OPIS-SECUMICAL ISJTORMAIIOH CS&SMi EPA- TOXICITY STUDIES OF SELECTED CHEMICALS TASK III: THE TOXICITY OF VINYLIDENE CHLORIDE IN MICE AND RATS AND ITS ALTERATION BY VARIOUS TREATMENTS JUNE 1977 DRAFT FINAL REPORT ENVIRONMENTAL PROTECTION AGENCY OFFICE OF TOXIC SUBSTANCES WASHINGTON, D.C. 20460 ------- Document is available to the public through the National Technical Information Service, Springfield, Virginia 22151. ------- TOXICITY STUDIES OF SELECTED CHEMICALS TASK III: THE TOXICITY OF VINYLIDENE CHLORIDE IN MICE AND RATS AND ITS ALTERATION BY VARIOUS TREATMENTS Final Report Prepared by Robert D. Short, Jr. Joseph M. Winston Jan L. Minor Chuen-Bin Hong Brett Ferguson Timothy Unger Mary Sawyer Cheng-Chun Lee Contract No. 68-01-3242 Joseph Seifter Project Officer Office of Toxic Substances U.S. Environmental Protection Agency Washington, D.C. 20460 June 1977 ------- PREFACE This report was prepared at Midwest Research Institute, 425 Volker Boulevard, Kansas City, Missouri 64110, under Environmental Protection Agency Contract No. 68-01-3242, MRI Project No. 4128-B, "Toxicity Studies of Selected Chemicals." The work was supported by the Office of Toxic Substances of the Environmental Protection Agency. Dr. William Marcus and Dr. Joseph Seifter are consecutive contract monitors for this project. the direction of Dr. William B. House between May 1, 1976 and June 1, 1977. The experimental work was supervised directly by Dr. Cheng-Chun Lee, Assistant Director, Biological Sciences Division for Pharmacology and Toxicology; assisted by Dr. Robert D. Short, Jr. (Senior Toxicologist), Dr. Joseph M. Winston (Associate Toxicologist), Mr. Jan L. Minor (Associate Toxicologist), Dr. Chuen-Bin Hong (Associate Pathologist), with the technical assistance of Mr. Brett Ferguson, Mr. Timothy Unger, and Ms. Mary Sawyer. Approved for: MIDWEST RESEARCH INSTITUTE This work was conducted in the Biological Sciences Division under W. B. House, Director Biological Sciences Division ii ------- NOTICE This document is a preliminary draft. It has not been formally released by EPA and should not at this stage be construed to represent Agency policy. It is being circulated for comment on its technical accuracy and policy implications. SUMMARY The toxicity of vinylidene chloride (VDC) was studied in mice and rats exposed to various concentrations of the vapors for 23 hr a day. In addition, the ability of various treatments to alter parameters of toxicity was evaluated. Mice were more sensitive than rats to the acute lethal, hepatotoxic and renal toxic effects of VDC. Disulfiram treatment reduced these toxic effects of inhaled VDC and reduced the levels of covalent bound radioactivity in the liver and kidney after the intraperitoneal administra- tion of ^C-VDC. Treatment with diethyldithiocarbamate and thiram also protected mice from the acute lethal effects of VDC. 1 ------- I. INTRODUCTION Vinylidene chloride (1,1-dichloroethyIene; VDC) is an intermediate used in the production of polymers and the synthesis of other chemicals. Since environmental contamination and human exposure are inevitable results of its widespread use, the risks of such exposures should be understood. 1 2/ The toxicity of VDC, which was recently reviewed,—1— has been studied in several mammalian species.—/ A continuous 90-day inhalation exposure to 189 mg/m^ (48 ppm) of VDC produced deaths in monkeys and guinea pigs but did not in dogs and rats. In addition, morphological changes occurred in livers from monkeys, dogs, and rats and in kidneys from rats. The hepatic lesions included focal necrosis, hemosiderin deposition, and fatty metamorphosis. The primary renal lesion was nuclear hypertrophy of the tubular epithelium. VDC toxicity was influenced by various parameters. Female rats were more sensitive than males to the oral toxicity of VDC.— The nutri- tional status also influenced toxicity. For example, starved rats were more sensitive to VDC^./ and the diurnal change in toxicity was correlated with hepatic levels of glutathione (GSH) .6./ Additional studies with diethyl- maleateZ/ and cysteine^/ pretreatment suggest that hepatic levels of GSH influence VDC toxicity. Metabolic studies indicate that (a) a major path- way for detoxification of VDC is by conjugation with GSH and (b) hepatotoxicity is associated with covalent binding of VDC metabolites in the liver.