Document XOQVEqXzOwjgOeb3ZGN77JmR4

FinalReport Salmonell-aEscherichicaoli/N4ammalian-Microsome ReverseMutationAssay withPFOS PREPARED FOR: 3M CorporateToxicology COVANCE STUDY NUMBER: 20784-0-409 C 0 V A14-@EC@- THE DEVELOPMEW SERVW.ESCOMPAW FINAL REPORT SALMONELLA ESCHERICHIACOLI/MANUAALIAN-MICROSOME REVERSE MUTATION ASSAY WITH PFOS AUTHOR Michael S.Mecchi,MS PERFORMING LABORATORY Covance LaboratorieIsnc.(Covance) 9200 LeesburgPike Vienna,Virginia22182 LABORATORY PROJECT IDENTIFICATION Covance StudyNo.: 20784-0-409 3M StudyNo.:T6295.17 SUBMITTED TO 3M CorporateToxicology 3M Center Building2.20-2E-02 St.Paul,Minnesota 55144-1000 STUDY COMPLETION DATE November 5, 1999 1 of 24 Covance20784-0409 QUALITY ASSURANCE STATEMENT Salmonella-Eschericcohliia/Mammalian-MicrosRoemveerseMutatioAnssaywithPFOS The reporthas been reviewed by the QualityAssurance Unit ofCovance LaboratoriesInc.,in accordancewith theGood LaboratoryPracticeregulationsas setforthinthe Environmental ProtectionAgency (EPA - TSCA), Title40 of theU.S. Code of FederalRegulationsPart792. The followinginspectionswere conducted and thefindingsreportedtothe Study Directorand studydirectormanagement. Writtenstatusreportsof inspectionsand findingsareissuedto Covance management accordingto standardoperatingprocedures. InspectionDates From To 9/9/99 9/9/99 Phase Preparationof S9 Nfix Dates Reported toStudy Directorand Study DirectorManagement 9/9/99 Auditor J.Crouch 10/21/99 10/21/99 DraftReport Review 10/21/99 K. Groeninger 11/04/99 11/04/99 FinalReport Review 11/04/99 K. Groeninger RepresentativeQ,ualiq Xssurance Unit Date -2- Covance 20784-0409 STUDY COMPLIANCE AND CERTIFICATION The describesdtudywas conducteidncompliancweiththeGood LaboratorPyracticreegulations as setforthintheEnvironmentalProtectioAngency (EPA - TSCA), Title40 of theU.S. Code of FederalRegulationsPart792, and with any applicableamendments. There were no significant deviationsfrom theaforementionedregulationosr the signedprotocolthatwould affectthe integritoyf thestudyor the interpretatiofn thetestresults.The raw datahave been reviewedby theStudy Director,who certifietshatthe evaluationof thetestarticlaespresentedherein representsan appropriateconclusionwithinthecontextofthe studydesignand evaluation criteriaA.ll testand controlresultsinthisreportare supportedby an experimentaldatarecord and thisrecordhas been reviewed by theStudy Director. Study Director: -wvl@ -@ Is @- Michael S. Mecchi, MS Geneticand CellularToxicology i Study Completion Date -3- TABLE OF CONTENTS Covance 20784-0409 Page No. ABSTRACT ...................................................6......... STUDY INFORMATION@ Sponsor TestArticle Assay Information Study Dates SupervisoryPersonnel ..........................................7.......... OBJECTIVE ..................................................7......... TEST SYSTEM RATIONALE .......................................7.......... MATERIALS AND METHODS .......................................8 ......... Test System TestArticle ControlArticles S9 MetabolicActivationSystem Dose RangefindingAssay MutagenicityAssay PlatingProcedures Scoring thePlates DATA .....................................................1.5......... Data Presentation Assay Acceptance Criteria Assay EvaluationCriteria RESULTS ...................................................1.6 ......... TestArticleHandling Dose RangefindingAssay MutagenicityAssay CONCLUSION ................................................17.......... RECORDS TO BE MAINTAINED ...................................1.8......... -4- TABLE OF CONTENTS (Continued) Covance 20784-0409 Page No. REFERENCES ................................................1.8 ......... DATA TABLES ...............................................1.9......... -5- Covance20784-0-409 ABSTRACT The objectiovfethistudywas toevaluattehetestarticlPeF,OS,fortheabilitoyinducereverse mutationseitherinthe presenceor absence ofmammalian microsomal enzymes at 1)the histidinleocusinthegenome of severalstrainsofSalmonellatyphimurium and at2) the tr)?tophanlocusof EscherichiacolistrainWP2uvrA. The doses testedin themutagenicityassaywere selectedbased on theresultsof a dose rangefindingassayusingtesterstrainsTAI 00 and WP2uvrA and ten doses oftestarticlreanging from 6.67to 5,000 pg perplate,one plateper dose,both inthepresence and absence of S9 mix. The testerstrainsused inthemutagenicityassaywere Salmonella typhimurium testerstrains TA98, TAIOO, TA1535, TA1537 and EscherichiacolitesterstrainWP2urvA. Theassaywas conducted inboth thepresence and absence of S9 mix alongwith concurrentvehicleand positive controlsusingthreeplatesper dose. The doses testedwith theSalmonella testerstrainswere 5,000,3,330,1,000,333, 100,and 33.3 gg per plateinthepresenceof S9 mix and 5,000,1,000, 333, 100,33.3,10.0,3.33,1.00,and 0.333 gg per plateinthe absenceof S9 mix. With tester strainWP2uvrA, the doses testedwere 5,000,3,330,1,000,333, 100,and 33.3 pg perplateboth in the presenceand absence of S9 mix The resultsof theSalmonella-Escherichiacoli/Mammalian-Microsome Reverse Mutation Assay indicatethatunder the conditionsofthisstudy,3M CorporateToxicology'stestarticleP,FOS, didnot cause a positiveincreaseinthe mean number of revertantpser platewith any of thetester strainseitherinthepresence orabsence of microsomal enzymes preparedfrom AroclorTm-inducedratliver(S9). -6- Covance20784-0409 STUDY INFORMATION Sponsor 3M CorporatTeoxicology testArticle Sponsor'sIdentificatioPnF:OS FC-95 Lot 217 Date Received: 08/19/99 PhysicalDescription:whitecrystallipnoewder StorageConditions: room temperature Assay Information Type ofAssay: Salmonella-Escherichcioali/Manirnalian-MicrosoRmeeverseMutationAssay ProtocolNo.: 409,Edition5 Covance Study No.: 20784-0-409 Study Dates InitiationDate: 08/20/99 Experimental StartDate: 08/24/99 Experimental Termination Date: 09/15/99 Supervisory Personnel Study Director: Michael S.Mecchi, MS Laboratory Supervisor: CarlosE. Orantes,BS OBJECTIVE The objectiveof thisstudywas to evaluatethe testarticlaend/oritsmetabolitesfortheirability toinduce reversemutationseitherinthepresence or absence ofmammalian microsomal enzymes at1)the histidinelocusinthe genome of severalstrainsof Salmonellatyphimurium and at2) the tryptophanlocus of EscherichiacolistrainWP2uvrA. TEST SYSTEM RATIONALE The Salmonella/Manunalian-microsome reversemutation assaydetectspointmutations,both frameshiftsand/orbase pairsubstitutionsT.he strainsofSalmonella typhimurium used inthis assayarehistidinaeuxotrophsby virtueof conditionalllyethalmutationsintheirhistidine operon. When thesehistidine-dependenctells(his-)areexposed tothetestarticlaend grown under selectivceonditions(minimalmedia with a traceamount of histidineo)nly thosecells -7- Covance 20784-0409 whichreverttohistidiinnedependenc(ehis+a)reabletoformcoloniesT.he tracaemountof histidinienthe media allowsalltheplatedbacteriatoundergo a few celldivisionsw,hich is essentiaflormutagenesistobe fullyexpressed.The his+revertantasrereadilydiscemableas coloniesagainstthe limitedbackground growth of the his-cells.By utilizinsgeveraldifferent testerstrainsb,oth basepairsubstitutiomnutationsand fi-ameshifmtutationscan be detected. The Ames Test has been shown tobe a sensitiver,apidand accurateindicatorof themutagenic activitoyf many materialsincludinga wide range of chemicalclasses. The EscherichiacoliWP2uvrA reversemutationassaydetectspointmutations,specificallbyase pairsubstitutionsT.he EscherichiacolitesterstrainWP2uvrA used inthisassayisa tryptophan auxotroph(trp-b)y virtueof a conditionalllyethalmutationata sitewhich blocksa stepof