Document 4JGvw5w6jLnzVozNJeXOyX82N

CORNINGHazleton MUTAGENICITY TEST ON T-6295 INAN IN VIVO MOUSE NHCRONUCLEUS 11 7?D@0@I..'9AA,i9YY62c@7 10996 1% >, 3m 0 CA, ASSAY FINAL REPORT AUTHOR HemalathaMurli,Ph.D. PEMORMING LABORATORY Coming HazletonInc.(CHV) 9200 LeesburgPike Vienna,Virginia22182 LABORATORY PROJECT IDENTIFICATION CHV StudyNo.: 17403-0-455 SUBMITTED TO 3M 3M CenterB,uilding220-2E-02 St.Paul,Minnesota55144-1000 s Y COMPLETION DATE May 23,1996 CHV StudyNo.: 17403-0-455 1 of26 CORNINGHazleton QUALITY ASSURANCE STATEMENT ProjeTcittlel:nVivoMouseMicronucleAusssay ProjectNo.: 20996 Assay No.: 17403 ProtocolNo.: 455 EditionNo.: 17 QualityAssuranceinspectionosf thestudyand reviewofthefinalreportof theabove referenced projectwere conductedaccordingtotheStandardOperatingProceduresoftheQualityAssurance Unit and accordingtothegeneralrequirementsoftheappropriateGood LaboratoryPractice regulationsF.indingsfrom theinspectionasnd finalreportreviewwere reportedtomanagement and tothestudydirectoorn thefollowingdates: InRection/Date FindingsReRgrted Auditor Harvest/03/21/1996 03/21/1996 C. Smith DraftReportReview/05/15,16/1996 05/17/1996 C. Orantes FinalReportReview/05/23/1996 05/23/1996 C. Orantes QualityA ---te nit d CHV StudyNo.: 17403-0-455 2 CORNINGHazleton STUDY COMPLIANCE AND CERNFICATION The describedstudywas conductedincompliancewith theGood LaboratoryPracticeregulations as setforthintheFood and Drug Administratio(nFDA) Title21 of theU.S.Code of Federal RegulationsPart58,issuedDecember 22,1978,(effectivJeune 20,1979) withany applicable amendments. There were no significandteviationfsrom theaforementionedregulationosr the signedprotocolthatwould affecttheintegritoyfthestudyortheinterpretatioofnthetestresults. The raw datahave been reviewedby theStudy Directorw,ho certifietshattheevaluationof the testarticlaes presentedhereinrepresentasn appropriatceonclusionwithinthecontextofthe studydesignand evaluationcriteria. Alltestand controlresultisnthisreportaresupportedby an experimentaldatarecordand this recordhasbeen reviewedby theStudy Director.Allraw data,documentation,recordsp,rotocol and a copy ofthefinalreportgeneratedas a resultofthisstudywillbe archivedinthestorage facilitioefsComing HazletonInc.foratleastone yearfollowingsubmissionofthefinalreportto theSponsor. Aftertheone yearperiod,theSponsormay electtohave theaforementioned materialrsetainedinthestoragefacilitioefsComing HazletonInc.foran additionapleriodof time,or senttoa storagefacilitdyesignatedby theSponsor. SubmittedBy: Study Director: Hemalatha Murli,Ph.D. Mammalian Cytogeneties Department of Geneticand CellulaTroxicology q Study CompletionDate CHV StudyNo.: 17403-0-455 3 CORNINGHazleton TABLE OF CONTENTS Page No. SUMNIARY ................................................6......... 1.0 SPONSOR .............................................7 ......... 2.0 NLATERIAL (TestArticle)...................................7......... 2.1 Client'Isdentification 2.2 Date Received 2.3 PhysicalDescription 2.4 GeneticsAssay No. 3.0 TYPE OF ASSAY ........................................7......... 4.0 PROTOCOL NO ..........................................7 ......... 5.0 STUDY DATES .........................................7......... 5.1 InitiatiDoante 5.2 ExperimentalStartDate 5.3 ExperimentalTerminationDate 6.0 SUPERVISORY PERSONNEL ................................7.......... 6.1 StudyDirector 6.2 LaboratorySupervisor 7.0 OBJECTIVE ...........................................7......... 8.0 MATERIALS ...........................................8......... 