Document 4v4Ez7nQYdQrvMGj0Jj2kpLje

In vimo vacaosouoorcas MuTAGENICITY Assas ZEST OF TM COMPANY COMPOUNDS T-2247 CoC AND T-2248 CoC F & 7R 3s3, Final Report 5 September 1978 8 EDs Ni coe ren TT id CX oe EERE oicorons Lavoraions a. dfn 333 Ravenswood Ave. Menlo Park, California 94025 01966 StnauARY SRI International examined 34 Company Compound T-2247 CoC and T-2248 CoC for mutagenic activity with strains TALS35, TALS37, TAIS38, TA98, and TALO0 of the bacteriun Salmonella typhimurium in the Ames Salmonella/microsome assay and with the yeast Saccharomyces cerevisiae D3. T-2247 CoC was also testeidn desiccators. Each assay was performed in the presence and in the absence of a metabolic activation system. T-2247 CoC and T-2248 CoC were not mutagenic or recombinogenic 1n any of the assays performed. 1 01967 INTRODUCTION SRI International examined 3M Company compounds T-2247 CoC and T-2248 CoC for mutagenicity byinvitro microbiological assays with five strains of the bacterium Salmonella typhimurium (TA1S3S, TALS37, TAI538, TA98, and TAL00) in the standard Ames Salmonella/microsome - assay, in desiccators, and with the yeast Saccharomyces cerevisiae D3. An Aroclor 1254-stimulated, rat liver homogenate metabolic activation system was included in the assay procedures to provide metabolic steps that the bacteria either are incapable of conducting or do mot carry out under. the assay conditions. The assay procedure with S. typhimurium has proven to be 80 to 90% reliable in detecting carcinogens as mutagens, and it has about the same reliability in identifying chemicals that are mot carcinogenic.! The assay procedure with S. cerevisiae is sbout 60% reliable in detecting carcinogens as agents that increase mitotic recombination.' The combination of the two assay procedures significantly enhances the probability of detecting potentially hazardous chemicals. However, because the systems do not always provide 100% correlation with careinogenicity investigations in animals, neither a positive nor a negative response conclusively proves that a chemical is hazardous or nonhazardous to man. 2 01968 METHODS Salmonella typhimurium Strains TAI535, TA1537, TA1538 TASS, and TAL00 The Salmonella typhimurium strains used at SKI sre all histidine auxotrophs by virtue of mutations in the histidine operon. Vhen these histidine-dependent cells are grovn on minimal medium petri plates containing a trace of histidine, only those cells that revert to histidine independence (his) are able to form colonies. The small asount of histidine allows all the plated bacteria to undergo a few divisions; in many cases, this growth is essential for mutagenesis to occur. The his' revertants are easily scored as colonies against the slight background growth. 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. typhimurium strains from Dr. Bruce Ames of the University of California at Berkeley.'TM In addition to having mutations in the histidine operon, all the indicator strains have a mutation (zfa) that leads to a defective lipopolysaccharide coat; they also have a deletion that covers genes involved in the synthesis of vitamin biotin (bic?) and in the repair of ultraviolet (uv)-induced DNA damage (uvrBT). The rfa mutation makes the strains more permeable to many large aromatic molecules, thereby increasing the mutagenic effect of these molecules. The uvrBTM mutation decreases repair of some types of chemically or physically damaged DNA and thereby enhances the strains' sensitivity to some mutagenic agents. Strain TALSIS is reverted to his' by many mutagens that cause base-pair substitucions. TAL00 4s derived from TAIS3S by the introduction of the resistance transfer factor plasnid pKMIOL. This plasmid is believed to cause an increase in error-prone DNA repair that leads to many mors mutations 3 01959 