Document zQd128LGbLBLMRr57ya23xK86

MANUFACTURING CHEMISTS ASSOCIATION 1925 CONNECTICUT AVENUE, N.W. WASHINGTON, O.C. 20009 (202) 483-0126 July IX, 1974 TO: Technical Task Group on Vinyl Chloride Research SUBJECT: Manuscript of Dow Report on VCM Metabolism Gentlemen: Distributed herewith are copies of the manuscript of the subject paper presented by Dr. Gehring at the May 10-11 meeting of the New York Academy of Sciences. Sincerely, KDJ/mb Enclosure Kenneth D. Johnson, Ph.D. Secretary Technical Task Group on Vinyl Chloride Research RSV 0000912 Preliminary Studies of the Fate of Inhaled Vinyl Chloride Monomer (VCM) in Rats Hefner, R. E. Jr., Watanabe, P. G. and Gehring, P. J. Dow Chemical U.S.A., Health and Environmental Research Midland, Michigan 4K640 INTRODUCTION Vinyl chloride monomer (VCM), extensively used for the production of polyvinyl chloride and other plastics, has been associated with the development of angiosarcoma and portal cirrhosis of the liver as well as other un toward effects in workers exposed to unknown but undoubtedly high concentrations of VCM. Angiosarcomas, zymbal gland carcinomas, and nephroblastomas developed in rats exposed to concentrations of VCM ranging from 50 to 10,000 ppm, 4 hours per day, 5 days per week for 12 months, and sub sequently maintained and observed until death . Refer ences to studies of the untoward effects of VCM in man and laboratory animals are located in this volume of The Annals of the New York Academy of Sciences. No information is available on the fate of VCM in the mammalian organism. Such information is essential for elucidating the toxicodynamics of VCM. This would provide a rationale to assess the potential hazard of exposure to low levels of VCM. Results of preliminary studies on the fate of VCM in rats exposed via inhalation are reported herein. Prior to undertaking these studies it *SV 0000913 was conceived that VGM might be metabolized via the alcohol dehydrogenase pathway, and if so, this pathway would very likely be saturable and that conjugation of VCM or its metabolites with glutathione and cysteine might be expected* Therefore, much of the work conducted to date has been directed at evaluating these possibilities. METHODS Kinetic Studies of the Uptake (Metabolism) of Inhaled Vinyl Chloride Monomer_____ Male Sprague-Dawley albino rats of Spartan strain weighing from 165 to 200 grams were exposed to initial concentrations of VCM gas ranging from 50.5 to 1167.0 ppm (0.13 to 2.99 mg/). Exposures ranged from 52.5 to 356.3 minutes. Figure 1 depicts the closed, recirculating 4.7& inhalation apparatus in which a group of four rats were concurrently exposed. To minimize contamination of fur and skin, only the nares of the rats protruded through a rubber membrane into the chamber. Expired carbon dioxide was continuously removed from the chamber by absorption on an Ascarite column in the recirculating system. As carbon dioxide was removed from the system, the pressure drop was detected by a mercury manometer fitted with a photoelectric cell. This activated a solenoid valve and dual syringe pump to inject makeup oxygen into the system. The chamber atmosphere was continuously analyzed for VCM (10.9u) by an in line Miran-I infrared analyzer (Wilks). RSV 0000914 -3- Using known concentrations of VCM prepared in 1002. Saran bags, the absorbance versus concentration adhered to the Beer-Lambert Law. During the exposures, routine checks for carbon dioxide in the chamber atmosphere were made at 4.26u* Oxygen consumption for each experiment was determined directly by measuring the oxygen metered into the system using a dry test meter (American Meter Co.). Prior to each exposure, the desired initial concentra tion of VCM was generated in the empty recirculating system and the decline in concentration was followed for a time equal to or exceeding that of the intended experiment. The decline in concentration was in accordance with first order kinetics as described by dC/dt a -K. C 1o In