tr@ptophanbiosynthesispriortothe formationof anthraniliaccid. Sincethetargetsitefortrue back mutationisan ochrenonsense mutation,bwtophan-independent revertant(strp+)can arise eitherby a base change atthe siteofthe originalalteratioonrby suppressionby specific suppressormutationsata second siteinTRNA genes (BrusicketaL, 1980). When the tryptophan-dependentcells(trp-)areexposed tothe testarticleand grown under selective conditions(minimal media with a traceamount of tryptophan)only thosecellswhich revertto uwtophan independence(trp+)are abletoform colonies.The traceamount oftryptophaninthe media allowsallthe platedbacteriato undergo a few celldivisionsw,hich isessentiaflor mutagenesistobe fullyexpressed.The trp+ revertantsarereadilydiscemable as coloniesagainst thelimitde background growth ofthetrp-cells.While thetrpreversionsystemrespondstomost alkylatinaggents,base-analogmutagens and certainmetals(i.es.olublechromates),frameshift mutagens would not be expected tobe detectedby thissystem. MATERIAI,S AND METHODS The experimentalmaterialsm,ethods and proceduresarebased on thosedescribedby Ames etaL, (1975) and Green and Muriel (1976). Test System Salmonella typhimurium. The testerstrainsused were theSalmonellatyphimurium histidine auxotrophsTA98, TAIOO, TA1535, and TAl5j7 asdescribedby Ames etal, (1975). Thetester strainsinuse atCovance were receiveddirectlyfrom Dr. Bruce Ames, Department of Biochemistry,Universityof CaliforniaB,erkeley. The specificgenotypesof thesestrainasre shown inthe followingtable. TesterStrainGenotypes HistidinMcutation hisG46 hisC3076 hisD3052 TA1535 TA1537 TAIOO TA98 AdditionaMlutations LPS Repair R Factor rfa uvrb rfa uvrb + Covance20784-0409 Inadditiotnoa mutatioinnthehistidionpeeront,hetestesrtraicnosntaitnwo additional mutationwshichenhancetheisrensitivtiotsyomemutagenicompounds.The rfawallmutation resultsinthe lossof one of the enzymes responsibleforthesynthesisofpartofthe lipopolysaccharidbearrierthatforms the surfaceof thebacteriaclellwall. The resultincgellwall deficiencyincreasespermeabilitytocertainclassesofchemicals such as thosecontaininglarge ringsystems(i.eb.enzo[a]pyrene)thatwould otherwisebe excludedby a normal intactcellwall. The second mutation,a deletionof the uvrb gene,resultsina deficienDtNA excisionrepair system which greatlyenhances the sensitivitoyfthesestrainstosome mutagens. Since the uvrb deletionextendsthroughthe bio gene,allof thetesterstrainscontainingthisdeletionrequirethe vitaminbiotinforgrowth. StrainsTA98 and TA 100 alsocontainthePKM 101 plasmid which furtherincreasesthe sensitivitoyf thesestrainstosome mutagens. The mechanism by which thisplasmid increases sensitivityomutagens has been suggestedtobe by modifying an existingbacteriaDlNA repair polyrnerasecomplex involvedwith themismatch-repairprocess. TesterstrainsTA98 and TA1537 arerevertedfrom histidindeependence (auxotrophy)to histidiniendependence(prototrophy)byframeshiftmutagens.TA1535 isrevertedby base substitutiomnutagens and TA 100 isrevertedby mutagens which causeboth fi-ameshiftasnd base substitutions. Escherichia coli.The testerstrainused was thetr)?tophanauxotrophWP2uvrA as describedby Green and Muriel (1976).The testesrtraininuse atCovance was receivedfrom The National Collectionof IndustriaBlacteria,Torrey Research StationS,cotland(UnitedKingdom). In additiontoa mutationinthe tryptophanoperon,thetesterstraincontainsa uvra DNA repair deficiencywhich enhances itssensitivityo some mutagenic compounds. This deficiencyallows the strainto show enhanced mutabilitysincethe uvra repairsystem would normallyactto remove the damaged pailoftheDNA molecule and accuratelyrepairitafterwards. TesterstrainWP2uvrA isrevertedfrom twtophan dependence (auxotrophy)totwtophan independence (prototrophyb)y base substitutiomnutagens. Frozen Permanent Stocks. Frozen permanent stocksof thetesterstrainwsere preparedby growing freshovernightculturesa,ddingdimethylsulfoxid(eDMSO, 0.09mI-/mL of culturea)nd freezingsmallaliquots(0.5-1.m5L) at 70'C. Master Plates.Masterplatesof thetestesrtrainwsere preparedby streakinegachtestesrtrain from a frozenpermanent stockonto minimal agarappropriatelsyupplemented with 1)for -9- Covance 20784-0-409 Salmonellatyphimurium,an excessof histidinea,nd biotin,and fortesterstrainTsA98 and TA 100,ampicillin(25 gg/mL), toensurethe stablemaintenanceof thepKM 101 plasmid;and 2) forEscherichiacoli,an excessof tr@ptophan.Testerstrainmaster plateswere storedat 5 30C. Inoculationof Overnight Cultures. Overnightculturesforuse in alltestingprocedureswere inoculatedby transferrinagcolony from the appropriatemasterplatetoa flaskcontainingculture medium. Inoculatedflaskswere placedina shaker/incubatowrhich was programmed tobegin operation(shaking,125 25 rpm; incubation3,7 2'C) so thatthe overnightcultureswere in log phase or latelog phase when turbiditmyonitoringbegan. Harvest of Overnight Cultures. To ensurethatcultureswere harvestedinlatelog phase,the lengthof incubationwas determinedby spectrophotometrimconitoringof cultureturbidity. Cultureswere harvestedonce a predeterminedturbiditwyas reached asdetermined by a percent transmittanc(e%T) readingon a spectrophotometerT.his targetturbiditeynsuresthatcultures have reached a densityofatleast0.5 X 10' cellsper n-iLand thatthecultureshave not overgrown. Overgrown (stationaryc)ulturesmay exhibitdecreasedsensitivittyo some mutagens. Cultureswere removed from incubationwhen thetarget%T was reached and were placed at 5 30C. Confirmation of Tester Strain Genotype. Testerstraincultureswere checked forthefollowing geneticmarkers on theday of theiruse inthemutagenicityassay. rfaWall Mutation. For the Salmonella typhimurium testerstrainculturest,he presenceofthe r.fawall mutation was confirmed by demonstrationof thesensitivitoyf theculturetocrystal violet.An aliquotof an overnightcultureof each strainwas overlaidonto platescontaining selectivmeedia and an antibiotiscensitivitdyiskcontaining10 gg of crystavlioletwas added. Sensitivitwyas demonstratedby inhibitioonf bacteriaglrowth in a zone immediately surroundingthedisk. pKM101 Plasmid. The presenceof thePKMIOI plasmid was confirmed fortheappropriate testesrtrainculturesby demonstrationof resistancteoampicillin.An aliquotof an overnight cultureof each strainwas overlaidonto platescontainingselectivmeedia and an antibiotic sensitivitdyiskcontainingampicillinwas added. Resistancewas demonstratedby bacterial growth inthe zone immediatelysurroundingthedisk. CharacteristicNumber of Spontaneous Revertants. The number of spontaneousrevertants -perplateinthe vehiclecontrolsthatischaracteristoifc therespectivestrainswas demonstrated by plating100 @iLaliquotsof theculturealongwith the appropriatevehicleon selectivmeedia. -10- Covance20784-0409 CulturinBgroth.The brothusedtogrowovernighctultureosfthetestesrtrainwsas Vogel-Bonner saltsolution(Vogeland Bonner, 1956)supplementedwith 2.5% (w/v)Oxoid NutrientBrothNo. 