Document wq7qQ7y0ZgL467rj4db9z8E4

Mutation Reaeareh, 67 (1979) 173--182 S El*ovi#r/North-HoUand Biom*dical Prw MUTAGENICITY OF VINYL CHLORIDE, VTNYLIDENE CHLORIDE AND CHLOROPRENE IN V79 CHINESE HAMSTER CELLS C. DREVON and T. KUROK1 Unit of Chemical Careinogtntsu, International Agency for Research on Cancer, ISO court Albert Thoma*, 69372 Lyon CMex 2 (France) (Received 5 June 1978) (Revision received 2 January 1979) (Accepted 16 January 1979) Summary The mutagenicity of vinyl chloride, vinylidene chloride (1,1 -dichloroethylene) and chloroprene (2-chloro-l,3-butadiene) was tested in V79 Chinese hamster cells in the presence of a 15 000 Xg liver supernatant from ptaenobarbitone-pre-treated rats and mice. Mutations in terms of 8-azaguanine and ouabain resistance were induced in a dose-related fashion by exposure to vapour of vinyl chloride in the presence of liver supernatant from phenobarbitone-pre* treated rats. Vapours of vinylidene chloride and chloroprene induced a doserelated toxicity in the presence of liver supernatant from phenobarbitooe-pretreated rats, but these two compounds were not mutagenic in V79 Chinese hamster cells under the present assay conditions. The results are discussed with regard to the metabolic activation of the compounds and to the correlation with their carcinogenicity in man and experimental animals. Chlorinated hydrocarbons, such as VCM, VDC and chloroprene, are pro duced in large quantities and are widely used in the production of plastics and synthetic rubbers. There is unequivocal evidence that VCM is carcinogenic to man, as well as to a number of other animal species [15]. VDC, a structurally related compound and a copolymer of VCM, is carcinogenic to mice and rats, but no data are available on its carcinogenicity to man [ 15]. Chloroprene has so far not been found to be carcinogenic in mice and rats, although some human ftiat Iflrlrm- Dvpartmam of Cucu CC Ktmtrrh, lonituu of Modlnl Sci*nc. UnlwnltT at Tokyo, Skfcokanodai. MlnattHcu. Tokyo 101 (Jopon). ASOmfetton*.- AZA1, Uuiuuw tvsnooet: res. footal nit ono; OtfAr. otuMip rokfttnc*. SIS. 15 000 X , auponutut fraction; VCM, vinyl eblohdo (monomv); VDC, vinyllrfano ehlooai (l.l-dfefaloiootayiono). SL 066632 studies revealed an increased cancer incidence and a high incidence of chromo somal aberrations in peripheral lymphocytes of workers exposed to this sub stance [15]. All three of these chlorinated hydrocarbons have been reported to be mutagenic in microbial systems [1,5,6,10,11,22--24,26,28,30,31]. The adverse biological effects of these compounds, as of many other chemical car cinogens, are attributable to the formation of reactive metabolites by micro somal mixed-function oxidases. In recent years, several short-term tests using mammalian cells have been developed: of these, the mutagenicity test in V79 Chinese hamster cells has been relatively well evaluated, using a series of direct-acting carcinogens and a number of those requiring metabolic activation, e.g. N-nitrosamines [8,19,20], polycyclic aromatic hydrocarbons ([13,17] and unpublished) and aflatoxins ([17,20] and unpublished). In mutagenicity assays of some ester derivatives of W-hydroxy-2-aminofluorene [18] and of praziquantel, a new anti-schistosomal drug [3], this system was found to be useful, when incorporated into a battery of short-term tests, in increasing the predictive value of these tests for carcino genicity. We now report on the mutagenicity of VCM, VDC and chloroprene in V79 Chinese hamster cells in the presence of liver post-mitochondrial frac tion plus cofactors. The results are discussed in correlation with the carcino genicity of these substances in man and experimental animals. Materials