this equation, C(ppm) equals the concentration of VCM at time t(min), CQ (ppm) equals the initial concentration of VCM, and (min is the rate constant for the decline in VCM concentration from the empty recirculating system. Regression analysis of the logarithm of the concentration of VCM versus time yielded K^. Initially values for were determined before and after inclusion of rats in the chamber. These values were reproducible, therefore was not redetermined after removal of the rats in every subse quent experiment. Between experiments, the system was disassembled, cleaned, and reassembled using a new Ascarite RSV 0000915 a f -4 - column. The for each reassembled system was unique, with in certain limits, which necessitated its redetermination for each experiment. After determining the rate of decline of VCM concen tration in the empty inhalation system, four rats were placed in the chamber. -The chamber was then charged with the desired concentration of VCM, and the rate of decline of VCM concen tration was determined as previously described. The rate of decline, Kg, followed apparent first order kinetics. After an initial equilibration of the tissues of the rats, the de cline of VCM concentration from the closed system was assumed to occur via metabolism of VCM plus the background loss from the system. Since oxygen consumptions were within 10% for all experiments described herein respiratory parameters were not significantly changed. Therefore, respiratory parameters were not a factor influencing the rates of metabolism. The rate of metabolism, Kc, would therefore be given by the equation cel To assess the effects of potential inhibitors of VCM metabolism, rats were pretreated by irtraperitoneal injection with 320 mg/kg pyrazole (1,2-diazole) 1 hour prior to exposure, 5 ml/kg ethanol 1.5 hours prior to exposure, or 75 mg/kg SK&F-525-A (&-diethylaminoethyldiphen}lpropylacetate) 0.5 hour prior to exposure. Without clearing or reassembling the inhalation system, both untreated control rats and rats treated with the chosen potential inhibitor were exposed to the desired concentration of VCM. The percent inhibition was calculated from the equation RSV 0000916 -5- K (control) - K treated --------------------------* % inhibition Kc control Effects of VCM on Liver Sulfhydryl Levels Groups of male Sprague-Dawley albino rats of Spartan strain weighing from 193 to 250 grams at initiation of the experiment were exposed to nominal concentrations of 15,000 ppm vinyl chloride for 5 days, 5000 or 500 ppm 5 days per week for 1, 3, or 7 weeks, and 50 ppra for 1 hour, 7 hours, or 5 days. The exposures were carried out in a glass-walled 16Q chamber under dynamic conditions with the VCM being metered into the chamber airstream. For repeated daily exposures, 7 hour exposures were conducted on the first four of five consecutive days each week. On the fifth day, if the rats were to be sacrificed, the duration of exposure was reduced to 5 to 6 hours? however, if the rats were being continued on exposure, they received a full 7 hour exposure. The body weights and food consumption of rats exposed to 500 and 5000 ppm VCM were determined before each daily exposure and the rats were observed periodically for signs of toxicity. Between 1 and 2 PM, immediately following the fifth exposure of the designated week, the rats were killed by cervical dislocation and the livers removed and prepared for assay of sulfhydryl content. Gross patholog ical examinations were conducted. RSV 0000917 0- 6- - The method used for the sulfhydryl assay was a modi- 12 fication of that described by Sedlak and Lindsay Exactly 500 mg of liver from each rat was homogenized for 1 minute in a Dounce tissue homogeni2er containing 8 ml of 0.02M disodium EOTA. For the total sulfhydryl assay, a 0.5 ml aliquot of each homogenate was mixed with 1.5 ml of 0.2M tris HC1 pH 9.2 buffer, 0.1 ml of 0.01M DTNB (5,5'- dithiobis(2-nitrobenzoic acid)), and 7.9 ml of methanol. A reagent blank without liver