Document a12KBROwG99XkJLjwe2nyg14y

COVA-N'C-E-.@@ THE DMU FINAL REPORT CHROMOSOMAL ABERRATIONS IN HUMAN V%RHOLE BLOOD LYMPHOCYTES WITH PFOS AUTHOR Hemalatha Murli,PhD PERFORMING LABORATORY Covance LaboratoriesInc.(Covance) 9200 Leesburg Pike Vienna,Virginia22182 LABORATORY PROJECT IDENTIFICATION Covance Study No.: 20784-0-449 3M CorporateToxicologyStudy No.: T6295.18 SUBMITTED TO 3M CorporateToxicology 3M Center Building220-2E-02 St.Paul,Minnesota 55144-1000 STUDY COMPLETION DATE October 25, 1999 1 of 25 Covance20784-0-449 QUALITY ASSURANCE STATEMENT ChromosomaAlberratioinnHsuman WholeBloodLymphocyteWsithPFOS The reporthas been reviewedby theQualityAssuranceUnit of Covance LaboratorieIsnc.,in accordancewith theGood LaboratoryPracticeregulationasssetforthintheEnvirorunental ProtectioAngency (EPA -TSCA), Title40 of theU.S.Code of FederalRegulationsPart792, withany applicablaemendments. The followinginspectionwsere conductedand thefindings reportedtotheStudy Directorand Study DirectorManagement. Writtenstatusreportsof inspectionasnd findingsareissuedto Covance Management accordingtostandardoperating procedures. InspectionDates 09/01/99 Phase Dosing Dates ReportedtoStudy Directorand Study DirectorManagement 09/01/99 Auditor P.Ciceres 10/13,14/99 DraftReportReview 10/14/99 P.Ciceres 10/25/99 FinalReportReview 10/25/99 P.Ciceres RepresentativeQ,u@lityAssurance Unit I5ate -2- Covance20784-0-449 STUDY COMPLIANCE AND CERTIFICATION The describesdtudywas conducteidncompliancweiththeGood LaboratorPyracticregulations as setforthintheEnvironmentalProtectionAgency (EPA - TSCA), Title40 of theU.S. Code of FederalRegulationsPart792,with any applicableamendments. There were no significant deviationsfrom the aforementionedregulationosrthe signedprotocolthatwould affectthe integritoyf thestudyor theinterpretatioonfthe testresultse,xcept thatthecontrolsubstances were not fullycharacterizedT.he raw datahave been reviewedby the Study Director,who certifietshattheevaluationof thetestarticlaes presentedhereinrepresentsan appropriate conclusionwithinthecontextof thestudydesignand evaluationcriteriaA.ll testand control resultsinthisreportaresupportedby an experimentaldatarecordand thisrecordhas been reviewed by the Study Director. Study Director: i@ 0AUlam Hemalatha Murli,PhD Geneticand CellularToxicology I 01,2j-lq@ Study Completion Date -3- Covance20784-0-449 TABLE OF CONTENTS PageNo. ABSTRACT ...................................................6......... STUDY INFORMATION Sponsor TestArticle Assay Information Study Dates SupervisoryPersonnel ..........................................7.......... OBJECTIVE ..................................................7......... TEST SYSTEM RATIONALE .......................................7.......... MATERIALS AND METHODS ......................................8.......... Test System Media and CellCultureConditions TestArticle ControlArticles S9 MetabolicActivationSystem Chromosomal AberrationsAssay DATA .....................................................1.0......... Data Presentation Assay Acceptance Criteria Assay EvaluationCriteria RESULTS ...................................................12.......... TestArticleHandling Chromosomal AberrationsAssay Without MetabolicActivation Chromosomal AberrationsAssay With MetabolicActivation CONCLUSION ................................................14.......... PROTOCOL DEVIATIONS ........................................1.4 ......... RECORDS TO BE MAINTAINED ....................................14......... -4- Covance 20784-0-449 TABLE OF CONTENTS (Continued) Page No. REFERENCES ................................................14.......... DATA TABLES ...............................................1.5 ......... HISTORICAL CONTROL DATA ....................................2.2......... DEFINITIONS OF CHROMOSOME ABERRATIONS FOR GIEMSA STAINED CELLS 23 Covance 20784-0-449 ABSTRACT The objectiovfethisinvitraossaywas toevaluatteheabiliotfyPFOS toinducechromosomal aberrationisnhuman whole blood lymphocyteswith and withoutmetabolicactivation. Due totoxicityconstraintst,he highestconcentrationtestedintheassaywas 599 jig/mLwithout metabolicactivatioannd 449 gg/mL with metabolicactivationT.he testarticlweas dissolvedin dimethylsulfoxid(eDMSO) forthe assay.The stocksolutionand itsdilutionwsere dosed usinga dosingvolume of I% (10.0gLJmL) and the vehiclecontrolcultureswere treatedwith 10.0 gLJmL ofDMSO. Inthechromosomal aberrationsassay,thetreatmentperiod was for3.0hours with and without metabolicactivationa,nd cultureswere harvested22.0hours from theinitiatioonf treatment. Replicateculturesof human whole blood lymphocyteswere incubatedwith 12.5,24.9,49.7, 99.3,149,199,249, 299,349, 449, and 599 gg/niL withoutmetabolicactivatioannd 12.5,24.9, 49.7,99.3,149,199,249,299, 349, and 449 Ag/mL withmetabolicactivationC.ulturestreated with concentrationosf 199,249, 299, and 349 Ag/mL withoutmetabolicactivationand 99.3,149, 199,and 299 Ag/mL with metabolicactivatiownere analyzedforchromosomal aberrationsN.o significanitncreaseincellswith chromosomal aberrationsp,olyploidy,or endoreduplicatiownas observed inthe culturesanalyzed. PFOS was considerednegativeforinducingchromosomal aberrationisnhuman whole blood lymphocyteswith and withoutmetabolicactivation. -6- Covance20784-0-449 STUDY INFORMATION Sponsor 3M CorporaTtoexicology TestArticle Sponsor'IsdentificatFiCo-n9:5(PFOS)L,ot#:217 DateReceivedA:ugust19,1999 PhysicaDlescriptioWnh:itec,rystallpionweder StoragCeonditionRso:om temperature AssayInformation TypeofAssay:ChromosomaAlberratioinHsuman WholeBloodLymphocytes ProtocoNlo..:449,Editio1n4 CovanceStudyNo.:20784-0-449 StudyDates InitiatDiaotne:August20,1999 ExperimentSatlarDtate:Septembe0r1,1999 ExperimentTaelrminatioDnate:Octobe0r1,1999 SupervisoPreyrsonnel StudyDirectorH:emalathMaurliP,hD LaboratorSyupervisoErm:iliCe.BeckelhimeBrS, OBJECTIVE TheobjectiovfethiisnvitraossawyastoevaluattheeabiliotfyPFOS toinduccehromosomal aberrationisnhuman whole blood lymphocyteswithand withoutmetabolicactivation. TEST SYSTEM RATIONALE Aberrationsarea consequenceof failuroermistakesinrepairprocessessuchthatbreakseither do notrejoinor rejoininabnormalconfiguration(sEvans,1962,1976).Descriptionosf thetypes of aberrationasreprovidedinthisreport.Structuraalberrationmsay be oftwo types, chromosome orchromatid: Chromosome aberrations:tructuraclhromosome damage expressedasbreakage,orbreakage followedby reunion,ofboth sistecrhromatidsatan identicaslite. -7- Covance20784-0-449 Chromatidaberratiosnt:ructurcahlromosomedamageexpresseadsbreakagoefsingle chromatidsorbreakagefollowedby reunionbetween chromatids.This isthemost common typeof structuraalberration. Most known chemicalclastogens(chromosome-breakingagents)requirea periodof DNA synthesistoconvertinitiaDlNA damage intochromosome alterationtshatbecome visibleat mitosis.The lymphocytesinblood do not usuallydivide,but theywere stimulatedtodividein cultureby exposuretophytohemagglutinin(PHA). At predeterminedintervalasfterexposureto the testarticlet,he lymphocytes were treatedwith a metaphase-arrestinsgubstance,Colcemi&, then were harvestedand staineda,nd metaphase cellswere