Cell Biology and Toxicology

, Volume 1, Issue 3, pp 159–179 | Cite as

Activities of chlorinated ethane and ethylene compounds in the Salmonella/rat microsome mutagenesis and rat hepatocyte/DNA repair assays under vapor phase exposure conditions

  • Tomiko Shimada
  • Albertina F. Swanson
  • Philip Leber
  • Gary M. Williams

Three chlorinated ethane and ethylene solvent products were examined for their genotoxicity in the Salmonella/microsome mutagenesis and hepatocyte primary culture DNA repair assays using vapor phase exposures. The positive control in this study, monochloroethylene (vinyl chloride), induced reversion mutation of Salmonella tester strains TA100 and TA1535 with enhancement by an exogenous activation system and elicited unscheduled DNA synthesis in rat hepatocytes in culture. Exposures to 1,1,1-trichloroethane (methyl chloroform) or 1,1,2-trichloroethylene samples which contained stabilizers resulted in increased recovery of revertant colonies of Salmonella at concentrations causing greater than 96% cell killing. However, these stabilized materials did not induce DNA repair and low-stabilized trichloroethylene did not induce reversion mutation or DNA repair. Exposure of Salmonella tester strains and hepatocytes to highly toxic vapor concentrations of technical grade 1,1,2,2-tetrochloroethylene, low-stabilized and stabilized, increased reversion mutation and elicited DNA repair. Tetrachloroethylene of high purity was not genotoxic. With all of these test products, the presence of an Aroclor-induced rat liver subcellular enzyme preparation in the mutagenesis assay did not have any effect on the results. These observations suggest that stabilizers or unknown impurities normally present at low concentrations in these products are responsible for the positive responses observed at the high exposure concentrations achievable under in vitro test conditions.

Key words

hepatocyte/DNA repair Salmonella mutagenicity 1,1,1-trichloroethane 1,1,2-trichloroethylene 1,1,2,2-tetrochloroethylene 



