Document 3aq8GLx7zOVaVQMz220BQg1D

OCT-81990 October 4, 1990 TO: Vinylidene Chloride Panel I have enclosed three (3) documents provided to me by Bill Hayes of Dow. These documents include: (1) A meeting report describing a research proposal on vinylidene chloride. The research will involve a study on the mechanisms of vinylidene chloride nephrotoxicity, which will be sponsored by a group of European companies (BASF, ICI, Solvay, Dow Europe). The study is expected to start in the 4th quarter of 1990; however, a study report may not be available until 1992. The study will be performed at the University of Wurzburg. (2) A study protocol for the mechanism study. (3) A published article by Trevor Green on the potential mechanisms of carcinogenicity for chloroethylenes. Vinylidene Chloride is discussed. These documents are simply for your information and no action is required. If you have any questions, please do not hesitate to contact me (phone: 202/887-1189; fax: 202/887-1237) or my staff assistant, Amy Kosko (phone: 202/887-1345). Sincerely, Jonathon T. Busch Manager, Vinylidene Chloride Panel SL 064650 MEETING WITH: HEALTH & ENVIRONMENTAL SCIRECEIVED MEETING REPORT MAR 1 l 1990 DOW CONFIDENTIAL gnuimnmBntal Affairs (full name, abbreviation, address) BASF Ludwigshafen/Solvay/ICI WRITTEN BY: Jan Wilmer DATE MET: 02/27/90 WRITTEN: 03/05/90 gffSSenf,. Gelbke, Hoffmann, Munk, Jaeckh and Druschke (BASF), Christ de Rooy, Solvay and Trevor Green, (ICI), Wilmer (DOW Europe) Guest: Dr. W. Dekant (University of Wuerzburg) Purpose: As a result of a review of the toxicology of VDC by the German BUA, more data are needed to explain the mechanism of the kidney tumor induction in male mice and to explain differences between species. The purpose of the meeting was to discuss a research proposal on VDC submitted by Dr. Dekant. Main conclusions of the discussions taken place at BASF were: - the studies should focus on the following aspects: a) glutathione//3-lyase pathway: formation of GSH adducts b) epoxidation of VDC in the liver followed by transportation of metabolites to the kidney c) P-450 mediated metabolic activation in the kidney d) studies will be started with Swiss mice. Depending on the results obtained in mice, species differences will be investigated in rats and human tissues (risk assessment). e) experiments will be carried out along the most appropriate route of exposure (inhalation). - the 4 companies expressed their willingness to support this 3-year research proposal (after revision of the present draft) - Dr. Dekant will submit revised proposal by the end of March which will then be discussed at the June meeting of the PVDC group - Dr. Hoffmann or Dr. Jaeckh (BASF) will be the monitoring scientist. BASF will help the Univ. of Wuerzburg to introduce Good Laboratory Practice (on a limited scale) - conditions and finances: standard contract between BASF and Univ. of Wuerzburg. BASF will recharge the other companies - proposal of BASF to share the costs (200,000 to 240,000 DM) on a equal base was accepted by ICI and Solvay, but not by Dow (proposal: "name plate capacity"). According to BASF's business manager Druschke, this was in contrast to what was agreed within the PVDC group (David Russell, Chris Dyball please clarify) - Dow USA will be asked to inform about status of VDC in the U.S EPA and ongoing activities within CMA (action: J. Wilmer) SL SA65i 1 C Abteilung Toxikologi Department of T xicology 6700 Ludwigshafen West Germany hdh-kb: 1024 STUDY PROTOCOL Studies on the mechanism of the nephrotoxicity of 1,1-Dichloroethene Testing facility: Institut fur Toxikologie und Pharmakologie der Universitat Wurzburg Head of the Institute: Prof. Dr.med. Henschler C Head of the Research group: Priv. Ooz. Or. W. Oekant This protocol consists of 12 pages. SL 064652 Abteitung Toxikolog' Department of Toxicology GENERAL Purpose of the study The objective of the study is to provide information on the biochemical mechanism of the highly species and sex specific nephrotoxicity and carcinogenicity of 1,1-Dichloroethene (OCE). Details on the scientific background for these experi ments are given in the Appendix. Signatures Name Study Director: Head of Institute: Study assistants; Quality Assurance: Monitoring Scientists: Signatur to be signed after approval of the proj ct Time Schedule Start of study: Estimated completion date: Estimated date of Draft Report: Final Report Dates to be set upon approval of the project P SL 064653 BASF Abteilung Toxikologie Department of Toxicology 2 1.4. 1.5. Archiving During the study all data are recorded and stored in the laboratory. The signed study protocol, the signed final report and all raw data are archived at BASF Aktiengesellschaft, Ludwigshafen (Dept, of Toxicology). All samples of biological material generated in the course of this study will be disposed of 4 months from the date of the final report, unless an extended storage period is requested in the protocol or by amendment or further storage in the institute is not appropriate. Quality assurance Performance of study: The study will be performed in compliance with the principles of Good Laboratory Practice*. Amendments: This protocol can be amended at the discretion of the study director. Detailed descriptions of all amendments will be signed by the study director. The amendment will be added to the original signed protocol and will be distributed according to the Distribution Schedul . * Note: The institute is mainly engaged in independent basic research. Procedures according to GLP will be established with the help of BASF toxicology to the extent which is possible in such a facility. SIj 64654 2. 2.1. MATERIAL AND METHODS* Test Article Name : Chemical Structure: Molecular Fu mula Origin: Batch Lot No. : Purity: Aggregate State: Stability: Storage: Precautions: Preparations of test article: Abteilung Toxikologie Department l Toxicology 1,1-Dichloroethene (DCE) "Vinylidene Chloride" (VDC) H CL C=C C 2 H z CL 2 Note to be continued for other test materials (eg. labelled substances) upon approval of the project. * Details will, be giv n upon approval of the project. SL 064655 2.2. Test System Rodents Anima1s : Strain: Origin : Sex : Age : Weight; Rationale; Husbandry: Room: Caging; Identification; Diet: Wat0r: BASP Abteilung Toxikologie Department of Toxicology 1 ) Rats 2 ) Mice 1) 2) about ... weeks Rats: about 150 Mice: 20 - 22 g (at start of acclimatization weight will be checked prior to dosing) Study data have to be set in relation to other rat and mouse toxicity data. Rat and mouse were found to be the most appropriate species for such experiments. Animals will be held in a climatized area of .............. During acclimatization in macrolon cages, type 3 During experiment in.a special all-glass closed in halation system (see figure in Appendix) Individual number (tattooing Pellet standard diet (Altrain, Lagel Tap water ad libitum SL 064656 BASF Abteilung Toxikologie Department of Toxicology Analysis of Food: Selection of Animals: In view of the aim and dura tion of this study contam inants occuring in this com mercial feed ought not to influence the results. The same applies for the tap wa ter. Selection will be done randomized from the study pool. Cell Systems Renal tubular epithelial cells: Animals: Strain: Origin: Sex: Age: Cell isolation: Bacteria for the Ames Test: Strains: Origin: isolation of epithelial cells will be done as described by Vamvakas et 1989 Cl] al 064657 SL BASF Abteilung Toxikologie Department of Toxicology 6 2.3. 