EDWARD JOSEPH NIKA WITZ.
the residue was distilled under a high vacuum
WILLIAM S. GUMP.
(5 mm.), 45 grams of 2,4,5-trlchloro phenol being
80
obtained thereby.
REFERENCES CITED
The foregoing illustrates the practice of this in­
references are of record In the
vention, which however, is not to be limited fileTheof following
this patent:
thereby but is to be construed as broadly as per­
missible in view of the prior art and limited solely
FOREIGN PATENTS
68
by the appended claims.
Number
Country
Date
We claim:
349,794
G
erm
any__________
July
29,1914
1. The process for preparing 2,4,5-trichlorophenol, which comprises heating at l60°-200‘ C.
OTHER REFERENCES
In the following proportions: 1 gram molecular
weight of 1,2,4,5-tetrachloro benzene and at least 80 Harrison et al., "Polyhalogeno—Derivatives."
J. Chem. Soc. (1943). pages 235-7 (3 pages,
2 gram molecular weights of an alkali metal
pages 235 and 236 are pertinent).
hydroxide in the presence of a t least 450 grams
Spielmann, "Richter’s Organic Chemistry," vol.
of at least one material from the group consist­
L published by P. Blaklston's Son ft Co., Philadel­
ing of ethylene glycol and propylene glycol, the
reaction being conducted under a pressure with­ 85 phia (1921), pages 98, 99 (2 pages).

I
r-' f

•?

■>.U nited States Patent Office
*

;

I

1

'

:

2,799,713

Patented July 16, 1957
2

preferably from 2 to 8 , percent by weight of the alkali
metal hydroxide; and (c) carrying out of the hydrolysis
2,799,713
under a pressure at least as high as the autogenous
METHOD OF MAKING TRICHLOROPHENOLS
pressure
of the mixture of materials. The hydrolysis
FROM TETRACHLOROBENZENES
6 is carried out in a reaction time of not more than 120
A kxuder H. Whliger, Jr„ Midland, Mike L. Aaron, minutes, preferably from about 6 to 90 minutes.
The invention concerns a process for hydrolyzing tetra­
Hemlock, and Gordon F. Dugan, Beaverton, Mkh.,
amlfann to The Dow Cbemicai Company, Midland, chlorobenzenes which involves employing each of the
MidL, a corporation of Delaware
reaction conditions set forth in items (a), (b) and (c)
10
just
mentioned and results in good yields of trichloro­
No Drawing. Application January 24, 1955,
phenols. The invention utilizes a readily available, in­
Serial No. 483,798
expensive, non-flammable reaction medium, and permits
5 Claim*. (CL 260—623)
carrying out of the alkaline hydrolysis of tetrachloro­
benzenes to produce trichlorophenols in a manner suitThis invention concerns an improved process for 15 able for the manufacture of trichlorophenols, batchwise,
hydrolyzing tetrachlorobenzenes to produce trichloro­ or in continuous manner, on an economically commer­
phenols. It relates more particularly to improvements cial scale.
in a process for carrying out the hydrolysis of a tetraSodium hydroxide is the preferred alkali metal hy­
chlorobenzene to produce a trichlorophenol.
droxide to be employed in the hydrolysis. Other alkali
United States Patent No. 2,615,923 forms trichloro- 20 metal hydroxides such as potassium hydroxide, or lithium
phenols by reacting tetrachlorobenzenes with an alkali hydroxide, or mixtures of any two or more alkali metal
metal hydroxide dissolved in, or mixed with, methyl al­ hydroxides can be used.
cohol at elevated temperatures and pressures. 0 . S.
The alkali metal hydroxide is employed in amount
Patent No. 2,509,245 makes 2,4,5-trichlorophenol by corresponding to from 1.8 to 4.5, preferably from 2 to
healing 1,2,4,5-tetrachlorobenzene at temperatures be­ 25 4 gram molecular proportions per gram molecular equiva­
tween 170* and 190* C. at atmospheric to 20 pounds lent proportion of the tetrachlorobenzene employed, and
pressure with an alkali metal hydroxide dissolved in, or in admixture with sufficient water to form an aqueous
mixed with ethylene glycol or propylene glycol.
solution of the alkali metal hydroxide in from 2 to 10 ,
There are several objections to the methods hereto­ preferably from 2 to 8 , percent by weight concentration.
fore proposed for tbe preparation of trichlorophenols 30 The hydrolysis is carried out with agitation at tem­
from tetrachlorobenzenes particularly when applied to peratures between 225* and 300* C., preferably from
the alkaline hydrolysis of tetrachlorobenzenes to produce 240* to 280* C., and under a pressure at least as high
trichlorophenols on a commercial scale. The principal as the autogenous pressure of the mixture of the ma­
disadvantage is that of employing an organic liquid or terials. Pressures of from about 400 to 1500 pounds
solvent in the hydrolysis reaction. The employment of . per square inch are satisfactory and pressures of from
an organic liquid in the hydrolysis results in the forma­ 400 to 3000 pounds per square inch gauge, or higher can
tion of byproduct ethers and increases the problems of, be used.
separation and recovery of the final product.
The hydrolysis proceeds readily under the aforemen­
When methyl alcohol is employed in the hydrolysis tioned reaction conditions to produce the trichloroconsiderable quantities of the alcohol are consumed in 40 phenols or salts thereof in appreciable yield in a re­
forming ethers, e. g. dimethylether, or methyl ether of action time of from about 6 to 120 minutes. The rate
trichlorophencl, and the alcohol must be replaced by new at which the reaction occurs is dependent in part upon
supplies. The employment of a glycol in the reaction the concentration of the alkali metal hydroxide in tbe
increases the problems of separation and recovery of the 4 - aqueous starting solution and the reaction temperature
final product. Also, the employment of an organic liquid employed, within the ranges here specified. The degree
or solvent in the hydrolysis takes up valuable reactor space of hydrolysis is dependent upon the reaction time, under
and lowers the capacity for reactants of a given reaction otherwise similar reaction conditions. At the highqr re­
vessel, is expensive and adds to the cost of the final action temperatures, e. g. 250* to 300* C., herein speciproduct.
50 lied, the hydrolysis reaction proceeds to completion at
It has now been found that a tetrachlorobenzene hav­ a faster rate and in a shorter time than at lower tem­
ing the empirical formula CsHsCls, e. g. 1,2,4,5-tetra­ peratures. The mixture of materials should not be heated
chlorobenzene, or a mixture of isomeric tetrachloroben­ at the reaction temperatures herein specified for pro­
zenes, can readily be hydrolyzed, whereby a single chlo­ longed periods of time such as to result in the removal
rine atom is replaced by a hydroxyl group or tbe cor- 5 ,-, cf more than one chlorine atom from a molecule of the
. responding metaloxy group to produce trichlorophenols tetrachlorobenzene starting material, or result in de­
| or salts thereof in good yield, by heating the tetrachloro­ terioration of the trichlorophenol product.
benzene in admixture with an alkali metal hydroxide and
In practice, the tetrachlorobenzene, the alkali metal
water at elevated temperatures and pressures as herein­ hydroxide and water, or an aqueous solution of the alkali
after described.
00 metal hydroxide, are placed in a suitable pressure resist­
More specifically, it has been discovered that the ant vessel, e. g. an iron, steel, or stainless steel autoclave,
alkaline hydrolysis of tetrachlorobenzenes to produce tri- in the desired proportions. The mixture is agitated and
chlorophenols in good yield can readily be carried out heated at reaction temperatures between 225* and 300*
under tbe following set of reaction conditions: (a) carry­ C. under at least the autogenous pressure of the mixture
ing out of the hydrolysis reaction at temperatures be­ 05 of materials for a reaction time of from about 6 to 120
tween 225* and 300* C., preferably from 240* to 280* minutes, then cooled. The hydrolysis mixture is removed
C.; (b) employing the alkali metal hydroxide in amount from the reactor. In the instance where the tetrachloro­
corresponding to from 1.8 to 4-5, preferably from 2 to 4, benzene starting material, e. g. 1,2,4,5-tetrachlorobenzene,
gram molecular proportion^ of the alkali metal hydrox­ is a crystalline material at qrdinary temperatures the unide per gram molecular equivalent proportion of the 70 reacted tetrachlorobenzene is usually recovered by filter­
tetrachlorobenzene and in admixture with sufficient water ing the hydrolysis mixture. The filtrate, or aqueous hy­
to form an aqueous solution containing from 2 to 10 , drolysis liquor containing the trichlorophenol in the form