hepatocyte primary culture


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. AMES, B., McANN, J. and YAMASAKI, E. (1975). Methods for detecting carcinogens and mutagens with the Salmonella/ mammalian-microsome mutagenicity test. Mutat. Res. 31:347–364.Google Scholar
  2. ANDERS, M.W. (1982). Aliphatic halogenated hydrocarbons. In: Metabolic Basis of Detoxification: Metabolism of Functional Groups (W.B. Jakoby, J.R. Bend, and J. Caldwell, eds.), Academic Press, New York. pp. 29–49.Google Scholar
  3. BADEN, J.M., KELLY, M.J., MAZZE, R.I. and SIMMON, V.F. (1979). Mutagenicity of inhalation anesthetics, nitrous oxide, cyclopropane, trichloroethylene, and divinyl ether. Br. J. Anaesth. 51: 417–421.Google Scholar
  4. BARTSCH, H., MALAVEILLE, C., MONTESANO, R., and TOMATIS, L. (1975). Tissuemediated mutagenicity of vinylidine chloride and 2-chlorobutadiene in salmonella typhimurium. Nature 255: 641–643.Google Scholar
  5. BARTSCH, H., MALAVEILLE, C., BARBIN, A. and PLANCHE, G. (1979). Mutagenic and alkylating metabolites of haloethylenes, chlorobutadienes and dichlorobutenes produced by rodent or human liver tissue. Evidence for oxirane formation by P-450-linked microsomal monooxygenases. Arch. Toxicol. 41: 249–277.Google Scholar
  6. BERGMAN, K. (1983). Interaction of trichloroethylene with DNA in vitro and with RNA and DNA of various mouse tissues in vivo. Arch. Toxicol. 54: 181–193.Google Scholar
  7. BLOCK, J.B. (1974). Angiosarcoma of the liver following vinyl chloride exposure. J.A.M.A. 229: 53–54.Google Scholar
  8. BOLT, H.M. and FILSER, J.G. (1977). Irreversible binding of chlorinated ethylenes to macromolecules. Environ. Health Perspect. 21: 107–112.Google Scholar
  9. BYINGTON, K.H. and LEIBMAN, K.C. (1965). Metabolism of trichloroethylene in liver microsomes. II. Identification of the reaction product as chloralhydrate. Mol. Pharmacol. 1: 247–254.Google Scholar
  10. CALLEN, D.F., WOLF, C.R., and PHILPOT, R.M. (1980). Cytochrome P-450 mediated genetic activity and cytotoxicity of seven halogenated aliphatic hydrocarbons in Saccharomyces cerevisiae. Mutat. Res. 77: 55–63.Google Scholar
  11. CASCIOLA, L.A.F. and IVANETICH, K.M. (1984). Metabolism of chloroethanes by rat liver nuclear cytochrome P-450. Carcinogenesis 5: 543–548.Google Scholar
  12. CERNA, M. and KYPENOVA, H. (1977). Mutagenic activity of chloroethylenes analysed by screening system tests. Mutat. Res. 46: 214–216.Google Scholar
  13. CHU, K.C. and MILMAN, H.A. (1981). Review of experimental carcinogenesis by compounds related to vinyl chloride. Environ. Health Perspec. 41: 211–220.Google Scholar
  14. CREECH, J.A. and JOHNSON, M.N. (1974). Angiosarcoma of liver in the manufacture of polyvinyl chloride. J. Occup. Med. 16: 150–151.Google Scholar
  15. DANIEL, J. W. (1963). The metabolism of C136-labelled trichloroethylene and tetrachloroethylene in the rat. Biochem. Pharmacol. 12: 795–802.Google Scholar
  16. DIPAOLO, J.A. and DONIGER, J. (1982). Neoplastic transformation of Syrian hamster cells by putative epoxide metabolites of commercially utilized chloroalkenes. J. Natl. Cancer Inst. 69: 531–534.Google Scholar
  17. DRENZO, A.B., GANDOLFI, A.J. and SIPES, I.G. (1982). Microsomal bioactivation and covalent binding of aliphatic organohalogens to DNA. Toxicol. Lett. 11:243–252.Google Scholar
  18. ELCOMBE, C.R. (dy1985). Species differences in carcinogenicity and peroxisome proliferation due to trichloroethylene: a biochemical human hazards assessment. Arch. Toxicol. in press.Google Scholar
  19. GARRO, A.J., GUTTENPLAN, J.B. and MILVY, P. (1976). Vinyl chloride dependent mutagenesis: effect of liver extracts and free radicals. Mutat. Res. 38: 81–88.Google Scholar
  20. GREIM, H., BONZE, G., RADWAN, Z., REICHERT, D., and HENSCHLER, D. (1975). Mutagenicity in vitro and potential carcinogenicity of chlorinated ethylene as a function of metabolic oxirane formation. Biochem. Pharmacol. 24: 2013–2017.Google Scholar
  21. HENSCHLER, D., BONZE, G. (1977). Metabolic activation of chlorinated ethylenes: dependence of mutagenic effect on electrophilic reactivity of the metabolically formed epoxides. Arch. Toxicol. 39: 7–12.Google Scholar
  22. HENSCHLER, D., EDER, E., NEUDECKER, T., and METZLER, M. (1977). Carcinogenicity of Trichloroethylene: Fact or Artifact? Arch. Toxicol. 37: 233–236.Google Scholar