and methods VCM (purity 99.9%) was generously provided by Rhone-Progil (Lyon, France); it was contaminated with ethanel (30 ppm), water (20 ppm), methyl chloride (<20 ppm) and non-volatile substances (<5 ppm). VDC, containing 0.3% 4-methoxyphenol as antioxidant, was obtained from Merck-Schuchardt (Darmstadt, Federal Republic of Germany). Chloroprene (2<hloro-l,3-butadiene; purity 99%) was provided by Distugil (Le Pont de Clair, Fiance); it was contaminated with 0.8% l-chioro-l,3-butadiene, crotylchloride at <200 ppm and 2-chlorobutene-l and 2-chlorobutene-2 at 80 ppm. 8-Azaguanine (Pfaltz and Bauer, Flushing, NY, UJS.A.), ouabain (Sigma Chemical Co., St. Louis, MO, U.S.A.) and agar (special agar Noble, Difco Lab., Detroit, MI, U.S.A.) were purchased. Autoclavable Eagle MEM was obtained from Flow Laboratories (Irvine, Ayrshire, Great Britain). FCS was purchased from Grand Island Biologi cal Co. (Grand Island, NY, U.S.A.). Mutagenicity in V79 Chinese hamster cells Mutations in terms of resistance to 8-azaguanine, a purine analogue, and to ouabain, a specific inhibitor of Na*/K*-activated ATPase in cell membranes, were recorded in V79 Chinese hamster cells. Experimental procedures were a modification of the assay system for nitrosamines reported previously [8,19]. V79 Chinese hsmster cells were plated at a concentration of 1.5 X 10* cells/ 60 mm petn dfch and were cultured overnight in Eagle MEM, supplemented with 10% FCS. They were then incubated at 37*C for 5 h or more in 2.5 ml of the reaction mixture, either in liquid suspension or suspended in 0.3% agar. The reaction mixture consisted of 0.75 ml SI5 post-mitochondrial fraction from the livers of BDVT male rats or OF-1 male mice, which had been treated with phenobarbitone (1 mg/i** Sdrensen phosphate buffe MgClj 6HjO per ml); t ymol glucose 6-phosph: MEM and 0.25 ml FCS. < vitamins from Eagle ME? ensure survival of the cr agar was added to modii. The petri dishes were the vapour of the chemii the desiccator, which b levels corresponding to 5 *-he pressure was adjust/ which are liquids at lov. volume of vapour (on th. desiccator. The desiccate min at 37*C to allow tl atmospheric pressure v foil, was then incubated nations of the compour mined previously, using period, the vapour was were washed twice acontrol cells were txei. chemicals or in reactit (glucose 6-phosphate ; reason, controls for wh plating efficiency as co those for which the SI 5 The cells were then ; mutagenicity induced. 60 mm petri dish (4 di genesis, 2 X 10* and 101 respectively (8 dishes frtion drugs were added 1 raM ouabain. The met. later; for the 8-ezaguani non-essential amino acs stained with Giemsa at frequency was calculate plating efficiency bein; confirmed by repeated , twice with chloroprene refer to the variability OUAT col nies was dei which were cultured _ used for selection [8,19 SL 066633 henobarbitone (1 mg/ml) in drinking water for 7 days; 0.75 ml modified drensen phosphate buffer (0.055 M, pH 7.4, containing 0.9% NaCl and 1.6 mg lgCl, - 6HsO per ml); and 0.75 ml phosphate-buffered saline containing 12.5 mol glucose 6-phosphate, 2 vmol NADP\ amino acids, vitamins from Eagle 4EM and 0.25 ml FCS. (Components of culture medium, such as amino acids, -jtamin* from Eagle MEM and FCS, were included in the reaction mixture to nsure survival of the cells for long incubation periods.) For agar suspensions, igar was added to modified Sprensen phosphate buffer at 1.0%. The petii dishes were then placed in a desiccator (10--15 1) and exposed to he vapour of the chemicals being tested. For VCM, the gas was introduced into he desiccator, which had been evacuated, with the aid of a manometer, at evels corresponding to 5, 10, 20 and 