homogenate, and a sample blank, without DTNB, were also prepared. The color generatec via the release of nitromercaptobenzoic acid anion was allowed to develop for 15 minutes, and the samples were centrifuged for 15 minutes at 4,000 g. Absorbance of each sample was read against the respective sample blank at 412 nm with a Beckman DB spectrophotometer. Subsequently, the molar concentration of total sulfhydryl in the sample was calculated using an extinction coefficient determined from standards of known concentrations of glutathione or cysteine. A plot of absorbance versus the concentration of cysteine or glutathione coincided with that reported by Sedlak 12 Lindsay nd The non-protein sulfhydryl content of liver was determined after precipitating out the protein by addition of 1 ml of 50% trichloroacetic acid to a 5 ml sample of liver homogenate. Each sample was diluted with 4 -'1 of distilled water and after 15 minutes centrifuged at 4,000 g. A 2 ml aliquot of the supernatant was mixed with 4 ml 000091* RSV -7- of 0.4M tris HC1 pH 8.9 buffer. Immediately before read ing the absorbance against a reagent blank, 0.1 ml of 0.01M DTNB was added. Subtraction of the non-protein sulfhydryl content from the total sulfhydryl content yielded a value for protein-bound sulfhydryl. Urine samples collected from rats exposed to 5000 ppm vinyl chloride for 4, 5, and 7 weeks were analyzed for the presence of S(2-chloroethyl)cysteine, S(2-hydroxyethyl)cysteine, and S (2-carboxymethyl)cyst sine (5 and 7 week samples only). Urine was collected for analysis by applying pressure to the posterior abdomen between the fifth and sixth hour of the fifth daily exposure on the designated week. Urine collected on the same cay was pooled. For the three aforementioned compounds, 2 to 15 yl of urine were spotted directly on a 5 by 20 cm Baker-flex* silica gel plate. Also spotted were samples of urine collected from control rats and standard aquecus solutions as well as control urine to which approximately 1 ug/ul of each of the compounds had been added. The chromatograms were developed for 5 hours in a sealed glass tank containing nbutanol, acetic acid, water 80:10:10 or 60:20:20. After being air dried, the plates were sprayed with Ninspray ninhydrin reagent and heated for 2 minutes at 809C. The color of the spots and their Rf values .were used to identify the compounds. A urine sample collected from rats exposed to 5000 ppm vinyl chloride for 9 weeks was analyzed for the presence ftSV 0000919 2- IL 8-- - o chloroacetic acid. The 10 ml urine sample was acidi fied with 0.1 ml of 50% v/v H2S04. Subsequently, the urine sample was extracted 3 times with 2 ml of diethyl ether. The diethyl ether extract was evaporated to 0.1 ml and 2 to 15 ul of the concentrated extract and an aqueous standard containing 5% w/v chloroacetic acid were spotted on an Eastman fluorescent silica gel plate. The chromato grams were developed in a sealed glass tank for 5 hours using the aforementioned solvent systems. After air drying the plates, they were examined under ultraviolet light to determine the location of spots. Subsequently, the plates were treated with ninhydrin as described previously. RESULTS Kinetic Studies on the Metabolism of Inhaled VCM Typical declines in the concentration of VCM in the inhalation apparatus containing 4 rats and initial concen trations of approximately 50 or 1000 ppm are shown in Figure 2. Also shown are the corresponding declines in the concentration of VCM from the unoccupied chamber. As indicated in the methods, the rate of these declines, Ke and K^/ respectively, were determined by regression anal ysis. The rate of metabolism, Kc* was assumed equal to Ke~K^. For seven separate exposures to concentrations of VCM ranging from 50 to 105 ppm, the mean and standard deviation for the apparent first order rate constant, K_, V were -8.04 xlO ^ + 3.40x10 ^ min This corresponds to a one-half life (tjy2) minutes. RSV 0000920 -9- For five separate exposures to