Document 4QYYVDdpMEB7ZBv6n46wYnzrN

Cid = Zsgmney IOFN TVHIRTEREO M3ICRCOOBMPIAONLYOGCIOCMAPLOUMNUDTSAGENICITY ASSAYS TY einal Repore spi 1978 By: GVriengMcoiercnytrob7F..ialSShieGmpeohnnee,rtdi,cPshM.iPDcr.roogbrMiaaomnlaoggeirst Prepared for: MG2eednieccroaamllparODefefpiacretsmen S3c. CePnautle,r Minnesota 55101 Atcencion: MJ.anaEg.erL,ongT,oxiScco.lD.ogy Services SRI Project LSC 4ad2-16 approved: -- yr n Deparen nencd of TRoTviFcoileoegrsor Ah I GY n LifeWXscSikeinmcmeesr,EDxiveissuiroinve Divevior RiEemEssEonal (543135)Ra5v2en5su.o6o-d2CaA0bvel0.e SMTeAnNlRoEPSa,rk,MaCralloopriaa-5rT40Wk2:5 910-373-1248 000056 SuARY SRI Incernacional examined 3M Company compounds T-2136 CoC, T-2138 i, and T-2140 IN for mutagenic activity with strains TALS3S, TALS37, TALS38, TA98, and TALOO of the bacterium Salmonella typhimurium in the standard Ames Salmonella/microsome assay and with the yeast Saccharomyces cere ovfisaiameetD3a.boliEcacahctaisvsaatyiownassypsetrefmo.rmed in the presence and in the absence None of the compounds was mutagenic in either the Salmonella/microsome assay or the S. cerevisiae assay. 1 000057 INTRODUCTION SRI Internacional examined compounds T-2136 CoC, T-2138 IM, and T-2140 I for mutagenicity by in viero microbiological assays with Salmonella typhimurium strains TALS3S, TALS37, TALS3S, TA98, and TAL00 and with the yeast Saccharomyces cerevisize D3. An Aroclor 1254-stimutlhaeceda,ssaryacprloicveedrurheosmogtoenaptreovimdeetamboeltiacbolaicctivsattepisonthsaytstethmewabsactienrcilauded in either are incapable of conducting or do not carry out under the assay conditions. The purpose of this study was to determine whether these compounds elicited a mutagenic response in microorganisms. The assay procedure with S. typhimurium has proven to be 80 to 90% reliable in detecting carcinogens as mutagens, and it has abou the same reliability in identifying chemicals chat are mot carcinogenic.' The assay procedure with S. cerevisise is about 60% reliable in detecting carcinogens as agents chat increase mitotic recombination.' The combinacion of che two assay procedures significantly enhances the probability of detecting potentially hazardous chemicals. However, because che systems do not always provide 100% correlation with the resules of carcinogenicity investigations in animals, neither a positive nor a megacive response proves conclusively that a chemical is hazardous or nonhazardous to man. 000058 ETHODS TSAa9l8m,oneanldlaaltyopohimurium Strains TASIS, TALS37, Tal538 The Saluonella typhimurium strains used ac SRI are all hiscidine haiusxcoitdroipnhes-debpyenvdiernttueceolflsmuatraetiognrsowninonthemihniismtaildinmeedoipuemronp.etriWhpelnactehsese containing a trace of histidine, only those cells that revert to histidine independence (hisTM) are able to form colonies. The small amount of histidine allows all the placed bacteria to undergo a few divisions; in many cases, this growth is essential for mutagenesis to occur. The his" revercants are easily scored as colonies against the slight background growch. The spontaneous mutation frequency of each strain is relatively constant, but when a mutagen is added to the agar the mutation frequency is increased 2- to 100-fold. We obtained our S. cvphimurius strains from Dr. Bruce Ames of the University of California ac Berkeley.'TM