2 (drypowder). Agar Plates.Bottom agar(25mL per 15 x 100 mm petridish)was Vogel-Bonner minimal medium E (Vogel and Bonner, 1956),supplementedwith 1.5% (w/v)agarand 0.2% (w/v) glucose. Overlay Agar for Selectionof Revertants. Overlay (top)agar was prepared with 0.7% agar (w/v)and 0.5% NaCl (w/v)and was supplementedwith 10 ML of 1)0.5mM histidine/biotin solutionper 100 mL agarforselectionofhistidinreevertantso,r 2) 0.5 mM tryptophansolution per 100 mL ofagarforselectionoftryptophanrevertantsW.hen S9 mix was required2,.0 mL of the supplemented top agarwas used in theoverlay.However, when S9 mix was not required, water was added tothe supplemented top agar(0.5mL ofwater per 2 mL of supplementedtop agar)and theresultin2g.5 mL of dilutedsupplemented top agarwas used forthe overlay.This dilutionensured thatthefinaltop agarand amino acidsupplement concentrationrsemained the same both in thepresence and absence of S9 mix. Test Article The Sponsor was responsibleforthedeterminationofthetestarticlsetabilitaynd the testarticle characteristiacssdefinedintheGLP regulations. Control Articles VehicleControls. Vehiclecontrolswere platedforalltesterstrainsboth inthepresenceand absenceof S9 mix. The vehiclecontrolwas plated,usinga 50 gL aliquotof vehicle(equaltothe maximum aliquotoftestarticldeilutiopnlated)a,longwith a 100 gL aliquotof theappropriate testerstrainand a 500 jiLaliquotof S9 mix (when necessary)o,n selectiveagar. PositiveControls. The combinationsofpositivecontrolsa,ctivationconditionand testerstrains platedconcurrentlywith theassayare indicatedinthefollowingtable. Covance 20784-0-409 PositiveControls TesterStrain TA98 TA98 TAIOO TAIOO TA1535 TA1535 TA1537 TA1537 WP2uvrA WP2uvrA S9 Mix + + + + + PositiveControl Conc. per Plate benzo[a]pyrene 2.5 gg 2-nitrofluorene 1.0Ag 2-aminoanthracene 2.5 ILg sodium azide 2.0pg 2-aminoanthracene 2.5jig sodium azide 2.0pg 2-aminoanthracene 2.5pg ICR-191 2.0gg 2-aminoanthracene 25.0 pg 4-nitroquinoline-N-oxide 1.0ag The sourcesand gradesof thepositivecontrolarticleasreas follows: benzo(a]pyrene(CAS #50-32-8),Sigma Chemical Co.,purity,-:!98% 2-aminoanthracene(CAS #613-13-8),Sigma Chmnical Co.,purity2:97% 2-nitrofluoren(eCAS #607-57-8),AldrichChemical Co., @t98% sodium azide(CAS #26628-22-8),Sigma Chemical Co.,purity.9-8!% ICR-191 (CAS #1707-45-0),Sigma Chemical Co.,purity@!98% 4-nitroquinoline-N-oxi(dCeAS #56-57-5),Sigma Chemical Co.,purity2:99%. SterilitCyontrols. The most concentratedtestarticldeilutiownas checked forsterilibtyy platinga 50 pL aliquot(thesame volume used intheassay)on selectivaegar. The S9 mix was checked forsterilibty plating0.5mL on selectivaegar. S9 Metabolic ActivationSystem S9 Homogenate. Livermicrosomal enzymes (S9 homogenate) were purchased from Molecular Toxicology,Inc.,Batch 0972 (42.8mg ofproteinper mL). The homogenate was preparedfrom male Sprague-Dawley ratst,hathad been injected(i.p.w)ith AroclorTm 1254 (200 mg per mL in com oil)at500 mg/kg asdescribedby Ames etaL, (1975). S9 Mix. The S9 mix was preparedimmediatelypriorto itsuse inany experimentalprocedure. The S9 mix containedthecomponents indicatedinthe followingtable. -12- S9Mix Components Component H20 IM NaH2pO4/Na2BPO4, pH 7.4 0.25M Glucose-6-phosphate 0.1OM NADP 0.825M KCI/0.2M mgCI2 S9 Homogenate Amount 0.70mL 0.10mL 0.02mL 0.04niL 0.04mL 0.10 ML 1.00ML Covance20784-0-409 Dose Rangefinding Assay The growth inhibitoreyffect(cytotoxicitoyf)thetestarticlteothe testsystem was determinedin orderto allow theselectionof appropriatedoses tobe testedinthemutagenicityassay. Design. The dose rangefindingassaywas performed usingtesterstrainTsAIOO and WP2uvrA both inthepresence and absence of S9 mix. Ten doses of testarticlweere testedatone plateper dose. The testarticlweas checked forcytotoxicituyp to a maximum concentrationof 5 mg per plate. Rationale.The cytotoxicitoyf thetestarticloebserved on testesrtrainTAIOO isgenerally representativoefthatobserved on the othertesterstrainsand because ofthe comparativelyhigh number of spontaneousrevertantpser plateobservedwith thisstraing,radationsof cytotoxicity can be readilydiscernedfrom routineexperimentalvariation.The Escherichiacolitesterstrain WP2uvrA does not possessther.fawallmutation thatthe Salmonella typhimurium strainshave and thus,a differenrtange ofcytotoxicitmyay be observed. Also,thecytotoxicitiynduced by a testarticlienthepresence of S9 mix may varygreatlyfrom thatobserved inthe absence of S9 mix. Therefore,thiswould requirethatdifferenttestarticldeose rangesbe testedinthe mutagenicityassaybased on thepresenceor absence of the S9 mix. Evaluation of the Dose Rangefinding Assay. Cytotoxicityisdetectableas a decreasein the number of revertantcoloniesper plateand/orby a thinningor disappearanceofthebacterial background lawn. Selectionof the Maximum Dose for the Mutagenicity Assay. Cytotoxicitywas observedin the dose rangefindingstudyand thehighestconcentrationof testarticluesed inthesubsequent mutagenicityassaywas a dose which was expected toexhibita thinningof thebacterial background lawn. Mutagenicity Assay Design. The assaywas performed usingtesterstrainsTA98, TAIOO, TA1535, TA1537, and VvT2uvrA both inthepresence and absence of S9 mix along with theappropriatevehicleand -13- Covance 20784-0409 positivecontrols.The doses oftestarticlweere selectedbased on theresultsof thedose rangefindingassay. Frequency and Route of Administration. The testerst-aimwere exposed to the testarticlveia theplateincorporationmethodology originalldyescribedby Ames etaL, (1975) and Maron and Ames (1983). Thismethodology has been shown todetecta wide range of classesofchemical mutagens. Inthe plateincorporatiomnethodology, thetestarticlet,hetesterstrainand theS9 mix (where appropriatew)ere combined inmolten agarwhich was overlaidonto a minimal agar plate.Followingincubationr,evertantcolonieswere counted. All dosesof thetestarticlet,he vehiclecontrolsand thepositivecontrolswere platedintriplicate. PlatingProcedures These procedureswere used inboth the dose rangcfindingassayand themutagenicityassay. Each platewas labeledwith a code which identifietdhetestarticlet,estphase,testerstrain, activatiocnonditionand dose level.The S9 mix and dilutionosf thetestarticlweere prepared immediatelypriorto theiruse. When S9 mix was not required,100 gL oftesterstrainand 50 gL of vehicleortestarticldeose were added to2.5mL ofmolten selectivteop agar(maintainedat45 2'C). When S9 mix was required,500 gL of S9 mix, 100 gL oftesterstrainand 50 ;LLof vehicleor testarticldeose were added to 2.0mL of molten selectivetop agar.Aftertherequiredcomponents had been added, the mixture was vortexedand overlaidonto thesurfaceof 25 niL of minimal bottom agarcontained ina 15 x 100 mm petridish. Afterthe overlayhad solidifiedt,heplateswere invertedand incubatedfor52 4 hr at37 2'C. Positivecontrolarticlewsere platedusinga 50 gL plating aliquot. Scoring the Plates Plateswhich were not evaluatedimmediatelyfollowingthe incubationperiodwere heldat 5 3 *C untilsuch time thatcolonycountingand bacteriablackground lawn evaluationcould takeplace. BacterialBackground Lawn Evaluation. The conditionof thebacterialbackground lawn was evaluatedboth macroscopicallyand microscopicall(yusinga dissectinmgicroscope)for indicationosf cytotoxicitaynd testarticlperecipitateE.vidence of cytotoxicitwyas scored relativteo thevehiclecontrolplateand was recordedalong with therevertantcountsforallplates atthatdose level.Lawns were scoredas 1)normal,2) slightlryeduced,3)moderatelyreduced, 4) extremelyreduced,5) absent,or 6)obscuredby precipitateI.fpresenton theplates, macroscopic precipitatweas scoredas slightm,oderate orheavy. -14- Covance20784-0409 CountingRevertantColonies.The numberofrevertacnotloniepserplatfeorthevehicle controlsand allplatescontainingtestarticlweere counted manually. The number of revertant coloniesperplateforthepositivecontrolswere countedby automated colonycounterwith the exceptionof thepositivecontrolsfortesterstrainTA98 inthepresenceof S9 mix inExperiment 20784-B I,which were counted manually. DATA Data Presentation For allreplicatpelatingst,he mean revertantpser plateand the standarddeviationwere calculated.The resultsof thesecalculationasrepresentedintabularform intheData Tables sectionof thisreport. Assay Acceptance Criteria