2.3.1. Study Design General Comments Due to the character of the study only a broad outline of experiments can be given in the following sections. Any experimental detail will be recorded in the raw data. Note: some additional details will be set after approval of the project prior to the start,of the ex periments in cooperation with the monitoring scientists. The study is divided in two main sections with several subsections. - Mechanistic Studies In this section experiments will be performed to investigate the extent and rate of DCE metabolism in cluding S-conjugation in mice. If such metabolism is found and elucidated in mice further experiments will be added to investigate sex specificity in mice and possibly its species specificity between mice and rats. If metabolism including S-conjugation is not found to occur to a relevant extent then the formation of reactive metabolites by renal cytochrome P-450 will be investigated in mice. Studies on the genotoxicity and cytotoxicity of DCE metabolites If in the first section S-con3ugated metabolites found and identified then in this section of the their possible genotoxicity or cytotoxicity will vestigated. are study be in In the following short descriptions of the above mentioned sections and subsections are given. SL 064658 Abteilung Toxikoloyie Department of Toxicology 7 * 2.3.2. Mechanistic studies Formation of S-conjugates a) Are mercapturic acids indicative for the formation of toxic S-conjugates excreted in urine after inha lation of ,4C-DCE to mice? Male Swiss mice are sensitive to DCE-induced renal car cinogenicity after longterm inhalation. In this first series of experiments, Swiss mice will be exposed to DCE by inhalation in a closed inhalation syst m. DCE uptake from the chamber atmosphere will be determined by monitoring DCE-ccncentrations in the gas phase by GC. The mice will tr n be transferred to metabolic cages and urine and feces will be collected for 72 h (Dekant et al., 1986a). Urinary metabolites indicative for the biosynthesis of toxic glutathione conjugates C S-(1-chlorovinyl)-N-acetyl-L-cystein, $-(2-chloroacetyl)-N-acetyl-L-cysteine] will be identified and quantified using GC/MS; structure confirmation will be performed with synthetic reference compounds. The sensitivity and specificity of the analytical methods used will be determined with synthetic S-(1-chlorovinyl)-N-acetyl-L-cysteine and S-(2-chloroacetyl)-Nacetyl-L-cysteine (Vamvakas et al., 1989). b) Are toxic glutathione conjugates of DCE formed by conjugation of DCE with glutathione in vitro? To further investigate the conditions for the formation of S-conjugates DCE is incubated in the presence of microsomal and cytosolic enzymes obtained from liver and kidney of male Swiss mice with asS-labeled gluta thione. The incubations mixtures (Dekant et al., 1987) will be processed and separated by high pres sure . liquid chromatography with diode array detection. Glutathione conjugates formed can be clearly differentiated from 35S-glutathione using these methods; S-<1-chlorovinyl) glutathione can be detected by its specific UV-sp ctra. The conjugates formed will then be isolated and their structure will be determined by mass spectrometry (thermospray and GC/MS), NMR and UV spectrometry (Vamvakas et al., 1989). Reference compounds will be produced by chemical synthesis to confirm th proposed structures. If the experiments conducted demonstrate e biosynthesis of toxic glutathione conjugates of DCE male mice, the following topic will be addressed: SL 064659 BASF Abteilung Toxikologie Department ot Toxicology 8 c) What is the ratio of the metabolic pathway via glutathione conjugation to the total metabolization in vivo? Are there species and sex differences? d) Is the proportion of DCE which is toxified via glutathione conjugation dose-dependent? To determine dose, sex and species specific differences in the formation of toxic glutathione conjugates and its competition with P-450 dependent biotransformation, metabolites representative for both pathways will be quantified by GC/MS after inhalation of different doses of DCE in male and female mice and rats. * Formation of reactive intermediates by renal cytochrome P-450 in mice In case that no indications for the biosynthesis of toxic glutathione conjugates will be obtained, the program will be modified to include experiments to address the problem of P-450 bioactivation in the kidney selectively in male mice. For this purpose, the covalent binding of 14C-DCE derived radioactivity to renal macromolecules will be determined in vivo after inhalation of 14C-0CE and the sex and species dependent extent of binding will be determined. Urinary meta bolites will also be identified by GC/MS (Dekant et al., 198Gb; Dekant et al., 1986c). To ascertain the involvement of P-450 dependent bioactivation reactions in the covalent binding of DCE-derived radioactivity in vivo, amino acid adducts derived from the reactive metabolite formed, chloroacetyl chloride, will be attempted to identify using GC/MS and HPLC-methods after hydrolysis of the isolated proteins obtain d from mice kidney. Reference compound can be synthesized by the chemical reaction of chloroacetyl chloride with selected amino acids containing nucleophilic sites not participating in the peptide bond wich are likely reaction sites of the electrophile in proteins (histidine, lysine, serine, cysteine). To determine sex and species differences in the extent of renal cytochrome P-450 dependent bioactivation of OCE, the extent of metabolism of 14C-DCE by P-450 will be quantified in kidney microsomes obtained from male and female rats and mice. Products possibly formed (chloroacetic acid, dichloroacetaldehyde, phospholioid adducts, heme alkylation; Dekant et al., 1987) will be quantified by gas chromatography, covalent binding of 14CpDCE derived reactive intermediates will be quantified by liquid szintillation counting (D kant et al.. 1986c). SL 064660 BASF Abteilung Toxikologie Department of Toxicology 9 * 2.3.3. Studies on genotoxicity of metabolites formed In case that potentially toxic glutathione conjugates are formed in mice, the following questions will also be addressed: Are the glutathione and cysteine conjugates that ar formed genotoxic and cytotoxic? On the basis of the results of the studies mentioned before glutathione and cysteine S-conjugates of OCE produced synthetically will be investigated in the Ames- Test ''ekant et al., 1986b) and in LLC-PKl cells (Varn is et al., 1989). a cell line from pig kidneys, for r agenicity or for the induction of unscheduled DNA synthfc .is as a measure of genotoxic effects. The cyto toxicity of these compounds will be determined in freshly isolated renal tubular epithelial cells (Vamvakas et al., 1909). SL 064661 BASF Abteilung Toxikologie Department of Toxicology 1 3. REPORTING Results will be reported and presented in a study report including a short summary, a description of the used methods and a short evaluation of the results. If appropriate data will be presented in tables and graphs. Appropriate statistical methods will be used. SL 064662 BASF Abteilung Toxikologie Department of Toxicology 4. LITERATURE Costa, A.K. and Ivanetich, K.M. (1962): Vinylidene chlorides: its metabolism by hepatic microsomal cytochrome P-450 in vitro. Biochem. Pharmacol., il, 2083 - 2092 Dekant, W., Metzler, M. and Henschler, D. (1986a): Identification of S-1,2,2-trichlorovinyl-N-acetylcysteine as a urinary metabolite of tetrachloroethylene: Bioactivation through glutathione conjugation as a possible explanation of its nephrocarcinogenicity. J. Biochem. Toxicol., J., 57 - 72. Dekant, W.. Vamvakas, S., Berthold, K., Schmidt, S., Wild, D. and Henschler, D. (1986b): Bacterial 8-lyase mediated cleavage and mutagenicity of cysteine conjugates derived from the nephrocarcinogenic alkenes trichloroethylene, tetrachloroethylene and hexachlorobutadiene. Chem.-Biol. Interactions, , 31 - 45 Dekant, W., Schulz, A., Metzler, M. and Henschler, D ( 1 986c) : Absorption, elimination and metabolism of trichloro ethylene: a quantitative comparison between rats and mice. Xenobiotiea, _1_fi., 143 - 152 Dekant, W., Martens, G., Vamvakas, S., Metzler, M. and Henschler, D. (1987): Bioactivation of tetrachloroethylene - Role of gluta thione S-transferase-catalyzed conjugation versus cyto chrome P-450-dependent phospholipid alkylation. Drug Metab. Dispos., 15. 