for a given dose of most mutagens.' In addition, plasmid pRMIOL confers resistance to the antibiotic ampicillin, which is a convenient marker to detect the presence of the plasnid in the cells. The presence of this plasaid also makes strain TAI00 sensitive to some frameshift nutagens (e.g., ICR-191, benzo(a)pyrene, aflatoxin Bi, and 7,12 dimethylbenz(a)anthracene). Strains TALS37 and TALS3S are reverted by many frameshift mutagens. TALS37 is more semsitive than TALSIS to mutation by some acridines and benzanthracenes, but the difference is quantitative rather than qualitative. Strain TAS is derived from TAI538 by the addition of the plasmid pKMIOL, which makes it more sensitive to some mutagenic agents. ALL indicator strains are kept at 4 C on mininal medium plates, supplemented with a trace of biotin, and an excess of histidine. The plates with che plasmid-carrying strains contain in addition ampicillin (25 ug/al), to ensure stable maintenance of the plasaid pRMIOL. New stock culture plates are made every two months from single colony reisolates that were checked for their genotypic characteristics (his, xfs, wrB, blo) and for the presence of the plasmid. For each experiment, an inoculum from the stock culture plates is grown overnigat at 37 C 4n nucrient broth (Oxoid, CM67). After stationary overnight grovth, the cultures are shaken for 3 to 4 hours to ensure optinal growth. Aroclor 1254-Stinulated Metabolic Activation System Some carcinogenic chemicals, either of the aromatic amino type or polycyclic hydrocarbon type, are inactive unless they are metabolized to active forms. In animals snd man, an enzyme system in the liver or other organs (e.g., lung or kidney) is capable of metabolizing a large number of these chemicals to carcinogens.'`"* Some of these intermediate metabolites are very potent mutagens in the S. typhimurium test. Ames has described the liver metabolic activation system that ve use.' In brief, adult male rats (250 to 300 g) are given a single 500-ng/kg intraperitoneal injection of a polychlorinated biphenyl, Aroclor 1254. This treatment enhances the synthesis of enzymes involved in the metabolic 4 01970 c4sonvreermsoivoend obfutchdermiisclailnsg. vatFeorurdisaypsroavfitdeerd tahed ilinbjietcutmi.on Otnhe tahneimfailfst'h dfaoyo,d the wets are killed, ant the Live howogenate fo prepared as folie. The Livers are removed aseptically and placed in a preveighed sterile glass beaker. The organ weight is deteratned, and all subsequent operations are conducted in sn foe bath. The livers are vashed in an eval volume of cold, sterile 0.15 M KCL (1 ml/g of wet organ), minced with | sterile surgical scissors in three with a Potcer-Elvehjen apparacus. volumes of 0.15 M The homogenate is KcCeln,trainfudgehdomofgoerni10zed atnutes at 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 aixture for each experisent consists of, for 10 a1: + 1.00 a of 5-9 Fraction + 0.20 ml of MgCl, (0.4 M) and KCl (1.654) + 0.05 ml of glucose-6-phosphate (1 M) ++ 05..4000 mall ooff NsAoDdPium(0p.h1oMs)phate (0.2 1, PE 7.4) + 3.35 ml of Ei0. : 01971 Assays in Agar To a sterile 13 x 100 m test tube placed in a 43 C heating block, we add in the following order: (1) 2.00 ml of 0.6% 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 negative controls, we use steps (1), (2), and (4) (optional) and 0.05 al of the solvent used for the test chemical. Because the majority of organic compounds ate not sufficiently water soluble---- particularly at the higher concentrations--we routinely use dimethylsulfoxide (DSO). Other solvents that are occasionally used are water, ethanol, or benzene. For positive controls, we test each culture by specific mutagens known to revert each strain using steps (1), (2), (3), and (4) (optional). This mixture is stirred gently and then poured onto minimal ager plates." after the top agar has set, the plates are incubated at 37C for 2 days. The number of his' revertant colonies is counted and recorded. * 0.6 % agar contains 0.05 wf histidine, 0.05 mf biotin, and 0.1 NaCl. + Mofinigmlaulcosaeg,ar0p.l2atgesOfcMognSsOi.s+t7Ho:f0,, pe2rg liotferc,itr15icgaocfidagmaorn,chy1d0ra3te, 10 g of K.HPOL, and 3.5 of NaHNH.POL+4Hz0. 