Before assaydatawere evaluated,the criterifaora validassayhad tobe met. The following criteriwaere used todeterminea validassay: TesterStrainIntegrity. rfaWall Mutation. To demonstratethepresenceof ther.fa wallmutation,Salmonella typhimuriumtestesrtraicnulturesexhibitedsensitivittoycrystavliolet. pKM101 Plasmid. To demonstratethepresenceofthe PKMIOI plasmid,culturesof the appropriatteesterstrainsexhibitedresistancteo ampicillin. CharacteristicNumber of Spontaneous Revertants. To demonstratetherequirementfor histidinoer tryptophan,the testerstrainculturesexhibiteda characteristniucmber of spontaneousrevertantpserplatewhen platedalong with thevehicleunder selectivceonditions. The acceptableranges forthe mean vehiclecontrolswere as follows: TA98 TAIOO TAI 535 TA1537 WP2uvrA 8 - 60 60 - 240 4 - 45 2 - 25 5 - 40 Tester StrainCulture Density. To demonstratethatappropriatneumbers ofbacteriareplated, the densityof testerstraincultureswere greaterthan or equal to 0.5x IC@ bacteriaper mL and/or had reacheda targetlevelofturbiditydemonstratedtoproduce cultureswith a densitygreater than orequalto 0.5x 10'bacteriaper mL. 15- Covance20784-0409 PositivCeontrolValuesintheAbsenceofS9 Mix. To demonstratethatthe testerstrainswere capableof identifyinagmutagen, the mean value ofa positivecontrolfora respectivetester strainexhibitedatleasta 3-foldincreaseover themean valueof thevehiclecontrolforthat strain. PositiveControl Values in the Presence of S9 Mix (S9 Mix Integrity).To demonstratethat the S9 mix was capableofmetabolizinga promutagen to itsmutagenic form(s),themean value ofthe positivecontrolfora respectivetesterstraininthepresenceof theS9 mix exhibitedat leasta 3-foldincreaseoverthe mean value of thevehiclecontrolforthatstrain. An acceptablepositivecontrolinthepresenceof S9 mix fora specificstrainwas evaluatedas having demonstratedboth the integritoyfthe S9 mix and the abilitoyf thetesterstraintodetecta mutagen. Cytotoxicity.A minimum of threenon-toxicdoseswere requiredto evaluateassaydata. Assay Evaluation Criteria Once thecriterifaora validassayhad been met, responsesobserved intheassaywere evaluated as follows: Tester StrainsTA98, TAI 00, and WP2uvrA. For a testarticlteobe consideredpositivei,thad toproduce atleasta 2-foldincreaseinthemean revertantpser plateof atleastone ofthesetester strainsover themean revertantpser plateofthe appropriatevehiclecontrol.This increaseinthe mean number ofrevertantpser platehad tobe accompanied by a dose responsetoincreasing concentrationosf thetestarticle. Tester StrainsTA1535 and TA1537. For a testarticlteobe consideredpositivei,thad to produce atleasta 3-foldincreaseinthemean revertantpser plateof atleastone of thesetester strainsover themean revertantpser plateofthe appropriatevehiclecontrol.This increaseinthe mean number of revertantsper platehad to be accompanied by a dose responseto increasing concentrationsof thetestarticle. RESULTS Test ArticleHandling The testarticleformed unworkable,non-homogeneous suspensionsinwater atconcentrationosf 500,399, 300,200, 99.9,and 50.0 mg/mL. In dimethylsulfoxidet,he testarticlfeormed a non-homogeneous suspensionat501 mg/mL and a homogeneous suspensionat401 mg/mL. For thisreason,dimethylsulfoxid(eDMSO, CAS# 67-68-5,Acros Organics,Lot No. A012649701) was used asthe vehicle.At 100 mg per mL, which was the most concentratedstockdilution prepared forthemutagenicityassay,thetestarticlfeormed a translucentwhite suspension.The -16- Covance20784-0-409 tesatrticfloermeda solutioant2.00mg/mL andremaineda solutioinnallsucceedindgilutions prepared forthe mutagenicityassay. Dose Rangeflnding Assay Doses testedinthemutagenicityassaywere selectedbased on the resultsofthe dose rangefindingassayconducted on thetestarticluesing testerstrainTsAIOO and WP2uvrA inboth thepresence and absence of S9 mix with one plateper dose. Ten doses oftestarticlef,rom 6.67 to 5,000 gg per plate,were testedand theresultsarepresentedin Tables 1 and 2. These data were generatedinExperiment 20784-Al. Indicationosf cytotoxicitwyere observedwith tester strainTAI 00 at10.0 gg per plateand above inthe absence of S9 mix asevidenced by the thinningofthe bacteriablackground lawn. No cytotoxicitwyas observed with testerstrain TA 100 inthepresence ofS9 mix orwith testerstrainWP2uvrA ineitherthepresence orabsence of S9 mix asevidencedby no decreaseinthenumber ofrevertantpser plateand a normal background lawn. Mutagenicity Assay The mutagenicityassayresultsforPFOS arepresentedinTables 3 through 5. These datawere generatedinExperiment 20784-B I. The dataarepresentedasindividualplatecounts (Tables3 and 5)and as mean revertantpser plate:kstandarddeviation(Tables4 and 5)foreach treatmentand controlgroup. The resultsof thedose rangefindingstudywere used toselectthe doses testedin the mutagenicityassay. The dosestestedwiththe Salmonella testerstrainswere 5,000,3,330,1,000, 333, 100,and 33.3 gg per plateinthepresenceof S9 mix and 5,000,1,000,333, 100,33.3,10.0, 3.33,1.00,and 0.333 gg per plateinthe absence of S9 mix. With testerstrainWP2uvrA, the doses testedwere 5,000,3,330,1,000,333, 100,and 33.3 gg per plateboth inthepresenceand absence of S9 mix. Inthe mutagenicityassay(Experiment20784-B 1,Tables 3,4 and 5),alldatawere acceptableand no positiveincreasesinthemean number ofrevertantsperplatewere observed with any ofthe testerstrainseitherinthepresenceor absence of S9 mix. All criterifaora validstudywere met. CONCLUSION The resultsof theSalmonella-Escherichicaoli/Mammalian-Microsome Reverse Mutation Assay indicatethatunder theconditionsofthisstudy,3M CorporateToxicology'stestarticleP,FOS, did not cause a positiveincreaseinthemean number ofrevertantpser platewith any ofthetester strainseitherinthepresenceor absence ofmicrosomal enzymes prepared from AroclorTm-inducedratliver(S9). -17- RECORDS TO BE MAINTAINED Covance20784-0409 Allrawdatad,ocumentatiorne,cordst,heprotocoaln,dthefinarleporgtenerateadsa resulotf thisstudywillbe archivedinthe storagefacilitioefsCovance-Vienna foratleastone year followingsubmissionofthe finalreporttothe Sponsor. Aftertheone yearperiod,the Sponsor may electtohave the aforementionedmaterialsretainedinthe storagefacilitioefs Covance-Vienna foran additionalperiodoftime or senttoa storagefacilitdyesignatedby the Sponsor. REFERENCES Ames, B.N., McCann, J.,and Yaznasaki,E.,"Methods fordetectingcarcinogensand mutagens with theSalmonella/N4ammalian-Microsome MutagenicityTest." Mutation Research, 31:347364(1975). Brusick,D.J.,Simmon, V.F.,Rosenkranz,H.S.,Ray, V.A. and StaffordR,.S.,"An evaluationof theEscherichiacoliWP2 and WP2uvrA reversemutation assay."Mutation Research, 76:169190(1980). Green, M.H.L. and Muriel,W.J.,"Mutagen testingusingtrp'reversioninEscheilchiacoli." Mutation Research,38:3-32(1976). Maron, D.M. and Ames, B.,"Revised methods fortheSalmonellaMutagenicityTest."Mutation Research,113:173-215 (1983). Vogel,H.J.and Bonner, D.M., "AcetylomithinaseofE. coli:Partiaplurificatioannd some properties.J" BioL Chem. 218:97-106(1956). -18- Covance 20784-0-409 DATA TABLES -19- Covance 20784-0-409 TABLE I : DOSE RANGEFMING ASSAY RESULTS TEST ARTICLE ID:PFOS EXPERIMENT ID:20784-Al VEHICLE: DMSO DATE PLATED: 24-Aug-99 DATE COLNTED: 27-Aug-99 pgtPLATE 0.00(Vehicl5e0,pL) TAI00 REVERTANTS w7rH S9 REVERTANTS PER PLATE BACKGROUND LAWN EVALUATION* 98 1 PER PLATE WITHOUT S9 REVERTANTS PER PLATE BACKGROUND LAWN EVALUATION* 86 1 TestArticle 6.67 117 1 83 1 10.0 100 1 73 2 33.3 % 1 79 2 66.7 108 1 86 2 100 94 1 97 2 333 93 1 89 2 667 77 1 82 2 1000 95 1 87 2 3330 76 1 85 2 5000 80 1 79 3 BackgroundLawn EvaluatioCnodes: I = nonnal 2 = slightrleyduced 