Document 85Re5vaQj9eJ7JBej3oBkdXkZ

FinalReport Unscheduled DNA SynthesisinRat LiverPrimary CellCultures With PFOS PREPARED FOR: 3M CorporateToxicology COVANCE STUDY NUMBER: 20784-0-447 C 0 V A- -C'-E-.'@-' THEDENU UNSCHEDULED FINAL REPORT DNA SYNTHESIS IN RAT LIVER PRDAARY CELL CULTURES WITH PFOS AUTHOR Maria A. Cifone,PhD PERFORMING LABORATORY Covance LaboratorieIsnc. 9200 LeesburgPike Vienna,VA 22182 LABORATORY PROJECT IDENTIFICATION Covance StudyNo.: 20784-0-447 SponsorStudy No.: T6295.19 SUBMITTED TO 3M CorporateToxicology 3M Center Building220-2E-02 St.Paul,MN 55144-1000 STUDY COMPLETION DATE November 9,1999 Page I of 20 Covance 20784-0-447 QUALITY ASSURANCE STATEMENT Unscheduled DNA SynthesisInRat LiverPrimary CellCultures With PFOS The reporthas been reviewedby the QualityAssurance Unitof Covance LaboratoriesInc.,in accordance with theGood LaboratoryPracticeregulationsassetforthin'the Environmental ProtectionAgency (EPA-TSCA), Title40 of theU.S. Code of FederalRegulationsPart792, and with any applicableamendments. The followinginspectionswere conducted and thefindings reportedtothe Study Directorand studydirectormanagement. Writtenstatusreportsof inspectionsand findingsare issuedtoCovance management accordingto standardoperating procedures. InspectionDates Phase Dates Reported to Study Directorand Study DirectorManagement Auditor 08/31/99 11/1,2/99 11/09/99 Weighing of TestArticle Protocoland DraftReport Review FinalReport Review 08/31/99 11/2/99 11/09/99 P. Ciceres P. Ciceres C. Smith .....p........i..@.@., RepresentativeQ,ualityAssurance Unit Date -2- Covance 20784-0-447 STUDY COMPLIANCE AND CERTIFICATION The describedstudywas conducted incompliance with theGood LaboratoryPracticeregulations assetforthintheEnvirorumentalProtectionAgency (EPA-TSCA), Title40 of theU.S. Code of FederalRegulationsPart792,and with any applicableamendments. There were no significant deviationsfrom the aforementionedregulationosr the signedprotocolthatwould affectthe integritoyfthe studyor theinterpretatiofn thetestresults.The raw datahave been reviewed by the Study Director,who certifietshatthe evaluationof the testarticlaespresentedherein representsan appropriateconclusionwithinthe contextofthe studydesignand evaluation criteriaA.lltestand controlresultsinthisreportaresupportedby an experimentaldatarecord and thisrecordhas been reviewed by the Study Director. Study Director: M'gia A. Cif6ne,PhD Genetic and Cellular4@xicology Study Completion Date -3- Covance 20784-0-447 TABLE OF CONTENTS ABSTRACT .................................................................5.................... STUDY INFORMATION Sponsor TestArticle Assay Information Study Dates SupervisoryPersonnel ......................................................6................... OBJECTIVE .................................................................6................... TEST SYSTEM RATIONALE .................................................6.................... MATERIALS AND METHODS Test System ControlArticles ................................................7................... EXPERIMENTAL DESIGN ...................................................8.................... Dosing Procedure UDS Assay Assay Acceptance Criteria Assay EvaluationCriteria RESULTS ..................................................................1.2................... CONCLUSION ..............................................................1.3................... RECORDS TO BE MAINTAINED ............................................1.4.................... REFERENCES ...........................I.........................................................1.4.................... DATA TABLES .............................................................1.5................... HISTORICAL CONTROL DATA ..............................................1.9................... -4- Covance 20784-0-447 ABSTRACT The testarticleP,FOS, was testedinthein vitroassayforunscheduled DNA synthesisinratliver primarycellcultures.PFOS was insolubleinwaterand Williams'Medium E. In dimethylsulfoxid(eDMSO), thetestarticlfeormed a suspensionwith precipitattehatsettletdo thebottom of thetube at501 mghnl. The precipitatweas absentat401 mg/mL. DMSO was chosen as thevehicle.Inthe assaydescribedinthisreport,rathepatocyteswere exposed tothe testarticlpereparedinDMSO and dosed intomedia. Inmedium, a suspensionwith fine precipitatweas observedat4010 4g/mL Based on solubilitiynDMSO and medium, a concentrationof 4000 gg/ml was selectedasthe top dose forthe invitroUDS assay.FifteenconcentrationosfPFOS were applied,rangingfrom 0.250 jig/mLto4000 jig/mL. Cytotoxicitwyas determinedby comparing the celldensityofthe treatedplatestothe celldensityof the controlplatesapproximately23 hours afterinitiatioonf treatment.The testarticlweas excessivelycytotoxicatand above 50.0 gg/ml,weakly cytotoxic at25.0gg/ml, and noncytotoxicatand below 10.0Rg/ml. The cellulamrorphology was suitable foranalysisatand below 25.0 gg/ml. No evidence foran increaseinUDS was observed. None ofthe dose levelsshowed an elevationinmean netnucleargrains(NNG) or inthe number of nucleicontaining5 ormore net grains.The testarticleP,FOS, was thereforeevaluatedas negativein theinvitroassay forunscheduledDNA synthesisinratliverprimary cellcultures. Covance 20784-0-447 STUDY INFORMATION Sponsor 3M CorporateToxicology Test Article Sponsor's Identification:PFOS (FC-95),Lot 217 Date Received: August 19, 1999 Physical Description: White crystallinpeowder Storage Conditions: Ambient temperature Assay Information Type ofAssay: Unscheduled DNA Protocol No.: 447 Edition17 Covance Study No.: 20784 3M Study No.: T6295.19 SynthesisinRat LiverPrimary CellCultures Study Dates InitiationDate: August 20, 1999 Experimental StartDate: September 1,1999 Experimental Termination Date: September 22, 1999 Supervisory Personnel Study Director: Maria A. Cifone,PhD Laboratory Supervisor: Agnes Nourbakhsh, BS OBJECTIVE The objectiveofthisin vitroassaywas toevaluatetheabilityofthe testarticleP,FOS, or a metabolite,toinduceDNA damage inratprimary hepatocytesby measuring unscheduledDNA synthesis(LJDS). TEST SYSTEM RATIONALE This assayaims toestabliswhhether thetestarticloer itsmetabolitesinteractwsith ratliver primarycell(hepatocytec)ulturestoinduceDNA damage followedby DNA repairmeasured as UDS. Hepatocyteswere isolatedfrom liverand exposed invitrotothetestarticlaend tritiated thyrnidine(3H-TdR). Primary hepatocyteshave sufficienmtetabolicactivittyoeliminatethe need fortheadditionof a microsomal activatiosnystem. An autoradiographitcechnique describedby Williams (1977)was used tomeasure incorporatioonf3 H-TDR intoDNA. Because only a smallpercentageof thecellsenterS-phase (replicativDcNA synthesisd)uringthe exposure period,the