Document vB20nQv4yXerJd35OVZ5e5EvE

ARLG-O133 Final Report Salmonella - Escherichia coli/Mammalian-Microsome Reverse Mutation Assay with PFOS -- EL 01923 COVANCE FINAL REPORT SALMONELLA - ESCHERICHIA COLIMAMMALIAN-MICROSOME REVERSE MUTATION ASSAY WITH PFOS AUTHOR Michael S. Mecchi, MS PERFORMING LABORATORY Covance Laboratories Inc. (Covance) 9200 Leesburg Pike Vienna, Virginia 22182 LABORATORY PROJECT IDENTIFICATION Covance Study No.: 20784-0-409 3M Study No.: T6295.17 SUBMITTED TO 3M Corporate Toxicology 3M Center Building 220-2E-02 St. Paul, Minnesota 55144-1000 STUDY COMPLETION DATE November 5, 1999 1of24 . 01924 -_ CoCvoavannccee22007784-0044009 QUALITY ASSURANCE STATEMENT Salmonella-Escherichia colitMammalian-Microsome Reverse Mutation Assay with PFOS a`TchceorredpaonrctehwaistbhetehnerGeovoidewLeadbobryatthoeryQuParlaicttyicAesrseugrualnatcieoUnnsiatsosfeCtofvoratnhcien tLhaeboErnavtiorroinemseInntc.a,lin TPrhoetefcotliloonwiAnggenincsype(cEtPioAns- wTeSrCeA)c,onTdiutclete4d0oafntdhteheUf.iSn.diCnogdseroefporFteeddertaoltRheegSutluatdiyDoinsrePcatrotr7a9n2d. Cstouvdayndcieremcatonramgaenmaegnetmaecncto.rdWirnigtttoenstsatnadtuasrdreoppoerrtastoifnignsprpoeccetdiuorness.and findings are issued to Inspection Dates From To 9999 919/99 Phase Preparation of $9 Mix Dates Reported to Study Director and Study Director Management ___ Auditor 919/99 J. Crouch 1021/99 1021/99 Draft Report Review 1021/99 K. Groeninger 11/04/99 11/04/99 Final Report Review 11/04/99 K. Groeninger i < Representative, Qualify Assurance Unit ufoy[35 Date le 01325 Covance 20784-0-409 STUDY COMPLIANCE AND CERTIFICATION The described study was conducted in compliance with the Good Laboratory Practice regulations as set forth in the Environmental Protection Agency (EPA - TSCA), Title 40ofthe U.S. Code of Federal Regulations Part 792, and with any applicable amendments. There were no significant deviations from the aforementioned regulations or the signed protocol that would affect the integrityofthe study or the interpretationofthe test results. The raw data have been reviewed by the Study Director, who certifies that the evaluation ofthe test article as presented herein represents an appropriate conclusion within the contextofthe study design and evaluation criteria. All test and control results in this report are supportedbyan experimental data record and this record has been reviewed by the Study Director. Study Director: Wet LS Wheel Michael S. Mecchi, MS Genetic and Cellular Toxicology 1-5-9% Study Completion Date 3. 01926 TAOFBCOL NTEE NTS Covance 20784-0-409 Page No. STUDYINFORMATION oie] Sponsor TestArticle AssayInformation StudyDates Supervisory Personnel OBIICTIVE 1sssumtiossssasessormnmnssnpmctsnttmpimessssstnistammntasasiesd TEST SYSTEMRATIONALE reer SE ns MATAENDR METI HODA S.LS o.oo 8 Test System TestArticle ControlArticles 59 Metabolic Activation System Dose Rangefinding Assay Mutagenicity Assay PlatingProcedures ScotrheiPlnatges DATA ssissigessonssssescsssmsmscessnrinesssprsscsstsrtisssssssnicessnssss1s Data Presentation Assay Acceptance Criteria Assay Evaluation Criteria Test Article Handling Dose Rangefinding Assay Mutagenicity Assay CONCLUSION iii eee 17 REC TOO BEMR AIND TAINSED ............ccooomiieinnniinnnesinnnnnnn 18 Co 01927 TABLE OF CONTENTS (Continued) Covance 20784-0-409 PageNo. os. } 01923 -_-- CoCvoavnacnece2027078844.-0-44009 ABSTRACT ``Tmhuetaotbijoencsteiivteohefrtihnisthsetpurdeyswenacsetooreavbasleuantceetohfemtaesmtmaartliiclaen, PmFiOcrSo,sofomraltheenazbiylmietystaoti1n)dtuhcee reverse htirsytpitdoipnhealnocluoscuisnoftEhescgheenroimcehoifa scoelviersatlrasitnraWiPn2soufvSraAl.monella typhimurium and at 2) the rTahnegedfoisnedsintgesatsedsaiynutshienmgutteasgteernisctiratiynsasTsAay10we0raensdelWePc2teudvbraAseadnodntetnhedroesseusltosfotefastdaortsiecle ranging from 6.67 to 5,000 pg per plate, one plate per dose, both in the presence and absenceof$9 mix. T`TAh9e8t,esTteArLs0t0ra,inTsAuIsSe3d5,inTtAhe15mu3t7agaenndicEistcyhearsiscahyiwaecroeliStaelsmtoernesltrlaaintyWpPh2iumruvrAi.um Ttehsetaersssatryawinass ccoonntdruocltseudsiinngbotthhretehpelaptreessepnecredoasned. aTbsheencdeosoefs St9esmteidxwailtohngthweiStahlcmoonnceulrlraenttesvteerhisctlreaiannsdweproseitive 35,3030,01,030,,33303,.3,1,1000.00,,333.33,3,1010.,00a,nadnd330..333u3gppgepreprlatpelaitnetihnetphreeasbesnceenocefoSf9Sm9imxixa.ndW5i,t0h00t,es1t,e0r00, strain in the WP2uvrA, the doses tested presence and absence of $9 were mix 5,000, 3,330, 1,000, 333, 100, and 33.3 ug per plate both iTnhdeicraetseultthsaotfutnhdeerStahlemocnoenldliat-iEosncsohefrtihcihsiastcuodyl,i/3MMamCmoarlpioarnat-eMiTcorxiocsoolmoegyR'esvetersstearMtuitclaet,iPoFnOASs,say sdtirdainnosteciatuhesreianptohseitpirveeseinnccreeoarseabinsetnhceemoefamnicnruomsboemraolferenvzeyrmteasntpsrpeeprarpeldatferwoimth anyofthe tester AsoclorTM-induced rat liver ($9). "8s : 01929 Covance 20784-0409 STUDY INFORMATION Sponsor 3M Corporate Toxicology Test Article Sponsor's Identification: PFOS FC-95 Date Received: 08/19/99 Lot217 Physical Description: white crystalline powder Storage Conditions: room temperature Assay Information `TypeofAssay: Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay Protocol No.: 409, Edition 5 Covance Study No.: 20784-0-409 Study Dates Initiation Date: 08/20/99 Experimental Start Date: 08/24/99 Experimental Termination Date: 09/15/99 Supervisory Personnel Study Director: Michael S. Mecchi, MS Laboratory Supervisor: Carlos E. Orantes, BS OBJECTIVE `The objectiveofthis study was to evaluate the test article and/or its metabolites for their ability to induce reverse mutations either in the presence or absenceofmammalian microsomal enzymes at 1) the histidine locus in the genomeofseveral strainsofSalmonella typhimurium and at 2) the tryptophan locusofEscherichia coli strain WP2uvrA. TEST SYSTEM RATIONALE `The Salmonella/Mammalian-microsome reverse mutation assay detects point mutations, both frameshifts and/or base pair substitutions. The strains ofSalmonella typhimurium used in this assay are histidine auxotrophs by virtue of conditionally lethal mutations in their histidine operon. When these histidine-dependent cells (his-) are exposed to the test article and grown under selective conditions (minimal media with a trace amountofhistidine) only those cells .7- . 