  23. HENSCHLER, D., REICHERT, D., and METZLER, M. (1980). Identification of potential carcinogens in technical grade 1,1,1-trichloroethane. Int. Arch. Occup. Environ. Health 47: 263–268.Google Scholar
  24. International Agency For Research On Cancer. (1983). Approaches to Classifying Chemical Carcinogens According to Mechanism of Action. IARC Internal Technical Report No. 83/001. IARC, Lyon, France.Google Scholar
  25. ONES, B.K., and HATHWAY, D.E. (1978). Tissue mediated mutagenicity of vinylidene chloride in Salmonella typhimurium TA 1535. Cancer Lett. 5: 1–6.Google Scholar
  26. KLINE, S.A., MCCOY, E.C., ROSENKRANZ, H.A. and Van DUUREN, B.L. (1982). Mutagenicity of chloralkene epoxides in bacterial systems. Mutat. Res. 101: 115–125.Google Scholar
  27. LAIB, R.J. (1982). Specific covalent binding and toxicity of aliphatic halogenated xenobiotics. In: Reviews on Drug Metabolism and Drug Interactions (A. H. Bechett and J.W. Gottod, eds.) Vol. IV, pp. 1–48, Freund Publishing House Limited, London.Google Scholar
  28. LAIB, R.J., STOCKLE, G., BOLT, H.M., and KUNZ, W. (1979). Vinyl chloride and trichloroethylene: comparison of alkylating effects of metabolites and induction of preneoplastic enzyme deficiencies in rat liver. J. Cancer Res. Clin. Oncol. 94: 139–147.Google Scholar
  29. LANE, R.W., RIDDLE, B.L., and BORZELLECA, J.F. (1982). Effects of 1,2-dichloroethane and 1,1,1-trichloroethane in drinking water on reproduction and development in mice. Toxicol. Appl. Pharmacol. 63: 409–421.Google Scholar
  30. MACDONALD, J.R., GANDOLFI, A.J., and SIPES, I.G. (1982). Covalent binding of 14C-1,1,2-Trichloroethane to hepatic proteins following acetone pretreatment. Drug and Chem. Toxicol. 5: 233–247.Google Scholar
  31. MALAVEILLE, C., BARTSCH, H., BARBIN, A., CAMUS, A.M., and MONTESANO, R. (1975). Mutagenicity of vinyl chloride, chloroethylene oxide, chloroacetaldehyde and chloroethanol. Biochemic. Biophys. Res. Commun. 63: 363–370.Google Scholar
  32. MCCANN, J., SIMMONS, V., STREITWEISER, D., and AMES, B.N. (1975). Mutagenicity of chloroacetaldehyde, a possible metabolic product of 1,2-dichloroethane (ethylene dichloride), chloroethanol (ethylene chlorohydrin), vinyl chloride and cyclophosphamide. Proc. Natl. Acad. Sci. (U.S.A.) 172: 3190–3193.Google Scholar
  33. MILLER, R.E. and GUENGERICH, F.P. (1983). Metabolism of trichloroethylene in isolated hepatocytes, microsomes, and reconstituted enzyme systems containing cytochrome P-450. Cancer Res. 43: 1145–1152.Google Scholar
  34. NESTMANN, E.R., LEE, E.G-H., MATULA, T.I., DOUGLAS, T.R., and MEULLER, J.C. (1980). Mutagenicity of constituents identified in pulp and paper mill effluents using the Salmonella/ mammalian-microsome assay. Mutat. Res. 79: 203–212.Google Scholar
  35. NESTMANN, E.R., OTSON, R., KOWBEL, D.J., BOTHWELL, P.D. and HARRINGTON, T.R. (1984). Mutagenicity in a modified Salmonella assay of fabric-protecting products containing 1,1,1-trichloroethane. Environ. Mutagen. 6: 71–80.Google Scholar
  36. OTTENWAELDER, H. and BOLT, H.M. (1980). Metabolic activation of vinyl chloride and vinyl bromide by isolated hepatocytes and hepatic sinusoidal cells. J. Environ. Path. and Toxicol. 4: 411–417.Google Scholar
  37. PARCHMAN, L.G. and MAGEE, P.H. (1982). Metabolism of [14C] trichloroethylene to 14CO2 and interaction of a metabolite with liver DNA in rats and mice. J. Toxicol. and Environ. Health 9: 797–813.Google Scholar
  38. PEROCCO, P., and PRODI, G. (1981). DNA damage by haloalkanes inhuman lymphocytes cultured in vitro. Cancer Lett. 13: 213–218.Google Scholar
  39. RANNUG, U., GOETHE, R., and WACHTMEISTER, C.A. (1976). The mutagenicity of chloroethyleneoxide, chloroacetaldehyde, 2-chloroethanol and chloroacetic acid, conceivable metabolites of vinyl chloride. Chem. Biol. Interact. 12: 256–263.Google Scholar
  40. RANNUG, U., SUNDVALL, A., and RAMEL, C. (1978). The mutagenic effect of 1,2dichloroethane on S. typhimurium. I. Activation through conjugation with glutathione in vitro, Chem. Biol. Interact. 20: 1–16.Google Scholar
  41. REICHERT, D. (1983). Biological actions and interactions of tetrachloroethylene. Mutat. Res. 123: 411–429.Google Scholar
  42. SCHUMANN, A.M., QUAST, J.T. and WATANABE, P.G. (1980). The pharmacokinetics and macromolecular interactions of perchloroethylene in mice and rats as related to oncogenicity. Toxicol. and Appl. Pharmacol. 55: 207–219.Google Scholar
