Document Ex2p4Q2VDmdjVx72voyQr731R

AR RSE 0649 PFOS Genotoxicity P - Y u00048  aS = J) 0 = FBER mas -- asin-- Snes Sansary 1981 By: YaDolEtyeRLoiaEnlaTanEsrereners ne Z -- (SSEeHrrRraIs) fJ--p--sa--teen: SonariShEeppEe 2h camntner rvs 3L . Feyo iten .h SSeI S SRI Project 150-8956 serene wpe DsPeoovitsdto. nCI.4isommferbrannbirecor QL Chmtfsna [. rA. eScgimeir,mExeecuntive Director 333 Ravenswood Ave. Menla o Park, CalCifronieaS84t025ers 000049 Susur SKI International examined M Company's Compound T-2997C5C for mutagenic activity vith strains TALS3S, TALS3?, TALS, TAS, and TALOD of Salmonells typhimurium in the standard Anes Salzonella/microsone assay and with the yeast Saccharomyces cerevisiae D3 using FDA-approved GLP procedures. Each assay vas performed in the presence and in che absence of a ra liver mecabolic activation system. Compound T-2997CoC 7a not mutagenic wi recosbinogenic in any away performed. a 000050 covets SUMARY LL... om QUALITYASSURANCE STATEMENT . . . . oo .... 1v INTRODUCTION . eee eee 1 MATERIALS oe eee 2 METHODS LL 3 RESULTSANDDISCUSSION . . . . . ........... 9 anes Table lo... 10 TleZ..................... om Tabled... xm Tabled. iii... 1 ss 000051 QUALITY ASSURANCE UNIT Final Repor Statement SRI Tncernational assures the quality and integrity of this study, the Anes Salmonella Microsone assay snd the Saccharcsyces cerevisiae D3 reconbinogentc assay of the 3H Company's compounds T-2997 CoC. The study was inspected on December 12 and 22, 1980 during che atlucion, placing, and plate counting of the tvo assays. The findings of the Quality Assurance Unie Inspector vere reported at the tine of each fnspeccion to the Study Director. SRI management vas also inforsed of the inspection results on December 12 and 22, 1980, respectively. This final repore accurately describes the methods and standard eperating procedures and reflects the av data of the study. fay deviations from the spproved protocol and standard operating procedures were made wich proper authorization and documentation. _/ / om Toaticy Aghoriice omit[Titi Bate 24, 194, = 000052 INTRODUCTION SRI International examined 3M Company's Compound T-2997CoC for mutagenicity by in vitro microbiological assays with strains TALS3S, TALS37, TALS3S, TA9S, and TALOD of the bacterium Salmonells typhimurium in the standard Anes Salmonella/microsome assay and with the yeast Saccharonyces 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 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 derecting carcinogens as agents that increase mitotic recombination. However, because the assay systems do not alvays provide 100% correlation with carcinogenicity investigations in anivals, neither a positive nor a negative response conclusively proves that a chemical is carcinogenic or noncarcinogenic to man. The assays with Compound T-2997CoC were begun on 12 December 1980 and completed on 7 January 1981. Copies of the final report will be kept in our files (Building 28, Room 213) and in SRI's Records Center. The laboratory notebook will be retained in Building 284, Room 10 for one year after it is filled, and then will be stored in SRI's Record Center. ALL that is left of Compound T-2997CoC will be kept for six months in our chemical storage room (Building 28, Room 217), then returned to the 34 Company. 