analyzedmicroscopicallyforthe presence of chromosomal aberrations. Many mutagenic chemicalsdo not actdirectlyon DNA but do so afterbeing convertedtoactive intermediatebsy enzymes found inliver.Human lymphocyteshave only a limitedcapacityto metabolizesome testarticless,o an exogenous metabolicactivationsystem (ratliverS9 homogenate) isincludedwith a seriesof treatmentstoenhance thedegree of conversionand the abilityof theassaytodetectclastogenicm,etabolicintermediates. Numerical aberration(sachange inthenumber of chromosomes from thenormal number of46 forhuman lymphocytes)were not determined.However, theoccurrenceofpolyploidyor endoreduplicationw,hich were scored,might indicatethatthe testarticlheas thepotentiatlo induce numericalaberrations. MATERIALS AND METHODS Test System Human venous blood from a healthy,male, adultdonor (nonsmoker withouta historyof radiotherapyc,hemotherapy,or drug usage,and lackingcurrentviralinfectionsw)as drawn into sterileh,eparinized"vacutainers"W.hole blood cultureswere initiateidn 15 mL centrifugteubes by adding 0.6 ml of freshheparinizedblood to 9.4mL ofculturemedium. Media and CellCulture Conditions Culturesin 15 ml tubeswere incubatedwith loosecaps at37*C 2*C ina humidified atmosphere of 5% 1.5% C02 inair.The medium was RPMI 1640 supplemented with approximately15% fetalbovine serum (FBS),penicilli(n100 units/mL),streptomycin (I00,ug/mL),L-glutwnine(2 mM) and I% phytohemagglutiniMn (PHA-M). The totavlolume foreach culturewas 10 mL. Test Article The testarticlweas a white,crystallinpeowder thatwas storedatroom temperature.The test articlweas dissolvedinDMSO. -8- Covance20784-0449 ControlArticles Negativeand VehicleControls.Inthenonactivatiaosnsaysv,ehiclceontrolwserecultures containingthevehicleforthetestarticleD,MSO, atthehighestconcentrationused intest cultures1,0.0pL/niL. Inthe activatioanssays,the vehiclecontrolswere the same as describedin thenonactivationassaysbut with the S9 activatiomnix included. PositiveControl Agents. The positivecontrolagentswhich were used inthe assayswere mitomycin C (MMC) forthenonactivatiosneriesand cyclophosphamide(CP) inthemetabolic activatiosneries.Mitomycin C (CAS# 50-07-7,Sigrna,Lot 88H2508) isa clastogenthatdoes not requiremetabolicactivationC.yclophosphainide(CAS 6055-19-2,Sigma, Lot # 73HO846) does not actdirectlbyut must be convertedtoactiveintermediatebsy microsomal enzymes. In the chromosomal aberrationasssays,threeconcentrationosf MMC (1.0,1.5,and 2.0 gg/mL) and CP (25,50,and 75 gg/mL) were used toinducechromosomal aberrationsO.ne ofthe concentrationwsas analyzed ineach of theaberrationassaysbased on thecurrentinformationon thepositivecontrolresponseinthelaboratory.Both MMC and CP were dissolvedinwater. S9 Metabolic ActivationSystem The invitrometabolicactivatiosnystem (Maron and Ames, 1983) consistedof a ratliverpostmitochondrialfraction(S9)and an energy-producinsgystem(NADP plusisocitriaccid).Various hepaticP450 isoenzyme levelsareincreasedby treatmentof theratswith Aroclor" 1254 (single concentratioonf 500 mg/kg and sacrifice5ddays later(MolecularToxicology,Inc., Lot No. 929).The S9 fractionp,reparedinsucroseorinpotassium chloridew,as retainedfrozen at<-70*C untiluse. Aliquotsof S9 were thawed immediatelybeforeuse and added totheother components to form theactivationsystem describedas follows: S9 ActivationSystem Component NADP (sodiumsalt) Isocitriacid Homogenate (S9 fraction) ConcentrationinCultures 1.5mg/mL (1.8mM) 2.7mg/mL (10.5inM) 15.0AL/mL* (1.5%) This concentratioonfratS9,obtainedfrom MolecularToxicologyInc.,Boone, NC, has consistently causedCP tobe highlyclastogenifcormany differenltots. Chromosomal Aberrations Assay In the chromosomal aberrationsassays,replicatecultureswere used at each concentrationand the vehiclecontrolsand foreach ofthreeconcentrationosfthepositivecontrol.Inthe aberrations assaywithoutand with metabolicactivation2,2.0-hour harvestswere conducted. Thisharvest time correspondsto 1.5times thecellcycletime of approximately15 hours afterthe lymphocytes areinduced todivideby the additionof PHA-M (Galloway etal.,1994). -9- Covance20784-0-449 AberrationAsssayWithoutand With MetabolicActivationC.ulturewsereinitiatweidth 0.6mL ofwhole blood in9.4 mL of freshculturemedium. For thenonactivationassay,2 days aftercultureinitiatiotnh,ecellswere incubatedat=37'C with the testarticlien freshmedium at predeterminedconcentrationsf,or3.0 hours. For theactivationassay,2 days afterculture initiatiotnh,ecultureswere incubatedat=37'C for3.0hours inthepresenceof thetestarticle and the S9 reactionmixturein freshRPMI 1640 culturemedium withoutFBS. The cultures were thenwashed with bufferedsaline,refedwith complete RPI@U 1640 culturemedium and incubatedfortherestof thecultureperiodup tothetime ofharvestwith 0.1 [ig/mL Colcemid" presentduringthe last2.0hours ofincubation.The cultureswere thenharvested. Harvest Procedure. The cultureswere centrifugedt,he supernatantdiscarded,and thecells were swollenwith 75 mM KCI hypotonicsolution.The cultureswere then fixedwith an absolutemethanol:glacialaceticacid(3:1,v/v)fixative. SlidePreparation and Staining.Slideswere preparedby droppingtheharvestedcultureson clean,glassslidesand air-driedT.he slideswere stainedwith 5% Giemsa solutionforthe analysisofmitoticindex and chromosomal aberrationsA.ll slideswere then air-driedr,insedin xylene,and mounted permanently. DATA Cellswere selectedforgood morphology and only cellswith thenumber of centromeresequalto themodal number 46 were analyzed. One hundred cellsi,fpossible,from each replicatfelaskfrom fourconcentrationosf thetest articlea,nd from thevehicleand one dose of thepositivecontrolcultureswere analyzedforthe differenttypesof chromosomal aberration(sEvans,1962,1976). At least25 cellswere analyzed from thoseculturesthathad greaterthan25% ofcellswithone or more aberrations.Mitotic index was evaluatedfrom the vehiclecontroland a range ofconcentrationbsy analyzingthe number ofmitoticcellsinatleast1000 cellsper cultureand theratioexpressedas a percentage of mitoticcells.Percentpolyploidyand endoreduplicatiownere alsoanalyzedby evaluatingat least100 metaphases per culture,ifavailablea,nd tabulated.For controlof bias,allslideswere coded priortoanalysis.Cellswith aberrationswere recordedon thedatasheetsby the microscope stagelocation. Data Presentation Data were summarized intablesshowing mitoticindex, percentpolyploidy,percent endoreduplicationt,he numbers of cellsanalyzed,typesof aberrationsfound,frequenciesof aberrationpser cell,and percentagesofcellsbearingaberrationsC.hromatid and isochromatid gaps were noted but were not added intothetotalsforaberrationassessmentsincethey arenot consideredtorepresenttruebreaks.The historicaclontroldataarepresentedafterthedatatables. -10- Covance20784-0-449 AssayAcceptancCeriteria An assawyasconsideraecdceptabfloerevaluatiooftnesrtesulotnslyifalolfthefollowing criteriwaere satisfiedT.he two treatmentconditionsareindependentunitsand would be repeatedindependentlya,s needed,tosatisftyheacceptancecriteria. Acceptable Controls. The vehiclecontrolculturesmust containlessthanapproximately5% cellswithaberrationsT.he positivceontrolresulmtust