incorporationof3 H-TDR intoDNA isused as a measure of therepairof DNA damage caused by treatmentwith thetestarticleT.he existenceand degree ofDNA -6- Covance 20784-0-447 damage isinferredfrom an increaseinnucleargraincounts intreatedhepatocytescompared to nucleargraincounts from untreatedhepatocytes.The typesof detectableDNA damage are unspecifiedbut must be recognizableby thecellularepairsystem and resultinthe incorporation of nucleicacidbases including3H-TDR intotheDNA. MATERIALS AND METHODS Test System The indicatocrellsforthisassaywere hepatocytesobtainedfrom a singleadultmale,Fischer344 rat,weighing 214.0 grams. The animal was purchased from CharlesRiver LaboratoriesR,aleigh, NC, and was housed ina shippingcrateand receivedfood suppliedforshippingby the vendor and water ad libitum.The ratwas used forthesurgicalprocedurewithin2 days of receipta,nd was anesthetizebdeforesurgerywith about 60 mg/kg sodium pentobarbitably intraperitoneal injectionand exsanguinatedduringtheprocedure. The cellswere obtainedby perfusionof theliverinsituwith a collagenasesolutionW,MEC (see CellCollectionand Cultureinthesectionon theUDS assay).Monolayer cultureswere establisheodn plasticcoverslipsinculturedishesand were used the same day forinitiatioonf the UDS assay. Media and Cell Culture Conditions. Williams'Medium E supplemented with 2 mM L-glutamine,100 @Lg/mlstreptomycinsulfatea,nd 150 @Lg/mlgentamicin(WMEI) was thebase culturemedium, and was modified foreach specificrequirement.The hepatocyteswere obtained by perfusionof liversinsituwith HBS S/EGTA followedby WMEC:Hanks' balancedsalts (Ca++-andMg++-free)containing0.5 mM ethyleneglycol-bis(O-aminoetehtyhler)-N,N-tetraaceticacidand 50 mM HEPES bufferatpH 7.2(HBSS/EGTA), and WMEI containing 50-100 units/mlof collagenaseand 50 mM HEPES bufferatpH 7.2(WMEC). The cultures were establisheidnWMEI supplementedwith 10% fetalbovineserum (WME+). Allcell cultureswere maintainedasmonolayers in a humidifiedincubatorat35 to37.5*C inan atmosphere of4 to6% C02 in air.Aftertheestablishepderiod,the culturelabelingwas initiated usingWMEI containing10 PCi/Ml 3H-TDR at40 to60 Ci/mMole (WME-treat). Control Articles VehicleControlArticle.The vehiclecontrolculturerseceiveda concentratioonfthevehicle equivalenttothevehicleinthetestarticle-treatceudlturesand were subjectedto allof the experimentalmanipulationthatthetestarticle-treatceudlturesreceived.Dimethylsulfoxide (DMSO; CAS 67-68-5,Lot #128H3460, Sigma Chemical Co.)ata concentratioonf 1% was used foralltrials. PositiveControl Article.The positivecontrolcompound isknown toinduceUDS inrat hepatocyteprimarycellcultures.2-Acetylaminofluoren(e2-AAF, CAS# 53-96-3,Lot#57HO293, SigrnaChemical Co.)was dissolvedinDMSO and diluted1:100 intoWME-treat fora final concentratioonf 0.10 pg/ml (4.48x 10-7M). -7- Covance20784-0-447 EXPERIMENTAL DESIGN DosingProcedure A preliminasroylubilitteyswtas performewdiththetestarticlPeF,OS,inwater, Williams'Media E and DMSO. The testarticlweas insolubleinallvehiclesbut formed an acceptablesuspensioninDMSO at401 mg/mL. DMSO was thereforechosen as thevehicle. When dosed intomedia formammalian cellst,hetestarticlfeormed a suspension/ftmslucent liquidwith very fineparticleosfprecipitataet4010 @tg/mL. Based on thisinformation,a concentrationof4000 gg/ml was selectedasthetop dose. A freshpreparationof testarticlienthevehiclewas used forthe assay.A suspension/solutioonf testarticlienDMSO was serialldyilutedwith DMSO to obtainthe finaldesiredconcentrations of testarticleD.MSO stockswere dosed 1:100 intomedia tocreatethe desireddosing concentrations. The sponsorisresponsibleforthe determinationand documentationof the analyticaplurityand compositionofthe testarticlaend the stabilitaynd strengthofthe dosingsolutions. UDS Assay Dose Determination. A concur-renctytotoxicitayssessmentwas used to selectappmpriatedoses fortheparticularf,reshprimary cultureofhepatocytesused inthisstudy.A range of fifteen concentrationfsrom 0.25 gg/ml to4000 gg/ml was appliedtothe cells.A viablecellcount, based on trypanbluedye exclusion,was thenobtainedapproximately23 hours afterinitiatioonf the treatments.This informationwas used to choose sixconcentrationfsoranalysisofnuclear labelingstartinwgith thehighestdose thatresultedina sufficienntumber ofsurvivingcellswith intactmorphologies and proceedingtosuccessivelylower doses. CellCollectionand Culture. This assaywas based on theproceduresdescribedby Williams (1980),and Butterworthetal.(1987). The hepatocyteswere obtainedby perfusionof liver insituforfourminutes with HBSS/EGTA thenfor II minutes with WMEC. The hepatocytes were obtainedby mechanical dispersionof excisedlivertissueina culturedishcontaining WMEC. The suspended tissueand cellswere thenallowed to settlteoremove cellclumps and debrispriorto collectionT.he collectedcellsuspensionwas centrifugedand the cellpellet resuspendedinWME+. Afterobtaininga viablecellcount,a seriesof35 mm culturedishes(at least5 per dose level)were inoculatedwith approximately0.5 x 106 viablecellsin 3 ml of WME+ per dish.Culturedishesused fortheUDS analysiscontainedround plasticoverslips, while dishesused toassessattachmentefficiencaynd toxicityhad no coverslips. To establisthhe cellculturesc,ellswere allowed an attachmentperiodof 2 hours at35 to 37.5*C in a humidifiedatmosphere containing4 to 6% C02. Unattached cellswere thenremoved and the cultureswere refedwith WMEI. The LJDS assaywas initiate1d.4hours laterby replacing the media intheculturedisheswith 2.5ml WME-treat containingthe testarticlaetthedesired -8- Covance 20784-0-447 concentrationE.ach treatment,includingthepositiveand vehiclecontrolsw,as performed on at leastfiveculturest,wo ofwhich were used forcytotoxicitmyeasurements. Termination and CytotoxicityMeasurement. Afteran exposureperiodof 19.6to20 hours, theUDS assaywas terminatedby washing the cellmonolayers twicewith WMEI. The cultures used forthecytotoxicitmyeasurements were refedwith WMEI and returnedtothe incubator.At approximately23 hours aftertheinitiatioonfthe treatmentsc,ytotoxicitwyas assessedas cellular morphology and cellsurvivalrelativteothevehiclecontrol.Viablecellcountswere obtained usingtrypanbluedye exclusion. The triplicatleabeledcellculturesforUDS analysiswere refedwith WMEI containingI mM thyrnidineand incubatedfor30 minutes. The nucleiinthecellswere swollenby additionof I% sodium citratteo thecoverslips(containintghecellmonolayers)for 12 minutes,and thenthe cellswere fixedwith coldaceticacid:ethano(l1:3)and driedovernight.The coverslipwsere mounted on glassslidesd,ippedin a solutionof Kodak NTB2 emulsion and deionizedwater,and dried.The coatedslideswere storedfor6 days at2 to8*C inlight-tighbtoxes containingpackets ofDrieritee.The emulsions were thendeveloped