30% in volume; after 20--30 min at 37*C he pressure was adjusted to atmospheric levei. For VDC and chloroprene, vhich are liquids at low temperatures, the volume calculated to give a known /olume of vapour (on the basis of Avogadro's law) was frozen and placed in the iesiccator. The desiccator was partially evacuated and incubated for 20--30 min at 37*C to allow the compound to vaporize; air was then introduced until atmospheric pressure was obtained. The desiccator, covered with aluminium foil, was then incubated in a water-bath at 37*C for 5,10 or 15 h. The concen trations of the compounds in aqueous phase under these conditions were deter mined previously, using gas-liquid chromatography [6,24], After the exposure period, the vapour was removed under vacuum and replaced by air; the cells were washed twice and incubated for 2--3 h in fresh culture medium. The control cells were treated in the same way, either simply in the absence of the chemicals or in reaction mixture from which the SI 5 fraction or cofactors (giucose 6-phosphate and NADP*) had been omitted. For some unknown reason, controls for which cofactors were omitted repeatedly yielded a lower plating efficiency as compared with cells incubated in the complete mixture or those for which the S15 fraction was omitted (see Table 2). The cells were then plated for the determination of the cytotoxicity and mutagenicity induced. Cytotoxicity was determined by plating 100 cells/ 60 mm petri dish (4 dishes for each point) and cultured for 7 days. For muta genesis, 2 X 10* and 10* cells/60 mm dish were plated for A2A1 and OUA1, respectively (8 dishes for each point). After an expression period of 48 h, selec tion drugs were added to give final concentrations of 20 vg/ml 8-azaguanine or 1 mM ouabain. The media containing the drugs were changed once, 5--7 days later; for the 8-azaguanine medium, FCS was replaced by dialysed FCS and non-essential amino adds were added to 0.1 mM. The cultures were fixed and stained with Giemaa at 12 days for A2Ar and at 14 days for OUA1. Mutation frequency was calculated per 10* survivors, the number of cells plated and the plating efficiency being taken into account. Reprodudbility of the results was confirmed by repeated experiments; 5 times with VCM, 4 times with VDC and twice with chloroprene. Means and standard errors in the figures and tables refer to the variability in a single experiment. The stability of the A2A' and OUA1 colonies was demonstrated in 10 and 11 isolated colonies, respectively, which were cultured for more than one month in the absence of the drug used for selection [8,19]. 176 Results Mutagenicity of VCM To determine the optimal conditions for detecting the mutagenicity of these gaseous compounds, V79 cells were composed to 20% (v/v) VCM in air for 5, 10 or 15 h in the presence of liver SI 5 from phenobarbitone-pre-treated rats; the cells were suspended either in liquid (left column of Fig. 1) or in 0.3% agar (right column of Fig. 1). Agar suspension was used, since VCM has been reported to induce mutations in Salmonella typhimurium only when tested in the agar-incorporation assay and not in the liquid-suspension assay [5]. Postmitochondrial fractions from phenobarbitone-pre-treated rats or mice have been shown to increase VCM-induced mutagenicity in S. typhimurium over that obtained with fractions from untreated animals [5,24]. As seen in Fig. 1, the reaction mixture, in either suspension, was not by itself toxic to the V79 cells within 10-h incubation time; 15-h incubation in liquid LIQUID AGAR caused a decrease in pla agar suspension caused o mutagenicity when adde^ courses of mutation indi. in liquid incubation: witn were observed at 5 h and