Document 3Nq7m1dXM29nzEK7BOVyX0gDD

-- nevrs, A ------. "3M TOMPANY'S COMPOUND T-3290CoC ~oeETs Final Report November 1982 se | LSEivEDY i ifaNd i ) (c E= C= (-- " Deb L. Uenihasas Debra E. Verbaere, Microbiologlist pSTSeae--Ll--Boe--re r2ns sccenons p51L1pscoommiese ies 1 pease 34s S-- Bo tn WUiAAS HSehne ((fmeern GL oRTaT D === sRoN sMoAs 0222 SUMMARY SRI International examined 3 Company's Compound T-3290CoC for mutagenic activity with strains TAIS3S, TALS37, TALS3, TAS, and TAL00 of bacteriun Salmonella typhimurium in the standard Ames Salmonella/microsome in vitro mutagenicity assay. Coapound T-3290CoC was also screened for recombinogenic activity with the yeast Saccharomyces cerevisiae D3 assay. Both assays were performed in the presence and absence of a ratLiver metabolic activation system. Compound T-3290CoC was found to be neither mutagenic nor recombinogenic when tested using these procedures. 1 600223 contents SUMMARY.ceeeeensennnnnnnneennannnnennnsnnananannes 41 INTR++sO svssD sssU snnnC nnnnT nnnnInnnO nnnnN nnnn. nne 1 MATERIALS +eveeeerenereeneesssannnnnesennenennnnns METHODS +vveeerennnnneeeesennnnnneaenesaananaaes 3 RESULTS AND DISCUSSION.ssveevvussreveeeneeasaeeaes 9 TABLES Table Luvvvsernnsssssssssssnnnsnsseessesneees 10 Table 2uvvuvsnnnnnnnnnnnnnnnnnninnnnieninne 11 Table 3ueeerrrrrrreeeeeennnnnneeeeenaanaeaees 12 Table huvrrrrrrrrersreeserssneereereerneeeees 13 111 00224 INTRODUCTION SRI International examined 3M Company's Compound T-3290CoC for mutagenicity by invitro microbiological assays with strains TALS3S, TALS37, TALS38, TA%, and TALOO of the bacteriua Salmonella typhimurium in the standard Ames Salmonella/microsose assay 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 microorganisms either are incapable of conducting or do not 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 not carcinogenic. The assay procedure with S. cerevisiae is about 60% reliable in detecting carcinogens as agents that increase mitotic recombination. However, because the assay systems do not always provide 100% correlation with carcinogenicity investigations in animals, neither a positive nor a negative response conclusively proves that a chemical is carcinogenic or noncareinogenic to man. 1 00225 MATERIALS Zest Compound - Mame: 1-3290CoC - Date Received: 8 October 1982 - Description: Yellow-amber quid - Storage Conditions: Room temperature - Special Testing Conditions: Nome Indtcator organisms - Species: SSaaclcmhoanreolnlyacest.yphceirmeuvriisuimae172 - Strains: 5TA.LS3tSy,phTiAsLuSr3i7u,n; TADL3SIfSo,r ST.ASSc,eraenvdisiTaAeLOO for * Metabolic Activation Aroclor 1254-induced rat-Liver $-9; SRI Batch E-8; ~ 31 ag/al protesn. o Solvent Used Sterile vacer 0026 veTHODS Salmonella typhimurium Strains TALS3S TALSI7, TALSI8, TASS, and TALOO The Salmonella typhimuriun strains used at SRI are all histidine auxotrophs by virtue of utatfons in the histidine operon. When these histidine-dependent cells are grown on minimal sedius ager plates contatning a trace of histidine, only those cells that revert to histidine independence (his*) are able to form colonies. The small amount 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* revertancs are easily visible as colonies against the slight background grouth. The spontaneous mutation frequency of each strain is relatively constant, but vhen a mutagen is added to the agar, the mutation frequency is tncreased, usually in a