inKodak D19, fixed,and stainedusing Williams'modified hematoxylinand eosinprocedure. SlideAnalysis. The cellswere examined microscopicallaytapproximately150OX magnificationunder oilimmersion and thefieldwas displayedon thevideo screenof an automaticcounter.UDS was measured by countingnucleargrainsand subtractingtheaverage number of grainsinthreenuclear-sizecdytoplasmicareasadjacenttoeach nucleus (cytoplasmic count).Thisvalue isreferredtoasthe netnucleargraincount. The coverslipswere coded to preventbiasin graincounting. The netnucleargraincount was determinedforfiftyrandomly selectedcellson each coverslip. Only nucleiwith normal morphologieswere scored,and any occasionalnucleiblackenedby grainstoo numerous to countwere excluded as cellsinwhich replicativDeNA synthesis(Sphase)occurredratherthanrepairsynthesis.Normally, 150 cellsper dose levelwere read (50 from each of thetriplicatceoverslips)T.he complete number of 150 cellsper dose may not be availabledue totoxicitoyr qualityofpreparation.Graincountdataobtainedfora given treatmentisacceptableaspartof theevaluation-iofbtainedfrom atleasttwo replicatecultures and atleast100 cellsper dose. The mean netnucleargraincount isdetermined from the triplicatceoverslipsforeach treatmentcondition. Assay Acceptance Criteria An assaynormallyisconsideredacceptableforevaluationof thetestresultsonlyifallof the criterilaistedbelow aresatisfiedT.his listinmgay not encompass alltestsituationst,husthe studydirectorexercisesscientifjiucdgment inmodifying thecriterioar consideringeventsthat might affectassayreliabiliatnyd acceptance. CellCulture Conditions. The viabilitoyf thehepatocytescollectedfrom theperfusionprocess normallyexceeds 70%. A varietyof factorscan affectcellyieldand viabilitys,o valuesbelow -9- Covance 20784-0-447 70% arenot uncommon nor necessarildyetrimental.A lower limitforacceptabilitiyssetat50% inordertoavoid thepossibleuse of a damaged, unrepresentativseample of cells. The viabilitoyfthemonolayer cellculturesused forthe assaytreatmentsmust be 70% or greater. Normally,theviabilitoyf attachedcellsisabout90%. The number ofviablecellsinthevehiclecontrolculturesshouldremain reasonablystableover the experimentaltime periodbecauserapidlydeclining(dying)culturemsay not respondina representativmeanner tothetestarticltereatments.Therefore,theaveragenumber of viablecells inthevehiclecontrolculturesmust not be lessthan 50% of thecellnumber atthebeginningof thetreatmentperiod. Acceptable Controls. The averagenetnuclearlabelinginthevehiclecontrolculturesmust be in therange of -5.00to 1.00.In additionn,o more than 10% ofthe cellsshouldcontainfiveormore netnucleargrains.Failuretomeet eitherof thesecriteriwaillinvalidattehe assay. The positivecontrolisused to demonstratethatthe cellpopulationemployed was responsiveand themethodology was adequateforthedetectionof UDS. For testarticlecsausingweak orno LJDS activityt,he averageresponsetothepositivecontroltreatmentsmust exceed both criteria used to indicatUeDS (seeAssay EvaluationCriterisaectionof thisreport).For a testarticle clearlycausingdose-relateUdDS activitya,n assay isacceptableintheabsence of a positive controllostfortechnicalreasons. Acceptable High Dose. Itisnot necessaryto includetoxicdoses (lessthan95% survivali)nthe LTDS analysisbut itisnecessarytoshow thatan increaseinconcentrationresultsinexcessive toxicityasmeasured by eithersurvivalsbelow 50% orobservationofpoor cellulamrorphology. Highly toxicdoses can resultinreduced incorporatioonf 3H-TDR resultingindecreasedgrain countsor poor cellulamrorphology and may leadtoartifactdsuringgrainanalysis.The highest analyzeddose must approach excessivetoxicityo,r resultintestarticliensolubilitoyr,reachthe highestapplicabledose of 5 mg/ml. Acceptable Number ofDoses. A minimum of sixdose levelsmust be analyzedfornuclear graincounts. Repeat trialnseed onlyauginentthe number of analyzeddose levelsinthe firstrial toachievea totalof sixdifferenctoncentrations. Grain count dataobtainedfora given treatmentisacceptableaspartofthe evaluationifobtained from atleasttwo replicateculturesand atleast100 cellsper dose. Assay Evaluation Criteria Severalcriterihaave been establishewdhich, ifmet, willprovidea basisforevaluationof a test articlaesactivein theLTDS assay.The criterifaora positiveresponsearebased on a statistical analysisof Covance historicadlataand calculatioonf therequiredminimum increaseinnuclear labelingusing an approach describedby Casciano and Gaylor (1983). -10- Covance 20784-0-447 The testarticliesconsideredactivein theLTDS assayatappliedconcentrationsthatcause: An increaseinthemean net nucleargraincount to atleastfivegrainsper nucleusabove the concurrentvehiclecontrolvalue,and/or; An increaseinthenumber of nucleiwith fiveormore net grainssuch thatthepercentageof thesenucleiintestculturesis10% above thepercentageobserved invehiclecontrolcultures. Generally,ifthe firsctonditionissatisfietdh,e second oftenwillbe met. However, satisfaction of onlythe second conditioncan alsoindicateUDS activityW.hen netnuclearlabelingis elevatedabove thevehiclecontrollabelingbut does not reach the statisticalsliygnificant miniminn, theoverallpatternof theresponseisconsideredinthe evaluation.DifferentDNAdamaging'agentscan givea varietyofnuclearlabelingpatterns,and weak agentsmay strongly affectonlya minorityof thecells.Therefore,both ofthe above conditionsareconsideredinan evaluation. A dose-relateidncreaseinLTDS foratleasttwo consecutiveappliedconcentrationsisalso desirabletoevaluatea testarticlaes activeinthisassay. Insome cases,LTDS can increasewith dose and thendecreasetonear-zerowith successivelyhigherdoses. Ifthisbehaviorisassociated with increasedtoxicityt,hetestarticlcean be evaluatedasactive.Ifan isolatedincreaseoccurs fora treatmentfarremoved from thetoxicdoses,the LTDS isconsideredspurious. The testarticliesconsiderednegativeifnone ofthe above criteriaaremet. When, inthejudgment ofthe studydirectorr,esultsareneitherclearlypositivenor clearly negative,thepresence of a dose response,thefi-equencydistributioonf cellularesponses,and thereproducibilitoyf dataamong slidesisconsidered. The positivecontrolnuclearlabelingisnot tobe used asa referencepointtoestimatemutagenic or carcinogenicriskassociatedwith theLTDS activitoyf thetestarticleU.DS elicitebdy test agentsinthisassay isprobablymore dependenton thetypeof DNA damage inflicteadnd the availablerepairmechanisms thanon thepotency ofthe testagent asa mutagen or carcinogen. Some forms of DNA damage arerepairedwithoutthe incorporatioonfnew nucleicacids.Thus, thepositivecontrolsare used todemonstratethatthe cellpopulationemployed was responsive and themethodology was adequate forthedetectionof UDS. Covance 20784-0-447 RESULTS A preliminarysolubilittyestwas performed