Document O1wjkNXGmXpexw654eM36q1JX

\ /nt. J. Cancer: 15, 429-437 (1975) HtpilnitJ hoiti the Jnifthttih'nul Jmit/hil >*/ Cuntrr (g) tnlcmnliimul Union At^iutl Cancer ; ,j<Hcl in Switzerland___________ HUMAN, RAT AND MOUSE LIVER-MEDIATED MUTAGENICITY OF VINYL CHLORIDE IN S. TYPHIMUIUUM STRAINS International Agency for Research on CafiCcr, Unit of Chemical Carcinogenesis, ISO Cours Albert Thomas, 69008 Lyons, France ' Exposure ofS. typhimurium strains TA 1580, TA 1535 and G-46 to vinyl chloride increased the number of J/is+ rcverianislplate 16, 12 or 5 times over the spontaneous mutation rate. After 6 h of exposure to vinyl chloride, the mutagenic response for TA 1530 strain was enhanced 7-, 4- or 5-fold when fortified postmitochondrUd liver fractions from humans, rats or mice were added. The enzyme-mediated vinyl chloride mutagenicity was dependent on an hiA DRH generating system and the enzyme activity was localized in a liver microsomalfraction: 9,000 'Ay, liver supernatant was three times more active than microtomes, while liver cytosol or alcohol dehydrogenase did not affect the mutagenicity. Phcnobarbitone pretreatmenl of rats and mice increased the mutagenic response by up to 15-40% as compared to untreated controls. The relative mutagenic activities of VCM, taking the value from mouse liver as WO, for TA 1530 strain mediated by 9,000 X g tissue fractions were: rat liver, 80; mouse and rat kidney, 20 `""Z. v.* ttv*r ttrnm tour tuoD.iv specimensJ, 170, 64, 70 and 46. Chhroacetahbhydc and ch/oroacefic acid, a urinary metabolite of VCM, showed toxic effects, while chloroelhanof was weakly mutagenic for TA 1530 strain. Vinyf chloride monomer (VCM) is extensively used for the production of polyvinyl chloride and other plastic material, and also as a propellant for aerosols. Carcinogenicity in rats, when exposed to 30,000 ppm VCM in air, was first reported by Viola vt af, (1971). Subsequently, Malloni and Lcfcminc (1974) showed that angiosarcomas of the liver, as well as other tumours, were induced in rnts-aml mice exposed to various doses of VCM by inhalation. Recently, eases of angiosarcoma of the liver have been found in workers exposed to VCM during the polymcrizalion process and a causal relationship between VCM exposure and this type of tumour in man has been established (Creech and Johnson, 1974; Heath et at., 1974; Lee and Harry, 1974; I ARC, 1974). The systemic action of (his carcinogen and its low chemical reactivity suggest that the biological effects of VCM arc dependent upon its metabolic activation. In these studies, we report the mutagenicity of VCM and its presumed metabolites in S. lyphimurium strains, mediated by liver and other tissue fractions of rat, mouse and human origin, mateuial and methods . .. ennea s VCM (purity 99.9%) was generously provided by Rhonc-Progil, Lyons, France, contaminated wilh ethanol (30 ppm), water (20 ppm), riiclhylchloride (<20 ppm) and non-volatile substances Received: October 16, 1974, 429 t 0 9 ^6 1 "W ft * Zj'-'t .r . .* i .'I ,. r. M VI -! ?; ~ r ' .*/*'' i*r'1 /* f.-"ft, f- '"v >*' .*Vi f? fi-'h <- V /'r *'"' r\ i, u t! v. >! I; o e -.r-j l '`o H h i! k. y i.( v.-' : - h * ; -rO\ H f; an * " i *>> i. /*>, **.-*<> ,. -, -:<**. i. i' !i v.