dose-related manner. Ve obtained our S. typhimurium strains from Dr. Bruce Anes of the University of California at Berkeley. In addition to having mutations tn the histidine operon, all the indicator strains have a mutation (rfa) that leads to a defective lipopolysaccharide coat; they also have a deletion that covers genes involved in the synthesis of the vitamin biotin (bio) and in the repair of ultraviolet (uv)-tnduced DNA damage (urd). The rfa mutation makes the strains more permesble to many large molecules, thereby increasing the mutagenic effect of these molecules. The wrb mutation renders the bacteria unable to use the accurate excision repair mechaniss to remove certain cheatcally or physically induced DVA lestons and thereby enhances the strains' sensitivity to sose mutagenic agents. Strata TALSIS 1s reverted to his* by many mutagens that cause base-pair substitutions. TALO0 1s derived from TALSIS by the introduction of the resistance transfer factor, plasaid pR1OL. This plassid is belteved to cause an 3 00227 increase fn error-prone DNA repair that leads to many more mutations for a given dose of most mutagens. In addition, plasaid pRMIOL confers resistance to the antibiotic ampicillin, which is a convenient marker to detect the presence of the plasaid in the cell. The presence of this Plasaid also makes strain TALOO sensitive to some frameshift mutagens [e-g., ICR-191, benzo(a)pyrene, aflatoxin B,, and 7,12-dimethylbenz~ (a)anthracene]. Strains TALS and TALS38 are reverted by many frameshift autagens. Strain TAS is derived from TASB by the addition of the plasmid pRNIOL, which makes it more sensitive to some mutagenic agents. All fndicator strains are kept frozen in nutrient broth supplemented with 10% sterile glycerol at -80C in 1-ul samples containing about 10 cells. New frozen stock cultures are made every 3 months from single colony isolates that have been checked for their genotypic characteristics (his, rfa, uveB, bio) and for the presence of the plasmid. For each experiment, the frozen l-al samples are allowed to thaw at room temperature before fnoculatfon fn 50 ml of glucose minfmal liquid medium supplemented with an excess of biotin and histidine. The cultures are grown at 37C, unshaken for 4 hours, then gently shaken (100 rpm) for 11 to 14 hours. ALL strains are genetically analyzed whenever experiments are performed. Aroclor 1254-Stinulated Metabolic Activation System Some carcinogenic chemicals (e.g., of the aromatic amine type or the polyeyelie hydrocarbon type) are inactive unless they are metabolized to active forms. In animals and 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 Sprague-Dauley rats (200 to 250 g) are given a single 500 mg/kg intraperitoneal injection of Aroclor 1254 (a mixture of polychlorinated biphenyls). This treatment enhances the synthesis of enzymes involved in the metabolic conversion of cheatcals. Four days after the injection, the " 00R28 antsals' food is removed but drinking water is providedad libitwn. On the fifth day, the rats are killed and the liver homogenate is prepared as follous. The livers are removed aseptically and placed in a preveighed sterile glass beaker. The organ veight is determined, and all subsequent operations are conducted in an ice bath. The livers are washed with an equal volume of cold, sterile 0.15 M KC minced with sterile surgical scissors in three volumes of 0.15 KCl, (3 ml/g of wet