and the testarticleP,FOS, was insolubleinwater and Williams'Media E. In DMSO, thetestarticlfeormed a translucenltight-yelloswuspension with a few undissolvedpiecesof testarticloen thebottom ofthe vialata concentrationof 501 mg/mL. At 401 mg/mL, a translucenltight-yelloswuspensionwith a smallamount offoam on top was observed. When dosed intomedia formammalian cellst,he testarticlfeormed a suspension/tr-mslucelnitquidwith veryfineparticleosfprecipitataet4010 gg/mL. At concentrationfsrom 253 mg/mL to 15.6mg/mL inDMSO, PFOS formed a translucenotff-white towhite homogenous suspensionwith a smallamount of foam on top. When dosed intomedia, 312 @tg/mL formed a transparentmedia-coloredsolution.Based on thisinformation,a concentrationof 4000 @ig/mL was selectedas thetop dose. Inthe UDS assay,thetestarticlfeormed a translucenltight-yellohwomogeneous suspension with a small amount of foarnon top inDMSO at400 mg/mL. The stockbecame a transparent colorlessolutionat50.0mg/mL. In media priorto treatment,a precipitatweas presentfrom 4000 jig/mLto 1000 pg/niL. Fifteenconcentrationosf PFOS were applied,rangingfrom 4000 4g/mL to0.250 @Lg/mL. The testarticlweas excessivelycytotoxicatand above 50.0 @Lg/mL,weakly cytotoxicat25.0 gg/mL, and noncytotoxicat and below 10.0 @Lg/mL. The cellulamrorphology was suitableforanalysisat and below 25.0 @Lg/mL. The hepatocyteswere collectedata calculatedviabilitoyf92.3% based on tr)?anblueexclusion. The treatmentswere initiatewdithin3 hours with cellmonolayers thatwere 96.7% viable,with an attachmentefficiencoyf94.5%. Afteran additiona2l2.7hours inculture(which encompassed the 19.6-hourtreatmentperiod),theaverageviablecellcount inthe controlcultures was 86.0% oftheviablecount atthebeginningofthetreatments.This stabilitiyncellnumber and thenormal morphologicalappearance ofthecellsindicatedthatthehepatocytecultureswere ingood metabolicconditionfortheLJDS assay. The vehiclecontrolvalueswere a mean netnucleargraincount of -4.74and a mean percent nucleicontainingfiveormore netgrainsof2.67%. These valueswere used todeterminethe minimum criterifaora positiveLJDS response.-For a treatmenttobe consideredpositive,there must be an increaseinthemean netnucleargraincount toatleastfivegrainsper nucleusabove theconcurrentvehiclecontrolvalue,and/oran increasein thenumber of nucleiwith fiveormore net grainssuch thatthe percentageofthesenucleiintestculturesis10% above thepercentage observedinvehiclecontrolcultures.The criterifaora positiveresponseinthisassaywere a mean net nucleargraincount exceeding 0.26and /oratleast12.67% ofthenucleicontainingfive or more netgrains.None of thetreatmentswith thetestarticlseamples caused a positive responseineitherthemean nucleargraincount or inthe nucleicontaining.fivoermore net grains. -12- Covance 20784-0-447 The 2-AAF treatmentinduced largeincreasesinnuclearlabelingthatexceeded both criteriuased toindicateLTDS. Thispositiveresponseconfirmed thatthe hepatocytecultureswere metabolicallyactiveduringthe LTDS assay. Heavily-labelednucleithatwere blackenedwith numerous grainsrepresentcellsundergoing DNA replicatio(nS-phase)as opposed toDNA repair.The number presentinthisstudywas low and did not interferweith theassay. CONCLUSION The testarticldeid not induce unscheduled DNA synthesisa,smeasured by mean netnuclear graincountsorthe percentageof nucleicontainingfiveor more net grains.Doses were chosen foreach assaybased on cellcounts determined at20-24 hours afterdosing and the analyzed treatmentscovered a good rangeof cytotoxicitfyorevaluationof LTDS. The testarticleP,FOS, was thereforevaluatedasnegativeinthe invitroassayforunscheduledDNA synthesisinrat liverprimarycellcultures. -13- Covance 20784-0-447 RECORDS TO BE MAINTAINED All raw data,documentation,records,protocoland the finalreportgeneratedas a resultof this studywillbe archivedinthe storagefacilitioefsCovance-Vienna foratleastone yearfollowing submissionof the finalreporttotheSponsor. Afterthe 1-yearperiod,the Sponsor may electto have the aforementionedmaterialsretainedinthe storagefacilitioefsCovance-Vierma foran additionapleriodof time,or senttoa storagefacilitdyesignatedby theSponsor. REFERENCES Butterworth,B.E.,J.Asby, E. Bermudez, D. Casciano,J.Marsalis,G. Probstand G. Williams (1987) A protocoland guideforteinvitrorathepatocyteDNA-repair assay,Mutation Research, 189,113-121. Casciano,D.A. and D.W. Gaylor (1983) Statisticcarliterifaorevalutatincghemicalsaspositive ornegativeinthehepatocytesDNA repairassay,Mutation Research,122:81-86. Williams,G.M. (1977) Detectionofchemicalcarcinogensby unscheduledDNA synthesisinrat liverprimary cellculture,Cancer Research,37:1845-1851. Williams,G.M. (1980) The detectionof chemicalmutagens-carcinogensby DNA repairand mutagenesisinliverculturesi,n: F.de Serresand A. Holaender (Eds.),Chemical Mutagens, Plenum Press,N.Y, Vol. 6,pp. 61-79. -14- . . Covance 20784-0-447 DATA TABLES -15- Covance 20784-0-447 TABLE 1. SUMMARY OF DATA FROM THE RAT HEPATOCYTE TEST ARTICLE: PFOS COVANCE STUDY NO.: 20784-0-447 TRIAL =IATION DATE: I-September-99 UDS ASSAY Test Condition Concentration Mean Net Nuclear Grains (NNG) (1) % Cells w/ 2t5 Mean NNG (2) Mean Cyto. Grains (3) Survival At 23.3 hours (4) VehicleControl- DMSO 1.00 % -4.74 2.67 19.03 100.0 PositiveControl 2-AAF 0.100 jig/mL 19.35 96.00 16.17 88.5 Test Material 25.0 10.0 5.00 2.50 1.00 0.500 pg/mL pg/mL gg/mL lig/mL @Lg/mL pLg/mL -2.81 -3.71 -4.51 -3.17 -4.04 -4.87 6.67 20.81 65.2 6.00 22.61 103.3 3.33 18.77 96.8 5.33 15.84 109.3 0.67 17.39 96.3 1.33 19.29 102.2 (1) Average ofnetnucleargraincountson triplicactoeverslip(s150totalcells)N.et nucleargrains= Nuclear graincount - Average cytoplasmicgraincount. (2) Average percentageof cellswith greaterthanor equalto5 netnucleargrainson triplicactoeverslips(150 totalcellsy. (3) Average of cytoplasmicgraincountson triplicactoeverslip(s150 totalcells). (4) Survival= Number ofviablecellsper unitarearelativteothesolventcontrol. 