702 - 709. Dekant, W., Vamvakas, S. and Anders. M.W. (1989): Bioactivation of nephrotoxic haloalkenes by glutathione conjugation: Formation of toxic and mutagenic inter mediates by cysteine conjugate 0-lyase. Drug Metab. Rev., 2JL. 43 - 83 SL 064663 BASF Abteilung Toxikologte Department of Toxicology Liebler, O.C. and Guengerich, F.P. (1983): Olefin oxidation by cytochrome P-450: evidence for migration in catalytic intermediates formed with vinylidene chloride and trans-1-phenyl-1-butene. Biochemistry, 2JL< 54 82 - 54 89 group Liebler, O.C., Meredith, M.J. and Guengerich, F.P. ( 1 985 ): Formation of glutathione conjugates by reactive meta bolites of vinylidene chloride in microsomes and isolated hepa tocytes. Cancer Research, .5., 186 - 193 Liebler, D.C., Latwesen, D.G. and Reeder, T.C. (1988): S-( 2-chloroacetyl)glutathione, a reactive glutathione thiol ester and a putative metabolite of 1,1-dichloroethylene. Biochemistry, 23. > 3652 - 3657 Okine, L.K. and Gram, T.E. (1986): In vitro studies on the metabolism and covalent binding of [ 1 4C]1,1-dichloroethylene by mouse liver, kidney and lung . Biochem. Pharmacol., , 2789 - 2795 Reichert, D., Werner, H.W., Metzler, M. and Henschler, 0. ( 1 979 ) : Molecular mechanisms of 1 , 1-dichloroethylene toxicity.excreted metabolites reveal different pathways of reactive intermediates. Arch. Toxicol., i_2., 159 Vamvakas, S., Dekant, W. and Henschler, D. (1989): Assessment of unscheduled DNA synthesis in a cultured line of renal epithelial cells exposed to cysteine S-conjugates of haloalkenes and haloalkanes. Mut. Res., 222., 329 - 335 Vamvakas, S., Kremling, E. and Dekant W. (1989): Metabolic activation of the nephrotoxic haloalkene 1,1,trichloro-3,3,3-trifluoro- 1 -propene by glutathione con^uga tion. Biochem. Pharmacol., _2_&, 2297 - 2304 SL 064664 BASF Abteilung Toxikologie Department of Toxicology hdh-kb; 1025 APPENDIX State of Research; Scientific background for this study 1,1-Dichloroethene (DCE) is an important reactive intermediate in industrial syntheses and is also often used as a copolymer in th production of plastics. DCE is the compound with the severest toxic effects among the chlorinated ethenes, and even low doses result in liver and kidney lesions after repeated administration of DCE. The carcinogenicity of DCE is still open to debate. DCE is mutagenic in the Ames test, it caused tumors in the liver in son^| long term studies; in one study, it selectively induced renal tumors in male mice. Some of the bioactivation mechanisms which lead to hepatotoxic effects have been elucidated. The bioactivation mechanism pre sented in Figure 1 to explain the hepatotoxic effects has be n supported by the experimental results (Costa and Ivanetich, 1982; Liebler and Guengerich, 1 983; Liebler et al. , 1985)*. * References are given in Section A of the study protocol SL 064665 BASF Abteilung Toxikologie Department of Toxicology 0 Cl '\ Cl 0 Cl Cl Phospholipids Heme +SSH // GS OH G3 QH flerkaotunc acids Fig, i; Metabolism of 1,t-dichloroethene by cytochrome P-450 SL 064666 BASF Abteilung Toxikologie Department of Toxicology DCE is a substrate for cytochrome P-450 dependent monooxlgenases. Chloroacetyl chloride is formed as a product of this reaction possibly via an epoxide-intermediate. Chloroacetyl chloride is a highly reactive acylation agent and reacts with cellular macromolecules such as proteins and phospholipids; toxic effects may be induced bythis covalent binding. All other metabolites which have been identified up to now can also be attributed to reactions of chloroacetyl chloride with water, glutathione and other nucleophiles. Indications of a reaction of chloroacetyl chloride with phospholipids were obtained by the identification of an etanolamine conjugate (Reichert et al, 1979) as a DCE metabolite in vivo. Bioactivation mechanisms which could explain the selectiv nephro toxicity and carcinogenicity of DCE in mice have, however, not yet been defined. The administration of DCE results in lesions at the proximal tubules; tubular adenocarcinomas were induced by long-^^ term administration to mice. This observation may provide one w^y of elucidating the mechanisms responsible for the organ specif icity. Identical toxic effects on the kidney with regard to both acute toxicity and carcinogenicity were found for several struc turally related halogenated alkenes, such as hexachlorobutadiene, dichlorodifluoroethene , tetrachloroethene , tnchloroethene , tetrafluoroethene, trichlorotrifluoropropene and dichloroacetylene (overview in Dekant et al., 1989). The mechanisms of organotropic carcinogenicity and toxicity for these compounds have already been elucidated well, including substantial contributions from our own group. The following hypothesis which could explain the organ specificity of these compounds (Fig. 2): SL 064667 BASF Abteilung Toxikoiogie Department of Toxicology GSH S-transferase 1 -5G - slutathionyl 1 1 Cl 0 2 C1 NH 3 * 0-lyase N-acetyl-transferase Cl 0 3 Cl nh CCCH 3 45 Acylation of cellular nacronolecules Fig, 2: Mechanism of haloalkene bioactivation by the glutathione congugate/cysteine congugate 0-lyase pathway SL 064668 BASF Abteilung Toxikologie Department of Toxicology 5 In the liver, trichloroethene and many other substances from thi: group which are nephrotoxic and/or nephrocarcinogenic are conju gated by glutathione S-transferases with glutathione. The gluta thione conjugates formed are transported, either intact of after enzymatic degradation by jf-glutamyl transpeptidase and dipeptidases, to the kidney and concentrated there. The cysteine S-congugates that are formed after degradation are converted further into mercapturic acids by N-acetyl transferases. These are excreted in the urine. However, cysteine S-conjugates of halogenated alkenes are also substrates for enzymes from the group of the cysteine conjugate B-lyases which cleave these S-conjugates with the forma tion of pyruvate, ammonium ions and reactive intermediates, thioacyl fluorides and thioketenes. These electrophilic and highly toxic intermediates cause the primary lesion at DNA which'may 1 ad to tumor formation and they cause acute toxic effects by the re action with other macromolecules. Q-lyases and the enzymes of the mercapturic acid biosynthesis which are responsible for the degra dation of glutathione conjugates are present in the proximal tuq^^ bules of the kidney in high concentrations. This topographical distribution and the ability of the kidney to concentrat amino acids and their derivatives may explain the organ-specific ffect. Most of these steps have been validated (Dekant et al., 1989). Toxic glutathione conjugates may also be formed within the metab olism of DCE. Investigations in which the influence of various pretreatments on nephrotoxicity and the covalent binding of ,4C-DCE metabolites in the kidney was determined indicate that precursors of the toxic metabolites are formed in the liver rather than in the kidney and reach the kidney via transport mechanisms (Okine and Gram, 1986). These observations agree with the concept of the formation of toxic glutathione conjugates in the metabolism of DCE. SL 064669 BASF Abtetlung Toxikologie Department of Toxicology 6 Besides the metabolism via the glutathione con 3ugate / cysteine con jugate 13-lyase pathway, the formation of direct reactive gluta thione conjugates is also conceivable with DCE. Chloroacetyl glutathione, a reactive thiol ester and an alkylating agent with a half-life of 4 hours under physiological conditions has been postulated as a metabolite of DCE (Liebler et al., 1988); this compound is possibly formed by the reaction of chloroacetyl chlor ide with one molecule of glutathione and may, especially after the depletion of glutathione by high concentrations of electrophilic DCE metabolites, excreted from the liver, reach the kidney and induce toxic effects there by reacting with macromolecules (Fig. 3). At higher concentrations of glutathione, a detoxifica tion of chloroacetyl glutathione with a further molecule of gluta thione is, however, likely in liver. SL 064670 BASF Abteiiung Toxikoloqie Department of Toxicology 0 < Cl GS'rl LIVER GSH GS SG Cl SG transpor t to Kidney^ -f Nuc1eopni1ic tlacromo 1 ecul es 0 KIDNEY Nu. SG Fig . Possible formation of the alkylating agent S-I2-chloroacetyl)glutathione