organ), and homogenized with a PotterElvehjem apparatus. The homogenate is centrifuged for 10 minutes at 9000 x g, and the supernatant, referred to as the S-9 fraction, is quickly frozen on dry ice and stored at ~80C. The metabolic activation mixture for each experiment consists of, for 10 m1: 4 1.00 al of 5-9 fraction 0.20 ml of MgCl, (0.4 M) and KCL (1.65 4) 0.05 ml of glucose-6-phosphate (1 ) 0.40 ml of NADP (0.1) 5.00 ml of sodium phosphate buffer (0.2 , pH 7.4) 3.35 al of BO. Plate Incorporation Assay Prior to testing, the test article is serially diluted from an initial stock. The dose levels are based on the results of a preliminary rangefinding experiment. The article is usually tested over a minimum of six dose levels, the highest nontoxic dose level being 10 mg/plate unless solubility, mutagenicity, or toxicity dictates a lower upper limit. All assays are repeated at least once on a separate day. s 00229 The plate incorporation assay is performed in the following way. To a sterile 13 x 100 m test tube placed in a 43C heating block ve add: (1) b2.i0o0timn1anodf 00..60%5 waigarhisctoindtianiening 0.6% NaCl, 0.05 my (2) 0.05 ml of indicator organisms (about 10 bacteria) (3) 0.05 ml of a solution of the test article (4) 0.50 al of metabolic activation mixture (if appropriate). This mixture is stirred gently and then poured on plates containing about 25 ul of minimal glucose agar. After the top agar has set, the plates are incubated for 48 hours at 37C. The number of his* revertant colonies is counted using a BloTran II automated colony counter when possible. When accurate counts cannot be obtained (e.g., because of precipitate), the plates are counted manually using an electric probe colony counter. Concurrent sterility, negative (solvent), and positive controls are run with every experiment. Sterility controls include plating out separately steps (3) and (4). For negative controls, we use steps (1), (2), (4), and 0.05 al of the solvent used for the test article. For positive controls, ve test each bacterial culture with the following mutagens using steps (1), (2), ), and (4): TSAoLd0i0u.m szide for the base-pair substitution mutants TAIS3S and 9-Antnoacridine for the frameshift mutant TALSI7. e 2-Nitrofluorene for the frameshift mutants TASS and TASS. a2e-tAanbtohlriacminaectifvoartiaolnl. tester strains, in the presence of Saccharomyces cerevisiae D3 The yeast S. cerevisiae D3 is a diploid microorganisn heterozygous for a mutation leading to a defective enzyme in the adentne-metabolizing pathway. When grown on mediua containing adenine, cells homozygous for this mutation produce a red pigment. These homozygous mutants can be generated froa the heterozygotes by mitotic recombination. The frequency of this recombinational event may be increased by incubating the organisms with various carcinogenic 6 (0030 or recombinogente agents. The recombinogentc activity of a compound or its metabolite is deterained fron the number of red-pigmented colonies appearing on test plates. A stock culture of 5. cerevisiae is stored at 4C. For each experiment, broth contatatng 0.05% MgSO, 0.15% KiyPO;, 0.45% (NH,),S0, 0.35% peptone, 0.5% yeast extract, and 2% dextrose is inoculated with a loopful of the stock culture and incubated overnight at 30C with shaking. The in vitro yeast mitotic recombination assay in suspension is conducted as follows. The overnight culture {a centrifuged and the cells