5941

A

V

i

M,7VV,713

s

0
0
1

I
f•
Ml1

3
4
*'*. of its alkali metal salt, b thereafter aririiflrd with an
TabU I
add such as sulfuric acid, or hydrochloric add, prefer­
ably the latter. The trichloropbenol is separated from
Products
Reaction
tionsCondi­
the aqueous liquor in usual ways, e. g. by decanting or
filtering if the product is a solid, or by extraction oi the 6
Conver­
Run No.
1 .3,4,6- 3,4,6-Trlcblorosion.
aqueous
liquor with a water-immisdble solvent
Trtrapbenol
eentPer- Cblorosuch as benzene, toluene, or chlorobenzene to dissolve
Ttms,
rrçp
.
Benxene,
Min.
the trichloropbenoL The trichloropbenol Is recovered
Yield,
Oms. Oms. Percent
from the solvent in usual ways, e. g. by distillation.
The hydrolysis of the tetrachlorobenzene can be carried 10
out in continuous manner by feeding the tetrachloro­ I.............. 130
83,
3
It10.46 IS.
SI
64
76J
10.3
to
*40
n
benzene and a solution of the alkali metal hydroxide and S.................
e
ss
»si
7.6
300
water in the desired proportions to a reaction zone, e. g. s..............
7S6
«.............. to
7.6 10.8
IS 4
10
X»
a tubular reactor or other suitable pressure resistant
vessel, wherein the materials are agitated and heated in 15
EXAMPLE 3
admixture with one another under pressures at least as
In each of a series oi experiments, a charge of 1,2,4,5high as the autogenous pressure and at temperatures and
similar to that employed in Example
for reaction times as previously described, and thereafter tetrachlorobenzene
sodium hydroxide in proportions corresponding
cooling and discharging the hydrolyzed mixture from the 2to, and
one gram molecular proportion of the tetrachloro­
reaction zone, after which the trichlorophenol product is 20 benzene
and two gram molecular equivalent proportions
recovered in usual ways.
the sodium hydroxide, and sufficient water to form an
The following examples illustrate ways in which the of
aqueous solution of the sodium hydroxide in concentra­
principle of the invention has been applied but are not tion
as stated in the following table, was placed in an
to be construed as limiting its scope.
25 iron bomb. The mixture was agitated and heated at a
EXAMPLE 1
temperature of 275* C. under the autogenous pressure of
A charge of 113.4 grams (0.525 mole) of 1,2,4,5- the materials for a period of 10 minutes, then cooled.
tetrachlorobenzene having a freezing point of 138* C., The trichlorophenol was recovered from the reacted mix­
42 grams (1.05 moles) of sodium hydroxide and 900 ture employing procedures similar to those described in
cc. of water, was placed in an iron bomb. The mixture SO Example 2. Table II identifies the experiments and gives
was agitated and heated at a temperature of 250* C. under the concentration of the sodium hydroxide in percent by
the autogenous pressure of the mixture of materials for weight in the aqueous starting solution. The table gives
a period of 20 minutes, then cooled. The bomb was the percent conversion, the weight in grams of the re­
1,2,4,5-tetrachlorobenzene, the weight in grams
opened and the contents removed. The mixture was fil­ covered
tered to recover the unreacted 1,2,4,5-tetracfalorobenzene, 35 of 2,4,5-trichlorophenol obtained and the percent yield
of said product, based on the 1,2,4,5-tetrachlorobenzene
which was washed with water and dried. There was ob­ consumed
in the reaction. *'
tained 38.3 grams of unreacted 1,2,4,5-tetrachlorobenzene.
The conversion was 66 percent, based on the tetrachloro­
T a b U II
benzene starting material. The filtrate, i. e. the aqueous
hydrolysis liquor, was acidified with dilute hydrochloric 40
Hydrolysis
Products
Solution
acid. The acidic mixture was extracted three times, each *9
with a 100 cc. portion of benzene, and the benzene solu­
Conver­
Ban No. Concentra­
sion, 1A4.6tions combined. The solution was distilled to separate
3,4,5-TrlehloroPercent
tion
ot
Tetrapbenol
the components. There was obtained 56.3 grams (0.28
Sodium
ChloroHydroxide
mole) of 2,4,5-trichlorophenol having a freezing point 45
Benzene,
InPercent
Water,
Oms. Oms. Percent
Yield,
of 63.5* C and 1.8 grams of residor. The yield of 2,4,5trichloropbenol product is 80.5 percent, based on the
1.2.4.5- tetrachlorobenzene consumed in the reaction.
sSO
00
so
81«
ISO
S-----------------78
SO 30.4
n
to...«....,,....,..
10
EXAMPLE 2
M
40.0
3S0
SS7
60
In each of a series of experiments, a charge of 37.8
EXAMPLE 4
grams of 1,2,4,5-tetrachlorobenzene (freezing point 131*
C ), 14 grams (0.35 mole) of sodium hydroxide and
A charge oi 43.2 grams of crude tetrachlorobenzenes
300 cc. of water was placed in an iron bomb. The consisting oi 75 percent by weight of 1,2,3.4-tetrachloromixture was agitated and heated at a temperature and 66 benzene, 18 percent of 1,2,4,5-tetrachIorobeiizene and 7
for a reaction time stated in the following table under percent of 1,2,4-trichlorobenzene was placed in an iron
the autogenous pressure of the materials, then cooled. bomb. A charge of 16 grams of sodium hydroxide and
The reacted materials were removed from the bomb.
304 cc. of water were added. The mixture was agitated
The mixture was filtered to separate the unreacted tetraand heated at a temperature of 250* C. under the autog­
chlorobenzene. The filtrate was acidified with aqueous 60 enous pressure of the materials for a period of 20 minutes,
hydrochloric add and the acidic mixture extracted with
then cooled. The bomb was opened and the contents
benzene to separate the trichlorophenol product from the
removed. The reacted materials were extracted with
aqueous liquor. The product was recovered by heating
benzene. The aqueous and organic layers were separated.
the benzene solution to distill and separate the benzene The organic layer was distilled. There was obtained 16.4
from the 2,4,5-trichlorophenoL The product was ob­ 65 grains of unreacted tetrachlorobenzenes. The aqueous
tained as residue after distillation of the benzene. Table
hydrolysis liquor was made acidic with hydrochloric add,
I identifies the experiments and gives the temperature
then extracted with benzene. The benzene solution was
and the time in minutes for which the hydrolysis reaction
heated to distill the benzene. There was obtained as
was carried out. The table gives the percent conversion
residue or product 22.8 grams of trichloropbenols. The
based on the tetrachlorobenzene starting material. The 70 product was analyzed and found to consist of 65 percent
table also gives the weight in grams of the unreacted
by weight of 2,3,6-trichlorophenol, 18 percent of 2,4,5-tri1.2.4.5- tetrachlorobenzene and tjhe' weight of the 2,4,5- chloropbenol, and 17 percent of 2,3,¿trichloropbenol.
trichlorophenol product obtained and the yield of said
EXAMPLE 5
product, based on the 1,2,4,5-tetrachlorobenzene con­
sumed in the reaction.
76
In each of a aeries of experiments, a charge of 1 ,2,4,5-

584

9 ,700,718

i

6
. tetrachlorobenzene (freezing point 131* C.) and »odium
alkaline hydrolysis of a tetrachlorobenzene, the steps
which consist in hydrolyzing 1 ,2 ,4 ,5 -tetrachlorobenzene by
hydroxide in proportion as stated in the following table,
together with sufficient water to form an aqueous 5 weight
heating a mixture of one gram molecular proportion of the
percent solution of the sodium hydroxide, was placed in
1,2,4,5-tetrachlorobenzene, from 1.8 to 4.5 gram molecular
an iron bomb. The mixture was agitated and heated at a 3 equivalent proportions of sodium hydroxide and sufficient
water to form an aqueous 2 to 10 weight percent solution
temperature of 250* C. for a period of 20 minutes, then
cooled. The reacted materials were removed from the
of the sodium hydroxide, at reaction temperatures between
bomb. The products were recovered employing pro­ 225* and 300* C. under at least the autogenous pressure
of the mixture of materials for a reaction time of from
cedures similar to those described in Example 2. Table
III identifies the experiments and gives the gram molecular 10 6 to 120 minutes, then cooling the mixture and recovering
proportions of i,2.4,5-tetrachlorobenzene and sodium
2.-t.5-trichlorophenol from the hydrolyzed mixture.
hydroxide employed in the hydrolysis reaction. The table
4. A process as claimed in claim 3, wherein the sodium
hydroxide is employed in amount corresponding to from
gives the time and temperature conditions under which
2 to 4 gram molecular proportions per gram molecular
the hydrolysis was carried out. The table also gives the
percent conversion, based on the tetrachlorobenzenc start­ 13 equivalent proportion of the tetrachlorobenzene starting
ing material, the yield of 2,4,5-trichlorophenol obtained,
material.
5. In a process for making a trichlorophenol by the
based on the 1,2,4,5-tetrmchlorobenzene consumed in the
alkaline hydrolysis of a tetrachlorobenzene, the steps which
reaction, and the freezing point of the trichlorophenol.
consist in hydrolyzing a tetrachlorobenzene by continuTabU IU
20 ously feeding a tetrachlorobenzene and sodium hydroxide
dissolved in sufficient water to form an aqueous 2 to 10
Reaction
Con­ Con­ 2,4,0-Trlchloweight percent solution of the sodium hydroxide, in pro­
ditions
Sodium
roptvnol
1-2.4.9Kim
ver­
Chloro- Hydrosportions corresponding to from 1.8 to 4.5 gram molecular
sion,
No. Bentrne,
proportions of the sodium hydroxide per gram molecular
Temp.,
Per­
Mole Mtriis Ttme,
Yield,
PreesPer­ Point,
Mm. •O. cent cent
ta* 25 equivalent proportion of the tetrachlorobenzene, to a
reaction zone wherein the materials are heated in admix­
•c.
ture with one another at temperatures between 225* and
300* C. under at least the autogenous pressure of the
6»
80
99.7
1 ........
2
20
2»
1
SI
2»
70
2........
20
90.
S
1
2.9
mixture
of materials for a reaction time of from 6 to 120
290
t1
S.0 20
a........
continuously withdrawing the reacted mixture
SO SO
4.0
290 S4
7» 99.1
20
92.S 80 minutes,
4........
from said reaction zone, and separating the trichloro­
phenol from the hydrolyzed mixture.
We claim:
*t
1. In a process for making a trichlorophenol by the
References Cited in the file of this patent V alkaline hydrolysis of a tetrachlorobcnzene, the step which
consists in hydrolyzing a tetrachlorobenzene by beating 35
UNITED STATES,PATENTS
a mixture of one gram molecular proportion of a tetra­
2,059,245
Nikawitz et aL ---------------May 30,1950
chlorobenzene, from 1.8 to 4.5 gram molecular equivalent
FOREIGN PATENTS
proportions of an alkali metal hydroxide and sufficient
.«rater to form an aqueous 2 to 10 weight percent solution
1,049,023 France_________________Aug. 12,1953
of the alkali metal hydroxide, at reaction temperatures
(Corresponding Great Britain, 718,779, Nov. 17, 1954)
between 225* C. and 300* C. under at least the autog­
OTHER REFERENCES
enous pressure of the mixture.
Hinaji:
Chem.
Abstracts, voL 48 (1954), Col 2774
2. A process as claimed in claim 1, wherein the alkali
( 1 page).
metal hydroxide is sodium hydroxide.
3. In a process for making a trichlorophenol by the w

/
i

594 "

3

I
11

3

SRC U L E S P O W D E R C O M P A N Y

August 2, 1962

JACKSONVILLEj ARKANSAS
OPERATIONS BOOKLET

t
This booklet gives information on the chemistry,
properties, equipment, method of manufacture etc.
for the various items produced at the
Jacksonville, Arkansas plant. It is a sussaxy of
the operations as they currently exist.
It should be noted that Jacksonville personnel
helped considerably in compiling the data
presented in the booklet.

A

11
2,4-DICHLOROPHENOXYACETlC ACID (2.4-D)

2,4-dichlorophenoxyacetic acid is prepared by coupling monochloracetic
acid with dichlorophenol as follows:
CHgCIC-OH

+ NaOH

A '

— ^ CHgCIC

^N a

sodium hydroxide
MW 1+0

NaMCA
ONa

+ NaOH

2,4 DCP
MW 163

+

Cl

OC l

Caustic
MW 1+0

NaCl

+

0

Cl

“

< -s-®

2,4-D acid
MW 221
MP H+1°C

with HC1.

+ %0

/r

OCHgC?

G f
Cl

+ NaCl
6odium chloride
MW 5 8 .5

sodium 6alt of
2,l+-dichlorophenoxyacetic acid
MW 243

This is essentially a. Williamson synthesis.
produces the sodium salt of 2,4-D acid.

11

sodium glycolate, etc.

sodium 2,4-dichlorophenate

©
o -c h 2 c -o h

+ HgO + CHgOHC-ONa + NaCl

The coupling process

The 2,4-D i6 released by acidifying

Final product is a white to tan granular solid and contains about 1^

impurities including NaCl.
EQUIPMENT:
One 1,500 gallon stainless steel, agitated and jacketed coupling reactor.
One 1,200 gallon steel, agitated and Jacketed coupling reactor which is
generally used with 'the recovered DCP and TCP.
v

MCA, DCP and HC1 are stored in tanks as mentioned in previous sections.

/

t

- 12 50$ Caustic storage tank, steel - 12,000 gallons.
Weigh tank to measure DCP, glassed steel on platform scales-300 gallons.
Caustic drop tank, steel - 350 gallons♦
Rotary vacuum drum filter.
Auxiliary items such as vacuum pump, etc.
Rotary drum filter feed tank, wood, with agitator - 5,000
gallons.
Slurry tank to hold cake from rotary drum filter, heated and
agitated.
Filtrate tank for liquor from rotary drum filter - 7,500 gallon.
Acidification equipment including:
Small stainless steel mix tank with agitator - 75 gallons.
Cooling tank, steel - 350 gallons.

t

Large vacuum filter, Cypress wood, similar to a large Buchner
funnel.
Carts to convey wet 2,U-D acid to drying area.
Atmospheric tray dryers. (Screen bottom trays)
Miscellaneous pumps, etc.

METHOD OF MANUFACTURE:
DCP, water, and half of the caustic pumped to coupling «actor.
Initial temperature held at less than 55*0.
MCA and the remainder of the caustic then added with coding, pH of
10.0 to 10.5 important.
Temperature allowed to go to 75-85*0.
Mixture now contains various products including sodium chloride, sodium
glycolate, sodium dichlorophenate, sodium salt of dichlorophenoxyacetic acid.
The coupling reaction takes approximately 8 hours.