61930 Covance 20784-0-409 h`iwshtiicdhinreevienrtthteo mhiesdtiiadianlelionwdseaplelntdheenpcleat(ehdisb+a)ctaerreiaabtloe utnodfeorrgmo caolfoenwiecse.ll Tdhiveistiroancse,awmhoiucnhtiosf . essential for mutagenesis to be fully expressed. The his+ revertants are readily discemable as tceosltoenritersaiangasi,nbsottthhebalsiemiptaeidr bsaucbsktgirtouutinodn gmruotwatthioonfsthanedifsr-amceelslhsi.ftBmyutuatitliioznisngcsaenvebrealdedtiefcfteerde.nt a`cTthievAitmyeosf mTeasntyhmaastbereieanlsshioncwlnudtionbgeaawisednesirtainveg,eorfacpihdemaincdaalccculraastseesi.ndicatorofthe mutagenic `The Escherichia coli WP2uvrA reverse mutation assay detects point mutations, specifically base apauixrotsurbospthit(u1t7ipo-n)s.byTvhiertEusecohfeariccohnidaitcioolnatlelsytelretshtarlaimnuWtaPt2iuonvratAaussietde wihnitchihsbalsoscakysisaastterpypotfophan tryptophan biosynthesis prior to the formation of anthranilic acid. Since the target site for true back mutation is an ochre nonsense mutation, tryptophan-independent revertants (irp+) can arise cither by a base change at the siteofthe original alteration or by suppression by specific suppressor mutations at a second site in tRNA genes (Brusick et al., 1980). When the tryptophan-dependent cells (1rp-) are exposed to the test article and grown under selective conditions (minimal media with a trace amountoftryptophan) only those cells which revert to tryptophan independence (7+) are able to form colonies. The trace amountoftryptophan in the `media allows all the plated bacteria to undergo a few cell divisions, which is essential for mutagenesis to be fully expressed. The trp+ revertantsarereadily discemable as colonies against the limited background growthofthe trp- cells. While the trp reversion system responds to most alkylating agents, base-analog mutagens and certain metals (i.e. soluble chromates), frameshift `mutagens would not be expected to be detected by this system. MATERIALS AND METHODS The experimental materials, methods and procedures are based on those described by Ames et al., (1975) and Green and Muriel (1976). Test System Salmonella typhimurium. The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TALS3S, and TALS37as described by Ameetasl, (1975). The tester strains in use at Covance were received directly from Dr. Bruce Ames, Department of Biochemistry, UniversityofCalifornia, Berkeley. The specific genotypesofthese strains are: shown in the following table. Tester Strain Genotypes Histidine Mutation HsG46 hisCI0T6 hisD30S2 TAIS3S TALS} "Additional Mutations LPS Repair R Factor da wrB - -8- 0191 -_-- Covance207840 Covan4 ce 200 784-0 0409 `Imnuatdadtiitoinosnwthoiachmuetnahtainocne itnhetihre sheinsstiitdiivnietyopteorsoon,methmeutteasgteernistcrcaionmspcoounntdaisn. tTwhoeadrdfiatwiaolnallmutation results in the lossofoneofthe lipopolysaccharide barrier that enzymes forms the responsible for the synthesisofpart of surfaceofthe bacterial cell wall. The the resulting cell wall rdienfgicsiyesntceymisnc(ir.eea.sbeesnpzeor[maelapbyirleintey)ttohcaterwtoaiunldclaostsheesrowficsheebmeicexacllsudsuecdhbaysatnhoosremaclonitnatianctincgelllarwgalel. sTyhsetseemcwohnidcmhutgraetaitolny,eanhdealnecteisontohfetshenesiutrivBitygeofnteh,erseseulsttsraiinnsatdoefsiocmieentmuDtNagAenesx.ciSsiionnceretphaeiruvrB vdeilteatmiionnbeixotteinndfsorthgrroowutghh.the bio gene, alofthe tester trains containing this deletion require the Strains TA98 and TA100 also contain the PKMI01 plasmid which further increases the sseennssiittiivviittyyotfotmhuetsaegsetnrsaihnasstobeseonmesumgugteasgteednst.o bTehbeymmeocdhiafnyiisnmg bany ewxhiiscthintghibsacptleraisamliDd NinAcreraepsaeisr polymerase complex involved with the mismatch-repair process. Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. TAIS35 is reverted by base substitution mutagens substitutions. and TA100 is reverted by mutagens which cause both frameshifis and base Escherichia coli. The tester strain used was the tryptophan auxotroph WP2uvrA as described by C`GorleleenctainodnoMfuIrnideulst(r1i9a76l)B.actTehreiat,esTtoerrrsetyraRiensienaurscehaSttaCtoivona,ncSceowtalasnrde(cUeniivteeddfKrionmgTdhoem)N.ational In addition to a mutation in the tryptophan operon, the tester strain contains a uvrA DNA repair tdheefisctireanicnytowhsihcohweennhhaanncceesditmsutseanbsiiltiitvyitsyitnocestohmeeumvurtAagreepnaiicr csoymsptoeumnwdosu.ldTnhoirsmdaelfliyciaecntctyoallows remove the damaged part of the DNA molecule and accurately repair it afterwards. iTensdteeprenstdreaninceW(Pp2routvortrAopihsyr)evbeyrtbeadsefrsoubmsttirtyupttioopnhmauntdaegpeennsd.ence (auxotrophy) to tryptophan gFrroowzienngPferresmhaonveenmtiSgthotckcusl.tuFrerso,zeanddpienrgmdainmeentthysltsouclkfsooxfitdhee(tDeMstSeOr ,str0a.i0n9swmeLr/emprLoepfacrueldtubrye) and freezing small aliquots (0.5-1.5 mL) at <~70C. Master Plates. Master platesofthe tester strains were prepared by streaking each tester strain from a frozen permanent stock onto minimal agar appropriately supplemented with 1) for 9. . 01932 Covance 20784-0409 `STaAl1m0o0n,elalmapitcyiplhliinmu(r2i5umu,g/amnLe)x,cteosseonfsuhriesttihdeinset,abalnedmbaiionttine,naanncdefoofr tthesetperKsMtr1a0in1s pTlAa9sm8ida;ndand 52)23forCE.scherichia coli, an excessoftryptophan. Tester strain master plates were stored at iInnooccuullaatteidonbyoftrOavnsefrenrirignhgtaCcuolltounryesf.roOmvtehmeiagphptrocpurlitautreesmafosrteursepliantealltotaesftliansgkpcrooncteadiunriensgwceurlteure o`pmeerdaituimo.n (Isnhoackuilnagt,ed12fl5as+ks25weprme;pliancceudbaitnioans,h3ak7e+r/2incCu)bastootrhwathitchehowvaesrnpirgohtgrcualmtmueredstwoebreegiinn log phase or late log phase when turbidity monitoring began. lHeanrgvtehosftoinfcuObvaetrinoinghwtasCudlettuerremsi.neTdobeynsspuercettrhoapthcoutlotmuertersiwcemroenihatrovreisntgeodficnullattuerelotgurpbhiadsitey,.the tCrualntsumrietstwanecree(h%aTrv)esrteeaddionngcoenaparsepdeectterropmhionteodmetturebri.diTthyiwsatsarrgeeatcthuerdbiadsitdyeteensrumriensedthbatyaculpteurrceesnt have reached a densityofat least 0.5 X 10 cells per mL and that the cultures have not Couvletrugrreoswnw.erOevreregmroovwend (fsrtoamtiionncaruyb)atciuolntuwrehsemnatyheetxahrigbeitt%deTcrweaasserdesaecnhseidtiavnitdywteorseopmleacmeudtaagtens. 53C. Confirmation of Tester Strain Genotype. Tester strain cultures `genetic markers on the dayoftheir use in the mutagenicity assay. were checked for the following #fa Wall Mutation. For the Salmonella typhimurium tester strain cultures, the presenceofthe vfiaolewta.llAmnutaaltiiqouontowafasncoonvfeimrimgedhtbcyudletumroenosfteraacthionsotfratihnewasesnsoivteirvliatiydofontthoe pclualtteusrecotnotcariynsitnagl selective media and an antibiotic sensitivity disk containing 10 pgofcrystal violet was added. Sensitivity was demonstrated by inhibitionof bacterial growth in a zone immediately surrounding the disk. PKMI01 Plasmid. The presenceofthe pKM101 plasmid was confirmed for the appropriate tester strain cultures by demonstrationofresistance to ampicillin. An aliquotofan overnight cultureof each strain was overlaid onto plates containing selective media and an antibiotic sensitivity disk containing ampicillin was added. Resistance was demonstrated by bacterial `growth in the zone immediately surrounding the disk. Characteristic Number of Spontaneous Revertants. The numberofspontaneous revertants. per plate in the vehicle controls that is characteristicofthe respective strains was demonstrated by plating 100 uL aliquotsofthe culture along with the appropriate vehicle on selective media. -10- 01933 Covance 20784-0409 VCuolgteulr-iBnognnBerrotsahl.t sTohleutbiroont(hVuosgeedl taongdrBoownnoevre,rn1i9g5h6t)csulutpuprleesmoefnttheedtweisttehr2st.r5a%ins(ww/avs) Oxoid Nutrient Broth No. 2 (dry powder). Agar Plates. Bottom `mediumE (Vogel and agar (25 Bonner, mL per 15 x 100mm 1956), supplemented petri with dish) 1.5% was Vogel-Bonner minimal (w/v) agar and 0.2% (w/v) glucose. O(wvievr)laanydA0g.a5r%fNoarCSlel(ewc/tvi)onaonfdRweavserstuapnptlse.meOnvteerdlwaiyt(hto1p0) magLaorfw1a)s p0.r5epmarMedhwisittihdi0n.e/7b%ioatgianr solution per 100 mL agar for selection ofhistidine revertants, or 2) 0.5 mM tryptophan solution `tpheer