concentrations of VCM ranging from 220 to 1167 ppm, K was -2.65x10 3 + 1.35x10 min 1. This corresponds to a 1: m^nutes* As indicated by the standard deviations, there was little variation of Kc within the indicated range of concentration, Furthermore, there was no consistent trend for the values of Kc within this range. Administration of 320 mg/kg pyr&zole, an inhibitor of alcohol dehydrogenase., xanthine oxidase and other enzymes3, 1 hour before exposure to 65 and 123`i ppm VCM resulted in 71.2 and 86.9% inhibition of metabolism of VCM, respectively (Figure 3). Because of the lack of specificity of pyrazole as an enzyme inhibitor, 5 ml/kg 95% ethanol was administered to rats in an attempt to specifically inhibit alcohol dehydro genase activity. In rats exposed to initial concentrations of 56 and 97 ppm VCM, ethanol pretreatment caused 96.0 and 82.9% inhibition of VCM metabolism, respectively. When rats were pretreated with ethanol and exposed to initial concentrations of 1025 and 1034 ppm ^CM, inhibition of metabolism was 46.5 and 35.7%, respectively. Figure 4 illustrates the inhibition for separate experiments con ducted at extremes of initial VCM concentrations. Pretreatment of rats with 75 mg,'kg SK&F-525-A, an inhibitor of some types of microsomal oxidases* 3 resulted in no inhibition RSV 0000921 -10- o metabolism of VCM in rats exposed to initial concen trations of 65 ppm and an 18.8% inhibition in those exposed to 1038 ppm (Figure 5). Effect of Inhaled VCM on Liver Sulfhydryl Levels No antemortem or postmortem signs of toxicity were noted in rats exposed to any concentration of VCM used in these experiments. Analysis of food consumption data for rats exposed to nominal concentrations of 500 or 5000 ppm VCM, and unexposed controls revealed no statistically signifi cant differences. In addition, no significant difference was found in the water consumption of rats exposed to 5000 ppm and controls. Throughout the durations of observation, the mean body weights of rats exposed to the various concen trations of VCM were essentially the same as those of the respective controls. No gross pathological lesions related to exposure of VCM, were found in any of the rats. Table 1 shows the non-protein sulfhydryl content of the liver of rats exposed to 50, 500, 5000 or 15,000 ppm VCM for the indicated durations. Values for concurrent controls are also given. In Figure 6, these data are summarized and expressed as the percent depression of the non-protein sulfhydryl content of the liver as a function of exposure concentration and duration of exposure. Significant reductions in the levels of non-protein sulf hydryl occurred in rats exposed to 50 ppm VCM for 7 hours, 500 and 5000 ppm for 1 and 3 weeks, and 15,000 ppm for 1 RSV 0000922 -11- week. There is no definitive association between exposure concentration and the degree of depression. The degree of depression decreases with continued exposure which suggests compensatory mechanisms are responding to relieve this biochemical effect induced by VCM. The protein bound sulfhydryl content of the liver of rats exposed to 50, 500, 5000 or 15,00C ppm VCM for the indicated durations were not significantly different from those of the concurrent controls. .This was not unexpected because the sulfhydryl groups of protein have been shown not to be readily akylated unless denaturation of the protein renders them available for alkylation1u - Effect of Ethanol Administration of VCM Induced Depression of Non-Protein Sulfhydryl in Liver_____________________________________ In this experiment, four rats pretreated with 5 ml/kg ethanol and four untreated control rats were exposed to 1070 ppm VCM. Following 105 minutes of exposure, the concen tration of non-protein sulfhydryl in ths liver was deter mined. For those pretreated with ethanol, the depression of non-protein sulfhydryl was 77.0 + 12.8%, while for controls it was 95-0 + 3.4%. These levels of depression were signifi cantly different as determined by Student's "t" test, p<0.05. 