9.0 SOLUBILITY AND STABILITY ................................8 ......... 10.0 DOSE RANGEFINDING STUDY I ..............................9......... 10.1 Dose Selection 10.2 DosingInformation 10.3 Resultsand Interpretation 10.4 Conclusion CHV StudyNo.: 17403-0-455 4 CCRNINGHazleton 11.0 DOSE RANGEFINDING STUDY II .............................I.I ......... 11.1 Dose Selection 11.2 DosingInformation 11.3 Resultsand Interpretation 11.4 Conclusion 12.0 MICRONUCLEUS STUDY ..................................1.3 ......... 12.1 Dose Selection 12.2 MicronucleusAssay DosingInformation 13.0 BONE MARROW HARVEST, SLIDE PREPARATION AND ANALYSIS .......14. 14.0 EVALUATION CRITERIA ..................................1.5......... 14.1 General 14.2 Data Presentatioannd Interpretation 15.0 RESULTS AND INTERPRETATION ............................1.5......... 16.0 CONCLUSION .........................................1.7 ......... 17.0 REFERENCES .........................................17.......... 18.0 DEVIATIONS FROM THE SIGNED PROTOCOL ....................1.7...... 19.0 EXPERIMENT DATA TABLES ...............................1.8 ......... CHV StudyNo.: 17403-0-455 5 CORNINGHazleton SUMMARY MutageniciTteystonT-6295inanInVivoMouseMicronucleAusssay TheobjectiovfethiisnvivoassawyastoevaluatheeabiliotfythetesatrticTl-e6,295t,oinduce micronucleinbone marrow polychromaticerythrocyteosfCrl:CD-I*(ICR)BR mice. In thedose selectiosntudy,thetestarticlweas suspendedindeionizedwaterand dosed by oral gavage at500, 1630,2750,3880 and 5000 mg/kg, and thedose selectiosntudywas repeated testingdoselevelsof 700, 1000,and 1300 mg/kg. Six animals(threemales and threefemales) were assignedto each dose group. Animals were observedforthreedays afterdosingfortoxic signsand/ormortality. Based -ontheresultsofthedose rangefindingstudy,themaximum toleratedose was estimated as 950 mg/kg. Inthemicronucleusassay,thetestarticlweas suspendedindeionizedwaterand dosedby oralgavage at237.5,475,and 950 mg/kg. Ten animals(fivemales and fivefemales) were randomly assignedtoeachdosefiiuvesttime group.Vehicleand positivceontrolgroups, euthaiiizeadpproximately24 hoursafterdosing,were includedintheassay.The animalsdosed with thetestarticlweere euthanizedapproximately24,48 and 72 hours afterdosingfor extractioonf thebone marrow. The testmaterialT,-6295,didnotinducea significanitncreaseinmicronucleiinbone marrow polychromaticerythrocyteusnder theconditionsofthisassayand isconsiderednegativeinthe mouse bone m=ow micronucleustest. CHV StudyNo.: 17403-0-455 6 CORNINGHazleton MutagenicTietsytonT-629i5nanInVivoMouseNficronucAlsesuasy 1.0 SPONSOR:3M 2.0 MATERIAL (TestArticle) 2.1 Client'IsdentificatiTo-n6:295 2.2 DateReceived:January22,1996 2.3 PhysicalDescriptionO:ff-whitemixtureofpowder and flakes 2.4 GeneticsAssay No.: 17403 3.0 TYPE OF ASSAY: In VivoMouse MicronucleusAssay 4.0 PROTOCOL NO.: 455,Edition17 5.0 STUDY DATES 5.1 InitiatiDoante:January22,1996 5.2 ExperimentaSltartDate:March 13,1996 5.3 ExperimentaTlerminationDate:April18,1996 6.0 SUPERVISORY PERSONNEL 6.1 StudyDirectorH:emalathaMurli,Ph.D. 6.2 LaboratorySupervisorM:onica Vegarra,B.S. 7.0 OBJECTIVE The objectivoefthisinvivoassaywas toevaluatteheabilitoyfthetestarticlTe-,6295,to inducemicronucleinbonemarrow polychromatiecrythrocyteosfCrl:CD-I*OCR) BR mice. Thisstudywas conductedusingmodificationosftheproceduresuggestedby Heddleetal.