suspension, mutation fre that microsomal enzyir. Malaveille et ai. [25]. h number of resistant coioOUA', obtained at 5 h i Consequently, incubation the following experiment! VCM induced dose-re tions of between 5 and (v/v), mutation trequenc PU. 1. TVs* nmillTT of tb* induction of cytotoxicity and at mutation* by ponin to 20* <!) VCM la ob <> m reaction mixtun contaiaia* SIS ttvor traction (tom pb*nobwMtonw'pn-tnnted mti pbu otbotoa, in dlMr liquid mipindnn (l*ft column) or in 0.3* iri *u*p*n*ion (ztqbt column). Control* (O) won n--nil m tb* mm* way, but without VCM. Cytotoxicity woo d*lmiuin*d tram tb* plotlac Wakey and m ntnatd at p*m*at** of platin* cfficiaaey la control*. Mutation tnquancy i* aapnmid a* tb* numb** of r*n*tant eoloaio* pat 103 nirvtyon, takint into aosouat th* aumbor of call* plated and tb* platiaq tfflctency. Bad: SE. comcsntpatic yi*. 2. vCM-tadue*d cytotoxii ab (*, y/y). o, eompteu raact-. tat* ph` cofaetoim. o, nactiot ttpinnd u d**ebb*d ia tb* lo* SL 066635 177 caused a decrease in plating efficiency of about 50%; and 15*h incubation in agar suspension caused only slight toxicity. VCM induced cytotoxicity and mutagenicity when added in liquid suspension or in agar suspension. The time courses of mutation induction were much more gradual in agar suspension than in liquid incubation: with liquid incubation, the highest mutation frequencies were observed at 5 h and were followed by a sharp decline; whereas with agar suspension, mutation frequencies reached almost a plateau after 5 h, suggesting that microsomal enzymes are stabilized in this suspension, as reported by Malaveille et al. [25]. In both the agar and liquid incubations, the highest number of resistant colonies per 10* survivors was about 30 for A2A' and 3 for OUA', obtained at 5 h in liquid incubation and at 10--15 h in agar incubation. Consequently, incubation for 5 h in a liquid reaction mixture was chosen for the following experiments. VCM induced dose-related mutations and toxicity when given at concentra tions of between 5 and 30% (v/v) in air (Fig. 2). With a concentration of 30% (v/v), mutation frequencies were 10--20 times that of the spontaneous back- COMCZNTIIATION OF VCM (% in air] FI*. 1. vCMdadueod cytotoxicity end aut**xaieity is V7* edit h * function of concentration o( VCM in air (%, vfv). , complete mention nixtun contemn* SIS liver fraction from pbenoPerbitoa^pix-neaiad reti phu cotecum, o, mectlon mixture .-`-'-t no SIX Cytotoxicity end nutation frequency ere txpnned ae deeciibed in tile legend to FI*. 1. SL 178 ground: 51 AZAr and 4 OUA1 colonies were obtained per 101 survivors. Neither toxicity nor mutation was induced when the SI 5 fraction was omitted from the reaction mixture (Fig. 2). Mutagenicity of VDC The mutagenicity of VDC in V79 Chinese hamster cells was tested by exposing them to 2.0 and 10.0% of the vapour in air for 5 h in reaction mixture containing SI5 fraction of liver from phenobarbitone-pre-treated rats and mice. SI 5 fraction from mice was used because VDC was mutagenic in S. typhimurium only when tested with mouse*liver homogenate and was almost inactive when tested with liver homogenate from rats [6], although VDC has been shown to be carcinogenic in both species [15]. These results axe summarized in Table 1. A dose-related toxicity was induced when cells were incubated in reaction mixture containing S15 fraction from rat liver plus cofactors, but VDC was not toxic with the SI 5 fraction from mouse liver*, this suggests that a toxic metabolite(s) was formed by rat microsomal.enzymes. However, no increase in the appearance of AZAr and OUA1 colonies was observed after incubation in the presence of liver SI 5 fraction from either rats or