ICNI UUUEIIT iaih' 4 IR & D REPORT rHBRATamnntTCTT'Egfir HET K-1711-(21) BATE llldfr-------i_“£----- DOW CHEMICAL U.S.A. RESTRICTED! far w*« within Tha Daw Chaaiical Canpany an If. Sept. 28, 1976 uKTRirwLru us.'- Health and Environmental Research 1 ? .7 °|0|° » 7 |1 ,5,5 TtTLS COMPARISON OF THE PATE OF VINYL CHLORIDE FOLLOWING SINGLE AND REPEATED PAGES IN FULL REPORT CRI NUMBER EXPOSURE IN RATS AUTl'Aft III-----------------------------------------------------------------------— P. G. Watanabe, J, A. Zempel and P. J. Gehrlng 'AUTW6B III IlgMATUHl III---------------------------- ^JL(' _ _) report t ^7 and mainly: *£| DATA REFERENCES (bdok and page): (Refer also to earlier related reports and publications.) PATENT STATUS: Q disclosure submitted tU case filed C3no patent action required DESCRIPTIVE SUMMARY WITH CONCLUSIONS: Rats were exposed to 5000 ppm nonlabeled vinyl chloride (VC) 6 hours/day, 5 days/week for 7 weeks. On the last day of exposure 14C-labeled VC was used and the fate of the 14C-VC was followed for 72 hours and compared with the fate in rats subjected to a single 6-hour exposure to 5000 ppm 14C-VC. The routes and rates of excretion of Re­ activity were the same for the two experimental groups. The activity of microsomal enzymes, as reflected by aniline hydroxylase and £-nitroanisole O-demethylase of 9000 x g liver supernatants was essentially the same in rats exposed once, repeatedly or in nonexposed control rats. Covalent binding to hepatic macromoxecules was greater in rats repeatedly exposed when compared to those subjected to a single exposure. The hepatic nonprotein sulfhydryl level of the repeated and single exposed groups immediately following exposure was 79% and 39% of control, respectively. These results indicate that repeated exposure to VC does not induce its biotrasnformation. However, the increase in hepatic macromolecular binding indicates that repeated exposure augments the reaction of electrophilic metabolites with macromolecules, and this may be ex­ pected to enhance potential toxicity including carcinogenicity. I DISTRIBUTION: See Back Page i _ .mm ** m r* i i t DI-6560 9-8-75 DO 137685 OONF TOFNT T AL COMPARISON OP THE FATE OF VINYL CHLORIDE FOLLOWING SINGLE AND REPEATED EXPOSURE IN RATS by P. G. Watanabe, J. A. Zempel and P. September 28, J. Gehring 1976 Toxicology Research Laboratory Health and Environmental Research Dow Chemical U.S.A. DO 137686 O.QNFIDFNTT AL. COMPARISON OF THE FATE OF VINYL CHLORIDE FOLLOWING SINGLE AND REPEATED EXPOSURE IN RATS P. G. Watanabe, j. A. Zempel and P. J. Gehring ABSTRACT Rats were exposed to 5000 ppm nonlabeled vinyl chloride 6 hours/day, exposure 14 5 days/week for 7 weeks. (VC) On the last day of C-labeled VC was used and the fate of the 14 C-VC was followed for 72 hours and compared with the fate in rats subjected to a single 6-hour exposure to 5000 ppm The routes and rates of excretion of same for the two experimental groups. microsomal enzymes, 14 14 C-VC. C-activity were the The activity of as reflected by aniline hydroxylase and £-nitroanisole O-demethylase of 9000 x g liver supernatants was essentially the same in rats exposed once, or in nonexposed control rats. repeatedly Covalent binding to hepatic macromolecules was greater in rats repeatedly exposed when compared to those subjected to a single exposure. The hepatic nonprotein sulfhydryl level of the repeated and single exposed groups immediately following exposure was 79% and 39% of control, respectively. These results indicate that repeated exposure to VC does not induce its biotrans­ formation. However, the increase in hepatic macromolecular DO 1.37687 00NFTDENTTA1 -2- binding indicates that repeated exposure augments the reaction of electrophilic metabolites with macromolecules, and this may be expected to enhance potential toxicity in­ cluding carcinogenicity. 