4 - extrenierleyduced 5 = absent sp = slighptrecipitate mp - niodemteprecipitate (requirheasndcount) 3 = moderatelryeduced 6 = obscurebdy precipitate hp - heavyprecipitate (requirheasndcount) -20- Covance 20784-0409 TABLE 2: DOSE RANGEF71NDING ASSAY RESULTS TEST ARTICLE ID:PFOS EXPERIMENT ID:20784-Al VEHICLE: DMSO DATE PLATED: 24-Aug-99 DATE COLTNTED: 27-Aug-99 pg/PLATE 0.00 (Vehicle,50 pL) WP2uvrA REVERTANTS PER PLATE WITH S9 REVERTANTS PER PLATE BACKGROUND - LAWN EVALUATION* WITHOUT S9 REVERTANTS PER PLATE BACKGROUND I-AWN EVALUATION* 21 1 18 1 TestArticle 6.67 16 1 11 1 10.0 14 1 9 1 33.3 21 1 18 1 66.7 21 1 10 1 ]()0 23 1 9 1 333 26 1 10 1 667 17 1 15 1 1000 9 1 12 1 3330 20 1 16 1 5000 22 1 12 1 Background Lawn EvaluationCodes: I = normal 2 = slightlryeduced 4 = extremelyreduced 5 = absent sp = slighptrecipitate mp = moderateprecipitate (requirehsandcount) 3 = moderatelyreduced 6 - obscuredby precipitate hp = heavyprecipitate (requirehsand count) -21- Covance 20784-0409 TABLE 3: MUTAGENICITY ASSAY RESULTS -INDIVEI)UAI,PLATE COUNTS TEST ARTICLE ID:PFOS EXPERIMENT ID:20794-BI DATE PLATED: 09-Sep-99 DATE COLJNTED: 13-Sep-99,14-Sep-99 VEHICLE: DMSO PLATING ALIQUOT: 50 gL DOSE/PLATE MICROSOMES: RAT LIVER VEHICLE CONTROL TEST ARTICLE 33.3 pg 100 pg 333 pg 1000 gg 3330 ILg 5000 gg POSITIVE CONTROL** MICROSOMES: NONE VEHICLE CONTROL TEST ARTICLE 0.333pg 1.00 gg 3.33 gg 10.0 jig 33.3 Mg 100 pg 333 99 1000 pg 5000 pg POSITIVE CONTROL*** TA98 1 23 23 23 24 17 22 30 20 24 31 17 29 37 14 19 27 20 23 23 13 18 28 365 440 453 12 20 24 12 19 22 12 15 18 19 26 27 20 22 23 17 23 25 14 15 21 13 15 16 8 15 19 6 12 13 186 195 210 REVERTANTSPERPLATE TAIOO 1 23 TA1535 123 95 98 113 10 13 15 75 98 99 86 89 95 111 112 118 64 74 81 97 95 104 90 90 % 8 12 12 11 12 14 6 9 12 6 9 14 4 9 11 7 11 12 835 997 1055 102 117 121 88 94 95 77 88 94 67 77 % 79 87 98 72 81 83 73 79 110 79 94 93 63 69 73 83 83 84 80 88 99 671 716 722 9 9 12 8 8 11 6 7 10 9 13 17 8 14 14 6 14 15 9 13 18 10 10 is 9 13 14 5 7 15 644 688 694 TA1537 123 5 6 10 2 5 12 3 5 12 5 6 10 7 9 10 4 56 7 89 159 173 183 BACKGROUND LAWN* 1 1 1 1 1 1 1 1 38 8 1 45 8 1 124 1 7 9 10 1 24 6 1 2 59 1 4 9 11 1 4 5 12 1 458 1 34 7 2 443 507 546 1 TA98 TAIOO TA1535 TA1537 bmo[a]pymne 2-aminoanthracenc 2-aminoanthracene 2-aminoanthmcene 2.5pg/plate 2.5pg/plate 2.5pgtplate 2.5pg/plate Background Lawn EvaluationCodes: I = nom-tal 2 - slightlryeduced 4 = extremelyreduced 5 - absent sp = slighptrecipitate mp - moderateprecipitate (requirehsand count) TA98 TAIOO TA1535 TA1537 2-nitrofluoTene 1.0pgtplatc sodium azide 2.0pg/plate sodium azide 2.0pg/plate ICR-191 2.0pg/plate 3 = moderatelyreduced 6 = obscuredby precipitate hp = heavyprecipitate (requirehsand count) -22- Covance 20784-0409 TABLE 4: MUTAGENICrrY ASSAY RESULTS -SUMMARY TEST ARTICLE ID:PFOS EXPERIMENT ID:20784-BI DATE PLATED: 09-Sep-99 DATE COUNTED: 13-Sep-99,14-Sep-99 VEHICLE: DMSO PLATING ALIQUOT: 50 pL DOSE/PLATE MICROSOMF-S: RAT LIVER VEHICLE CONTROL TEST ARTICLE 33.3 gg 100 gg 333 gg 1000 pg 3330 pg 50M pg POSITIVE CONTROL** MICROSOMES: NONE VEHICLE CONTROL TEST ARTICLE 0.333pg 1.00 pg 3.33 pg 10.0 pg 33.3 pg 100 jig 333 gg low gg 5000 pg POSITIVE CONTROL*** MEAN REVERTANTS PER PLATE WITH STANDARD TA98 MEAN S.D. 23 1 23 7 25 6 28 10 20 7 22 2 20 8 419 48 TAIDO MEAN S.D. 102 10 91 14 90 5 114 4 73 9 95 9 92 3 929 113 TA1535 MEAN S.D. 13 3 11 2 12 2 93 10 4 94 10 3 113 10 DEVIATION TA1537 MEAN S.D. 73 65 75 73 92 51 81 172 12 19 6 IS 5 is 3 24 4 22 2 22 4 17 4 15 2 14 6 10 4 197 12 92 4 86 9 80 15 88 10 79 6 87 20 85 7 68 5 83 1 89 10 703 28 10 2 92 &2 13 4 12 3 12 5 13 5 12 3 12 3 95 675 27 63 62 22 92 42 53 84 74 62 52 499 52 TA98 TAIOO TA1535 TA1537 benzo[alpyrene 2-aminoanthracene 2-aminoanthracene 2-aminoanthracene 2.5gg/plate 2.5gg/plate 2.5pgtplate 2.5pg/plate Background Lawn EvaluationCodes: I - normal 2 - slightiyteduced 4 = exti.,,ielyTeduced 5 = absent sp = slighptrecipitate mp - moderateprecipitate (requirehsand count) TA98 TAIOO TA1535 TA1537 2-nitrofluoTene 1.0pg/plate sodium azide 2.0pglplate sodium azide 2.0pglplate ICR-191 2.0pg/plate 3 - moderatelyreduced 6 = obscuredby precipitate hp - heavy precipitate (requirehsand count) BACKGROUND LAWN* 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 -23- Covance 20784-0409 TABLE 5: MUTAGENICITY ASSAY RESULTS -INI)IVEI)UAIPLLATE COUNTS AND SUMMARY TEST ARTICLE ID:PFOS EXPERIMENT ID:20784-BI DATE PLATED: 09-Sep-99 DATE COUNTED: 13-Sep-99,14-Sep-99 VEHICLE: DMSO PLATING ALIQUOT: 50 pL DOSEIPLATE MICROSOMES: RAT LIVER VEHICLE CONTROL TEST ARTICLE 33.3 ILg 100 pg 333 pg 1000 pg 3330 pg 5000 pg POSITIVE CONTROL** MICROSOMES: NONE VEHICLE CONTROL TEST ARTICLE 33.3 pg 100 ps 333 iLg 1000 pg 3330 pg 5000 pg POSITIVE CONTROL*** REVERTANTS PER PLATE WP2uvrA 1 23 9 17 22 10 12 15 14 18 18 IS 20 30 17 21 22 I1 18 21 14 15 20 213 243 247 18 19 21 9 11 20 16 17 19 10 16 25 12 17 19 11 14 18 9 14 16 157 274 288 MEAN REVERTANTS PER PLATE WITH STANDARD DEVIATION WP2uvrA MEAN S.D. 16 7 12 3 17 2 23 6 20 3 17 5 16 3 234 19 BACKGROUND LAWN* 1 1 1 1 1 1 1 1 19 2 1 13 6 1 17 2 1 17 9 1 16 4 1 14 4 1 13 4 1 240 72 1 WP2uvrA 2-aminoanthracene 25.0pg/plate Background Lawn EvaluationCodes: I - nonmi 2= 4 = exuamlyreduced 5- sp = slighptrecipitate mp = slightlyteduced absent moderateprecipitate (requirehsand count) WP2uvrA 4-nivNuinoline-N-oxide1.0pgtplate 3 - niodemtelyreduced 6 = obscuredby precipitate hp - heavyprecipitate (requirehsand count) -24- COVAWCE-@ THE Dmwpmapff SEIMCas r-ompoxy el COVANCE STLTDY c PROTOCOL 409 EDITION 5 SALMONELLA - ESCHERICHIA COLJ/MAMMALIAN-MICROSOME MUTATION ASSAY REVERSE Covance LaboratorieIsnc.(Covance)willconductthisstudyincompliancewithGood LaboratoryPractice(GLP) regulationasnd standards.Thisprotocola,tleastone criticaplhaseof thework inprogressa,nd thefinalreportwillbe auditedby QualityAssurancein accordance with SOPs atCovance. This studywillbe conductedat9200 LeesburgPike,Vienna,Virginia 22182 (Covance-Vienna). PART 1. SPONSOR INFORMATION AND APPROVALS 1.0 SPONSOR IDENTIFICATION Company Name: Address: 3M Corporation 3M Center Building220-2E-02 St.Paul,NIN 55144-1000 2.0 TEST ARTICLE IDENTIFICATION FO, s /@Z 3.0 TEST ARTICLE ANALYSIS Determinationof thetestarticlsetabilitaynd thetestarticlceharacteristiacssdefinedin theGLP regulationisstheresponsibiliotfytheSponsor. 4.0 NOTIFICATION OF REGULATORY SUBMISSION In ordertocomply with GLP regulationasnd standardsc,onsultinglaboratoriemsust be notifiedifallor partof a studyisintendedforregulatorsyubmission.Covance maintains a masterscheduleof studieswhich fallunder regulatoryreview.Pleaseindicatewhich agency,ifany,might receivetheresultosf thisstudy: Undetermined 1:1FDA MAFF 11 MOHW 12/98 M EPA-TSCA EPA-FEFRA 1:1OECD 0 OTJ[IER 1 Of 18 C 0 V A-NrC--E.