mice. / C/ / f S I Mutagenicity of chloroprene V79 cells were exposed to 0.2,1.0, 2.0 and 10% (v/v) chloroprene vapour in air for 5 h in reaction mixture containing SI 5 fraction of livers from phenobaxtoitone-pre-treated rats, plus cofactors. The results are summarized in Table 2. Chloroprene was toxic to the cells, in the absence of a metabolic activation system, at concentrations of more than 1%. This toxic effect could be a direct action of chloroprene or that of one of its enzymic (from the V79 cells) or non-enzymic breakdown products. An enhanced toxic response was observed when the cells were incubated with SI5 fraction plus cofactors; this suggests TABLE 1 CYTOTOXICITY AND MUTAGENICITY OF VDC IN V79 CHINESE HAMSTER CELLS IN THE PRES ENCE OF POST-MITOCHONDRIAL FRACTION (SIS) OF LIVER FROM PHENOBARBITONE-PRETREATED RATS AND MICE Concentration in air <%) Incubation Cytotoxicity * Mutagenicity b A2Ar OUA* 0 2.0 10.0 0 2.0 10.0 0 aja 10.0 ' compute* with rmt S15 complete, wllb mourn SI $ minua SIS 63.0 1S.3 2.3 S8.3 61.3 64.4 6X8 49. S 60.3 4.5 0.4 4,2 0 00 6,4 0.4 3.1 0 3.9 0.2 1.0 0.4 3.6 0.3 2.1 0.6 * Eapmaaod aa plating elBaency: the percentage of Ml)* that forma* colonial. b Mutation frequency la imnatd u tha number of mutant colon!n per 10s nintvon, the number of eaili Plata* and plating efficiency bdn| talnn into account. table: CYTOTOXICITY AND MUTAGE: Of THE PRESENCE OF POST-Mf BITONE-PRX-TREATED RATS Concentndos In air <%) Incubation 0 complete 0.2 1.0 2.0 10.0 Q 0.2 1.0 2.0 10.0 twirme cola- a The tacuKt of two independent Expt. I and at 0. 2.0 end 10-0 etmtue value. wart obtained in 6 Expnaaad u plating efficiency: t * Mutation frequency la eapreaaed cdll platad and Pleant elflaen enzymic formation of a mutations,-in terms of A'7 presence or absence of m Discussion Table 3 summarizes chloroprene in man and graphs [15], and juxtape* and, from the present st been shown to be carcu. produced tumours at diffe tested by the oral rout TABLE 3 SUMMARY OF DATA ON CAl BOPRENE Compound Evidence o c*remo(*n VCM VDC Chloroprene Man ? a CatcinognlMty data art taken ti SL 066637 r* >-.3 ..-ftp -* 179 ABLE 2 YT0T0X1CJTY AND MUTAGENICITY OP CHLOROPRENE IN V79 CHINESE HAMSTER CELLS N THE PRESENCE OP POST-MITOCHONDRIAL FRACTION (SIS) OF LIVER PROM FHENOBaR:;TONI*PRI-TREATED RATS * ^aeamnaea & air (%) Incubation Cytotoxicity b Muuiraitdty 5 A2Ar OUA' 0 0.2 1.0 2.0 10.0 compliu 64.S 28.8 0 0 0 2.9 0.8 2.8 1.4 00 00 00 0 0.2 1.0 2.0 10.0 minua co factor! 31.3 23.8 9.0 U 0 2-1 0.8 T.9 1.0 7.1 0 00 00 1 Tb multi of two utdtpindrat uptnisiou ut eembinid: edloroprra* * t**td it 0, 0-2 and 1.0% u> Expt. I tod it 0, 2.0 tad 10.0% In Expt. n. Viluu lot control! (0%) in ultra traa Expt. L tltfeouch limilir vtluM won obuinid In Expt. II. 6 ExpmMd plxtlns tfldanr: thi ptremtM* < rail* tbit formed colonin. c MuttOon tmjumcr u txpraud u to* aumbtt at rwUust soloiua pn 10s nuvtvoa, the aumbtt of e*Ui pltud ind pittint ffleiincr Peut ttkto into locount. enzymic formation of a toxic metabolite(s) from chloroprene. However, no mutations, in terms of AZAr and OUAr, were induced by this compound in the presence or absence of metabolic activation systems. Discussion Table 3 summarizes the data on the carcinogenicity of VCM, VDC and chloroprene in man and experimental animals, taken from the LARC Mono graphs [15], and juxtaposes these with data on mutagenicity in S. typhimurium and, from the present study, with that in V79 Chinese hamster cells. VCM has been shown to be carcinogenic in man as well as in experimental animals: it produced tumours at different sites, including angiosarcomas of the liver, when tested by the oral route or inhalation exposure in rats, mice and hamsters. TABLE 