7 Johnson has reported that ethanol aloni* does not affect the non-protein sulfhydryl content of the liver. RSV 0000923 A -12- Metabolites of VCM Preliminary results which must be viewed with con siderable reservation have been obtained from studies in which rats were exposed to 5000 ppm unlabeled VCM for 4, 5 or 7 weeks. Chromatograms of urine collected from rats after each of these exposure durations were comparable. S(2-hydroxyethyl}cysteine (R^ 0.26-0 28 in 80:10:10 nbutanol, acetic acid, water) appeared to be present. S(2chloroethyl)cysteine and S(2-carboxynethyl)cysteine were not detected; however, it is conceivable that the latter compound was not adequately resolved from the urine back ground. In another experiment, chromatograms of urine from rats exposed to 5000 ppm VCM for 9 waeks revealed the presence of monochloroacetic acid (R^. 0.74 using the aforementioned solvent system). The spots for monochloro acetic acid were visualized under ultraviolet light on the fluorescent plates and did not develop with ninhydrin reagent. Studies of the metabolism of VCM were terminated until methods were developed to synthesize 14 C-VCM. 14 CVCM in a liquid state polymerized even when an inhibitor was present and a temperature of -70C was maintained. Recently, a method has been developed to synthesize 14 CVCM in a gaseous state from 1,2-dichloroethane. In the 14 gaseous state C-VCM is relatively stable. Currently, RSV 0000924 -13- the methodology is being refined to allow synthesis of 14 C-VCM with reproducible specific activity. Using 14C-VCM, three 180 g. mal-i rats have been exposed in the closed recirculating system for 65 minutes. The initial concentration of *"4C-VCM with a specific activity of 1.155 mCi/mmole was 49 ppm. Assuming equiva lent uptake of VCM by the 3 rats, each rat received 0.49 mg/kg VCM. Immediately following exposure, the rats were removed from the chamber and placed in Roth type metabo lism cages which allow separate collection of urine, feces, expired carbon dioxide and any expired VCM. Within 15 hours post exposure, a mean of 58.0% of the 14 C activity had been excreted in the urine, 2.7% m the feces, and 9.8% as expired carbon dioxide. By 75 hours post exposure, 67.1% had been excreted in the urine, 3.8% in the feces, and 14.0% as expired cirbon dioxide. Only a trace, 0.02% of the dose, was expired as VCM and trapped on activated carbon. After 75 hours, 1.6% of the dose remained in the liver, 3.6% in the skin, 0.2% in the kidneys and 7.6% in the remaining carcass. The mean percent of the assumed dose recovered from the threes rats was 97.9%. Various chromatographic techniques are being evaluated for separation and identiflcation of the 14 C labeled urinary metabolites. S(2-hydroxyethyl)cysteine and S(2carboxymethyl)cysteine and their respective N-acetyl derivatives appear to be likely metabolites. Ultimate verification of metabolites will be determined by gas RSV 0000925 l -14chromatographic - mass spectroscopic analysis. In the experiment just described, monochloroacetic acid has not been detected. In summary, metabolism studies have demonstrated tentatively the following: a) VCM is quite readily metabolized to polar metabo lites which are excreted predominantly in the urine of rats exposed via inhalation to an initial concentration of 50 ppm. Smaller amounts of VCM are excreted in the e:cpired air as carbon dioxide and in the feces. Vory little is excreted in expired air as unchanged VCM. b) A significant but small amount is retained in tissue, particularly liver, as long as 75 hours post exposure. c) Metabolites excreted in the urine appear to be conjugated with glutathione and/or cysteine through covalent linkage to :he sulfhydryl group. This is consistent with the 'eduction of the non protein free sulfhydryl leva is in the livers of exposed rats. Preliminary in vitro experiments have shown that direct conjugation of vinyl chloride with cysteine or glutathione in aqueous solutions