—' The purpose of this study was to determine the acute toxicity of continuously inhaled VDC and evaluate the effect of various treatments on this parameter. The treatments were selected to alter the metabolic activa- tion of VDC or promote the detoxification of VDC. In addition, adrenergic blocking agents were used since VDC exposure sensitized rat hearts to cate- cholamines II. METHODS A. Animals CD rats and CD-I mice (Charles River Breeding Laboratories, North Wilmington, Massachusetts) were housed in our animal quarters for at least 7 days prior to use. These quarters were maintained at 24 ± 2°C with a relative humidity of 50 ± 10% and a 7 AM to 7 FM photoperiod. Rats were generally housed two per cage and mice six per cage. Animals were given free access to rodent chow (Wayne Lab-Blox, Allied Mills, Inc., Chicago, 2 ------- Illinois) and tap water. During the exposure period, animals were housed in stainless steel cages with wire mesh bottoms. When feed consumption was determined, the animals were given powdered rodent chow in stainless steel feeders (Hoeltge, Cincinnati, Ohio) which were designed to minimize spillage. B. Exposure to VDC 1. Duration: Animals were exposed to VDC for 22 to 23 hr a day for 7 days. Control animals were similarly housed and exposed to room air. 2. Chamber design: Five stainless steel cubical type animal exposure chambers were used in these studies. Three chambers were of 3.5 m^ volume and two chambers were 4.5 m^ in volume. The contaminants entered the inlet air stream and were mixed in a plenum at the top of each chamber. Air was exhausted at the bottom of the chamber. 3. Chamber air supply and flow rate: The air supply to the chambers was drawn from a stack on the roof of the building. Chamber air passed through a coarse filter and then over coils for heating, cooling and dehumidifying. Chamber temperature was maintained at 75°F ± 5° and humidity at 50% ± 10%. Air flow rates were measured at the exhaust side of the chambers with orifice plates connected to magnehelic gauges. Chamber air flows were calibrated using an Autotronics 100-ssx air flow transducer. Air flow rates were adjusted to maintain 10 to 15 air changes per hour in each of the chambers. 4. Chamber safety: All chambers were operated at a slight nega- tive pressure (0.1-0.2 in. of water) to prevent escape of the contaminant into the room atmosphere. All air from the chambers traveled under negative pressure to an incinerator operated at a temperature of 1700° to 1800°F. 5. Generation of VDC vapor: Vinylidene chloride with a purity of 99% was obtained from the Aldrich Company. VDC vapor was generated by bubbling nitrogen into VDC contained in a glass flask. A stream of inlet air was directed through the flask and carried the VDC vapor into the plenum of the chamber. The rate of VDC generation was determined by the flay. of nitrogen into the VDC flask. The nitrogen flow was controlled by th_e use of rotameters. 6. Chamber atmosphere monitoring: VDC was quantified with, a Varian 2700 gas chromatograph equipped with a flame ionization detector and a stainless steel column (6 ft x 1/8 in.) packed with 0.4% Carbowax 1500 on 3 ------- Carbopak A. The injector, column, and detector temperatures were 135°C, 95°C and 170°C, respectively. A Varian CDS-111 chromatography data system was used to determine chamber concentrations. The CDS-111 calculates chamber concentration by integration of peak areas. The CDS-111 was calibrated with VDC standards at the beginning of each day and provided a direct printout of chamber concentration in parts per million. VDC standards were prepared by serial dilution (weight/volume) of VDC in carbon tetrachloride. The desired final concentrations in a 4 jUl injection were determined by the following procedure: mg/liter (fig/ml) = Ppm * ^ 24450 The amount of VDC (mol wt = 96.94) in a 1-ml sample from chambers that contain 10, 50 and 100 ppm VDC is 0.04, 0.20 and 0.40 g, respectively. For 1 Ml of standard, these same amounts would be obtained from solutions of 40, 200 and 400 mg/liter; but since 4 fil of standard were injected, the final dilutions of VDC in carbon tetrachloride were 10, 50 and 100 mg/ liter. Each point on the calibration curve was the mean of three determina- tions. Each chamber was equipped with 10 sampling ports on two sides of the chamber. Preliminary studies were conducted to determine the uniformity of the concentration of the test material at different points in the chamber. The concentration at the various sampling points was compared to a reference point in the center of the chamber. It was found that the average concen- tration at the various points was ± 3% of the desired chamber concentration. The concentration at the reference point was 98.4% to 100.4% of the average chamber concentration. Therefore, the chambers were routinely sampled at the reference point for determination of chamber concentrations. C. Treatments to Alter VDC Toxicity Groups of mice were treated with one of several compounds in order to alter the toxicity of VDC. The chemical structure of some of these com- pounds is shown in Figure 1. The compounds used in this study were: disulfiram (0.10% in feed 2 to 3 days before and during exposure), diethyldithiocarbamate (0.12% in feed 3 days before and