be significanthliygher(p< 0.01)thanthe vehiclecontrols. AcceptableHigh Dose. Iftheaberratiornesultasrenegativeand thereisno significant reduction(approximatel2y:50%)inmitoticindex,theassaymust includethehighestapplicable dose (10mM or 5 mg/mL, whicheverislower)or a dosewhere a well-dispersesduspensionin culturemedium was obtainedthatdidnotsettlreapidly. AcceptableNumber of Doses. The assaymust includeatleasthreeanalyzableconcentrations. Assay EvaluationCriteria The followingfactorsaretakenintoaccountinevaluationofthetestarticldeata: Percentageofcellswith aberrations. Percentageofcellswith more thanone aberration. Evidenceforincreasingamounts of damage with increasindgose,i.e.d,ose relatedincrease. The experimentalunitisthecell,and therefortehepercentageofcellswith structuraalberrations was thebasisforevaluation.Statisticaanlalysiesmployed a Cochran-Armitagetestforlinear trendand Fisher'Esxact Test(Thakuretal.,1985)to compare thepercentageof cellswith aberration(sand,ifapplicablet,hepercentageof cellswithmore thanone aberrationi)ntreated cellsto.theresultosbtainedforthevehiclecontrols. Statisticaanlalysiwsas alsoperformedforcellsexhibitinpgolyploidyand/orendoreduplication inordertoindicatseignifican(tp:SO.01)increaseisntheseeventsasindicatorosfpossible inductionofnumericalaberrationsh;owever,thetestarticle-waesvaluatedonly forstructural aberrationasnd notfornumericalaberrationbsy thisprotocol. Evaluation ofa PositiveResponse. A testarticlweas consideredpositiveforinducing chromosomal aberrationisfa significanitncrease(thedifferencweas consideredsignificawnhten p:@0.01)inthenumber of cellswith chromosomal aberrationissobservedatone ormore concentrationsS.tatisticeavlaluationofthepercentageof cellswithmore thanone aberration providedan indicatioonftheseveritoyfthepositivreesponseobserved.The lineartrendtest evaluatedthedoseresponsiveness.Ifa significanitncreasewas seenatone or more concentrationsa,dose-responseshouldhave been observed. Covance20784-0449 Evaluatioonfa NegativeResponse.A tesatrticwlaes considerendegativfeorinducing chromosomal aberrationisfno significanitncreasewas observed inthenumber ofcellswith chromosomal aberrationsatany ofthe concentrations. Equivocal Evaluation. Although most assaysgave clearlypositiveor negativeresultsi,nrare casesthe datasetwould precludemaking a definitivjeudgment about the activitoyf thetest articleR.esultsmight remain equivocalorquestionableregardlessof thenumber of timesthe assayisrepeated. Although the evaluationcriteriparovidedherenormallywas sufficient,he StudyDirectormight use additionalconsiderationtsoobtaina finalevaluationof thetestarticlbeased upon the Study Director'scientifijcudgment. RESULTS Test ArticleHandling Solubilityand Dose Determination. PFOS was insolubleinwater atconcentrationosf 50.0to 500mghnL. InDMSO, a translucentl,ight-yelloswuspensionwith some undissolvedtestarticle was obtainedata concentratioonf 501 mg/mL aftervortexing.A translucentl,ight-yellow suspensionwas obtainedat401 mg/mL. The suspensionat401 mg/mL was dosed inthe absence of cellsusinga I% (10 gL/mL) dosingvolume inRPMI 1640 culturemedium. At a dosed concentrationof4010 jig/mL,a translucenstuspensionwith fineparticleosfprecipitatweas observedand thepH was 8.0(pH ofRPMI 1640 culturemedium was 8.0).DMSO (Acros Organics,lot# A12649701) was selectedasthevehicleforthisassay.Aftersonicatingfor = 5 minutes,a translucentl,ight-yelloswuspensionwas obtainedata concentrationof 400 mg/mL. Alldosingwas achievedwith a 1.0% (10.0liL/mL)dilutionoftheDMSO stock solutionsa,nd thevehiclecontrolculturewas treatedwith 10.0gL/mL of DMSO. Concentrationsof 27.2,38.8,55.4,79.1,113, 161,230, 329,470, 671,959, 1370, 1960, 2800, and 4000 jig/mL were testedwith a 3.0-hourtreatmentand a 22-hour harvestinthechromosomal aberrationsassayswith and withoutmetabolicactivationD.ue tothepoor growth of cells (Tables I and 4),theassaywas repeatedtesting.concentratiofns12.5,24.9,49.7,99.3,149, 199,249, 299, 349,449,and 599 pg/mL withoutmetabolicactivationand 12.5,24.9,49.7,99.3, 149,199,249, 299,349,and 449 gg/mL with metabolicactivatiownith a 3.0-hourtreatmentand a 22-hour harvest.The stabilitoyf thetestarticluender thepreparation and dosing conditionsused inthisassayistheresponsibilitoyfthe sponsor. -12- Covance20784-0-449 ChromosomalAberratioAnssayWithoutMetaboliAcctivation A summaryofthetreatmetnitmesforthechromosomaalberratiaosnssaiysgivenbelow. Summary ofChromosomalAberratioAnssayTreatmenStchedulienHours ActivatioTnestArticleWash Colcemid'Harvest Condition Added Added Started -s9 0 3.0 20.0 22.0 Reductionsof 38%,8%,15%,15%,12%,19%,42%,69% and 92% were observedinthemitotic indicesof theculturetsreatewdith 12.5,24.9,49.7,99.3,149,249,299,349,and 449 ggImL, respectivelays,compared with thevehiclecontrolculture(sTable2). Chromosomal aberrations were analyzedfrom theculturetsreatewdith 199,249,299,and 349 gg/mL (Table3).No significanitncreaseincellswithchromosomal aberrationpso,lyploidyo,r endoreduplicatiownas observedatthe concentrationasnalyzed. The sensitiviotfythecellcultureforinductionofchromosomal aberrationissshown by the increasedfrequencyofaberrationisnthecellsexposedtomitomycin C, thepositivecontrol agent.The testarticliesconsiderednegativeforinducingchromosomal aberrationsp,olyploidy, and endoreduplicatiounndernonactivatiocnonditions. Chromosomal AberrationsAssay With MetabolicActivation A summary of thetreatmentimesfortheinitiaclhromosomal aberrationasssayisgivenbelow. Summary of Chromosomal AberrationsAssay Treatment Schedule in Hours Activation TestArticle Wash Condition Added +s9 0 3.0 Colcemie Added 20.0 Harvest Started 22.0 Reductionsof 12%, 41%, 71%, and 53% inthemitoticindicesas compared with thevehicle controlcultureswere observedintheculturestreatedwith 49.7,199,249, and 299 Ag/mL, respectivel(yTable5). Chromosomal aberrationwsere analyzedfrom theculturestreatewdith 99.3,149,199,and 299 gg/mL (Table6).Due to severetoxicityo,nly27 and 4 metaphaseswere availablfeoranalysisfrom theculturetsreatewdith299 gg/mL. No significanitncreaseincells withchromosomal aberrationsp,olyploidyo,r endoreduplicatiownas observedatthe concentrationasnalyzed. The successfualctivatiobny themetabolicsystemisillustratbeydtheincreasedincidenceof cellswithchromosomal aberrationisnthecultureisnducedwithcyclophosphamide,thepositive -13- Covance20784-0-449 controalgent.The tesatrticliesconsiderendegativfeorinducincghromosomalaberrations, polyploidy,and endoreduplicatiounnder activatiocnonditions. CONCLUSION PFOS was considerednegativeforinducingchromosomal aberrationisnhuman whole blood lymphocyteswith and withoutmetabolicactivation. PROTOCOL DEVIATIONS Negativecontrolculturewsere setup forthefirstrialT.hishad no impact on thestudy. Polyploidyand endoreduplicatiownere evaluatedfrom >100 cellsfrom MMC- and CP-treated culturesT.his had no impact on thestudy. RECORDS TO BE MAINTAINED All raw data,documentation,records,protocoland a copy of thefinalreportgeneratedat Covance as a resultofthisstudywillbe archivedinthe storagefacilitioefsCovance-Vienna for atleastI yearfollowingsubmissionof the finalreporttotheSponsor. Afterthe I-yearperiod, the Sponsor