-v i! .! w.i . /"'iill LL^'I tili .,- kU*1(I i! *> b,t! V;/CU Vi.1;H il f* r ' ^ .7/ ' "i nnyy 1lJi RRaannnnuugg,, AA JJoohhatwnsson, C Rtwnel mil C A Wr.rhlrrioisler, **ia*w*^ t jo >'*> j nnvuuiir.-ionlal-To^xilcooltOQy Unit, Wallenberg Laboratory, University ol Stockholm, f '[., 1 J fMC`4 05 Stockholm, 8{Sweden AWBIO Vol. 3, No, 5, pp 194-197 (1974). ;* % '. j57 ><- ;v Vinyl chlorine fcns recently been shown to cause a mabruicnt liver tumor direct cfircinccenic compounds. In order dicense In mr.rt otter pcrtipnb'aivrl nsrtefurc in PVO n'ants. Thin aciiMli'os the to overcome that problem, combined I problem ol whether such hazards uonkl bo avomort or at Icar.l riiniinAhort in 1 lie* lest methods have been worked out v i:h I future by n screenInp lor mtitnn-'.rvcily cf chcrolcelr. iiseit in industricr. 'lilt? tnsplr. lor cuch ;i tciecinnn j-rs:.r-di:r" Is th.? tiere ncfftl.ntion t'e'wyon microor-ariis-ms and nmmiiiHls. 'ii"- rr,ieroorjT.nv.r.rs are used for die aeNrd c.f.rcip.oacnic and nv.knrin'iu Uk-niii of clicminsfc. 'Jipcnrr.onts v/illt Cd/nnnvUn niiitapenicity Irstiny, hut the.1 ac* ex L'ncteiin thov.vci tiinl th'' r.nreuioricnic hazard cf vii.l chlmide could have been posed to those metabolites u: the com trev.-d by means e! tmilcr.'Onicify tnsls. The data Indicate tiiat vinyl chloride is - not motr-genlc per &o l>ut t-ecomcc mutn^entc attor n metabolic cctivniion in the flyer. pound which arc formed in :-a nimet mammal thost mediated assay} (13' or in niftmniclijn liver microsomal syli::r.s i>: vitro (17, 14). Vinyi chloride has artrr.cicd a con-idcr* chls emovtni of public anoniion LccitiLsc of the rcccnl discovery of its carcir.Oi'cnic makes use of the close correlation be tween the carcinogenic and mutagenic properties of chemicals 14). This corre The present investigation of the gen etic effect of vinyl chloride Ha.v been performed on Suhnonctia typi.imuriut.i. action in nan. In several countries cases lation. its do other data, points to the The nuitaycnicity of the compoun'i nv of the rare maliiuiant cmriosar- fact that cliiinp.es in DMA arc ut leust n.*i unaly/cd by means of reverse mutaiiojis corna of the iiwn have l.-.Ln reported essential ratite of Crtncnl' indt'Cliort. Tl; in the histidine locus. The..effect-of tuviCDR vori.eiR who have been exposed testing for mutagenicity is much cheaper tnei.ibolic scfivatioti wax studied by `:til*o- to viiij'i chloride in plastic itutiiMrie* OJ. M*h(;ni has fmthsrtnori: sqMMcfl on ina.'rasr of .ii_v,ioj.irc<ji:ia in rats af'er and far less time eonMiniia. .mu funl er- morc, Hu? results can in many cases be interpreted in mnlcculiw wok ing rat liwr inicrcsoutal sy-it.-ms td rt:e b.tcrcrial cultures accordmi* to She metiiOfl x.-nrl.'il r>.' l-y _*-' ( ! 1) ' t, Jio nnm vri.vl riro>iS^ Of l,r?ct rcit-lis iiida.:circiiiO".;nic effects it rtiil tower c'orcs (3). The hazard ol vinyl chlorice primarily concerns workers in the piastre industries. v. !;o pel a particnSarly Jiiyh stspusure in difr'crcrl can- *:"'** *r !ke chr.n^;i o:v-;ri..r, ... 7?^*. This obviously is of importnrec fur m belter iinderstandinj of the h'otn^ic.il action of suspect compounds. One major obstacle in u-.ir.j muta genicity tests, with iTiicroory-misms for T'.v ohjstiira vi me present invcsiit:;*tion her; primarily been to ur- vinyl chloride us an example of a cornr^und cjiteinogc-iMC to mcr, in order in siudv the cppltceciliiy of n.ui.