occurs to a small degree but very slowly. d) Monochloroacetic acid also appears to be a metabolite of VCM, when rats were exposed to 5000 ppm for an extended tint';. RSV 0000926 -15- DISCUSSION Because of the preliminary nature of the studies reported herein, the results and subsequent discussion must not be considered conclv- Justification for premature publication of some of the data contained herein is the magnitude of the impact of recently revealed toxi cological effects of VCM and the urgent need for com munication of even preliminary results. The studies reported herein have demonstrated re liably in some instances and tentatively in others the following: a) Rats exposed to concentrations of VCM below 100 ppm metabolize the compound ;:airly readily and in accordance with first ordesr rate kinetics, t^/2 * 86 minutes. b) When exposed to a concentration of VOi exceeding 200 ppm, its rate of metabolism was reduced, tjy2 " 26* minutes. This indicates that the predominate pathway for metabolism of VCM by rats exposed to 100 or less is saturable. Residual metabolism at concertrations exceeding 200 ppm may be via this pathvay in conjunction with additional pathway(s). c) Pyrazole inhibits the metabolism of VCM suggest ing that metabolism of VCM is via alcohol dehydro genase . RSV 0000927 2 -16- t d) Stronger evidence for the metabolism of VGM by alcohol dehydrogenase was its inhibition by the administration of etharol. This inhibition was less pronounced in rate exposed to 1000 ppm than in rats exposed to 100 ppu or less, suggesting that metabolism via pathways other than that of alcohol dehydrogenase occurs in rats.exposed to 1000 ppm VCM. e) SK&F-525-A does not affect the metabolism of VCM by rats exposed to 65 ppm VCM but there is an indication that it slightly depresses metabolism by rats exposed to 1038 ppm VCM. This suggests that in. rats exposed to concentrations of VCM which exceed the capacity of the alcohol dehydro genase pathways metabolism may occur via oxidases in the microsoroes. f) Monochloroacetic acid was found in urine of rats exposed to 5000 ppm VCM daily for 9 weeks. g) Exposure to VCM reduces the non-protein sulfhydryl concentration of liver. This reduction is not definitively associated with the exposure concentration of VCM in a range of 50 to 15,000 ppm. There is a tendency for the reduction to become less pronounced with repeated daily exposures. These results are consistent with a 0ooog^a -17- saturable mechanism for the metabolism of VCM fol lowed by conjugation of the metabolites of VCM with glutathione and/or cystsine. h) The administration of ethanol significantly re duces the depression of the concentration of non protein sulfhydryl in the livers of rats caused by exposure to 1000 ppm VCM for 105 minutes. i) In rats exposed to 49 ppm, VCM is metabolized to polar products which are axe reted predominantly in the urine. These products appear to be derived following initial metabolism of VCM and subsequent conjugation of the products with glutathione and/or evsteine through covalent binding with the suij.--*dryl. A small but significant fraction of VCM :.s metabolized to C02 and expired. An even smaller but signifi- 14 cant amount of C activity appears to be retained in the liver primarily, but also in other tissues as long as 75 hours post exposure. Considering the results in toto, we hypothesize that in rats exposed to concentrations or. VCM below 100 ppm, VCM is predominantly metabolized via. sequential oxidation to 2-chloroethanol, chloroacetaldehyde, and monochloroacetic acid by the alcohol dehydrogenase pathway. C1HC = CH, C1H-C-CH.0H -------------- 2 2 dehydrogenase cih2c-cho cih2c-ccoh RSV 0000929 -18- Only small amountsr if any, of monochloroacetic acid are formed at low doses because chloroacetaldehyde reacts rapidly g with the sulfhydryl of glutathione and cysteine . This conjugation accounts for the lack of dose related, reduction in the non-protein sulfhydryl content of the liver of rats exposed