1 : 000053 MATERIALS + Test Compounds - Name: T-2097CoC. - Date Received: 8 December 1980. - Description: Waxy amber solid. - Stability: Stability assured by client. - Storage Condition: Stored at room temperature. - Special Testing Conditions: None. + Indicator Organisms - Species: Scearlemvoinseilalea. typhimurium LT2 and Saccharomyces - Strains: ST.ALSt3ySp,himTuArLiSu3m7;, DT3AISo3f8,5.TAc9e8r,eviasnidaeT.ALOO of + Mecavolic Activation ANIrEoHcSlorRLI102O534,-i*ndu3c0edmg/rmalt lpriovteerin5.-9; SRI Batch D7, D8, and + Solvent Used Dimethylsulfoxide (DMSO). 2 000054 METHODS TSAaIl5m3o7,nellTAaIStIyS,phiTmAu9r8i,usanSderTaAinLeODTALS3S, The Salmonella typhimurius strains used at SKI are all histidine auxocrophs by virtue of mutations in the histidine operon. When these histidine-dependent cells are grown on minimal medium agar places containing a trace of histidine, only those cells that revert to histidine independence (nis") are able to forn colonies. The small amount of histidine allous all the plated bacteria to undergo a fev divisions; in many cases, this growth is essencial for mutagenesis to occur. The his' revertants are easily visible as colonies against the slight background growth. The spontaneous mutation frequency of each strain is relatively constant, but vhen a mutagen is added to the agar, the mutation frequency is increased, usually in a dose-related manner. We obtained our S. typhimurium strains from Dr. Bruce Ames of the University of California at Berkeley. In addition to having mutations in the histidine operon, all the indicator strains have a mutation (zfs) that leads to a defective lipopolysaccharide coat; they also have a deletion that covers genes involved fn the synthesis of the vitamin biotin (bio) and in the repair of ultraviolet (uv)-induced DNA damage (urd). The rfa mutation makes the strains more permeable to many large molecules, thereby increasing the mutagenic effect of these molecules. The uved outation causes decreased repair of some types of chemically or physically damaged DNA and thereby enhances the strains' sensitivity to some mutagenic agents. Strain TALS3S 4s reverted to his' by many mutagens that cause base-pair substitutions. TALOD is derived from TAI535 by ie futroduction of the resistance transfer facvor, pissaid PRUOL. This plasaid is believed to cause an increase in error-prone DNA repair chat leads to many more mutations for a given dose of most mutagens. In addition, plasmid PALO] confers resistance to the anti biotic ampicillin, which is a convenient marker to detect the presence 5. . 000055 of the plasmid in the cell. The presence of this plasmid also makes strain TAL00 seasitive to some frameshift mutagens [e.g., ICR-191, benzo(a)pyrene, aflatoxin B,, and 7,12-dimethylbenz(a)anthracene] Strains TALS37 and TALS3S are reverted by many frameshift mutagens. Strain TASS 1s derived from TALS38 by the addition of the plasnid pRHIOL, which makes it more sensitive to some mutagenic agents. ALL indicator stratus are kept at 4C on minimal agar plates supplemented vith an excess of biotin and histidine. The plates vith the plasmid-carrying strains also contain ampicillin (25 ug/ml) to ensure stable maintenance of the plasmid pRMIOL. New stock culture plates are Bade every & to 6 vaske from single colony fsolates that have been Checked for their genotypic characteristics (his, Ifa, uvrB, bie) and for the presence of the plasmid. For each experiment, an inoculum from the stock culture plates 1s grown overnight at 37C in nutrient broth (oxom, a67). Aroclor 1254-Stimulated Metabolic Activation Syste Some carcinogenic cheaicals (e.g., of the aromatic amino type of the polycyclic hydrocarbon type) are inactive unless they are metabolized to active forns. 