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Arph. Toxicol. 42, 159--169 (1979) Archive* of TOXICOLOGY Spnntiet -Vcrkig 1979 Original Investigations Molecular Mechanism of 1,1-Dichloroethylene Toxicity: Excreted Metabolites Reveal Different Pathways of Reactive Intermediates D. Reichert, H. W. Werner, M. Metzler, and D, Henschler Institut fur Pharmakologie und Toxikologie, Universitat Wurzburg, Versbacher Landstrasse 9, D-8700 Wurzburg, Federal Republic of Germany Abstract. The excretion and biotransformation of [14C] 1,1-dichloroethylene (vinylidene chloride, VDC) after administration of a single oral dose has been investigated in female rats. Seventy-two hours after a dose of 0.5, 5.0, and 50.0 mg/kg, 1.26, 9.70, 16.47%, respectively, are exhaled as unchanged VDC, and 13.64, 11.35,6.13% as UC02. The main pathway of elimination is through renal excretion with 43.55, 53.88, 42.11% of the administered radioactivity. Through the biliary system, 15.74, 14.54, 7.65% of the activity are eliminated. The isolation of the main metabolites of VDC from 24 h urine is accom plished through the combined application of solvent extraction, ion exchange chromatography and thin layer chromatography. Then gas chromatography and mass spectrometry are used for their identification. Three metabolites have been identified: thiodiglycolic acid, N-acetyl-S-(2-carboxymethyl)cy$teine and methylthio-acetylaminoethanol. .In addition, three smaller unidentified radioactive peaks have been found. Thiodiglycolic acid is the main metabolite in VDC metabolism. The simultaneous formation of an ethanolamine- and a cysteineconjugation product points to different reaction pathways of the postulated in termediate reactive epoxide; ethanolamine probably originates from membrane lipids, which react with VDC-epoxide and/or its derivatives. This pathway could explain, in part, the parenchyma damaging effect of VDC. Key words: 1,1-Dichloroethylene (Vinylidene chloride) -- Pharmacokinetics -- Biotransformation -- Mercapturic acid -- Molecular toxicity. Introduction 1,1-Dichloroethylene (vinylidene chloride, VDC) has been employed for decades as a monomere in the manufacture of plastic materials. Not until 1977 was the carci nogenic effect of this substance demonstrated (Viola and Caputo, 1977). The initial suspicion of such a toxic property has been expressed with the recognition of the structurally closely related vinyl chloride as a carcinogen in humans (Creech and Johnson, 1974; Lee and Harry, 1974) and animals (Maltoni and Lefemine, 1975). 0340-5761/79/0042/0159/S 02.20 SL 037403 160 D. Reichert et al. Since then, numerous reports have appeared concerning the possible carcinogenic potential of all chlorinated ethylenes. In bacterial test systems, after metabolic activation, a mutagenic effect has been proven for vinyl chloride, VDC and trichloroethylene. However, tetrachloroethylene and 1.2-dichloroethylenes (cis and trans) are not mutagenic (Bartsch et al., 1975; Greim et al,, 1975). Previously unknown reactive metabolites are responsible for the mutagenic and carcinogenic effect of VDC (Bonse et al,, 1975). The rate of metab olism of this substance is dose-dependent and saturable, as shown in experiments with isolated perfused rat livers (Reichert and Henschler, 1978) and whole animals (Jones and Hathway, 1978), However, it is suppressed by inhibitors of microsomal mixed-function-oxidases and through a lowered glutathione concentration in the liver (Reichert and Henschler, 1978; Reichert et al., 1978). Studies concerning the fate of VDC in rats have recently been reported (Jones and Hathway, 1978; McKenna et al., 1978). Although we largely confirm the results of McKenna et al. (1978) with regard to