may electtohave the aforementionedmaterialsretainedinthe storagefacilitioefs Covance-Vienna foran additionapleriodoftime,or senttoa storagefacilitdyesignatedby the Sponsor. REFERENCES Evans, H.J.,"Chromosomal aberrationsproduced by ionizingradiation."InternationalReview of Cytology,13:221-321 (1962). Evans, H.J.,"CytologicalMethods forDetectingChemical Mutagens." Chemical Mutagens, Principlesand Methods for theirDetection,Hqllaender,A. (ed.),Vol. 4, pp. 1-29,Plenum Press: New York and London (1976). Galloway,S.M.,Aardema, M.J.,IshidateM,., Jr.,IvettJ,.L.,Kirkland,D.J.,Morita,T.,Mosesso, P.,and Sofuni,T.,"Report from working group on invitrotestsforchromosomal aberrations." Mutation Research,312(3):241-261(1994). Maron, D.M., and Ames, B.N.,"Revised methods forthe Salmonellamutagenicitytest." Mutation Research,113:173-215 (1983). Thakur,A.J.,Berry,K.J.,and Mielke,P.W.,Jr.,"A FORTRAN program fortestingtrendand homogeneity inproportions."Computer Programs inBiomedicine,19:229-233(1985). -14- Covance 20784-0-449 DATA TABLES -15- Covance 20784-0449 ASSESSMENT TABLE 1 OF TO)UCITY FOR CHROMOSOMAL ASSAY ABERRATIONS Assay No.: 20784 TrialNo.:I Date: 09/01/99 Lab No.: CY8249 CorMound: PFOS MetabolicActivation:-S9 Treatnient VEHICLE CONTROL DMSO 3.0 hour treatinent2,2.0 hour harvest % % Average % Mitotic Mitotic % Mitotic Index Index Mitotic Index A cultureB culture Index Reduction 1O.OpUmL 0.7 0.6 0.7 0 TEST ARTICLE 271 pg/mL 0.8 0.6 0.7 0 38.8pg/mL 0.4 0.6 0.5 29 55.4gg/niL 0.5 0.5 0.5 29 79.1 gg/mL 0.5 0.6 0.6 14 113 pgynil 0.4 0.7 0.6 14 161 pgtnil 0.4 0.5 0.5 29 230 pg/niL 0.7 0.6 0.7 0 329 pgtml 0.4 0.6 0.5 29 470 ILg/mL 1.9 1.6 1.8 0 DMSO Dimethylsulfoxide Covance 20784-0-449 TABLE2 ASSESSMENT OF TO)UCITY FOR CHROMOSOMAL ASSAY ABERRATIONS Assay No.:20784 TrialNo.:IA Date:09/15/99 Lab No.:CY9079 Compound: PFOS MetabolicActivation-:S9 TTeati.lte. VEHICLE CONTROL DMSO 3.0hourtreammt, 22.0hour harvest % I % lavcmge % MitoticMitotic % Index Index Mitotic A cultutBecultureIndex 10.0;iUmL 2.2 3.0 2.6 0 TEST ARTICLE 12.5gg/mL 1.9 1.3 1.6 38 24.9pg/mL 2.5 2.3 2.4 a 49.7pg/mL 2.2 2.2 2.2 15 99.3pg/mL 2.2 2.1 2.2 15 149pgtffl 2.5 2.0 2.3 12 199pginil 3.7 3.8 3.8 0 249 pg/niL 1.6 2.5 2.1 19 299 jig/mL 1.4 1.6 1.5 42 349jig/mL 1.2 0.4 0.8 69 449 pg/mL 0.2 0.2 0.2 92 DMSO Dimethylsulfoxide -17- TABLE3 CHROMOSOME ABERRATIONS IN HUMAN LYMPHOCYTES CellsFixed22.0Hours AfterInitiatiofnTreatment,3.0Hour Treatment Assay No.: 20784 Trial IA Date:09/15/99 Lab #:CY9079 Compound: PFOS CONTROLS VEtliCLE: DMSO POSITIVE: mmc TEST ARTICLE DMSO=Dimethylsulfoxide 1O.OpUmL 1.50pg/mL 199 pg/mL 249 pgtml 299 pg/mL 349 pgtml MMC=Mitomycin CELLS SCORED NOT COMPUTED NUMBER SIMPLE AND TYPE OF ABERRATION COMPLEX R A 100 2 B 100 2 A+B 200 2 2 A 75 13 B 50 5 1 A+B 125 6 4 A 100 2 B 100 2 A+B 200 4 A 100 2 B 100 2 A+B .200 4 A 100 2 1 B 100 A+B 200 2 1 A 100 3 3 B 100 1 A+B 200 4 3 2 2 6 10 1 10 4 11 3 1 6 1 10 21 3 2 16 1 2 2 1 1 1 Significantlgyreaterthan the solventcontrols,p!50.01. MetabolicActiva # OF % ABERRA- CELLI TIONS WITH OS?TiH@E@ PER ADERR : CELL TIONI 0.00 0.0 0.02 1.0 0.01 0.5 0.36 29.3 0.52 36.0 0.42 32.0* 0.00 0.0 0.00 0.0 0.00 0.0 0.00 0.0 0.00 0.0 0.00 0.0 0.00 0.0 0.02 2.0 0.01 1.0 0.00 0-.0 0.02 2.0 0.01 1.0 Covance 20784-0-449 TABLE4 ASSESSMENT OF TO)UCITY FOR CHROMOSOMAL ASSAY ABERRATIONS Assay No.:20784 TrialNo.:I Date:09/01/99 Lab No.:CY8249 Compound: PFOS MetabolicActivation+:S9 Treammt VEHICLE CONTROL DMSO 3.0hour treatment2,2.0hourharvest % I % lavemgclM % M itoticMitotic % itotic Index Index Mitotic Index A culturBe cultureIndex Reduction 1O.OpUmL 0.6 0.5 0.6 0 TEST ARTICLE 27.2jig/mL 0.2 0.2 0.2 67 38.8pg/niL 0.2 0.2 0.2 67 55.4pg/niL 0.2 0.3 0.3 50 79.1pg/niL 0.2 0.4 0.3 50 113pglmL 0.3 0.4 0.4 33 161pglnil 0.1 0.1 0.1 83 230pg/mL 0.2 0.0 0.1 83 329lig/mL 0.1 0.4 0.3 50 470 ILg/mL 'No cellpsresenotn slide DMSO Dimethylsulfoxide -19- Covance 20784-0449 TABLE5 ASSESSMENT OF TO)ACITY FOR CHROMOSOMAL ASSAY ABERRATIONS Assay No.:20794 TrialNo.:IA Date:09/15/99 Lab No.:CY9079 Compound: PFOS MetabolicActivation+:S9 Treat.,itc. VEHICLE CONTROL DMSO TEST ARTICLE 3.0hourtreat=t,22.0hourharvest Av7sel M,io/totoi, Mitotic Mitotic Index Index Mitotic Index A c0u/l0tureB c0u0ltuteIndex 1 Reduction 10.0pLImL 1.6 1.7 1.7 0 12.5Itg/mL 2.0 1.9 2.0 0 24.9pgtml 2.5 2.6 2.6 0 49.7 ILg/mL 1.4 1.6 1.5 12 99.3gg/mL 1.8 3.2 2.5 0 149pg/mL 2.0 2.4 2.2 0 199ligImL 1.0 1.0 1.0 41 249 pg/mL 0.5 0.5 0.5 71 299 pgtnil 0.9 0.7 0.8 53 DMSO Dimethylsulfoxide -20- TABLE6 CHROMOSOME ABERRATIONS IN HUMAN LYMPHOCYTES CellsFixed22.0Hours AfterInitiatiofnTreatment,3.0Hour Treaftmnt Assay No.: 20784 Trial TA Date:09/15/99 Lab #:CY9079 Mctabol Compound: PFOS CONTROLS VE141CLE: DMSO POSITIVE: CP TEST ARTICLE 1O.OpUmL 50.0pg/mu 99.3pg/mL 149 pg/mL 199 pg/mL 299 pgtmo CELLS SCORED A 100 B 100 A+B 200 A 25 B 25 A+B 50 A 100 B 100 A+B 200 A 100 B 100 A+B 200 A 100 B 100 A+B 200 A 27 B4 A+B 31 NOT COMPUTED 2 6 8 32 33 65 1 2 3 1 31 41 4 2 6 NUMBER SIMPLE AND TYPE OF ABERRATION COMPLEX 2 2 8 2 9 17 2 3 3 6 3 1 4 # OF % IABERRA- CELLS TIONS WITH PER ABERR, DF GT CELL TIONS 0.00 0.0 0.02 2.0 0.01 1.0 0.40 28.0 0.36 32.0 0.38 30.0* 0.00 0.0 0.00 0.0 0.00 0.0 0.03 3.0 0.03 2.0 0.03 2.5 0.03 2.0 0.01 1.0 0.02 1.5 0.00 0.0 0.00 0.0 0.00 0.0 Only 70 metaphase cellsfrom theB culturewere scoredforpolyploidyand endoreduplication. Too few nietaphasecellsavailableforanalysisof polyploidyand endoreduplication. DMSO=Dimethylsulfoxide CP=Cyclophosphamide *Significantlgyreaterthanthesolventcontrolsp,5O.Ol. Covance 20784-0449 EIISTORICAL CONTROL DATA CONTROL DATA OF CHROMOSOME ABERRANONS IN HUMAN 1/97THROUGH 9/98 20 -24 HOUR HARVEST LYMPHOCYTES NeptiveControl 3 HourTreatment VehiclCeontro(lPooled) 3 HourTreatment PositivCeontro-lMMC 3 HourTreatment NeptiveControl ContinuousTreatment VehiclCeontro(lPooled) ContinuousTreatment PositivCeontro-lMMC ContinuousTreatment NeptiveControl 3 HouTTreannent VehicleControl(Pooled) 3 HourTreatment PositivCeontro-lCP 3 HourTTeam=t N = Number oftrials. MMC = MitomycinC CP - Cyclophosphamide Activation % ofCells % ofCells % Poly- % Endore- % with With>I ploid duplicatedMitotic AberrationsAberrations Cells Cells Index Without MIN 0.0 MAX 2.5 AVG 0.45 SD (:E) 0.60 N 19 0.0 0.0 0.0 1.2 0.5 1.5 0.0 6.2 0.03 0.11 0.00 2.86 0.12 0.36 0.00 1.54 19 19 19 19 Without MIN 0.0 MAX 1.5 AVG 0.61 SD (*) 0.54 N 19 0.0 0.0 0.0 1.1 0.0 0.5 0.5 6.9 0.00 0.05 0.08 2.79 0.00 0.16 0.19 1.77 19 19 19 19 Without MIN 20.0 1.3 0.0 0.0 - MAX 62.0 26.0 0.5 0.0 - AVG 40.97 12.87 0.08 0.00 - SD (:E) 10.64 6.95 0.19 0.00 - N 19 19 19 19 - Without MIN 0.0 MAX 1.5 AVG 0.42 SD (*) 0.40 N 32 0.0 0.0 0.0 0.9 0.0 1.5 0.5 6.7 0.00 0.20 0.02 3.28 0.00 0.38 0.09 1.63 32 32 32 32 Without MIN 0.0 MAX 2.5 AVG 0.56 SD () 0.62 N 33 0.0 0.0 0.0 1.1 0.5 1.0 0.0 6.7 0.03 0.14 0.00 3.15 0.12 0.29 0.00 1.67 33 33 33 33 Without MIN 11.5 MAX 64.0 AVG 31.06 SD (*) 11.66 N 33 1.5 0.0 0.0 1.8 28.0 0.5 0.0 4.9 7.93 0.08 0.00 3.12 5.72 0.18 0.00 1.58 33 33 33 3 With MIN 0.0 MAX 2.0 AVG 0.48 SD (:L) .0.59 N 45 0.0 0.0 0.0 0.7 0.5 2.0 0.5 9.4 0.01 0.22 0.01 3.22 0.08 0.43 0.08 1.83 45 45 45 45 wit', MIN 0.0 MAX 2.5 AVG 0.44 SD (*) 