in the metabolism of 1 , 1 -dich1oroethen (Liet^ler et al., I 9 60 ) SL 064671 BASF Abteilung Toxikologie Department of Toxicology 8 Cytochrome P-450 dependent bioactivation might also account for species and sex specific nephrotoxicity. P-450 isozymes catalyzing the activation of OCE to electrophiles ( 1 , 1 -dichlorooxirane and chloroacetyl chloride) may be expressed specifically m the kidney of male mice thus causing organ specific toxicity and carcinogen icity by P-450 activation reactions of DCE in situ. The subject of the investigations is to elucidate the mechanisms of the selective nephrotoxicity of DCE in male mice focusing on the formation of toxic glutathione conjugates and the marginal conditions of their formation, especially the dependenc on species, sex and dose. On the basis of the detection of such reac tions in the metabolism of DCE, a molecular mechanism is to be established to explain the specific nephrotoxicity and nephrocarcinogenesis. In case toxic glutathione S-conjugates will not b identified, the experiments will concentrate on determining the contribution of P-450 dependent bioactivation reactions to OCE nephrotoxicity in mice. The results obtained are to contribute to an improved assessment of the hazard for man by exposur to DCE. SL 064672 BASF Abteilung Toxikologie Department of Toxicology * Working Hypothesis Based on theoretical considerations, the nephrotoxicity of DCE in male mice may toe the consequence of a selective bioactivation by one of the following reaction sequences: - Metabolism of DCE by glutathione S-transferase to S-11-chlorovinyl)glutathione, processing of this glutathione S-conjugate to S- chlorovinyl)-L-cysteine which is cleaved in part by cys ts conjugate 0-lyase in kidney to reactive intermediates. Act nation of S-(1-chlorovinyl)-L-cysteine results in the urinary excretion of S-(1-chlorovinyl)-N-acetyl-L-cyste$ne. 1 - Formation of t2-chloroacetyl)glutathione by formation o| chloroacetyl chloride, reaction of this acetyl chloride to th elec trophilic thioester (2-chloroacetyl)glutathione which is selec tively concentrated in the kidney. - Cytochrome P-450 dependent bioactivation of DCE selectively in the kidney of male Swiss mice resulting in high concentration of reactive intermediates in the target organ. The aim of the study is to elucidate the molecular mechanisms of the nephrotoxicity of DCE. SL 064673 Annu Rev Pharmacol Toxicol 1990. 30.73^9 Copyright t 1990 by Annual Reviews Inc. AU rights reserved NOTICE von*oruv CHLOROETHYLENES: A Mechanistic Approach to Human Risk Evaluation Trevor Green ICI Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, SK10 4TJ, United Kingdom KEY WORDS: carcinogenicity, species differences, peroxisomes, hyaline droplets, epoxides INTRODUCTION The four chlorinated ethylenes, vinyl chloride (chloroethyiene), vinylidene chloride (1.1 -dichloroethylene), 1,1,2-trichloroethylene and 1,1,2,2tetrachloroethylene form a group of structurally related chemicals with prop erties invaluable to modem industrial society. Polymers and copolymers of vinyl chlonde and vinylidene chloride have become the most important synthetic resins in use today, with worldwide production of PVC exceeding 18 million tons in 1988. The solvent properties of trichloroethylene and tetrachloroethylene have resulted in their widespread use in metal degreasing, dry cleaning, and a wide variety of industrial applications. These chemicals have now been in common use for more than 50 years. During this time workers have been exposed to a wide range of concentrations, in some cases for periods of 20 years or more, which has allowed a significant data base to be compiled about the short- and long-term effects of these chemicals on human health. In the last 20 years this information has been supplemented by numerous studies in laboratory animals. The chloroethylenes are either gases or volatile liquids and the principal route of exposure is by inhalation. Exposure to high concentrations results in CNS depression and narcosis (1-4); trichloroethylene, for example, has been used as an anesthetic for many short-duration surgical procedures (5). Other effects have been reported, ranging from acro-osteolysis. part of a phenom enon known as vinyl chloride disease (6), to liver, kidney, and lung changes 0362-1642 '90/0415-0073S02.00 SL 064674 I AR-PHARMACOL TOXICOU0173W3 74 GREEN iDTO (7, 8).^^n|pt^^(iflpidi^;\acrcM3Steolysis in laboratory animals led to the discovert ofjbe cjtfcupgfiypity of vinyl chloride. In a study exposing rats to vinyl ctudijde ^^afcaiauqp, Viola (9) and Viola et al (10) reported an increased' incidence of skin, lung, and bone tumors. Maltoni et al (11) confirmed these findings and also reported an increased incidence of an giosarcoma of the liver. In the same year, 1974, Creech &. Johnson (12) reported three deaths from angiosarcoma of the liver among workers em ployed on a vinyl chloride plant in the USA. The rarity of this type of tumor and the results of Maltoni'$ animal studies immediately established a connec tion between exposure to vinyl chloride and human liver cancer. This led to one of the most extensive epidemiological and toxicological studies of any industrial chemical. One consequence of the work on vinyl chloride was the subsequent testing of the other chloroethylenes for the ability to cause cancer. That the structural ly similar chloroethylenes would have properties similar to vinyl chloride appeared to be confirmed when each was shown to cause cancer in laboratory animals. There were, however, significant differences. Whereas vinyl chlo ride was established as a human carcinogen and was found to be mutagenic and carcinogenic in all tests used to measure these responses, a marked lack of mutagenicity and species-, strain-, and sex-dependent responses in two-year cancer studies characterized the evaluation of the other chloroethylenes. Furthermore, there was no evidence from occupationally exposed populations of any increase in any type of human cancer. Consequently, the risks to man from exposure to these chloroethylenes were much less well defined than for vinyl chloride. The variable results in the cancer bioassays and the lack of activity in many mutagenicity tests spawned a series of mechanistic studies on the differences between vinyl chloride and the other three chemicals in this group. According to these studies, at least two, if not all three, polychlorinated ethylenes act in an entirely different manner from vinyl chloride and involve mechanisms that were unknown in 1974 when the carcinogenicity of vinyl chloride was first discovered. Consequently, the risks to man are better understood and a scientific basis now exists for the control and use of these chemicals. This paper reviews the mechanisms of action of each of the chlorinated ethylenes and considers the relevance of the animal studies to people exposed to these chemicals. The cancer studies and mutagenicity assays have been reviewed by others and are only considered if relevar `o the mechanisms involved in the development of cancer. VINYL CHLORIDE By 1986. twelve epidemiology studies had identified 120 cases of vinyl chlonde-mduced angiosarcoma of the liver in populations employed in CHLOROETHYLENE CARCINOGENICITY 75 the manufacture of vinyl chloride (13), compared to an incidence in the general population of 0.10 per million. Increases in other tumors including liver, brain, lung, thyroid, lymphatic tissue, and skin were noted but a relationship could not be established between them and vinyl chloride expo sure. Hemangiosarcoma of the liver was also induced in rats, mice, and hamsters exposed to vinyl chloride (11). As in man, such tumors are extremely rare in these species. Other tumors associated with vinyl chloride exposure included zymbal gland tumors in rats and hamsters, nephroblastomas in rats, pulmo nary and mammary gland tumors in mice, and forestomach papillomas and melanomas in hamsters. The same range of tumors was seen in rats following either oral administration or inhalation exposure. Tumors were