00 137688 CONF T DFNT T Al -3- INTRODUCTION While many pharmacokinetic and metabolic studies have been conducted on vinyl chloride (VC), most of these investi­ gations have concentrated on the fate of VC during and fol­ lowing single exposure. Since cancer has been induced by long term repeated exposure of both experimental animals and man to VC, it is important to consider alterations in the disposition of VC in the body which may occur after repeated exposure. In preliminary studies monochloroacetic acid was tentatively identified as a major urinary metabolite following repeated exposure (6 hours/day, nonlabeled VC 5 days/week for 9 weeks) (Hefner et al., 1975). However, to 5000 ppm this meta­ bolite has not been found in rats given a single exposure to 14C-VC (Watanabe et al., 1976a). This raised the question whether the biotransformation of VC may be altered upon repeated exposure. A change in the biotransformation of VC after repeated ex­ posure is particularly important in assessing the hazard of exposure to VC since evidence suggests that VC is metabolized to a reactive metabolite which is ultimately responsible for DO 1.37689 CONFIDENTIAL -4- its toxic manifestations. This concept is supported by studies showing an enhancement of the mutagenic activity of VC to bacteria when microsomal and soluble enzymes are added to the system to provide for metabolic transformation (Bartsch et al^, al. 1974). 1975; Malavielle et al., Rannug et In addition, recent reports demonstrated that liver microsomal enzymes, in vitro, mediate the binding of VC metabolites to the microsomes protein sulfhydryl groups, adenosine 1975; (Barbin et al., RNA (Kappus et al., (Bolt et al., 1975). 1975), 1975), and That the biotransformation of VC is involved intimately in its toxic response was further substantiated when Reynolds et al. (1975a) showed that acute hepatotoxicity could be produced by VC if the animals were pretreated with phenobarbital or Arochlor 1254, inducers of microsomal enzyme activity. (1975b) Reynolds et al. also reported that a single 6 hour exposure to 5% VC deactivates cytochrome P-450 and other components of the mixed function oxidase system. Since there is evidence suggesting that repeated exposure to VC may alter its biotransformation and since the metabolism of VC is associated with its toxicity, the objective of the current study was to determine whether the fate of VC is 00 137690 OONFTDFNTTAL -5- indeed altered with repeated exposure. This was accomplished by exposing rats to nonlabeled VC for 7 weeks and on the last exposure to ^C-labeled VC. The fate of the 14C-VC was then followed for 72 hours and compared to animals receiving a single exposure. METHOD Material. Vinyl chloride gas (Matheson Gas Products) 99.9% minimum purity was used throughout the study. labeled VC was synthesized from (New England Nuclear, prior to use 14 14 (1,2 Lot #819-292, (Wagner and Muelder, C) 14 C- 1,2-dichloroethane 4.8 mCi/mmole) 1975) . of immediately The synthesized C-VC has been reported to be 95-96% radiochemically pure (Wagner et al., ducts) 1975). Nonlabeled VC was mixed with the specific activity. 14 (Matheson Gas Pro- C-material to obtain the desired helium gas mixture was injected into a 10 liter Saran bag (Anspec, containing the desired quantity of nonlabeled VC. Animals. tory) study. 40 ml of the 14 C-VC, Inc.) Typically, Male Sprague-Dawley rats (Spartan Research Labora­ with an initial weight of 160-180 g were used in the All animals were housed in rooms in which a constant humidity, temperature and 12 hour light-dark cycle were maintained. (8 AM-8 PM) Pood and water was provided ad libitum DO l37^TAl -6- except during the exposure. tween 9:00 AM and 4:00 PM Exposure. Exposures were conducted be­ (EST). All rats were exposed under dynamic conditions to a nominal concentration of 5000 ppm VC (control) in 30 £ glass inhalation chambers. into the chamber air flow pump. (treated) (~6 1/min) or room air VC was metered with a dual syringe The nominal concentration of VC was determined from the ratio of the rate at which the VC gas was dispensed and the total chamber air flow. The analytical concentration of VC was monitored continuously by recirculating a fraction of the chamber atmosphere through an infrared spectrophotometer (Wilks) set at 10.6 u. The rats repeatedly exposed to VC were exposed 6 hours/day, posures in 44 days). 