during exposure), thiram (0.10% in feed 3 days before and during exposure), cysteine CO.10% or 0.50% in feed 3 days before and during exposure), methionine (0.10% or 0.50% in feed 3 days before and during exposure), N-acetylcysteine (1,200 mg/kg orally every day during exposure), SKF 525-A (50 mg/kg ip every day during exposure), cobaltous chlor- ide* 6 H2O (60 mg/kg ip daily for 2 days before exposure), BAL (50 mg/kg sc daily during exposure), phenoxybenzamine (10 mg/kg ip daily for first 2 days of exposure), propranolol (10 mg/kg ip daily during exposure), Vitamin C 4 ------- (100 mg/kg ip daily during exposure), DL-a-tocopherol acetate (Vitamin E, 750 mg/kg orally once 2 days before exposure and on first day of exposure), ethanol (15% in drinking water 2 days before and during exposure), sodium phenobarbital (0.1% in drinking water 7 days before and during exposure), or selenium (1 ppm in feed 10 days before and during exposure). D. Observations 1. General: Mortality, general well being, and in some cases, feed consumption were determined for animals exposed to VDC. 2. Histopathology: Livers, kidneys, and hearts from male mice and rats exposed to VDC were examined for microscopic lesions. The tissues were fixed in neutral buffered 10% formalin, processed in an Autotechnicon and stained with H and E stain. 3. Serum enzymes: Serum glutamic-oxaloacetic transaminase (SGOT)—^ and serum glutamic-pyruvic transaminase (SGPT)i?/ were determined in cardiac blood from mice and aortic blood from rats. 4. Hepatic non-protein sulfhydryl concentrations: A colorimetric method—^ was used to measure this parameter in: (a) male and female rats exposed to 0 and 75 ppm VDC for 1, 2, 4, 8, and 16 days. As the result of logistic problems, these assays were conducted 3 to 4 hr after the animals were removed from the chamber; and (b) male mice that received various treat- ments for a total of 10 days (i.e., 3 days before exposure and 7 days during exposure to room air). 5. Glucose 6-phosphate dehydrogenase activity: Male rats were exposed to 0 and 75 ppm VDC for 16 days. The rats were sacrificed by de- capitation and the liver was removed, weighed, and homogenized in 16 volumes of Buffer A (55 mM Tris buffer that contained 3.3 mM MgC^ with a pH of 7.8). The homogenate was centrifuged at 20,000 g for 15 min. The enzyme assays contained 2.7 ml Buffer A, 0.1 ml of 6 mM NADP, 0.1 ml of 100 nM glucose 6-phosphate, and 0.1 ml of the liver supernatant. The assay blank contained everything except glucose 6-phosphate. The rate of reaction was measured by the production of NADPH which was followed spectrophotometrically at 340 nm. The protein concentration-^/ was determined on the supernatant. 6. Covalently bound radioactivity: Covalent bound radioactivity was measured in male mice after the intraperitoneal administration of 3 mg/kg ¦^C-VDC (20 fiCi/kg) which was obtained from New England Nuclear (Boston, Massachusetts) with a specific activity of 0.652 |iCi/nimole. The ^C-VDC was slowly bubbled into the peanut oil vehicle with nitrogen. The tissues were homogenized in cold water and macromolecules were precipitated with an equal volume of 1 N perchloric acid (PCA). The precipitate was washed with 5 ml 5 ------- of 0.2 N PCA, 0.2 N PCA, 95% ethanol saturated with sodium acetate, absolute ethanol, ethanol:ether (3:1), heated 1 hr at 37°C in 4 ml of 0.5 N sodium hydroxide, and afterwards 3 ml of 30% thichloroacetic acid (TCA) was added. The precipitate was washed with 5 ml of 5% TCA and heated in 5% TCA for 20 min on a boiling water bath. The pellet was washed with 5 ml of 5% TCA and dissolved in 10 ml of 0.3 N sodium hydroxide. Radioactivity and proteinic/ were determined on this fraction and the results were expressed as DPM/mg protein. E. Statistics Mortality data were evaluated in terms of the concentration of VDC required to kill 50% of the animals (LC^q)—^ and the time required to kill 50% of the animals at 20 ppm of VDC (LT^q)^ Other data were analyzed by the two sample rank testiZ/ or Fisher exact probability test—^ with a level of significance selected as p 0.05. Data are reported as the mean + S.E. III. RESULTS A. Toxicity of VDC in Mice and Rats 1. General: VDC was more toxic, as measured in terms of lethality (dead/exposed) and hepatotoxicity (SG0T and SGPT), in mice than rats (Table 1). Since the cause of death is not known, the toxicity of VDC, as discussed in this report, is subdivided into lethality and organ toxicity. Organ toxicity is measured in terms of serum enzymes (SG0T and SGPT) for the liver and histo- pathology for the liver, kidney and heart. 