Document 2qyaNyzE9O7BdkK0DeaN8RzGp

INSTITUTFOR PHARMAKOLOGIE UND TOXIKOLOGIE DER UNIVERSITAT WURZBURG Yorstand: Prof L>- D. Henschltr Dr. Philip G. Watanabe Toxicology Research Laboratory DOW Chemical 1803 Building Midland, Michigan 48640 7*. tX f, cS<*^u*sc' 8700 Wurzburg, den 5. Mai 1980 \ eribacher MrjKc V Telclon i OV 31,201 3V 8: *dtr 2ZI 39 M He/g Dear Dr. Watanabe, Thank you for your letter of April 25, 1980. BMS&Uftfl U^^^oetbylene, stabiUse4,wit.h. U * i&.,ooli K 12 with sUsb^^e^iye, results after 4flaiigJL2*S?^f ^ microsomes -Ugprin,t_enclo,sedj . The same. WiiJHHLfSKS?..?*** in Salmonella t^phimurium TA 100 (see other reprintL&&.In TA 1539 (unpublished,. .^erlorjned mutagenicity tests with epichloxohydrin tri. This has been done by Loprienoj he sent us a preliminary report of his findings, I have not (yet) rseen a published paper. Our conclusion that epichlorohydrin and 1 epoxibutane might be responsible for the carcinogenicity of technical grade tri, especially in the NCI-bioassay, is based Jon the testing of these compounds as such, and on the literature i. report on their mutagenicity and/or carcinogenicity. Sincerely yours, UlAAm (D,Henschler) SL 034287 Arch Toncol 37. 333-236 (1977) TOXICOLOGY < b> Sprir.f-cr Yctiaf |v7" Short Communication rcipogcnimfv of TiichlQroclhylfciitt Fact.or_Arlifact? F.. Eder. T, Ncudccker, and M. Metzlcr Institut fur Toxikolopc dcr Univcrsilat Wiirzhurj:. Vcrshachcr LandxtraGc 9, D 8700 Wiirzburg. Federal Republic of German) Abstract. Technical trichloroethylene has been found carcinogenic in mice after high daily doses per os. A GC-MS analysis of this technical sample rescaled the presence of considerable amounts of epichlorohydrin and 1.2-epoxibutanc as stabilizers. These epoxides are highly mutagenic in the Ames test and are. most probably responsible for the carcinogenic effect found in mice. The question whether trichloroethx lene is carcinogenic or not remains open. Key words: Trichloroethylene -- Epichlorohydrin -- 1.2-Epoxibutane -- Card nogenicity -- Mutagenicity. Zusammcnfassung. Technisches Trichlorathvlen envies sich nach hohen. tagh chcn oralen Dosen an Mausen als carcinogen. Eine GC-MS Analyse des be nutzten technischen Praparates ergab die Gegenwart bctrachtlicher Gehalic an Epichlorhydrin und 1.2-Epoxibutan. die im Ames Test stark mutagen sind: dicse Epoxide tragen hochstwahrscheinlich die carcinogcne Wirhung in dem fur den Carcinogeneseversuch verwendeten technischen Produki, wo sic als Stabilise toren zugesetzt werden. Die Frage. ob Trichlorathvlen carcinogen ist oder mcht. bleibt offen. Trichloroethylene has been reportet to produce high incidences of hepatocellular carcinomas in both male and female mice but not in rats after high daily oral doses in corn oil for 18 months (Memorandum DHEW, 1975). This report resulted in a variety of warnings and precautious measures, especially in the working enxiron ment where considerable exposure to this solvent is prevalent, inasmuch as specula lions on the similarity with the notorious carcinogen, vinyl chloride with regard to chemical structure and possible metabolic activation have been forwarded (van Duuren and Bancrjec. 