2-AAF = 2-acetylaminofluorene DMSO = Dimethyl sulfoxide -16- Covance 20784-0-447 TABLE 2. INDIVIDUAL SLIDE DATA ASSAY NO. 20784-0-447 TRLKL RIITIATION DATE: 09/01/99 SlideCode Dose Mean Net Standard Mean (gg/ML) Nuclear Grains Deviation Cytoplasmic Grains 8 vc -4.38 16.86 6 vc -4.16 17.44 4 vc -5.68 22.80 Dose Average 4.74 0.82 19.03 Cellsin S-Phase 0/1500 screenedforS-Phase = 0.00% 9 PC 23.60 7 PC 20.66 2 PC 13.78 18.06 14.58 15.86 Dose Average 19.35 5.04 16.17 Cellsin S-Phase 7/1500 screenedforS-Phase =0.47% 5 25.0 -1.42 15.88 3 25.0 -3.32 21.64 1 -3.68 24.90 Dose Average -2.81 1.21 20.81 Cellin S-Phase 4/1500 screenedforS-Phase = 0.27% 18 10.0 -3.30 21.38 17 10.0 -2.72 22.72 16 10.0 -5.12 23.74 Dose Average -3.71 1.25 22.61 Cellsin S-Phase 5/1500 screenedforS-Phase = 0.33% 15 5.00 -5.94 20.06 14 5.00 -4.94 22.94 13 5.00 -2.66 13.32 Dose Average 4.51 1.68 18.77 Cellsin S-Phase 2/1500 screenedforS-Phase = 0.13% % Cellsw/ ?:5 Net Nuclear Grains 2:oo 4.00 2.00 2.67 98:oo 100.00 90.00 96.00 8.00 8.00 4.00 6.67 4.00 6.00 8.00 6.00 4.00 4.00 2.00 3.33 -17- Covance 20784-0-447 TABLE 2. INDMDUAL SLIDE DATA (Continued) ASSAY NO. 20784-0-447 TRLKL EQITIATION DATE: 09/01/99 SlideCode Dose Mean Net Standard Mean % Cellsw/ @ 5 (gg/mL) Nuclear Deviation Cytoplasmic Net Nuclear Grains Grains Grains 21 2.50 -3.66 13.92 4.00 20 2.50 -4.30 17.48 2.00 19 2.50 -1.54 16.12 10.00 Dose Average -3.17 1.44 15.84 5.33 Cellsin S-Phase 4/1500 screenedforS-Phase = 0.27% 24 1.00 -4.38 16.08 0.00 23 1.00 -3.96 16.56 0.00 22 1.00 -3.78 19.52 2.00 Dose Average 4.04 0.31 17.39 0.67 Cellsin S-Phase 0/1500 screenedforS-Phase = 0.00% 12 0.500 -4.14 19.98 2.00 11 0.500 -3.46 15.02 2.00 10 0.500 -7.02 22.86 0.00 Dose Average 4.87 1.89 19.29 1.33 Cell in S-Phase 1/1500 screened forS-Phase = 0.07% TotalCellsinS-Phase 23/12000 screenedforS-Phase 0.19% VC VehicleControl PC PositiveControl HISTORICAL CONTROL DATA HISTORICAL VEHICLE CONTROLS DV VITRO UNSCHEDULED DNA SYNTHESIS ASSAY Covance 20784-0-447 Study Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Average Range: Low High UDS Grains/ Nucleus SD -3.53 -2.68 -0.37 0.74 -0.74 -1.24 -1.41 -0.86 -0.47 -0.87 -0.99 0.16 -1.45 -0.80 -0.73 -1.36 -2.78 -1.83 -2.67 -4.63 -1.43 1.28 % ofNuclei with @@5 Net Nuclear Grains b 0.0 2.7 6.7 8.0 6.0 7.3 1.3 8.0 .5.3 5.3 9.3 9.3 7.3 6.7 6.0 3.3 0.7 0.7 4.0 3.3 5.06 2.91 Average Cyto Grains b 15.44 16.17 8.78 10.59 13.28 17.47 16.87 14.54 10.30 21.57 14.69 10.73 14.62 12.95 18.63 11.29 11.50 10.49 16.49 20.05 14.32 3.54 -3.53 0.74 0.0 8.29- 9.3 21.57 Number of datapointsis20. Data includedfrom 5/90to 1/99 'Average of netnucleargraincountstriplicaotre duplicatecoverslips (150 cellsanalyzed) b Average valuesfortriplicatoerduplicatecoverslip(s150 cells)analyzed SD = Standarddeviation -19- Covance 20784-0-447 HISTORICAL CONTROL DATA (continued) HISTORICAL POSITIVE CONTROLS IN VITRO UNSCHEDULED DNA SYNTHESIS ASSAY Study Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Averagge- Range: Low High LJDS Grains/ Nucleus SD % of Nuclei with @ 5 Net Nuclear Grainsb Average Cyto GIrai.ns b 29.45 100.0 25.71 15.53 92.0 11.52 7.35 63.3 12.53 12.35 88.7 11.31 14.18 97.3 16.25 14.45 92.7 16.05 16.09 100.0 15.73 9.51 78.7 15.98 13.17 90.7 14.27 16.92 97.3 10.51 17.38 86.7 15.68 4.46 46.0 10.89 15.59 99.3 10.73 10.69 84.8 11.68 13.79 92.7 14.09 14.66 89.3 17.85 14.33 91.3 12.22 14.63 96.7 11.50 16.81 98.7 10.49 17.51 97.3 16.49 14.44 4.89 89.19 13.36 14.07 3.65 4.46 29.45 46.00 100.00 10.49 25.71 Number of datapointsis20. Data includedfrom 5/90to 1/99 The positivecontrolused is 10 @Lg/ml2-AAF 'Average of netnucleargraincountstriplicatoerduplicatecoverslips(150 cells) analyzed b Average valuesfortriplicatoerduplicatecoverslips(150 cells)analyzed SD = Standarddeviation -20- C 0 V A-NFC E-' IHE DEVELOPMENI SERVICES COMPANY AMENDMENT TO THE STUDY PROTOCOL Page Iof I STUDY TRTLF-: UnscheduledDNA SynthesisinRat LiverPrimaryCellCultures PROTOCOLNO.: 447Editionl? COVANCE STUDY NO.: 20784-0-447 Amendment #1. I Section4.2.3Terminationand CytotoxicitMyeasurement The thirdsentenceinparagraph3 ischanged from "Me cellswillbe fixedwithcold aceticacid:ethano(l1:3)and driedforatleast24 hours."to"rhe cellswillbe fixedwith coldaceticacid:ethano(l1:3)and driedatleastovernight." Reason: Overnightallowsforsufficiendtrying. 2. Section4.2.3Terminationand CytotoxicitMyeasurement The thirdsentenceinparagraph4 ischangedfrom "rhe emulsion-coatedslideswillbe storedfor7 to 10 daysat2 to 80C inlight-tigbhotxes containinga desiccant.t"o "rhe emulsion-coatedslideswillbe storedfor6 to9 days at2 to8*C inlight-tigbhotxes containinga desiccant." Reason: Previousstudiesindicatedthata shorterexposureperiodwas needed. 3. The referencetoMirsalisT,yson and Butterworth,1982 isdeletedfrom Section4.2.2of theprotocol: Reason: The referencerefersto proceduresforan invivolinvitroUDS assay. Study Director: h-, (Ma.4@-A.Cifone,PK. Date t'e'@etiacnd Cellul oxicology caV THE DEVELOPMENT SERVICES COMPANY AMENDMENT TO THE STUDY PROTOCOL Page I of I STUDY TITLE: UnscheduledDNA SynthesisinRat LiverPrimaryCellCultures PROTOCOL NO.: 447 Edition17 COVANCE STUDY NO.: 20784-0-447 Amendment #2. 1. Section1.0 SponsorIdentification The name ofthesponsorischanged from 3M Corporationto3M CorporateToxicology. Reason: Sponsor request. Study Director: fone,Ph.p. Date CellulaziToxicology C 0 V AW'C-E-.-'*@' THE MKLCWMENT SERVICESCOUMNY COVANCE STUDY NO. PROTOCOL NO. 447 EDMON 17 UNSCHEDULED DNA SYNTHESIS IN RAT LIVER PRIMARY CELL CULTURES Covance LaboratorieIsnc.(Covance)willconductthisstudyincompliancewithGood LaboratoryPractice(GLP) regulationasnd standards.This protocola,tleastone criticaplhaseof thework-inprogressa,nd thefinalreportwillbe subjectto auditby QualityAssurancein accordancewithStandardOperatingProceduresmaintainedatCovance. The studywillbe conductedby Covance at9200 LeesburgPike,Vienna,Virginia22182 (Covance-Vienna). PART 1. SPONSOR INFORMATION AND APPROVALS 1.0 SPONSOR IDENTIFICATION Company Name: Address: 3M Corporation 3M Center Building220-2E-02 St.Paul,MN 55144-1000 2.0 TEST ARTICLE IDENTIFICATION 3.0 TEST ARTICLE ANALYSIS The Sponsor willbe responsiblfeorthedeterminationand documentationofthe analyticaplurityand compositionof thetestarticlaend thestabilitaynd strengthofthe dosing solutions. 4.0 NOTIFICATION OF REGULATORY SUBMISSION In ordertocomply with GLP regulationasnd standardsc,onsultinglaboratoriemsust be notifieidfallor partof a studyisintendedforregulatorsyubmission.Covance maintains a masterscheduleof studieswhich fallunder regulatoryreview.Pleaseindicatewhich agency,ifany,might receivetheresultosfthisstudy: 0 Undetermined 0 FDA WEPA-TSCA 0 MAFF 0 MOHW 0 OECD 0 None 13 EPA-FIFRA 11 Other 1/98 1 of 13 COVA44'C-E'."N THE WMWPMFPff SEWIRCFS COMPAW 5.0 STUDY DATES Proposed ExperimentalStartDate: Proposed ExperimentalTerminationDate: 6.0 APPROVAL OF STUDY PROTOCOL Study Director. A. Cifone,Ph.D TestingFacilitMyanagement: (:-ftaqt^, BrianC.MyhrPhJ). AssociateDirector Sponsor'sAuthorizedRepresentative: -/rce@ Marvin T. Case,D.V.M., Ph.D. PROTOCOL NO. 447 EDITION 17 9 5@ Date: 5-/XO/,2 Date: el- Date: 1/98 2 of 13 CTH0EV A44'C-E-.-"\ OMLOPMEoff SERVICES COMO%NY PROTOCOL NO. 447 EDITION 17 PART 2.STUDY PROTOCOL UNSCHEDULED DNA SYNTHESIS IN RAT LIVER PRIMARY CELL CULTURES 1.0 OBJECTIVE The objectivoefthisinvitraossayistoevaluatteheabilitoyfa testarticloer,a metabolitet,o induceDNA damage in ratprimaryhepatocytesby measuringunscheduled DNA synthesi(sUDS). 