s1u0p0pmleLmoefntaegdartofporagsaerlewcatisonuosfetdryinptthoephoavenrlraeyv.ertHaontwse.veWrh,ewnhSen9 mSi9xmiwxaswaresquniortedr,eq2u.i0remd,L of `water was added to the supplemented top agar (0.5 mLofwater per 2 mLof supplemented top daiglaurt)iaonndentshuerreedsutlhtaitntgh2e.5finmalLotfodpaiglautreadndsuapmpilnemoeanctieddstuoppplaegamrenwtascounsceendtfroartitohnesorveermlaaiyn.edThtihse same both in the presence and absence of S9 mix. Test Article `The Sponsor was characteristics as responsible for the defined in the GLP dreetguelramtiinonast,ionof the test article stability and the test article: Control Articles Vehicle Controls. Vehicle controls were plated for all tester strains both in the presence and `ambsaexnicmeuomfaSl9iqmuioxto.fTtehsetvaerhtiicclleedciolnuttriooln wplaastepdl)a,taedl,onugsiwnigtha 5a010p0LuaLliaqluioqtuooftvoefhitcheleap(perqouparlitaotethe tester strain and a 500 kL aliquot of$9 mix (when necessary), on selective agar. Positive Controls. The combinationsofpositive controls, activation condition and tester strains plated concurrently with the assay are indicated in the following table. on 01934 Positive Controls Covance 20784-0-409 Tester Strain TA9S TA98 TA100 TA100 TALS3S TAIS3S TAI537 WTAPI2Su3r7A WP2uvrA 59 Mix + Positive Control benzofalpyrenc Conc_perPlate 2518 = + 22-naimtionfolaunotrhernaecene 10g 25pg = + sodiumazide 2aminoanthracene 204g 254g = + 2saomdiinuomaanztihdreacene 204g 251g +-ICR2-Iam9in1oanthracene 204g 250ug = dnitroguinolineN-oxide 10g `The sources and gradesofthe positive control articles are as follows: 2be-nazmoi[naoJapnytrhernaece(nCeA(SC#A5S0-#3621-38-)1,3-S8i)g,mSaiCghmeamiCchaelmiCcoa,l pCou.r,itpyu2ri9t8y%297% 2-nitrofluorene (CAS #607-57-8), Aldrich Chemical Co., 298% sodium azide (CAS #26628-22-8), Sigma Chemical Co, purity 298% ICR-191 (CAS #1707-45-0), Sigma Chemical Co, purity 298% 4-nitroquinoline-N-oxide (CAS #56-57-5), Sigma Chemical Co., purity 299%. pSltaertiilnigtya CSoOnktrLolasli.quTohte(tmhoesstacmoencveonlturmaeteudsteedstianrttihcelaesdsialyu)toionn wsealseccthieveckaegadr.forTshteerSil9itmyibxywas checked for sterility by plating 0.5 mL on selective agar. $9 Metabolic Activation System T$o9xHicoomlooggey,naItnce.., BLaitvcehr0m9i7c2ro(s4o2m.8almegnofzpyrmoetse(iSn9pheormmoLg)e.naTteh)e hweormeogpeunracthaesweadsfprroempaMroeldecfurloamr male Sprague-Dawley com oil) at 500mg/kg rats that had as described been injected (i.p.) with by Ames ef al, (1975). AroclorTM 1254 (200 mgpermL in T$h9eMi$x9.miTxheco$n9tamiinxedwtahse cproemppaorneednitmsmienddiiactaetleydpriniotrhteofoiltslouwsienigntaabnlye.experimental procedure. -12- 01935 $9 Mix Components Covance 20784-0-409 CHOomponeAm0n .o7u0 nmt Lt_ 0I2M5NMaHGl,uPcOo/seN-aG;-pHhPoOsp,hpatHe 7.4 ~~ 0.10 0.02 mL mL 0.10M NADP 004mL 0.825M KCU0.2M mgCly 004ml $9 Homogenate 010ml 100 mL Dose Rangefinding Assay `The growth inhibitory effect (cytotoxicity)ofthe test article to the test system was determined in order to allow the selectionofappropriate doses to be tested in the mutagenicity assay. Design. The dose rangefindingassaywas performed using tester strains TA100 and WP2ivrA both in the presence and absence of S9 mix. Ten dosesoftest article were tested at one plate per dose. The test article was checked for cytotoxicity up to a maximum concentration of5 mg per plate. Rationale. The cytotoxicityof the test article observed on tester strain TA100 is generally representativeofthat observed on the other tester strains and because ofthe comparatively high `numberofspontaneous revertants per plate observed with this strain, gradationsof cytotoxicity can be readily discemed from routine experimental variation. The Escherichia coli tester strain WP2uvrA does not possess the rfa wall mutation that the Salmonella typhimurium strains have and thus, a different rangeofcytotoxicity may be observed. Also, the cytotoxicity induced by a test article in the presence of S9 mix may vary greatly from that observed in the absence of S9 mix. Therefore, this would require that different test article dose ranges be tested in the `mutagenicity assay based on the presence or absenceofthe S9 mix. Evaluation of the Dose Rangefinding Assay. Cytotoxicity is detectable as a decrease in the `numberofrevertant colonies per plate and/or by a thinning or disappearanceofthe bacterial background lawn. Selection of the Maximum Dose for the Mutagenicity Assay. Cytotoxicity was observed in the dose rangefinding study and the highest concentrationoftest article used in the subsequent `mutagenicity assay was a dose which was expected to exhibita thinningofthe bacterial background lawn. Mutagenicity Assay Design. The assay was performed using tester strains TA9E, TA100, TAS35, TALS37, and WP2uvrA both in the presence and absenceofS9 mix along with the appropriate vehicle and .3- . 01936 -_-- CCoovvaannccee2200778844:-00409 positive controls. The rangefinding assay. dosesoftest article were selected based on the resultsofthe dose tFhreepqluaetenciyncaornpdorRaotuiotnemoeftAhdomdionliosgtyraotriigoinn.alTlyhdeetsecsrtiebresdtrbayinAsmweerseefexaplo,s(e1d97to5)thaendteMstaarrotinclaenvdia mAumteagsen(1s9.83)I.n tThheipslamteetihnocdoorploorgaytihoansmbeetehnodsohloowgny,tothdeetteecstt aarwtiicdlee, rtahnegteeostfecrlsatsrsaeinsoafndchtehemiSc9al pmliatxe.(wFhoelrleoawpipnrgopirnicautbea)tiwoenr,ercevoemrbtianntecdoilnonmioelstweenraegacrouwnhtiecd.h wAalls doovserelsoaifdtohnetoteastmairntiicmlea,ltahgear `vehicle controls and the positive controls were plated in triplicate. Plating Procedures `These procedures were used in both the dose rangefinding assay and the mutagenicity assay. aEcatcihvaptliaotnecwoandsiltaiboenlaedndwidtohsea lceovdele.wThhicehSi9demnitxifiaenddtdhieltuetsitonasrotifclte,hetetsetstphaartsiec,letewseterresptrraeipna,red immediately prior to their use. `When 9 mix `were added to was not required, 2.5 mL ofmolten 100 iL oftester strain and 50 selective top agar (maintained uL of at 45 vehicle + 2C). or test article dose When S9 mix was aredqdueidretdo,25.000muLLooffmSo9ltmeinx,se1l0ec0tiuvLeotfotpeasgtaerr.stArfatienratnhde5r0eqpuLiroefd vceohmipcolneeonrttseshtaadrtbiecleendaodsdeedw,erteh:e i`mniaxt1u5rexw1a0s0vmormtepxeerdiadnidsho.veArfltaeird othnetoovtehrelasyurhfaadceosofli2d5ifimeLd,ofthmeipnliatmeaslwebroettionmveargtaerd caonndtained incubated aliquot. for 52% 4 hr at 37+ 2C. Positive control articles were platedusing a 50 L plating Scoring the Plates Plates which were not evaluated immediately following the incubation period were held at 5:+3C until such time that colony counting and bacterial background lawn evaluation could take place. eBvaacltueartieadl bBoatchkmgarcoruosncdopLiacawlnlyEavanldumaitciroons.coTphiceaclolnyd(iutsiionngofatdhiessebaccttienrgimalicbraocskcgorpoeu)nfdorlawn was rienldaitciavteiotnostohfecvyethoitcolxeicciotntyraonldpltaestteaarntdiclweapsrerceicpiotradteed. aElvoingdewnictehotfhecyrteovteorxtiacnittcyowuanstsscfoorreadll plates 4at) tehxattrdeomseelylerveedlu.ceLda,w5n)sawbseernet,scoorr6e)doabssc1)urneodrmbayl,pr2e)cispliitgahttel.y rIefdpurceesde,nt3)onmotdheerpaltaetelsy,reduced, macroscopic precipitate was scored as slight, moderate or heavy. 14. 