-.cenici'v u's'.irn' for the detection of carcinogen* in the rertrens, but thi cccupationnl health ' frcckm i.'.sv hsvc rani.tications for the y fcsncrat public. N'inyl chlorirk* hrs. for insttnee, been u.cd as a propellant for hi'.ir sprnys and rcsticities. Mcncnvcr. the pylyrnrri7.tr! ptchi'-ct of vinyl cUUmuIl-, PVC pbstic. is tvM .til over the world far a. variety of product:-, antcas other tilings for t!ic v ir.ypinr, of varimw fondstuffs. It seem' inevitable that list pop- preliminary screening of cav.ir.ovcnic effects is the fact that many compounds behave as carcinogens only after an ac tivation in tbs mammalian body. This activation, or in other casts deactivation, of a foreign compound, is principally performed by the liver and involves oxi dation, reduction, hydrolysis and con jugation. A Witte variety ot oxidative re lictions cjf C- and N-oxyjv.n.ition, O*. human environment. We arc likely 10 be confronted wi'h this kind of problem at nil accetcraied rate in the future and the necessity cf fituling r. Ncrsible solu tion to the screening of carcinogenic hazards ol chemicals is imminent. Considerif.;' tiie vinyl chloride probletn. one could )*>. ilits fnllrnxmj ti-.iev.mTiL Had this genetic t.-st method been nvaiTuidu and been uv:d lot screening cf vr-tyl t Ofation Is expired to ilre r.mnoiii'.r via N-, and S-dcidl.vlatton and epnxidatinn chloride wh .-:i il was introduced for in sttclt sources, altltocp'i the doses aic most are mediated by the drug mctr-.lioli/.lnp. dustrial production, wculi! such a screen likely wnaU. system located in the membranes nt the ing have fiver* a wunbiv, of the cv.rcmo- Tice carcinoi'cnic effects of vinyl chlo cndnplasr.i.V.ie tcticulnm of the liver nvnicity of 'his compound? ride focus attention ci; the car;*.l and cells i5, <i, 7, S). This mixed-function fundarnenta! pr-d>U'ni of how such ha?.- oxygenase mechanism can utilize reduced MATERIALS AND .METHODS . 'ardi tan be avoided or at host diminish nicptinainid? adenine dir.uclrotijc phos l:otr hisl`dinc-r Jiurinr. Mt.iins of Snlrno* ed in die future---and above: al! how cr.r- phate (NADI1!!) as a.n electron donor ttvllti tyi>li:i''u'i'iri (VA !,'.*5. TA J.'.iS, C>'.t9f,i*nic. chcniicak surh ns viovl 'h!o- There reactions alto t;>he place in TA 1537. m.u T A l5aS'i -*cve i;-co I-.t tk'^s Gem be sputwd %pcTwv-cT>taliy irt the vitif utter the addition of N'/D'dl-itcn- this investi t.itirn. These strains have he:n first place. Consirlrtin!*. ihu ruimhcr of chemicals in iniJii'.iri-i. it i*. hardly realistic to rt !y on c-.nccr mduc- ricn tests on i:sico viih LiiiM.ijrcfr.cnt of ur.fif JiC^\Kn:y; \-nii 1cm\ iai.e :i ie?*. two years. The only fcasihiv pro-;-du*c i:i present seems to hr a sysic-o which enitinp system to nticm-.oT.il <10, 11, l?.j. Aidn'Ui'h mici'O.srjtanistns are paiticularly suitabii- f.orn a pruvltcsl pen-tic pom; r.f view for nnriat-.citcitv `Csline, t.'iey do no: perutr.n jho same metahtilic activation as mnmmafs and they will tlictciore not pici; up Mich in slescri'n.:tl detail hy Arnes et i:i f3 5>. Tii.v :.r< u-.-.il in r bnck-r.iutation systern. whao rcsei>.;Oii to lu-.tul-ir; independ ence occurs CaM' 1.V ft i.-vu'il .