pharmacokinetic data obtained after oral application (Reichert and Werner, 1978), differences continue to exist in the qualita tive and quantitative characterization of the metabolites appearing in the urine. McKenna et al. (1978) have isolated four main metabolites from rat urine, two of which they have identified: thiodiglycolic acid and the mercapturic acid N-acetyl-S(2-hydroxyethyl)cysteine. Jones and Hathway (1978) have found two main metabo lites: besides thiodiglycolic acid, they postulate a cyclic product as an additional main metabolite. In addition, they have shown chloroacetic acid, dithioglycolic acid and thioglycolic acid in lesser concentrations. With regard to these discrepancies, further studies concerning the metabolism of VDC appear to be necessary. In this paper, we report on the metabolism of VDC after oral administration in rats, which together with the discovery of previously undescribed metabolites, expands knowledge of the behavior of this substance in organisms and demonstrates new possibilities of reaction mechanism(s). Materials and Methods Chemicals. 1.2-[,4C) VDC was purchased from New England Nuclear Corp. and had a specific ra dioactivity of 0.475 mCi/mmole. Its radiochemical purity exceeded 98%, as shown by radiogas-chromatography. The l4C-labeled VDC was solubilized in tricapriline. Thiodiglycolic acid was purchased from E. Merck, Darmstadt, FRG, S-(2-carboxymethyl)-L-cysteine from EGA-Chemie, Steinheim, FRG. N-acetyl-S-(2-carboxymethyl)-L-cysteine was prepared by acetylation of S-(2-carboxymethyl)-L-cysteine with acetic anhydride according to Ozawa (1963). Its identity was confirmed by GC/MS. Methylthio-acetylaminoethanol (MAAE) was prepared as follows: mercaptoacetic acid (EGA-Chemie, Steinheim, FRG) (3 mmol, 276.3 mg) was methylated with an ethereal solution of diazomethane. The reaction mixture was then treated with 2-aminoethanol (3 mmol, 183 mg) in a water bath (80 C) for 20 min. The reaction product was characterized by GC/MS. Further derivatisations of MAAE were achieved by reaction with propionyl chloride and with trifluoroacetic anhydride: 50 pi of dry MAAE was reacted with an excess of propionyl chloride in a water bath at 80 C for 15 min and subsequently evaporated to dryness in a stream of dry nitrogen. This synthesis was in analogy to a similar reaction between colamine and propionyl chloride, described by Jenden et al. (1972). Trifluoroacetylation was performed by established methods (Kaiser et al., 1974). For esterification of carboxylic acids, an excess of freshly prepared n-butanol-3N HC1 was added to the sample. Butylation was then performed in a water bath (80 C/15 min) as described by Kaiser et a). (1974). SL 037404 1,1-Dichloroethylene Metabolism 161 Animals and Treatments. Female Wistar rats (180--220 g), Institut fur Versuchstierzucht, Hannover, FRG were used in all studies. Pairs of rats were administered single doses of [14C] VDC (as a solution in tncapriline, 3 ml/kg) by stomach tube between 9 and 10 a m. The animals were transferred into an all-glass metabolism cage immediately after dosing. A constant rate of dry air (500 ml/min) was drawn through the cage. Standard diet (Altromin) and water were supplied ad libitum. Sampling and Measurement of Radioactivity. Excreted [14C] activity was followed for 72 h. Urine and feces were collected at 6 h intervals, the cage being rinsed with a total volume of 100 ml water in several portions. (I4C| VDC and l4C02 were collected in 0.5 h and 3 h intervals by drawing the air from the metabolism cage through a series of traps. Before entering the first trap, the moisture of the air was removed with Drierite (W. H. Hammond, Drierite Co.). Following the procedure described by Watanabe et