01927 -_ CCoovvaannce 220778844-00-4409 cCoonutnrtolisnagnRdeavlel rptlaatnetsCcoolnotnaiiensi.ngTtheset narutmicbleerwoefrreevceorutnatnetd cmoalnounailelsyp.erTphleatneufmobr etrhoefverheivcelretant ecoxlcoenpiteisonpoefr ptlhaetepofsoirtitvhee cpoonstirtoilvse fcoornttreosltserwsetrraeincoTunAt9edibnythaeutpormeasteendcecoofloSn9ymcioxunitnerExwpiethritmheent 20784-B, which were counted manually. DATA Data Presentation cFaolrcualllatreedp.liTcahtee rpleastuilntgsos,ftthheesmeeacanlcruelvaetritoannstsarpeerprpelsateentaendditnhteasbutlaanrdafrodrdmeviniatthieonDawteareTables sectionofthis report. Assay Acceptance Criteria Before assay data were evaluated, the criteria criteria were used (0 determine a valid assay: foar valid assay had to be met. The following Tester Strain Integrity. #fa Wall Mutation. To demonstrate the presence of the rfa wall mutation, Salmonella typhimurium tester strain cultures exhibited sensitivittoy crystal violet. `PaKppMr1op0ri1atPeltaessmteird.strTaionsdeemxohinbsittreadteretshiestparnecseetnoceaompfictihleliPnK. MI01 plasmid, cultures ofthe hCihsatriadcitneeroirsttriycpNtoupmhabne,rthoeftSepstoenrtsatrnaeionucsulRteuvreesrteaxnhtisb.iteTdoadcehmaornacstterraitsetitchneurmebqeuirroefment for Tsphoentacacnecpotuasblreevrearntagnetssfpoerrthpelamteeawnhevnehpilcalteedcoanltoronlgswwietrhethaes vfeohlilcowlse:under selective conditions. TA98 8-60 TAI00 60 - 240 TAISIS 4-45 TAISY 2. 25 WP2uwrA 5 - 40 t`TheesdteenrsSittryaoifntCeustletrusrteraDinencsuilttyur.esTwoedreemogrnesattrearttehtahnatorapepqruoaplritaot0e.5nuxmb10ersboacftbearicatepreiramarLe apnladt/eodr, thhaadnroeracehqeudalattoar0g.e5txle1v0elobfatcutrerbiiadipteyrdmeLm.onstrated to produce cultures with a density greater -15- 01938 Covance 20784-0-409 Positive Control Values in the Absence of $9 Mix. capable of identifying a mutagen, the mean valueof a To demonstrate positive control that the tester strains were for a respective tester strain strain exhibited at least a 3-fold increase over the mean valueofthe vehicle control for that tPhoesiSt9ivmeiCxonwtarsoclaVpaabllueeosfimnetthaeboPlriezsienngcae pofroSm9utMaigxen(5190 iMtis xmuItnatgeegnriitcy)f.orTm(os)d,emtohensmteraantevtahlaute ofthe positive control for a respective tester strain in the presenceofthe S9 mix exhibited at least a 3-fold increase over the mean value ofthe vehicle control for that strain. hAanviancgcedpetmaobnlsetproastietdivbeoctohnttrhoeliinntetghreitpyroefstehneceSo9fmSi9xmainxdftohreaabsipleictiyfoifctshteraitneswtaerssetrvaailnuatoteddetaesct a `mutagen. Cytotoxicity. A minimumofthree non-toxic doses were required to evaluate assay data. Assay Evaluation Criteria Once the criteria for a valid assay had been met, responses observed in the assay were evaluated as follows: Tester Strains TASS, TA100, and WP2uvrA. For a test article to be considered positive, it had 10 produce at least a 2-fold increase in the mean revertants per plateofat least oneofthese tester strains over the mean revertants per plateofthe appropriate vehicle control. This increase in the mean numberofrevertants per plate concentrationsofthe test article. had to be accompanied by a dose response to increasing `Tester Strains TA1535 and TA1537. Foratest article to be considered positive, it had to produce at least a 3-fold increase in the mean revertants per plateofat least oneofthese tester strains over the mean revertants per plateof the appropriate vehicle control. This increase in the `mean numberofrevertants per plate had to be accompanied by a dose response to increasing `concentrationsofthe test article. RESULTS Test Article Handling `The test article formed unworkable, non-homogeneous suspensions in water at concentrations of 500, 399, 300, 200, 99.9, and 50.0 mg/mL. In dimethylsulfoxide, the test article formed a non-homogeneous suspension at 501 mg/mL and a homogeneous suspension at 401 mg/mL. For this reason, dimethylsulfoxide (DMSO, CAS# 67-68-5, Acros Organics, Lot No. A012649701) was used as the vehicle. At 100 mg per mL, which was the most concentrated stock dilution prepared for the mutagenicity assay, the test article formed a translucent white suspension. The -16- } 01939 Covance 20784-0-409 test article formed a solution at 2.00 prepared for the mutagenicity assay. mg/mL and remained a solution in all succeeding dilutions Dose Rangefinding Assay Doses tested in the mutagenicity assay were selected based on the resultsofthe dose trhaengperfeisnednicnegaansdsaaybcsoenndcueoctfedS9onmithxewtietsht aornteiclpelautseipnegrtedsotseer.stTreainnsdoTsAe1so0f0taesntd aWrPti2clue,vrfrAomin6b.o6t7h 105,000 ug per plate, were tested and the resultsarepresented in Tables 1 and 2. These data `were generated in Experiment 20784-A1. Indications ofcytotoxicity were observed with tester strain TA100 at 10.0 ug per plate and above in the absence of S9 mix as evidenced by the thinning ofthe bacterial background lawn. No cytotoxicity was observed with tester strain `TAL00 in the presenceofS9 mix or with tester strain WP2uvrA in either the presence or absence of$9 mix as evidenced by no decrease in the numberof revertants per plate and a normal background lawn. Mutagenicity Assay `The mutagenicity assay results for PFOS are presented in Tables 3 through 5. These data were `generated in Experiment 20784-B1. The data are presented as individual plate counts (Tables 3 and 5)and as mean revertants per plat+e standard deviation (Tables 4 and 5) for each treatment and control group. `The resultsofthe dose rangefinding study were used to select the doses tested in the `3m3u3t,ag1e0n0i,caitnyda3s3s.a3y.pgThpeerdpolsaetsetiensttehde wpirtehsetnhceeSoaflmSo9nemlilxaatnedste5r,0s0t0r,ain1s,0w0e0r,e3353,,00100,03,,3333.03,, 11,00.000,, 3.33, 1.00, and 0.333pgperplate in the absence of S9 mix. With tester strain WP2uvrA, the doses tested were 5,000, 3,330, 1,000, 333, 100, and 33.3 pg per plate both in the presence and absence of S9 mix. In the mutagenicity assay (Experiment 20784-B1, Tables 3,4 and 5), all data were acceptable and 10 positive increases in the mean number ofrevertants per plate were observed with anyofthe tester strains either in the presence or absenceof S9 mix. All criteria for a valid study were met. CONCLUSION `The resultsofthe Salmonella-Escherichia coliMammalian-Microsome Reverse Mutation Assay indicate that under the conditionsofthis study, 3M Corporate Toxicology test article, PFOS, did not cause apositive increase in the mean number of revertantsperplate with anyofthe tester strains either in the presence or absence of microsomal enzymes prepared from AroclorTM-induced rat liver (S9). 17- 01910 -_-- CoCvoavnanccee220077844-004-4p099 RECORDS TO BE MAINTAINED tAhlilsrsatwuddyatwai,lldboceuamrecnhtiavteidonin, trheecosrtdosr,atgheefapcrioltiotcieoslo,fanCdovtahnecfein-aVlireenpnoartfgorenaetrlaetaestd oasnea yreesaurlt of following `may elect submissionofthe final report to the Sponsor. to have the aforementioned materials retained After in the theone yearperiod, storage facilities of the Sponsor Covance-Vienna Sponsor. for an additional periodof time or sent to a storage facility designated by the REFERENCES `Awmitehst,hBe.SN.a,lMmconCealnlna,/MJ.a, mamnadliYaanm-aMsiackrio,soE.m,e"MMuettahgoednsicfiotrydTeetsetc.t"inMguctaartciionnogReensseaarncdh,mu3t1a:g3e4n7s3641975). tBhreusEiscckh,erD.iJc.h,iSaicmolmionW,P2V.Fa.n,dRWosPe2nukvrarnAz,reHv.eSr.s,eRmauyt,atVi.oAn.aasnsdayS.t"afMfuotrda,tiRo.Sn.R,e"seAanrcehv,alu7a6t:i1o6n9-of 190 (1980). Green, M.H.L. and Mutation Research, Muriel, W.J., "Mutagen 38:3-32 (1976). testing using trp" reversion in Escherichia coli." Maron, D.M. and Ames, B., "Revised Research, 113:173-215 (1983). methods for the Salmonella Mutagenicity Test." Mutation Vogel, HJ. and Bonner, D.M., "AcetylomithinaseofE. properties." J. Biol. Chem. 218:97-106 (1956). coli: Partial purification and some 18. 