2. Lethality: The lethality of VDC inhaled by mice and rats for various days is presented in Table 2. Mice had a reduced feed consumption (data presented below in part B), weight loss, rough coats, and were lethargic. In addition, the body temperature of debilitated mice was reduced by 5 to 7°C. The feed consumption and body weights of rats was also reduced; however, there was no effect of 60 ppm VDC on body temperature. Mice were more sensitive to the acute lethal effects of VDC than rats and most of the deaths occurred within the first 3 days of exposure. Male mice were more sensitive to 19 and 41 ppm of VDC than females; however, this effect was not significant at 80 ppm. 3. Organ toxicity: a. Serum enzymes: The serum enzymes of male mice exposed to 0, 15, 30 and 60 ppm of VDC as a function of time are presented in Table 3. After 1 exposure day, there was a dose-related increase in both SGOT and SGPT. 6 ------- Towards the end of the 5 days exposure period, these values tended to decline. No mice survived a 3-day exposure to 60 ppm of VDC. The serum enzymes in male rats were elevated after exposure to 60 ppm of VDC (Table 4). This change was not as dramatic as in mice simi- larly exposed and required a longer exposure period before it was evident. b. Histopathology: Microscopic lesions observed in male mice exposed to 15, 30 and 60 ppm of VDC as a function of time are pre- sented in Table 5. Although there was no hepatic midzonal necrosis at 15 ppm of VDC, the severity of this lesion increased as the VDC concentration was raised from 30 to 60 ppm. However, hepatocellular degeneration, which con- sisted of centrilobular cloudy swelling and/or cytoplasmic vacuolization, was evident at all of the VDC concentrations. An increased number of mitotic figures in hepatocytes, was observed at both 15 and 30 ppm of VDC. A very high incidence of tubular nephrosis was observed in the kidneys of mice ex- posed to 15, 30 and 60 ppm of VDC. In addition, there was evidence of tubu- lar regeneration in mice exposed to 15 ppm of VDC for 5 days. A mild focal myocardial degeneration was observed in a few mice exposed to 15 and 30 ppm of VDC. None of these lesions were observed in control mice at any of the observation periods. The microscopic lesions observed in rats exposed to 60 ppm of VDC for various days are presented in Table 6. Centrilobular degenera- tion and/or necrosis and mild hyperplasia of the bile duct occurred in the rats exposed to VDC. In addition, mild focal myocardial degeneration occurred in one rat exposed to 60 ppm of VDC for 3 days. None of these lesions were present in the control group at any of the observation periods. B. Effect of Various Treatments on the Lethality of VDC in Mice Groups of mice were given one of several compounds during initial studies to identify compounds that altered the toxicity of VDC. Ethanol and BAL increased the lethality of 20 ppm VDC in male mice (Table 7). in contrast, phenobarbital treatment reduced the mortality of 74 ppm VDC in female mice. Selenium did not alter the toxicity of VDC. Additional studies were conducted to identify compounds that modi- fied the lethal effects of VDC. Some of these compounds were incorporated into the feed. The feed was given prior to VDC exposure, since mice ex- posed to VDC had a reduced feed consumption (Table 8). Although, mice that received cysteine (0.1% in diet), methionine (0.1% in diet) and disulfiram had a reduced feed consumption during VDC exposure, they did consume the diets containing these compounds in the absence of VDC. Disulfiram was the only one of these compounds that altered the LC^q value of VDC, which was calculated 7 ------- after the first exposure day (Table 9). In addition, cobaltous chloride did not alter this measure of toxicity. The LCjq of VDC, which was calculated after the second day of ex- posure, was increased in mice pretreated with disulfiram, diethyldithio- carbamate (DDC), and thiram (Table 10). However, treatment with N-acetyl- cysteine, methionine (0.5% in diet), cysteine (0.5% in diet), propranolol, Vitamin C, and Vitamin £ did not alter this parameter. If lethality was expressed in terms of the time required to kill 50% of the mice exposed to 20 ppm of VDC (LT^g), then disulfiram diethyl- dithiocarbamate (DDC), and thiram provided protection (Table 11). In addi- tion, this presentation of the data suggested that methionine (0.5% in diet) and cysteine (0.5% in diet) also provided a degree of protection. None of the other treatments presented in Table 11 protected mice against the lethal effects of VDC. C. Effect of Disulfiram Treatment on the Organ Toxicity of VDC in Mice 1. Serum enzymes: The hepatotoxicity of VDC, as measured by ele- vated SGOT and SGPT, was reduced in male mice treated with disulfiram (Table 12). This protective effect was evident after the first, but not the second or third, exposure days. No control mice survived 3 days in an atmo- sphere containing 60 ppm VDC. In contrast, disulfiram treated mice survived a similar exposure to VDC. Serum enzymes in control mice exposed to room air are presented in Table 3. Treatment with disulfiram did not alter these values. 