-

13

-

Material pumped, to rotary vacuum drum filter feed tank along with more
vater.
Slurry filtered on rotary vacuum drum filter.
Filtrate containing unreacted DCP, 6odium chloride, degraded
monochloroacetic acid and some dissolved sodium salt of 2,4-D acid pumped to a
storage tank.

The 2,4-D and DCP are recovered by pri adjustment and toluene

extraction.
Cake from the rotary drum filter washed and dumped into a slurry tank.
This contains the sodium salt of 2,4-D acid.
The cake slurry is pumped to a mix-tank and adjusted with HC1 on a
continuous basis to maintain a pH of less than 1.

This releases the 2,4-D acid.

The adjusted slurry fed by gravity to a large Buchner type filter.
Filtrate goes to wash tank.

pH adjusted.

Secondary filtrate to sewer.
t

The cake is washed to remove most of the NaCI and the wet 2.4-D acid
shoveled into carts.
The carts are moved to the drying areas and the 2,4-D acid dryed for use
later in the esterification operation.

Wet product is used when formulating

amines.
Obtain about 4,000 pounds 2,4-D acid per batch.
CAPACITY;
Currently about 200,000 lbs./month.
UNIT QUANTITIES (per 100 lbs. 2,4-D)
Theory

Actual

DCP

73-8

*105

MCA

42-7 ^

64

NaOH

(50$)

73

HC1 as 32/0 (by-product from DCP & MCA operations^
Toluene (gal)
f' 1
♦This should drop to 82 assuming 9 0 $ receovery of DCP.
♦♦Excess caustic used.

Ai'6. 67
14. ^
$0t 0

♦ * 12 5

165
4 .3

<$ ^
v „V»•

(

1*

5948

- lU 2 , k ,5-TRICHLOROPHEHOXYACEnC ACID (2,U,5-T)

CHEMISTRY:
2 iU,5-Trichlorophenoxyacetic acid is prepared by coupling
oonochloroacetic acid with trichlorophenol (in a sodium salt solution).

The

reaction is summarized below:

CH2C1C-0H

©

+ NaOH

ChgCICf-ONa

+ HgO

sodium MCA

MCA
MW 9 U . 5

ONa

Cl
Cl

Cl

sodiûm 2,k 5-trichlorophenate
MW 219.5
(TCP MW 197.5)

&
0

O-CHpC-OH
\

•D

H rP
HC1

NaCl

OCH J
4
'ONa

Cl

= ,0

+ NaCl -

Cl

sodium salt of
2,U,5-Trichlorophenoxyacetic acid
MW 277.5
^

2,Uy5-T acid
MW 255.5
MP 153SC

■ '

j.

i.t.J-T ’ r*>- 7'
As with the 2jk-D reaction this is essentially a Williamson synthesis for
amyl-alkyl ethers.

The coupling process produces the sodium salt of 2,4,5-T acid.

The 2 , h 5-T is released by acidifying with HC1.
solid and contains about

2%impurities such as tetrachlorobenzene and sodium

chloride.

EQUIPMENT:

Final product is a tan granular

-------- - -I

One 750 gallon stainless steel

agitated and Jacketed coupling reactor.

5949

I
- 15 -

MCA, HC1 and caustic are stored in tanks as mentioned in 2,^-D section.
Rotary vacuum drum filter with auxiliary equipment such as vacuum pump,
feed tank, filtrate tank, and cake slurry tank.
Acidification equipment including:
Cypress wood mix tank with agitator - 100 gallons.
Cypress wood cooling tank - 200 gallons.
Large vacuum filter, cypress wood - similar to a large Buchner
funnel.
Carts to convey wet 2,U,5-T acid to drying area.
Same atmospheric tray dryers as used with 2,U-D.
Miscellaneous pumps, etc.
METHOD OF MANUFACTURE:

f

Essentially the same as 2,L-D except less caustic required due to
excess amount already in the TCP slurry.
w

Cycle time for coupling about 8 hours.
Separation made as with 2,L-D.
Obtain about 1,100 pounds 2,L,5-T acid per batch.
CAPACITY:
Currently about 70,000 lbs./month.
UNIT QUANTITIES (per 100 lbs. 2,U,5-T)
Theory

Actual

TCP

77-3

*1 1 1

MCA

37

60

HC1 (as 32$)

-

150

Toluene (gals)

-

NaOH (50$)

( ,

A'jo y'
i* t,n
44, ez
4Z.1-! y

6 .0

50

c 2_. 7

■•This should drop to 86 assuming 90$ recovery of :TCP.

5950

5951

: 2 .2 s

T U X K 'o m C .Y

(II'

IIIC IM lIl'IDK S

/)cfKirlnn‘tit of ì ’inirnmrottnf!/ nini To.rirolnft;/, Ixoijnl V oh iiin tnj nini . 1f// iritffimil ('olirti!-,
( fij/ir/f/ffff/rr«, t h i i i / n u k

T a III.U o r C lIN T U N T H
I. I ni rullili') imi

11. Inorganic herbicides
A. A I'H C lli I I'M .
M. Sodium chlorate.
C. Sulphuric acid . .
III. Organic herbicides .........................................................................
A. C hloriiialed phcrioxy-aeids.................................................
II. Chlorinated aliphatic acids and their sodium salts
C. Carbam ates and allyl alcoh ol............................................
D . Substituted u rea s.....................................................................
E. T riazin os.......................................................................................
F. S ubstituted p h en ols................................................................
G. M iscellaneous organic herbicides.....................................
1. T ributyl phosphorotrithioate (D E F)
2. M aleic hydrazide
3. Endothal sodium
• 4. D iquat (K cglonc)
5. A m inotriazole
IV. Conclusions

231
234
230
235
240
241
241
243
243
244
244
215

I. INTRODUCTION

In their endeavours to raise productivity, agriculturists have, made use of
chemicals to a steadily increasing extent For the purpose, of controlling injurious
insects, fungi, weeds, etc., a great number of pesticides has been developed,
among which the insecticides,' especially the organophosphate cholinesterase
inhibitors, have attracted considerable toxicological interest.. Relatively little
has been written about herbicides, i.e., compounds which have been found useful
for weed control. Many of the toxicological data underlying assessments of the
risk involved by using them in practice, originate from confidential, non-published
reports placed at the disposal of the authorities concerned. Such data have not
been included in the present survey. Of a number of compounds, the toxicology
of which has been elucidated in relation to their use for purposes other than as
herbicides, only a brief account will be given with references to relevant literature.
Regarding the practical use of herbicides, reference, is made to handbook
literature (70, 80, 119, 120). Weedkillers usually are. classified into two main
groups, selective and non-selcctive. The selective subslances can be used on
crops without damaging the. cultivated plants, whereas the noil-selective types
kill all vegetation. Substances of the latter group may therefore be used also for
destroying potato haulm and for desiccation of green parts of plants which may
unduly delay harvesting, in which case they are often called desiccants or de­
foliants. The herbicides arc grouped, according to their mode of distribution in
225

5952

i ;ui.. :
Ini-i n--. m - u ** *. i.-. i .<*. .
from >wpo?*cd parts of foliage or rool>, ami residual herbicides, which arc spri-ad
on or in the soil and arc effective mainly against germinating seeds.
•
For the present survey of the toxicology of the herbicides, a classification
according to chemical configuration has been chosen. Inasmuch as common
names, abbreviations, and registered trade marks ® are used indiscriminately in
the literature, wc have, as far as possible, included the common names recom­
mended for pesticides by the British Standards Institution, marked (*), names
approved by the British Weed Control Council (f), and those approved by the
Weed Society of America (J).
ir. .in o r g a n ic h e r b ic id e s

Prior to the Second World War, mainly inorganic compounds were used for
chemical weed control. However, as their actions are not very selective and they
arc often very persistent in the soil, so that damage to cultivated crops has been
difficult to avoid, they have been replaced by organic compounds to a steadily
increasing extent. Yet, various familiar inorganic compounds arc still used as
herbicides, e.g., calcium cyanamidc, cupric sulphate, ferrous sulphate, mercurous
chloride, potassium cyanutc, and sodium tetraborate (borax). The toxicological
data on these arc well known from the handbook literature. The same is true
of arsenites, sodium chlorate, and sulphuric acid, but these compounds will
nevertheless by described briefly, because their use as herbicides has introduced
special problems of toxicity.
A , Arsenites
Arsenites are used as non-selcctive herbicides, especially for destruction of
potato haulm, as aqueous solutions of potassium-ortho-arsenite (Kj.VsjOs) and
sodium ortho-arsenite (Xa*Asj03). The preparations may also contain metaarsenitc and pyro-arsenite alkali salts.
In rats, the LU50 for alkali arsenites has been found to be 70 mg 'kg when
administered by mouth and 150 ing/kg when applied dennally (32). In domestic
animals (40) and man (103) the toxicity is considerably higher, fatalities having
occurred in the horse, cow, and man after oral ingestion of 2 lo 10 mg/kg. The
solubility, preparation, and purity have a considerable influence on the toxicity
of arsenic compounds (48); the toxic properties, actions on the animal organism,
manifestations of poisoning, etc., have been excellently summarized recently
(10S).
The use of arsenites as weedkillers has occasioned an extensive series of acute,
often fatal cases of poisoning in domestic animals, especially cattle, which have
eaten contaminated crops or residues of sprays (40). Concentrations toxic to innii
have been found in milk from cows fed on crops contaminated by arsenites Mil).
Ca-es of direct human poisoning have also been observed. An instance has been
described where one woman died and four others fell ill after drinking water from
a reservoir which had been contaminated through a leaking pump valve in the

iii 1iii-iit Britain resulted in a voluntary agreement between iudu-try .aid 'in;
authorities for discontinuing the use of arsenite herbicides (32 i.

C. Sulphuric acid

Sulphuric acid, which in certain countries, e.g.. Great Britain, is commonly
used for potato haulm destruction, is often applied in high ennc.-mr . l ir.Accidcnls with severe skin burns and eye burns may therefore occur i:',2 . Inhalatiop of sulphuric acid may also exert a toxic effect, as indicated by studies
on guinea pigs (3, 4) and on volunteer human subjects (5).
ti t. ORl'SAMC HF.nBICIOIfs

A. Chlorinated phcnoxy-acids
Chlorinated compounds of phenoxy-acetic acid, propionic acid, and butyric
acid, have been very extensively used within the past 15 years as herbicides
under the common name of "hormone weedkillers.'' Tln v stimulate pars of
susceptible plants, particularly within the group of dicotyledon*, to excessive,
uncontrolled growth, which causes the plants to die GOP). The most imporan:
compounds, which in the forms of water-soluble salt- or lipoid—ohihlc enters
constitute the active components of commercial preparation-, are listed in
Table 1, where the LD50 values iu rats are also given, in addition n> ill" • mpounds listed in the table, p-chlorophcnoxyacetic acid ( 1-CPAi. -odium 2- 2.4dichlorophcnoxy)cthyl sulphate (2,4-DK.S-sodinm, Sesone, SMSd and y-f2.4.3trichlorophcnoxy)butyric acid (2,4,o-TI5, 4-(2,4,5-TB'i> ire usi d as heihi. ites
of this type.

5953

B. Sodium chlorate

Sodium chlorate and other chlorates are extensively used for killing all vegeta­
tion of farmyards, railway tracks, roadsides, etc., as well as for destruction of
potato haulm. Chlorates possess no particularly great acute loxieitv, the 1.1X0
for sodium chlorate administered by mouth to rats having been stated to be 12r0
mg/kg (32). Nevertheless, oral ingestion of both sodium chlorate and potassium
chlorate, used in oral hygiene, has caused numerous cases of poisoning 2i man
(21, 33, 105). In domestic animals, too, cases of acute poisoning have been ob­
served after oral ingestion of chlorates (10). The mechanism of the poisoning—
methemoglobin production and its consequences—has been well studied '21. 33.
106).
A special problem is that of the high inflammability of sodium chlorate. This
can be reduced in commercial preparations by admixture with calcium chloride
or sodium chloride, but after dissolution in water and spraying, dried residues
may become ignited. In Denmark, a tractor driver whose clothes had '• come
impregnated with sodium chlorate during spraying, died of severe bums after
they were ignited by a red-hot exhaust pipe.