(1983). CHV StudyNo.: 17403-0-455 7 CORNINGHazleton 8.0 MATERIALS Adult male and femalemice,strainCrl:CD-I*(ICR)BF, were purchasedfrom Charles River LaboratoriesP,ortageNH. This healthy,random bred strainwas selectedto maximize geneticheterogeneityand atthesame time assureaccessto a common source. The protocolforthisstudywas approved by the CHV-ACUC priortothe initiationf dosing. Animals were housed seven per cage during quarantine,and housed fiveper cage at randomization.The temperatureand relativheumiditywere maintainedat72 6 'F and 55 15%, respectivelye,xcepton March 10 and 16,1996, forthe second dose rangefindingstudy,when the relativehumidity was recordedas 36.1% and 39.0%, respectivelyA. 12-hourlight/12-houdrarkcyclewas maintained.A connnercialdiet (PudnO CertifieLdaboratoryPellet"s # 5002) and waterwere availablAed libitumfor thedurationof the study.The feedwas analyzedby the manufacturerforconcentrations of specifiedheavy metals,aflatoxinc,hlorinatedhydrocarbons,organophosphates,and specifiednutrients.The waterwas analyzedon a retrospectivbeasisforspecified microorganisms,pesticidesa,llWinity,heavy metals,and halogens. Sanitizedcagingwas used forhousing the animals.Personnelhandlinganimalsor working withintheanimal facilitiweesre requiredtowear suitableprotectivegarments and equipment. Animals were quarantinedforseven days beforebeing placedon study.Animals were randomly assignedto studygroupsand were individuallwyeighed priortodosing.All animalswere dosed based upon theindividualbody weights.Animals were uniquely identifiebdy eartag.Dose or treatmentgroups were identifiebdy cage card/label. At theterminationofthe studyallsurvivinganimalswere euthanizedby C02 inhalation, followedby penetrationofthethorax.Any extraanimalsnot used forthe studywere euthanizedby C02 inhalationf,ollowedby penetrationofthe thorax. 9.0 SOLUBILITY AND STABILITY The testarticleT,-6295, was suppliedas a off-whitemixture ofpowder and flakes.The solubilitoyf thetestarticlweas evaluatedincom oil,0.5% high viscositycarboxymethyl cellulosea,nd acetone:com oil,20%:80%, v:v. Suspensionssuitablefordosingwas not obtainedwith thesevehicles.The testarticlweas crushed ina mortar and pestleand a translucento,ff-whitesuspensionwas obtainedindeionizedwater,thatpassed easily throughan 18G needleaftermixing ina Tissumizerl.Deionizedwaterwas the vehicle of choiceforthisassay.The stabilitoyfthetestmaterialunder the dosingconditionsof thisassayistheresponsibilitoyf thesponsor. CHV StudyNo.: 17403-0-455 8 CORNINGHazleton 10.0 DOSE SELECTIONSTUDY I 10.1 Dose Selection Dose levelsof500,1630,2750,3880 and 5000 mg/kg were administerebdy oral gavageforthefirsdtoserangefmdingstudy. 10.2 DosingInforination The animalsusedinthefirsdtoserangefindinagssaywere dosedon March 13, 1996. The weightrangeoftheanimalsusedinthefirsdtoserangefindingassay was 30.8-40.3and 24.4-30.8grams,forthemales and females,respectively. Dosing solutionwserepreparedjustpriortodosingandwere preparedby making a 250 mghnl stockforthehighdose(5000mg/kg ).Thiswas preparedby adding deionizedwater(Lot# 19,preparedatCHV) to3.5025g ofT-6295 up toa volume of14.0ml,resultinigna cloudycrearncoloredsuspension.Thiswas mixed for=2 minuteswitha TissumizerO.Dilutionosfthisstockwereprepared forthe3880, 2750,1630 and500 mg/kg doselevelsA.lldosingstockswere placedon magneticstiprlatesduringthedilutioannd thedosingprocedure. Dosingwas achievedusinga 20.0ml/kg dosingvolume. Allanimalswere nine weeks and two daysoldatthetimeofdosing.An outlinoefthedosingscheme is foundinthefollowintgable. DosingScheme forDose RangefindingAssay I Treatment T-6295 500 mg/kg 1630 mg/kg 2750 mg/kg 3880 mg/kg 5000 mg/kg Male Female 3 3 3 3 3 3 3 3 3 3 A totalof30 animalswas usedinthisassay.Alldosesgivenwereon an acute (one-timoenly)basis. 