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. Eyphipurius test. Ames has described the liver metabolic activation system that we use. In brief, adulc male racs (250 to 300g) are given a single 500 ng/kg intraperitoneal injection of Aroclor 1254 (a mixture of polychlorinated biphenyls). This treatment enhances the synthesis of enzymes involved 4n the metabolic conversion of chemicals. Four days after the injection, the animals' food is removed but drinking vater is provided ad libitum. On the fifth day, the rats are killed and the liver homogenate is prepared as follovs. The livers are removed aseptically and placed in a preveighed sterile glass beaker. The organ weight is determined, and all subsequent operations are conducted fn an ice bath. The livers are washed ` . 000056 with 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 KCl, and homogenized with a Potter-Elvehjem apparatus. The homogenate is centrifuged for 10 minutes at 9000 x g, and the supernatant, referred to as the 5-9 fraction, is quickly frozen in dry ice and stored at -80C. The metabolic activation mixture for each experiments consists of, for 10 ml: + 1.00 ml of 5-9 fraction 0.20 ml of MgCla (0.4 ) and KCL (1.65 4) + 0.05 ml of glucose-6-phosohate (1 ) + 0.40 ml of NADP (0.1 M) * 5.00 ml of sodiun phosphate buffer (0.2 N, pH 7.4) + 3.351 of Bao. Assays in Agar To a sterile 13 x 100 mm test tube placed in a 43C heating block, we add in the following order: (1) 2.00 ml of 0.6% agar" (2) 0.05 ml of indicator organisms (3) 0.50 ml of metabolic activation mixture (if appropriate) (4) 0.05 ml of a solution of the test chemical. This mixture is stirred gently and then poured onto mininal agar 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. For negative controls, we use steps (1), (2), and (3) and 0.05 ml of the solvent used for the test chemical. Dimethylsulfoxide (DMSO) was used as the solvent for T-2476ChR. For positive controls, we test each "The 0.6% agar contains 0.05 aM histidine, 0.05 wf biotin, and 0.6% NaCl. "M0i.2nsogalofaMggaSr0.p+l7aHt,e0s, c2onsgisotf coif,tripceracliidterm,ono1h5ydgraotfe,aga1r0,g10ofgK,oHfPOgLl,ucoasned, 3.5 of NaNH,HPO,+4H,0. 5 000057 culture by specific autagens known to revere each strain, using sceps W, @, 3), ad @. Saccharoavees cerevisise D3 The yeast S. cerevisiae D3 1s a diploid microorganism heterozygous for a mutation leading to a defective enzyme in the adenine-mecabolizing pathuay. When grown on medium concaining adenine, cells homozygous for this mutation produce a red piguent. These homozygous mutants can be generated from the heterozygotes by mitotic recombination. The frequency of this recombinational event may be increased by incubating the organisms #1si. vailous carcinogenic or recostincgenic agents. The recwsbiwpewic activity of a compound or its metabolite is determined from the number of red-pigaented colonies appearing on test plates. 