0197a2 Assays in Desiceators for Volatile Compounds The standard Anes plate test is not entirely suitable for the testing of highly volatile chemicals, so we have modified the procedure to conduct such testing. The Saloonella plates are prepared as described for the assays in agar, but no test chemical is added. The plates, the 11ds having been removed, are placed side by side on a perforated shelf in a 9-liter desiccator. A known volume of the test chemical is added to a glass petri plate that is placed in the center of and attached to the bottom of the shelf. A control cheatcal is tested similarly fn each experiment. The desiccator is sealed and placed on a magnetic stir plate 1n a roon maintained at 37 C. A magaeric stirrer with vanes, placedin the base of each desiccator, ensures adequate dispersion of the chemical. After incubation for hours, the plates are removed from the desiccators, their 11ds are replaced, and they are incubated at 37 C for an additional 42 hours. The musber is his revertants is counted and recorded. 7 01973 DESICCATOR ASSAY c= == PETRI PLATES Le WITH SALMONELLA PORCELAIN --Se GLASS PETRI C1 {/----~iate For V FAN ------ a b TEST CHEMICAL - 7] STIRRER 8 0197q 4 Saccharomyces cerevisize D3 The yeast . cerevisise D3 isa diploid microorganism heterozygous for a mutation leading to a defective enzyme in the adenine-metabolizing pathway.' When grown on medium containing adenine, cells homozygous for this mutation produce a red pigment. These homozygous mutants can be generated from the heterozygotes by mitotic recombination. The frequency of this recombinational event may be increasedby incubating the organisus with various mutagens. The degree of mutagenicity of a compound or of its metabolite is deternined from the number of red pigmented colonies appearing on the plates.' The 5. cerevisiae tester strain is stored at -80 C. For each experiaent, the tester strain is inoculated in 1% tryptone and 0.5% yeast extract and grown overnight at 37 C with aeration. The fn vitro yeast mitotic recombination assay in suspension is conducted as follows. The overnight culture is centrifuged, and the cells are resuspended at a concentration of 10 cells ml/in a 67 mt phosphate buffer (pi 7.4). To a sterile test tube are added: + 1.30 al of the organisms + 0.50 al of either the metabolic activation mixture or buffer + 0.20 ul of the test chemical. : Because many organic chemicals are not appreciably water soluble, dinethylsulfoxide (DHSO) is used routinely as che solvent for the cest chemical. Other solvents that are used occasionally are ethanol, benzene, or water. Several doses of the chemical (up to 5%, w/v or lv) are tested in each experiment, and appropriate controls are included. The suspension mixture is incubated at 30 C for & hours on a roller drum. The sample is diluted serially in sterile physiological saline, and a volune of 0.2 al of the 107% and 107% dilutions is spread on tryptone-yeast agar plates; five plates are used for the 10-* dilution and three plates are used for the 10% dilution. The plates are incubated for 2 days at 30 C, followed by 2 days at 4 C to enhance the development of the red pigment indicative of adenine- 9 01975 deficient homozygosity. a dissecting microscope Plates at 10 X mofagntihfeic1a0t7i%ond,iluatndionthearneussbcearnneodf with red colonies surviving orfrraecdtiosnectoofrsorg(amniitsoutsic isrecdoemtbeirnnainntesd) is from retcheordneuds.berThofe