5 days/week for 7 weeks (32 ex­ The mean analytical concentration of VC over the 7 week exposure period was 4775±908 (SD) ppm. On the final day of exposure the animals were subjected to a 6 hour exposure to 14 C-labeled VC generated in the same manner as described above. On this final day of exposure, the chamber atmosphere was also analyzed at hourly intervals by gas chromatography, and at the same times the 14 C- activity was determined by bubbling 1 ml aliquots of the chamber atmosphere into a scintillation solution containing Concifluor (Mallinckrodt Chemical), 2-methoxyethanol, toluene DO 1 376*3? CONFT DFNT T AL -7- (6:11:83) (Watanabe et al., 1976a). The radioactivity was determined by counting in a liquid scintillation spectro­ meter. The mean analytical concentration determined on the final exposure day when the animals were exposed to Re­ labeled VC was 4600 ppm ± 311 (SD). The specific activity was 50 dpm per microgram VC. The inhalation chamber was operated in a laboratory fume hood to prevent contamination of the working environment. After transit through the inhalation chamber the absorbed on activated charcoal. 14 C-VC was These traps were disposed of as radioactive waste according to standard regulations. Procedure. Eight rats were exposed repeatedly to VC as described previously. On the last day, 5 additional'un­ exposed rats and the 8 rats exposed repeatedly were exposed to 5000 ppm posure to 14 14 C-VC for 6 hours. C-VC, Following this final ex- 3 of the 8 exposed repeatedly and 2 of the 5 exposed once were placed in glass Roth-type metabolism cages for the collection of urine, feces and expired air. Room air was drawn through the cages at 400-500 ml/min. The exiting air was passed through a series of traps to collect the expired 14 14 C-VC and . COj. The air leaving the chamber DO 137693 CONFIDENTIAL was passed first through a glass tube containing about 40 g of Drierite (W. A. Hammond Drierite Co.) to remove moisture. Subsequent transit through a series of two cold finger traps containing 50 ml of toluene, 2-methoxyethanol (80:20), and a single trap containing 120 ml of 5 M ethanolamine in 2methoxyethanol enabled the collection of respectively. 14 C-VC and C02» The cold finger traps were immersed in 2- methoxyethanol, periods. 14 dry ice baths throughout the collection The trap for CC>2 was maintained at room temperature. Samples of excreta were collected for 72 hr after termination of exposure and analyzed for C activity. VC was collected at 0.5 hr intervals for 3 hr; and urine receptacle 24 hr. At the termination of the study kidney, liver) were changed and feces were collected every were killed by a blow to the head, (fat, the COj trap (immersed in dry ice bath) at 12 hr intervals for 72 hr; Expired (72 hr) the animals and samples of tissues were collected for analysis of 14 C activity. The remaining carcass was skinned and homogenized (50% w/v) in distilled water and analyzed for 14 C activity. The samples of excreta and tissue were prepared for scintil­ lation counting as described previously (Watanabe et ad., 1976a). DO 137694 OONF T DENT T AL -9- Carbon 14 activity was determined by counting in a Mark II or Mark III liquid scintillation spectrometer. External standard channel ratios were used to determine the counting efficiency. The counts per minute were converted to dis­ integrations per minute