al. (1976), we separated the [14C1 VDC and 14CO; in four consecutive traps. The first two contained 50 ml toluene/2-methoxyethanol solution (8 : 2, v/v) each, and were cooled in a dry ice bath. The remaining two traps contained 100 ml of diethanolamine/2-methoxyethanol (1:1, v/v) each. The traps for 14C02 were maintained at room temperature. Samples from the traps and urine samples were transferred directly in Rotiszint 22 liquid scintil lation medium (C. Roth, Karlsruhe, FRG) for counting the radioactivity. The carcass was weighed and digested in 20% KOH. Portions of the digests were added to scintillation counting vials. The activity in each vial was measured in a Packard liquid scintillation counter. Separation of Urinary Metabolites. The collected 24 h urine of two rats was freeze-dried, the residue was extracted with methanol at room temperature until it contained less than 1% of the radioactivity. The extract was evaporated to a small volume at 40 C under reduced pressure. One part of this solution was analysed by thin-layer chromatography. Aliquots were spotted on silica gel plates (0.25 mm layers of silica gel, F 254, E. Merck and developed in n-butanol: acetic acid : water solution (40: 15 : 15, v/v/v). Compounds were detected by spraying with ninhydrin or with a reagent for bivalent sulfur compounds (Knight and Young, 1958). The radioactive zones were located with a Berthold radiochromatogram scanner, and removed by scraping off and extraction of the silica gel with methanol (50 ml). These fractions were analysed by GC/MS. Another part of the methanolic solution was evaporated to dryness, dissolved in pyridine-formiate buffer (0.05 M, pH 3) and passed through a 200 x 6 mm column of Aminex A 5 (Bio Rad Lab.). Operating pressure was 10--15 bar at 50 C. The eluate was collected in 1 ml fractions. Single fractions were evaporated and derivatized as described below. Detection and Identification of Urinary Metabolites. Radioactive substances from zones on the thinlayer plates and fractions eluted from the cation exchange column were butylated following the proce dure of Kaiser et al. (1974). GC/MS was performed with a Varian CH 7 mass spectrometer combined with a Varian 2700 gas chromatograph, and Varian SS 100 MS data system. Mass spectra were taken at 70 eV. Fractions containing radioactive metabolites were analyzed by radio gas chromatography using a Varian 2700 GC equipped with a flame ionization detector and a Berthold RGC 170 radiogas detector. The separation was carried out on a 6 ft glass column packed with 3% OV 225 on Gas ChromQ, 100/120 mesh. Carrier gas He 30 ml/min. Temperatures: column oven 80--270 with 4C/min, injector 250 C, detector 270 C. Results Pharmacokinetics of [l4CJ VDC after Oral Administration In Table 1, the elimination pathways of t14C] activity after a single oral application are compiled. The main portion of the [14C] (42--53%), in a dose-range from 0,5 to 50.0 mg/kg, is recovered from the urine. Both, in urine and in feces, only the non volatile radioactivity has been determined. SL 037405 162 D. Reichert et al Table I. Excretion of radioactivity following oral single-dose administration of 1|4C] VDC to rats Specimen Exhaled air VDC CO, Total Urine Total Feces Total Carcass and tissues Cage rinse Total Time (h) 0-72 0-72 0- 6 6-12 12-24 24-48 48-72 0-12 12-24 24-48 48-72 Dose 0.5 mg/kg 5.0 mg/kg % of administered activity 50.0 mg/kg 1.26 0.39 13.64 + 4.72 14.90 4.76 18.40 3.93 14.59 1.73 7.97 0.55 1.99 0.69 0.60 0.02 43.55 4.59 0.11 + 0.07 8.52 + 2.68 5.86 0.52 1.25 0.47 15.74 3.32 5.57 + 0.60 9.80 3.13 89.56 3.74 9.70 + 1.26 11.35 2.65 21.05 3.69 16.39 2.20 22.79 1.87 11.14 2.25 3.00 1.52 0.56 0.36 53.88 4.14 1.68 1.23 6.24 3.02 6.24 1.89 0.38 + 