1976). Pure trichloroethylene (Tri) has been found slighih mutagenic in a modified Ames mutagenicity testing system (Greim et ah. 1975). Mechanistic evaluations of the possible pathways of metabolic activation and deactivation of this compound have, however, revealed that the postulated electrophilic intermediate, trichloroethyl 234 D. Hcnschlcr el a! Table I. Contaminants i f a technical grade sample of inchlorocthilcnc a1 idemtficd h> GC MS The typical mass fragments were completely identical for the fractions in tl.. s.niplc and a r, gr jde samples for comparison Compound Epichlorohydrin Epoxihuiane Carbon tetrachloride Chloroform 1.1.1 Tfichlorocthane Diisobutylcne (2.2.4 Trimcthylpcmcne-1) F.th)lacclatc Pcntanol-2 Butanol-2 Main mass peaks m/e % w/w Molecular ion Base peak 0.22 0.20 0.05 0.01 0.035 0-020 * 92 72 -- 118 -- 112 57 42 117 83 97 57 0.052 0.015 0.051 88 - 74 45 45 45 Other t> pical peaks 1 II III 27 31 49 39 41 57 35 47 82 35 47 - 27 61 117 41 69 97 29 43 31 59 31 59 59 60 73 ene epoxide, may be bandied differently in different biological systems: a Lewis acid type catalytic rearrangement to the non-reactive chloral hydrate is likely to occur in the mammalian liver cell (Bonse and Henschler, 1976). a reaction which mighi not necessarily prevail in microorganisms such as the tester strains used in the in \itro mutagenicity assays. Technical grade Tri contains several impurities and must be stabilized, for use as a degreasing agent, by antioxidants. Up to now not much attention has been paid to the biological reactivity of these contaminants. We therefore submitted a sample') of the Tri used in the bioassay experiment for carcinogenicity (Memorandum DHLW, 1975) to an extended chemical analysis by gas chromatography -- mass spectrom etry. The results are presented in Table 1. The proportion of all identified contaminants amounts to 0.65%. Two of these constitute strong monofunctional alkylating agents, the mutagenicity and/or carci nogenicity of which has previously been described, or suspected, rcspecitvely: epichlorohydrin (Fishbein, 1976; l.A.R.C. series, 1976) and 1.2-epoxibutane (van Duuren et al., 1967). We reinvestigated the mutagenic potential of these compounds and of another candidate, diisobutylene, in the Ames system (Fig. 1). The results with the most sensitive strain used, 5. typhimurium TA 100 confirm the high muta genic activity for both epichlorohydrin and 1.2-epoxibutane, in contrast to a very low activity for diisobutylene, whereas the effect of trichloroethylene is questionable. Carbon tetrachloride, chloroform, and 1.1.1-trichloroethane were found inactive both with and without addition of induced rat liver microsomes. It is concluded from these results that the carcinogenic effect of the technical sample of Tri used in the bioassay experiment (Memorandum DHEW. 1975) is most probably predominantly, if not exclusively, due to the epoxides which are added to some (Steehrs, 1957) but by no means to all brands of Tri. This view is supported by 1 Kindly obtained and supplied by the Trichloroethylene Toxicology Subcommittee of the Manu facturing Chemists Associations, USA SL 034289 Fatcinogcnicitj of Trichloroethylene: Fact or Artifact 235 Fig, 1. Mutagenicity in the Ames in vitro system using S. lyphimuriurr. TA 100 (Ames ei al.. 1975) of trichloroethylene and contaminants identified in a technical sample. (") Epichlorohydrin; () Epoxibufane; (A) Diisobutvlene; (O) Trichloroethylene. Open symbols: without, filled s> r.bols: with addition of PCB (Aroclor 1254) induced rat liver microsomes. The bacteria were grown in nutrient broth, shaken for 12 h at 37CC, and 0.1 ml was then added to the molten top agar (2 ml), with and without 0.5 ml of "S-9-mix". This mix contained per ml; 8 mM MgClj, 33 mM KC1. 5 mM glucose 6 phosphate. 4 mM NADP, 100 mM sodium phosphate (pH 7.4) and 0.3 ml of liter homogenates (S 9) (9000 x g superna tant) from male Wistar rats (of about 250 g each) which were induced by a single i.p. injection of a polychlorinated biphenyl (PCB) mixture (Aroclor 1254), diluted in corn oil to a concentration of 200 mg/ml. A dosage of 500 mg/kg was given to each rat 5 days before sacrifice. The solutions tested were added directly to the top agar. Triplicate petri plates containing Vogcl-Bonncr E medium were overlayed with this mixture and incubated at 37C. After 48 h the revertant colonies were counted. Sponta neous back mutation rate 138 1 8 (without microsomes) and 242 10 (with microsomes) colonies per plate the fact that carcinomas were not produced in rats but only in mice, a species with a comparatively low activity of epoxide hydrase (Oesch. 