@ THP DEWLOPMINT SERMES COMPAW PROTOCOL 409 EDITION 5 5.0 STUDY DATES Proposed Experimental StartDate: Proposed ExperimentalTerminationDate: 6.0 APPROVAL OF STUDY PROTOCOL Study Director. -A-k4lv(Michael S.Mecchi, M.S. TestingFacilitMyanagement: Date: Brian C. Myhr, Ph.D. AssociateDirector Sponsor'sAuthorized Representative: T Marvin T. Case,D.V.M., Ph.D. Date: C, Date: 12/98 2 of IS c 0 v A-Nr@EC@-- THRDMWPMI!Psfafnv=sCOMPMV PROTOCOL 409 EDITION 5 PART 2. STUDY PROTOCOL SALMONELLA - ESCHERICHIA COLJ/MAMMALIAN-MICROSOME MUTATION ASSAY 1. OBJECTIVE REVERSE The objectiveof thisstudyisto evaluatethe testarticlaend/oritsmetabolitesfortheir abilitytoinduce reversemutationseitherinthe presenceor absence of mammalian microsomal enzymes at1)the histidinleocusin thegenome of severalstrainsof Salmonella typhimurium and at2) the tryptophanlocusof Escherichiacolistrain WP2uvrA. 111. TEST SYSTEM A. Salmonella typhimurium: The Salmonella/Maminalian-niicrosomreeverse mutation assay detectspointmutations,both frameshiftsand/orbase pair substitutionsT.he strainsof Salmonella typhimurium used inthisassay are histidinaeuxotrophsby virtueof conditionalllyethalmutationsintheirhistidine operon. When thesehistidine-dependenctells(his-)areexposed to thetestarticle and grown under selectivceonditions(minimalmedia with a traceamount of histidineo)nly thosecellswhich revertto histidiniendependence(his+)areableto form colonies.The traceamount of histidinienthe media allowsalltheplated bacteriato undergo a few celldivisions,which isessentiaflormutagenesistobe fullyexpressed.The his+ revertantsarereadilydiscemable as coloniesagainstthe limitedbackground growth of thehis-cells.By utilizinsgeveraldifferenttester strainsb,oth base pairsubstitutiomnutationsand frameshiftmutationscan be detected.The Ames Testhas been shown tobe a sensitiver,apidand accurate indicatorof the mutagenic activitoyf many materialsincludinga wide rangeof chemical classes. B. Escherichiacoli:The EscherichiacoliWP2uvrA reverse-mutationassaydetects pointmutations,specificallbyase pairsubstitutionsT.he Escherichiacolitester strainWP2uvrA used inthisassayisa tryptophanauxotroph (ir7@'b)y virtueof a conditionalllyethalmutation ata sitewhich blocks a stepof tryptophan biosynthesispriortothe formationof anthraniliaccid.Sincethe targetsitefor trueback mutation isan ochre nonsense mutation,tryptophan-independent revertants(trp*c)an ariseeitherby a base change atthe siteof the original 12/98 3 of 18 C 0 V ArM-"EC@-. THI!DMU PROTOCOL 409 EDITION 5 alteratioonr by suppressionby specificsuppressormutations ata second sitein TRNA genes (Brusicketal,1980).When thetryptophan-dependenctells(trp-)are exposed to the testarticlaend grown under selectiveconditions(minimal media with a traceamount of tryptophan)only thosecellswhich revertto tryptophan independence (trp+)areabletoform colonies.The traceamount of tryptophanin the media allows alltheplatedbacteriatoundergo a few celldivisionsw,hich is essentiaflormutagenesisto be fullyexpressed.The trp+ revertantsarereadily discemable as coloniesagainstthe limitedbackground growth of the trp-cells. While the irpreversionsystem responds tomost alkylatingagents,base-analog mutagens and certainmetals (i.es.olublechromates),frameshiftmutagens would not be expected to be detectedby thissystem. Ill. MATERIALS A. TesterStrains 1. Salmonella typhimurium: the testerstrainsto be used will be the Salmonella typhimurium histidine auxotrophs TA98, TAIOO, TA1535, and TA1537 as describedby Ames etal(1975).The specificgenotypesof thesestrainsareshown inTable 1. TABLE 1. TESTER STRAIN GENOTYPES I-listinde Mutation AdditionalMutations hisG46 hisC3076 hisD3052 LPS Repair R Factor TA1535 TA1537 rfa uvrb TAIOO TA98 rfa uvrb +R 12/98 In additiontoa mutation inthe histidinoeperon,the testesrtrainscontain two additionalmutationswhich enhance theirsensitivityo some mutagenic compounds. The rfawallmutation resultsinthe lossofone of the enzymes responsibleforthesynthesisof partof thelipopolysacchaiide barrierthatforms thesurfaceof thebacteriac)ellwall.The resultincgell wall deficiencyincreasespermeabilitytocertainclassesofchen@calssuch as thosecontaininglargeringsystems(i.eb.enzo(a)pyrene)thatwould otherwisebe excluded by a normal intactcellwall. 4 of 18 COVArM-C@@E.@ 'r"Famu PROTOCOL 409 EDITION 5 The second mutation,a deletionof the uvrb gene,resultsina deficient DNA excisionrepairsystem which greatlyenhances thesensitivitoyf thesestrainsto some mutagens. Since the uvrb deletionextendsthrough the bio gene,allof thetesterstrainscontainingthisdeletionalsorequire the vitaminbiotinforgrowth. StrainsTA98 and TA,100 alsocontaintheR-factorplasmid,pKMI01, which furtherincreasesthe sensitivitoyf thesestrainsto some mutagens. The mechanism by which thisplasmid increasessensitivittyomutagens has been suggestedtobe by modifying an existingbacteriaD]NA repair polymerase complex involved with the mismatch-repairprocess. TesterstrainsTA98 and TA1537 are revertedfrom histidinedependence (auxotrophy)tohistidiniendependence(prototrophyb)y frameshift mutagens. TA1535 isrevertedby base substitutiomnutagens and TAIOO isrevertedby mutagens which cause both frameshiftsand base substitutions. 2. Escherichia coli:the testerstrainto be used willbe the tryptophan auxotrophWP2uvrA asdescribedby Green and Muriel (1976). In additionto a mutationin the tryptophanoperon,the testerstrain containsa uvra DNA repairdeficiencywhich enhances itssensitivityo some mutagenic compounds. This deficiencyallowsthe straintoshow enhanced mutabilitiyftheuvra repairsystem would normallyactto remove thedamaged partof the DNA molecule and accuratelyrepairit afterwards. TesterstrainWP2uvrA isrevertedfrom tryptophandependence (auxotr-ophyt)otryptophahindependence (prototrophyb)y base substitutiomnutagens. 3. Source of TesterStrains a. Salmonella typhimurium: the testerstrainsin use at Covance were receiveddirectlfyrom Dr. Bruce Ames, Department of Biochemistry,Universityof CaliforniaB,erkeley. 12/98 5 of 18 C 0 V AW-@C- E@- THE DMLOPMEoff SIMM COMMNY PROTOCOL 409 EDITION 5 b. Escherichiacoli:testerstrainWP2uvrA inuse atCovance ivas receivedfrom The NationalCollectionof IndustriaBlacteria, TorreyResearch Station,Scotland(UnitedKingdom). 4. Storaeeof theTesterStrains a. Frozen Permanent Stocks Frozen permanent stockswillbe preparedby growing fresh overnightculturesa,dding DMSO (0.09m@n-d of culture)and freezingaway appropriatelvyialedaliquots.Frozen permanent stocksof thetesterstrainswillbe storedat 70 0C. b. Master Plates Master plateswillbe preparedby streakingeach testerstrainfrom a frozenpermanent stockonto minimal agar appropriately supplemented with eitherhistidinaend biotin(and forstrains containingtheR-factor,ampicillino)r tryptophan.Testerstrain master plateswillbe storedat5 :t3 OC. 5. PreRarationof Overnight Cultures a. Inoculation Overnightculturesforuse in alltestingprocedures,willbe inoculatedby transferrinagcolony from the appropriatemaster plateto a flaskcontainingculturemedium. Inoculatedflaskswill be placedina shaker/incubatowrhich willbe programmed tobegin operation(shaking,125 t 25 rpm; incubation3,7 2*C) so that theovernightculturesarein logphase or latelog phase when turbiditmyonitoringbegins. b. Harvest To ensurethatculturesareharvestedin latelog phase,the lengthof incubationwillbe determinedby spectrophotometrimconitoringof cultureturbidity.Cultureswillbe harvestedonce a predetermined turbiditiysreached as detenrninebdy a percenttransmittanc(e%T) 12/98 6of 18 C 0 V Alt'-C@E. 7"EDMU PROTOCOL 409 EDITION 5 6. 12/98 readingon a spectrophotometer.This targetturbiditeynsuresthat cultureshave reached a densityof atleast0.5 X 10'cellsper ml and thatthecultureshave notovergrown. Overgrown (stationary) culturesmay exhibitdecreasedsensitivittyo some mutagens. Cultureswillbe removed from incubationwhen the target%T is reached and placed at 5 3 *C. Confirmation of TesterStrainGenoty2gs a. Salmonella typhimurium: testerstraincultures will be checked for the followinggeneticmarkers on the day of theiruse inthe mutagenicityassay: r.faWall Mutation ne presenceof ther.fawallmutationwillbe confirmedby demonstrationof theculturessensitivittyocrystalviolet. (2) pKM101 Plasmid The presenceofthePKMIOI plasrnidwillbe confirmedfor culturesof testerstrainTsA98 and TAIOO by demonstrationof resistancteo AmpiciIIin. (3) Spontaneous Reversion The number of spontaneousrevertantsper plateinthe vehiclecontrolsthatischaracteristiocf the respective strainswillbe demonstratedby platingaliquotsofeach culturealongwith the appropriatevehicleon selective medium. b. Escherichia coli:testerstrainWP2uvrA willbe checked forthe followinggeneticmarker on theday of itsuse in themutagenicity assay: (1) Spontaneous Reversion The number of spontaneousrevertantsper plateinthe vehiclecontrolsthatischaracteristoifcWP2uvrA willbe 7 of 18 C 0 V ArNrCE. THE MVE" PROTOCOL 409 EDITION 5 demonstratebdy platinagliquotosfeachculturaelongwith theappropriatevehicleon selectivemedium. 