0.56 N 46 0.0 0.0 0.0 0.5 1.0 0.5 0.5 6.9 0.03 0.07 0.02 2.90 0.16 0.17 0.10 1.52 46 46 46 46 With MIN 9.0 MAX 54.0 AVG 32.52 SD (*) 10.39 N 46 0.0 0.0 0.0 0.4 34.0 1.6 0.0 5.9 11.71 0.07 0.00 2.53 8.11 0.26 0.00 1.95 46 46 46 6 -22- Covance20784-0-449 DEFINITIONS OF CHROMOSOME ABERRATIONS FOR GIEMSA STAINED CELLS Simple TB ChromatiBdreak: An achromaticregioninone chromatid,largerthanthewidth of a chromatid.The associatefdragmentmay be partialloyr completelydisplaced. SB Chromosome Break: Chromosome has a clearbreak,formingan abnormal (deletedc)hromosome withan acentrifcragmentthatis dislocatedT.hisclassificatinonw includestheacentric ftaginen(tAF).An AF isdifferenftrom a SB onlyinthatit can notbe relatedtoany specifichromosome. DM "DoubleMinute"Fragment: These aresmalldoubledots,which may representerminalor interstitdiealetionso,r evensmallrings.These possible originsarenot distinguishable. Complex ID InterstitDiealletion: Lengthof chromatid"cutout"from midregionofa chromatid,resultinigna smallfragmentor ringlyingbesidea shortenedchromatidora gap inthechromatid. TR Triradial: An exchange between two chromosomes, or one chromosome and an acentricfragment,which resultisna three-armedconfiguration. QR Quadriradial: An exchangelikea triradiablu,tresultinigna four-armed configuration. CR Complex Rearrangement: An exchange among more thantwo chromosomes or fragments,causedby theinductionof severalbreaks. D Dicentric: An exchangebetween two chromosomes which resultisna chromosome withtwo centromeres.Thisisoftenassociated withan acentricfragmentinwhich caseitisclassifieadsDF. DF Dicentricwithfragment. -23- Covance20784-0-449 Complex(continued) TC Tric@ntric: An exchangeinvolvingthreechromosomes and resultinigna chromosome withthreecentromeres.Often associatewdith two tothreeAF. Such exchangescan involvemany chromosomes and arenamed asfollows: QC Quadricentric: PC Pentacentric: HC Hexacentric: fourcentromeres,up to fourAF fivecentromeresu,p to fiveAF sixcentromeresu,p tosixAF R Ring: A chromosome which forms a circlceontaininga centromere. Thisisoftenassociatewdith an acentricfragment,inwhich caseitisclassedas RF. RC Ring Chromatid: Singlechromatidring(acentric). RF Ring with associateadcentricfragment. Cl Chromosome Intrachange: Exchange withina chromosome; e.g.,a ringthatdoes not includethe entirechromosome. T Translocation: Obvious transfeorf materialbetween two chromosomes resultinigntwo abnormalchromosomes. When identifiable, scoredas "T" not"2AB". AB Abnormal: Abnormal monocentricchromosome. Thisisa chromosome whose morphology isabnormalforthekaryotype,and often theresultofa tmslocation,pericentricnversione,tc. Classificatiuosned ifabnormalitycannotbe ascribedto,e.g., a reciprocal.translocation. -24- Other GT GreatetrhanFive: PP PolyploiCdell: E Endoreduplication: Abs >1 Ab Not Computed TG ChromatidGap: SG Chromosome Gap: UC UncoiledChromosome: Covance20784-0-449 Greaterthan5 aberrationsA: cellwhich containsmore than 5 aberrationsH.eavilydamaged cellswillbe analyzedto identiftyhetypesof aberrationbsecausemultiplefragments, such asthosefound associatewdith a tricentridco,not count asindependentaberrations. A cellcontaininmgultiplecopiesofthehaploidnumber (n) ofchromosomes. A failuroef chromosomes toseparater,esultinigna 4n cell. Cellswithchromosomal aberrations Cellswith> 1 chromosomal aberrations ("tigdap").An achromati(cunstainedr)egioninone chromatid,thesizeofwhich isequaltoor smallerthanthe widthof a chromatid.These arenotedbutnot usually includedinfinaltotalosf aberrationasstheymay notallbe truebreaks. ("isochromatigdap,IG").Same as chromatidgap but atthe same locusinboth sistecrhromatids. Failureof chromatinpacking.Probablynota trueaberration. -25- COVA-Nr@CE. ME DWEWPMEW SERVIMS COMPAW AMENDMENT TO THE STUDY PROTOCOL Page I ofI STUDY TITLE: chromosomal AberrationsinHuman Whole Blood Lymphocytes PROTOCOL NO.: 449,Edition14 COVANCE STUDY NO.: 20784-0-449 Amendment #3 The Sponsorwillbe identifieads3M CorporateToxicologyand notas 3M Corporation. Reason: Sponsor'srequest. StudyDirector: ,Ip ,,, ja-, HemalathaMurli,Ph.D. Date Mammalian Cytogenefics DepartmentofCellulaarnd GeneticToxicology c 0 V Alg--EC@-- THE MWWPUENT SEIMCU COMPMY COVANCE STUDY NO. c@0 PROTOCOL NO. 449 EDMON 14 CHROMOSOMAL ABERRATIONS IN HUMAN WHOLE BLOOD LYMPHOCYTES Covance LaboratoriesInc.(Covance)willconductthisstudyincompliancewith Good LaboratoryPractice(GLP) regulationsT.his protocola,tleastone criticaplhase ofthework in progress,and thefinalreportwillbe subjecttoauditby QualityAssurance inaccordancewith StandardOperatingProceduresmaintainedatCovance. The study willbe conducted by Covance at9200 Leesburg Pike,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,NIN 55144-1000 2.0 TEST ARTICLE IDENTIFICATION dF, C.-@ Li ,3/), I Ive 3.0 TEST ARTICLE ANALYSIS The Sponsor willbe responsibleforthe determinationand documentation of the analyticaplurityand compositionof thetestarticlaend the stabilitaynd strengthofthe dosingsolutions. 4.0 NOTIFICATION OF REGULATORY SUBMISSION In orderto comply with GLP regulationsc,onsultinglaboratoriemsust be notifiedifallor partof a studyisintendedforregulatorysubmission.Covance maintainsa master scheduleof studieswhich fallunderregulatoryreview. Pleaseindicatewhich agency,if any,might receivetheresultsof thisstudy: 11 Undetermined 11 FDA MAFF MOHW 2/98 /taEPA-TSCA OECD EPA-FIFRA OTBER 1 of 15 C 0 V A-NRC E.' 'f"l!DMLOPMENT SEIMCU MMPAta 5.0 STUDY DATES Proposed ExperimentalStartDate: Proposed ExperimentalTerminationDate: 6.0 APPROVAL OF STUDY PROTOCOL Study Director: Hemalatha Murli,Ph.D. TestingFacilitMyanagement: @) c - intk BrianC. Myhr, Ph.D. AssociateDirector Sponsox'sAuthorized Representative: Mar@in T. Case, D.V.M., Ph.D. PROTOCOL NO. 449 EDMON 14 Date: C, Date: Date: -L-'4k4aaZ &f4r 2t98 2 of 15 C 0 V A-NrC'E--'-N THE DMTOPMEM SERMMS COMPAW PROTOCOL NO. 449 EDITION 14 PART 2. STUDY PROTOCOL CHROMOSOMAL ABERRATIONS IN HUMAN WHOLE BLOOD LYMPHOCYTES 1.0 OBJECTIVE The objectiveof thisinvitroassayistoevaluatetheabilitoyf a testarticlteoinduce chromosomal aberrationsinculturedhuman lymphocytes with and withoutan exogenous metabolic activatiosnystem. 2.0 TEST SYSTEM RATIONALE Aberrationsare a consequence of failureor mistakesinrepairprocessessuch thatbreaks eitherdo notrejoinor rejoininabnormal configuration(sEvans,1962, 1976). Descriptionsofthe typesofaberrationasreprovidedin Section10.0.Structural aberrationmsay be oftwo types,chromosome or chromatid: Chromosome aberration:structuraclhromosome damage expressedas breakage, orbreakage followedby reunion,of both sisterchromatidsatan identicaslite. Chromatid aberrations:tructuraclhr-omosome damage expressedas breakage of singlechromatidsorbreakagefollowed by reunionbetween chromatids.This is the most common typeof structuraalberration. Most known chemical clastogens(chromosome-breaking agents)requirea periodof DNA synthesistoconvertinitiaDlNA damage intochromosome alterationtshatbecome visible atmitosis. The lymphocytes inblood do not usuallydivide,but theyare stimulatedto dividein cultureby exposuretophytohemaggluiinin(PHA). At predeterminedintervals afterexposure to thetestarticlet,he lymphocytes aretreatedwith a metaphase-arresting substance,Colcemid*, then areharvestedand stained,and metaphase cellsare analyzed microscopicallyforthepresenceof chromosomal aberrations. Many mutagenic chemicals do not actdirectlyon DNA but do so afterbeing convertedto activeintermediatebsy enzymes found in liver.Human lymphocytes have only a limited capacityto metabolizesome testarticless,o an exogenous metabolic activatiosnystem (ratliverS9 homogenate) isincludedwith a seriesoftreatmentstoenhance thedegreeof conversionand theabilitoyf theassayto detectclastogenicm,etabolicintermediates. Numerical aberrations(achange inthenumber ofchromosomes from the normal number of46 forhuman lymphocytes)arenot determinedby thisprotocol.However, the 2/98 3 of 15 c 0 v A-Nr'-EC@. THE DEVELOPMENT SERVICES COMPAW PROTOCOL NO. 449 MrrION 14 occurrenceofpolyploidyor endoreduplicatiownh,ich willbe scored,may indicattehat thetestarticlheas the potentialtoinduce numericalaberrations. 