0.06 14.54 + 1.18 2.83 + 0.42 7.51 + 0.31 99.81 + 1.21 16.47 + 4.24 6.13 0.33 22.60 + 4.37 4.76 1.39 12.83 + 2.20 19.65 2.41 4.06 0.83 0.81 + 0.25 42.11 3.53 0.75 0.48 2.71 1.17 3.69 0.47 0.50 0.18 7.65 1.47 2.77 0.34 5.91 0.79 81.04 3.90 In the exhaled portion, unchanged [14C] VDC and UC02 are identified. After application of 0.5 mg/kg, only 1.2% of the total radioactivity is exhaled as un changed [14C] VDC, 13.6% as 14C02. A reversal in the proportion of VDC to C02 has occurred after a dose of 50.0 mg/kg. In this case, about three times more VDC than C02 is excreted. The elimination of [14C] VDC as a function of time after 0.5, 5.0, and 50.0 mg/kg is shown in Fig. 1. Figure 2 represents the biphasic pattern of 14C02 exhalation after a dose of 0.5 and 50.0 mg/kg over a period of 72 h. In Table 2 are the calculated elimination half-lives of radioactivity listed, excreted as [14C] VDC, 14C02, and urinary activity. From both, figures (1 and 2) and tables (1 and 2), the biphasic elimination is evident at all dose levels by pulmonary and urinary excretion. Isolation and Identification of Urinary Metabolites The isolation of the main metabolites of VDC in the urine of rats is accomplished through the successive procedures of solvent extraction, ion exchange chromatog raphy and thin layer chromatography. Finally the metabolites are separated and identified by means of combined gas chromatography and mass spectrometry. Thin layer chromatography has revealed 5 radioactive, clearly resolved zones (A, B, C, D, and E). The least polar fraction E (Rf 0.54, about 12% of the activity) on the SL 037406 1,1-Dichloroethylene Metabolism 163 Fig. 1. Expired l'*Cj VDC expressed as percentage of the administered dose (0.5, 5.0, and 50.0 mg/kg) versus time (h). In this figure and Fig. 2 each value is the mean S.D. from three experiments Fig. 2. Semilogarithmic concentration-time curves of HC02 in the expired air following doses of 0.5 and 50.0 mg/kg [,4CJ VDC orally Table 2. Half lives of elimination of [,4C] VDC, 14C02 and activity in urine after oral administration of [l4CI VDC Phase of elimination Dose 0.5 mg/kg 5.0 mg/kg 50.0 mg/kg Rapid Slow Rapid Slow Rapid Slow VDC Urine 43 min 4 h 30 min 4 h 30 min 35 h 18 h 30 min 18 min 4 h 45 min 3 h 45 min 2 h 54 min 24 h 14 h 27 min 2 h 45 min 5 h 15 min 6 h 21 min 20 h 45 min 25 h SL 037407 oo 164 p. Reichert et al. plate has been scraped ofT and eluted with methanol. An initial analysis with GC/MS has indicated that the active material could be methylthio-acetylaminoethanol (MAAE). Therefore, we have synthesized this substance. The synthetic product and metabolic fraction E have shown identical retention times on columns with different stationary phases (SE 30 and OV 225). The mass spectra are in agreement with respect to the molecular ion and the typical fragments (Fig, 3). For further charac terization, the metabolite and synthetic product were then converted into two differ- 100 > 50 M UlL "c e ioo s= 44 50 |I_J7u|0 i, cCH3-5 --CH2-- " NH-CHj-CH2OH MAAE h9 s M 85 K 3 4____ 200 Metabolite LLJI_LlL131 L149_______. 100 200 m/e 100-j 50 .5 49 103 ll .l 100 50- 100 CH3-S"CH2-C - NH-CH^CH^O-C-CFj MW 245 ' MAAE *TFAA/CH2Ct2 11 24* u M _____________________ ,_________________1_____________________ 200 250 m/e '99 Metabolite* TFAA/CH2CI2 21$ _________ 1, ISO 200 250 m/e (3 O CHj-S-CHj-C- NH-CHfCH^O-C - CHj-CHj 100 MW 205 MAAE * propionyl chloride 11150 nil .Li 100 100* fit 50- I J---------- 150 2H - M1- 200 250 m/e Metabolite* propiooyl chloride .. . Jl Ml U 204 l_____________________ 200 250 m/e Fig. 3. Mass spectra of the metabolite in TLC zone E and synthesized methylthio-acetylaminoethanol. Trifluoroacetyl- and propionyl-derivatives of metabolite and authentic substance are compared SL 037408 1,1-Dichloroethylenc Metabolism 165 ent derivatives by esterification of the primary alcohol group with trifluoroacetic anhydride and propionyl chloride. The conversion has proceeded smoothly in both cases. The trifluoroacetyl and propionyl esters from the synthetic product and meta bolic fraction E show the same retention times, and their mass spectra are identical (Fig. 3). This indicates that the fraction E metabolite is identical with MAAE. The main activity (approx. 