7. TesterStrainMedia a. CulturingBroth The brothused to grow overnightculturesof the testerstrainswill be Vogel-Bonner saltsolution(Vogel and Bonner, 1956) supplementedwith 2.5% (w/v)Oxoid NutrientBroth#2 (dry powder). b. NfinimalBottom Agar Plates Bottom agar(25 ml per 15 x 100 mm petridish)willbe VogelBonner minimal medium E (Vogel and Bonner, 1956) supplementedwith 1.5% (w/v)agarand 0.2% (w/v)glucose. C. Top Agar forSelectionofRevertants Top (overlay)agarwillbe preparedwith0.7% agar(w/v)and 0.5% NaCl (w/v)and willbe supplementedwith 10 ml of 1)0.5niM histidine/biotsionlutionper 100 ml agarforselectioonf histidine revertantso,r 2) 0.5 mM tryptophansolutionper 100 ml agarfor the selectionof tryptophanrevertants. When S9 isrequired,2.0 mi ofthe supplemented top agar isused fortheoverlay.However, when S9 isnot required,water isadded tothe supplemented top agar(0.5mi of waterper 2 ml of supplemented top kgar)and 2.5mi of the dilutedsupplemented top agarisused forthe overlay.This dilutionensuresthatthe finaltop agar and an-@noacid supplement concentrationsremain the same both inthepresence and absence of S9 mix. 12/98 8 of 18 C 0 V AW-@C- E@. THE OMU PROTOCOL 409 EDITION 5 B. LiverMicrosomal Enzvme ReactionMixture (S9 Mix) 1. S9 Homo2enate Liver n-@crosomalenzymes (S9 homogenate) willbe purchased commercially and willbe preparedfrom male Sprague-Dawley ratsthat have been injected(i.p.w)ithAroclor:rm1254 (200mg/nilincom oil)at 500 mgtkg as describedby Ames etal,1975. 2. S9 Nfix ne S9 mix willbe preparedimmediatelypriortoitsuse in any experimentalprocedure.The S9 mix willcontainthecomponents indicatedinTable 2. TABLE 2. S9 MIX COM[PONENTS H20 1M NaH2PO4/Na2BPO., pH 7.4 0.25M G)ucose-6-phosphate O.IOM NADP 0.825M KCI/0.2M MgC'2 S9 Homogenate 0.70ml 0.10mi 0.02 ml 0.04 ml 0.04 ml 0.10 ml 1.00ml C. Controls 1. Vehicle Controls Appropriatevehiclecontrolswillbe platedforallstrainsin thepresence and absence of S9 mix. Vehicles compatiblewiththistestsystem include but willnotbe limitedto:Deionized H20, dimethylsulfoxid(eCAS #67-68-5),ethanol(CAS #64-17-5),and dimethylformamide(CAS #68-12-2). 12/98 9 of 18 C 0 V A-N'C TME MWU PROTOCOL 409 EDITION 5 2. PositiveControls The combinationsofpositivecontrolsa,ctivatiocnonditionand tester strainsplatedconcurrentlwyith theassayareindicatedinTable 3. Tester Strain TABLE 3. POSITIVE CONTROLS S9 Mx PositiveControl Conc. R;rPlate TA98 + benzo[a]pyrene 2.5jig TA98 2-nitrofluorene 1.0/1g TAIOO + 2-arninoanthracene 2.5jig TA100 sodium azide 2.0,ug TA1535 + 2-aminoanthracene 2.5 jug TA1535 sodium azide 2.0jig TA1537 + 2-aminoanthracene 2.5jig TA1537 ICR-191 2.0jig Wp2uvrA + 2-aminoanthracene 25.0jig WP2uvrA - 4-nitroquinoline-N-oxide 1.0/1g 3. SteriliCtoyntrols a. TestArticle The most concentratetdestarticldeilutionwillbe checkedfor sterilibtyyplatingan aliquotofthesame volume used intheassay on selectivaegar. b. S9 Nfix The S9 mix willbe checkedforsterilibtyyplating0.5mi on selectiveagar. 12/98 10 of 18 C 0 V ArNr-C--E.@ THC OEVELOPMENT SEWCES CoMpANy PROTOCOL 409 EDrrION 5 IV. METHODS A. Dose Rangefinding Study The growth inhibitoreyffect(cytotoxicitoyf)thetestarticlteo thetestSystem will be determined inorderto allow theselectionof appropriatedoses tobe testedin the mutagenicityassay. 1. DesigLi The dose rangefindingstudywillbe performed usingtesterstrainsTAIOO and WP2uvrA both inthe presenceand absence of S9 mix. A minimum of ten doses of testarticlweillbe testedatone plateper dose. Unless otherwisedirectedby the Sponsor,the testarticlweillbe checked for cytotoxicituyp to a maximum concentrationof 5,000 Ag per plateif solubility/miscibipleirtmyits.Ifthetestarticleexhibitslimited solubility/miscibiliittwyi,llbe testedforcytotoxicituyp to themaximum workable concentrationattainablien thevehicleof choice. a. Rationale The growth inhibitoreyffect(cytotoxicitoyf)thetestarticloen testerstrainTAIOO isgenerallyrepresentativoefthatobservedon the otherSalmonella typhimurium testerstrainsand because of TAIOO's comparativelyhigh number of spontaneous revertantpser plate,gradationsof cytotoxicitcyan be readilydiscernedfrom routineexperimentalvariationT.he Escherichiacolitesterstrain WP2uvrA does not possesstherfawa mutationthatthe Salmonella typhimurium strainshave and thus,a differenrtangeof cytotoxicitmyay be observed.Also,thecytotoxicitiynducedby a testarticlienthe presenceofmicrosomal enzymes may vary greatlyfrom thatobservedinthe absence of microsomal enzymes. Therefore,thiswould requirethatdifferentestarticldeose ranges be testedinthe mutagenicityassaybased on the presence or absence of the microsomal enzymes. 12/98 11 of 18 C 0 V ArNf@C--E.@ THI!am" PROTOCOL 409 EDITION 5 2. Evaluationof theDose RaneefindingStudy Cytotoxicityisdetectableas a decreaseinthe number of revertantcolonies per plateand/orby a thinningor disappearanceof the bacteriablackground lawn. 3. Selectionof theMaximum Dose fortheMutagenicityAssay a. CytotoxicityObserved When cytotoxicitiysobserved in thedose rangefindingstudy,the highestconcentrationof testarticlteobe used in the subsequent mutagenicityassaywillbe thatwhich givesa detectablereduction of revertantsper plateand/ora thinningor disappearanceofthe bacteria]background lawn. b. No CytotoxicityObserved Ifno cytotoxicitiysobserved inthe dose rangefindingstudythen the highestdose oftestarticlteobe used inthe mutagenicityassay willbe thesame as thattestedinthe rangefindingstudyunless1) the Sponsor specifieasn alternatmeaximum dose,2) thereis limitedavailabiliotfythetestarticlaend/or3) thetestarticle precipitatehseavilyinthe top agar. B. MutagenicityAssay 1. Design The assaywillbe performed usingtestesrtrainsTA98, TAIOO, TA1535, TA1537, and WP2uvrA, both inthe presenceand absence of S9 mix. Unless the Sponsor specifiesdoses tobe tested,the doses of testarticle willbe selectedbased on the resultsof the dose rangefindingstudy.If cytotoxicityhas been demonstratedin thedose rangefindingstudy,a minimum of sixdosesof testarticlweillbe testedalongwith the appropriatevehicleand positivecontrols.Ifno cytotoxicithyas been demonstrated,a minimum of fivedoseswillbe tested. 