} huse-pair M-hslttuti;'.: i l'A 1J3M or L-itc-p-.a a.lUiliOLi s.r iMction (TA I5 3*j--1 ,\ 15'*). i'ulin'ui v.'i'jir pruwr. ovcrnii;ni m cum- 194 zwntA vn i wo s URL 19481 7T URL 19462 _|>!ctc medium (Difco, Antibiotic medium 3). Platings with the toft ajar technique v/crc made on complete ma!i"tn tCA plates) and on minimal medium (MA plates) described by Vogel and Bonner (16) with supplements according to Ames (17) . The soft agar coiwiMed of 0.9 per cent NaCl and 0.0 percent agar. Undiluted bacteria (U.l ml) were added to soft agar (2 ml) and poured onto MA plates and 0.1 ml of appropriate dilutions correspondingly onto CA plates. Dilu tions were made in 0.9 percent NaCI. Colonies on MA plates were counted After two days' incubation at 37 - C, giv ing the number of mutants. Thu number of viable cells (surviving cells) were counted on CA plates after one day's -incubation. Male rats (Spraguc-Dawlcy. Anticimex) maintained on normal diet were starved 16--20 hours before they were sacrificed by decapitation and the liver was homogenized. The homogenate was centrifuged at 9000 Xg for ten minutes. The supernatant containing (he micro tomes was mixed with a NADI'II-pcncrating system consisting of NADP, glucose-6-phosphatc. phosphate buffer (pH 7.4), MgCl3 and KCl in the pro portions given by Ames ei nl (14). When microsomal systems arc mentioned in the text below or in the cables it includes the 9000Xg supernatant containing the mierosomes and the NADPH-penernting system. The negative (iniirol without NAD? is Aerefore designated "micro- - N AriP" }.mninnnnfhrri. CttlftC Vd> Uicv u> T!;v compound induces muiatlons after mi crosomal activation in all the strains used (14). The vinyl chloride used (AB Aerosol Packing Co, Vallemuna. Sweden) has been snzlyicd with gas clirpmatc'g-apliy end mass spectrometry for impurities fcy S Jensen (IE). The purity was very iigh and only trace amounts of isoproprt- nol were found. When nothing else is stated the procedure of Ames ct vl (14) was followed in the experiments. Tires different types of treatment with vinyl chloride were used in preliminary experiments v/ith TA 1535. A water solution of vinyl chloride was prepared by bubbling vinyl chloride gas through eater in a test tube for a couple of min utes. This solution was added In the soft egar together with the microsomal system and bacutia befote pterim;. An cttetr.pt was also made to dissolve vinyl chloride directly in the microsomal sus- .. pension "by bubbling it through tor half ft miaule prior, te the addition to soft egar fit the third procedure the bacrcna were first plated together with the mi crosomal system end then treated 75 miniPcs in a vinyl chloride-containing fitrccnphcrefll percent v/v) at room tem perature (23 C). This last procedure was adopted in the following main experi ments and will therefore be described in some detail. After plaiinc of the bacieria" with and whithnm microsomal systems the petri dishes were placed in a closed vessel (standard vacuum desiccator, volume It) iV equipped whh two tcilon tubes for inlet and outlet of gas. The heavy vinyl chloride gas was pressed into the glass vessel via one of the ;nhcs which ended near (he bottom. lh:s ndd:* tion caused an expulsion through the other lube (at the top) of a corresponding volume of air, which could he measured. The atmospheric concentration of vinyl chloride measured in this way was during the main experiments 20 percent. When