observed over a wide range of doses (x 103) from as low as lOppm and, in some studies, after short exposures (14). The wide range of effects in several species was considered characteristic of a genotoxic carcinogen. This view was supported by the short-term mutagenicity tests. Vinyl chloride was mutagenic and clastogenic in vivo, and in vitro when in the presence of an appropriate metabolic activating system (8). Clastogenicity has also been reported in workers exposed to high concentrations of vinyl chloride (15). There seems little doubt that vinyl chloride is mutagenic and carcinogenic as a result of its metabolism by microsomal mixed function oxidases (cytochromes P-450) to chloro-oxirane (chloroethylene oxide) (16, 17). This highly electrophilic epoxide is a potent mutagen when tested directly or when generated from vinyl chloride in the presence of an appropriate metabolizing system (18, 19). DNA-alkylation products that lead to mispainng, depurination, and fragmentation have been isolated from animals exposed to vinyl chloride and from a variety of in vitro systems (20, 21). These products are consistent with chloro-oxirane being the ultimate carcinogenic form of vinyl chloride. The other known mutagenic metabolite of vinyl chloride is chloroacetaldehyde, the rearrangement product of chloro-oxirane (19). Although less mutagenic than the epoxide, chloroacetaldehyde is known to react with DNA to give the same alkylation products and could therefore have a role in the carcinogenicity of vinyl chloride (20). The major detoxification pathway for these two mutagenic metabolites is by conjugation with glutathione leading to the excretion of S-(2hydroxyethyl) cysteine and thiodiglycolic acid in urine (Figure 1) (22, 23). The epoxide also appears to be a substrate for epoxide hydratase and gives an unstable diol that is further metabolized to become incorporated into the citric acid cycle and yield carbon dioxide in vivo. That vinyl chloride metabolism is a saturable process (24) is reflected by the incidences of angiosarcoma and the reduction of the slope of the dose-response curve to zero at higher dose levels (14). There are no known major species differences in the metabolism of vinyl chloride. 76 GREEN H Cl H H Cl DNA adducts CANCER Detoxification pathway* [GSH, glutathiona] Figurt i The metabolic activation of vinyl chloride. The vast amount of information available about vinyl chloride, which is very briefly summarized here, corroborates the mutagenicity and carcino genicity of this chemical. This conclusion is consistent with its metabolism to a mutagenic epoxide and the development of cancer by a mechanism involv ing alkylation of DNA. Vinyl chloride is therefore a classical genotoxin causing cancer by somatic mutation. THE MUTAGENICITY AND CARCINOGENICITY OF VINYLIDENE CHLORIDE, TRI- VD TETRA-CHLOROETHYLENE Vinylidene chloride, trichloroethylene, and tetrachloroethylene are all metab olized by cytochrome P-450 to products consistent with the formation of an epoxide intermediate (25, 26) and are identical in this respect to vinyl chloride. Although the stability and reactivity of the epoxides have been intensively investigated (27-30), there is no direct evidence that they exist as free chemical species in biological systems. It has been suggested that these epoxides never leave the enzyme site as free species (31), unlike vinyl chloride epoxide, which has been detected spectroscopically and trapped with a variety of nucleophiles (32, 33). A further indirect measure of the formation and reactivity of these epoxides is the mutagenicity of the parent chemical in vivo, and in vitro in the presence of suitable metabolic activation. Again, these results provide little evidence that these epoxides exist in mammalian systems. Weak responses have been seen for vinylidene chloride in prokary otic and some eukaryotic systems (34. 35). The responses are highly de pendent upon species or the source of the metabolic activation used in the in vitro tests, with mouse tissues the most active (36, 37). A similar pattern is seen for trichloroethylene where the responses are perhaps even weaker, e.g. no evidence of point mutations in bacteria (38. 39), some in yeast (40. 41), but chromosomal effects in the mouse micronucleus assay (42). Tetrachlor oethylene appears not to be mutagenic or clastogenic either in vivo or in vitro CHLOROETHYLENE CARCINOGENICITY 77 in tests where pure material was used (39, 43). DNA*binding studies have been reported for each of the chloroethylenes. although there is little evidence of binding except with vinyl chloride where specific adducts have been identified (44-47). There is therefore a clear gradation from a potent mutagen and clastogen, vinyl chloride, through to tetrachloroethylene, which appears to have no genotoxic properties in short-term tests. This conclusion is based on an overview of a complex area that in detail is beyond the scope of this review. Many of the mutagenicity tests reported in the literature are con founded by the use of impure material and the presence of mutagenic stabiliz ers that have been used in some commercial grades of these chemicals. Nonetheless, the conclusion is valid when properly conducted tests of pure material are considered. The pattern of inconsistent responses seen in the mutagenicity tests is repeated in the two-year cancer studies. The carcinogenicity of vinylidene chloride, the closest analog of vinyl chloride and therefore the one that might be expected to most resemble vinyl chloride in its properties, has never been fully resolved. Of eighteen chronic studies only one reported a carcinogenic effect (48). Male Swiss mice exposed to 23ppm by inhalation had a low incidence of renal adenocarcinoma. The effect was not seen at lOppm, nor in female mice, in other species at higher dose levels, or in mice of other strains. In light of these negative results, this apparently real but unique response fails to resolve the issue of whether or not vinylidene chloride is a carcinogenic hazard to man. A further bioassay is unlikely to resolve this issue. Trichloroethylene and tetrachloroethylene have also shown marked spe cies-. strain- and sex-specific responses in two-year studies (43,49-54). Both chemicals are clearly carcinogenic in the liver of B6C3F1 mice in several studies by either inhalation or ingestion (32, 49-51). However, trichloro ethylene did not induce tumors in Swiss mice (52) nor did either chemical cause liver tumors in rats (43, 49-51). A low incidence of kidney tumors was reported in male rats exposed to tetrachloroethylene in two studies (43, 53), and lung tumors have been observed in CD-I mice exposed to trichlo roethylene in another study (54). Thus, in contrast to vinyl chloride where there are consistent responses in the cancer bioassays and a clear genotoxic mechanism, the other chloro ethylenes fail to give a clear indication of their genotoxic potential in short term tests and the cancer studies fail to give a clear indication of the hazard to man. Many of these issues have largely been resolved by mechanistic studies that explained the species and sex differences, and linked the relevance of the animal studies to exposed human populations. Vinylidene Chloride-Induced Mouse Kidney Tumors Perhaps the least satisfactory explanation of a species-specific response among the chloroethylenes is found for vinylidene chloride. This may reflect 78 GREEN the degree of uncertainty over its carcinogenicity. Kidney tumors were found in only one study that used Swiss mice and then in only two out of eighteen surviving male*; '48). Severe nephrotoxicity was a feature of the study at the dose level wr. tumors were seen but not at lower dose levels where no tumors occurn Of the species and strains used in the eighteen cancer studies,jthe male Swiss mouse was more susceptible than rats, hamsters, or other strains of mice to the nephrotoxic effects of vinylidene chloride. Thus, it is tempting to make a connection between the kidney damage and the low incidence of tumors seen in these mice. Other possible