In addition to having musations in the histidine operon, all the indicator strains have a musasion (227) that leads to a defective lipopolysaccharide coat; they also have a deletion that covers genes involved in the synchesis of vitamin biotin (bie") and in che repair of ultraviolec (uv)-induced Ola damage (urd). The rfa mutation makes che scrains more sersesble o many large aromatic molecules, thersby increasing the mutagenic effect of these molecules. The uvrd" mutation decreases repair of some types of chemically or physically damaged DNA and chereby enhances she strains' sensitivity to some mutagenic agents. Strain TALSIS is reverted to ais" by many mucagens that cause base-pair substitutions. TALCO is derived from TALSIS by the introduction of the resistance eransfer factor plasmid pKMIOL. This plasmid is believed to cause an increase in error-prone DNA repair chat leads to many more mutations 3 000059 for a given dose of most mutagens. In addition, resistance to the aneibiotic ampicillin, which is plasmid pRMIOL confers a convenient marker th0isdeptleacstuicdhealpsroesmeankceesofscrtahienpTlAaLs0n0idseinnsitthieveceltols.someThefrapmreessheinfcte of sutagens (e.g., ICR-191, benzo(a)pyrene, aflatoxin B., and 7,12- dimechylbenz(a)anthracene). by many frameshift autagens. Strains TAIS37 TALS37 and TALS38 is more sensitive are reverted than TALS38 to mutation by some acridines and benzanthracenes, but che difference is quantitative rather than qualitative. Strain TA98 is derived from TA1538 by the addition of the plasmid pKMIOL, which makes it more sensitive to some mutagenic agents. AL indicator strains are kept at 4C on nininal medium plates, supplemented with a trace of biotin, and an excess of histidine. The Places with the plasmic-carrying strains contain in addition ampicillin (25 ug/al}, to susure stable maintenance of the plasmid pRMIOL. New stock culture plates are made every two months from single colony ! reisolates that were checked for their genotypic characteristics (his, rfa, uveB, bio) and for the presence of the plasaid. For each experiment, an inoculum from the stock culture plates is grown overnight at 37 C in nucrient broth (Oxoid, C67). After scatiomary ovemight growch, the cultures are shaken for 3 to 4 hours to emsure optimal growch. Aroclor 1254-Stizulated Metabolic Activation Svsten Some carcinogenic chemicals, either of the aromatic amino type or polyerelic hydrocarbon type, are inactive unless they are metabolized to active forms. In animals and 3an, an enzyme system in the liver or other organs (e.g., lung or kidney) is capable of metabolizing a large umber of these chemicals to carcinogens.'*TM' Some of these facermedfate etabolices are very potent mutagens in the S. typhimurium test. azes has described the liver metabolic activation system that we use. In brief, adult male rats (250 to 300 g) are given a single 500-mg/kg intraperitoneal injection of a polychlorinated biphenyl, Aroclor 1254. This creacaent enhances the synthesis of anzymes fmvolved in the metabolic ` 000060 conversion of chemicals. Fourdays after the injection the animals' food is removed but drinking water is provided ad libitum. On the fifth day, the rats are killed, and the liver homogenate is prepared as follows. The livers are removed aseptically and placed in a preweighed