225

DALGA ARD'MIKKKLSEN AND rOULSEN

TAUU5 1

LO
CO
LO

TOXICOLOGY OF HERBICIDES

229

The acute toxicity following oral administration to a number of experimental
animals is modcn.ie. The LD50 ns n rule is of the order of .'100 to 700 mg per
kilogram for the species examined (89), excepting dogs, which seem to be more
susceptible, where the LD50 for both (2,4-diehlorophenoxy)acctic acid (2,4-D).
and 2,4,5-T luis been found to be 100 mg/kg (27). Experiments with oral
administration of various salts and esters of 2,4-D as pure chemicals and as
commercial preparat ions showed no significant difference in toxicity to a number
of small animals fnm that of the free 2,4-D acid (53, 89).
Short-term studies on rats revealed no signs of reduced intake of food or
inhibition of growth in response to admixture of 2,4-D at 400 p.p.m. in the fodder
for 30 days or 1000 p.p.m. for 14 days. Subcutaneous injection of 50 to 100 mg
2 .4-D per kg to mice daily for 90 days had no demonstrable-effect on the general
condition, fertility, or tissue histology (53). In Table 2 are recorded the results
of feeding phehoxy-compounds to larger animals. Except for the rather consider­
able toxic effect on sheep of 2,4,5-TP, administered in the. form of propylene
glycol butyl ether esters, these experiments showed that the large herbivores
sooin to tolerate prolonged ingestion of phenoxy-compounds to the same extent
as rats. In dogs, on the other hai\d, these compounds were found to have a pro­
nounced toxic action: 20 mg/kg given for 2 to 3 weeks produced severe, fatal
poisoning.
Parenteral and oral administration of toxic doses of 2,4-D to experimental
animals brings about a characteristic complex of signs and symptoms (18,27, 53),
which has been studied particularly in dogs. After a few hours the animals
display a disinclination to move. This passivcncss is gradually aggravated, and a
picture of myotonia develops with rigidity of the skeletal muscles and ataxiu.
The condition may improve transitorily with movement. In severe cases the
animals show progressive apathy, depression, and muscular weakness, especially
of the hindlegs, with periodic, clonic spusms, and, finally, coma. The muscular
signs ure accompanied by marked anorexia; frequent!}’, irritation of the nose and
eyes is indicated by scratching reactions. Further, bleeding from the nose and
mouth may occur, as well us diarrhea with blood-stained stools. Local irritation
of the alimentary tract often causes vomiting. However, this sign may be absent,
even after oral administration to dogs (27). Autopsy may reveal necrotic ulcers of
the oral mucosa and signs of irritation with histologicully demonstrable inflam­
matory changes and necrosis of the small intestinal mucosa, as well as focal
necrosis in the liver (27, 53) and degeneration of the renal tubules.
When given by mouth to dogs, even in fatal cases 2,4,5-T has produced only
weak signs in the forms of ataxia and stiff movements of the hindlegs (27).
As foi; the human response, n report is available (9, 75) of a man who in a
srlf-ex-pf.nrnont ronsumed .7H) imr of 2 .-1-D daily for 3 neck wi»h »to n* r*

TA IJLU 2

Chlorinated phenoxy-acids; short-term studies in doQ, sheep, and cow

Compound
MCI’A.
2,4-0
—
—
2,4,5-T
—
—
2,4,5-TP

Daily Dote,
Animal
Retult
Specie«
Bodymü/ka
Weight
Tolerated without detectable symptoms during
Con30
21 days
Tolerated without detectable symptoms during
100
Fbeep
35 days
Dog
2-10 Tolerated without detectable symptoms during
00 days
D or
3 of 4 animals died, 18th to 49th day
20
Tolerated without detectable symptoms during
Sheep
100
35 days
Dog
2-10 . Tolerated without detectable symptoms during
90 days
20
Dog
4 of 4 animals died 11th to 75th day
Sheep
Lethal after 11 doses
100

Ref.
Ko.
(24)
(85)
(27)
(27)
(85)
(27)
(271
(85)

The action of ingested phcnoxy-acid herbicides on muscular function, which is
reminiscent of that following administration of halogenatcd acetic acid com­
pounds (22. 23), suggests an interference with carbohydrate metabolism. Two
cases of a transitory diabeliform condition observed in spraying personnel
following work with chlorinated phenoxy-acid herbicides point in the same
direction. However, hyperglycemia and glycosuria could not be reproduced with
certainty in rabbits; only one out of five animals responded in this way in ex­
ploratory experiments, where the doses ranged from 125 to 500 mg/kg and the
period of administration from G to 50 days (08).
The results of a long series of investigations into the toxicity to domestic
animals and game of hcrbicidal preparations and treated crops suggested that
acute poisoning caused by consumption of crops from sprayed fields is unlikely
to occur (lt>, 24, 4t>, 75, 87), excepting that “grass-eating” dogs may be poisoned
by newly sprayed lawns. Human beings and domestic animats arc presumably
liable to poisoning only by oral ingestion of highly concentrated preparation? or
spray solutions.
À special problem in connection with the use of chlorinated phenoxy-acid
compounds is the lendmirj» of the preparations—even in extremely low concen­
trations—to impart to water, milk, and other nutrients a very persistent chlorophonoMike odor and taste. Spilling of highly concentrated preparations close to
wells or water courses has in several instances, by percolation through the strain,
given a disagreeable taste to the water for long periods afterwards. Administra­
tion of 2,4-1) to dairy cattle does not seem to result in excretion of biologically
demonstrable amounts in the milk (75).

B. Chlorinated aliphatic acids and their sodium sa'.'-.

The chlorinated aliphatic acids possess more general phytov.xic properties.
Somo of these arc used, therefore, for defoliation and dcsiccaii' r* of rulti-ated
plants prior to mechanical harvesting. The most important f . mpounds are:
sodium trichloro-acctatc (TCA-fJ sodium), sodium monochloro-a .-’arc 'SMC'A),
and sodium-dichloropropionate (Du!apon-sodium*fi), as weil as ’he com': ined
compound, 2-(2,4,5-triehlorophcnoxy)-ethyl-r2',2'-dichloropp. r. :onate Erbonff).
Studies on the acute toxicity of sodium trirhlwn-natatc have .-how» -ha* 'bis
substance is not very toxic (118), the LDoO after oral admini-raTon 1-Ung 320
mg/kg for rats and 4970 mg.'kg for mice. The animals quickly g-,> into an a *sthesia-Iike state, which lasts for 30 hours, to lx* succeeded by <sorr.a ar.d d-«.rh.
or by awakening and survival. Sodium trichloro-acetare ha* a :‘ar less lo.-alirritating action than the free acid, which can corrode the ski:. and mucous
membranes.
Sodium monorhloro-acctate fSMCA) is considerably more
It* a«-?-,.*:, on
bacteria and in the animal organism is reminiscent of that of other x.'inohalogensubstituted acetic acid compounds, such as monoiodo- and monobromac^tic
acid, the enzyme-inhibiting and bacteriostatic proponies of *.\h: h have K-en
thoroughly studied (vide 22). The LD5G after oral admini.-tra'y'n bis r.-«Q I•■ :::d
to be 70 mg/kg for rats, and SO mg kg for guinea pigs (US . and of the same
order for geese (20.), whereas for mice the toxicity is somewhat !<v.v*r, the LD50
being 2o5 mg/kg (118). In the small experimental animals apir.hy and loss of
weight were noticed, and in the fatal cases death occurred wi*h:r. 3 days. The
fatal oral dose for young cattle was found to lie within the ra:.ir- : 10Q to '.30
mg/kg. Colicky-like restlessness and incoordination were nb-*rv»::i. developing
in the course of 4 to 5 hours into universal fascicular twitching?-. gr.i-hinc of -he
teeth, anxiety, dyspnea, and tachycardia. Coma and death foll-.v.v-.'i dior 0 he •:rs
(23). This picture, which is probably referable to blocking of ox: la v e iu *al • hv
processes, is reminiscent of that scon in pigs 22) and dogs p .-oned wvh
njonobromacetic acid. The gross and microscopic findings . .-op-y w-re
likewise identical with those described for monobromacotic a-id-r honed pig«
2

( 22).

Sodium 2,2-dichloropropinnate (Dalapnn't has become of p;.r*: ■■m‘mAr auer^s!
owing to its hcrbicidal action on monocotyledons, such a* gni-'-v. :n relatively
small quantities, therefore being useful as a selective herbicide cer jin crops of
cultivated plants. Dalapon is absorbed by plants and then trandoci*-*d. On tki*

5955

Very few toxicological data arc available regarding other chlorinated an made
acids, introduced alone or in combination as pre-cmergener- and .¡■ elective herbi­
cides for agricultural crops. 2,3,6-Trichlarophcnyl-acotic avid h-naci). 2.3.0trichlorobenzoic acid (2,3,6-TBAft, TBA. TCB) and .’^amir.o-S.o-dicLJorobenzoic acid (amibenj.) are used for these purposes. The LDoO of 2.3,8-TBA
after n single oral dose to rats was stated to be within the rang- of 700 to I’OO
mg/kg (32).

-rlU

-ud»

IA

lli ^.«

toxicity ui the substance has been studied from .sovrrul n.-|><vis ¡brumdi ru;:
prohensive series of experiments (81). Administration of single doses to a mimbor
of small experimental animals has given LD50 values for rats, mice, guinea pigs,
rabbits, and chickens within the range of 4000 to 9000 mg/kg. Two young cattle
survived 1000 mg/kg given by mouth daily for 10 days. Beyond transitory
symptoms in one of these (anorexia, diarrhea, indisposition), no signs of a toxic
action «ere found by clinical or pathological examination. After oral administra­
tion of 15, 50, and 100 mg/ kg daily to dogs for one year, blood and urine analyses,
as well as liver function tests and histological examination of tissues revealed no
sign.* of a toxic action, beyond an increased weight of the kidneys following the
dose of 100 mg/kg. Examination of tissue specimens from the dogs showed up
to “S p.p.m. of Dalapon in kidney and liver tissues (81).
Feeding of rats with Dalapon for 2 years in doses of 100, 300, and 1000 p.p.m.
in the fodder, corresponding to about 5, 15, and .50 mg/kg daily, was tolerated
with no signs of a toxic action, apart from a minor increase in kidney weight on
the largest dose, fn these experiments with 1000 p.p.m., 10 to 30 p.p.m. of Dala­
pon was fourni on chemical analysis of the liver and kidney, and 20 p.p.m. in
milk. Thus, cumulation docs not take place. In rats, reproduction and lactation
proved to be uninfluenced through three generations with daily administration
of 300 to 3000 p.p.m. of Dalapon in the fodder (81).
The local-irritating action of Dalapon has been studied on rabbits, the skin of
which was exposed daily for 10 days to a 10% aqueous solution. No more than
signs of a mild, transitory irritation was seen (81).
.1

»

C. ('nrhnmairs and aUtjl alrohol

Within this group of herbicides, which, as shown in Table 3, comprises enrhu­
mâtes, thiocarbamatos, and dithiocarbamates, is a number of compounds which
are used especially as pre-emergence or selective herbicides on certain crops.
Propham and eMorprophum have also been used to prevent potatoes intended
for consumption from sprouting while stored. The toxicity of these compound«
with prolonged ingestion is therefore of particular interest.
Propham (O-Awpropyl N-phonyl carbamate, JFC). Various investigations
have shown that carbamate esters possess a carcinogenic action. Thus intraperi­
toneal injection of isopropyl carbamate raised the frequency of lung tumors in a
strain of mice from 17 to 90% (00). The idea of inquiring into a possible cam»»*
gonic action of Propham, which splits off aniline by acid hydrolysis, therefore
suggested itself. However, with prolonged oral, intramuscular, ami intrapleural
administration of Propham to rats and mice, no signs of a carcinogenic action
were found (50). This is in agreement with the results of experiments on group»*
of four rats each, given 400, 800, and 1000 p.p.m., respectively, daily in the fodder
for 3 months. No signs were demonstrable here of a toxic action on the. general
condition, growth, or fertility, and no pathological changes were seen at autop.\V
or on histological examination of the tissues (101).

a n d i.iln- •>, J . J ’-*0 l>\ i.>r;ti miiiiiii. t..i. .