1973). the enzyme which detoxifies epoxides. Further studies in intact animals with exposure to nonepoxide stabilized Tri, both by inhalation and gavage, are needed to substantiate or reject the above assumption. Epidemiological investigations should also take into considera tion the possibility that epoxide additives of high vapour pressure might be involved as carcinogenic factors. Until otherwise proven, positive evidence for a carcinogenic risk can only be based on epoxide-stabilized samples of trichloroethy lene. SL 034290 236 References D. Hcnschlcr cl a] Ames, B. N,, Purston. W. E., Yamasaki, E., Lee, F. E.: Carcinogens are mutagens A simple test system combining liver homogenates for activation and bacteria for detection Proc nat Acad. Set. (Wash.) 70. 2281 -2285 (1973) Bonse, G., Hcnschlcr, D.: Chemical reactivity, biotransformation. and toxicity of polychlorinated ali phatic compounds. CRC CriL Rev. Toxicol. 4, 395--409 (1976) Fishbein, L.: Industrial mutagens and potential mutagens. 1. Halogcnatcd aliphatic derisaloes. Muta tion Res. 32, 267 307 (1976) Greim, H., Bonse, G., Radwan, Z., Reichert, D., Htnschlcr, D.: Mutagenicity in vitro and potential carcinogenicity of chlorinated ethylcnes as a function of metabolic oxtrane formation. Biochem. Pharmacol. 24, 2013-2017 (1975) I.A.R.C.: Monograph series on evaluation of carcinogenic risks of chemicals to man 11, 131-139 (1976) Memorandum, Dept, of Health, Education & Welfare, Washington (20. 3. 1975) Oesch. F.: Mammalian epoxide hydrases: inducible enzy mes cataly sing the inactixstior of carcinogenic and cytotoxic metabolites derived from aromatic and olcfmic compounds Xenobiotica 3, 305 -340 (1973) Stechrs. F. W.: U.S. Patent N 2,818,446 December 31, 1957 Van Duurcn. B. I.., Langscth, L., Goldschmidt, B. M., Orris. L.: Carcinogenicity of epoxides, lactones, and pevoxy compounds. VI. Structure and carcinogenic activity. J. nat. Cancer Inst. 39, 1217-1226 (1967) Van Duurcn. B. L., Banerjcc, S.: Covalent interaction of metabolites of the carcinogen trichloroethyl ene in rat hepatic microsomes. Cancer Res, 36, 2419-2422 (J976) Received April 20, 1977 SL 034291 Biochemical Pharmacology, VoL 24. pp. 2013-2017. Pngimon Pmi, 1973. Printed in Gnat Bmam. MUTAGENICITY IN VITRO AND POTENTIAL CARCINOGENICITY OF CHLORINATED ETHYLENES AS A FUNCTION OF METABOLIC OXIRANE FORMATION H.Greim, G.Bonse, Z.Radwan, D.Reichert, D.Henschler Departments of Toxicology of Gesellschaft fur Strahlenund Umweltforschung, 8042 Miinchen-Neuherberg, and the University 8700 Wiirzburg, Versbacher Landstr.9, West-Germany (Received 10 July 1975; accepted 19 August 1975) All chlorinated ethylenes undergo biotransformation in mammalian organisms, the main pathway being an oxidation to oxiranes, as the first step. The stabilitj of these oxiranes varies widely and depends on the number of chlorine substitu tions and on the relative position of the substituents in the molecule. Symmetr ically substituted oxiranes from the tetrachloro- and the isomeric 1,2-dichloro- ethylenes seem to be rather stable . In the case of vinyl chloride and tri chloroethylene the polarity of the unsymmetrical oxiranes exerts lesser stabilitj and induces intramolecular rearrangement ' . Attempts to prepare the oxirane with the highest polarity from 1.1-dichloroethylene (1.1-DCE) remained unsuc cessful till now. In the reaction