2. Hlstopathology: The microscopic lesions observed in control and disulfiram treated male mice exposed to 60 ppm VDC for various days is presented in Table 13. None of these lesions were observed in either control or disulfiram treated mice that were exposed to room air. As a result of the high mortality in control mice exposed to 60 ppm VDC, it is valuable to com- pare the disulfiram treated group (Table 13) with the various concentrations of VDC previously presented (Table 5). No midzonal necrosis was observed in mice treated with disulfiram and exposed to VDC for 1 day. However, midzonal necrosis was observed in this group after the second exposure day. Disulfiram also reduced the severity of renal tubular nephrosis early in the exposure period; however, as the duration of exposure increased, the severity of this lesion also increased. D. Biochemical Observations on the Toxicity of VDC and the Effect of Various Treatments 1. Covalent binding: Radioactivity, covalently bound to protein, was measured in the liver and kidney at 4 and 24 hr after the intraperitoneal 8 ------- administration of ^C-VDC (Table 14). The level of bound radioactivity was reduced at both times in mice pretreated with disulfiram. 2. Hepatic non-protein sulfhydryl concentration in rats exposed to VDC: The non-protein sulfhydryl concentration in the livers from male and female rats exposed to 75 ppm of VDC for various days is presented in Table 15. These values increased in a biphasic fashion over the 16-day observation period. Since these observations were made 3 to 4 hr after ex- posures were terminated, these values do not represent the non-protein sulfhydryl concentration during exposure. 3. Hepatic non-protein sulfhydryl concentration in the male mice after various treatments: This value was measured to determine if various treatments elevated the hepatic non-protein sulfhydryl concentra- tion. Male mice exposed to room air and fed diets that contained disul- firam, diethyldithiocarbamate, thiram, cysteine (0.5% in feed), or methionine (0.5% in feed) for 10 days did not have elevated concentrations of hepatic non- protein sulfhydryl groups (Table 16). 4. Hepatic glucose 6-phosphate dehydrogenase activity in male rats exposed to VDC: The activity of this enzyme was increased about four-fold in the liver of male rats exposed to 75 ppm of VDC for 16 days (Table 17). IV. DISCUSSION The results of this study demonstrate that (1) mice are more sensi- tive than rats to the lethal, hepatotoxic and renal toxic effects of VDC, (2) disulfiram reduces the acute lethal and organotoxic effects of inhaled VDC and reduces the levels of covalent bound radioactivity in the liver and kidney after ^C-VDC, and (3) diethyldithiocarbamate, thiram and, to a lesser extent, methionine and cysteine also protect mice from the lethal effects of VDC. Microscopic examination of the liver and kidney revealed that renal tubular nephrosis was the lesion that occurred most frequently at the concen- tration of VDC studied. In addition, there was evidence that this lesion was reversible, as demonstrated by the presence of tubular regeneration, after a 5-day exposure to 15 ppm. However, there was no similar evidence of reversi- bility in mice exposed to 15 ppm for 1, 2 and 3 days or 30 and 60 ppm for any of the times studied. Renal lesions are of interest in view of the occurrence of kidney tumors in mice exposed to VDC.—^ However, no kidney tumors were observed in CD-I mice, the strain used in the present study, during a 12-month exposure to 55 ppm of VDC.—^ The cause of death is a topic for speculation. The data presented in this report suggest that death is due to hepato-renal failure. However, other mechanisms involving, for example, the cardiovascular or respiratory system may also be involved. 9 ------- Disulfiram, diethyldithiocarbamate, and thiram are structurally related dithiocarbamates. Disulfiram is used clinically in alcohol therapy programs and thiram is used both in the agricultural and rubber industry. Disulfiram is metabolized to diethyldithiocarbamateZl/ and both compounds alter the metabolism of xenobioticsi-l/ and protect against several types of drug-induced toxicities.^3,24/ in addition, members of the dithiocarba- 25/ mate class have radioprotective properties.—' Although the mechanisms by which the dithiocarbamates tested pro- tected against VDC toxicity is uncertain, speculations may be offered con- cerning such mechanisms. For these speculations, it is assumed that VDC is metabolized by the hepatic mixed function oxidase system to a compound that produces organotoxicity which results in death. If disulfiram reduces the metabolic activation of VDC, then SKF 525-A—^ and cobaltous chloride^!