2.0 TEST SYSTEM RATIONALE Thisassayaimstoestabliswhetherthetestarticloeritsmetabolitecsan interacwtith mt liverprimarycell(hepatocytec)ulturetsoinduceDNA damage followedby DNA repair measured asUDS. Hepatocyteswillbe isolatefdrom liverand exposed invitrotothetest articlaend tritiattehdymidine('H-TdR). Primaryhepatocytehsave sufficienmtetabolic activittyoeliminatetheneed fortheadditionof amicrosomal activatiosnystem.An automdiographictechniquedescribedby Williams (1977)isusedto measure incorporatioonf -H-TDR intoDNA. Because onlya smallpercentageof thecellswill enterS-phase(replicatiDvNeA synthesisd)uringtheexposureperiod,theincorporation of 3H-TDR intoDNA can be used as a measure of therepairof DNA damage caused by treatmentwith thetestarticleT.he existenceand degreeof DNA damage willbe inferred from an increaseinnucleargraincountsin treatedhepatocytescompared tonucleargrain countsfrom untreatedhepatocytes.The typesof detectablDeNA damage areunspecified butmust be recognizableby thecellularrepairsystem and resultintheincorporatioonf nucleicacidbasesincluding3H-TDR intotheDNA. 3.0 MATERIALS 3.1 Test System The indicatocrellsforthisassaywillbe hepatocytesobtainedfrom a singleadult, male,Fischer344 mt (weighing150 to300 g),which willbe purchasedfrom the CharlesRiverBreedingLaboratoriesI,ncorporateodr anotherqualifiedealer. Animals willbe receivedby animal carepersonnel,exan-dned,and housed accordingtoapplicablCeovance SOPS. Food and waterwillbe suppliedad libitumT.he animalswillbe used forthesurgicaplrocedurewithintwo daysof receipt.Animals willbe anesthetizepdriortosurgerytoobtainthehepatocytes (sodiumpentobarbitaltabout60mgtkg by intraperitoneianljectiona)nd exsanguinatedduringthe procedure. 1/98 3 of 13 C 0 V A-Nr@C-E@. THE OMLOPMI!W SERVICIES COMPMF PROTOCOL NO. 447 EDITION 17 The cells will be obtained by perfusion of the liver in situ with a coliagenase solutionW,MEC, (seeSection4.2.LJDS ASSAY). Monolayer cultureswillbe establishedon plasticoverslipsinculturedishesand used thesame day for initiatioonf the LTDS assay. 3.1.1 Media and Cell Culture Conditions Williams'Medium E supplemented with 2 mM L-glutamine,100 Ag/mi streptomycinsulfatea,nd 150 tlgtmlgentamicin(WNIEI) isthebase culturemedium, and ismodified foreach specificrequirement.The hepatocyteswillbe obtainedby perfusionof liversinsituwith BBSS/EGTA followedby V;MEC: Hanks'balancedsalts(Ca++-and Mg++-free) containing0.5mM ethyleneglycol-bis(O-aminoetehtyhler)-N, N-tetra-acetiacidand 50 mM BEPES bufferatpH 7.2(BBSS/EGTA), and WMEI containing50 to 100 unitstnfolf coliagenase(WMEC). The culturesareestablisheidn V;NM supplemented with 10% fetalbovine serum (WME+). All cellculturewsillbe maintainedas monolayersina humidifiedincubatorat35 to 37.5'Cinan atmosphereof 4 to6% C02 in air.Aftertheestablishmentperiodtheculturelabelingisinitiateudsing WMEI containing10 iLCi/ml'H-TdR at40 to 60 Ci/mMole (WUE-treat). When wateristhe vehicleof choice,the concentratioonf -H-TDR willbe adjustedso thatthefinalconcentrationof labelinthemedium (aftear 1:10 dilutionof thewater stocks)willbe about 10 iLCi/ml. 3.2 Test Article Solidor liquidtestarticleasresuitableforthisassay.The testarticliesidentified inPartI of thisprotocol.Storageconditionswillbe as specifiedby the Sponsor. 3.3 Control Articles 33.1 Vehicle Control Article The vehiclecontrolarticlewillbe the vehicleselectedforthetestarticle. The vehiclemay be specifiedby the Sponsor or selectedby theprocess describedinSection4.1,Test ArticleHandling. The vehiclewillbe used inthe vehiclecontrolculturesata concentratioenquivalenttothe vehicle inthe testarticle-treatceudltures.The vehiclecontrolcultureswillbe handled identicalltyothe testarticltereatedcultures.An untreated (negative)control,consistingof assayproceduresperformed on cells exposed to WME-treat only,willbe includedwhen acetoneisused as the vehicle. 1/98 4 of 13 C 0 V Alt CE."@" THE licalopmefff SEMKI!S COMPAW PROTOCOL NO. 447 EDITION 17 3.3.2 PositiveControlArticle The positivecontrolarticle2,-acetylaminofluore(n2e-AAF; CAS 53-96-3),isknown toinduceUDS inrathepatocyteprimarycellcultures. The 2-AAF willbe dissolvedinDMSO and diluted1:100 intoWNE-treat fora finalconcentratioonf 0.10 ug/ml (4.48x lOr7M). 4.0 EXPERIMENTAL DESIGN 4.1 TestArticleHandling Ifsolubilitiynfonnationisnot providedby theSponsor,a preliminarsyolubility testwillbe carriedoutwith serum-fireculturemedium or sterildeeionizedwater, dimethylsulfoxide(DMSO; CAS #67-68-5),'ethan(oClAS #64-17-5),oracetone (CAS #67-64-1).Ifthevehicleofchoiceisculturemedium, thetestarticlweill be dissolvedatthehighestdesiredconcentratioinnVrNM-treat.Stock solutions inwaterwillbe diluted1:10,inDMSO or ethanolwillbe diluted1:100,and in acetonewillbe diluted1:200inWNM-treat. The vehicleselectedwillbe theone which givesthebestsolubilitaynd dispersiocnharacteristiacfsterdilutionin culturemedium. Insome cases,testarticleasrra,pparentlyinsolubleinvehicles thatarecompatiblewithtissueculture.In such cases,thevehiclechosen and the highestdose testedwillbe thatin which an evenlydispersedsuspensioncan be prepared. 4.2 UDS Assay 4.2.1 Dose Determination and Culture Conditions The maximum dose willbe determinedon a caseby casebasistakinginto accountboth solubilitaynd any relevantcytotoxicitiynformationavailable on thetestarticleT.he highestdose testedwillbe 5 mg/ml, unlesshigher dosesarespecifiebdy theSponsor.Relativeliynsolubletestarticlewsill be testedup to approximately2 timesthesolubilitlyimiti,finsoluble articldeoes not interferweiththeenumerationof nucleargrains. The top dose testedforliquidtestarticlewsillbe 5 mg/ml, which may be determinedby weightorcalculatefdrom thespecifigcravity.Solubility testingwillbe thesame as describedabove. 1/98 5 of 13 C 0 V A49--EC@-. THE DMLC PROTOCOL NO. 447 EDITION 17 Ifmarked pH changes arenoticedduringthesolubilittyest,neutralization, with HCI or NAOH, willbe performedto maintaina normal culturepH range (6.8to 7.4)only afterconsultatiownith theSponsor. A concurtentcytotoxicitayssessmentisan integralpartof the LTDS assay used to selectappropriatedoses fora particularf,reshprimary cultureof hepatocytes.A rangeof 15 concentrationswillusuallybe appliedtothe cells.Fewer concentrationmsay be initiatewdhen toxicityinformationis avaflable.A viablecellcount (trypanbluedye exclusion)willthenbe obtained20 to24 hoursafterinitiatioonf the treatments.At leastsix concentrationswillbe chosen foranalysisof nuclearlabeling,startinwgith the highestdose thatresultsina sufficienntumber of survivingcellswith intactmorphologies and proceedingtosuccessivelylower doses. Repeat trialsmay containas few as 5 dose levelsatthediscretionofthe study director.The testarticlseolutionwillbe preparedinunediatelybeforeuse. The maximum finalconcentrationosf vehicleinV;NE-treatwillbe up to 10% water, 1% DMSO or ethanol,or 0.5% acetone. 