using a standard quench curve. The remaining rats in the groups exposed repeatedly and singly to VC, 5 and 3 respectively along with a group of 4 controls exposed to room air for 6 hours were killed by a blow to the head immediately following exposure. A piece of liver was sampled and used for determining hepatic non­ protein sulfhydryl content by a modification of the method of Sedlak and Lindsay (1968). Another piece of liver was used to prepare a 9000 x g supernatent in 1.15% KCl in order to determine aniline hydrolyxlase nitroanisole O-demethylase vity. (LaDu et al., (Kinoshita et al., 1971) 1966) and j>- acti­ Macromolecular binding of radioactivity to hepatic tissue was determined by the method of Jollow et al. (1974). The carcass was analyzed for total radioactivity as des­ cribed above. RESULTS Excretion of 14 C-activity within 72 hours after a single or repeated exposure to 5000 ppm ^C-VC is shown in Table 1. DO 137^ confidential -10- The percentage of 14 C-activity excreted by each route as well as the total mg equivalents VC recovered were essen­ tially identical for the singly and repeatedly exposed groups. The majority of 14 C-activity eliminated was expired as VC per se. The time course for expiration of and urinary excretion of 14 14 C-VC per se C-activity (Figure 2) (Figure 1) were also essentially identical for the singly and repeatedly exposed rats. The curves were fit by linear regression analysis of the logarithmically transformed data. The estimate of the apparent first order rate constant for expiration of VC was 0.023 min-1 + 0.01 30 minutes. biphasic. (SD) which corresponds to a half-life of The elimination of urinary 14 . . C-activity was An estimate of the apparent first order rate constant for the initial portion of the urinary excretion curve from 12-36 hours was 0.155 hr ^ + 0.002 corresponds to a half-life of 4.47 hours. (SD) which The data for the slow phase of urinary excretion were extremely variable; therefore no attempt was made to estimate the excretion rate. Less than 1 percent of the radioactivity excreted in the urine occurred during the slow phase. Urinary 14 C-activity was separated by thin layer chromato­ graphy in n-butanol, acetone, I^O (50:20:30) on cellulose DO 137696 CONFI DENT T Al -11and n-butanol, acetic acid, H2O (80:20:20) on silica gel. The profile of radioactivity for rats exposed repeatedly or singly were qualitatively similar and no significant radioactivity was associated with the value of a standard of monochloroacetic acid. The concentration of radioactivity detected in tissue 72 hours after exposure revealed no statistically significant difference between rats exposed once or repeatedly to VC (Table 2). It does appear that in those exposed repeatedly more radioactivity may have been retained in the liver and skin; however, the number of animals used does not provide for an adequate statistical evaluation. The effect of VC on xenobiotic drug metabolism by liver 9000 x g supernatents as reflected by aniline hydroxylase and £nitroanisole-O-demethylase activity is presented in Table 3. Neither single or repeated exposures to 5000 ppm VC altered discernibly the enzyme activity in either system when com­ pared to air exposed controls. The total amount of VC biotransformed, molecular binding of 14 the hepatic macro- . . C-activity and the hepatic nonprotein sulfhydryl content following single and repeated exposure 00 137697 CONFTDFNTTAl. -12are shown in Table 4. The total amount of VC biotransformed was not significantly different between the two groups. However the hepatic macromolecular binding appears to be increased in the rats exposed repeatedly. When the protein binding was corrected for the amount of VC biotransformed (B/A x 100) a statistically significant increase was found. This parameter indicates that a larger fraction of the biotransformed VC reacts