sterile glass beaker. The organ weight is determined, and all subsequent operations are conducted in an ice bath. The livers are washed in an equal volume of cold, sterile 0.15 M KCl (1 ml/g of wet organ), minced with sterile surgical scissors in three volumes of 0.15 M KCl, and homogenized with a Poccer-Elvehjem apparatus. The homogenate is centrifuged for 10 minutes ac 9000 x g, and the supernatant, referred to as the S-9 fraction, is quickly frozen in dry ice and stored at -80 C. The metabolic activation mixture for each experiment consists of, for 10 ml: + 1.00 al of $-9 Fraction + 0.20 al of MgCl, (0.4 ) and RCL (L.65M) * 0.05 al of glucose-6-phosphate (1 ) + 0.40 ml of NADP (0.1 M) * 5.00 ml of sodium phosphate (0.2 , pE 7.4) 3.35 al of K:0. 5 000061 Assays tn agar To a block, we sterile 13 add in the x 100 mm test tube following order: placed in a 43 Cheating (1) 2.00 al of 0.67 agar* (2) 0.05 al of indicator organisms (3) 0.05 ml of a solution of the test chemical (4) 0.50 ml of metabolic activation mixture(optional). For aad 0n.e0g5atialveofconttherolsso,lvewnet usuesedstefposr (1), the (2), test and (4) chemical. (optional) 3ecause the aajoricy of organic compounds are mot sufficieacly water soluble-- particularly ac che higher concentracions--we routinely use dizechylsulfoxide (DMSO). Other solvents used are water, ethanol, or benzene. For that are positive occasionally controls, we test each culture by specific mutagens known to using steps (1), (2), (3), and (4) (optional). revert each strain This atxcure is stirred sencly and then poured onto minimal agar places." After the cop agar has sec, the places are incubated at 37C for 2 days. recorded. The number of his revertant colonies is counted and * 0.6 agar contains 0.05 mf histidine, 0.05 mf biotin, and 0.1 H Nacl. oM1f0inigmloauflcosXaeig,HaPrO0L.p,2latagensdof3c.o5gnSs0i3.s+to7fHo:Nf0,a,RNpeH2r.5PlOoiLfteeirca,i0t.r1i5cgacoifdagmaerm,shv1d0z3ate, 6 000062 Saccharomyces cerevisiae D3 The yeast S. cerevisiae D3 is a diploid microorganism heterozygous for a mutation leading to a defective enzyme in the adenine-getabolizing pathway.' When grown on mediua containing adenine, cells homozygous for this mucation produce a red pigment. These homozygous mutants cam be generated from che heterozygotes by mitotic recombination. The frequency of this recombinational event may be increasedby incubating the organisms wich various mutagens. The degree of mutagenicity of a compound or of its metabolite is determined from the number of redpigmented colonies appearing on the plates.' The S. cerevisiae tester strain is stored at -30 C. For each experiment, the tester strain is inoculated in 17 tryptone and 0.5% yeast extract and grown overnight at 37 C with aeration. The in vitro yeast mitotic recombination assay in suspensicn is conducted as follows. The overnight culture is cencrifuged, and the cells are resuspended at a concentration of 10 cells al/in a 67 = phosphate buffer (pH 7.4). To a sterile test cube are added: + 1.30 al of the organisms + 0.50 ml of either che metabolic activation mixture or buffer + 0.20 al of che test chemical. Because many orzanic chemicals are aot appreciably water soluble, dizechylsulZoxide (DMSO) is used routinely as the solvent for the test chemical. Other solvents chat are used occasionally are