/ should also have provided protection. If treatments protected by providing additional sulfhydryl groups for the detoxification of VDC epoxides, then doses of N-acetylcysteine that protect mice from acetaminophen toxicity^/ should also have protected against VDC toxicity. Although cysteine and methionine, which are also sulfhydryl containing compounds, provide a degree of protection, the effect is not as dramatic as with the dithiocarbamates. The failure of these compounds to alter the hepatic nonprotein sulfhydryl concentration may be due to (1) pharmacokinetic properties of the compounds and/or (2) adaptation of the liver to an increased supply of sulfhydryl containing compounds. The results suggest that disulfiram protects against toxicity by a mechanism that involves more than an inhibition of VDC activation or an increase in VDC detoxification. Possibly, disulfiram is superior to the other nondithiocarbamate compounds because both mechanisms are operating simultaneously. In other words, disulfiram and its metabolisms may not only reduce the activation of VDC but also increase the extent of detoxification. In this regard, dithiocarbamates may serve as more effective molecules for detoxifying VDC metabolites than some of the sulfhydryl containing compounds that were tested. 10 ------- REFERENCES 1. Haley, T. J. Vinylidene chloride: A review of the literature. Clin. Toxicol. 8:633 (1975). 2. Huffman, R. D., and Desai-Greenaway, F. Health and environmental impacts: Task 1, vinylidene chloride. U.S. Department of Commerce, National Technical Information Service, Washington, D.C. (1976). 3. Prendergast, J. A., et al. Effects on experimental animals of long-term inhalation of trichloroethylene, carbon tetrachloride, 1,1,1-trichloro- ethylene. Toxicol. Appl. Pharmacol. 10:270 (1967). 4. Jenkins, L. J., Trabulus, M. J., and Murphy, S. D. Biochemical effects of 1,1-dichloroethylene in rats: dissociation of its hepatotoxicity from a lipoperoxidative mechanism. Toxicol. Appl. Pharmacol. 24:457 (1973). 5. Jaeger, R. J., Trabulus, M. J., and Murphy, S. D. The interaction of adrenalectomy, partial adrenal replacement therapy, and starvation with hepatotoxicity, and lethality after 1,1-dichloroethylene intoxication. Toxicol. Appl. Pharmacol. 24, abs. No. 133 (1973). 6. Jaeger, R. J., Conolly, R. B., and Murphy, S. D. Diurnal variations of hepatic glutathione concentrations and its correlation with 1,1-dichloro- ethylene inhalation toxicity in rats. Res. Comm. Chem. Pathol. Pharmacol. 6:465 (1973). 7. Jaeger, R. J., Conolly, R. B., and Murphy, S. D. Effect of 18 hr fast and glutathione depletion of 1,1-dichloroethylene-induced hepatotoxicity and lethality in rats. Exp. Mol. Pathol. 20:187 (1974). 8. Jaeger, R. J., et al. Biochemical toxicology of unsaturated halogen monomers. Environ. Health Perspect. 11:121 (1975). 9. McKenna, M. J., et al. The fate of [j^c] vinylidene chloride following inhalation exposure and oral administration in rats. Sixteenth Annual Meeting, Society of Toxicology, Toronto, Canada (1977). 10. Siletchnik, L. M., and Carlson, G. P. Cardiac sensitizing effects of 1,1-dichloroethylene: enhancement by phenobarbital treatment. Arch. Int. Pharmacodyn. 210:359 (1974). 11. Amador, E., and Wacker, W. E. C. Serum glutamic-oxaloacetic transaminase activity: A new modification and an analytical assessment of current assay techniques. Clin. Chem. 8:343 (1962). 11 ------- 12, 13, 14, 15, 16, 17, 18, 19, 20 21 22 23 24 Henry, R. J., et al. Revised spectrophotometry methods for the determina- tion of glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase, and lactic dehydrogenase. Am. J. Clin. Path. 34:381 (1960). Sedlak, J., and Lindsay, R. H. Estimation of total, protein bound, and non-protein sulfhydryl groups in tissue with Ellman's reagent. Anal. Biochem. 25:192 (1968). Lowry, 0. H., et al. Protein measurements with folin phenol reagent. J. Biol. Chem. 193:265 (1951). Weil, C. S. Tables for convenient calculations of median-effective dose (LD^g or ED^q^ and instructions in their use. Biometrics 8:249 (1952). Litchfield, J. T. A method for rapid graphic solutions of time-percent effect curves. J. Pharmacol. Exp. Therap. 97:399 (1949). Goldstein, A. Biostatics on Introductory Test. The MacMillan Co., New York (1969). Siegel, S. Nonparametric Statistics, McGraw-Hill, New York, pp. 96-104 (1956). Maltoni, C., et al. Recent findings on the carcinogenicity of chlorinated olefins. Environ. Health Perspect. In press. Lee, C. C., et al. Toxicity and carcinogenicity of vinyl chloride com- pared to vinylidene chloride. Environ. Health Perspect. In press. Stromme, J. H. Metabolism of disulfiram and diethyldithiocarbamate in rats with demonstration of an in vivo ethanol-induced inhibition of the glucuronic acid conjugation of the thiol. Biochem. Pharmacol. 