3.0 MATERIALS 3.1 Test System Human venous blood from a healthydonor willbe drawn intosterileh,eparinized "Vacutainers".Whole blood cultureswillbe initiatebdy adding0.6ml of fresh heparinizedblood to 9.4 ml of culturemedium ineach 15 nilcentrifugetube. 3.1.1 Media and CellCulture Conditions Cultureswillbe incubatedwith loosecaps atabout37 *C t 2 *C ina humidifiedincubator,in an atmosphereof about5% :t1.5% C02 in air. The medium willbe RP@E 1640 supplemented with approximately15% fetalbovine serum (FBS),penicilli(n100units/ml)s,treptomycin (100gg/ml),L-glutaniin(e2mM) and I% phytohemagglutini(nPRA-M). The totalculturevolume willbe 10 ml. 3.2 Test Article Solidorliquidtestarticleasre suitablfeorthisassay.Volatiletestarticlemsay be used in cultureswith tightlcylosedcaps. The testarticliesidentifieidnPartI of thisprotocol.Storageconditionswillbe as specifiedby the Sponsor. 3.3 Control Articles 3.3.1 VehicleControlArticle The vehiclecontrolarticlweillbe thevehicleselectefdorthetestarticle. The vehiclemay be speci@iedby the Sponsor or selectedby theprocess describedinSection4.1(TestArticleSolubilitTyesting).The vehicle concentratioinnthe vehiclecontrolcultureswillbe equivalenttothe vehicleconcentrationin thetestarticle-treatceudltures. 3.3.2 PositiveControl Articles Known clastogenic(chromosome-breaking)agentswillbe used for structuraalberrationsanalysis. 2/98 4 of 15 COVA-Nr-C-E-.-"' THE DEVELOPMENT SEWCES COMMMY PROTOCOL NO. 449 EDITION 14 WithoutS9 activationN:fitomyciCn(N4MC;CAS #50-07-7)a, clastogenthatdoes not requiremetabolicactivationw,illbe dissolvedin water and used ata finalconcentrationbetween 0.025 to3.0 Ag/ml. With S9 activation:Many clastogensdo not actdirectlyon DNA but must be metabolicallyconvertedto clastogenicintermediatesby a cofactor-supplementepdost-mitochondriaflraction(S9)preparedfrom liver.An example iscyclophospbamide (CP; CAS #6055-19-2),used here asthe positivecontroltodemonstratethe activitoyf the S9 metabolic activatiosnystem. CP willbe dissolvedinwater and used ata final concentratiobnetween 10 to300 iLg/ml. 3A S9 Metabolic ActivationSystem The invitrometabolicactivatiosnystem (Maron and Ames, 1983) consistsof a rat liverpost-mitochondriaflraction(S9)and an energy-producingsystem (NADP plusisocitriaccid).Priorto collectinlgiversfrom male Sprague-Dawley rats, variousP450 isoenzyrnelevelswillbe increasedby an acutetreatmentof therats withAroclor:rm1254 at500 mglkg. The S9 fractionwillbe prepared approximately5 days later.The S9 fractionp,reparedinsucroseorinpotassium chloride,ispurchased from a commercial supplierand isretainedfrozen at--@70*C untiluse. Aliquotsof S9 willbe thawed immediatelybeforeuse and added totheothercomponents toform the activationsystem describedas follows: Component NADP (sodiumsalt) Isocitriaccid Homogenate (S9 fraction) Concentrationin Cultures 1.5mgtmi (I-8 mM) 2.7mg/ml (10.5mM) 15.0jul/ml*(1.5%) This concentratioonf ratS9, obtainedfrom MolecularToxicology Inc.,Boone, NC, has consistentlcyaused CP tobe highlyclastogenicformany differenltots. 4.0 EXPERIMIENTAL DESIGN 4.1 Test ArticleSolubilityTesting Ifsolubilitiynformationisnot providedby theSponsor,a preliminarysolubility testwillbe carriedout with sterildeeionizedwater,serum-freeculturemedium, dimethylsulfoxide(DMSO; CAS #67-68-5),ethanol(CAS #64-17-5),or acetone (CAS #67-64-1).The vehiclesofchoice arewateror serum-freeculturemedium. TestarticlseolutionsinDMSO or ethanolwillbe diluted100-foldinculture 2/98 5 of 15 C 0 V Alt CE.@-N THEDEVEU PROTOCOL NO. 449 EDITION 14 medium, and testarticlseolutionsinacetonewillbe diluted1:200in culture medium. The solubilitaynd dispersioncharacteristiocfsthe testarticlweillbe determined,and the vehicleselectedwillbe theone which givesthe best solubilitoyr dispersionafterdilutioninmedium. In some cases,testarticleasre apparr,ntliynsolublein vehiclesthatarecompatiblewith tissueculture.In such cases,the vehiclechosen and thehighestdose testedwillbe thosegiving an evenlydispersedsuspension.For liquidtestarticlest,he dosing stocksmay be preparedby weight or be calculatedfrom the specificgravity. The top dose testedfornon-toxictestarticlesw,here the molecular weight is unknown, willbe 5 mg/ml. Ifthemolecularweightisprovidedby theSponsor and thetestarticlheas a purityof 95% or greatert,he maximum dose willbe 10 mM or 5 mg/ml, whichever islower. 4.2 Dose Rangerinding Dose rangefindingwillbe conducted asan integraplartof the chromosomal aberrationsassays.A range of doses willbe applied,asappropriateforeach treatmentcondition,to achievea wide toxicitryange,a range of solubleand insolubledoses,or a maximum dose of 10 mM or5 mg/ml (whicheverislower) fornontoxictestarticles. 43 Test ArticleHandling Test articlceoncentrationswillbe expressedasweight per milliliteorf culture medium inthe cellcultures. For testarticletshatareknown to be markedly volatilet,he cultureswillbe incubatedwith tightlyclosedcaps untilthetestarticlieswashed off.To compensate forthe lackof C02 circulatioBnE,PES buffer(25 mM final concentrationw)illbe includedinthemedium tocontrolthepH. Ifmarked pH changes arenoticed(i.e.c,olorchanges inmedium) duringthe solubilittyestB,EPES bufferwillbe includedinthemedium to attemptto counteractthepH change. Furtherneutralizati(oen.g.w,ith HCI or NAOH) will be performed tomaintaina normal culturepH range(approximately6.8to7.4) only afterconsultationwith the Sponsor. 4.4 Chromosomal Aberrations Assay The chromosomal aberrationasssaywillbe conductedwith about 3-hour 2/98 6 of 15 c 0 V A- N rC-E'".' THE OMLOPMENT SERVR-" COMPANV PROTOCOL NO. 449 EDITION 14 exposurestotestarticlientheabsenceand presenceof theS9 metabolicactivation system. In the absenceof any toxicitiynformation,a wide rangeof testarticle concentrationwsillbe applied,startinagbove the observed solubilitlyimitin culturemedium, or atapproximately10 mM or 5 mg/ml (whicheverislower)for solubletestarticlesS.uccessivelylower doseswillusuallybe determinedby using 70% of theprecedingdose. A singleharvesttime of approximately22 hourswillbe used. This harvesttime correspondsto 1.5times thecellcycletime of approximately15 hours afterthe lymphocytes areinduced todivideby the additionofPHA-M (Galloway etal., 1994). Lymphocyte cultureswillbe initiatewdith 0.6ml of whole blood in9.4ml of freshculturemedium. Duplicateflaskswillbe used foreach dose levelv,ehicle, and foreach of threedosesofthe positivecontrolcompounds. Vehiclecontrol cultureswillbe dosed with thevehicleforthetestarticlaetthe same or the maximum concentrationused inthe testcultures. 