to 50 through 15,000 ppm VCM. Alternate pathways of VCM metabolism which may be involved at high doses are at this time purely speculative. However, at 200 ppm, metabolism by the more rapid alcohol dehydrogenase pathway appears to be saturated, and metabolism via oxidation of the accumulating 2-chloroethanol may occur as follows: C1H-C * CH,0H * H22 ^ catalase ClH-C-----CH,OOH-->C1H2C z* * CHO Carter, et. al.3 have demonstrated that microsomal oxidation of ethanol in vitro proceeds via the formation of hydrogen peroxide and catalase which subsequently forms a peroxide of ethanol. Acetaldehyde is the end product of this oxidation. It is conceivable that a similar oxidation of 2-chloroethanol occurs. In addition to this series of reactions, a direct expoxidation of VCM may occur as follows: RSV 0000930 -19- ClHC ai. oxidase > cih2c-cho ----------------------- > cih2c-cooh Zicf/ et. al. 1 s have r.jjown that chloroethylene oxide spon taneously rcsrrunoiis to ehloroccetaldehyoie. Such mechanisms would explain why SIC&F-525-A causes a slight inhibition of metabolism in rats exposed to 1038 but not 65 ppm VCM. Also it may explain why mcnoohloroaectic acid may be excreted by rats exposed to 5000 ppm but not 50 ppm VCM. In the former case, chloroacetaldchyde is produced by each of the hypothesized pathways which may result in a greater amount being oxidized to monochloroacetic acid than being conjugated with gluta thione and/or cysteine. Inferences from the results of these studies about the toxicodynamics of VCM are premature, but worth mentioning. First, the saturation of a primary metabolic pathway for VCM degradation and redirection through other pathways provides some hope that a threshold concentration for the untoward effects of VCM may exist. Metabolites of VCM formed only via the alternate pathways may constitute the ultimate toxin and carcinogen. RSV 0000931 5 -20It has been reported that the administration of cysteine or glutathione provides protection against the untoward effects of various aliphatic and aromatic mustards, triethylenemelamine, X-rays# and ionizing radiation.1 <* '5'10'1 k Conceivably# cysteine and glutathione may provide a natural defense against tumor producing free radicals generated within the body, as well as synthetic or naturally occurring alkylating agents which are absorbed into the body. There fore# reduction of the non-protein sulfhydryl content of the liver in animals exposed to VCM may constitute predisposi tion to toxicity and carcinogenicity mediated via other materials. Finally, the speculated formation of chloroethylene oxide seems particularly pertinent insofar as postulation of the mechanism of carcinogenesis. This compound is undoubtedly a very active difunctional alkylating agent. It is most interesting that inorganic arsenicals have been reported to cause untoward hepatic effects, portal cirrhosis and angio sarcoma, like those reported for VCM.11 The mechanism of toxicity for arsenic has been shown to occur via its reac tion with 6,8-dithiooctanoic acid (a-lipoic acid).6 In this reaction arsenic forms a stable bridge between the two sulfhydryl groups. If chloroethylene oxide were formed, it would readily react with a-lipoic acid, bridging the sulf hydryl groups like arsenic. ASV 0000932 -21- CH + Cl CH-------CH--------CII------- (CH-) -A------ COOH Iz I 24 SH SI2 CII2----- C: j i------ {CH2) ^-------COOH S \ CH--CH OH Although this postulated mechanism in highly speculative/ the rarity of materials knov/n to produce untoward hepatic effects like those of VCM must bo given some weight. ACKNOWLEDGMENTS Those studies were supported in part by a grant from the Manufacturing chemists Association. The technical advise of Dr. U.K.J. T.eong in designing the inhalation apparatus and the critical review of the manuscript by Dr. V. K. Rowe is gratefully acknowledged. 6/21/74 RSV 0000933 X. -22- REFERENCES 1. Ball/ C. R. 1966. Biochom. Pharmacol. 15:809-816. 