40%) on the thin layer plate is found in zone D (Rf 0.37). The column chromatographically purified acid fraction is converted to the butyl ester with n-butanoI-3N HC1. Radio GC of this derivatized sample reveals a major peak cochromatographing with the dibutylester of authentic thiodiglycolic acid. Moreover, the mass spectrum of the butylated metabolite is identical with that of the reference compound, thus proving that the metabolite in fraction D is thiodi glycolic acid. In addition to MAAE and to thiodiglycolic acid, three additional radioactive compounds were demonstrated by radio GC in the eluate of the ion exchange col umn after butylation. There is reason to suggest that one of the components might be a mercapturic acid. The mass spectra of one of the metabolites proved to be identical with that of the butyl ester of N-acetyl-S-(2-carboxymethyl)cysteine (Fig. 4). Cochromatography of the synthetic products with radioactive biological material has verified the identity of both substances. At this point it has been certain that N-acetyl-S-(2-carboxymethyl)cysteine is a metabolite of TLC zone A, B, or C. A comparison of the RF-value of the synthetic product and radioactive material from the thin layer plate is interpreted to mean that fraction B contains this mercap turic acid. A lack of agreement in retention time and mass spectrum has been found in a further comparison to other mercapturic acids (hydroxyethyl- and methyl-mercapturic acid). The mass spectra of the remaining two urinary metabolites are shown in Fig. 5. The spectra imply that the metabolites may have closely related structures, because the type of fragmentation appears to be similar. The ion at m/e 57 and the difference of 100 between the ions with the highest mass (presumably the molecular ion) may indicate that the metabolites are mono- and dicarboxylic acids. However, no un equivocal structure could be assigned to these metabolites so far. 100-1 50- B8 105 us n ml. 111 L iii 0 C02-C4H9 c4h9-o-c-ch2 -s-ch2-ch nh-co-ch3 MW 333 M 200 250 300 m,e 100-1 Metabolite * C4H9OH HCl 50- 159 172 .It. 1* .Li 2 >2 260 274 -J---- ---------------- L_ _L 100 150 200 250 300 m/e Fig. 4. Mass spectra of the butyl esters of N-acetyl-S-(2-carboxymethyl)cy$teine and metabolite rel intensity SL 037409  166 D. Reichert el al. Discussion The results of this investigation confirm the report by McKenna et al. (1978) con cerning the pharmacokinetics of VDC after a single oral application in rats. In a dose range of 0.5--50.0 mg/kg the orally administered VDC is quickly and com pletely absorbed. This is substantiated on the one hand by the initial part of the pulmonary excretion of VDC and C02 and on the other hand through a rapid and extensive renal and biliary excretion of metabolic end products. However, differences exist in the qualitative and quantitative interpretation of the metabolic end products of VDC metabolism. The reason for this disparity could stem from different analytical techniques. McKenna et al. (1978) have employed high pressure liquid chromatography, Jones and Hathway (1978) thin layer and gas chromatography. Thiodiglycolic acid has been confirmed as the main metabolite in urine. Starting From chloroacetic