colonies appearing on the plates of the 10-* dilution. The number of mitotic recombinants is calculated per 10 survivors. A positive response in this assay is indicated by a dose-related increase of more than threefold in the absolute number of mitotic recombinants per =i11ilicer as well as in the relative nusber of mitotic recombinants per 10 survivors. Io 01976 RESULTS AND DISCUSSION Table 1 presents the results of testing T-2247 CoC in the Ames Salmonella/microsome assay. The data are an average of two assays perforned on separate days. The compound was tested over a wide range of concentrations, from 10 to 5000 ug/plate, both with and without metabolic activation. Because no. dose-related increase in the number of mutants over the background count was observed, we conclude that T-2247 CoC was.not mutagenic in S. typhimurium. As Table 2 shows, T-2248 was tested at dose levels of 10 to 5000 ug/plate. Toxicity was observed in strain TAIS38 at 1000 ug/ plate and in strains TAIS35, TALS37, TALS38, TA98, and TAL0O at 5000 ug/plate without metabolic activation. T-2248 CoC was toxic at 1000 ug/plate in strain TA1537 and at 5000 ug/plate in all strains when tested with activation. No mutagenic activity was observed with T-2248 CoC in the Ames Salmonella/microsome assay. Table 3 presents the averaged results of testing T-2247 CoC in the desiccator assay on strains TA98 and TAl00. The assay was conducted in duplicate (two plates per strain per dose) with an exposure of 8 hours. The sample was exposed to a wide range of doses of from 0.1 to 5.0 ml per desiccator. No mutagenicity or toxicity was observed. Tables 4 and 5 present the results of assays on T-2247 CoC with S. cerevisiae D3. T-2247 CoC was tested at concentrations of from 0.1 to 5.0% (Table 4) and at concentrations of from 1.0 to 5.0% (Table 5). The compound was not toxic in these assays and did not cause a dose related increase in the nusber of mitotic recombinants. Therefore, we conclude that T-2247 CoC was mot recombinogenic in . cerevisiae D3. Tables 6 and 7 present the results of testing T-2248 CoC with S$. cerevisiae D3. A slight toxic and apparent mutagenic response was seen at 5.0% without metabolic activation (Table 6). T-2248 CoC was retested at concentrations from 1.0 to 5.0% (Table 7). No dose- un 01977 related increase in the nusber of mitotic recombinants was observed; therefore, we conclude that T-2248 CoC was not recombinognic to S. cerevisiae D3. We conclude that T-2247 CoC is not toxic or mutagenic in either S. typhimurium or S. cerevisiae D3. T-2248 CoC is toxic at higher doses in the Salmonella/microsome assay and is not mutagenic in either S. typhimurium or S. cerevisiae D3. 12 01978 REFERENCES 1. Jc.arcMicnCoangne,nsE.asChmouit,ageEn.sYaimnastahkei,SaalnmdoneB.llNa./miAmcerso.someDetteesctt:ion of Assay of 300 chemicals. Proc. Nat. Acad. Sci. USA, 72, 51355139 (1975). 2. B. N. Ames, Cazcinogens E. as G. Gurney, frameshift Jm.utaA.genMsi:llerM,etaabnodli#.tesBaratnsdch. daemriinveatciavrecsinoogfen2s-.acePtryolca.niNnaotf.luoArceadn.e aScuid.oUtShAe,r 6a9r,oma3t1i2c8 3132 1972). 3. bB.a.cNt.eriAamels,fesF.t D. Lee, system faonrd tWh.e Ed.eteDucrtsitoonn.andAncliamspsriofviecdation of autagens and carcinogens. Proc. Nat. Acad. Sci. USA, 10, 782-786 (1973). 4: B. N. Ames, Carcinogens Wa.reE.muDtuargsetnosn:, AE. sYiammpalseaktie,stansdysFt.emD.coLmebei.ning liver hNoatm.ogeAncaadt.esScfio.r aUScAt,iv1a0t,ion22a8n1d-22b8a5cte(r1i9a73)f.or detection. Proc. 5. JD.etMeccCtainonn, oNf. cEa.rScpiinnoggaernas, asJ.mKutoabgoernis,: andBacB.terN.ialAmetse.ster strains with R factor plasmids.' Proc. Nat. Acad. Sci. USA, 12, 979-983 (1975). 6. L.A. Poirier relationships and and Vt.heF.rolSeimoofn.mutMaugteangiecniscc-rceaernciinngogteensitcs for carcinogenicity. Clin. Toxicol, (5), 761-771 (1976). 