all vinyl chloride had been added, the tubes were closed and a magnetic stirrer was started to ensure homoge neous distribution of the gas. Three different times of exposure to the gas were used. 3D, 60 and 90 minutes. Two experiments with TA 1535 were made in this way. An experiment with all four strains TA 1535--TA 153S was finally performed with a treatment time of 90 minutes. given directly to the bacteria with gase ous vinyl chloride bru.es about a higher exposure tl-'sc than a tuutmcnt with vi nyl chloride previously dissolved in water or in the liver microsomal sus pension. In another set of experiments (Table 2 and Figure 1), the treatment was given with 20 percent gaseous vinyl chloride. The rcsulrs were in gcud accordance with the previous ones, that is, a significant increase of mutants was obtained with liver microsomal systems. Without liver Figure 1. TA 1S3S (base substitution) ircatod In an atmetphoie of air containing 20 percent (v/v) vinyl chloride (VC), Experiment t in Table 3. RESULTS The results of the preliminary experi ments with TA 1535 and liver micro* somes is presented in Table 1, As can be seen, no mutagenic effect could be traced after the treatment with vinyl chloride dissolved in water or in the microsomal suspension. After sn exposure to 11 per cent vinyl chlonde gas, on (he other hand, there was a significant increase of It hniilH hp nnintnrf mil that the survival of the bacteria was not affected in any detectable way by the treatment and therefore the results cannot be due to a selective survival. The fact that only the treatment with vinyl chlo ride gas had any effect on the mutation tats indicates that a continuous treatment - VINYL CHLORIDE MICflOSOMES - CONTROL MlCnOSOVtS * Toble l; -Tho-etfecl en-TA IMS^ba; ab:U!ut!on) alter different type* ol treatment with vinyl chloride (VC) at the presence or liver microtome*. Series 1-2 an<J 3-5 respective)*- r/ero per formed tmuiiHneously. Sls'itticol analyse* vnth t-test. Signiiicnnco level*: 0.01 <p<0.0S; * 0.00t<p<0.01: p<0.CC1. Scries Type of treatment Number of . viable cells plite XI0'# Mean value of 7. plates Number of mutants per plate Mean value of 5 plates Number of mutants per 10 surviving cells iSE t VC dissolved in wafer 2 Control 3 VC dissolved in microsomal suspension 4.1 4 11 " VC in atmosphere 4.5 5 Control 4.3 19.2 18.0 27.2 78 2 23.0 6.60,4 I7.6i2.6** 5.3 0.2 195 Tr.l'fr ?. ir, 1T:5 `i't* tr.-r!-i In cn atmosphere rl rtr ccntjtnl.vg ?C prrcen; (v/vj r,,-i[mi5 vinyl r.iu-uij* (V^l. 7h-> tllt-tJ :.(MS'rco M munbci -it nii:'.j<fi'.* per 1C1 Servian;} ee!h. Ptatlstical .vri.vtjsiv, Tr*?,i t. F.xpj:n Mi-.NTI F.m:RlMl-NT u Type of treatment Number-of Number of viable cells mutants, per pblc Mean value XIO'* of 5 plates Mein value of 3 plates. Numho af mutants per 10'*M,nmriR fells-.S E Number of liable cells per plate xio'* Mean value Of 3 rlatcs. Number of mutants. Mean value cf 5 glutts Number of mutants per 10 survSring cells-S E - Tims of treatment (minute:-) VC-h microsomal system VC CtmtrcNf microsomal system VC-t microsomal system VC Control-J- microsomal system VC+ microsomal system VC Control-)-microsomal system 4.2 3.1 4.1 4.1 3.4 4.1 4.0 3.7 4.1 69.2 19.2 45.0 72.2 I P.4 39.4 115(8 22.8 37.4 16.3d: 2.0 6.2 0.7 10.9t,| I7.81.3r4* 5.40.8 9.60.4 29.01.J"* 7J1.0 9.0 