acid as an intermediate in VDC metabolism, the formation of thiodiglycolic acid can be explained (Fig, 6): chloroacetic acid has been identified as a metabolite from VDC in liver tissue (Reichert and Bashti, 1976) and in urine (Jones and Hathway, 1978). Yllner (1970) has shown thiodiglycolic acid in the urine of mice after application of monochloroacetate. Chloroacetic acid reacts with glutathione in an enzyme-catalyzed reaction, to form carboxymethyl glutathione (Fig. 6). Cleavage of the glycine and glutamine fragments yields carboxymethylcysteine. Apparently, this compound is preferentially deaminated and subsequently decarboxylated. The main metabolite thiodiglycolic acid is the end product of this reaction sequence. Up to now, the intermediate product, carboxymethylcysteine has not been shown in VDC metabolism. However, the identification of the N-acetyl derivative of this compound can be considered as indirect evidence. N-acetyl-S-(2-carboxymethyl)cysteine is the only mercapturic acid we have found in VDC metabolism. In our studies, there was no indication of the formation of the hydroxyethylmercapturic acid (McKenna et al., 1978). We cannot account for this pathway, which would involve a reductive dechlorination of VDC as a major step in metabolism. The great importance of glutathione in the metabolization rate and the toxicity of VDC has been stressed previously (Reichert et al., 1978; Jaeger et al., 1974). SL 037410 * M-Dichloroethylene Metabolism Cl Cl H . Cl H. w7i I II Cl -- C -- C -- Cl I x3>- 0 a a -c Cl-CH2-CO-NH-CH2-CH2-OH * HI 0M Cl-C-C -- OH H HOOC - CH2-- S - CH^CH -COOH ^ V * NH2 o HjC-S-CHj-CO-NH-CHj-CHj-QH ____________________________ HOOC-CH^S-C^-COOH HOOC --CH^S-CHj-CH - COOH ____________________ ________________ NH-CO-CH3 Fig. 6. Metabolic pathways of VDC. Identified metabolites underlined However, in addition to the described reaction sequence involving glutathione conju gation and the elimination of S-containing end products, another totally different reaction mechanism must exist which leads to the formation of ethanolamine deriva tives, The formation of this unusual metabolite is not yet clarified in detail. An initial step could be the formation of chloroacetic acid chloride (Fig. 6). This electrophilic intermediate product arises from the very unstable epoxide by an intramolecular chloride shift. The largest part is hydrolyzed to chloroacetic acid, which, as de scribed above, conjugates with glutathione. A smaller portion, however, evidently remains bound to the membrane and can directly react there with phosphatidylethanolamine, an obligatory constituent of lipid membranes. The subsequent enzymatic cleavage of the phosphatidyl residue leads to the ethanolamine derivative of chloro acetic acid. Additional support for this metabolic pathway is the fact that chloro acetic acid chloride is electrophilic enough to react directly with nucleophiles, e.g., functional SH-groups (Reichert and Werner, unpublished). A similar mechanism has recently been postulated by Cohen et al. (1975) in halothane metabolism. They Si 374li 168 D. Reichert et al. presume that trifluoroacetic acid, a major halothane metabolite, can react directly with phosphatidylethanolamine. The enzymatic cleavage then results in N-trifluoroacetylethanolamine. Now the question remains as to how the methylthio-group is introduced into the intermediary product chloroacetyl aminoethanol. Two ways are conceivable: direct nucleophilic attack of a methylthio-group (i.e., of methionine), or formation of a glutathione conjugate. To conclusively answer this question, further investigations are required. The increasing number of metabolites with the insertion of the methyl thio-group on an electrophilic