3 SUMMARY OP DATA ON CARCINOGENICITY AND MUTAGENICITY OF VCM. VDC AND CHLO ROPRENE Compound Svidanct of fiormaofonltdtr 4 in Eridraea at auUcralMty in Mill Animal* S. typhimurium V79 VCM VDC Chkuopira* ? -- + A y * -- ** * Cirunoimicity diu in Ultra from IARC Monofliph VoL 19 (151. $* wxt tat ddtail*. SL 066638 180 Epidemiological studies have shown that exposure to VCM results in an increased carcinogenic risk to man, involving liver, brain, lung and the lymphato-haemapoietic system. The data in experimental animals, also indicative of a carcinogenic effect of VDC, are inadequate to allow an evaluation of the carcinogenicity of VDC or chloroprene. Occupational exposures to chloroprene have, however, been reported to be associated with a variety of toxicological effects, including chromosomal aberrations among exposed workers. In one study, an excess of lung and skin cancers was related to occupational exposure to chloroprene. These observations raise the possibility that chloroprene is a human carcinogen. The present study confirms previous results on the mutagenicity of VCM in S. typhimurium [1,5,10,24,26,28], Escherichia coli K12 [11], Schizosaccharomyces pombc [22], Saceharomyces cerevisiae [22] and Drosophila melanogaster [23,30]. This subject has been reviewed by Bartsch and Montesano [7], Bartsch et al. [4], Fishbein [9] and the IARC Monograph [15], Recently, Styles [29] reported VCM-induced transformation in BHK-21 cells in the presence of a microsome preparation. Although VCM induced mutations in S. typhimurium in the absence of a metabolic activation system, a much higher mutagenic response was observed when a 9000 x g supernatant from rat liver was added [1,5,26]. Pre-treatment of rats with phenobarbitone increased the mutation rate over that with a fraction from untreated rats [5,24], Mutations have also been observed in the presence of liver homogenates from mice [5,10, 22,24] and from human biopsies [5,24] as well as in the host-mediated assay in mice [22]. These results, together with those from the present study, indicate enzymic formation of a mutagenic metabolite(s) from VCM. Among the possible metabolites that have been tested, chioroethylene oxide, an obligatory epoxide in metabolism by microsomal mixed-function oxidases, was the strongest mutagen in V79 Chinese hamster cells [12] as well as in microbial assays [14,21,24,27]; chloroacetaidehyde was mutagenic [5,12,21,24, 26,27], but chloroacetic acid was not mutagenic, in such assays [5,12,24,26, 27]. The above data strongly suggest that chioroethylene oxide is die metabo lite of VCM that is principally responsible for the various advene biological effects of the parent compound. VDC and chloroprene may follow a metabolic pathway similar to that of VCM [2,16]. VDC and chloroprene have been shown to be mutagenic in microbial assays: VDC produced reverse mutations in S. typhimurium and in E. coli in the pres ence of a metabolic activation system [6,111 ^chloroprene induced reverse muta tions in hi. typhimurium in the absence of metabolic activation, but an increased mutagenic response was observed when liver homogenate from mice was added to such assays [6], In Drosophila, recessive lethal mutations were induced by feeding male flies with chloroprene [31]. In V79 Chinese hamster cells1 how ever, these two chemicals induced a dose-related_toxicity_in the, presence of liver 615 fraction from phenobarbitone-pre-treated rats but were not mutagenic within the constraints of the present. ay. conditions. This suggests that there is at least enzymic formation of toxii tabolites from VDC and chloroprer by microsomal enzymes. Acknowledgements The authors thank Mx with gaseous compounds for editorial assistance; the critical reading of t NCI Contract 1CP-55630 References I Andrews. A.W., E.S. ZawUto vinyl eWorld* end mathyl cl 3 Baibin. A., H. BrdsL A. C miarosoma-medlated forma. Biopan. Cemntu.. *7 ( 3 Baruch. H.. T. Kuroki, C. RiCnMl E. Vocal and A. ctaistoeomsl drut la bactr 143. 