4.4.1 Assay without S9 activation Two days aftercultureinitiatiotnh,eculturemedium willbe replacedwith freshmedium and thecellswillbe treatedwith the testv,ehiclecontrol, and positivecontrolarticlesT.he cultureswillbe returnedtothe incubator at37 2 *C foran exposure periodof about 3 hours. The culturesthen willbe washed with phosphatebufferedsaline(PBS) toremove the test articlea,nd freshmedium willbe added. Incubationwillbe continuedfor about 18 hours,withColcenii& (0.1ilglml)added forthelast2 :t0.5 hours of incubation. 4.4.2 Assay with S9 activation Two days afterculturein:ltiaticounl,turemedium willbe replacedwith freshmedium withoutfetalbovine serum (FBS) and thecellswillbe treatedwith thetest,vehiclecontrola,nd positivecontrolarticlesT.he S9 metabolicactivatiosnystem alsowillbe includedin allcultures.The cultureswillbe returnedtothe incubatorat37 2 *C foran exposure periodof about 3 hours. The shortincubationtime isused because prolonged exposure tothe S9 mixture istoxictocells,and enzyme activity islostrapidlyat37*C. Serum isomittedtoavoidpossibleinactivatio(nby bindingto serum proteins)of short-livedh,ighlyreactiveintermediates produced by the S9 system. Afterexposure,thecellswillbe washed at 2/98 7 of 15 c 0 v A-Nr -CE@. THE DEnLOPMEPff SEIMCES CompAmy PROTOCOL NO. 449 EDMON 14 leasttwicewith PBS, and normal culturemedium containing15% FBS willbe added. Incubationwillbe continuedforabout 18 hours,with Colcen-@& (0.1itgtml)added forthelast2 t 0.5hours of incubation. 4.4.3 Summary of assay schedule Schedule (hours)* Test TestArticle Exposure Wash Colcemi& Harvest Condition Added Completed Completed Added Started -s9 0 +s9 0 3 4 20 22 3 4 20 22 Times given areapproximate and may varywith the sizeofthe assay. 4.4.4 Assay termination The cultureswillbe centrifugedt,he supernatantdiscarded,and the cells willbe swollenwith 75 mM KCI, fixedinmethanol:glacialaceticacid (3:1,v/v),dropped onto glassslidesa,nd airdried.The slideswillbe stainedwith 5% Giemsa and airdried.Afterdrying,the slideswillbe rinsedinxyleneand mounted per-manently.For controlof bias,the slides willbe coded foranalysis.One thousand(1000)cellswillbe analyzedfor mitoticindex (percentageofcellsin mitosis)from each cultureinthe dose range inclusiveof the toxicityrange obtainedas wellas thenegativeand vehiclecontrolcultures. The doses selectedforanalysiswillbe based on theobservationsof niitotiicndexreduction,and the maximum dose selectedwillbe the highestdose atwhich a sufficienntumber of analyzablemetaphase cells areexpected. Ifpossible,the highestdose selectedwould suppressthe mitoticindexby about 50%, but not greaterthan60% reduction. Cellswillbe selectedforscoringon thebasisofgood chromosome morphology, and only cellswith thenumber of centromeresequal tothe modal number 46 willbe analyzed.Normally, 200 metaphases per dose level(100 from each oftheduplicatecultures)i,favailablew,illbe read. If200 metaphases arenot availablefrom each ofthesedoses,lower doses may be evaluated.The complete number of 100 metaphases per culture may not be availabledue totoxicityorqualityof preparation.At least25 2/98 8 of 15 c 0 V Alg-'-C-E.@ THE DEVELOPMENT SEWCES COMPAW PROTOCOL NO. 449 MITION 14 metaphasewsillbe analyzefdromthoseculturetshathavegreatetrhan 25% of cellswith one or more aberrationsP.ercentpolyploidyand endoreduplicationwillalsobe analyzedby evaluating100 metaphases per culture,ifavailablea,nd tabulated. Standardforms willbe used torecordanalyses.For each cellbeaiingan aberrationt,he microscope stagelocation(vernierreadings)willbe recordedso thatthecellmay be relocatedifnecessary.The complete list of aberrationscoredand theirdefinitionasreprovidedin Section 10.0. Gaps arerecordedseparatelyand reportedbut willnot be includedinthe totalaberrationfrequency. 5.0. DATA 5.1 Data Presentation Data willbe summarized in tablesshowing mitoticindex,percentpolyploidy, percentendoreduplicationt,henumbers ofcellsanalyzed,typesof aberrations found, frequenciesof aber-rationpser cell,and percentagesof cellsbearing aberrationsC.hromatid and isochromatidgaps willbe notedbut willnot be added intothetotalsforaberrationassessment sincetheyarenot consideredtorepresent truebreaks. 5.2 Assay Acceptance Criteria An assaywillbe consideredacceptableforevaluationof testresultsonly ifallof thefollowingciiteriaaresatisfiedT.he metabolicactivatioannd nonactivation sectionsof the aberrationsassaysareindependentunitsand willbe repeated independently,asneeded,to satisfythe acceptancecriteria. 5.2.1 Acceptable Controls The vehiclecontrolculturesmust containlessthan approximately5% cells with aberrations. The positivecontrolresultmust be sipificantlhyigher(p :50.01)thanthe vehiclecontrols.Ifthepositivecontrolresultinthe testwith S9 is adequateinan assay where activationand nonactivationassaysarerun concurrentlyb,ut thepositivecontrolinthenonactivationassayfailst,he testneed not be repeatedbecause the S9 activationpositivecontrol demonstratesthesensitivitoyf the cells. 2/98 9 of 15 C 0 V A-K'C-E'.@-\ THE DEVELOPMENT SEIMCU COMPAW PROTOCOL NO. 449 EDMON 14 5.2.2 Acceptable High Dose Ifthe aberrationresultsarenegativeand thereisno significanrteduction (approximately;-5>0%) inmitoticindex,the assaymust includethehighest applicabldeose (atargetdose of 10 mM or 5 mg/ml, whicheverislower) ora dose exceedingthesolubilitlyimitinculturemedium. Testingwillbe conducted atinsolubledose levelswhen a well-dispersedsuspensionin culturemedium isobtainedthatdoes not settlerapidly. 5.2.3 Acceptable Number ofDoses Each assaytreatmentconditionmust includeatleastthreeanalyzabledose levels. 5.3 Assay Evaluation Criteria Aftercompletion of microscopic analysis,datawillbe decoded. The following factorsaretaken intoaccount inevaluationof thetestarticldeata: Percentageof cellswith structuraalberrations. Percentageof cellswith more thanone structuraalberration. Evidence forincreasingamounts of damage with increasingdose,i.e.a, dose- relatedincreasein aberrations. The experimentalunitisthe cell,and thereforethepercentageofcellswith structuraalberrationwsillbe thebasisforevaluation.Statisticalnalysiswill employ a Cochran-Armitage testfo.-lineartrendand Fisher'sExact Test (Thakur etal.,1985) tocompare the percentageofcellswith aberrations(and,if applicablet,he percentageof cellswith more thanone aberrationi)n treatedcells to theresultsobtainedforthevehiclecontrols. Statisticaanlalysisalsowillbe performed forcellsexhibitingpolyploidyand/or endoreduplicatioinn ordertoindicatesignifican(tp -@0.01)increasesinthese eventsasindicatorsofpossibleinductionof numericalaberrationsh;owever, the testarticlweillbe evaluatedonly forstructuraalberrationasnd not fornumerical aberrationbsy thisprotocol. 