7. Lac.tDi.vitKiyeri,n Ec.igYaarmeatstaekis,moakendcBo.ndeN.nsaAtmeess.. DPerotce.ctNiaotn. ofAcamdu.tagSecin.ic USA, 71, 4159-4163 (1974). 8. B. N. Ames, carcinogens Ja.ndMcmCuatnang,ensanwditE.h YtahmesaSkail.monMeeltlhso/dnsammfaorlidsent-emcitcirnogsome mutagenicity test. Mutation Res., IL, 347-364 (1975). 9. Fco.nKv.ersZiiomnmewrimtahnnnaintdrouR.s Schwater. Induction of mitotic acid, l-methyl-3-nitro-1- gene ncietrrevoissoigauea.nidiMonle. anGedn.othGeermeta.l,ky1l0a0t,ing63-a6g9ent(s196i7n).Saccharomyces 13 01979 10. DS.irJe.sniBnrgusitcekchnainqdueV.s Ww.ithMayyeera.st. NeEwnvdiervoenl.opmHeenatlsthinPemruspteacgteinviecsi,ty 6, 83-96 (1973). 1 01980 compound Negative control (4:0) PosSiociivne caornitsrols 5Zhihnmicithnreooaafnciiruiondreiennes w 247 co 322 2R2 Tate1 IN VITRO ASSAYS TW1IT2H47SALNDNELLA TYPHMRIUN HAocttiavbaotlitocn -: :- 3:: - : +: :i i: AddofeEdeCkopptoorpmonPlaate 00.05 5i0o 2esd 2s 151000 51050000000 510 15000 51000000 TAvAeIraSgeSso THAisLWtiSsdHtiandeineTRAeveLrtanSteRevertaTntsSppeer TPAAPlIace n5 osswwonnowow w@ 8 msn now how wes mm ae Buoowsooamow w yo s Boononono1uoam wvoonw o ou uown wo7 o s aBm ona wom 5sow5 xa o7m mow 585 s wum omommw Compound. Negative control (1,0) PoSsoedtivuem cacoindterols 2-hiaLtenroosfclruiodriennes 2 ntheanine 5 7-2248 coc 3 5 T Twn o 8 IN VITRO ASSAYS WTI2T2H48SACLoYCOVELLA TYPHINURIVY AMecttaibvoaltlieon +::: +: + -:: zz : +++ ++ + MoifctCoogmepaonusnd Added per Place 500.05 5L.o0 p2es} 2s 1105000 1050000 5000 5100 510000 13000000 TAvAeLraSge THAaItSiYdineTRAevSertanTtsAOper TPAlIaOtOe n5 osswwoom wom wn now [ 268 w ns w ow mas dw w2 onou B ow wnwow a5 1wwowon ooww 0 wo7r TToowrxom nsous om o6z womw 2 ss s m 1 mmaws r soTm 13Totom wmor Table 3 INVITED ASSATY-S2W2I4T7HCSoAc;LMO8N-EHLOLUAR TEYIPRHOISNIRUERLUK IN DESLCCATORS. Compound Negative control (1:0) PosTtLetDviechcloonrtoraotlhylens T2247 coc 5 MJeettaibvoaltiicon +-: ::: ++ ii iAnmoDuensttcoefacCoorapoalu)nd BTeAvSeBrtmtaT/APLlDaDte B7 oom T10o Conasmus 0o'.s1 n nooonme s1o0 137 u7s 0o'.s1 u5e7 0so 7F-- 238 2I&] Compound Negative control (1,0) Pos1t,e2t,v3e,4c-oDnlteepoolxybotane T2207 oc " 5 Table 4 IN VITRO ASSAYS WITT2H24SA7CCCIoAcROHYCES CEREVISIAE MAecttaibvoaltiicon +: +-:: : ++ ++ CoPnecrencternattion GelIsSpuorrviavfors (vor vi) _ (e107) percent 62 w1o0 00..002255 epts o%w 0oT'.so1 5esi.se8 o0an 50 so nsw o0'.s1 e 6s m107 shloo 6s0i wws F Mitotie c Recor sbPienranm t0s7 (107) Sarvivors 335s 5517 uao 050 513000 a1e7 8s "on "0 33..00 wwse 22.00 331s -32 8 ie compound. Nerative control (1:0) Pos1t,e2t,v3e,4c-oDnlterpoolxsbutane T2207 oC 5 Table s IN VITRO ASSAYS W1I-TH22S7ACCCaIcARONYCES CEREVISIAE MAcectlavbaotlitoen + -+ -z :: ++ :i C(onvPcoeernrcternvatjt)ion_Cell(s1S0opco7vr)ivaolrePercent a M(it0o1t0i)c RecombiSnuarnva ti,vors s8s 010 3255 53.02 00..002255 6790 ww m sew aa00 a12o00 65s7 ownu 2r.0o 2r9a ws 72 lo e1 50 em 30 wo 12.00 s5i3 10o1 22.l00 2348 wsoo bila 8 20lo0 527 23 2a2 ] Table 6 IN VITRO ASSAYS WTI2T2H4S8ACCCoI.AROHYCES CEREVISIAE Compound Negative control (1,0) Pos1t,e2t,v3e,4c-oDlneepoolrybocane 7-248 coc 8 MAecctaibvoaltdieon +-+ -: :++ ++ ConcPeenrtcernattion ColSlurpvoirvaolrs (vorv/) _ (e107) percent 6e2r 1w0o 00..002255 epst w%m 0o.s1 5e.s6 0s T5%o s3o a0 o0l.s1 270 3mws sToo Pes H.)0 MitottweeRecombinantas (107) Survivors 333 55.77 u0s aE0n 3308 5s.i4s m5.o0 122 o1o3 LEes 3100 w1s3 o3Q 2&@ Conpount Negative control (0) Son1t1t2,ive cDolnetprooxlybutane 2248 coc ate 7 INYTTRD ASSAYS VI2T2HS0A8CCcHoAcRONCESCERBVISIAE iWevtesbtoilteen ;Z -B .: : +:: ii CColnPcoeacnaetrnrattyion 0o.0o25n 010 a53500 215000 i5o0 _GR_ITsS0u0pe7rt)vatipwerreaem MtToGet1ai0ls)RecosblSoumrnvtiev,o_rs es oiw 2i3 wioz wSe om6 om 1100000 64 se a9meo si1oo0 15i65e 37s0 ameo 730 IiH s se2 on6om easoh uipsl i55s0 w 8 ia 1s3e 322 g