01911 Covance 20784-0-409 DATA TABLES 19} 01942 Covance 20784-0-409 TABLE 1 : DOSE RANGEFINDING ASSAY RESULTS TEST ARTICLE ID: PROS. EXPERIMENT ID: 20764-A1 VEHICLE: DMSO "DATE PLATED: 2-Aug99 "DATE COUNTED: 27-Aug:99 bePLATE 000 (VehicleS041) "TAIO0 REVERTANTS PER PLATE wins WITHOUTS) REVEPRETRANTS BACLKaGwROnUND. REVEPRETRANTS BACKLGARWONUND, PATE EVALUATION PATE EVALUATION E) v w% 1 Tes Aric: oer w ' ' 100 0 ' " 2 ns 9% ' 2 wr 108 ' % 2 0 u ' " 2 " ' 2 wn " ' = 2 100 9s ' w 2 EN) 7% ' 5s 2 sao = ' 3 B4a=c7kgnoroorummnadlLeawnn Evsaluation Coe2Ss:==sbieghnyreduced pn Sigprepiaie mp - m{roeidreeshparncdicuotune) 3 == Gmodsemcelbyuyrperrdecceeidpd bp =(breequyirpesreacnpdraceou) -20- 01943 - coCvovmacnecel2o07m8e4-o0u-4n09 TABLE 2: DOSE RANGEFINDING ASSAY RESULTS TEST ARTICLE 0: FrOS EXPERIMENT ID: 078401 DATE PLATED,24Aug59 venicLE puso DATE COUNTED: 27:Aug9 nate Sov Torani pr -- EIorSwm Broicsn ae evibamow . : REoIo Smousmoornmton Wee van - ; 5s ; wo , M , . , wo . , " \ s erm oneetca o3E2 gat SIT e ----p -- g+=n-- pmrteimiloreaasts a. 01944 Covance 20784-0-409 TABLE3 : MUTAGENICITY ASSAY RES-UINDLIVITDUSAL PLATE COUNTS TEST ARTICLE ID: PFOS. EXPERIMENT ID: 20784-51 "DATE PLATED: 09-5p-99. "DATE COUNTED: 13-5p-99, 14-5699 VEHICLE: DMSO PLATING ALIQUOT: 50 hL. DOSEPLATE VMEIHCIRCOLSEOMCEOSN:TRRAOTLLIVER TESTARTCLE 033 ug 1f0ried 3200 uugg POSITIVE CONTROL** MVEIHCIRCOLSEOCMOENS:TNROONLE TEST ARTICLE o10n0 sug 130303 busg w533ubg 0505 wupg 00 ug POSITIVE CONTROL** REVERTANTSPER PLATE. TTags s TToAI: 7Ta2ss3 Tas 1 27 BoB ou sows 0B 5s 60 0m0o2monnssowwoews wsonwr 32 ssoonn m"9omNeowm as e6 o9w257 651100 2BowmBBwwoswo 47m9mH 47s86 Hs M0 4 ms Eos 2 Wm 1% me 2M 2P1osw 28 2 oB n 1Mo3ds2%a BBooaass 6ds 613 6 195 20 mows 8 92 38 8 ome ommoos Mmoem ow 19 osmoas 88 omos7m momo 86 s7u0 s5nM 96 mowas d9 i0s3 os 70s a1 s2 s4 7220954s8 4ss9nm 3454 87 en Te To ee e443 507 sie STTAAIO bZeanmcilnaclapnyiernaecene 2235ppgppitne TTAAISIITS ZZaammiinnccaenniibbraacceennee 2233uupppp * BTac=kgnroomunadlLawn Evaation Code2s=:sighiyreduced p4n= osixgpyrcrpeidaceed mp5== ambosdeknnttepreciptie equines and count) TTAAI sZaoidvuomfalozrdcene TTAAIISTS IsCoRdIu9m1aide 63+= moodbereilbycyrperudeuccirpeidadic bp =h(eaevypqreuchiapnridceousm) 2100gppgipnee 2200mign GBRAOCUKN.D Lave ' 11 ii 'i ' ' '' i'' i1 2i ' -2201915 Covance 20784-0-409 TABLE 4: MUTAGENICITY ASSAY RES-USULMMTARSY TEST ARTICLE ID: PFOS EXPERIMEINDT: 2084.81 "DATE PLATED: 09-Sep-99 "DATECOUNTED:13-Sep-99, 14-Sep-99 VEHICLE: DMSO PLATING ALIQUOT: 50 4 TTDOSEPLATE VMEIHCIRCOLSEOMCEOSN:TRROALT LIVER TESTARTICLE w333u4g 1300 uugg 0prog 4s POSITIVE CONTROL** VMEIHCIRCOLSEOCMOENS:TNROONLE. TESTARTCLE 0103034ss 130303 4ug w33uug 13050 uugg 00 bg POSITIVE CONTROL*** MEAN REVERTANTS PER PLATE WITH STANDARD DEVIATION WEWTasssb MEWTAIs0o WWTassso WENTisssb no 0 Bos 7a Fna o 0an 13 2no 2 76ss 220 170 mnooae 05 3 0732 02 82 25 39 05 34 5so 1 as as 929 113 wow mou ve 24 02 63 1w5o3s s8 1s5 [5I 2 :6 22 2a 42 8 160 2no3s +5 22 2nos4 75 07 nBooss s[a 3 H1o2e B@o 5 2no33 672 os Ea 95 52 won wow as 1 ar STTAAIO bZeanmoilnaclapnyierniecene 2255 ppppilnee TTAAIISSHSTS ZZaammiinncaanniivvaacceennee 2255 ppgpple * Bac=kgnrooumnad Lawn EvalusionCode2s=:sighiyreduced p4n= scixgepmrecyprieadtuceed mp5 == mabsoendte precipice eguires andcom) TTAAIO Zsaoidvuomfaunoirdeene TTAAIISTS IsCoRdIu9m1aide 63%= omobderselcbyyuperdrecdeipdi bp = h(easvyperehqcainpidcoeunst) 2010uppglpienee 2200upi)ne GBRAOCUKN.D Awe 1 iT i1 '' ' ' i' 11| ii 21 ' -2301916 - Covance20dum TABLES: MUTAGENICITYASSAYRESULTS INDIVIDUAL PLATECOUNTS AND SUMMARY TESTARTIIC: LFOES EXPERIMENT ID: 2075451 DATE PLATED: 09-5ep99 VEHICLE DMSO DATECOUNTED: 1350p99,14:50p9 PLATING ALIQUOT: 50 41 ------ee------------------------------------------------ pm oseriure EVERTANLISEFENRFLATE A I WT STANDhAeReDnORATION CS--RoSnA i MicRosouES: RATLVER on om w , -2 wn BHHI RE]R 3FvI o8l i;i rosmvecontro sictrosoowes:rnove TARE 35 as as aa --- 5 now PY , wa \ noe \ [---- 0 NPBA Zamnombrcee 250ys pBiB=tkiLupnnEoeonsonn odls3s:tyrenReRR ----tay wn \ [EN 432SomooyteLnotse o_o) "2- 01947 ANCE COVANCE. COVANCE STUDY NO.: 20 784 -(-4C 7 PROTOCOL 409 EDITION SALMONELLA - MUTATION ASSAY ESCHERICHIA COLIIMAMMALIAN-MICROSOME REVERSE LCaobvoarnacteorLyabPorraacttiocreie(sGILnPc.) (rCegouvlaantcieo)nswialnldcsotnadnudacrtdsth.isTshtiusdypriontoccoomlp,laitalnecaestwointeh Good critical phase of the work in progress, and the final report will be audited by Quality Assurance in accordance `with SOPs at Covance. This study will be conducted at 9200 Leesburg Pike, Vienna,Virginia 22182 (Covance-Vienna). PART 1. SPONSOR INFORMATION AND APPROVALS 10 SPONSOR IDENTIFICATION ACdodmrpesasn:y Name: 33MM CCoernptoerration `Building 220-2E-02 St. Paul, MN 55144-1000 20 TEST ARTICLE IDENTIFICATION PFOS 3m Hed, No Te295,/7 30 TEST ARTICLE ANALYSIS Determination of the test article stability and the test article characteristics as defined in the GLP regulations is the responsibilityof the Sponsor. 40 NOTIFICATION OF REGULATORY SUBMISSION Inorderto comply with GLP regulations and standards, consulting laboratories must be notified if all or part ofa study is intended for regulatory submission. Covance maintains `a master schedule of studies which fall under regulatory review. Please indicate which agency, if any, might receive the results of this study: Oundeermined Ora Kepatsca 0 eparirra Omarr Owmonw Doeep Oomer 12198 Lof18 01918 Cc 0 VANCE. PROTOCOL 409 EDITION 5 50 STUDY DATES Proposed Experimental Start Date: Proposed Experimental Termination Date: 6.0 APPROVAL OF STUDY PROTOCOL Study Director: WAALS Wal Michael S. Mecchi, M.S. `Testing Facility Management: nC Brian C. Myhr, Ph.D. Associate Director Sponsor's Authorized Representative: Me TG e Marvin T. Case, D.V.M,, Ph.D. A. gost 999 October 1999 pae 3-pe-98 pa: __[20]29 yA N 12/98 ' 20f 18 01919 COVANCE> PROTOCOL 409 EDITION 5 PART 2. STUDY PROTOCOL SALMONELLA - ESCHERICHIA COLIMAMMALIAN-MICROSOME REVERSE MUTATION ASSAY 1 OBIECTIVE `aTbihleitoybjteocitnivdeucoef rtehivserssteudmyutiasttiooenvsaeliutahteer tihnettheestpraretsiecnleceanodr/aobrsietsncmeetoafbomlaimtmeaslfiorantheir Smailcmroonseolmlaalteynpzhyimmeusriautm1a)ntdheathi2s)titdhienetrlyopctuospihnanthleocguesnoofmEescohfesreivcehriaal sctorlaiisntsroaifn WP2uvrA. 0. IESTSYSTEM A. Salmonella typhimurium: The Salmonella/Mammalian-microsome reverse `sumbusttaittiuotnioansss.ayTdheetescttrsaipnosionftSmaultmaotinoenlsl,abtoytphhfirmaumreisuhmifutsaendd/inorthbiassaesspaayirare hoipsetriodni.neWahuxeontrtohpehsse bhiysvtiirdtiuneeo-dfecpoennddietnitonceallllsy(lheitsh-a)larmeuteaxtipoonssedintothtehier theissttiadritincele ahinsdtigdrinoew)nonulnydetrhsoesleeccetlilvsewchoindcihtireovnesrt(mtionhiimsatildimneediinadweiptehndaetnrcaece(haimso+u)natreoafble