14:393 (1965). Zemaitis, M. A., and Green, F. E. Impairment of hepatic microsomal drug metabolism in the rat during daily disulfiram administration. Biochem. Pharmacol. 25:1355 (1976). Lutz, L. M., Glende, E. A., and Recknagel, R. 0. Protection by diethyl- dithiocarbamate against carbon tetrachloride lethality on rats and against carbon tetrachloride-induced lipid peroxidation in vitro. Biochem. Pharmacol. 22:1729 (1973). Wattenberg, L. W. Inhibition of carcinogenic and toxic effects of poly- cyclic hydrocarbons by several sulfur-containing compounds. J. Nat. Cane. Inst. 52:1583 (1974). 12 ------- 25. Barnes, J. H., et al. Synthese et effects radioprotecteurs d'alkanebis- dithiocarbamates disodiques, d'acides u-aminoalkyldithiocarbamiques et de leurs derives N,N'-dimethyles. Eur. J. Med. Chem-Chimica Therap. 10:619 (1975). 26. Conney, A. H., et al. Enzyme induction and inhibition in studies on the pharmacological actions of acetophenetidin. J. Pharmacol. Exp. Ther. 151:133 (1966). 27. Tephly, T. R., and Hibbeln, P. The effect of cobalt chloride administra- tion on the synthesis of hepatic microsomal cytochrome P-450. Biochem. Biophys. Res. Comm. 42:589 (1971). 28. Piperno, E., and Berssenbruegge, D. A. Reversal of experimental para- cetamol toxicosis with N-acetylcysteine. Lancet, October 2:738 (1976). 13 ------- CHEMICAL STRUCTURES CI. CI' ^C = CH2 VINYLIDENE CHLORIDE c2H5v ^2^5 N-C-S-S-C-N C2H5' I I "c2h5 NH2-CH-CH2-SH 1 COOH DISULFIRAM CYSTEINE C2H5. C2H5' N-C-S" Na NH2-CH-CH2-CH2-SCH3 COOH DIETHYLDITHIOCARBAMIC ACID,Na METHIONINE CH3 CH3 /CH3 ^N-C-S-S-C-N^ ch3 Ac-NH-CH-CH2-SH COOH THIRAM N-ACETYLCYSTEINE ch2-ch2-ch2oh t I SH SH BAL Figure 1 - Chemical Structures of Some Compounds Used in the Study to Alter VDC Toxicity 14 ------- TABLE 1 TOXICITY OF 60 PPM VDC IN MICE AND RATS Mice Rats Days Exposed 1 2 1 2 Dead / Exposed 2/10 8/10 0/10 0/10 SGOT (IU/L) 1,946 + 211— 751 + 150 74+6 264 + 33 SGPT (IU/L) 3,043 + 209 1,112 + 226 44 + 7 198 + 29 a/ Mean + S.E. for 2 to 5 determinations. TABLE 2 LETHALITY OF VDC INHALED BY MICE AND RATS Species Sex Mice Male Rats Days Exposed VDC fppm) 19 41 oa/ 20 5(&/ 30 , 50^ 4C&{ 70^ 80 20 100 100 100 Female 19 0 0 0 0 41 0 0 0 0 80 10 70 80 80 Male 313 0 0 0 0 at Percent dead (10 animals/group). b/ Lethality in males significantly different from females (Fisher exact probability test). 15 ------- TABLE 3 EFFECT OF VDC EXPOSURE ON SERUM ENZYMES IN MALE MICE Days Serum Exposed Enzyme VDC Concentration (ppm) 15 30 60 \*U SGOT 56+8 (5)2/ 249 + 28 (4) 466 + 50 (5) 1,946 + 271 (4) SGPT 32 + 6 (5) 200 + 19 (4) 474 + 66 (5) 3,043 + 209 (4) SGOT 82 + 30 (4) 91+5 (5) 705 + 120(6) 751 + 150 (2) SGPT 38+4 (4) 189 + 26 (5) 675 + 81 (6) 1,112 + 226 (2) SGOT SGPT 64 + 14 (5) 29+2 (5) 138 + 16 166 + 33 (4) 448 + 179(4) (4) 514 + 200(4) No Survivors SGOT SGPT 60+5 (5) 32+2 (5) 118 + 18 47+5 (6) 294 + 50 (2) (6) 109 + 8 (2) No Survivors a/ Mean + S.E. (number of mice). TABLE 4 EFFECT OF VDC EXPOSURE ON SERUM ENZYMES IN MALE RATS Days Serum VDC Concentration (ppm) Exposed Enzymes 0_ 60 1 SGOT 66 + 10 (5)—^ 74+6 (5) SGPT 28 + 2 (5) 44 + 7 (5) 2 SGOT 63+4 (5) 264 + 33 (5) SGPT 34 + 4 (5) 198 + 29 (5) 3 SGOT 81 + 4 (5) 238 + 47 (5) SGPT 34+6 (5) 122 + 29 (5) a/ Mean + S.E. (number of rats). 16 ------- TABLE 5 MICROSCOPIC LESIONS IN ORGANS OF MICE EXPOSED TO VDC VDC (ppm) 15 30 Mitotic figures of hepatoctes Slight increase Moderate increase Hepatocellular degeneration 60 Exposure Days: 1 2 3 5 1 2 3 5 1 2 Number of Mice Examined 5 5 5 7 5 5 4 2 4 2 Liver lesions Midzonal necrosis Qa/ Mild 0 0 0 20 40 50 0 0 50 Moderate 0 0 0 0 0 0 0 0 50 50 Marked 0 0 0 0 0 0 0 0 50 0 0 60 40 0 0 0 0 0 0 0 0 0 0 0 0 0 25 0 0 0 20 40 20 86^/ 20 40 0 50 25 0 Kidney lesions Tubular nephrosis Moderate 0 0 0 57 0 0 0 0 0 0 Marked 0 0 0 43 0 0 0 0 0 0 Severe 10(£/ 100^/ 100^/ 0 10(£/ 100^/ 10(£/ ioo^./ 50 100^ Tubular regeneration 0 0 0 86^/ 0 0 0 0 0 0 Cardiac lesions Mild focal 0 0 0 28 0 0 0 50 00 myocardial degeneration a/ Percent of mice examined with the indicated lesion. None of these lesions were observed in the control group (five mice/day) exposed to room air. b/ Significantly different from control group (Fisher exact probability test). ------- TABLE 6 MICROSCOPIC LESIONS IN ORGANS OF RATS EXPOSED TO 60 PPM VDC Exposure Days 1 2 3 Number of Rats Examined 5 5 5 Liver lesions Centrilobular degeneration and/or necrosis Mild 40 o 00 Moderate 0 0 60 Mild hyperplasia of 60 40 0 bile duct Cardial lesions Mild focal 0 0 20 myocardial degeneration a/ Percent of rats examined with the indicated lesion. None of these lesions were observed in the control group (five mice/ day) exposed to room air. bJ Significantly different from control group (Fisher exact pro- bability test). TABLE 7 MORTALITY IN MICE EXPOSED TO VDC FOR 7 DAYS Sex Treatment VDC (ppm) % Dead Male None 20 40 Ethanol 20 100 BAL 20 100 Female None 74 90 a/ Phenobarbital 74 0— a/ Significantly different from non-pretreated group (Fisher exact probability test). 