^

.......

#

5956

i• ..vd'ii t tii

to be of the order of oOUO mg per kg for both species (I l'». Ir- feeding experi­
ments on male rats given 310 to 20,000 p.p.m. for 00 days, no efT-6*?? was cr a n ed
on growth. Doses of 12o0 p.p.m. and higher produced an increase in w*.-:,*ht o:
their livers, without histological changes being demonstrable i'll*' •.
In the light of the above investigations, the observation of th* «lcve!opn.*-nT of
skin tumors following painting of the back skin of Prophan.- an-: Chlorpropaamfed rats with croton oil is of considerable theoretical ¡mere4’ 34). However,
comprehensive feeding experiments (G7) with administration m 2C00 p.p.m.
through* 2 years to rats and 1 year to dogs revealed no car.inocenic crTcct.
Feeding at 20,000 p.p.m. provoked in both species signs of a to:.;'* action. which
was manifested in rats by retarded growth, increased m ortify of the male
animals, and increased liver and kidney weights, though wit ho ;; deir.onsirahle
histological changes. In dogs, retarded grotvth, increased weights of liver, kiiney.
and spleen, and splenic congestion were noted (fi7).
Barbane (4-chloro-2-butynyl-X-(3-chlorophcnylJcarbam3te:' di-play* a ^omewhat greater acute toxicity than the Prophnm compounds, the i.D"»U cio.r ora]
administration having been found to be GOO mg per kg for rhe rat and rabbit,
and 240 mg/kg for the guinea pig. Dermal application of 1*300 me kg ov-r a
period of 24 hours caused no deaths among rats. Oral administration o: 0. 1:*.5.
and 37 mg/kg daily for 22 days produced no toxic reaction, whereas 7-3 n.j kg
. over the same period efTocted loss of weight. Feeding esporin.-mts with rats
• showed no toxic action of loO p.p.m. for IS months (36). Barbane is a potent
akin-sensitizing agent in man, in whom allergic reaction with r^h dev-dor? at
subsequent contact. Protection against cutaneous contact i- 'her- ."re
:rv
during its use. Plastic (polyvinyl chloride) seems to be a more suitable protr-.-ive
. materiaf than rubber (36). '
^ . SMDC (sodium N-mcthyl dithiocarbamatc dihvdrate. merhan;--odiuni. Las a
. special sphere of application, hoing used for killing weed seeds. -• ;l u*?.'.&•> ies.
and the like by so-called soil sterilization. When applied in :he ~.»il it Iiber;.tes
gaseousmefAi//iso//itnc>/ano/e, the active substance, which is also available commer­
cially as an aqueous solution (Trapex) for similar purposes. The I.D-30 o: SMDC
by oral administration to rats has been stated to bo 820 mg kz 12'.« . Th~ tox; ity
Of methylisothiocyamle is considerably higher, the LD.30 bo;::g !>7 mg kz >2).
The main toxicological interest attaches, however, to the pronounced lo.--.ily
Irritating action of these substances on the skin and mucous mor..i»ran*s. • -pecially those of the respiratory* organs, as well as the possibility c>: ¿haorp::«:-:. of
toxic amounts by these routes. The LD.30 of SMDC after dermal ..ppi:•.-.i; to
rabbits has been found to !>»■ 800 mg kg (20). whereas daily ru: hi*_• -.f u.. *V\ - vihlocyanate in 10‘7? ethanol for 0 weeks into rabbit ears g:iv<- r. • nor-* ?h..\ a
Weak reaction (82).
•.. Allyl alcohol. This unsaturated alcohol has the same rang*» of
as
.SMDC and methylisothiocyanate; it is applied in solution to the ~ '.I. -vher ,;s

fi

5

i

5

%y p,

«£

>,
j .'

5960

5961

c ©

' t

/

/ ¿>

O*.

CHEM ICAL
MIDLAND, 'Michigan
March 2y, 1965

Riley
R. N. Smiley
L. B. Grant
C. 0. Hutchenreuther
F. C. Amstutz
W. P. Falsey
G. E. L y n n '
• W. M. Gill
M. G. Wiltse
D. E. Pletcher
W. L. Corbin
D. D. Irish
J. E. Peterson

019193

K
Ì . Rowe
Biochemical Research Laboratory
1701- Building
J. C. T u cker
J. W. Harris
H. W. F e i nauer
E. C- Staehling •
C. E. Otis
K. Y..Hansen
R. C. Hoff
W. J.’ M c C o y
J. D. Doedens
K. C . Barrons
H. R. Hoyle
B. B. Holder, M.D.
S. E. Sadek

REPORT ON THE CHLGRACNE"PROBLEM MEETING ON 3 /2 ^ /° 5
Present:

Dr. J. Wilkenfeld and
Mr. Raymond Verhoeze, Hooker Chemical C o r p o r a t i o n
Mr. Francis Kennedy and
Dr. Ed Chandler, Diamond Alleali C o m p a n y
Mr. C. L. Dunn and
Dr. John ?. Frawley, Hercules Povrder Company*

V. K. recaoped the Dov? situation in terms of the p r o b l e m
and the initial studies b y Toxicology and E n v i r o n m e n t a l
Research Laboratory regarding the in-plant situation.
He
expanded this in general terms to the study of end products,
ours and other peoples. He made reference to s y m m e t r i c a l
tetrachloro-p-dlbenzodloxln- He referred to the e v i d e n c e
for unknown acnegens. There were some questions f r o m t h e
group about the unknowns- W e (Dow) were not a b l e to a n s w e r
these questions except to review the evidence for t h e i r •
exiatence In the process samples and end product's.

CONFIDENTIAL - ELGJÙCT TO INJUNCTION
JXC, E.D. /.U. 4-4-73; COW/EPA AGREEMENT 9-79
g

PLAINTIFF'S
EXH IBIT

5962

V- K. Rowe

j£ 1-iarch 2g r 1965

Dr. Holder reviewed the medical side of the Dov; experience;
he said that we now have approximately 60 to 7 0 cases of
individuals w i t h chloracne ranging from tvjo severe cases to
some very mild cases that were difficult to diagnose. H e
showed slides of the more dramatic cases. The slides w e r e . ,
exclusively views of the faces of the individuals afflicted.
H e described in fair detail the appearance of the indivi­
duals mentioning the blackheads specifically. H e then
reviewed the clinical studies that are being m a d e on these people with emphasis on the liver function tests. H e m e n ­
tioned the single liver biopsy that has been taken and
studied in w h i c h the liver was normal although the man. had
a r£ther pronounced case of chloracne. Dr. H o l d e r also
.mentioned the incidence of fatigue among the afflicted~"D^oole
as" being the o n l y other significant .finding in these folks.
~~
H e touched b r i e f l y on treatment indicating that v arious
topical treatments were not particularly effective. H e
described the cycling of this disorder i n individuals w h o
had b e e n completely removed from exposure. He mentioned
that some fellows are approaching the end of their trouble
two cr two and one-half years after onset of the skin d i s ­
order. H e also described "acute chloracne“ w h i c h is a n a c u t e
inflammatory condition that appears considerably sooner t h a n
the normal chloracne in individuals and appear? after p r o - *
nounced single exposure. The acute chl’
o racne shows up within,
a few days of exposure. Dr. Holder mentioned five to eight
days specifically.. There w a s 'considerable discussion b y t h e
group on the skin disorder Itself. The Hooker representatives
related experience of skin condition thirty years after e x p o surel Their cases were more similar to the Dowiclde Dumps
vw Hl~dh D ow has experienced in that there were large Polls o F .
hnmns
'than one multitude or small blackheads
and eruptions wnich~~D5w is seeing IrT the" current, cases-.
Dr. S a dek showed slides of ears and livers of rabbits t h a t .
had b e e n exposed 00 zne symmetrical terrachloro-p-dlbenzodioxl
H e d i scussed the pathology in detail w h i c h I w i l l not a t t e m p t
to summarize. : ' *—
V. K. mentioned the studies in w h ich the rabbit ears have
been treated w i t h TCBD in benzene or corn oil and then
washed w i t h soap .and water at various time intervals l a t e r If exposure occurs for very long, w a s h i n g does little good.
H e also briefly mentioned the oral studies but without
detail.
Silver3tein described the plant study on w a s h i n g
of contamination from tools and surfaces. This study
Indicated that benzene, acetone and Chlorothene NU wer e
effective in removing the contaminant from tools and also
that'detergent and water with, scrubbing action.could clean
up tools and equipment.
Some discussion ensued on the use

5963

V. K. Rowe

-

3-

March 29, I955

of detergent-and w a t e r and the point was made aga i n that
strong scrubbing action was necessary for this a p p r o a c h
to be successful.
.
.•
Harold Gill then discussed the analysis for t e t r a c h l o r o- p — .
dibenzodioxin b y vapor phase chromatography. H e listed
the limit of sensitivity on various process materials. H e
mentioned the oil whi c h h e defined as a non-saponlfiable
mixture of chloro anisoles, tetrachlorobenzene and t r i chlorobenzene; the limit of sensitivity for T C B D in t h i s
material is 10 ppm. The limit is 1 ppm for 2 , 4 , 5 - t richlorophenol, and f o r 2,4,5-T Acid, either acetic or propio n i c .
Gill then defined 1 p p m as a very discernible, peak. ■ H e m e n ­
tioned that he-might estimate 0.5 p pm in some instances b u t
'to be conservative the analyst reports <1 p p m i f the p e a k
does h o t measure up to the quite identifiable level of 1 •
ppm. The analytical problem has not yet b e e n solved f o r the
.T-Acld esters. The general procedure used f o r the T-A c i a s
is to extract the sample (arbitrarily about 20 grams) w i t h
chloroform (about 40 milliliters), filter t h e - c h loroform
to remove solids and w a s h w i t h a n equal volume o f N/lO
caustic to remove any acidic materials* T h e chloroform
extract then is concentrated b y evaporation to"'one-tenth
the original volume; t h u s , 'the concentration of the d i o x i n
in the chloroform w i l l be ten times higher than in the
original sample. W h e n the analysis is conducted on t r i - .
chlorophenol, the material is dissolved in N / l caustic t o
the extent of 10^, and this solution is then extracted
w i t h the chloroform and handled as Indicated above.
A question was asked about the utilization o f detectors
other than the flame ionization which is specified i n t h e
Analytical Laboratory write-up for this analysis. Gill
h a s ‘not tried the micro coulometrlc detector becausq h e
is not set up to do so, but he has experimented w i t h
electron capture. He stated that theoretically this uriit
should not .provide a n y greater Increase in sensitivity.
In actuality he found a slight increase in s e n s i t i v i t y b u t
there are usually too m a n y chlorinated species p r e s e n t
w h i c h m a y saturate the electron capture cell w h o s e r e c o v e r y
is too slow to be of practical use. He summarized b y s a y ­
ing that the slight -increase in sensitivity' is n o t w o r t h
the effort -to switch from flame ionization t o e l e c t r o n
capture. A question was asked about how' the e x t r a c t i o n is
performed. Gill stated that it is performed in a w i d e
m o u t h bottle on a shaker for one hour.
(It w a s “n ot m e n ­
tioned, but it is the case that this is done a t r o o m
temperature.) He mentioned that spiked samples have b e e n