10.3 Resultsand Interpretation Allanimalswere examinedafterdosingand dailythroughoutthedurationofthe study(threedays)fortoxiceffectasnd/ormortalitieAsl.lanimalsappeared CHV StudyNo.: 17403-0-455 9 CORNINGHazleton normailmmediataefltyedrosinTgh.etoxicoibtsyervatairoensshowninthe followintgable: Animal ObservationsforToxicityforDose Rangefmding AssayI -TimeAfter Dose Dosing Level (hours)(mg/kg) 500 Normal Observations 1630 Slightlhyypoactive 1 2750 Hypoactivewithdyspnea 3880 Hypoactivewithdyspnea 5000 Hypoactivewithdyspnea,lacrimatioanndtremors 500 Normal 1630 Hypoactive =18 2750 Allanimalsfound dead,exceptfemale# 6978,which was hypoactivec,oldtothetouch,paleand had tremors 3880 Allanimalsfounddead 5000 Allanimalsfounddead 500 Normal =42 1630 Males 4 6963 and 6967,and female# 6984 founddead,all otheranimalsappearednormal 2750 Female # 6978 found dead 500 Normal =72 1630 Allremaininganimalsappearednormal The mortalitdyataforthisassayaresummarizedinthefollowingtable: Summar-y ofMortalitieWsithin3 Days inMice Dosed AcutelywithT-6295 forDose RangefindingAssayI Treatment 500 mg/kg 1630 mg/kg 2750 mg/kg 3880 mg/kg 5000 mg/kg Male Female 0/3 0/3 2/3 1/3 3/3 3/3 3/3 3/3 3/3 3/3 CHV StudyNo.: 17403-0455 10 CORNINGHazleton 10.4 Conclusion Based on theseresultst,hemaximum tolerateddose could notbe determined. 11.0 DOSE PANGEFINDING STLJDY II 11.1 Dose Selection Dose levelsof 700, 1000 and 1300 mg/kg were administeredby oralgavage for thesecond dose rangefindingstudy. 11.2 Dosing Information The animalsused inthesecond dose rangefindingassaywere dosed on March 16, 1996. The weightrangeoftheanimalsused inthesecond dose rangefinding assaywas 32.7- 39.8 and 25.0- 31.3grams,forthemales and females,respectively.Dosing solutionwsere preparedjustpriortodosingand were preparedby making a 65.0mg/rffsltockforthehighdose(1300 mg/kg ). Thiswas prepared by addingdeionizedwater(Lot# 19,preparedatCHV) to780.0mg ofT-6295 up to a volume of 12.0ml,resultinigna cloudycream coloredsuspension.Dilutions ofthisstockwere preparedforthe1000,and 700 mg/kg dose levels.Alldos'mg' stockswere placedon magneticstiprlatesduringthedilutionand thedosing procedure. Dosing was achievedusinga 20.0ml/kg dosingvolume. Allanimalswere nine weeks and fivedays oldatthetimeofdosing.An outlineof thedosingscheme is found inthe followingtable. Dosing Scheme forDose RangefindingAssay Il Treatment T-6295 700 mg/kg 1000 mg/kg 1300 mg/kg Male Female 3 3 3 3 3 3 A totalof 18 animalswas used inthisassay.Alldosesgivenwere on an acute (one-timeonly)basis. CHV StudyNo.: 17403-0-455 11 CORNINGHazleton 11.3 Resultsand Interpretation Allanimalswereexamined afterdosingand dailythroughouttheduratioonfthe study(thredeays)fortoxiceffectasnd/ormortalitieAsl.lanimalsappeared normalimmediatelyafterdosing.The toxicitoybservationasreshown inthe followingtable: Animal ObservationsforToxicityforDose RangefindingAssay 11 Time After Dose Dosing Level (hours)(mg/kg) Observations 700 Normal =1 1000 Slightlhyypoactive 1300 Hypoactiveand hunched 700 Normal --24 1000 Normal 1300 Male # 7259 founddead,allremainingappeared hypoactiveand hunched 700 Normal =43 1000 Female # 7255 founddead,allothersappearednormal 1300 Allremainingmalesandfemales#'s7263 and 7261 found dead,one survivingfemaleappearednormal 700 Normal --70 1000 Allremainingappearednormal 1300 Allremainingappearednormal The mortalitdyataforthisassayaresummarizedinthefollowingtable: Summary ofMortalitieWsithin3 Days inMice Dosed AcutelywithT-6295forDose RangefindingAssay II Treatment 700 mg/kg 1000 mg/kg 1300 mg/kg Male Female 0/3 0/3 0/3 1/3 3/3 2/3 CHV Study No.: 17403-0455 12 CORNINGHazleton 11.4 Conclusion Based on theseresultst,hemaximum tolerateddosewas determinedtobe 950 mg/kg. 12.0 L@UCRONUCLEUS STUDY 12.1 Dose Selection Based on resultfsrom thedose rangefindinsgtudy,doselevelsof237.5,475,and 950 mg/kg were selectefdortestinignthisstudy. 