to form colonies. The trace amount of histidine in the media allows all the plated bfualcltyereixaprteosusnedde.rgTohea hfiesw+creelvledritvainstisonasr,e wrehaidcihlyidsiesscseernntiaabllefoarsmcuoltoangieesneagsatiinosstbethe limited background growthofthe his- cells. By utilizing several different tester strains, bothbasepair substitution mutations and frameshift mutations can be detected. The Ames Test has been shown to be a sensitive, rapid and accurate cihndeimciactaolr oclfatssheesm.utagenic activity of many materials including a wide range of B. Escherichia coli: The Escherichia coli WP2uvrA reverse mutation assay detects point mutations, specifically base pair substitutions. The Escherichia coli tester strain WP2uvrA used in this assay is a tryptophan auxotroph (rp) by virtue of a conditionally lethal mutation at a site which blocks a step of tryptophan biosynthesis prior to the formationofanthranilic acid. Since the target site for true back mutation is an ochre nonsense mutation, tryptophan-independent revertants (trp) can arise either by a base change at the site of the original 12/98 3of18 01950 COVANCE> PROTOCOL 409 EDITION 5 talRteNrAatgioenneosr (bByrsuuspipcrkeestsialo,n1b9y80s)p.eciWfhicensutphperetsrsyoprtompuhtaant-idoenpseantdaenstecceolnlds s(ittrep-i)nare `ewxipthoasetdratcoetahmeotuesnttarotfictlreyapntodpgharno)wnonulnydtehrosseelceecltlisvewhciocndhitrievoenrst(tmoitnriympatlopmheadnia tihnedempeednidaenaclleo(wtsrpa+ll) tahree palbalteetdboafcotrermicaoltoonuiensd.erTghoeatfreacwecaelmloduinvtisoifontsr,ywpthoipchhanisin desissecnetimaalblfeoramsuctoalgoennieessiasgationbste tfuhlelyliemxiptreedssbeadc.kgTrhoeuntdrpg-r+orwetvehrtoafnttshea1rrep-recaedlills.y m`Wuhtialgeetnhse atnrpdrceevretrasiniomnestaylssteim.r.essoplounbdlsetcohrmoomsatteasl)k,ylfartaimnegsahgiefnttsm,utbaagsee-nasnawloougld not be expected tobedetected by this system. I. MATERIALS A. TesterStrains 1. SSaallmmoonneellllaa ttyypphhiimmuurriiuumm:htihsetitdeisnteeraustxroatirnospthosbTeAuSse,dTwiAl1l0b0e,tThAeLS35, and `TA1537 as described by Ames et al these strains are shown in Table 1. (1975). The specific genotypes of _-- TABLE I. TESTERSTRAINGENOTYPES Histidine Mutation __ AdditionalMutations hisG46 hisC3076 hisD3052 LPS Repair R Factor TAIS3S TAIS37 fa wrB - TAL00 TAS ofa wwrB +R _--_-- tInwoadaddidtiitoinontaolammuuttaattiioonns iwnhithcehheinsthiadnicnee otpheeirronse,nstihteivteisttyetrosstroamien.s contain t`hmeuteangzenyimcescormepsopuonnsdisb.leTfhoer trhfeaswyanltlhemsuitsaotfipoanrrteosfulttsheinlitpheoploolsyssaocfcohnaeriodfe w`baalrlrideerftihcaitefnocrymisnctrheeassuersfpaecremoefabtihleibtaycttoerciearltaceilnlcwlaalsls.esTohfecrheesmuilctailnsg cseulclh oatshtehrowsiesecobnetaeixncilnugdleadrgbeyrainngosrymsatleimnsta(ci.te.ceblelnwzaol(la.)pyrene) that would 12/98 4of 18 01951 COVANCE> PROTOCOL 409 EDITION 5 TDhNeAseecxocinsdimountraetpiaoinr,saysdteleemtiwohnicohf tghreeautvlyrBengheanne,cersestuhletsseinnsiatdievfitiycioefnt tthheesbeiostgreainnes, taolsoofmtehemuttesatgeernsst.raiSnisnccoentthaeinuivnrgBthdiesledteiloentieoxntaelnsdosrtehqruoiurgeh the vitamin biotin for growth. `SwthriaicnhsfTurAt9he8r ainncdreTaAse1s00thealsseonsciotnitvaiityn otfhethRe-sfeacsttorraipnlsatsomisdo,mpeKmMu1ta0g1e,ns. Thahsebmeeecnhsaungisgmesbtytewodhibcehbtyhimsopdliafsymiindg ianncrexeiassteisnsgebnascittievriitayl tDoNmAutaregpeanisr `polymerase complex involved with the mismatch-repair process. `(Taeusxtoertrsotprahiyn)s tToAh9is8tiadnindeTiAn1d5ep3e7ndaernecreev(eprrtoetdotfrroopmhyh)isbtyidfirnaemdesehpiefntdence misutreavgeerntse.d bTyAmLuSt3a5geinssrewvheirtcehdcbayusbeasbeotshubfsrtaimteusthioinftmsuatnadgebnasseand TA100 substitutions. 2. E`asucxhoetrriocphhiaWPco2liu:vtrhAe taesstdeerssctrriabiendbtoybGereuesend wainldlMbuertiheel t(r1y9p7t6o).phan cIonnatdadiintsioanutvoraAmDutNaAtiorenpaiinrthdeeftircyipetnocpyhwahniocphereonnh,antcheesteisttsesresntsriatiinvity to `seonmheanmcuetdamguetnaibcilciotympifotuhnedsu.vrTAhriespadierfiscyisetnecmy awloluolwds ntohermsatlrlaiynatcotsthoow rafeimeorwvaerdtsh.e damaged part of the DNA molecule and accurately repair it `(Taeusxtoetrrsotprahiyn)WtoP2truyvprtoAphisanreivnedretpeednfdreonmcetr(ypprtootpohtarnopdheyp)ebnydebnacsee. substitution mutagens. 3. Soou fTesr terc Strae ins. a SreaclemiovneedlldiaretcytplhyifmruormiuDmr:.tBhreutceestAermestsr,aiDnsepianrutsmeeanttCoofvance were Biochemistry, University of California, Berkeley. 1298 Sof 18 01952 COVANCE> PROTOCOL 409 EDITION 4. 5. 12/98 b. rEesccehievreidchfiraocmolTih:eteNsatteriosntarlainCoWlPle2cutivornAofinInudsuestartiaClovBaacntceeriaw,as Torrey Research Station, Scotland (United Kingdom). Storage oftheTesterStrains a Frozen Permanent Stocks Forvoezmeinghptercumlatnuernest,satdodciknsgwDilMl bSeOpr(e0p.a0r9emdibmyigroofwciunlgtufrree)sahnd freezing away appropriately vialed aliquots. Frozen permanent stocks of the tester strains will be stored at $-70C. b. Master Plates aMafsrtoezrenplpaetremsawnielnltbestporcekpaornetdo bmiynsitmraelakaignagreaapcphrtoepsrtieartestlryain from csounptpalienmienngttehdewRi-tfhacetiotrh,earmhpiisctiildliinne)aonrdtbriyopttionph(aann.d fToerststerrasitnrsain `master plates will be stored at 5 + 3C. PrepoafOvreranigthti Culoturnes a Inoculation Oinvoecmuilagthetd cbuyltturraenssffeorrriunsgeaincoalllotneysftirnogmprthoeceadpuprreosp,riwaitlel bmeaster pbleatpela1c0aed filnaaskschoankteari/innicnugbcautlotrurwehmicehdiwuimll.beInporcuolgartaedmmfeladsktso wbielglin othpeeroavteironnig(hsthackuilntgu,re1s2a5r=e i2n5lropgmp;haisnceuobratliaotne,l3o7g p+h2aseC)whseonthat turbidity monitoring begins. b. Harvest iTnocuebnastuiroentwhialtlcbuletdueretseramrienheadrvbeystsepdecitnrloaptheoltoogmepthraisce,motnheitloernigntgh ooff ctuurblideittyuirsbirdeiatyc.heCdualstudreetserwimlilnebde bhyaravepsetrecdenotntcreaanspmrietdteantceerm(i%nTe)d 6of 18 01953 COVANCE> PROTOCOL 409 EDITION 5 cruelatduirnegs ohnavaesrpeeaccthreodphaotdoemnestietry.ofTahtisletaasrtg0e.t5tuXrb1id0itcyelelnssupreersmtlhat caunldtutrheast mthaeycuelxthuirbeist hdeacvreenaostedovseenrsgirtoiwvint.y tOovseormgeromwunta(gsetnatsi.onary) Cultures will be removed reached and placed at 5 + from 3C. incubation when the target %T is 6. ConfoifTrestmerSatratinGienootypnes a. tShaelmfoonlellolwaintgygpehniemtuircimuamr:kteerssteornsttrhaeindcauylotfurtehseiwrilulsbeeicnhtehceked for `mutagenicity assay: (1) fa Wall Mutation `dTehmeonpsrtersaetnicoenooffthtehe1fcaulwtaulrlesmusetnastiitoivnitwyiltlobcerycsotanlfivriomleedt.by @ pPKMIO0I Plasmid `cTuhletuprreessoefncteeostfetrhsterapiKnsMT1A0918palnasdmTiAd1w0il0l bbye confirmed for demonstration of resistance to Ampicillin. (3) Spontaneous Reversion `vTehheicnluemcboentrroolfsstphoanttiasncehoaursacrteevreirsttainctosfptehreprleastpeeicntitvhee scturlatiunrse waillolnbgewditehmotnhsetrapaptreodprbiyatpelavteihnigcalleioqunotsesleocftievaec.h medium. b. fEoslclhoewriincghigaenceotlii:cemsatrekresrtroanintWhePd2auyvorfAitwsilulsebeincthheecmkuetdafgoernitchiety assay: (1) Spontaneous Reversion V`TehheicnluemcboentrrooflsstphoanttiasnechoaursacrteevreirsttainctospfeWrPp2luatveriAn the will be 12/98 Tof18 01951 COVANTCE> PROTOCOL 409 EDITION 5 demonstrated by plating aliquots of the appropriate vehicle on selective each culture medium. along with 7. Tester StrainMedia a Culturing Broth b`TeheVobgroetlh-Buosnendetrosgarltoswolouvteimoing(hVtogcuelltuarnedsBoofntnheert,es1t9e5r6s)trains will supplemented powder). with 2.5% (w/v) Oxoid Nutrient Broth #2 (dry b. Minimal Bottom Agar Plates BBoontntoemr maiganrim(a2l5 mmledpieurm15E(xV1o0g0elmamndpeBtroinndeisrh,)1w9i5ll6)be Vogelsupplemented with 1.5% (w/v) agar and 0.2% (wiv) glucose. . Top Agar for Selection of Revertants NTaoCpl(o(vwe/rvl)aya)ndagwairllwiblel sbueppprleepmaernetdewdiwtiht0h.170%malgaorf1()w/0v.)5amndM0.5% rheivsetritdainntes/,bioorti2n)s0o.l5utmioMn pterryp1t0o0phmaInasgoalrutfioornspeelrec1ti0o0nmolf ahigsatridfionre the selection of tryptophan revertants. f`oWrhethneSo9veirslarye.quiHroewde,v2e.r0,mwohfetnhSe9siuspnpoltemreeqnutierdedt,opwaatgearriiss audsdeedd s0uptphleesmuepnptleedmteonpteadgatro)paangdar2.