4 stock culture of S. cerevisiae is stored at 4C. For each experiment, broth containing 0.05% MgSO, 0.15% KHAPOL, 0.45% (NH.)aSO0., 0.35% peptone, 0.51 yeast extract, and 27 dextrose is inoculated vith a loopful of the stock culture and incubated overnight at 30C with shaking. The in viero 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/al in 67 m phosphate buffer (pH 7.4). To 3 sterile test tube are addsd: + 1.00 ml of the resuspended culture + 0.50 sl of either the metabolic activation mixture or buffer + 0.20 ml of the test chemical + 0.30al of butter. Several doses of the appropriate controls test chemical are included. are tested in each experiment, and The suspension mixture is incubated at 30C for 4 hours ona Toller drum. The sample is then diluted serially in sterile physiologic saline, and 0.2 ml of the 107% and 107% dilutions is spread on plates containing the same ingredients as the broth plus 2.0% agar; five places 6 , 000058 are spread with the 107 dilution and three plates are spread with the B10Y7"2 ddaiylsutiaotn.4CThtoe eplnahtaensce are the idnecvueblaotpeadenftorof2 days at the rad p3i0gmC,entfoilnldoiwceadtive anorufembaesdrceanonifnneemd-idtweoifttiihccieanrtedcioshmsobemicontzaiynngtgossimti(ycr.redoscPcoolplaeotneisaetsc1oo0nrxtamrieandginnigsfeictcthoaertsi1)o0n7,fs danidluttihoen recorded. The surviving fraction of organisms is decerntned fron che total number of colonies appearing on the plates of the 10TM* dilution. A posTihteivneusbreerspoonfsemitiontitchisreacsosmabyinaisntsind1iscactaeldculbyataeddopseer-r1e0latseudrviivnocrrse.sse So4fl1miolriecetrhaans 3v-eflolldastninthetheabsroelluatteivemmnbuesrberofomfimtiotroicticrerceocmobmibniannatnetspeprer. 10 survivers. nusberNoofstcaotilsotniiceaslapmpetehaordingvasonnetehdeedp.laceRsesauflttesrariencuabattaibounl.ation of the References 32Af10mr2dea6sm,7eo3st1hh33.ei2rfcN.a(,ur1oc9m7Ea2ag.)teG.in.cs:aGmuirnnMeeeyt,acbaJor.cliintAo.egseMniasln.lderd,ePrroiacvn.adtiFHa.vte.sBaAorctfaadc2.ho.aSceetCr.asriVcoeimhntoecgroei.nmsoraosne sAtmuiecosan,geannBs.d: bN.a,cAteWs.riimapE.lefoDrutresdstetotnes,cytsiEto.enm.YacmoPamrsboaickn.ii,nNgaatnl.diveAFcr.adDh..omSLoeegsee..natVeeCsnar7cf0io.rnog3be5e0nmisi.uaere 2285 (1973). PsArmyoescst.,emNB.afcorN.A,ctahdeF..deDS.ctie.LceteUi,SoAna7na0nd,d.7c6l3Ea.-s7s8iD6ufrisc(ta1ot9ni7.3o)n. oAfn miumtpargoevnesd banadctecrairaclinotpeesnts. Ms2munetdsa,tBiuotBnagNeR.ness. 3v3.i1t,MhcC3t4ah7ue-a3,S6a4"lanndo(n1e5E.7i5l)Ya.a/msassmamkail,isnhoemtihcordosscsfeorutdestpeecntiicnigtycarrcoirnnogens sBrcurseiecnki,ngD.teJc.h,iiqaunedsV.viUt.h an. yMeaaysetr.. NEnevwirdoenv.elHoepamletnhtsPefrnspmeucttaigveensic8i.ty63-86 ; . 000059 Kier, L. activity D., E. Yamasaki, and B. N. Ames. in cigarette smoke condensates. Detection Proc. Nat. of mutagenic Acad. Sci. USA 71, 4159-4163 (1974). McCann, gens as Jm.u,tagE.ensChoiin, tEh.e YSaamlamsoankeil,la/amndicrB.osoN.meAmteess.t: Detection Assay of o3f00ccahrecmiincoa-ls. Proc. Nat. Acad. Sci. USA 72, 5135-5139 (1975). McCann, J., carcinogens N. as mE.utaSgpeinnsg:arn,BacJ.terKioabolrit,esatnerd B. N. Ames. strains with Detection of R Factor plasmids. Proc. Nat. Acad. Sci. USA 72, 979-983 (1975). Mortelmans, property of Kp.lasEn.,id aRnd46B.fAr.oDm. itSstocukletrr.avioSleegtr-egpartoitoenctiofng tpherompuerttayt.or Mol. Gen. Genet. 167, 317-327 (1979). Zwiitwhdenrintarno,usT.aciKd.,, al-ametRXh. ylSc-h3v-aniietrr.o-l-Inaidturcoisiougauaonfimdiitnoetiacnd goetnheercoamlvkeyr-sivu lating agents 961). in Saccharomyces cerevisiae. Mol. Gen. Genet. 