2. Calcutta G., T. A. Connors, l. h. Elson and W.C.J. Pose. 1963. Biochcin. Pharmacol. 12:833-837. 3. Carter, E. A. and K. J. Ii'jelbacher. 1972. Lab. Invest. 27:28 3-28 6. 4. Dow Chemical Co. - Unpublished data. 5. Goldcnthal, E. I., M. U. Nndharni and P. K. Smith. 1959. Rad. Rgs. 5:571-583. 6. Gonsalus, I. C. 1953. J. Cell. Comp. Physiol. 41 (suppl. 1):133 . 7. Johnson, Ii. K. 1965. Biochem. Pharmacol. 14:1383. 8. Johnson, M. K. 1967. Biochera. Pharmacol. 16:185-199. 9. Maltoni, C. This Journal. 10. Patt, II. M. 1953 . Physiol. Rev. 33:35-76. 11. Popper, II. National Institute of Health, Bethesda, MD. Personal Communication. 12. Sedlak, J. and R. H. Lindsey. 1968. Anal. Biochem. 25:192-205. 13. Soliman, M. R.I., H. D. Johnson and A. E. Wade. 1974. Me tab . and Disposlition. 2:87-96. 14. Stacey, K. A. , M. Cobb, S. E. Consens and P. Alexander. 1958. Ann. N. Y. Acad. Sci. 68:657. 15. Zief, M. and C. H . Schramm. April 18, 1964. Chem. Ind. 660-661. RSV 0000934 Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Captions for Figure and the Table Closed Recirculating Inhalation Apparatus Used for Exposing Rats to VCM. Typical declines in VCM concentration with time at approximately 50 and 1000 ppm VCM ex-* posure concentrations. Also shown are the respective declines in VCM concentration for the unoccupied inhalation apparatus. Declines in VCM concentration with time at 65 and 1234 ppm VCM exposure concentrations for both pyrazole pretreated and untreated control rats. Also shown are the respective declines in VCM concentration for the un occupied inhalation apparatus. Declines in VCM concentration with time at 56 and 1034 ppm VCM exposure concentrations for both ethanol pretreated and untreated control rats. Also shown are the respective declines in VCM concentration for the unoccupied inhala tion apparatus. Declines in VCM concentration with time at 65 and 1038 ppm VCM exposure concentrations for both SK&F-525-A pretreated and untreated con trol rats. Also shown are the respective de clines in VCM concentration for the unoccupied inhalation apparatus. RSV 0000935 a t Figure 6 Table 1 -2- Percent depression of non-protein sulfhydryl in the liver of rats as a function of VCM ex posure concentration and duration of exposure. --0 Non-protein sulfhydryl content (1 x 10 moles SH/mg of liver) of rats exposed to SO, 500, 5000 or 15,000 ppm VCM and concurrent controls. RSV 0000936 600 " 500 VCM Exposure Concentration (ppm) Controls RSV 000093? 0000938 IHrQ VINYL CHLORIDE CONCENTRATION 7 Hr* c Weeks 50 ppm * 1 3 7 1 3 7] \ 500 ppm / s ]*j> 5,000 ppm 1 15,000 ppm I_________I_________I_________I_________I-------------L. 0 10 20 30 40 50 % Depression Of NPSH Mean Cone. -- Mean Cone. * Depression - Of &>"* O' Epo* Mean Cone. Of Controls s "Significantly depressed using Students "T" lift, p^0.09 _l_ 60 70 t IVCM Exposure Concentration (ppm) &SV OOOO939 VCM Exposure Concentration (ppm) | HSV 0000940 RSV 0 0 0 0 9 4 1 1 HrQ VINYL CHLORIDE CONCENTRATION 7 Hrs Q Weeks > 50 ppm ]* 1 3 * 500 ppm 7 \ 1 3 ft 5,000 ppm 7 / 1 15,000 ppm I_________I_________l_________I_________I________U 0 10 20 30 40 60 % Depression Of NPSH Mean Cone. -- Mean Cone. % Depression - 0* Controls Of Exposed Mean Cone. Of Controls s 'Significantly daprassad using Studtnt* *T* test, p<0,06 60 70 t 1 . nation of Exposure 15,000 ppm Concurrent Controls VCM CONCENTRATION 5,000 ppm Concurrent Controls 500 ppm Concurrent Controls +50 ppm Concurrent Controls 7 hours per day for 5 days *0.29 +0.08 0.42 +0.07 *0.33 +0.02 0.46 +0.03 *0.35 0.09 0.58 +0.06 0.34 +0.01 0.43 +0.09 7 hours per day 5 days per week for 3 weeks *0.41 +0.03 0.72 +0.03 *0.45 +0.06 0.61 +0.08 7 hours per day 5 days per week for 7 weeks 0.59 +0.08 0.70 +0.02 0.58 +0.06 0.64 +0.08 A single 1 hour exposure to 50 ppm VCM resulted in 0.64+0.05 and 0.65+0.06 for the concurrent controls. A single 7 hour exposure to 50 ppm VCM resulted In 0.17+0.07 and 0.44+0.08 for the concurrent controls. >* Significantly different using Students "t" test, p<0.05. RSV 0 0 0 0 9 4 2