  43. SCHUMANN, A.M., FOX, T.R., and WATANABE, P.G. (1982a). A comparison of the fate of inhaled methyl chloroform (1,1,1-trichloroethane) following single or repeated exposure in rats and mice. Fund. Appl. Toxicol. 2: 27–32.Google Scholar
  44. SCHUMANN, A. M., FOX, T. R., and WATANABE, P. G. (1982b). [l4C] Methyl chloroform (1,1,1-Trichloroethane): Pharmacokinetics in rats and mice following inhalation exposure. Toxicol. Appl. Pharmacol. 62: 390–401.Google Scholar
  45. SHAHIN, M.M. and VON BORSTEL, R.C. (1977). Mutagenic and lethal effects of a-benzene hexlachloride, dibutyl phthalate and trichloroethylene in Saccharomyces cerevisiae. Mutat. Res. 48: 173–180.Google Scholar
  46. SIPES, I.G. and GANDOLFI, A.J. (1982). Bioactivation of aliphatic organohalogens: formation, detection and relevence. In Toxicology of the Liver (G. Plaa and W.R. Hewitt, eds.), pp. 181–212. Raven Press, New York.Google Scholar
  47. STOTT, W.T., QUAST, J.F., and WATANABE, P.G. (1982). The pharmacokinetics and macromolecular interactions of trichloroethylene in mice and rats. Toxicol. Appl. Pharmacol. 62: 137–151.Google Scholar
  48. STOTT, W.T., REITZ, R.H., SCHUMANN, A.M., and WATANABE, P.G. (1981). Genetic and nongenetic events in neoplasia. Fd. Cosmet. Toxicol. 19: 567–576.Google Scholar
  49. TOWN, C. and LEIBMAN, K.C. (1984). The in vitro dechlorination of some polychlorinated ethanes. Drug Metabol. and Dispos. 12: 4–8.Google Scholar
  50. UEHLEKE, H., and Tabarelli-Poplawski, S. (1977). Irreversible binding of 14C- labelled trichloroethylene to mice liver constituents in vivo and in vitro. Arch. Toxicol. 37: 289–294.Google Scholar
  51. UPTON, A.C., CLAYSON, D.G., JANSE, J.D., ROSENKRANZ, H., and WILLIAMS, G.M. (1984). Report of ICPEMC Task Group on the differentiation between genotoxic and non-genotoxic carcinogens.Mutat. Research. 133: 1–50.Google Scholar
  52. VAN DUUREN, B.L. (1975). On the possible mechanism of carcinogenic action of vinyl chloride. Ann. N.Y. Acad. Sci. 246: 258–268.Google Scholar
  53. VAN DUUREN, B.L. and BANERJEE, S. (1976). Covalent interaction of metabolites of the carcinogen trichloroethylene in rat hepatic microsomes. Cancer Res. 36: 2419–2424.Google Scholar
  54. VAN DUUREN, B.L., KLINE, S.A., MECHIONNE, S., and Seidman, I. (1983). Chemical structure and carcinogenicity relationships of some chloralkene oxides and their parent olefins. Cancer Res. 43: 159–162.Google Scholar
  55. VIOLA, P.L., BIGOTT, A., and CAPUTO, A. (1971). Oncogenic response of rat: skin, lungs, and bones to vinyl chloride. Cancer Res. 31: 516–522.Google Scholar
  56. WATERS, E.M., GERSTNER, H.B., and HUFF, T.E. (1977). Trichloroethylene. 1. An overview. J. Toxicol. and Environ. Health 2: 671–707.Google Scholar
  57. WEISBURGER, E.K. (1977). Carcinogenicity studies on halogenated hydrocarbons. Environ. Health Perspect. 21: 7–16.Google Scholar
  58. WEISBURGER, J.H. andWILLIAMS, G.M. (1980). Chemical Carcinogens. In: Toxicology, The Basic Science of Poisons, 2nd edition (J. Doull, C.D. Klaasen, and M.O. Andur, eds.), pp. 84–138. Macmillan, New York.Google Scholar
  59. WILLIAMS, G.M. (1976). Carcinogen-induced DNA repair in primary rat liver cell cultures; a possible screen for chemical carcinogens. Cancer Lett. 1: 231–236.Google Scholar
  60. WILLIAMS, G.M. (1977). Detection of chemical carcinogens by unscheduled DNA synthesis in rat liver primary cell cultures. Cancer Res. 37: 1845–1851.Google Scholar
  61. WILLIAMS, G.M., BERMUDEZ, E., and SCARAMUZZINO, D. (1977). Rat hepatocyte primary cell cultures. III. Improved dissociation and attachment techniques and the enhancement of survival by culture medium. In Vitro 13: 809–817.Google Scholar
  62. WILLIAMS, G.M., LASPIA, M.F. and DUNKEL, V.C. (1982). Reliability of the hepatocyte primary culture/DNA repair test in testing of coded carcinogens and non-carcinogens. Mutat. Research 97: 359–370.Google Scholar

Copyright information

© Princeton Scientific Publishers, Inc 1985

Authors and Affiliations

  • Tomiko Shimada
    • 1
  • Albertina F. Swanson
    • 1
  • Philip Leber
    • 2
  • Gary M. Williams
    • 1
  1. 1.Naylor Dana Institute for Disease Prevention American Health FoundationValhalla
  2. 2.PPG Industries, Inc., Barberton Technical CenterBarberton

Personalised recommendations