V. K. Rowe

4-

-

r-Iarch 29> 1955

run this way-and the recovery ranged from SO to 100 p e r
cent. The ratio of solvent to the material being extracted
on this step is not critical according to Gill. -Their
t
standard .procedure Is 20 grains of sample'and 40 mil l i l i t e r s
of chloroform.
On trichlorophenol samples specifically, .20
grams of phenol Is converted to phenate — about 10 p e r cent
concentration in water. The phenate solution is extracted
w i t h 20 milliliters of chloroform in a single extraction.
The chloroform Is then concentrated so that the c o n c e n t r a t i o n
of the dioxin w i l l be ten times that In the original sample.
The question o f volatility of dioxin came up and H a r o l d G i l l
.stated that he*found he can distill o-dichlorobenzene a w a y
f r o m tetràchlorobensodioxinJ 'He said that i n his o p i n i o n
the secret.was to avoid distilling to dryness.
•

•

A m e m b e r of the group asked if samples of standard T C B D w e r e
available. ’ T h e answer w as yes and 100 mg samples trere p r o ­
vided to one of the representatives from each company.
(a
sample had previouslj»- been given to Dr. K e lly of konsanto.)
A question of laboratory safety in the analytical w o r k
up and the basic precaution of wearing vinyl gloves w a s
mentioned. Information relative to the gloves_.we used
w as provided to the group.
”
”
.
Disposal of contaminated, laboratory materials and p l a n t •
materials was discussed. W e mentioned that D o w burns some
small amounts of waste. - Harold Gill stated that his l a b ­
oratory study of combustion showed that 99*9o p e r cent of.-'
.the dioxin sample was burned at 800°C. We described w h y
w'e felt that our practice of burning small amounts of
d ioxin was a. safe one.
V. K. then outlined the project In w h i c h plant samples and •
products (not mentioned b y name) were spiked w i t h kn o w n .,
amounts of the TCBD. The spiked samples w e r e s p l i t . f o r t he
purpose of checking our analytical procedures f o r r e c o v e r y •
and correlating these results with the bio-assay method.
The question of specification, quality control s p e c i f i c a t i o n
that is, was raised and we were asked if ve could g i v e l e v e l s
of d ioxin contamination w h i c h were permissible limits.
V. K. mentioned that at present w e are using zero w i t h a
confidence of 1 p pm in process samples. There w a s some
discussion on the problem of customers using finished p r o - '
ducts under far less desirable conditions of h e a l t h c o n t r o l
than w e can provide our workmen in our own plant. T h e r e
seemed to be agreement among the group that we could n o t
afford to sell contaminated products.
I

5965

V. K. Rowe

-

5 -

l'ìarch 29 , 1955

\

Jack Peterson then discussed the data from animal experiments using pure symmetrical tetrachlorobenzodioxin. D o s e s
ranging from 2 parts per billion to 1000 parts pe r million..
of tetrachlorobenzodioxin in benzene had ,been administered
to the. rabbit ear. Dosage in most cases was 0.1 m l per day.
Both single and multiple exposures have been studied and multiple exposures administered on a five days p e r we e k •
basis'. The significant factors in the study a r e dose,, t h e
number of applications and. the days on exposure of the
animals- The response which is reported in the gross o b ­
servation of the condition of the r a b b i t ’s ear b y the
toxicologists. This does not include pathological findings —
there is not enough .data in this area to discuss. The .level.
_ response ranges from none through very slight,, slight,
slight to moderate, moderate, moderate to- severe, severe,
and extremely severe. Jack Indicated 'to the group that
there ‘is not a sharp definition between these categories o f
response and indicated also that there is some d i f f i c u l t y
in graphing this type of response. He .described th e r e s o o n s e
from single applications to the rabbit ear firstr at 100
parts p e r million there was a severe response i n eight days;
a t 40 parts per million there was a slight response in
eleven days; a t ‘20, 10, 7 and 4- parts per m i l l i o n there w a s
no response.
These tests were run on s i n g l e .rabbits and -without washing the material off. Jack then discussed t h e
.multiple application data which he took from his m a j o r
graph of this data. The. important points that h e m a d e f r o m "
this data were first that at the limit of .VPC sensitivity.
.a severe response m a y be produced. I n other words, e v e n if.
the VPC does not detect TCBD, an animal response m a y still
occur. His. second important point was that the Induction
period for response averaged about ten days o n t h e animals
in the studies.
There v:as a brief discussion then about the a i r samples'’t h a t
were taken in the plant. Silverstein mentioned t h a t some
air samples have shown activity on the animals. T h e d e g r e e
of response is slight and the number of samples t h a t show
activity is small out of the total number taken a n d the
amount of air that must be sampled is very much l a r g e r t h a n
the amount a m a n normally breathes in a n eight h o u r day.
-The meeting was'adjourned. The group then proceeded to t h e
Toxicology Laboratory to view some of the test animals.
They
v/ere shewn responses of varying intensity and these w e r e
described. This demonstration appeared to have c o n s i d e r a b l e

Biochemical Research Laboratory
1701 Building

LGSrsjl
5966

V. K. Rowe.

C

rch 29, 1 9 6 5

- 6 -

Postscript
All participants seemed to appreciate well the problem a n d all
indicated that they would return home and attempt to convince
their management to institute safety specifications (really Equality control) for- their various products i n this area. All
agreed that the industry should meet its o wn responsibility.
All were very appreciative of Dow's effort t o steer t h e m a w a y
from a danger area. Time xfill tell whether w e a c c o mplished
our mission, but as of now X feel satisfied w i t h our e f f o r t
and the reception it received.
.

. VKR

5967

»'¡
•:'
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K,

tv-

§

5968
8

T

DEFENDANT'S
EXHIBIT
iu a .3

KIHJUCD

J to « 2 4 . 1965
Boss Bulbo Hand
lu ip r
H o p ru d u cta
| m C h en lea l o f C*aad*
S a r n ia , Canada

DOW CONFIDENTIAL

i

•

t,4,5«TSZCSLOAOPSS(QL, TBS "T" ACIM , AXD ASSOCIATED ACBBODtS

jg

Z kftT* o o t boon n e g le c t in g your roq u oat f o r la f o n s a t io o t o uao
^
i a d is c u s s in g tb * s u b je c t p ro b i* « w ith Naugatuck and tb o Co-Op. ¿ Ì
Z bar« boon s t y u le d , how ever, b*ca u so tb o a n a l y t i c a l «otboda
^
h a r e boon changed and aro l a tb o procoa* o f b e la g c lo a r o d and
* raproducod. Z oxp oct tb o« any d a y , b u t r a tb o r th a n « a l t lo n g e r ,
Z th ou gh t X abou ld a d v is e you o f tb o a l t u a t l o a . Z «111 aood
you eo p lo a o f tb o a o «otboda aa aoon aa tb o y bocoao a v a i l a b l e .
Za regard t o tb o o r o r a l l p r o b le a , *o a r o a t t o a p t l a c t o do
e v e r y th in g p o a a lb lo t o a v o id tb o p o a a lb lo o eo u rro a co o f e h l o r a o o e l a any a p p lic a t io n s in v o lv in g tb o b a n d lla c o r « so o f t r i a b lo r o p h o n o l, tr lc b lo r o p b o o o z y a c o tle a c id and I t s d e r i v a t i v e s .
Aa you « o i l t o o « , «0 bad a s e r io u s a l t u a t l o a l a o u r o p e r a tin g
p la n t s b eca u se o f o o n ta a ln a tlo n o f 2 ,4 ,5 - t r l e h l o r o p h e o o l « l t b
l a p u r l t l c s , tb o « o a t a c t iv e o f « b le b l a 2 , 3 , 7 , 8 - t e t r a c h l o r o d lb o a z o d lo z la . T h is « a t t r l a l la o x e c p t lo n a l ly t o x i c ; I t baa a
trem endous p o t e n t i a l f o r p rodu cin g e h lo r a e n e and s y a t o a le I n ­
j u r y . I f I t la p r e s e n t l a tbo t r lc h lo r o p h e n o l, I t « 1 1 1 bo
a a r r lo d th rou gh I n to tb o T a c id and l a t a tb o o s t o r s and bonce
l a t o fo r m u la tio n s « h lc h aro t o bo s o ld t o tb o p u b l i c . Qdo o f
t b s th in g s « b le b wo « s a t t o a v o id l a tb o o c c u r r e n c e o f a ay
sen * la o o o s u s e r s . X aa p a r t ic u l a r ly co n cern ed boro w ith p e r ­
s o n s «bo a ro u s in g tb o s a t a r l a l on a d a l l y , r e p e a te d b a s i s such
a a o u a to « o p e r a to r * aay u se I t . I f t h i s a b o u ld o c c u r , tb o « b o le
In d u str y « i l l be fc^^d h i t and X w ould e x p e c t r e s t r i c t i v e
l e g i s l a t i o n , e i t h e r b a r r la g th e s a t a r l a l o r p u t t in g v e r y r i g i d
e o a t r o ls upon i t . T h is l a tbo « a m r e a so n «by we a r e s o e o o aern ed t h a t v e e le a a up our own bouse f r o « w it h in , r a th e r than
h a v in g a o « io n e f r o « w ith o u t do i t f o r u s . Xa t h i s w ay, we can
approach th e p r o b le a l a a a o r d e r ly « a a o e r . Z f th e p ro d u cer«
sa d h a n d le r s o f t a l s « a t e r l a l w i l l c o o p e r a t e , t h e r e l a b o
ro a so o why we ca n n o t g e t t h i s p r o b le « un der s t r i c t c o n t r o l and
th e r e b y h o p e f u lly a v o id r e s t r i c t i v e l e g i s l a t i o n ; l a o th e r w o rd s,
l e t ua p r a c t ic e good o lt lx e n a h lp . At th e p r e s e n t t i n * , we a r e
• f th e o p in io n th a t « a t e r l a l c o n ta in in g no te t r a o h lo r o d lb e n x o d lo x ln w ith a c e r t a i n t y o f 1 ppw d o es n o t p r e s e n t aa a p p r e c i­
a b le hazard t o consum ers; li k e w i s e , we do « o t b e l i e v e t h a t su ch
■ a t e r l a l c o n s t i t u t e s a s i g n i f i e s * h azard t o p e r so n s w ork in g l a

5969

DOW CONFIDENTIAL
1« M u lh o llu d

- 8 -

June 2 « , 1965

p '-a n ts h a n d lin g such p h e n o l, ? a c id , o r T a c id e s t e r * .

i

0-A K DOU

Z s i g h t add th a t we t r t c o n tin u in g our resea rch « * on t h i s
« A r t ic u la r p rob Ir a fr o a th a sta n d p o in t o f s tu d y in g th a o th a r
im p u r itie s w hich u r hara tha c a p a c ity to produce t h i s typa
o f r e a c t io n . A ls o , we a re s t t e n p t ln g to q u a n t it a t e th e a f ­
f e c t s o f th a taxewn acnagena whan added t o b ase a a t a r l a l s .
T h is work i s p r o g r e s s in g w a ll, but I t w i l l be s e v e r a l « o a th s
b e f o r e we hare a com p leted s t o r y .
Z would urge a g a in th a t I f your b ig custom ers such as Co-Op
arv< la u g a tu c k hare p a r t ic u l a r q u e s tio n s about t h i s p r o b ls e
t h a t you l s r l t e th ea t o cooe to Midland where we w i l l be
g la d to d is c u s s th e n a t t e r in d e t a i l w ith than and show th ea
srhat we h are le a r n e d . Ve a r e not l a any way a t t e a p t l o g to
h id e our p r o b le n under a heap o f san d , but we c e r t a i n l y do
n o t wane t o hare any s it u a t io n s a r i s e w hich w i l l c a u se th e
r e g u la to r y a g e n c ie s t o becone r e s t r i c t i v e . Our prim ary o l J a c t l r e i s t o a v o id t h i s .