(50.m5lmoifotfhweadtielrutpeedrs2upmplleomfented top aaggaarriasnudsaemdifnoor tahceidovseurplpalye.meTnhtiscodnicluetnitornateinosnusrersemthaaitntthheefsinaamletop both in the presence and absence of S9 mix. 12/98 . 8of 18 01955 COVANTCE> PROTOCOL 409 EDITION 5 B. Liver Microsomal Enzyme Reaction Mixture (S9 Mix; 1 5H9omogenate cLoimvemrermciicarlolsyoamnadl ewinlzlybmeespr(eSp9arheodmforgoemnamtael)ewSilplrabgeupeu-rDcahwalseeyd rats that have been 500 mg/kg injected (i.p.) as described with AroclorTM 1254 by Ames et al, 1975. (200 mg/ml in corn oil) at 2. $9Mix. e`TxhpeerSi9memnitxalwiplrlocbeedpurree.parTehdeiSm9memdiixatweillylpcroinotraitno tithseucsoemipnoanneynts indicated in Table 2. --_-- TABLE2.S9MIXCOMPONENTS HO IMNaH,PO/NaHPO, pH7.4 0.25M Glucose-6-phosphate 0.10M NADP 0.825M KCU0.2M MgCl, 9 Homogenate: _-- 070m! 010ml 002ml 0.04 ml 0.04mi 210ml 100ml C. Controls 1. Vehicle Controls aApnpdraobpsreinactee voefhSi9clmeicxo.ntrVoelhsicwliells cboempplaattiebdlfeorwiatllhstthriasintsesitnstyhseteprmeisnecnlcuede #b6u7t-w6i8l-l5)n,otetbhealniomlit(eCdAtSo:#D6e4i-o1n7-i5z)e,d aHn,d0,didmiemtehtyhyllfsourlmfaomxiiddee ((CCAASS #68-12.2). 12/98 : 90f 18 01956 COVANCE> PROTOCOL 409 EDITION 5 2. PositiveControls `sTthraeicnosmpbliantaedticoonnscuorfrepnostiltyiwvietchontthreoalsss,aaycatriveatiinodniccaotnedditiinoTnaabnlde t3.ester _-- Tester TABLE3.POSITIVECONTROLS Conc. Simin SOMix Positive Control perPlate TAS + TA - TAI00 + TA100 - TAIS3S + TAISIS TAISY? + TAIS37 ~ WP2rA + WP2urA - benzofalpyrene 2-nitofluorene 2-aminoanthracene sodium azide 2-aminoanthracene sodium azide 2-aminoanthracene ICR-191 2-aminoanthracene 4-nitroquinoline-N-oxide 25ug 10g 25ug 20g 25ug 20g 25g 20g 250g 10g 3. Sterility Controls a TestAnicle `The most concentrated test article dilution will be checked for sterility by plating an aliquotofthe same volume used in the assay on selective agar. bo S9Mix `The S9 mix willbe checked for sterilitybyplating 0.5 ml on selective agar. 12/98 : 100f18 01957 COVANCE> PROTOCOL 409 EDITION 5 IV. METHODS A. DRosaengefindiStnudgy b`Tehedegtreorwmtihneidnhiinbiotrodreyretfofeacltl(ocwyttohteosxieclietcyt)ioonfotfheaptperstoparritaictleedtoostehsett0esbtestyesstteemd iwnill the mutagenicity assay. 1. Design a`TnhdeWdPos2eurvarnAgefbiontdhiinngtshteudpyrewsielnlcbeeapnedrafbosremnecdeuosfinSg9tmesitxe.r sAtramiinsnTimAu1m00 ootfhteernwidsoesedsiroefctteesdt bayrttichlee Swpiolnlsboer,tetshteedteastt oanrteicplleatweilplebredocshee.ckUendlefosrs scoyltuobtiolxiitcyi/tmyisucpibtioliatympaexrimimtus.m Icfontcheenttersattairotnioclfe e5x,h0i0b0itusglipmeirtpeldate if `swolourbkialbiltey/cmoinscciebnitlriattyi,oint waitltlaibneabtleestiendthfeorvceyhtioctloexoifcicthyouicpet.o the maximum a Rationale t`eTshteegrrsotrwatihn TinAh1ib0i0toirsygeefnfeerctal(lcyytroetporxeisceinttya)toifvtehoef ttehsatt oarbtsieclrevoedn on `tThAe1o0t0h'esr cSoamlpmaornaetlilvaeltyyphhiigmhunruimumbetresotferssptornatinasneaonudsbreecvaeurtsaenotsf per rploauttei,negreaxdpaetriiomnesontfalcyvtaortioaxtiicoint.yTchane bEescrheeardiiclhyidaiscocleimteedstferrostmrain S`aWlPm2ounverlAladtoyepshniomturpoisusmessstrtaihnesrhfaavwealalnmdutthautsi,oandtihfaftertehnet range of tceysttotaorxtiicclietyinmtahye pbreeosbesnecreveodf.miAclrsoos,otmhaelceytnoztyomxiecsitmyaiyndvuacreyd by a `gTrheeartelfyofrer,otmhitshawtooublsderrveeqduiirne tthheatadbisfefnecreenotftemsitcarrotiscolmeadloseenzryamnegse.s be tested in the mutagenicity assay based absence of the microsomal enzymes. on the presence or 12/98 11of18 91353 COVANCE> PROTOCOL 409 EDITION 5 2. Evaluation of the Dose Rangefinding Study Cpyetroptloaxtieciatnyd/iosrdebtyecattahbilnenaisnga odrecdriesaaspepeianrtahnecenuofmbteher boafctreerviearltabnatcckoglroonuineds lawn. 3. SelectionoftheMaximumDosefortheMutagenicityAssay a. Cytotoxicity Observed `When cytotoxiciitsy observed in the dose rangefinding study, the `hmiugthaegsetnciocnicteynatsrsaatyiownilolfbteestthaarttwichliecthobgeiveusseaddientetchteasbulebsreeqduuecnttion of revertants per plate and/or a thinning or disappearance of the bacterial background lawn. b. No Cytotoxicity Observed If no cytotoxicity is observed in the dose rangefinding study then the highest dose of test articletobe used in the mutagenicity assay will be the same as that tested in the rangefinding study unless 1) the Sponsor specifies an alternate maximunn dose, 2) there is limited availability of the test article and/or 3) the test article precipitates heavily in the top agar. B. MutagenicityAssay I Design `TTAhLe5a3s7s,ayawnidllWPbe2upverrfAo,rmbeodthusiinntghetepsrteersestnrcaeinasnTdAa9b8s,enTcAe1o0f0S,9TmAiLxS.35, Unless the Sponsor specifies doses to be tested, the doses of test article will be selected based on the results of the dose rangefinding study. If cytotoxicity has been demonstrated in the dose rangefinding study, a minimum of six doses of test article will be tested along with the appropriate vehicle and positive controls.Ifno cytotoxicity has been demonstrated, a minimum of five doses will be tested. 12/98 : 120f18 01959 COVANCE> PROTOCOL 409 EDITION 5 2. Frequency and Route of Administration `iTnhceortpeosrteartisotnraminestwhioldlobleogeyxpoorisgeidnatlolythdeestecsrtiabretdicblyevAimaetsheetplaalte(1975) and M`wairdeonraanngde Aofmcelsas(s1e9s8o3f).chTehmiiscamletmhuotdagoelnosg.y hIansthbeeepnlastehoiwncnortpoodreatteicotna `methodology, the S9 mix (where appropriate), the tester strain, and the test aprltaitcel.eaFroellcoowmibnigniendcuibnamtoilotneanta3g7ar w2hiCchfoirs 5o2ve2rl4aihdr,onrtevoeartmaintnicmolaolniaegsar willbe counted. All doses of test article, vehicle controls and positive: controls will be plated in triplicate. C. PlatingProcedures `These procedures will be used in both the dose rangefinding study and the `mutagenicity assay. Eteasctehrpsltartaeinw,ialcltbiveatliaobnelceodndwiittihoan caonddedwoshei.chTihdeenSti9fimeisxthaendtesdtilaurttiioclnes,otfestthpehtaesset, article will be prepared immediately prior to their use. When article S9 mix is dose will not required, 100 1of tester be added to 2.5 ml of molten strain and 50 selective top 1] of vehicleortest agar (maintained at 450521Cof)v.ehiWchleenorSt9estmiarxtiicsleredqousierewdi,ll50b0e 1adodfedS9tom2i.x0,m1l0o0f mIoolftetnessteelrescttriavien taonpd agar. After the required components have been added, the mixture will be vionrate1x5exd a1n0d0 omvmerlpaeiudrodnitsoh.theAfstuerrfatchee oovfe2r5lamyihoafs msoilniidmifailedb,otthteopmlaatgeasrwciolnltbaeined inverted and incubated for 52 + 4 hr at 37 + 2C. When necessary, plating aliquots of other than 50 1 of test article/vehicle will be used. Positive control articles will be plated using a 50 1 plating aliquot. D. `ScothrePilantegs Plates which are not evaluated immediately following the incubation period will be held at = 3C until such time that colony counting and bacterial background lawn evaluation can take place. 