4 Baruch. H., C. Malavallla. . maMhoUsn of vinyl eWorld 5 Banach. H., C. MaJaveill* a vinyl ahiorida in 5. typhimi S Banarh. H-. C. Mnlaveille, R- ehloride and 3<hlorobudltt T Baruch. H., and R. Montei 33 (1975) 93-114. 3 Dmvon. C.. T. Kuroki and . Una hr varioua ehamieaU, . Toxleoloiy. Elsevier/North-1' 9 rubbain. L.. tnduatrial mu tion Res.. 33 (1974) 3*7-1 10 Gam. AJ. J.B. Guttenpiar axtracts and feat radicals. Mu II Grata. H.. G. Booaa. A. R. carcinoftnictty of chlorim Pharmacol.. 34 (1975) 301. 13 Hubarman. E,, H. Baruch ar vinyl chloride metabolites, ch 939--944. 13 Hubannan. E.. and L. Sac (ana. Int. J. Cancer. 13 (19 '. - 14 Huaaain. S.. and S. Ostanin matsbolltea. Chem.-Riol. In- 15 International Afency for , lanic of Chemicals to Lyon.1979. 1* Jonas. B.K.. and D.E. Hath* act.. 20 (1979) 27-41. 17 Knhn. D.F., and C. Heidi calls by polycvcui ivd.-- 19 Kuroki. T.. an-: Quoranc In V"' 19 Kuroki. T.. C. Drx- calls by varioua nitron. 30 Linftnbaefa, R-. HJ, f . with liver caietnofens. Proi 31 Loptiano. N., R. Barala. S. Nlexi. C. Lepodni. 0. Rosa vinyl ehlorida maubolitss t SL 066639 181 Ackn wledgements The authors thank Mr. C. Maiaveille for his guidance concerning treatment with gaseous compounds; Miss J, Mitchell for secretarial aid; Mrs. E. Heseltine for editorial assistance; and Drs. L. Tomatis, R. Montesano and H. Bartsch for the critical reading of this manuscript. This work was supported in part by US NCI Contract 1CP-55630 and CEC contract No. 190-77-1 ENVF. References 1 Andrawa, A.W,, E.S..2*wi*towki and C.R. Valaotina. A comKin of tha mutanic propartia* of vinyl chloride tod methyl cblorid*. Mutation Rat.. 40 (1978) 273--276. 2 Barbia, A.. H. Brdtil, A. Ctoicr. 9. Jaequipnoa, C. MalawilU. R. Mont--no and H. Bartach. Liver microaoma-madiatad formation of alkylatiap aaaota from vinyl bromide u4 vinyl ehlorida. Uwkna. Btophya. Ra. Comaun.. 67 (1975) $96--603. 3 Baruch. H.. T. KunU. C. MalavtiUa. N. Lopriano. R. Bamla, A. Ahboodandolo. S. Boaatti. C. Raktaldl. E. Votnl and A. Davit. Abaanoa of mutapamcity of praiiquantaL a new, afraotiva antiahiatoaonal drur in bacteria. yaaata, Inaaeta and aaaouliaa calk. Mutation Raa $1 (1978) 133-- 142. 4 Baruch. H_ C. Malavaflla. A. Barbia. K. BrfeU. L. Tomatia and R. Montaaaao. Mutafanialty aad mataboliam of vinyl ahloiida aad ralatad compound*. Environ. Haaitb Pvtvp.. 17 (1976) 193--196. $ Banach, R-. C. Malavadlla aad R. Monfaauio. Human, rat and mourn livtr madiatad mutapanidty of vinyl ahloiido in S. typhimurtum tttaiaa. 1st. 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EU*vir/North*Hallind I Short Communication CHEMICALS WHICH ft typhtmurium TESTER -c RICHARD H. McKEE, JAM Litron Laboratory, Ltd., (Received 8 August 1978) (Revieion received 16 Jenua. (Accepted 2 February 197 Previous uses of ary1 their photosensitive p been synthesized for su 4-fluoro-3-nitrophenyl ; inactivation of enzym amino acid transport azides can also photost the mechanism of ba (DNPA) were tested test system. The Amt. bacteria which contain the mutations carried frameshift mutations, tagenic event mediated chemicals have been 5" which induce raversio: Ames strains could prov experiments within the ferent laboratories. V DNPA induce the revv strains (TA98, TA100, Materials and method! The Salmonella typr tained from Dr. Bruce testing with these stzs for plate-incorporaticcedure is that Vogel--i concentration as in ti. SL 066641