18 ------- TABLE 8 FEED CONSUMPTION MALE MICE EXPOSED TO VDC VDC Concentration (ppm) Treatment 0 41 80 None 4.4 + 0.1—^ 1.5 + 0.3 1.7 + 0.1 Cysteine (0.1%) 4.6 + 0.2 1.8 + 0.6 1.8 + 0.3 Methionine (0.1%) 4.8 + 0.1 1.5 + 0.1 1.8 + 0.1 Disulfiram (0.1%) 4.2 + 0.1 3.3 + 0.3 2.8 + 0.1 a/ Gqi/mouse/day determined on a cage basis for a total of 10 mice housed 5 mice/cage. TABLE 9 ONE DAY LC?n (PPM) OF VDC IN MICE Treatment Male Female Control 98 (82-118)-' 105 (92-121) Disulfiram (0.10%) >320 >320 Cysteine (0.10%) 98 (76-127) 92 (74-113) Methionine (0.10%) 113 (93-138) 113 (93-138) CoCl, 113 (81-157) 123 (85-179) a/ LC50 in ppm (95% confidence limits) or approximation °f LCj0. 19 ------- TABLE 10 TWO DAY LCcQ (PPM) OF VDC IN MICE Treatment Male Control 35 (25-47)-' Disulfiram (0.10%) >160 DDC (0.12%) >160 Thiram (0.10%) >160 N-Acetylcysteine 20 (7-25) Methionine (0.50%) 38 (28-51) Cysteine (0.50%) 43 (31-58) SKF 525-A 26 (17-35) Phenoxybenzamine 35 (25-47) Propranolol 28 (19-37) Vitamin C 35 (25-46) Vitamin E 35 (25-47) a/ LC50 *n PP111 (957. confidence limits) or approximation of LC TABLE 11 LTgn (DAYS) AT 20 PPM OF VDC Treatment Male Control 4.0 (3.6-4.4)—7 Disulfiram (0.10%) >7 DDC (0.12%) >7 Thiram (0.10%) >7 Cysteine (0.50%) >7 Methionine (0.50%) 7 N-Acetylcysteine 1-2 SKF 525-A 2.4 (1.7-3.4) Phenoxybenzamine 3-4 Propranolol 2.7 (2.0-3.7) Vitamin C 2.6 (1.9-3.5) Vitamin E 3-4 a/ ^50 days (95% confidence limits) or approximation of LT^Q. 20 ------- TABLE 12 TOXICITY OF 60 PPM VDC IN CONTROL AND DISULFIRAM TREATED MICE Diet Control Disulfiram Days Exposed to VDC 1 2 3 1 2 3 SGOT (IU/L) 1,946+271 (4)-' 751 + 150 (2) No Survivors 140 + 38 (5) 784 + 332 (4) 2,292 + 965 (3) a/ SGPT (IU/L) 3,043 + 209 (4) 1,112 + 226 (2) No Survivors 66 + 11 (5) 1,236 + 668 (4) 3,182 + 1,488 (3) a/ Mean + S.E. (number of observations). 21 ------- TABLE 13 MICROSCOPIC LESIONS IN ORGANS OF MICE EXPOSED TO 60 PPM VDC AND TREATED WITH DISULFIRAM Treatment None Disulfiram^/ Exposure Days: 1 2 12 3 Number of Mice Examined 4 2 5 5 3 Liver lesions Midzonal necrosis Mild Ob/ 50 0 0 0 Moderate 50 50 0 0 0 Marked 50 0 0 60 0 Severe 0 0 0 0 67 Hepatocellular degeneration 25 0 0 20 33 Kidney lesions Tubular nephrosis Mild 0 0 20 40 0 Moderate 0 0 20 20 0 Marked 50 0 0 40 100 Severe 50 100 0 0 0 a/ Disulfiram (0.1%) in feed 3 days before and during exposure to VDC. b/ Percent of mice examined with the indicated lesion. None of these lesions were observed in groups (five mice/day) fed either control or disulfiram diets and exposed to room air. 22 ------- TABLE 14 COVALENTLY BOUND RADIOACTIVITY AFTER 14C-VPC IN CONTROL AND DISULFIRAM TREATED MICE Tissue Liver Hr after 14C-VPC 4 24 DPM/mg Protein Control 88 + lW 58 + 2 Disulfiram 31 + 7 23 + 5 Kidney 4 24 134 + 9 88 + 2 32+7 27 + 4 a/ Mean + S.E. for four determinations. TABLE 15 HEPATIC NON PROTEIN SULFHYDRYL CONCENTRATION IN RATS EXPOSED TO 75 PPM OF VDC Concentration—^ Days Exposed (7o of Control) To VDC Males Females 1 145 + 5^ 149 + 4 2 100+8 118 + 8 4 95+4 138 + 11 8 184 + 22 158 + 6 16 201 +9 170+8 a/ Determined 3 to 4 hours after rats removed from chamber, b/ Mean + S.E. for three to four de- terminations. 23 ------- TABLE 16 HEPATIC NON-PROTEIN SULFHYDRYL CONCENTRATION IN MALE MICE AFTER VARIOUS TREATMENTS Concentration Treatment—^ (% of Control) None 100 + . Disulfiram (0.10%) 102 + 2 DDC (0.127.) 100 + 3 Thiram (0.10%) 95+2 Cysteine (0.50%) 84+2 Methionine (0.50%) 87+1 a/ Mean + S.E. for four observations, TABLE 17 HEPATIC GLUCOSE 6 PHOSPHATE DEHYDROGENASE ACTIVITY IN MALE RATS EXPOSED TO VDC FOR 16 DAYS VDC (ppm) Activity—^ 0 20.2 + 1.6^ 75 80.7 + 4.1 a/ nMoles NADPH formed/min/mg protein. b/ Mean + S.E. for four deter- minations . 24 ------- TECHNICAL REPORT DATA (Please read fnurnctioiis on the reverse before completing) . REPORT NO. EPA- 3. RECIPIENT'S ACCESSIOI*NO. 4. TITLE AND SUBTITLE Toxicity Studies of Selected Chemicals Task III:The Toxicity of Vinylidene Chloride in Mice and Rats and its Alteration by Various Treatments 5. REPORT DATE June 1977 6. PERFORMING ORGANIZATION CODE 7. AUTHOR(S) R. D. Short, J. M. Winston, J. L. Minor, C. B. Hong, B. Ferguson, T. Unger, M. Sawyer, and C. C. Lee 8. PERFORMING ORGANIZATION REPORT NO. 9. Pfc R FORMING ORGANIZATION NAME AND ADDRESS Midwest Research Institute 425 Volker Boulevard Kansas City, MO 64110 10. PROGRAM ELEMENT NO. 11. CONTRACT/GRANT NO. 68-01-3242 12. SPONSORING AGENCY NAME AND ADDRESS Environmental Protection Agency Office of Toxic Substances Washington, DC 20460 13. TYPE OF REPORT AND PERIOD COVERED Draft Final 14. SPONSORING AGENCY CODE 15. SUPPLEMENTARY NOTES . , . - T7. 1 JJ r------- |