are resuspended at a concentration of 10 cells/ml in 67 uM phosphate buffer (pil 7.4). Toa sterile test tube are added: R 1.00 al of the resuspended culture 0.50 al of either the metabolic activation mixture or buffer 0.20 al of the test chemical + 0.30 al of butter. Several doses of the test chemical are tested in each experiment, and appropriate controls are included. The suspension mixture is incubated at 30C for 4 hours on a roller drua. The sample fs then diluted serially in sterile physiologic saline, and 0.2 al of the 105 and 1073 dilutions is spread on plates containing the same ingredients as the broth plus 2.0% agar; five plates are spread with the 10-3 dflucton and three plates are spread with the 1075 aflution. The plates are incubated for 3 days at 30C, followed by 1 day at 4C to enhance the developnent of the red pigaent indicative of adenine-deficent homozygosity. Plates containing the 1073 dilucion are scanned with a dissecting microscope at 10 x magnification, and the number of mitotic recombinants (red colonies or red sectors) is recorded. The surviving fraction of organisas is deternined fron the total number of colonies appearing on the plates of the 105 af1utton. 7 00231 The positive number of response mitotic in this recombinants is calculated per assay is indicated by a dose-rel 105 ated s urvivors. increase o f A ore than 3-fold fn the sbsolute nusber of mitotic recombinants per milliliter as well as fn the relative number of mitotic recombinants per 10 survivors. References AFnreasn,eshBi.fNt.,muE.tagGe.nsG:urneMye,tabJ.oliAt.esMilalnedr,derainvdatHi.veBsartofsch2.-acCeatryclianaoigneonfsluoarsene and oatshmer).aromatic amine carcinogens. Proc. Nat. Acad. Sci. USA 69, 3128-3132 mAnuetsa,genBs.: N.,A Ws.impEl.eDutressttons,ystE.emYacmoassbaiknii,nganldiveFr. Dh.omolgeee.nateCsarefionrogaecntsivaatrieon and bacteria for detection. Proc. Nat. Acad. Sci. USA70, 2281-2285 (1973). FAnoers,theB. deN.t,ectF.ionD. aLnede,claansdsiWf.icEa.tiDounrsotfonm.tagAennsimparndovecdarcbiancotgeernisa.l tePsrtoc.syNsatte.m Acad. Sci. USA 70, 782-786 (1973). Ases, B. N., J. McCann, and E. Yamasaki. Methods for detecting carcinogens ManudtatmuitoangeRness. w3i1t,h 3th4e7-S3a6l5m(o1n9e7l5l)a./mammalian-microsome mutagenicity test. Btreucshincikq,uesD.wiJt.,h aynedastV.. W.EnvMiaryoenr.. HeNaelwthdevPeelrosppmeecnttisvesin6,mut83a-g8e6nic(i1t8y73)s.creening Kciiegra,retLt.eD.s,uokEe. Ycaomnadseanksia,tesa.nd BP.rocN.. NAamte.s.AcaDde.tecSctii.onUSoAf 7m1u,tag4e1n5i9c-41a6c3tiv(i1t9y74)i.n McCann, J., E. Choi, E. Yamasaki, and B. N. Ames. Detection of carcinogens as mWuatt.ageAncsad.in Stcih.e USSaAlm1o2n,el9l7a9/-m9i8c3ros(o1m9e75)t.est: Assay of 300 chemicals. Proc. McCann, J., B. and N. Ames. Detection of carcinogens as mutagens in the PSraolcmo.neWaltl.a/mAicacdr.osoSmcei. tUesStA:73,Ass9a5y0-9o5f4 3(01097c6h)e.micals: Discussion. Voofrtpellaasamnisd,R4K.6 fE.r,omanidtsB.wltA.ravD.iolSetto-cpkerro.tectSienggregpartoipeorntyo.f tMhoel.muGteant.orGepnreotp.erty 167, 317-327 (1979). Zniiotmreorunsana,ctdF,. 1K.-,metanhdyl-R.3-nSicthruoal-elr-.nitIrnodsuocgtuiaonnidoifnemitaondticothgeernealckoynlvaetrisnigonawgietnhts in Saccharonjces cerevisiae. Mol. Gen. Genet. 100, 63-69 (1976). 