echamol, benzene, or water. Several doses of the chemical (up to 5%, w/v or v/v) are tested in each experiment, and appropriate controls are included. The suspeasion mixture is incubated at 30 C for 4 hours cm a roller drus. The sample is diluted serially in sterile physiological saline, and a volume of 0.2 ml of che 10TM* and 10TM* dilucions is spread on trypcone-yeast agar places; five plates are used for the 10-7 dilution and three places are used for che 10-7 dilution. The plates are incubated for 2 days at 30 C, followed by 2 days at & C to enhance the development of che red pigment indicative of adenine 7 000083 deficient homozygosity. Plates of the 10TM dilution are scanned with caodlionsiseesctoirngrmedicrsoescctoopres a(tmit1o0tiXcmargenciofmibicnaatnitosn), iasndretchoerduemdb.erThoef red surviving fraction of organisms is determined from the number of colonies appearing on the plates of the 10-* dilution. The number of mitotic recombinants is calculated per 10 survivors. 4 positive response in this assay is indicated by a dose-related increase of more than threefold in the absolute number of mitotic recombinants per @illilicer as well as ia the relative number of mitotic recombinants per 10 survivors. 8 000054 RESULTS AND DISCUSSION T-2138TabIl,esan1d tTh-r2o1u4g0h I3 prinesetnhte Atmhees rSeaslulmtosnelofls/tmeisctrinogsomTe-21a3s6sayCoCa,nd with the yeast S. cerevisiae D3. T-2136 CoC was dissolved in water. T-2138 IM and T-2140 IH were excracted in DMSO overnight; various volumes of the extract were used in che bacterial assay procedure and made up to 100 ul with DNSO (Tables 2 and 3). The negative control used for these two samples was 100 1 of DMSO. When kaown mutagenic compounds were added to the top agar of the positive control plates, the number of mutants above the background count was increased. When various concentrations of T-2138 | IM, or T-21%0 IM were added to the top agar, no dose-related increase in the number of mutants over the background count was observed, either with or without metabolic activation. These results lead to the con- clusion that T-2136 CoC, T-2138 IM, and T-2140 I are not mutagenic in che Salmonella/microsome assay. Tables 4 and 5 present the results of testing T-2136 CoC with . serevisiae D3. At various concentrations ranging from 0.1% cto 5%, this compound did not cause a reproducible, dose-related increase in che number of mitotic recombinants. Therefore, we conclude that this compound is not mutagenic in S. cerevisise DI. This assay was not conducted on T-2138 I or T-2140 IM because only limited quantities of these compounds were available. 9 000065 Compouns____ Negative control (1,0) PoIsShtoendiiivunemoaaccsoeinidtdertonles 5 Z2o0d0nitehrroafnilnueo.ren 7213 coc 232 S&a IN VATHO ASSAYS Table 1 WITH SALNOUELLA TyPHIMURION T2136 cac Shceipvaalttieon +- :. he+:< + ::: zz ++ ++ ++ oHficCroomgprouannd Added per Plate 5500,,000000 500.5 525 121105 5100 150000 13000000 s100 105000 10500000 TAAvIeSrTaSge TWAiaTtSiYdtToTeATReIvOeErtanatsaper aPlaatcee B a s ow8 219 ianx 2 C3 mn 2s 6 saes aa 13a 1139392 mies 2A Asnw5s omaGeo1g B A e8 Zoos w a0 D2 nn aags 7 Z ae 6sauww yagegqy a B nosawy on 38 wm 3% gggk ag ITED AS5AYS WrTTaesip2onta TRI compos [Te ---- fteecttlbvoililoen AddofedComurpouFnadce TeraEge AWirtTidinerReavercantas por aPltacee :- 110 01 a4 ow ow om FS[oiSEaeoritnoumosnncscroeinttgerionles oeoR po >- Fros om os as Thattranine : :: isis i w6 ut unuo 0u5s5 aw : 1 mo uk 2:- :: 2 i0s 00 oonm onw omom :: iHFS owmwommoooosdoooooonnoonowoBnBop o3 om `:: w:: :i 55 mBn2ooosoesdoonnmnoHwaono liem : I B moo0 s 0nonnoeda g: Feros oF TE Tpertacnts. 