1.0 3.8 2.0 3.7 3.9 2.3 3.9 4.2 3.6 3.9 49.8 10.6 2S.0 51.6 IS.fi 20.4 77.2 22.6 !3,2I.O" S,40.9 6.7 0.9 13,4J.O** 8.1 0.6 5.20.7 IS.40.9***` .20.9 -- 30 69 90 1 The number cf mutants from "Control + microsomal system" in this scries had to be excluded because or infection. I towever. lucre is a qood agreement between the control scries within both cxpcrmvnts I ami II, and therefore (he comparison of the series with St) minutes treatment has been made versus the pooled controls for 3Q and 60 minutes of experiment II. URL 19463 microtome!., vinyl chloride dirt not ex* Kibh any mutagenic cf;cn. Concerning uid auntoci ui ti:c cm . .is i.l law pi ifvious expep'rnent there v-.i', r.n s':gr. of a toxic effect by vinyl chloride together with liveT microsomal systems. It may be pointed out, however, -that the data in dicate seme toxic effect on the bacteria by vinyl chloride itself without liver microsomes. In order to elucidate the activation of vinyl chloride by the liver microjntncs. a series without NADP was included in this experiment. The remits, presented scpr.fately'iit'Table 3 together with the relevant data from Table 2, show that a mutagenic effect was only obtained with the liver microsomal svstem and HAD?, but not. without NADP. It should be mentioned that bucauvt of lack of ipncc in the container used for the treatment, the series me.ivurinn the survival of the bacteria had to be omitted for the treatment with the liver micro-jomal system without NADP. However, *Cd the basis o: the survival rale measured to the other expertrrertfal series, it can be concluded that tle number of mutants per plate should give :: suffichutly accu rate estimation of the mutation rate. In particular, the similarity of mnlniton fre quencies between the series with liver ffucrosmnal system* without NADP a\ compared to the ter.es with only vinyl chloride :i indicative for the inter pretation of the mechanism cf activaof vinyl chloride. As mentioned aoovc. this tact: ot mutagenic etreet v. iih'tut NADP iiiciie.ttcs i?ut in the test system used, cn active metabolism by the r.iicrosomcs is required for the muta genic effect. An experiment with nil four strains TA 1535--TA 153S was finally perform ed, in order to threw some light on ti>v molecular mechanism behind the mu tagenic effect of vinyl chloride. The re sults arc shown in Table 4. As in the previous experiments n mutagenic effect was obtained with TA 1535. responding to base pair substitutions, but no increase of mutations was caused in TA 1536-- TA I53S, responding to frame shift mutations. DISCUSSION From the experiments reported above, it can be concluded that vinyl chloride induces point mutr-tions. um oiuy after a metabolic activation. It can further more be established that vinyl chloride causes base pair substitution, but appar ently no insertion or deletion of base pairs in DMA. The latter kind of effect is hardly to be expected vmh a com pound like vinyl chloride. II is evident that vinyl chloride re sembles many other carcinogens in the respect that it nets as ? mutagen via s metabolite, which seems to be formed m the liver. It is reasonable to assume that the caTciiiof.enic action is also ric- rtf' ih* nm. Aikcncs, cyeloalkcucs as well as a variety of aromatic compounds are known to be metabolized. eg in the rai liver, via intermediate epoxides, formed in the NADPH-lcpcndcnt oxjf.vnaiion by mkrosomes |5. 