5 00R32 RESULTS AND DISCUSSION 3 Company's Compound T-3290CoC was screened for mutagenic activity with the standard Anes Salmonella/microsome in vitro mutagenicity assay using the five standard Ames strains of Salmonella typhimurium: TALS3S, TALS37, TALSIS, TASS, and TAL00. This compound vas assayed on two separate days,11 October and 18 October 1962, each time over a dose range of 10 to 5,000 ug/plate, with two plates per dose level, using sterile water as the solvent. All assays were performed both in the presence and in the absence of a rat-liver metabolic activation system. This compound foamed when vortexed; however, this did not appear to interfere with the testing. No dose-related fncrease fn the nusber of histidine-independent revertants vas observed in either of the two assays. Therefore, ve conclude that Compound T-3290CeC Ls nonmutagenic when tested by these Procedures. Data from these assays are presented in Tables 1 and 2. Compound T-3290CoC was also assayed for recombinogentc activity using the yeast Saccharomyces cerevisiae D3 assay for mitotic recombination. This assay was performed on tuo separate days, 11 October and 18 October 1982. This compound was tested twice over the dose range of 0.05% to 5.0%, both with and without a rat-liver metabolic activation system. Compound T-3290CeC vas found to be reproducibly nonrecosbinogentc when tested by these procedures. No dose-related increase in the number of mitotic recombinants per 103 survivors vas observed. Data from these assays are presented in Tables 3 and 4. 5 006233 pe 1m ven ass wir ones Tv EpecinenCtompuotuensd T11CGoCber 1982 pnt compo tle, ae TER I PE SFtretttet tGeostests :- wEowOSew RmoEm BwoEm BweEs Fodictivme sCetnec - Leo sor wo rcaeocitanee P5 ne ws ws wm 5 tance -s :i: m L:: oomoaw aasa d boaoa wow mmolm ;` gg ommeromec :::- =i1 Es BNwBaooowuposo1thmfs hwEoonmwoamoaowo wn mmaaimad nn obnomm o Do m B[A aaA d hE own ou .:: : 051 i sHEyEsosfe Pos mBwE OmoSaE mn By GwWe mm T[o5e yyoi da RmmRRBomE wimwi Tate2 IN YATHD ASSAYS WITH SALANBLLA TYPIINORLON Compouns T-3290000 Srpertaenc bates 18 october 1982 _compou_s oheociluatceion _CpoeCmhprbooeurlndwe TE_E__W_T__TseWvuerstianhtssgerm tPlaitE e diSo0no0e0 Tega ve Gomer Startle wer :- aeoSe 54s6 Bmows myoona mwoewy fosteive Controls Sodtun Artie - te wows man Solasmoncridtne s Zatrotloree so 5 10s woe mom 8g& Zetatheentne :-: s i1 es 4s s0 a2ne2 uoeu awwm Mon3e a : Wow a ow Componnd T0000 = : 1105000 H w Bowo ow sisos4 mS5o o%a bwwooowwn dwaoouws z:: 15005000000 Bnoonrooe403os Bho wnSowdn mmNooouwn eawaoems i:+ 15100 WS $o5n7oo6wmN8 of9 hmao owl mpmoooadm nadeowmms ::: 1S500000000 Gf S6ed o3s47 mhmooEmu 3Smoawwmn wmmoaoosw Ties yycep-- T0300---- pecinnt dees 11 ctor 1982 comm peetye oi rmerie t TETTsRurvivees r. sMiaesctf,eastsoncoensann, rerertieeter b wow 0 TnStraeeiruvtietGpnieesomtesptins + . 5.3 100 6 was sa wm 3 - Settatncyein gg mpm 103000 : w cowson4a ws "oz . 0. wow a " 8g: x :::: }ob: el oHHwoooonrws i i =B.beAl i;: : ootiufs ; wvGoaoooys 1 A 0a2 :} nos i Table 4 IN VITRO ASSAYS WITH SACCHAONCES CEREVISIAE DY Compound 73290000. Expertaent Date: 18 October 1982 Compound. MMeetbaibvoaltiicon_ CeolnsFcheetrniccveeonalttuiomne GeIIv(Sx upTroovTr)iavlorpsereemt TMi(c1o0r t7e) Re ecosTbe Sgeuarrnvti1n 05svors Negative Controls Stertle water ;- wwro 11000 35 w7e.5 Possetve Controle 1,2,3,4-Diepoxsbutane - 0.023 we 10 sue 2057 Stertgmatocystin :- 00..00000055 woss 110000 py3 9% 7.0 & & Compound 73290600 Eo. -z o0.r05 l3.e8 9s 5" 1106.75 ~gc ::: 5o1's 3p36ls 2a 55 255 1w3s9 53 +3 1 o0.r08 os wost 110000 wz 100 "` 2 99.31 we 1i 51 3% 1a00 i5 s3s