mechanisms have been considered. Vinylidene chloride is metabolized by a saturable pathway to a putative epoxide intermediate that rearranges to chloroacetyl chloride (25, 55). The major detoxification path ways for these intermediates are hydrolysis and conjugation with glutathione, resulting in a variety of metabolites and the depletion of hepatic glutathione levels. Studies have investigated the metabolism and pharmacokinetics of vinylidene chloride in different species and organs, seeking an explanation for the response seen exclusively in the mouse kidney. Alkylation of macro* molecules is highest in the mouse kidney (56-58) and is dependent on cellular glutathione levels (59). Oesch et al (36) compared the effes of vinylidene chloride on hepatic and renal enzymes and reported a rec ;ion in epoxide... hydratase and glutathione-S-transfeme activity in male mouse kidney that-- was not seen in female mice or in rats of either sex. Although a reduction in these detoxification enzymes is consistent with the outcome of the cancer studies, one enzyme, epoxide hydratase, apparently plays a very minor role in the metabolism of vinylidene chloride. In conclusion, higher metabolic rates, higher levels of covalent binding, and possible reductions in the levels of detoxifying enzymes are all consistent with the male Swiss mouse's susceptibility to kidney tumors. Implicit is the belief that vinylidene chloride is carcinogenic as a result of its metabolism to a reactive electrophile such as the epoxide. However, the crucial factor in determin ing the species and strain differences may be the susceptibility of the Swiss mouse to the cytotoxic effects of vinylidene chloride. There is some evidence that tumors are not seen in the absence of toxicity; this suggests that the weak alkylating effects of vinylidene chloride metabolites may not be sufficient to cause cancer. Kidney damage and the resulting cell division may facilitate the expression of the weak genotoxic potential of the metabolites or. alternatively, kidney damage may alone lead to cancer by a nongenotoxic mechanism. Whichever mechanism applies, the effects seem to be unique to the male Swiss mouse and to have little relevance to other species, including man. Tri- and Tetrachloroethylene Induced Mouse Liver Tumors Both trichloroethylene and tetrachlorcthylene cause a very similar increase in the incidence of hepatocellular carcinoma in B6C3FI mice following either SL 064679 oral ad term e\ prolifei prolifei basis o and sul some p to inch rats an two-ye mice it within livers c triflklpi strate t chemic Tricl rachlor 67-70) peroxr sponse chemic this ap oxisorr metabc range i Tr FiSurr (umou' CHLOROETHYLENE CARCINOGENICITY 79 oral administration or inhalation exposure for two years (43. 49-51). Short term exposure to these chemicals causes liver growth and marked peroxisome proliferation in mice but not rats (60-62). The link between peroxisome proliferation and cancer in rodents strongly suggests that this response is the basis of the species difference in carcinogenicity (63-65). The identification and subsequent cancer bioassay of the metabolite responsible for the peroxi some proliferation confirmed this hypothesis. Both chemicals are metabolized to trichloroacetic acid (Figure 2), which causes peroxisome proliferation in rats and mice in vivo and in rat and mouse hepatocytes in vitro (60). A two-year study in which trichloroacetic acid was administered to B6C3F1 mice in drinking water found a 32% incidence of hepatocellular carcinoma within 61 weeks of the start of the study (66). Tumors were not found in the livers of control animals at that time. There can be linle doubt therefore that trichloroacetic acid is the metabolite of trichloroethylene and tetrachloroethylene responsible for the liver tumors seen in mice. These studies also demon strate that the epoxide intermediates proposed in the metabolism of both chemicals have no role in the initiation or development of these tumors. Trichloroacetic acid is a common metabolite of trichloroethylene and tetrachloroethylene in most animal species including rats, mice, and man (26, 67-70). When trichloroacetic acid was administered to rats and mice hepatic peroxisome proliferation was seen in both species (60). However, this re sponse and liver cancer were seen only in mice and not rats when the parent chemicals were administered in the two-year cancer studies. The reason for this apparent anomaly is pharmacokinetic; trichloroacetic acid-induced per oxisome proliferation is a threshold phenomenon (60). In the mouse, the metabolism of trichloroethylene and tetrachloroethylene is linear over a wide range of dose levels and the threshold is easily exceeded at the high dose H Cl Cl Cl " Trichloroethylene </v\, CljCCHO -- CljCCOOH * Peroxieome* CANCER Cl Cl o' \i Cl Cl xc--/ / v/ N Cl o Cl Pcruxiiomes V Cl CC0CI -* Cl CC00H 33 Tetrochloroethylene Figurr 2 The metabolism of tn- and tetrachloroethylene in relation to the development of liver tumours in B^CtF, mice. 80 GREEN levels used in the cancer studies. In the rat. metabolism becomes saturated at relativ*' low dose levels and the threshold concentration of trichloroacetic acid r tred to induce peroxisome proliferation is not exceeded at any dose of the -ent chemical (26. 60, 71, The effects of this species difference in metatx. sm is clearly seen in the blood levels of trichloroacetic acid in the two species. For example, the peak in blood levels was seven times higher in the mouse than the rat when both species were exposed to 400ppm tetrachioroethylene for six hours (72). These experiments produced an entirely consistent explanation for the mechanism of action of these two chemicals and for the basis of the rodent species differences. The mechanism involves peroxisome proliferation medi ated by a common metabolite, trichloroacetic acid. The finding that trichloroacetic acid is a complete carcinogen appears to rule out any role for electrophilic epoxide metabolites in the hepatocarcinogenicity of these chemi cals. which is consistent with their lack of mutagenicity. Also consistent with a nongenotoxic mechanism is the nature of the threshold response for peroxi some proliferation, which is also the basis of the species difference between rats and mice and is derived from simple pharmacokinetic differences be tween the two species. Thus, mice are susceptible to liver cancer as a direct result of the high metabolic rates frequently found in this species and rats are protected by tewer rates and saturation of the metabolic pathway. The above experiments and bioassays all used the B6C3F1 strain of mouse, and for the trichloroethylene studies, oral gavage as the route of administra tion. The Swiss mouse exposed by inhalation is apparently not susceptible to trichloroethylene-induced liver cancer in the same way (52). although the reasons for this strain difference are unknown. Swiss mice metabolize trichlo roethylene at a similar rate and to a similar extent and are also susceptible to peroxisome proliferation (60, 71). Thus, other than differences in the dose and route of administration in the two cancer bioassays, and the known sensitivity of the B6C3F1 mouse to liver carcinogens, there is no clear explanation for this strain difference in carcinogenicity. The relevance of the mechanism identified in B6C3F1 mice for humans exposed to tri- and tetrachloroethylene has been assessed in a number of studies comparing metabolic rates m mice, rats, and man. and the response of human liver tissue to the key metabolite, trichloroacetic acid (60). The metabolism of both trichloroethylene and tetrachloroethylene is limited in man by saturation of the metabolic pathway at relatively low exposure levels (67, 73, 74). In this respect, man resembles the rat and may not be able to produce sufficient trichloroacetic acid to stimulate peroxisome proliferation. There is. however, an even more significant difference between mice, rats, and man in their response to trichloroacetic acid. The peroxisome prolifera tion seen in vivo in rats