carbon atom have been compiled recently by Jenner and Testa (1978). They conclude from the present data that, in most cases, the direct nucleophilic attack is the probable pathway. The results of this study show that the inactivation of VDC in vivo occurs by a large number of reactions, some of which could be demonstrated. The identification of the main metabolites from VDC in rats leads to an improved understanding of the toxic properties of this substance. Consequently, the covalent binding of chloroacetic acid chloride on membrane constituents (i.e., phosphatidylethanolamine) could explain, in part, the parenchyma damaging effect of VDC. Acknowledgements. This work was supported in part by BG Chemie, Heidelberg. We wish to thank Dr. E. Schiltz, Physiologisch-chemisches Institut, Wurzburg, for providing the column chromatography. The skilful technical assistance provided by Miss Ch. HolterhofT (animal work) and Mrs. J. Colberg (mass spectrometry) is gratefully acknowledged. References Bartsch, H., Malaveille, C., Montesano, R., Tomatis, L.: Tissue mediated mutagenicity of vinylidene chloride and 2-chlorobutadiene in Salmonella typhimurium. Nature 255, 641--643 (1975) Bonse, G., Urban, Th., Reichert, D., Henschler, D.: Chemical reactivity, metabolic oxirane formation and biological reactivity of chlorinated ethylenes in the isolated perfused rat liver preparation. Biochem. Pharmacol. 24, 1829-1834 (1975) ,Cohen, E. N., Trudell, J. R., Edmunds, H. N., Watson, E.: Urinary metabolites of halothane in man. Anesthesiology 43 392--401 (1975) Creech, J. L., Johnson, M. N.: Angiosarcoma of liver in the manufacture of polyvinyl chloride. J. Occup. Med. 16, 150-151 (1974) Greim, H., Bonse, G., Radwan, Z., Reichert, D., Henschler, D.: Mutagenicity in vitro and potential ,carcinogenicity of chlorinated ethylenes as a function of metabolic oxirane formation. Biochem. Pharmacol. 24 2013-2017 (1975) Jaeger, R. J., Conolly, R. B., Murphy, S. D.: Effect of 18 h fast and glutathione depletion on 1,1- dichloroethylene-induced hepatotoxicity and lethality in rats. Exp. Mol. Pathol. 20, 187--198 (1974) ,Jenden, D. I., Booth, R. A., Rock, M.: Simultaneous microestimation of choline and acetylcholine by gas chromatography. Anal. Chem. 44 1879--1881 (1972) Jenner, P., Testa, B.: Novel pathways in drug metabolism. Xenobiotica 8, 1-25 (1978) Jones, B. K., Hathway, D. E.; The biological fate of vinylidene chloride in rats. Chem.-Biol. Interact. 20, 27-41 (1978) Kaiser, F. E., Gehrke, C. W., Zumwalt, R. W., Kuo, K. C.: Amino acid analysis. Hydrolysis, ionex- change clean-up, derivatization, and quantitation by gas-liquid chromatography. J. 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[Suppl.J 293, R255 (1976) Reichert, D., Henschler, D.: Uptake and hepatotoxicity of l.l-dichloroethylene by the isolated bloodperfused rat liver, lnt. Arch. Occup. Environ. Health 41, 169--178 (1978) Reichert, D., Werner, H. W., Henschler, D.: Role or liver glutathione in l.l-dichloroethylene metab olism and hepatotoxicity in intact rats and isolated perfused rat liver. Arch. Toxicol. 41, 169-178 (1978) Reichert, D., Werner, H. W.: Disposition and metabolism of [MC] l.l-dichloroethylene after single oral administration in rats. Naunyn-Schmiedeberg's Arch, Pharmacol. (Suppl.) 302, R22 (1978) Viola, P. L., Caputo, A.: Carcinogenicity studies on vinylidene chloride. Environ. Health Perspect. 21, 45-47 (1977) Watanabe, P. C., McGowan, G. R., Madrid, E. O., Gehring, P. J.; Fate of [14C] vinyl chloride follow ing inhalation exposure in rats. Toxicol. Appl. Pharmacol. 37, 49--59 (1976) YUner, S.: Metabolism of chloroacetate-l-MC in the mouse. Acta Pharmacol. (Kbh.) 30, 69--80 (1970) Received February 26, 1979 SL 0374X3