%
cc

Z t r u s t t h a t you w i l l be v e ry J u d ic io u s l a y o u r u s e o f t h i s
I n f o r e s t io n . I t c o u ld be q u ite e w h a m s s in g I f i t wars m is­
in t e r p r e t e d o r n ls u s e d .

T O /jd
act

L. S i l r e r s t e l n
C. O tis
fr a d y Holdeman
F . A sstu tx
0 . O oergen
1 . B o y le
V. F a ls e r

▼ . 1 . ftswe ( 2 )

4

T 1 7 .* -1 2 -2 0 —-~
C orrespondence

F .3

flbder no c ir c u m sta n c e s nay t h i s l a t t e r be rep ro d u ced ,
show n, o r s e n t t o anyone o u t s id e o f Dow.

TO
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5971

0000005
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M r. U , S. Buckley • -¡.Technical D irector
. : Thompson Chemical Company
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5023 Locust S treet .
- f t . L ouis, M issouri 4 3 1 0 1 ? / ' i *'*>:-v«*>•>:. *'.L>.)v.

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an t to
our discussion
a •week o»r so. i _ago
about your
ehioraene
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«* * ••' *'Tv- *•' ■■P
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problem , X have had th ree published a rtic le s reproduced and am
; 'T
_ ••• - i
enclosing them herew ith, Two ot them a re tran slatio n s which we .
/* :
- have m ade. The tran slatio n s m ay not be p erfect, hut the im portant
~\ ? :
*points a r e th e re . One ie entitled "C linical O bservations about the
• O rigin oi C h lo racn s," and the other la antitied "Industrial Poisoning
1’; '* ^ ; ^ in the M anufacture ©i Chloropbenol Compounds, *. In thie la tte r
cj... ,'r .

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^•- . B iochem ical R esearch L aboratory
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McCoy J*. •
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T ran sl. MID. no.3728

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H autarst

Tel.10 so.3 p.126-129 (1939)

t o r»

C lin ic a l ob servations about tha o r ig in of
ehloracn e.
\

Author:

Braun, y .

Translator:
. Bata: -

Trans. Chess. Inc.
Hay 27, 1966

Requested by:
Murray, J io c h a a ., 1701 Bldg.
O riginal language: GasBtt~
T ran slation n o .:
66-3-46
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5973
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,4 ( 0 « »C • * ' C 3

M . A . ‘raves - R. C.
J. J. Ford - ?.. C.
A. I. Conner - R. C
A. D. Sidvell - Jacksonville

Wilmington, Delavare
November 18, 1965

TO:

C. L. Dunn - Synthetics Department

FROM:

E. E. Christofano - Medical Department

2,k,5-T

The following comments will summarize the investigations ve
contemplate on chloracne possibly associated with
esters.
1. Badische uses a bioassay technique to check each batch of TCP
purchased. Their test consists of feeding each of three rabbits with.
1 gm./kg. of TCP, with a determination of bromsulphalein retention after
5 days. More than 5$ of BSP retention is cause for rejection of the batch.
Sensitivity is claimed at 3 ppm.

[•
fc

2. Dov bioassays by a rabbJLt ear painting technique. The internal
surface of one rabbit ear is painted daily with 0 . 1 ml. of a chloroform
solution of test material (10 or 50$) of an extract of TCP in chloroform.
Unless severe irritation is noted 10 doses are applied. The ears are
observed for two additional weeks for the appearance of folliculitis and
cyst formation. They have noted fatalities from a single application and
liver damage has accompanied the chloracne reactions. Sensitivity is
claimed at about 1 ppm.
While the Dow testing may be more sensitive, the Badische method
is faster and less expensive. In order to evaluate both methods we recommend
that Hercules supply material to Badische for their testing and simultaneously
begin gathering confirming data in a commercial laboratory. Because of a
lack of information on the toxicity of in-process materials, these latter
studies should begin only after oral toxicity has been determined. For this
first stage the acute oral LD50 studies would cost approximately $3 0 0 .
Subsequent acnegen studies on these materials are expected to cost about
$6,000, however, an accurate estimate is not possible until Stage 1 is
complete. Preparation of the samples and subsequent analysis is estimated
to cost $5 0 0
t

^D E FE N D A N T S
t

t

EXHIBIT

2 -f

10

000034

ULES POWDER COMPANY

HER CULES POW DER COMPANY

C. L. Dunn - Synthetics

November iS, 1 9c 5

- 2 -

Dr. Oettel of Badische has agreed to bioassay several spiked samples
to permit comparison of results with the gas chromatographic determination of
dioxin and with the Dow ear painting procedure. To permit this study we
request that 25 g. of each of the following spiked samples be prepared:
a.

Standard 2,k,5-T acid spiked with 0, 2, 5, 10, 20 ppm dioxin.

b.

Plant grade 2,k,5-T acid with a known dioxin content.

c.

Plant grade sodium trichlorophenate spiked with 0,
dioxin (based on TCP equivalent).

d.

3, 5, 10 ppm

C P

Dioxin standard approximately 2 mg.
Plant grade pnapyl ester of 2,U,5-T acid as 6 lbs/gal.
solution in xylene spiked wit^ 0, 2^ 5, 10, 20 ppm dioxin.

U

a, i --

Approximately 1 0 ^gms. of each sample should be prepared for ship­
ment to Dr. Oettel and the remainder retained for future confirming tests
conducted by Bio-Test. Chemical analysis of the spiked samples would be
required after the biological testing demonstrates physiological activity.

• — ■— ^.Acute oral toxicity data on sodium trichlophenate, 2,^,5-T acid and
finished propyl ester formulation should be begun promptly. Approximately
'50 gjns. of•each material should be-sent to Dr. Joseph C. Calandra,
Industrial Bio-Test Laboratories, 1810 Frontage Road, Northbrook, Illinois.
Dr. Calandra has a supply of dioxin.
After these preliminary tests are conducted we will establish a more
comprehensive program for the evaluation of hazard during manufacture and
in the product as it would be used by the customer. Without these pre­
liminary data a test program would be unnecessarily expensive.

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HERCULES INCORPORATED

RESEARCH CENTER

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0000347
k-

V/ilmington, Delaware
June 15, 1966

MEDICAL DEPARTMENT
DIVI SION
FOR 3I0 ASSAY TESTS
In accordance with your letter of November 18,

1965 > the samples listed below are being sent to you
for transmittal to Dr. Oettel of Badische, for use in
the bioassay tests.
Approximately 10 grams of each sample (except for
the pure dioxin sample) is included. The samples are
packed in four shipping containers, and the contents of
each are listed on the outside of the container.
The
individual samples are also clearly identified with
an appropriate label.
I have- indicated which samples
have dioxin added to them, but have no t 1shown the
fortification-level on the label. If you wish to
provide Dr. Oettel with *this information, it is
included in the attached list. It would be useful to
point out to Dr. Oettel'that the samples from Group 3
and Group 6 are more easily sampled if heated as
indicated.

JJp/cbb
Attachment

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5977
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DEPOSITION
EXHIBIT

líX tfv A — )2 -

Mr. E. E. Chris toi¿no

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1

SAMPLES PREPARED FOR 3I0ASSAY TESTS
(1) Standard 2,4/5 T Acid With Added Dioxin
Sample Designation
XI4867-41-1
X14867-41-2
XI4867-41-5
XI4867-41-4
XI4867-41 -5

ppm. Dioxin Added

O ’
2
5

10
20

(2) Plant Grade 2,4,5 T acid with Known Dioxin
Sample Designation
X15280-56-8

XI 5280-56-9

ppm,. Dioxin

3

1

T

(5) Plant Grade Sodium Trichlorophenate with Added Dioxin
Fortification is based on a 35$? NaTCP Content.
Sample Designation
X15280-55-1
X15280-55-2
X15280-55-3
X15280-55-4
X15280-55-5

ppm, Dioxin -*0

t

0

1.2
2.4
5.7

1 3 .0

Heating the above samples to 65 °C. aids in sampling.
(4) Dioxin Sample - 2,3,7*8 tetrachlorodibenzo-p-dioxi.n Designated XI 528O- 56-IO.
(5) 2 Ethyl hexyl, ester of 2,4.5 T with added dioxin (Brush-Rhap LV-4-0)
Fortification is based on 44.Iff? 2,4,5 T acid.
Sample Designation
X15280-56-1
X15280-56-2
X15280-56-3

X I5 2 8 O -5 Ö- 4

X15280-56-5

ppm. Dioxin

0
2
5

10
20

(6 ) Non-saponifiable fraction from wash solvent still.
Designated
XI 5280- 56-7 . This sample contains unreacted tetrachlorobenzene
and relatives, anisol of trichlorophenol and related phenols,
toluene and a small amount of NaTCP.
Sidwell believes this is
where the dioxin would end up in our process.
We are storing
this material at the rate of 2-3 drums per day.
To melt,
heat this sample to 50°C.

5978

ì

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0000004

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T H O M P S O N C H E M I C A L C O M P A N Y , S T , L O U I S , M IS S O U R I

Oc F ebruary 2, 1767« W. J . McCoy Informed me that a Mr, M, S, . . .