19). Furthermore, tri chloroethylene is metabolised in the rat to trichloroacetic acid and trichloroethr.no!, which compounds are assumed to be formed via an intramolecular rearrange ment to nichlorof.cctaldchydc of a pri mary metabolite, trichloroethylene ovule (5). The most plausible primary meta bolite from vinyl chloride (l) in the present system hence would be chlorocthylcne oxide III). This compound In? been synthesized by several methods front rthylcne oxide (2D. 21) and is de scribed ns a liquid. l*p 65--67? C (211, v.hich is rapidly hydrolysed by water and even at room temperature slowly re arranges to chloroacctaldehyde (111). Cl CH-CH, I Cl 0-1,-010 1H V____ CH ci7 w\o/ R 156 *- A >' a-' ' -C r~ i . r-.v. fZ V' . ;yjK- 1 VihV- 3, FA 1136 'uYcMut'-v} IK-.Ki1 li n'mrs'.;,ir innia Irli'j ?,. p-.rccrt (r/'t) f--x<;niis vinyl ch`Ni-.b--- -r r-ripmt* p-jr pti'c, n-wn vn'U-r fit 5 pi.Hrx * A t'. Type cf tr-.'ittrnent Time cf trc.'.lnunt lixprriment I ?0 ptirt-.ites 61) minhtes 90 minutes VC+fn'crosom.il sjitcm VC + microsomal system -- NA DP VC Ccif.trr!+microsom.il system eiU-LG.S 20/*i 3.5 -15.fi 4.7 Experiment II 72.2iS3 IU.6--0.7 IS.4 i 2 (< 39.40:1.? 1I5.84.2 30.K1.3 22.R-1 3.1 37.4^4.3 VC+m'ciosom:t system VC+microiomnl sssinn-- NADP .VC Control-bmkrosoAtYl system 49.fi-3.9 12.4-1.9 I0.fi-fl.fi 75.0-3.5 5l.i3.9 I6.40/i lE.dz.1.5 20.4-2.7 77.2i3.f> 20.2-2.2 22.63.4 CMwocthylcnc oxi-Jc cnr> he cs peeled to reset as a bifimctionul alkylairnr. c^cnt. Moreover, it IvMnp.s to the I'lvup of highly reactive ^-chiormnitj aliphatic ethers, amoitj which, v:, chloronteihyl methyl ether (CMV.ID s*nd bi'tchinronteihyl) ether are reivpnizcd as strong carcitiogcns (22). Mi'tageniciiy tests of the metabolites discussed arc under wav. Vaz fret that vinyl chloride needs a mrtsboJic activation b.-i'tvc it acquires i B inuiagcnk property focuses attention on the necessity of usine a test svstcri whin)* *(,-* fftBr./'*'*' --a - Es possible mimics ijr't (tiotoboiistn per formed in the mamr-mli.>n hotly. Tin* liver microsotr.al system v.nh Jd'.-mmcf/u k a very useful tool in this n-'peei. It rhouUl, however, he pointed mu th.:t the way of distributing the comnntmd K essential. False negative result* c,*n easily be brotiph: about E>y a lo-v water solu bility and a high volatility as in the ...^present ease with vinyl chiori.le. The nuilanenic property of this compound would have been undetected if only wa ter solutions ha.i been tested. There is an increasing wealth of data which indicate that potential carcinogens can at least lie pointed out bv mean* of mmarsnieity tests i-i, 14) anJ the pre sent finding.'; with vinyl chloride arc in pood accordance v.nh such a view. The question posed above, whether the car cinogenic-effect of vinyl chloride could h-<<* been imli ;-:to'l earlier by n'.cati- .*? muuyenicity tmts, can therefore he answered with `vcs". The actual cr- was. however, the inverse--the rareinopenic efrect w;;v discovered he I .To .:nv mutagenicity test-, had been perform'd. This is clearly inisiiiislTanry: Jj ;> -t-.v listec teviinp procedure tor chemicals with respect to rmnoeeme and carcinogenic effects is to he Indli up. it '-cents that cooperation between povernmental au thorities, scientists nr.d industry is essen tial. 7= Tef'lr 4. Cli*rw TA 1(35 fbase *;r*'A:tt:!icn) nr.tf T.t iw.