12.2 MicronucieusAssay DosingInformation The animalsused inthemicronucleusassaywere dosedon March 19,1996. Cyclophosphamide(CAS # 6055-19-2;Sigma,Lot 4 44H0486), thepositive controlw,as solubilizeidnsterildeeionizedwater(Lot# 19,preparedatCHV) and was administerebdy oralgavageat80.0mg/kg. The vehiclecontrol, deionizewdater(Lot# 19,preparedatCHV), was administerecdoncurrentlwyith thetestarticlaeta volume of20.0ml/kg.The weightrangeoftheanimalsusedin themicronucleusassaywas 29.9-37.0and 23.1-29.2grams forthemales and females,respectivelyT.he dosingsolutionfsortheassaywere preparedby making a 47.5mg/ml stockforthehighdose(950mg/kg ).Thiswas preparedby addingthevehicleto2.3751g ofgroundup T-6295 up toa volume of50.0ml, stirrinwgitha spatuland a cloudycream coloredsuspensionwas obtained. Dilutionosf this'stowcekre preparedfortheremainingdoselevelsA.lldosing stockswere placedon magneticstiprlatesduringthedilutioannd thedosing procedure.A secondgroup ofanimals(designateSdecondaryDose Ciroup)was alsoassignedtothestudyand was dosedwiththehighdoseofthetestarticle. These animalswere onlyusedintheassayasreplacementsforany which diedin theprimarydosegroup. Ten animals(fivemalesand fivefemales)wererandomlyassignedtoeachdose/harvestimegroup.Vehicleand positivceontrolgroups,euthanized approximatel2y4 hoursafterdosing,were includedintheassay.The animals dosedwiththetestarticlweere euthanizeadpproximatel2y4,48 and 72 hours afterdosingforextractioonfthebone marrow. An outlineofthedosingscheme isfoundinthefollowingtable: CHV StudyNo.: 17403-0-455 13 CORNINGHazleton DosinSgchemeforMicronucleAussay T-62915 Treatment 237.5mg/kg NumberofAnimalAsssigned PrimaryDose Groups SecondaryDose 24 Hr 48Hr 72 Hr Group MF MF MF MF 55 55 55 475.0 mg/kg 55 55 55 950.0 mg/kg 55 55 55 55 Vehicle Control.,deionized water, 20.0 ml/kg 5 5 - - - - - PositivCeontrol,Cyclophosphamide,80.0mg/kg 5 5 a The animalsassignedtothesecondarydosegroupswere dosedand were onlyusedto replaceanimalswhich diedintheprimarydosegroupatthehighdoselevel.Allextra animalsnotusedas replacementwsere euthanizedatthecompletionofthetrial. A totalof 120animalswas usedinthisassay.The ageoftheanimalsatthetimeof dosingwas nineweeks and one day. Volumes dosedwere 20.0ml/kg (exceptpositivceontrola)nd werebasedupon individuaalnimalweights.Volumes dosedtopositivceontrowlere 10.0ml/kg. 13.0 BONE MARROW HARVEST, SLIDE PREPARATION AND ANALYSIS At theappropriathearvesttime,theanimalswere euthanizebdy C02inhalationfollowed by penetratioonfthethorax.The adheringsofttissueand epiphysesofbothfemorawere removed. The marrow was flushedfrom thebone andtransferretdocentrifugteubes containin3g-5 ml bovineserum(onetubeforeachanimal).Followingcentrifugatiton pellethetissuet,hesupernatanwtas removed by aspiratioannd portionsofthepellet were spreadon slideasnd airdried.The slidewsere fixedinmethanol,and stainedin May-Grunwald solutiofnollowedby Giemsa (Schmid,1975).The air-driesdlidewsere coverslippeudsingDepex* mountingmedium. The slideswere codedforanalysisa,nd scoredformicronucleaindthepolychromatic erythrocyt(ePCE) tononnochromaticerythrocyt(eNCE) cellratioS.tandardformswere usedto recordthesedata.One thousandPCEs peranimalwere scored.The frequencyof micronucleatecdellswas expressedaspercentmicronucleatecdellsbasedon thetotal PCEs presentinthescoredopticfield.The normalfrequencyofmicronucleinthis Crl:CD-III(ICBRR) straiinsabout0.0-0.4%. CHV StudyNo.: 17403-0-455 14 CORNINGHazleton ThefrequenocfPyCEsversuNsCEswasdeterminbeysdcoritnhgenumbeorfPCEsand NCEs observedintheopticfieldwshilescoringthefirs1t000erythrocytes. 