of 1.1-DCE with m-chloroperoxybenzoic acid,the corresponding oxirane could not be detected; instead, the chloroacetyl chloride was isolated as the product of oxirane-rearrangement 5) However, no direct relationship exists between chemical reactivity and biological effects because the thermal rearrangement of the oxiranes leads to different chemical species: either acyl chlorides or aldehydes which are suspect of causing quite different biological effects. Besides rearrangement, oxiranes may react directly with biologic nucleophiles. Thus, the mutagenic 2013 SL 034292 2014 Preliminary Communications activity of vinyl chloride (VCM) has boon claimed to be exerted by a direct alkylating action of the oxirane '' . VCM has been demonstrated to be carcinogenic in animals ' and in man (for references, see1C^).Quite recently, a carcinogenic activity of trichloro ethylene (TRI) has been reported in mice after long-term administration of rather high daily oral doses (0,5 or 1.0 g/kg). This report11^ in connection with the above considerations prompted us to determine the mutagenicity of the whole series of chlorinated ethylenes with a metabolic activating microsomal enzyme system in vitro. Materials and Methods. Tetrachloroethylene, trichloroethylene, cis- and trans-l,2-dichloroethylene, 1,1-dichloroethylene were obtained from Merck a Co., Darmstadt, as a.g. reagents; vinylchloride as a purified gas (> 99,9%) from BASF, Ludwigshafen. Mutagenic activity of the derivatives formed during micro- i5\ somal activation was tested in a metabolizing in vitro system ' with E coli K 12. One can use this bioauxotrophic strain in four mutation systems to test nutagenic-agents: In the three back mutation systems gal+, arg+, and nad+, and in the MTR system, where forward mutation leads to resistance to 5-nethyl-DLtryptophane 1 31' . For the experiments 6 to 9 x 108 cells of an overnight culture were suspended in 1.5 ml incubate containing 5 mg microsomal protein, isolated from mouse livers, and the NADPH generating system 5 mM MgClj, 16 rtM DL-isocitrate-Na^, 0.66 mM NADP-Na^, 20 pi isocitrate-dehydrogenase (20 milliunits/pl) in 0.1 M phosphate buffer pH 7.4, as well as different concentrations of the test compounds. These concentrations were selected from preliminary experiments so that they did not reduce cell survival by more than 20 per cent (Table 1). After 2 hours of incubation in a shaking water bath at 37, the reaction was terminated in ice. The incubate was diluted in saline and plated on appropriate selective media as described previously^^. Survival of the E coli K 12 strain was determined by plating on the complete medium. Mutagenicity is expressed as colony-forming units (efu) that were counted on the appropriate selective media per efu counted on the complete medium. Liver microsomes were isolated from male mice pretreated for 10 days with 0.1 per cent phenobarbital in the drinking water to increase microsomal enzyme activity 12) SL 034293 i Preliminary Communications 2015 Tab.1: Mutagenicity of chlorinated ethylenes after incubation in a metabolic activating microsomal system concentra- tsurvival tion in the of medium ,, bacteria at 31 C * of spontaneous mutation rate in different operons of E coli K,,, 12 gal+ arg + MTR nad + C12C = CC12 Tetrachloroethylene 0.9 99-1 100 100 100 100 C12C = CHC1 Trichloroethylene ci2c = ch2 1.1-Dichloroethylene Cl .Cl 'c-c' H ^ ^tt cis-1.2-Dichloroethylene Cl H VC = c/ h ^ 'Nn trans-l.2-Dichloro- ethylene 3.3 2.5 2.9 2.3 76-4 7 4-7 88-5 90-3 123-23 232-36 114-18 100 120-14 229-26 100 100 100 100 100 100 100 100 100 loo C1CH = CH, Vinyl chloride 10.6 72-3 231-20 663-141 172-35 148-24 x) determined by GC analyses after injection of 5 pi of the liquid