V 1 : - : Buckley of Thompson Chemical had called Pew indicating that they
* ¿V /'/.V had had an accident associated with the manufacture e ftrlc h ln ro - ’v'ú .

phenol, and that they had torn« men with what they thought was '
chloracne, They wondered If wó could advise them la regard to .
• • • /, medical practice, 1 contacted Dr, Holder, who then called Mr,
Buckley and discussed with him medical aspects of the problem, ' <t- ..
It is my hade rstaadlng from Dt , Hold«r that M r, Buckley'then •''.
started talking about the chem istry of this m aterial, and D r, Holder
.suggested
that he contact me In regard to anything along this lias,
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V/V<

On o r about F ebruary 7 M r, Buckley called mo to seo what we knew
about chloracne caused by contact with m aterials associated with
the manufacture of trichloropbenol from te tra cklorobonxtne, 1.
told him that under certain circum stances, which 1 did not describo,
the cauitic Insoluble oils could contain considerable amounts of a
▼cry highly toxic substance which we had Identified as 2 ,3 ,7 ,8«
tetrachlorodibensodloxin« 1 Indicated to him that this m aterial was
a c t only extrem ely toxic system ically, but it was elao an extrem ely
potent
cbjoracnogea, lie Indicated that they had been distilling some
’« e
« •
o ils or ta rs containing anisóles when the' operation got out of control,
and the m aterial either spilled or splashed onto a hot autoclave, .Ha
thought that tyo fumes and vapors of the anisóles had caused th eir
/• • » '/rf.1» --,c difficulty, '• 1 told him 1 did not think the acne was caused by the
T i*
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anisólet« but ra th e r by other m aterials present In the acicales, -***•.• ;; ■e*
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Apparently Mr. Buckley had asked Or. Holder how te clean up tha
. ‘equipment, etc.« and O r. Holder Indicated to him that be meat taka
*: * * , entrama precautions, Including tbo oao of rubber auita and glove a and
.*m - > .*» ••p iralo ro . Ha «comed aurpriaed that wo would recommend «uch
Severa a a a « o ro s. but Xreiterated that ouch precaution« « a ro « h at
..
. .Ora believed n*e«eaary. Ha than wanted to know if Xthought they
<■!; could cXaaa up the contaminated equipment with steam . 1 felt at thia
point an obligation to tall him Chat «team tree tha w orst thing ha could
use, «imply bacana# it would volatilise tha m aterial« causing it to ■.
/ . recryataU laa and deposit elsew here. Thia waa not tha way to get rid ...
•
.of it. Ke suggested acrubbing down the equipment with detergent e r .
•* / ’solvents. ~X indicated that thia coold ha done if be made aura that a ll ' •
*. neccaaary precaution« were taken to prevent contact with the people. ..
I auggeated that ha aaaay o r take wipe «ample« of the equipment to
~
determ ine the degree of contamination. 2 auggeetad that thia aheuld
be done before and after the clean-up. Ke eaked how auch aaaaya ;
could be mado. 1 indicated to him that we had developed a method of
| .* \ analyaU and alao uaed biological aaaay employing a rabbit e a r. He .
-v.i did not aak me for detalla ef cheat procedure«,' co 1 did not give them i
< . . t". ¡..f ¿r+- *' £■ «*»«■ •— ‘;
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lie then asked where he could find information about chloracne, and Z
volunteered to «end him coplea of pertinent a rtic le s from tha published
Xhad« had thia.•ha a. beea'donc.
■ literatu ree* which
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.h>* • r-^My coevaraationa with Mr« Buckley were within the framework of eu r .Vi:*
hi.#.** T-tirr
“ *dI«ct»aaion of the problem. The information on the phyaiologlcal
activity waa perhapa even lea« than waa given to tha other producers
of trichloropbenol over a year ago. This waa because it waa quickly
. . apparent that M r. Buckley had little understanding of tto toxicological
' tap ecta of bis problem , liad he aaked for method«, e tc ., Xwould have
agreed,to tend them to him. However, linca he did not, Xsaw no
• reason to volunteer. — •
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Xdon't know whether this information la of any value to you. but It
. will« a t le a st, keep you Informed. If you have any question« o r eug. geatlona for further action, piece« do not hcaitale to contact mo.

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5982

INCORPORATED

0000334
Dr. J . P. Prowl ey - Medical Dept.
|Wl!mington, Delaware
June 30, 1967

*

TO:

Mr. H. E. Wilder - Jacksonville

ROM :

J . M . Eagan

Dr. Frawley sent to me the attached copy of a report dealing with five
cases of cloracne in Colombia which developed from the use of phenoxy herbicides.
The inference in these reports is that some producers allowed contaminated 2,4,5-T
to enter sales channels in 1963 and 1964. No prior cases of cloracne outside of
manufacturing operations have come to-our attention but this report should remind
' all of us that we must be certain to continue to be on the alert for abnormal operating
conditions which might contribute to the presence of the chemical which is reputed
to be responsible for this type of cloracne.

JME/abk
Attachment

5983
i o h -a s

> 3

ì

/ 3

t>
T H E D O W C H E M I C A L . C O M P A N Y i*»' :****■ ■
S ep tem b er 2 6 ,

1967

C o l o n e l C . C , She»6 <SAO>
D i r e c t o r a t e o f A i r F o r c e A e r o s p a c e Fue l *
K e l l y A ir F o r c e B ase» T c u i
7b24t

|

-

*“ .

D ear C o lo n e l S h cad :
Uv h a v e b e e n a s k e d t o b i d o n tht- d o v i p ) , c o n v e r s i o n a n d « • p c r i t i o n • (
a n £ , 0 0 0 , 0 0 0 g a l l o n p e r > * a r " O r a n g e " p l a n t a ? l*»». < V v c r n ^ r r . t o v u i
c h e m i c a l p l a n t a t W# Id em S p r i n g , M i s s o u r i .
T h i s I s t o i n f o r t y o u t h a t we h a w a d v i s e d V . C . K r a f t , C o l o n e l ,
C o r p s o f E n g i n e e r s s n K a r . u * C i t s . M i s s o u r i , t h a t we do n o t u i l e n d
t o b i d o n t h e i r s o l i c i t * : ; ■: "•*. rWCAN^-bB-R-WK, I , d a t e d S e p t e m b e r
1967.
"U io b asii.
b elo w :

reaso n

f o r o u r c « «. . * •••

due

te «r it n a l

l u s t e r s m il lin e d

" fl u* t i n e a l l o t t e d i«»r s u b r : * - ;• ~
h i d * , Sc>U r?N -r £ ,
O c t o b e r 1 2 , 1 9 6 7 , i s n . i a —; % ;**r p r e p a r a t i o n cm h i d ? .
of th is n a g n itu d e .
“ H i e p r e t e n t a t ?«»n o t a l c e r r j f i v
Q r » i m a*
S p r i n g w e n p r e c l u d e d h e t r i ' i • i ttu* t i:r v u l t c % ’» c
a r e s p o n s i v e b i d i n a n u v v r t«- *» .t *«•! i t i t a t i -m .
**Uc w o u l d l i k e t o s u a u p * * ' v
t h i s tin»*, a s f o l l o w s :

fu r

r. . i* .i>

¡967 t o
a p ro ject

•**S«r t h a r l > « d c n
U r t 'e p a r a tio n of

t o r n o t f*r* s e r . t i n ; a b i d a t

1*

T h e W e l d o n S p r i n g s i t e , I h j t v d a s i t i s h% t v c v n t h e
U n i v e r s i t y . ( Mi
.• r i E p e n r x n t a l F a i ^ a n d t h e
A u g u st B usch R e s e r v e , p r e s e n t s to o m u h o f a h a r a r d t o
t h e p l a n t l i f e i n in«..** a r c u s , i n i u ‘ f «»f a n a c c i d e n t a l
r e l e a s e o f p r o d u c t s f r u o th e O range r i a n t .

2.

Ik e

3,

The l a b o r s i t u a t i o n
t o o many p r o b l e m a .

4,

I t w o u l d b e i l m > U i o g H * » » i h l e t o m ‘ I vl* t h e
p ro b le m s in th e a r e a .

ì

req u irem en t of

15 n i n t h s

in

th e

start

S t,

#\

up and

fu ll

y era-

L o u i » a r e a w o u l J #» r c > c a t .

p o llu tio n

•

’

5985

^ 01 »
C o i t f t c t C . G. S h cad

pi .-.nhef 2i*t t*#h7

S.

D i s p o s a l o l v a s t . « o n : ! . i :•«
a r r i v e a t a p o s i t i v e s. .♦.» ».-n M
f o r c o l l e t I*»n o j tit. j i j n t .

itt t i-w* t o
:< i*u- t : ^ a l l o t t e d

0.

The I h l o r - A c n e pi« » h l« a a hv\ u (<’<) w i t h tc* r a n u t a t t u r c
*»f T r i c h l « r p h v n o l io»»i«: ».»t be s o l v v d w t t h ' m t » '¡ r s t
r e c e i v i n g p e r m i s s i o n i r*ir a r u r o p c a n C h e a i c a l
M a n u fa c tu re r.
T h e C h l o r - A c n e p r o b l e m i s o n e o f Human H e a l t h v h i v h
D.tv h a s h a d t o co m b at i n t h e d e s i g n o f lt% *~**n p a n t s
a t i t s M idland o p e r a t i o n s .
S e v e r a l p h a s e s o f th e r a n u f a c t u r i n g p r o c e s s f o r 2,4-D
an d 2 , 4 , 3 * T : a v o * w D«ra k n o w h o w t h a t wc d o n o t w i s h
to lo se to our c o a ^ c t i t e r s .

9.

The p e o p l e t h a t a r e now e n g a g e d ,
the f a c to r y I c s ^ l .
i n t h e p r « * d u c t i o n o f C.O'iO.i'iO , a l l o n s / w a r o f O r a n g e
f o r t h e G o v e r n n e n t a r e fru »arn- l u o p l v ( h a t wou ld h a v e
t o b e a s s i g n e d t o t h e W« iw»* S p r i r g p r o j e c t .

" T h e ih*w C h e m i c a l Compart. m n u • *» t o h a v . a h i c h d e c r e e o f i n t e r e s t
i n th e p r o j e c t and r n , u 4 1 > ih.
a»
*.i/. t o r y *«r b i d d e r s l i s t t o r
a n ) f o l l o w i n g < o l ic H a l to n
i - • ■*•*** c t i«.n w i t h t h e ».apply
raw o r
lnh*m («JI«u< m a t e r i a l s .
*Vhvn t h e TVH RFP i s r e l e a * « - '1 -» no;.* : * h e stx t*
b i d d e r s l i s t , and
f e e l t h a t we W i l l be m a h e t t . .
* ; t * e t . •«* h i d *n t h t . p r o j e c t *
MIf vo»i h a v e a n y q u e s t i o n « » o n e •
I k*. ' ** b o s t o n w i t h r e g a r d t .
: u - . DACA-i 1-68-R -O OO1
. , p K a - e %**. ■«*« t n
C la r.ir.ilv

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O ire c to r of O vcrtunrnt A ffa irs

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5987

.T

0000343

October 16, 1963

Kr. V. K. Rowe
Biochemical Research Laboratory
The Dow Chemical Company
1701 Euilding
Midland, Michigan
Dear Mr. Rove:
As you may recall, Hercules has been attempting to develop a
bioassay technique to simplify detection of dioxin in herbicide formulations.
Based principally upon work initiated by Dr. Oettel at BAS?, the tests
measured liver function changes which result from feeding rabbits with
i
herbicide. We feel such a technique offers several advantages over rabbit *'
ear painting tests.
\
Unfortunately, ve have been unsuccessful. But to state it so
bluntly is really unfair. First, ye attempted to determine the sensitivity
of liver change by feeding test materials with knc*.m additions of dioxin.
The toxicity of test materials prevented use of liver function change as a
bioassay for dioxin even at 10 ppm. We then dosed animals vith 0.01 U 0.1 ng/kg
of dioxin suspended in c o m oil. Carbon tetrachloride vas administered as
a positive control at 100, 250 and 500 mg/kg. Clinical determinations
including SCOT, SG?T and ESP retention were conducted prior to administration
and every two to three days thereafter for at least lU days.
Significant elevations in one or more of the clinical determinations
vere found in scee but not all dioxin a n imals. The changes vere not veil
correlated vith dose nor vere they consistent vith regard to onset or duration.
Animals on the positive control test responded as would be expected with
significant blood chemistry changes only two days after dosing vith 100 mg/kg.
Although positive results vere obtained with dioxin, the usefulness
of this technique as a bioassay for the presence of dioxin is questionable.
At the Toxicology Roundtable I mentioned these results to Ken Olsen
vfao suggested that you might comment upon them. But the other reason for
writing is to solicit your opinion on the usefulness of presenting our data
at the Society of Toxicology meeting next spring. Certainly such a presentation
vould raise questions as to the reason for conducting an unsuccessful study.

5988

Mr. V. K. Rove

-2-

October 1 6 , 1968

On the other hand, someone night be spared useless effort if such negative
information vcre offered to the scientific corununity.
I'd like to have your consents before ve proceed.
Sincerely,

Bail E. Christofano
Industrial Hygienist
EEC :rba

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5989

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i
i

1
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5990

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5993
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