4.2.2 Cell Collectionand Culture This assay isbased on the proceduresdescribedby Williams,(1980), NfirsalisT,yson and Butterworth,(1982)and Butterworthetal, (1987). The hepatocyteswillbe obtainedby perfusionof liversinsituwith HBSS/EGTA forabout4 minutes followedby WNEC forabout II minutes. Depending on theconditionof thelivert,histime may be altered by :t2 minutes. The hepatocyteswillbe obtainedby mechanical dispersionof excisedlivertissuein a culturedishcontainingWNMC. The suspended tissueand cellswillthenbe abowed to settletoremove cell clumps and debrispriortocollectionT.he collectedcellsuspensionwill be centrifugedand thecellpelletresuspendedinWNE+. Afterobtaining a viablecellcount,a seriesof culturedishes(atleast5 per dose level)will be inoculatedwith approximately0.5x 106viablecellsin3 ml of Vlhffi+. Culture dishesthatwillbe used forLTDS analysiswillcontainround plasticcoverslipswhile dishesused toassessattachmentefficiencyand toxicitywillhave no coverslips. An attachmentperiodof 1.5to 2 hours at35 to37.5*C in a humidified atmosphere containing4 to6% C02 willbe used toestablishthecell cultures.Unattachedcellswillthen be removed and thecultureswillbe refedwithV;NM. 1/98 6 of 13 C 0 V AICC@E.@ THE DMLOPMttff SEMCES COMPAW PROTOCOL NO. 447 EDrrION 17 The LJDS assaywillbe initiatewdithinthreehours by replacingthemedia in theculturedisheswith2.5mi WME-treat containingthetestarticlaet thedesiredconcentrationE.ach controland testarticltereatmentwillbe performedwithatleastfivecultures.Three of thesereplicatceultureswill be used forUDS analysisand two replicatewsillbe used forthe cytotoxicitmyeasurements.Disheswillbe labeledperCovance SOP. 4.2.3Terndnation and CytotoxicityMeasurement Afteran exposureperiodof 18 to 20 hours,thetreatmentsand labeling willbe terminatedby washingthecellmonolayerswithmedium. The culturesused forthecytotoxicitmyeasurements willbe refedwithNVBM and returnedtotheincubator. At 20 to24 hoursaftertheinitiatiofnthetreatmentsc,ytotoxiciwtiyllbe assessedas cellulamrorphology and cellsurvivalrelativteothevehicle control.Viablecellcountswillbe obtainedusingtrypanbluedye exclusion. The triplicaltaebeledcellculturesfortheLTDS assaywillbe refedwith VRM containing1mM thymidine.The nucleiwillbe swollenby additionof 1% sodium citratteo thecoverslip(scontainintghecell monolayers)for8 to 12 minutes.The cellswillbe fixedwithcoldacetic acid:ethano(l1:3)and driedforatleast24 hours.The coverslipwsillbe mounted on glassslides. The cellsurvivaland morphology obtainedfrom thecytotoxicictuyltures willbe usetochose doseslevelsforautoradiographyS.lideswillbe dippedin an emulsionof Kodak NTB2 and water,and dried.The emulsion-coatedslideswillbe storedfor7 to 10 daysat2 to 8*C inlighttightboxescontaininga desiccant.The emulsionswillbe developedwith Kodak D19 developerand Kodak Rapid Fixer,thenstainedwith a modifiedhematoxylinand eosinprocedure. 4.2.4 SlideAnalysis The cellswillbe examined microscopicallaytapproximately1500x magnificatiounnderoilimmersion and thefielddisplayedon thevideo screenof an automaticcounter.UDS willbe measured by counting nucleargrainsand subtractintgheaveragenumber of grainsinthree 1/98 7 of 13 C 0 V A-WC'- THZ MVELOPMENT SFMF=S COMPANY PROTOCOL NO. 447 EDITION 17 nuclear-sizceydtoplasmiacreasadjacenttoeachnucleus(cytoplasmic count).This valueisreferredto asthenetnucleargraincount.The coverslipswillbe coded topreventbiasingraincounting. The netnucleargraincount willbe routinelydeterminedfor 50 randomly selectedcellson each coverslip.Only nucleiwith normal morphologies willbe scored,and any occasionalnucleiblackened by grainstoo numerous to countwillbe excluded as cellsin which replicativDeNA synthesisoccurredratherthan repairsynthesis.Normally, 150 cellsper dose levelwillbe read(50 from each of thetriplicatceoverslips)T.he complete number of 150 cellsper dose may not be availabledue totoxicity or qualityof preparation.Grain count dataobtainedfora giventreatment isacceptableas pan of the evaluationifobtainedfrom atleasttwo replicateculturesand atleast100 cellsper dose. The mean netnuclear graincount willbe determinedfrom thetriplicatceoverslipsforeach treatmentcondition.Occasionally,a coverslipmay be recountedata later dateor by a differenttechnician.Since a differenctellpopulationwill generallybe scored,the average count for50 cellswillbe used inthe calculatioonf themean forthe triplicatereatment. 5.0 DATA 5.1 Data Presentation The finalreportwillincludethefollowinginformationintabularform forthe vehiclecontrol,positivecontrol,and each analyzedtreatment: The mean netnucleargraincountfortriplicatceultureswith standard deviation(usuallya totalof 150 cells). The mean percentageofcells.havingfiveor morr.netnucleargrainsfor triplicatceultures. The mean cytoplasmicgraincount fortriplicatceultures(usuallythreeareas percellina totalof 150 cells). The mean survivalforduplicateculturesw,hich isthenumber of viablecells per unitareaintheculturer-elativteothe vehiclecontrolvalue x 100%. 1/98 8 of 13 C 0 V A-Nf'C-E.--@' THE DEVELOPMENT SERVICES COMPANY PROTOCOL NO. 447 EDITION 17 Informationre0(yardintgestarticlseolubilitiyntheculturemedium, viabilitoyf the hepatocyteculturesa,nd the percentof cellsinDNA replicativseynthesis(S- phase)willalsobe provided. 5.2 Assay Acceptance Criteria. An assaywillbe consideredacceptableforevaluationofthetestresultsonly ifall ofthe criterilaistebdelow aresatisfiedT.his listinmgay not encompass alltest situationst,husthestudydirectorexercisesscientifjiucdgment inmodifying the criterioar consideringeventsthatmight affectassayreliabiliatnyd acceptance. 5.2.1 CellCulture Conditions The viabilitoyf thehepatocytescollectedfrom theperfusionprocess normallyexceeds 70%. A varietyof factorscan affectcellyieldand viabilitys,o valuesbelow 70% arenot uncommon nor necessarily detrimental.A lower limitforacceptabiliitsysetat50% in ordertoavoid the possibleuse of a damaged, unrepresentativseample of cells. The viabilitoyf themonolayer cellculturesused forthe assay treatments must be 70% or greater.Normally, the viabilitoyfattachedcellsisa.bout 90%. The number of viablecellsin thevehiclecontrolculturesshould remain reasonablystableoverthe experimentaltime periodbecause rapidly declining(dying)culturesmay not respondin a representativmeanner to the testarticltereatments.Therefore,theaveragenumber of viablecellsin the vehiclecontrolculturesmust not be lessthan50% of the cellnumber atthebeginningof the treatmentperiod. 5.2.2 Acceptable Controls The averagenet nuclearlabelinginthevehiclecontrolculturesmust be in therange of -5.00to 1.00. In additionn,o more than 10% of thecells should containfiveor more netnucleargrains.Failureto meet eitherof thesecriteriwaillinvalidatethe assay. The positivecontrolisused todemonstratethatthecellpopulation employed was responsiveand themethodology was adequate forthe detectionof UDS. For testarticlecsausingweak orno LTDS activityt,he 1/98 9 of 13 C 0 V AACC'-E-.'