12/98 130f18 01960 COVANCE> PROTOCOL 409 EDITION 5 1. BaBcteariac l kgrLaownEuvalnuatd ion `The condition of the bacterial background lawn will be evaluated for evidenceof cytotoxicity and test article precipitate. Evidence of cytotoxicity will be scored relative to the vehicle control and recorded along with the revertant counts for that dose. 2. Counting Revertant Colonies Revertant colonies fora specific tester strain within a given test article: dilution series will be counted either entirely by automated colony counter orentirely by hand (with the exception of the dose rangefinding study). If there is sufficient test article precipitate on the plates at any dose which interferes with automated colony counting, then the plates at all dose levels for that specific strain and activation condition willbe counted `manually. E. AnalyofsDiatsa Forall replicate platings, the mean revertantsperplate and the standard deviation will be calculated. V. EVAL N T Before assay data can be evaluated, the criteria for a valid assay must be met. A. CriFot rAe Valridi Assaay `The following criteria will be used to determine a valid assay: 1. Tester Strain Integrity: Salmonella ryphimurium a fa Wall Mutation To demonstrate the presence of the rfa wall mutation, tester strain cultures must exhibit sensitivity to crystal violet. 12/98 140f 18 01961 COVANCE> PROTOCOL 409 EDITION 5 b. pKMIOI Plasmid `cTuoltduermeosnosftrteastteertshterapirnesseTnAc9e8ofatnhdeTRA-f1a0c0tomrupsltasemxihdi,biptKrMeIsi0s1ta,nce 10 ampicillin. ec. Characteristic Number of Spontaneous Revertants `To demonstrate the requirement for histidine, the tester strain `rceuvletrutraenstmspuesrtpelxahtiebiwthaecnhaprlaactteedriasltiocngnwuimtbherthoefvsephiocnlteanuenoduesr selective conditions. The acceptable ranges for the mean vehicle controls are as follows: TA98 TAL00 TAI535 TAIS37 8- 60 60-240 4.45 2.25 2. TesterStrainIntegrity: Escherichia coli a Characteristic Number of Spontancous Revertants `cTuoltduermeomnusstrtaetxehitbhietraeqcuhairraecmteenrtisftoirc tnruympbtoeprhaonf,stphoentteasnteeorusstrain revertantsperplate when plated along with the vehicle under s`veelheicctlievecocnotnrdoiltsioinss.5 toTh4e0 arcecveerpttaanbtlseperarnpgleatfeo.r the WP2uvrA mean 3. TesterStrain CultureDensity `To demonstrate that appropriate numbers of bacteria are plated, the density of tester strain cultures must per ml and/or have reacheda be greater than target level of tourrbeiqduiatly tdoem0o.5nsxtr1a0tebdatctoeria produce per ml. cultures with a density greater than or equal to 0.5x 10 bacteria 12/98 15018 041962 COVANCE> PROTOCOL 409 EDITION 5 4. Positive Control Values a. Positive Control Values in the Absence of 9 Mix `mTuotdaegmeonn,stthreatmeeathnatvathleueteostfaerpsotsriatiinsvearceonctarpoalbfloeroafriedsepnetcitfiyvieng a vteasltueer ostfrtaihne mveuhsitcleexhciobnittroalt lfeoarstthaat3-sftroalidn.increase over the mean b. Positive Control Values in the Presence of $9 Mix (9 Mix Integrity) pTroodmeumtoangsetnrattoeittshmatuttahegeSn9icmifoxirms(csa)p,atbhleemoefamnetvaalbuoeliozfinthgea Spo9simtiixvemcuosnttreoxlhifboirat artelsepaescttiav3e-tfeosltderinsctrraeianseinovtehretphreesmeenacenovfalthuee of the vehicle control for that strain. sAtnraianccweipltlabbeleevpoasliutaitveedcaosnthraovlining tdheemoprnessternacteedofboSt9h fthoerainstpeegcriiftiyc ofthe 89 mutagen. mix and the ability of the tester strain to detect a 5. Cytotoxicity A minimumof assay data. three non-toxic dose levels will be required to evaluate B. CriteriaForAPositiveResponse a`rOenceevatlhueactreidtearsiaffoolrloawsv:alidassay have been met, responses observed in the assay 1 `Tester StrainsTA98,TA100,and WP2uvrA. iFnocrreaatseestinartthieclmeetaobnerecvoenrstiadntesrepderpopsliattieveo,fiat mluesatstpornoeduocfethatesleeatsetstaer2-fold strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean numofrbeveertarnts per plate must be 12098 16018 01953 COVANCE> PROTOCOL 409 EDITION 5 `accompanied article. by a dose response to increasing concentrations of the test 2. TesterStrains TAIS35 and TALS37 For a test article to be considered positive, it must produce at least a 3-fold increase in the mean revertants per plate of at least one of these tester strainsoverthe mean revertants per plateofthe appropriate vehicle control. This increase in the mean number of reveriants per plate must be `accompanied by a dose response to increasing concentrations of the test article. VI REPOTRHTERIESNULGTS A reportofthe resultsofthis study will be prepared by Covance and describe all methods usedforthe generation and analysis of the data. will accurately Results presented in the report for this assay will include: The results of the dose rangefinding study Gif applicable) including the number of rtehveeirdteannttistypeorf plate and abacterial background lawn the bacterial tester strains used in the evaluation assay for each dose: dose levels at which the test article was tested individual calculated plate mean counts for all and standard treated, positive deviation for all and vehicle control plates replicate plate counts cvaluationofresults VIL CHAANNDRG EVIE SIOS NS . Any changes or revisions of this approved protocol will be documented, signed by the Study Director, dated, and maintained with this protocol. The Sponsor will be notified of any changes or revisions. 1298 170f18 0196% COVANCE> PROTOCOL 409 EDITION VII. RECORDS TO BE MAINTAINED All raw data, documentation, records, the protocol, and the final report generated as a result of this study will be archived in the storage facilities of Covance-Vienna, for at least one year following submission ofthe final report o the Sponsor. After the one year period, the Sponsor may elect to have the aforementioned materials retained in the storage facilities of Covance-Vienna, designated by the Sponsor. for an additional period of time or sent to a storage facility IX. REFERENCES Ames, BN., McCann, J. and Yamasaki, E. (1975). Methods for detecting carcinogens and mutagens with the _Salmonella/mammalian-microsome mutagenicity test. Mutation Research 31:347-364. Brusick, D.J., Simmon, V.F., Rosenkranz, H. S., Ray, V.A., and Stafford, R.S. (1980). An evaluationofthe Escherichia coli WP2 and WP2uvrA reverse mutation assay. Mutation Research, 76:169-190. Green, M-H.L. and Muriel, W.J. (1976). Mutagen testing using rp" reversion in Escherichia coli. Mutation Research 38:3-32. Maron, DM. and Ames B. (1983). Revised Methods for the Salmonella Mutagenicity `Test. Mutation Research 113173-215. Vogel, HJ. and Bonner, D.M. (1956). Acetylomithinase of Escherichia coli: Partial purification and some properties. J. Biol Chem. 218:97-106. 12/98 180f 18 01955