covalently with hepatic macro­ molecules in rats exposed repeatedly. This result strengthens the significance of the previous observation that 72 hours after the last exposure more radioactivity was found in the liver of rats exposed repeatedly than those exposed once. In contrast to the results of macromolecular binding, hepatic nonprotein sulfhdyryl content was depressed to a greater extent in rats receiving a single exposure than in those exposed repeatedly. DISCUSSION Repeated exposure of rats to 5000 ppm 14 C-VC did not alter discernibly the routes or rates of excretion of radio­ activity or qualitatively the excretory products formed from VC or VC per se. These results negate the previous preliminary observation that monochloroacetic may be a major biotransformation producer of VC (Hefner et al., 1975). DO 137698 CONFIDFNTTAl -13- No differences were found in the activity of the enzymes, aniline hydrolylase and £-nitroanisole O-demethylase, liver of rats exposed repeatedly to 5000 ppm VC, exposed once and in nonexposed rats. Thus, in the in rats exposure to VC at this concentration does not appear to influence micro­ somal metabolism. In contrast to this conclusion, findings of Reynolds et al_. (1975b) are the that the cytochrome P- 450 content and the oxidative N-demethylation of aminoantipyrine and ethylmorphine were markedly depressed in rats following exposure to 50,000 ppm VC for 6 hrs. However, the extremely high exposure concentration renders suspect the relevance of any conclusion based on the Reynolds study. A most significant finding in the study reported herein was a significantly increased amount of radioactivity bound co­ valently to macromolecules of rats exposed repeatedly to VC versus those exposed once. An associated observation was the retention of an apparently greater level of radio­ activity in the liver of repeatedly exposed rats 72 hours after exposure than those exposed once. indicate that toxic manifestations, These results including carcinogenicity, DO 137699 CONFIDENTIAL -14- associated with the reaction of reactive metabolites of VC with macromolecules may be enhanced by repeated exposure to VC. In rats exposed repeatedly, the enhanced covalent binding of VC with macromolecules occurred in spite of a less significant depression of the nonprotein sulfhydryl content of the liver when compared to those rats exposed once. Previous studies have demonstrated that detoxification of the reactive metabolites of VC occurs via their enzymatic conjugation with glutathione Watanabe et al., 1976c) (Watanabe et al., 1976b; and subsequent excretion. Glu­ tathione is reportedly the major constituent of the non­ protein sulfhydryl content of the liver. The foregoing results are somewhat perplexing because an increased covalent binding of radioactivity is normally thought to be associated with either an enhanced biotrans­ formation to reactive metabolites or a decreased detoxi­ fication of the reactive metabolites. The former pos­ sibility is negated by the absence of a qualitative or DO 137700 CONFIDENTIAL -15- quantitative change in the biotransformation of VC in rats exposed repeatedly as well as the lack of evidence that microsomal enzymes are induced by repeated exposure to VC. The finding that the hepatic nonprotein sulfhydryl content was reduced less in rats exposed repeatedly to VC suggests that detoxification of reactive metabolites formed from VC by conjugation with GSH should not be depressed. Therefore, both logical reasons for the increased covalent binding of reactive metabolites of VC to macromolecules appear non­ operative. However, not to be overlooked is the fact that the nonprotein sulfhydryl content of the liver rather than GSH content was measured. It is conceivable that the GSH level of the liver was actually decreased. true, If this were the binding of reactive metabolites to