12/98 12 of 18 C 0 V A-U--CE-@- THE DMLOONEW SEW#CES COMPANY PROTOCOL 409EDITION 5 FrequencayndRouteofAdministration The testerstrainwsillbe exposed tothetestarticlveiatheplate incorporatiomnethodologyoriginalldyescribedby Ames eta](1975)and Maron and Ames (1983).Thismethodology has been shown todetecta wide rangeof classesofchemicalmutagens. In theplateincorporation methodology,theS9 mix (whereappropriate)t,hetesterstraina,nd thetest articlaerecombined in moltenagarwhich isoverlaidontoa minimal agar plate.Followingincubationat37 20C for52 4 hr,revertanctolonies willbe counted.Alldosesoftestarticlev,ehiclecontrolsand positive controlswillbe platedintriplicate. C. PlatingProcedures These procedureswillbe used inboth thedose rangefindingstudyand the mutagenicityassay. Each platewillbe labeledwitha code which identifietshetestarticlet,estphase, testerstraina,ctivatiocnonditionand dose. The S9 mix and dilutionosf thetest articlweillbe preparedinunediatelpyriortotheiruse. When S9 mix isnotrequired1,00 ILIof testerstraiannd 50 uIof vehicleortest articldeose willbe added to 2.5ml of molten selectivteopagar(maintainedat 45 2*C). When S9 mix isrequired5,00 Al of S9 nlix,100 Al of testesrtraiannd 50 iLlof vehicleor testarticldeose willbe added to 2.0rn]of moltenselectivteop agar.Aftertherequiredcomponents have been added,themixturewillbe vortexedand overlaidontothesurfaceof 25 ml ofminimal bottom agarcontained ina 15 x 100 mm petiidish.Aftertheoverlayhas solidifietdh,eplateswillbe invertedand incubatedfor52 4 hr at37 2*C. When necessaryp,lating aliquotsof otherthan50 iLlof testarticle/vehicwliellbe used. Positivecontrol articlewsillbe platedusinga 50 lilplatingaliquot. D. ScoringthePlates Plateswhich arenotevaluatedimmediatelyfollowingtheincubationperiodwill be heldat5 3*C untilsuchtime thatcolonycountingand bacteriablackground lawn evaluationcan takeplace. 12/98 13 of 18 C 0 V A-NrC'-E.'-'\ THE DmLopmara sEnVICES compom PROTOCOL 409 EDITION 5 I BacteriaBlackground Lawn Evaluation The conditionof thebacteriablackgroundlawn willbe evaluatedfor evidenceofcytotoxicitaynd testarticlperecipitatEev.idenceof cytotoxicitwyillbe scoredrelativteothevehiclecontroland recorded alongwith therevertanctountsforthatdose. 2. CountingRevertantColonies Revertantcoloniesfora specifitcestesrtraiw'inthina giventestarticle dilutionserieswillbe countedeitherentirelbyy automatedcolonycounter orentirelbyy hand (withtheexceptionofthedoserangefindinsgtudy).If thereissufficienttestarticlperecipitatoen theplatesatany dose which interferewsithautomatedcolony counting,thentheplatesatalldose levelsforthatspecifisctrainand activatiocnonditionwillbe counted manually. E. Analysisof Data For allreplicatpelatingst,hemean revertantpserplateand thestandard deviationwillbe calculated. V. EVALUATION OF TEST RESULTS Beforr,assaydatacan be evaluated,thecriterifaora validassaymust be met. A. CriteriFaor A ValidAssay The followingciiteriwaillbe used to determinea validassay: I. Tester Strain Integfi :Salmonella typhimurium a. r.faWall Mutation To demonstratethepresenceof ther.fawallmutation,testerstrain culturemsust exhibitsensitivitoycrystavliolet. 12/98 14 of 18 C 0 V Alt-'CE-@. THE DMLCWMSMT SRFMCES COMPANY PROTOCOL 409 EDrrION 5 b. PKMIOI Plasmid To demonstratethepresenceof theR-factorplasmid,PKMIOI, culturesof testerstrainTsA98 and TAIOO must exhibitresistance to ampicillin. C. CharacteristiNcumber of SpontaneousRevertants To demonstratetherequirementforhistidinet,hetesterstrain culturesmust exhibita characteristniucmber of spontaneous revertantpserplatewhen platedalong withthevehicleunder selectivceonditions.The acceptablerangesforthemean vehicle controlsareas follows: TA98 TAIOO TA1535 TA1537 8- 60 60-240 4- 45 2- 25 2. Tester StrainIntegdly:Escherichia coli a. CharacteristNiucmber of SpontaneousRevertants To demonstratetherequirementfortryptophan,thetesterstrain culturemust exhibita characteristniucmber of spontaneous revertantpser platewhen platedalongwiththevehicleunder selectivceonditions.The acceptablerange fortheNVP2uvrA mean vehiclecontrolsis5 to40 revertantpserplate. 3. TesterStrainCultureDensity To demonstratethatappropriatneumbers of bacteriareplated,thedensity of testerstrainculturesmust be greaterthanor equalto0.5 x 109bacteria per mi and/orhave reacheda targetlevelof turbiditdyemonstratedto produce cultureswitha densitygreaterthanorequalto 0.5x 109bacteria perml. 12198 15 of 18 C 0 V A-N-C-E-@- THIE T)MWPMEMT SERA"CES COMPAW PROTOCOL 409 EDITION 5 4. PositiveControlValues a. PositiveControlValues intheAbsence of S9 Mix To demonstratethatthe testerstrainsare capable of identifyinag mutagen, the mean value of a positivecontrolfora respective testerstrainmust exhibitatleasta 3-foldincreaseover themean valueofthe vehiclecontrolforthatstrain. b. PositiveControlValues in thePresenceof S9 Mx (S9Mx Integrity) To demonstratethatthe S9 mix iscapableof metabolizinga promutagen to itsmutagenic forin(s)t,hemean value of the positivecontrolfora respectivetesterstrainin the presenceof the S9 mix must exhibitatleasta 3-foldincreaseover the mean value of the vehiclecontrolforthatstrain. An acceptablepositivecontrolinthe presenceof S9 for a specific strainwillbe evaluatedas having demonstratedboth the integrity of theS9 niixand the abilitoyf thetesterstrainto detecta mutagen. 5. czotoxicity A minimum of threenon-toxicdose levelswillbe requiredtoevaluate assay data. B. CriteriaFor A PositiveResRonse Once the criterifaora validassay have been met, responsesobserved intheassay areevaluatedas follows: I. TesterStrainsTA98, TA 100,and NNT2uvrA For a testarticlteo be consideredpositivei,tmust produce atleasta 2-fold increaseinthe mean revertantsper plateof atleastone of thesetester strainsover themean revertantsper plateofthe appropriatevehicle control.This increasein themean number ofrevertantsper platemust be 12/98 16 of 18 C 0 V A-M-@EC@- THE DMLOPMEKT SEWCES COMMNY PROTOCOL 409 EDrrION 5 accompaniedby a doseresponsteoincreasicnogncentratioonfsthetest article. 2. TesterStrainsTA1535 and TA1537 For a testarticlteobe consideredpositive,itmust produce atleasta 3-fold increaseinthe mean revertantsper plateof atleastone of thesetester strainsover the mean revertantsper plateof the appropriatevehicle control.This increaseinthe mean number of revertantpser platemust be accompanied by a dose response to increasingconcentrationsof the test article. VI. REPORTING THE RESULTS A reportof the resultsof thisstudywillbe preparedby Covance and willaccurately describeallmethods used forthe generationand analysisof thedata. Resultspresentedinthe reportforthisassaywillinclude: The resultosfthedoserangefindisntgudy(ifapplicablien)cludintghenumberof revertanptesrplataenda bacteribaa]ckgrounldawnevaluatifoonreachdose the identityof thebacteriatlesterstrainsused inthe assay dose levelsatwhich the testarticlweas tested individualplatecounts foralltreatedp,ositiveand vehiclecontrolplates calculatedmean and standarddeviationforallreplicatpelatecounts evaluationof results VII. -CHANGES AND REVISIONS Any changes or revisionsofthisapproved protocolwillbe documentecl signedby the Study Director,dated,and maintainedwith thisprotocol.The Sponsor willbe notifiedof any changes or revisions. 12198 17 of 18 COVA-Nr@C@E.@ THE DEVELOPMENT SIMCIS Commmy PROTOCOL 409 EDITION 5 VIU. RECORDS TO BE MAINTAINED All raw data,documentation,records,the protocol,and thefinalreportgeneratedas a resultof thisstudywillbe archivedinthe storagefacilitioefsCovance-Vienna, forat leastone yearfollowingsubmissionof thefinalreportto theSponsor. Aftertheone year period,theSponsor may electto have the aforementionedmaterialsretainedin the storage facilitioefsCovance-Vienna, foran additionalperiodof time orsentto a storagefacility designatedby the Sponsor. IX. REFERENCES Ames, B.N.,McCann, J.and Yamasaki, E. (1975). Methods fordetectingcarcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicitytest. Mutation Research 31:347-364. Brusick,DJ., Simmon, V.F.,Rosenkranz,H. S.,Ray, V.A.,and StaffordR,.S. (1980). An evaluationof theEscherichiacoliV#rP2and WP2uvrA reversemutationassay. Mutation Research,76:169-190. Green,M.H.L. and Muriel,WJ. (1976).Mutagen testingusingtV reversionin Escherichiacoli.MutationResearch38:3-32. Maron, D.M., and Ames B. (1983).Revised Methods fortheSalmonellaMutagenicity Test. Mutation Research 113-173-215. Vogel, H.J.and Bonner, D.M. (1956). Acetylomithinaseof Escherichiacoli:Partial purificatioannd some propertiesJ..Biol Chem. 218:97-106. 12/98 18 of 18