5.3.1 Evaluation of a PositiveResponse A testarticlweillbe consideredpositiveforinducingchromosomal aberrationisfa significanitncrease(thedifferencewillbe considered 2/98 10 of 15 C 0 V A4t'C-E.-'-'\ THE DEVELOPMENT SERVICES COMPANY PROTOCOL NO. 449 EDITION 14 significanwthen p :50.01)inthenumber of cellswithchromosomal aberrationsisobservedatone ormore dose levels.Statisticeavlaluation of thepercentageofcellswith more than one aben-ationprovidesan indicationof theseverityof thepositiveresponseobserved.The linear trendtestevaluatesthe dose-responsiveness.Ifa significanitncreaseis seen atone or more dose levels,a dose-responseshould be observed. 5.3.2 Evaluation of a Negative Response A testarticlewillbe considerednegativeforinducing chromosomal aberrationsifno significanitncreaseisobserved inthe number ofcells with chromosomal aberrationfsorany of thedose levels. 5.3.3 Equivocal Evaluation Although most assayswillgiveclearlypositiveor negativeresultsi,nrare casesthedatasetwillprecludemaking a definitivjeudgment about the activitoyf the testarticleR.esultsmay remain equivocalorquestionable regardlessof thenumber oftimes the assayisrepeated. Although theevaluationcriteriparovidedherenormally willbe sufficienth,e study directormay use additionaclonsiderationtsoobtaina finalevaluationofthe testarticle based upon the studydirector'scientifijcudgment. 6.0 REFERENCES Evans,H.J.(1962) Chromosomal aberrationpsroduced by ionizingradiation, InternationaRleview of Cytology,13: 221-321. Evans,H.J.(1976)CytologicalMethods forDetectingChemical Mutagens. In:Chemical Mutagens, Principleasnd Methods fortheirDetection,Vol. 4,Hollaender,A. (ed.), Plenum Press,New York and London, pp. 1-29. Galloway, S.M.,Aardema, M.J.,IshidateM,., Jr.,IvettJ,.L.,Kirkland,D.J.,Morita,T., Mosesso, P.,and Sofuni,T. (1994) Report from working group on in vitrotestsfor chromosomal aberrationsM,utationResearch,312 (3): 241-261. Maron, D.M., and Ames, B.N. (1983) Revised methods fortheSalmonellamutagenicity testM,utation Research,113: 173-215. 2/98 11 Of 15 C 0 V AW'C-E-.'-\ THE DEVftOPMENT SERVICES COMPANY PROTOCOL NO. 449 EDITION 14 'nakur,A.J.B,erryK,.J.a,ndMielkeP,.W.,Jr.(1985)A FORTRAN programfor testingtrendand homogeneity inproportions,Computer Programs inBiomedicine,19: 229-233. 7.0 REPORT FORMAT Covance employs a standardreportformat foreach assaydesign.The finalreportwill providethe followinginformation. Sponsor identification. QualityAssurance statement. Statementof GLP compliance. Signatureof study director. Test articliedentificatiaonnd Covance Study Number. A physicaldescriptioonf the testarticlaend dateofreceiptwillbe includedinthissection. Type of assay and protocolnumber. Dates of studyinitiatioannd completion. Identificatiofn studydirectorand seniortechnician. Methods. Evaluationcriteria. Interpretationf results. Conclusions. References. Test resultspresentedin tabularform. I-listoricaolntroldata. 8.0 CHANGES OR REVISIONS Any changes to or revisionsof thisapproved protocolwillbe documented, signedby the studydirectord,ated,and maintainedwith thisprotocol. 9.0 RECORDS TO BE MAINTAINED All raw data,documentation,records,protocol,and finalreportsgeneratedas a resultof thisstudywillbe archivedinthe storagefacilitioefsCovance-Vienna foratleastone year followingsubmissionofthe finalreporttothe Sponsor. Afterthe one yearperiod,the Sponsor may electto have the aforementionedmaterialsretainedinthe storagefacilities of Covance-Vienna foran additionalperiodof time or senttoa storagefacilitdyesignated by the Sponsor. 2/98 12 of 15 c 0 v A-Nr"C-E.@ THE DEVELOPTAENT SERVICES COMPAW PROTOCOL NO. 449 EDMON 14 10.0 DEFINITIONS CELLS OF CHROMOSOMAL ABERRATIONS FOR GIEMSA STAINED SUVIPLE TB Chromatid Break: An achromaticregionin one chromatid,largerthanthe width of a chromatid.The associatedfragment may be partialloyr completelydisplaced. SB Chromosome Break: Chromosome has a clearbreak,forming an abnormal (deletedc)hromosome with an acentricfragmentthatis dislocated.This classificatinoonw includesthe acentric fragment(AF). An AF isdifferenftrom a SB onlyinthat itcan not be relatedtoany specificchromosome. DM "Double NEnute" Fragment: These aresmall double dots,which may represent terminalor interstitidaeletionso,r even smallrings. These possibleoriginsarenot distinguishable. COMPLEX ID InterstitiDaelletion: I-engthof chromatid"cutout"from midregion of a chromatid,resultingina smallfragment or ringlying besidea shortenedchromatid or a gap inthe chromatid. TR Triradial: An exchange between two chromosomes, or one chromosome and an acentricfragment,which resultsina three-armedconfiguration. QR Quadriradial: An exchange likea tiiradiablu,t resultingina fourarmed configuration. CR Complex Rearrangement: An exchange among more thantwo chromosomes or fragments,caused by the inductionof severalbreaks. D Dicentric: An exchange between two chromosomes which resultsin a chromosome with two centromeres.This isoften associatedwith an acentricfragment inwhich caseitis classifieads DF. DF Dicentiicwith fragment. 2198 13 of 15 C 0 V A- N r'C-E.'-'N THE DEVELOPMENT SEWACES COMPANY PROTOCOL NO. 449 EDRFION 14 CONDLEX (Continued) TC Tricentiric: QC Quadricentric: PC Pentacentric: HC Hexacentric: R Ring: RC Ring Chromatid: RF Cl Chromosome Intrachange: T Translocation: AB Abnormal: An exchange involvingthreechromosomes and resulting ina chromosome with threecentromeres.Often associatedwith two to threeAF. Such exchanges can involvemany chromosomes and arenamed asfollows: fourcentromeres,up tofourAF fivecentromeres,up to fiveAF sixcentromeres,up to sixAF A chromosome which forms a circlecontaininga centromere.This isoftenassociatedwith an acentric fragment,inwhich caseitisclassedas RF. Singlechromatidring(acentric). Ring with associatedacentricfragment. Exchange withina chromosome; e.g.,a ringthatdoes not includethe entirechromosome. Obvious transferof materialbetween two chromosomes resultingintwo abnormal chromosomes. When identifiablsec,oredas"T" not"2AB". Abnormal monocentricchromosome. This isa chromosome whose morphology isabnormal forthe karyotype,and oftentheresultof a trans)ocation, pericentjiicnversione,tc.Classificatiounsed if abnon,nalitcyannot be ascribedto,e.g.,a reciprocal translocition. 2/98 14 of 15 C 0 V A- N -C'-E-.'* 'r"EDMU"ENT SEWirAS COUPAW OTHER GT GreaterThan five: PP PolyploidCell: E Endoreduplication: NOT COMPUTED TG ChromatidGap: SG Chromosome Gap: UC UncoiledChromosome: PROTOCOL NO. 449 EDrrION 14 Greaterthan5 aberrationsA: cellwhich containsmore than 5 aberrationsH.eavily damaged cellswillbe analyzedtoidentifythetypesof aberrationsbecause multiplefragments,such as thosefound associatedwith a tiicentridco, not count as independentaberrations. A cellcontainingmultiplecopiesof the haploidnumber (n)of chromosomes. A failureof chromosomes toseparate,resultingin a 4n cell. ("tidgap").An achromatic(unstainedr)egionin one chromatid,thesizeof which isequal to or smallerthan thewidth of a chromatid.These are noted but not usuallyincludedin finaltotalsof aberrationsastheymay not allbe truebreaks. ("isochromatigdap,IG"). Same as chromatidgap butat thesame locusinboth sisterchromatids. Failureof chromatinpacking. Probably not a true aberration. 2/98 15 of 15