88 | PE Yogaive contrat (m0) | FosCSttoeativvnaeosccrrointteironles u hfreeren 0m 83 288 LLTr e3 p Tom fHeeatbhovlaticn ::::B: : -: B:.s :ii : psioefdCpoomrpouPnhdase 110%01 503 50 i3is i 5: i 0052 FH5 1 TdOveSrage TWisTeidineARevertante per aPisece Booo wo8 owponom m os 6. usr ass "0s us wel uouk 5 BKvoooodsoeo ood oos wnou &Bnoo mmom 5B0o5d aon ooB iEmm i onvoy6o s nwwho aEo%noowe BwoodooonB d 3a M Tae 4 | I THO Assis Winn2succucnoocness cavisiae | Expertaens 1 conpouna Metabolic ConTcnotroction CETTEtpeer roys T ieorte e secr minnm s_ feoivatton Wow G0 perce aay ora Negative conceal (10) :- 19 wdee Sso0 o6a Fostsivie cioenocesablutane 1- 0o.o0k25 9EC 1 m-ea furii .u T2136 coc -: 50i51o so ie o%omw Sn pxSc}oo 52maan i:+ so a T00.o31 ew e s w alm 2S0o 1p4d i 50 BH B0i opeed g g22 Table 5 IN VITRO ASSAYS WITTH-2S1A%ccuomtonvces cenevisiae Expertaent 2 Compound AMecttavbaotliloen fCvoPonecrrecvemnittr)laotn CTTSurpvopivoofrs G107) erent MiP(etxro1mtil0c) BecosbpiSeenrcaen1itvs0e7rs Negative control (1,0) :- 6sse 010 2sl.o0 Bs.z2 Pos1t2e,t3v,e4-cDolnecrpoolrybucans -+ 00.0022s5 e6ss aloe desnoo m81l.s2 21% coc z-- 2lL0o Wo 673 0m100 wo 1 sl2.o0 70 l2e.0 70 520 as as 30 3s +++ + 2w10o00 wses ols oomm 59 7s2%.000 w2e3 a so we 10 70 ole <gg 2 3 REFERENCES 1. AcJs.asracMyicnCooafgnne,n3s00E.aschCehmmouiitc,aagleEs.n.sYaiPmnraostcah.kei,NSaatal.nodnAec3la.lds.N/.mSicAcis.reos.sUoSAm,eDe7ct2ee,sctt:i5o1n35-of 5139 (1975). 2. 3. N. ames, Carcinogens I. as G. Gurney, frameshift aJ.utaAg.enMsi:llerM,etaabnodlii.tesBaraensdch. admeirnievacciavrecsinoogfen2s-.acePtryolca.ntNnaocf.luAocraedn.e aScnid. oUtShAe,r 8a9r,oma3t1i2c6- 3132 (1972). 3. b5.actN.erimaels,tesF.t Ds.ystLeeem, foarndthW.e dE.eteDcutristoonn.andanclaismspirfoivceadtion of a7u8t2a-g78e6ns (a1n9d73)c.arcizogens. Proc. Vat. Acad. Sci. USA, 70, 4. 8. N. ames, Carcinogens W. E. Durston, are mutagens: AI. sYiammpalseaktie,stansdystF.emD.comLbeei.ning liver Nhaocm.ogeAcnaadt.esScfio.r aUScAt,iv7a0t,ion228a1nd-22b8a5cte(r1i3a73)f.or detection. Proc. 5. JD.etMeccCtainonn,ofN.ca=.rcSipnionggeanrsn,asJ.muKcoabgoernis,: andBac3.terN.ialAmetse.ster 9s7c9r-a9i8n3s w(i1c9h75)3. factor plasnids. Proc. Nat. Acad. Sci. USA, 72, 6. Lre.lAa.tioPnosihriipesr and and carcinogenicity. Vt.he F.rolSeimoofn.mutaMguetnaigceniscc-rceaernicnignogceensisc Clin. Toxicol., 3 (3), 761771 (1978). for 7. Lac.cDi.vitKyieri,n cZ.igaYraemcacsaekis,mokaendco5.ndeNn.saAtmeess.. PDreotce.ctNiaocn. oAfcamdu.tagSeein.ic USA, 11, 4159-2163 (1974). 8. c3.arc.inoAgmeesn,s aJ.ndMcmCuacnang,ensanwdicIh. YtahemsaSkail.zonMeeltlhao/dzsanmfaorliadne-cmeicctrionsgome aucagenicicy cesc. Mutation Res., JI, 347-364 (1975). 9. Fc.onvKXe. rsZiiomnewrimtahnniatnrdouRs. aSccihdw,atelr-.methIynld-udc-tniiotnroo-fl-mitotic gene aceircervoissoigauea.nidiYonle. aGnedn.ochGeemreta.l,ky1l0a0t,ing63a-6g9ent(s196i7n).Saccharomvees 15 000071 10. Ds.creJ.eniBnrgusitcekchnainqdueVs. wW.ithMayyeera.st. NewEnvdiervoenl.opmHeenaltcshinPermsupteacgteinviecsi,ty 5, 83-96 (1973). 16 000072