macromolecules may be enhanced inspite of an apparently normal hepatic nonprotein sulfhydryl content. It may be expected that nonprotein sulfhydryl compounds other than GSH will be less effective in detoxifying reactive metabolites of VC. In conclusion, the results of these studies show that re­ peated exposure to high levels of VC cause a persistent but partially compensated depression of the nonprotein sulfhdyryl 00 c 1.37701 ONFIDENTIAL -16- content of the liver. More significantly, the binding of reactive metabolites of VC with hepatic macromolecules appears to be enhanced by repeated exposure to high levels of VC. Associated with this may be expected an enhanced toxicity, including carcinogenicity. The reason for the enhanced covalent binding with repeated exposure is under investigation. WRITTEN BY: P. G. Watanabe, Ph.D. Toxicology Research Laboratory 1803 Building Toxicology Research Laboratory 1803 Building Direc/tor, Toxicology^esearch Laboratory Health and Environmental Research 1803 Building REVIEWED BY: L. W. Rampy, Ph.D./y Toxicology Research Laboratory Health and Environmental Resaerch 1803 Building DO 137702 CONFTDFNTTAl -17REFERENCES Barbin, A., Bresil, H., Croisy, A., Jacquignon, P., Malavielle, C., Montesano, R. and Bartsch, H. (1975). Liver microsome mediated formation of alkylating agents from vinyl bromide and vinyl chloride. Biochem. Biophys. Res. Coram., 67, 596-603. Bartsch, H., Malavielle, C., and Montesano, R. (1975). Human rat, and mouse liver mediated mutagenicity of vinyl chloride in Salmonella typhimurium strains. Int. J. Cancer, 15, 429-437. Bolt, H. M., Kappus, H., Buchter, A., and Bolt, W. Metabolism of vinyl chloride. Lancet, 1425. (1975). Hefner, R. E. Jr., Watanabe, P. G., and Gehring, P. J. (1975). Preliminary studies of the fate of inhaled vinyl chloride monomer (VCM) in rats, Ann, N.Y. Acad. Sci., 246, 135-148. Jollow, D. J., Thorgeirsson, S. S., Potter, W. Z., Hashimoto, M. and Mitchell, J. R. (1974). Ace­ taminophen-induced hepatic necrosis VI., Pharma­ cology, 12, 251-271. Kappus, H., Bolt, H. M., Buchter, A. and Bolt, W. (1975). Rat liver microsomes catalyze covalent binding of 14C-vinyl chloride to macromolecules. Nature, 257, 134-135. Kinoshita, F. K., Frawley, J. P. and DuBois, K. P. (1966). Quantitative measurement of induction of hepatic microsomal enzymes by various dietary levels of DDT and toxaphene in rats. Toxicol. Appl. Pharmacol., 9, 505-513. LaDu, B. N., Mandel, H. G. and Way, E. L. (ed.) (1971). Fundamentals of Drug Metabolism and Drug Disposition, pp. 566-569, The Williams and Wilkins Co., Baltimore, Maryland. Malavielle, C., Bartsch, H., Barbin, A., Camus, A. M. and Montesano, R. (1975). Mutagenicity of vinyl chloride, chloroethyleneoxide, chloroacetaldehyde and chloroethanol. Biochem. Biophys. Res. Comm., 63, 363-370. D0 V37703 CONFIDENTIAL. -18- Rannug, U., Johansson, A., Ramel, C. and Wachtmeister, C. A. (1974). The mutagenicity of vinyl chloride after metabolic activation, Ambio, 3^ 194-197. Reynolds, E. S., Moslen, M. T., Szabo, S., Jaeger, R. J. and Murphy, s. D. (1975a). Hepatotoxicity of vinyl chloride and 1,1-dichloroethylene. Am. J. Path., 81(1), 219-231. Reynolds, E. S., Maslen, M. T., Szabo, S. and Jaeger, R. J. (1975b). Vinyl chloride induced deactivation of cytochrome P-450 and other components of the liver mixed function oxidase system: on in vivo study. Res. Comm. Chem. Path. Pharmacol., 17(4), 685-693. Sedlak, J. And Lindsay, R. M. (1968). Estimation of total protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's Reaqent, Analyt. Biochem., 25, 192-205. Wagner, E. R. and Muelder, W. W. (1975). A procedure for preparing 14C-labeled vinyl chloride. Ann. N.Y. Acad. Sci., 246, 152-153. Wagner, E. R., Muelder, W. W., Watanabe, P. G., Hefner, R. E. Jr., Braun, W. H., and Gehring, P. J. (1975). Gas chromatographic method for the preparation of