and mice treated with trichloroacetic acid has been reproduced in vitro using mouse and rat hepatocytes (60). Under the same CHLOROETHYLENE CARCINOGENICITY 81 conditions, trichloroacetic acid failed to induce peroxisome proliferation in human hepatocytes. Consequently, the mechanism believed to operate in the mouse appears to be unique to that species. Only the mouse has the combina tion of high metabolic rates and the ability to respond to trichloroacetic acid as a peroxisome proliferator that results in cancer. The rat is limited by metabol ic saturation; man is limited by both metabolic saturation and a lack of trichloroacetic acid-stimulated peroxisome proliferation. These conclusions are perhaps not too surprising. The mouse as the smallest of the species of interest would be expected to have the highest metabolic rates. It is also well known that peroxisome proliferation differs according to species and is much more common in rodents than in any other species. Tetrachloroethylene Induced Rat Kidney Tumors A low incidence of renal tubular cell adenomas and adenocarcinomas was found in male F344 rats exposed to tetrachloroethylene by inhalation for a lifetime (43). The incidence was not statistically significant, but because adenomas are rare in control F344 rats and malignant kidney tumors are not normally observed at all, several studies have sought a mechanistic explana tion for their origin. Previous lifetime studies of tetrachloroethylene in rats have all been affected by poor survival within the dosed groups (51, 76). In each case there was evidence that survival was reduced as a result of chemically induced nephropathy distinguishable from the age-related nephropathy normally seen in this strain of rat. Of the species tested, the male rat was the most sensitive to these effects. Mortality was slight in the NTP inhalation study (43), but again there was evidence of kidney damage characterized by tubular enlarge ment and hyperplasia. There are therefore striking similarities between tet rachloroethylene and vinylidene chloride in that the species most sensitive to the nephrotoxic effects of the chemical has a low incidence of kidney tumors after two years of exposure. It is possible that a similar mechanism involving sustained cytotoxicity and cell division may apply. Other well-defined mechanisms have been proposed to account for these tumors. High oral doses of tetrachloroethylene (l-1.5g/kg/day) cause an accumulation of the protein alpha-2u-g!obulin (hyaline droplets) in renal proximal tubular cells of male rats (75. 77-79). Tubular casts were formed and focal areas of proximal tubular regeneration were apparent in these studies. The effects were specific to the male rat and were not seen in females or in mice of either sex. Hyaline droplet formation or protein droplet nephrop athy has been linked with a significant number of male rat-specific kidney carcinogens and is believed to be part of a cycle of necrosis and cellular regeneration that results in cancer (77. 80-83). Such a mechanism would explain the specificity of the tumors and would not be dissimilar in effect to the cytotoxicity and hyperplasia reported from the two-year studies. Howev- i SL 064682 82 GREEN er, although these responses were observed after high oral doses of tetrachloroethyiene, they were not seen after inhalation exposure for 28 days (75) at the dose levels (up to 400ppm) usee - the NTT inhalation study (43). Apparently, hyaline droplet formatic <e peroxisome proliferation and other nongenotoxic responses, is a th, Id phenomenon and the highest dose used in the NTP study is below the rshold. Further short-term studies using higher doses (lOOOppm) by inhalation did induce this response in male rats (75). Thus, although a well-established link exists between hyaline droplet formation and renal cancer in male rats, the lack of effect ax the top dose level used in the two-year inhalation study (43) questions the signifi cance of this phenomena in the development of tumors in that study. A contribution from hyaline droplet formation to the development of these tumors cannot entirely be ruled out based on the results of a 28-day study. The potential for causing this effect has clearly been established and hyaline droplet formation may still occur during a lifetime study, even at the lower dose levels used in the NTP study (43). Yet another mechanism has been proposed to explain the low incidence of male rat kidney tumors seen after exposure to tetrachloroethyiene. Unlike the previous mechanisms, this one involves the formation of a mutagenic metabo lite in the kidney. Tetrachloroethyiene is metabolized by a second minor pathway involving hepatic glutathione conjugation (75, 84) (Figure 3). The conjugate is metabolized by the mercapturic acid pathway and excreted in urine as the N-acetyl cysteine derivative. The precursor of this metabolite, the cysteine conjugate of tetrachloroethyiene, is a substrate for the renal enzyme /3-lyase and is mutagenic in the Ames bacterial mutation assay when activated by rat kidney fractions (75, 85, 86). The lack of response of tetrachloro ethyiene itself in this assay is probably due to a failure of the standard test to a a osh s eMethioneC-C ------------ -- aj a\ S-tramfarM aa >, / C-C a/ \sc _ ai ---- == -- aty a \ c-c 5-CHj-CH COOH N-acetyl transferase, oa \/ c-c NHCOCH i3 Cl S-CH.-CH Urine COOH a ci a \ / i+ C-CN <-* H- cI - c-s Cl SH Cl \ DMA adducts Fifurr J The metabolism of tetrachloroethyiene by glutathione conjugation and activation by renal 0-lyase in ihe rat. SL 064683 repli< puthx geno cons conn eithe Mgm A fume true the i not f den' non M turn* fom not high com tetr: has in r. 0-1 > sim rat tath ot i W' I rru ger di" tat; lor oc > 9 In e' CHLOROETHYLENE CARCINOGENICITY 83 replicate the number of steps involved in the activation of the chemical by this pathway. These studies have been taken as evidence for the involvement of genotoxicity in the development of these tumors. Activity by this pathway is consistent with the male rat as the susceptible species, since glutathione conjugation and B-lyase activity is greater in male than female rats or mice of either sex (75). The pathway is minor, even in the male rat. and only increases significantly when the major cytochrome P-450 pathway has saturated (75). A surfeit of explanations exists for what is actually a very low incidence of tumors in one sex of one species of laboratory animal. This may equally be true with most mechanisms of carcinogenesis in that many factors influence the initiation and development of tumors. In this case it is entirely possible, if not probable, that the overall mechanism involves a low level of genotoxicity derived from glutathione conjugation and activation by /3-lyase, in conjunc tion with cell division stimulated by cytotoxicity or hyaline droplet formation. Each apparently separate mechanism may in combination provide the initia tion and promotion steps believed to be essential for chemical carcinogenesis. Most aspects of the mechanisms believed to be responsible for the kidney tumors seen in male rats are in fact unique to the male tat. Hyaline droplet formation and the protein alpha-2u-globulin are found only in the male rat and not in other laboratory animals or in man. Even in the male rat the effect is highly dose-dependent. Cytotoxicity and kidney damage are features of high continuous exposures and are unlikely to occur during occupational use of tetrachloroethylene. The other mechanism involving glutathione conjugation has been investigated in a series of studies comparing the key metabolic steps in rat. mouse, and human tissues (75). Human kidney possesses the enzyme /3-lyase with activity towards the cysteine conjugate of tetrachloroethylene similar to that in the mouse and approximately 30-fold lower than that in male rat kidney. Experiments using human liver fractions failed to detect glu tathione conjugation of tetrachloroethylene and concluded, based on the limit of detection of the assay, that the difference in rate between the tat and man was