100, 63-69 8 400060 RESULTS AND DISCUSSION Compound T-2997CoC vas tested for mutagenicity with the Anes Salmonella/microsome assay and with the yeast Saccharomyces cerevisiae D3 assay in the presence and in the absence of a metabolic activation system. The compound was tested twice on separate days in both assays. Dimethylsulfoxide (DMSO) was used as the solvent in all assays. In the Anes Salmonella/microsome assay, Compound T-2997CoC was tested over a wide range of concentrations, from 10 to 5,000 yg/plate (Table 1). Because no toxicity occurred at 5,000 ug/plate, the second test was run using a higher dose range, from 50 to 10,000 ug/plate (Table 2). Still no toxicity or dose-related increase in the nusber of revertants per plate was observed in either assay. In the microbiological assays with S. cerevisiae D3, T-2997CoC was tested over a wide range of concentrations, from 0.05 to 5.0% w/v (Tables 3 and 4). No toxicity or dose-related increase in the number of mitotic recombinants was observed in either assay. We conclude that Compound T-2997CoC is not mutagenic with S. tvphimuriun or recombinogenic in S. cerevisiae. 5 000061 | | Table 1 | I vo sans wim sane TRIN comomo 299100 Experiment ace: 12 becener 1980 Compound Jveetrsivvoatticion Gaglpniatde) RSS IMAaciIatne I 1 En Tog [-- so -: BBooBas 7 mwo g wRoNw mosm mo Ponttve Contras FR Sotton ard - Lo 00 ae am 00 527 ww [Ee Zotac - 50 Lo : io ne ome avoas am om awawh Samse Hwee . :: 3Lemw as oaog Compound 2587000 wmWooo bmoooamboo3336dssiaas3 momm om3oR iRwbioeee SSooouessoelo mmmooonimoso o53& 3sos 43hodaa mdRooEnm oomaMmiiWnn <i} $ +i : PwE oos [roSE A A BN ma WJEEon mn1 g$s iSTooosma S0e0 aeS3 eBlhs o53aat5 ddSoomRwo RdouoHdRoon eB1% Wnbi Tanke 2 IN VITRO ASSATS WITH SALMONELLA TPM convounD 1-2997c0c Experiaent Date: 15 becesber 1980 Compound Negative control mso Fostetve contrata Sodom hate fo Smtesceitine F amtecotivorene P-- . Compound T299C0C g 28a Whecttaibvoatttieon :- -: :-: :: :I .: +:: bo Co"mppaoiunnd Gglyiace) ro s0.0 50 1100 055 wSo0o0 Loswoe dsooeo 10500.00 510,50000000...000 dls IASI oo IAWiSstIidiaeRIeveArtanStsIpor TPlAate "ooae se7 sa a8 sonodoaou a ae on os a a nou ws B5us1s 1us 6ow ama ost a1789 135 8snas 73 B9R1 84 bnosd ad dLe oL17 91s 62 82 16 a4 68 47 17ah um e Ts osse50 som 7 oe oswona 1s Mwa8 dwnm G6ss7e 1s0s46 1sawon mwuonan aia 1o0 mm0 ww m9 oon 19m0omtsean nsimb un wwsiooewn mwwo ows IN VITO ASSAYS WITHTaSstaecc3unmces cerevisiAE D3 . conoud 1-2097c0c Experiment Dace: 17 becenber 1980 Conpoun AMecttaibvoaltiicon C(ounupceeronrcternwat/tvion ToTTSSup1r0ev7i)voforspercent MFeGiratlIoe Dti)c Recr osbisnuarni vtisv.ers Negative Conerol so :- ea s mim p2s0 5208 Posttive Control . 1.2,3,4-tepoxybatane :- l0.o02z5 6ssz nsno ono 1100000..00 Goapound T-2997C0 :- hots 5 0672s0 5as a rLoo ro a1s is :: s1o0 piies aes 010 iies : +i+ k50ys a5150 o5s 1100 ped 1174 re +: 3i%o 5 m53 2il0o biee 228? | Compound Negative Concrol so Postetve control 1,2.3,4-Dtoporybutane compound 12997000 : Tove + I VITRO ASSAYS WITH SACCHARONYCES CEREVISIAE DY cowound 1-2997c0c. Expertnent Date: 7 January 1981 AMeettatbaotlitoen Conpceerncternattion (vor viv) GoTTGsISupOertv)iva1orsperemt RCMiGt1ot0ic) RecosbiSnearnstisvers -+ s swam 550s s5i i- 00.003525 ssiae 1s a50m0.o0 1188000..00 -: : k0ts is : 10 ie Pin o5"1 w32o00 &21 &5 10 1s : slo i+ 0kts so 7 6 1 m0 5 o2o0 12s 1323 +i+ briyos o8Ssi5 a55m 3750 w57o%7 :&