l4C-labeled vinyl chloride, J. Labeled Compounds, 11, 535-542. Watanabe, P. G., McGowan, G. R., Madrid, E. O. and Gehring, P. J. (1976a). Fate of 14C-vinyl chloride following inhalation exposure in rats. Toxicol, Appl. Pharmacol., in press. Watanabe, P. G., McGowan, G. R. and Gehring, P. J. (1976b). Fate of 14C-vinyl chloride after single oral adminis­ tration in rats, Toxicol. Appl. Pharmacol., in press. Watanabe, P. G., Hefner, R. E. Jr., and Gehring, P. J. (1976c). Vinyl chloride induced depression of hepatic nonprotein sulfhydryl content and effects on bromosulphthalein (BSP) clearance in rats. Toxicology, in press. DO 1.37704 CONFTDFNTTAl LEGENDS Figure 1 Expired vinyl chloride (mg) versus time following a 6 hour exposure to 5000 ppm 14 C-VC. Repeatedly exposed animals singly exposed animals Figure 2 14 (x) and (•)• . , C-activity excreted in the urine expressed as percentage of the recovered radioactivity versus time following a 6 hour exposure to 5000 ppm (x) 14 C-VC. Repeatedly exposed animals and singly exposed animal (i). 00 137705 OONFTDFNTTAI TABLE 1 Percentage 14 C-Activity Eliminated During 72 Hours Following Inhalation Exposure to 5000 ppm Vinyl Chloride3 Percent 14 . . b C-Activity Single_ _ _ _ _ _ _ Repeated Expired: as VC as C02 54.5+3.5C (14.0)d 53.7±2.le (12.94) 8.0±1.4 (2.05) 9.611.6 (2.27) Urine 27.112.1 (6.93) 25.711.4 (6.21) Feces 3.212.5 (0.80) 1.410.4 (0.32) Carcass and Tissues 7.312.5 (1.89) 9.711.6 (2.32) Total mg Equivalents VC Recovered (25.67) (24.07) Rats exposed to 5000 ppm VC for 6 hours/day, 5 days/week for 7 weeks (repeated) or one 6 hour exposure (single). b Expressed as percentage of the total cMean ± SD, 14 C-activity recovered. 2 rats. ^Milligram equivalents VC. eMean 1 SD, 3 rats. 00 107706 CONFIDENTT AL TABLE 2 Percentage 14 C-Activity per Gram Tissue 72 Hour Following Inhalation Exposure to 5000 ppm Vinyl Chloride3 Percentage 14 . . b C-Activity Single Repeated Liver 0.11910.022° 0.15710.028' Kidney 0.06210.026 0.07010.006 Tissue N.D.e Fat N.D. Skin 0.04610.015 0.080+0.019 Carcass 0.03010.014 0.03910.011 aRats exposed to 5000 ppm VC for 6 hours/day, 5 days/week for 7 weeks (repeated) or one 6 hour exposure (single). b 14 . , Expressed as percentage of the total C-activity metabolized. cMean 1 SD, 3 rats. -a aMean 1 SD, 2 rats. eNot detectable, detection limit of 3 yg VC equiv./g fat or 0.03 percent 14C-activity metabolized per g tissue. DO 1.37707 GONFTDENTTAl TABLE 3 Effect of Vinyl Chloride on Drug Metabolism By A 9,000 x g Supernatent Fraction of Livera uq Control (4)^ Single VC Exposure (3) Repeated VC Exposure (5) a product/g liver/hour Aniline Hydroxylase £-Nitroanisole Q-Demethylase 65±16c 226+22 71±7 254±45 83 + 10 217±31 Rats exposed to 5000 ppm VC for 6 hours/day, 5 days/week for 7 weeks (repeated) or one 6 hour exposure - (single). Animals were killed immediately following the last ex­ posure and enzyme activity assayed. ^Number rats/group CMean ± SD DO 137708 CONFTDFNTTAL TABLE 4 Total Metabolism, Hepatic Macromolecular Binding and Hepatic Nonprotein Sulfhydryl Levels Following Single Or Repeated Exposure to 5000 ppm Vinyl Chloride3 B A yg VC Equivalents Metabolized yg VC Equivalents Bound per g Protein B/A x lOcfr Hepatic GSH {% of Control) Single Exposure 9265±1467c 113.5±10.4 1. 12±0.13c 39e Repeated Exposure 87181895^ 123.8+10.3 1.43+0.16f e 79 aRats exposed to 5000 ppm VC for 6 hours/day, 5 days/week for 7 weeks (repeated) one 6 hour exposure (single). ^The ratio of B/A x 100 was calculated from the individual animal data. cMean ± SD, 3 rats ^rtean ± SD, 5 rats eStatistically significant from air exposed controls. CONFTC T] M 2 si H o M O 2> ^Statistically significant from the single exposure. Student t-test (P<0.05). or mg VC Expired FIGURE I 0 1 2 3 4 5 Time (Hours) DO 137710 CONFTDFNTTAL Percent o f Recovered 14C-Activity FIGURE 2 conftdenttai DO 137711