at least an order of magnitude. In conclusion, three possible mechanisms have been reported to explain the male rat kidney tumors; each is very much dependent upon dose, two exhibit genuine thresholds, and the third is based on a pathway that is minor at low dose levels. It is unlikely that these mechanisms will occur in man; glu tathione conjugation has not been detected in human liver, hyaline droplet formation is exclusive to the male rat and chronic toxicity is only likely to occur after exposure to high concentrations over a prolonged period. OTHER TUMORS In addition to the multiplicity of tumor types reported following vinyl chloride exposure, other tumors have also been reported for trichloroethylene and ,, 064684 ( 84 GREEN tetrachloroethylene. An increased incidence of lung adenomas and adenocar cinomas has been reported in mice exposed to trichloroethylene by inhalation (54) and an increase in mononuclear cell leukemia was seen in F344 rats exposed to tetrachloroethylene (43). A recent NTP study (87) exposing four strains of rat to trichloroethylene also reported a low incidence of kidney tumors. Np satisfactory explanations for the mechanisms involved in the develop ment of these tumors are presently available, although two of the results may be features of the strain of animal used and the conduct of the study rather than a true carcinogenic response to the chemical. The incidence of mononuc lear cell leukemia in control F344 rats in the study (43) was 56% (males) and although this was statistically increased by exposure to tetrachloroethylene, its relevance for man is questionable. This type of tumor was not seen in other strains of rat, is known to be of high and variable incidence in the F344 rat, and is not found in man. The low incidence of kidney tumors reported in rats exposed to trichloro ethylene appears to be largely a result of chronic toxicity rather than from a more specific mechanism. Although reported, this study (87) was considered inadequate for assessing carcinogenicity because of poor survival within the dosed groups. Nephrotoxicity was again a feature of the study and the lack of dose-, sex-, or strain-dependence on the distribution of the kidney tumors suggests that toxicity may well be the cause of the low incidence observed. Other mechanisms, detected with tetrachloroethylene, apparently are either absent or present at much lower levels. CONCLUSIONS The discovery of the carcinogenicity of vinyl chloride led to the assumption that the other chloroethylenes would be similarly carcinogenic. This belief was well founded in that each is carcinogenic in at least one animal species. \t this point the similarities between vinyl chloride and the other chlormethylenes end. The expectation that they act in the same way as vinyl :hloride through mutagenic epoxide metabolites has not been fullfilled. New nechanisms have been discovered involving peroxisomes, hyaline droplets, tnd new metabolic pathways that were unknown in the 1970s when vinyl rhlonde was first investigated. Many of the new mechanisms are believed to ic nongenotoxic; they exhibit thresholds and in many cases show marked pecies-specificity. Chrome toxicity features in all studies where kidney umors have been observed. That a variety of mechanisms can operate within . close structural group of chemicals is perhaps the most significant conclu- ion from these studies. Within this finding the species-, sex- and target-organ pecificity of these chemicals has been explained and the need clearly illus trated for this The known n The quest assessment, structurally s grouped toge species sugg trichloroethy associated w others marke Quantitative occur betwe metabolite u hyalinij^k pharmSWn into account more generv the cancer s ethylene, th developmer In-depth their infanc Clearly, ho' the high-ris gens that m Table I kiv Chemicals Vinyl chlona Vinylidene c Tnchloroeih Tctrachlorcs SL 064685 CHLOROETHYLENE CARCINOGENICITY 85 trated for this level of understanding to adequately evaluate the risks to man. The known mechanisms for the chloroethylenes are summarized in Table 1. The question remains of the use of this type of work in human-risk assessment. The chloroethylenes illustrate clearly the pitfalls of assuming that structurally similar chemicals act by the same mechanism and can therefore be grouped together for risk assessment. Studies comparing mechanisms and species suggest that the risks to man from exposure to vinylidene chloride, trichloroethylene and tetrachloroethylene are considerably less than those associated with vinyl chloride. In some cases there are qualitative, and in others marked quantitative, differences between laboratory animals and man. Quantitative risk assessment is inappropriate where qualitative differences occur between species in response to peroxisome proliferators such as the metabolite trichloroacetic acid, or differences in tetrachloroethylene-induced hyaline-droplet formation. Where quantitative differences in metabolism and pharmacokinetics do exist, such procedures may be appropriate if they take into account those differences in the calculation of the risk to man. Even if the more generic weight-of-evidence approach is taken, the species differences in the cancer studies of vinylidene chloride, trichloroethylene and tetrachloroethylene. their lack of mutagenicity, and the mechanisms involved in the development of the tumors, all suggest the risks to man are minimal. In-depth mechanistic investigations and species comparisons are still in their infancy and have yet to play a major role in human risk assessment. Clearly, however, such an approach is important and can assist in separating the high-risk chemicals such as vinyl chloride from species-specific carcino gens that may pose little or no threat to man at occupational or environmen- Table 1 Known mechanisms for the carcinogenicity of the chloroethylenes Chemicals Vinyl chloride Vinylidene chloride Trichloroethylene Carcinogenic Metabolite Epoaide/chioraacctalde - hyde Epoxide/acid chloride? Unknown Trichloroacetic acid Tetrachloroethylene Trichloroacetic acid Cysteine conjugate Unknown Unknown Mechanism Tumors DNA alkylation Multiple All species DNA? Male mouse kidney Chronic toxicity Mate mouse kidney Peroxisome proliferation Mouse liver Peroxisomes Mouse liver DNA adducts Rat kidney Hyaline droplets Rat kidney Chronic toxicity Rai kidney i * 86 GREEN tal exposure levels. As this area develops and our understanding of these mechanisms improves, the challenge for regulatory authorities will be to incorporate such data into the risk-assessment process, rather than rely sc ;ely on inconsistent animal bioassays and short-term tests. Literature Cited 1. Selikoff. I. J., Hammond. E. C., eds. 1973. Toxicity of vinyl chlondepolyvinyl chloride. Ann. NY Acad. Sci. 246 2. Torkelson, T. R,, Rowe. V, K. 1982. Vinylidene chloride. In Patty't In dustrial Hygiene and Toxicology, ed. G. Clayton. F. Clayton, p. 3543. New York: McGnw-Hill 3. Browning. E., 1965. Trichloroethylene. In Toxicity and Metabolism ofIndustrial Solvents, pp. 189-212. Amsterdam: Elsevier 4. Rowe. V. K.. McCollistcr. D. D.. Spencer, H. C., Adams. E. M.. Insh. D. D. 1952. Vapour toxicity of tetrachlotoethylene for laboratory animals and human subjects. Am. Med. Assoc. Arch. Ind. Health 5:566-79 3. Hunter. A. R. 1962. Inhalation an aesthetic agents. Br. J. Anaethesia 34: 224-28 6. Cordier. S. M.. Fievres. C., Lefeve, M. J., Sevnn. A. 1966. Acro-osteoiyse et lesions cutandes usocides chez deux ouvners affectds au nettoyage d'autoclaves. Cah. Med. Trav. 4:3-39 7 IARC. 1989. 1ARC Monographs on the evaluation of carcinogenic nsk of chemi cals to humans. Vol. 19. Some monom ers. plastics and synthetic elastomers and acrotein. Lyon. France: IARC 8. IARC. 1979. IARC Monographs on the evaluation of carcinogenic risks of chemicals to humans. Vol. 20. Some halogenated hydrocarbons. Lyon, France: IARC 9. Viola. P L. 1970. Pathology of vinyl chloride. Med. Lav. 61:174--80 10. Viola. P L,. Bigotu. A.. 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