-iMS (Iranw sir'll heated tnr 6U minute i fcl rn eirw.-pherc rl air centaining C*i pei.'rr.t tv/vj ecsecaf vinyl cnlfliiJ* (V'C|. St.-tiii.'eal dalysej, see Table 1. Tyj* of treatment Sfmirt * N't-mb?r ef v; iW cells per J Vtc XJ.0"# Mean value cf ' p'ntcs Npnli'.T of miituiilc pgr plnlc Mf.'.t jhts of $ r.utcs Number f mutants per 10" sunhin.-; fells. -SE rtetcre-iee-: rrni Notes 1. } I. Crcre'i unit M N Jeim-'in. Journal of Me.linnr IS tfu 2. C Miiltoni anti Cl Itfi -unr. yfiWtrtmt) itcliu Chme ili Sell-"-- /im/if. ftctie r ntiriirn.'i Srr. H t.VI. J.-.nc. > ttV"i4|, J, Tcchfioler.v review. A'e- inVu/ui 62, 5-IS im-u. t, F. C Miller and J A M-lkr. tn Cbfniral Mmoi-mi, 1'iiiteiplrs ami Stcihiuh for their Vol 1. A I lotlacndtr hd. (I'irnum I'resi, Sew Veil.--Luiutun, i1)!!) pp (<?-!*>. 5. D V Pari.?. The ftiorheiiiiitrv of Foreign Coinpoiuitft (Percamsn Press. Oxford HOW. 6. H O Maude), in Fwiilametn/tls of Ding /llttfiliutiini end Drug Dnfiuuuan, II N U On. H G Msndct. I. Wav Ldt. (V.'avertv Press. Inc, Dslumore. 1971) pp M9.-1H6 7. It ft llrpJie. J Asflrnd. i R Cooper. L Oamlcii. H N La Du C Miionn and S Utlrnfru-n.;. Science 121. 6u.t <tv?S). t. O Hayai'hi, ProctetUngi of the Fourth //MwiiiKir-Mnf Coin; r,ji o/ /iiyt-Jirm/jfr/ l V B. J.t, p Ji (liJ). V. G Arrheniii>, Arim/nciun 1. JS7 tldTI). ]0. O C Mueller and J A M>IV. Journal of /Nsdni-rVn! Ch:ni:iln 180. Illy *T-S<)s. U. Arrhenius, ChriHttO'Undogicnl Inter aeiinns t. Ml HOW VUl. 12. H V Mailing. Mutation Ktsrruch 13. 423 (1071). 13. M G Cahru'e* and M S f.enaior. Pro- eeediitci of the Soetetr f' Eifinmciiial PuiloKf uiuf Afeificmr 130. HM IISJMI, U. 0 N Ames, \V F. Durston. F I'nmasati and F I) l^e. Pitre -cJtngs of the Xatiouat dcrufrnry of Seirncrr. USA 70, 2281 IWT.Vi. is. U N Aires. F D I.cc and W E Durston. FtoteeiUnes of the Sutmiml Aru.trm)' of Sritnes, USA 70. 781 1)0731. 16, It J Yorcl and IJ ht llnnncr. Journal 01 Bioitun ut Cl riiutri y 218. 77 t W5i'<>. ii. u ,y ,-srr.es, ir, Mi.ucnii Fnn- drift end Methods fvr Dctfition. Vel 2. A Hullacttticr. d. il'ltm-.rn Press, New Vnrk--London, 1^711 pp 2i:7--2S2. lit. Personal contniuiueat.pn, S Jensen. Wat- lenherc Labo:a(Ory. Gnsironr.wniat Tosico'oyy Unit. University of StiKlhofm. Sweden. 10. i \V Dab. I> M Jerina and C VViikor. Fxpenenna 2F. 1127 <IV72I. 2(>. Ch Waiting and P S Frederick. Journal of Anirnriitt Chemical Soufte Cl, 3326 1(1621. 21. 11 Gross i-.iid 3 Freiberg. Journal Jiir Prahtisrhe Chrrue 311. *03 <W)I. 22. ft IC J l.eonp. H N Msefsrljnd and W It'--llee'e, ArcWi'M of FnuiOnntenrol Health 22. 663 <1171;. 23. This iim-idVation lies been supported by grants (tnm the Swedish Hoard for Tech nical Development. The ouih.'is are grate ful to Dr h N Antes, University of Cali fornia. reiUntcy. for supplying the S ttphirnur.itm strains arn! for vj'uuhlc adv-cc and to Drs Arrhenius. S Jensen and D Jeiiss-.n (or helpful discussions. 24. Keccivcd Jure 13. 1V74. VC+ microrcmnl system Control-f micrpsomal ly-tem TA 1535 TA 1556 TA 1T'7 TA I..2 TA 1535 TA 1536 TA 1517 TA !5?y 4.J 1.2 1.7 3.7 4.3 I.I 1.7 4.0 <1.2 10.10.4et* 0,5 0.7i0.3 13.6 7.9 1.1 20.4 5.5O.G 15.2 3.5*07 O.f; O.fi*0.4 IfiA 7.7 *0.0 22.2 J.0J-CU AitDlD. 1974 197 Sr- URL! 9464 i ( l r iil