compounds; except vinyl chloride where the gas was introduced by bubbling through the liquid at 15 C. Results and Discussion. The results of the experiments are listed in Table 1. Cytotoxicity of the chlorinated ethylenes varies widely. To obtain 80-100 per cent survival of the tester strain, only 1 nM of trichloroethylene could be used but 10 mM of vinyl chloride. No mutagenic activity of the chlorinated ethylenes was detected in the test system without microsomal enzyme activity. When the complete incubate with metabolically active microsomes was used, conversion of VCM, 1.1-DCE as well as TRI induced mutations, the latter compounds being less mutagenic than VCM. The highest mutation rates were detected in the arginine genes, whereas reversibili iiiLii iH i!: ;il j !i__ SL 034294 2016 Preliminiiy Communications ty in the gal+ ond nad+ systems and the forward mutation to MTR resistance were loss sensitive to the mutagenic effects of the metabolites. Tetrachloroethylene and the cis- and trans-isomers of dichloroethylene were not metabolized to mutagens at all. Direct comparison of the mutagenic activity of the chlorinated ethylenes is not possible since different substrate concentrations had to be used to minimize cell death of the tester strain. However it is evident that mutage nicity of VCM is several times higher than that of 1.1-DCE and TRI. Our results are indicative of a conspicuous correlation between the stability of the oxiranes, as outlined earlier, and the mutagenicity of all six chlorinated ethylenes: those forming very unstable oxiranes (VCM, 1.1-DCE,TRI) induce mutations in the test system, whereas the others (Per, cis- and trans1.2-DCE) forming much mor stable oxiranes, do not. The mutagenicity of trichloroethylene, though only slight in extent in the gal+ system, which is known as very sensitive, has not been anticipated. Tri chloroethylene is metabolized in vitro and in vivo to the scarcely reactive chloral hydrate and furtheron to trichloroethanol and trichloracetic acid15^'16^ The latter compounds are not known to induce cytotoxic or genetic effects. Con version of TRl-oxi ".ne to chloral in vivo is a quite unexpected reaction because thermal .rearrangement in vitro entirely forms dichloroacetyl chloride 17) . This different behaviour should be further investigated. The results of such ex periments may contribute to the better understanding of the mutagenic and potentially carcinogenic properties of trichloroethylene. Acknowledgement: The skilful technical assistence of Mrs. Hesse and Steinhilber is gratefully acknowledged. SL 034295 Preliminary Communications 2017 REFERENCES 1. i.M.Frankel, C.E.Johnson, H.M.Pitt, J.org.Chem. 22, 1119 (1957) 2. K.Griesbaum, R.Kibar, B.Pfeffer, Liebigs Ann.Chem. 1975, 214 3. H,Gross, J.Freiberg, Journ.f.Prakt.Chem. 311, 506 (1969) 4. G.Bonse, Th,Urban, D.Reichert, D.Henschler, Biochem.Pharmacol.(1975) in press 5. G.Bonse, D.Henschler, unpublished results 6. U.Rannug, A.Johansson, C.Ramel, C.A.Wachtmeister, Ambio 3^ 194 (1974) 7. H.Bartsch, C.Malaveille, R.Montesano, Int.J.Cancer 1_5/ 429 (1975) 8. P.L.Viola, A.Bigotti, A.Caputo, Cancer Res, 31^, 516 (1971) 9. C.Maltoni, G.Lefemine, Environm.Res. 7, 387 (1974) 10. J.W.Lloyd, J.occup.Med. J6, 809 (1974); 17, 333 (1975) 11. Memorandum, Dept.of Health, Education & Welfare, Washington, 20.3.1975 12. P.Czygan, H.Greim, AJ Garro, F.Hutterer, F.Schaffner, H.Popper, 0.Rosenthal, DY Cooper: Cancer Res. ^3, 2983 (1973) 13. J.Ellenberger, G.Mohn, Arch-Toxicol. 33, 225 (1975) 14. G.Mohn, J.Ellenberger, D.McGregor,'Mutation Res. 25^ 187 (1974) 15. K.C.Leibman, Mol-Pharmacol. 1_, 247 (1965) 16. J.W.Daniel, Biochem.Pharmacol. 1_2, 795 (1963) 17. J.Derkosch, personal communication (1974) SL. 03A296