-N THE DMLOPMEPff SERVICES COMPANY PROTOCOL NO. 447 EDITION 17 average responsetothe positivecontroltreatmentsmust exceed both criteriuased toindicateLJDS (seeSection5.3).For a testarticlcelearly causing a dose-relatedLTDS activitya,n assaywillbe acceptableinthe absence of a positivecontrollostfortechnicalreasons. 5.2.3 Acceptable High Dose Itisnot necessarytoincludetoxicdoses (lessthan 95% survival)inthe UDS analysisbut itisnecessarytoshow thatan increaseinconcentration resultsinexcessivetoxicityasmeasured by eithersurvivalsbelow 50% or observationofpoor cellulamrorphology. Highly toxicdosescan resultin reduced incorporatioonf 3H-TDR resultingindecreased graincountsor poor cellulamrorphology and may leadto artifactdsuringgrainanalysis. The highestanalyzeddose must approach excessivetoxicityo,r resultin testarticliensolubilitoyr,reachthe highestapplicabledose of 5 mg/ml. 5.2.4 Acceptable Number of Doses A minimum of sixdose levelswillbe analyzedfornucleargraincounts. Repeat trialnseed only augment thenumber of analyzeddose levelsinthe firstrialto achievea totalof sixdifferenctoncentrations. Grain count dataobtainedfora given treatmentisacceptableaspartof the evaluationifobtainedfrom atleasttwo replicatceulturesand atleast 100 cellsper dose. 5.3 ASSAY EVALUATION CRITERIA Severalcriterihaave been establishedwhich, ifmet, willprovide a basisforevaluationof a testarticleas activein theUDS assay.-Thecriterifaora positiveresponse arebased on a statisticaanlalysisof Covance historicadlataand calculatioonf the requiredminimum increasein nuclearlabelingusing an approach describedby Casciano and Gaylor (1983). The testarticlweillbe consideredactivein theLJDS assayatappliedconcentrationtshat cause: An increasein themean net nucleargraincount toatleastfivegrainsper nucleus above theconcurrentvehiclecontrolvalue,and/or, 1/98 10 of 13 C0V THE DEVELOPMENT SEIMCES Comftmy PROTOCOL NO. 447 EDITION 17 An increaseinthenumber ofnucleiwithfiveormore netgrainssuchthatthe percentageof thesenucleiintestculturesis10% above thepercentageobservedin vehiclecontrolcultures. Generallyi,fthefirsctonditionissatisfietdh,esecondoftenwillbe met. However, satisfactiofnonlythesecondconditioncan alsoindicateUDS activityW.hen net nuclearlabelingiselevatedabove thevehiclecontrollabelingbutdoes notreachthe statisticaslilgynificanmtinimum, theoverallpatternof theresponseisconsideredinthe evaluation.DifferentDNA-damaging agentscan givea varietyof nuclearlabeling patternsa,nd weak agentsmay stronglyaffectonlya minorityof thecells.Therefore, bothof theabove conditionswillbe consideredin an evaluation. A dose-relateidncreaseinLTDS foratleasttwo consecutiveappliedconcentrationissalso desirableto evaluatea testarticlaesactiveinthisassay.In some cases,LTDS can increase with dose and thendecreaseto near-zerowithsuccessivelhyigherdoses. Ifthisbehavior isassociatedwith increasedtoxicityt,hetestarticlcean be evaluatedas active.Ifan isolatedincreaseoccursfora treatmentfarremoved from thetoxicdoses,theLTDS will be consideredspurious. The testarticliesconsiderednegativeifnone of theabove criteriaaremet. When, intbejudgement of thestudydirectorr,esultasreneitherclearlypositivneor clearlynegative,thepresenceof a doseresponse,thefrequencydistributioonfcellular responses,and thereproducibiliotfydataamong slidesisconsidered. The positivecontrolnuclearlabelinwgillnotbe usedas a referencepointtoestimate mutagenicor carcinogenirciskassociatewdiththeUDS activitoyf thetestarticleU.DS elicitebdy testagentsinthisassayisprobablymore dependenton thetypeof DNA damage inflicteadnd theavailablreepairmechanisms thanon thepotencyof thetest agentas a mutagen or carcinogen.Some -formsof DNA damage arerepairedwithout theincorporatioonf new nucleicacids.Thus,thepositivecontrolswillbe usedto demonstratethatthecellpopulationemployed was responsiveand themethodologywas adequateforthedetectionof UDS. 1/98 11 of 13 C 0 V AW'C-E-.--@' THIEDEVELOPMENT SEIMCES COMPANY PROTOCOL NO. 447 EDrrION 17 6.0 REFERENCES Butterworth,B.E.,J.Ashby, E. Bermudez, D. Casciano,J.Nbrsalis,G. Probstand G. Williams(1987) A protocoland guide forthein vitromt hepatocyteDNA-repair assay, Mutation Research,189,113-121. Casciano,D.A. and D.W. Gaylor (1983) Statisticcarliterifaorevaluatingchemicalsas positiveor negativeinthe hepatocytesDNA repairassay,Mutation Research,122:81-86. Williams,G.M. (1977)Detectionofchemicalcarcinogensby unscheduledDNA synthesisinmt liverprimarycellculture,Cancer Research,37:1845-1851. Williams,G.M. (1980) The detectionofchemicalmutagens-carcinogenbsy DNA repair and mutagenesisin liverculturesi,n: F. de Seriesand A. Hollaender(Eds.)C,hemical Mutagens, Plenum Press,NY, Vol. 6,pp. 61-79. 7.0 REPORT FORMAT Covance employs a standardreportformatforeach assaydesign.71befinalreportwill providethefollowinginformation. Sponsor identification. QualityAssurance statement. Statementof GLP compliance. Signatureof study director. Test articliedentificatiaonnd Covance Study Number. A physicaldescriptionof the testarticlaend date ofreceiptwillbe includedinthissection. Type of assayand protocolnumber. Dates of studyinitiatioannd completion. Identificatiofn studydirectorand seniortechnician. Methods. Evaluationcriteiia. Interpretationf results. Conclusions. References. Test resultspresentedintabularform. Ffistoriccaolntroldata. 1/98 12 of 13 C 0 V AM-C'-E'. THE DMLOPMENT SERVICES COMPANY PROTOCOL NO. 447 EDrrION 17 8.0 CHANGES OR REVISIONS Any changesor revisionosf thisapproved protocolwillbe documented,signedby the studydirectord,ated,and maintainedwith thisprotocol.The Sponsor willbe notifieodf any changesor revisions. 9.0 ANIMAL CARE AND USE STATEMEENT In theopinionof theStudyDirectorn,o alternativteestinmgethods areappropriatet,he studydoes notduplicateany previouswork with thistestarticlea,nd thenumber and speciesselectedareappropriateI.bis protocolwillbe reviewedby theCovance-IACUC forcompliancewith replatoryguidelinecsoncerningthecareand use of animals.Ifnot incompliance,a modificatiownillbe required.Any changesorrevisionosfthis approved protocolwillbe senttotheCovance-IACUC fortheirreview. 10.0 RECORDS TO BE MAINTAINED Allraw data,documentation,records,protocol,and thefinalreportgeneratedas a result ofthisstudywillbe archivedinthestoragefacilitioefsCovance-Viennaforatleastone yearfollowingsubn-dssioonfthefinalreporttotheSponsor. Aftertheone yearperiod, theSponsor may electto have theaforementionedmaterialrsetainedin thestorage facilitioefsCovance-Vienna foran additionapleriodof timeor sentto a storagefacility designatedby theSponsor. 1/98 13 of 13