14.0 EVALUATION CRITERIA 14.1 General T'hecriterfioartheidentificatoifomnicronuclewiere thoseofSchmid (1976). Micronucleiwere darklystainedandgenerallryound,althoughalmond and ringshapedmicronucleoiccasionalloyccurred.Micronucleihad sharpborders and were generallybetween 1/20and 115thesizeofthePCE. The unitofscoring was themicronucleatecdelln,otthemicronucleust;hustheoccasionaclellwith more thanone micronucleuswas countedasone micronucleatePdCE, nottwo (or more)micronucleiT.he staininpgrocedurepermittetdhedifferentiatbiyoncolor ofPCEs andNCEs (bluish-graenydred,respectively). 14.2 Data Presentatioannd Interpretation Data aresummarized by sexand dosegroupsforthedifferenttimepoints. Individuaalnimaldataarealsopresented.The analysisofthesedatawas performedusingan analysiosfvariance(Winer,1971)on eitheurntransformed (when varianceasrehomogeneous)andranktransforme(dwhen variancesare heterogeneousp)roportionosf cellwsithmicronuclepieranimal.Iftheanalysis ofvariancewas significa(npt<0.05)a,Dunnett'ts-tes(tDunnet@1955;1964)was usedtodeterminewhich dosegroups,ifany,were significantdliyfferenftrom the negativecontrol.Analyseswere performedseparatelfyoreachharvesttimeand sexcombination.The criterifaordetermininag positivreesponseinvolveda statisticaslilgynificadnotse-relateidncreaseinmicronucleatePdCES, orthe detectioonfa reproduciblaend statisticaslilgynificapnotsitivreesponseforat leastonedoselevel.A testarticltehatinducedneithera statisticaslilgynificant doseresponsenora statisticaslilgynificaanntd reproducibliencreaseatone dose levelwas considerednegative.Ineithecrase,thefinaldecisionwas basedon scientifjiucdgment. 15.0 RESULTS AND INTERPRETATION Allanimalswere observedimmediatelyafterdosingand periodicaltlhyroughouthe duratioonftheassayfortoxicsymptoms and/ormortalitieAsl.lanimalsinthevehicle and positivceontrolgroupsappearednormal afterdosingand remainedhealthyuntilthe appropriathearvestimes.Alltestarticldeosedgroupsappearednormalimmediatelyand CHV StudyNo.: 17403-0-455 15 CORNINGHazleton aboutonehouraftedrosing.Thetoxicitayndmortaliotbyservatioonfsthetestarticle dosed animals areshown inthe followingtable: Animal Observations forToxicity/Mortality Time After Dose Dosing Level Observations (hours) (mg/kg) 237.5 Normal 475.0 Normal 950.0 Males 4 7304 (24 hour harvest)7,314 (48 hour harvest)a,nd 7305 z22 (secondary)a;nd females# 7369 (48hour harvest)a,ll72 hour harvest,and 7332 (secondary)were found dead. Two males went intoconvulsionsupon opening oftheircages but recoveredaftera few minutes. All othersremainingappearednormal. 237.5 Normal 475.0 Female # 7343 (48 hour harvest)found dead,allremaining appeared normal =46 950.0 Males # 7300 (48 hour harvest)and 7286 (secondary)a,nd females # 7368 (48 hour harvest)and 7379 (secondary)were found dead. All remaining appearednormal. 237.5 Normal 475.0 All remaining appearednormal =71 950.0 Male # 7282 (secondary)found dead,allremaining appeared normal The testarticleT,-6295, induced no significanitncreasesinmicronucleated polychromaticerythrocyteosver the levelsobservedinthevehiclecontrolsineithersex or atany ofthe harvesttimes. Bone mar-rowtoxicitywas manifestedby the significant reductioninthe PCE/NCE ratiosof the48 and 72 hour males from the237.5 and 950 mg/kg dose groups,72 hour males from the475 mg/kg dose group,48 hour females from the475 mg/kg dose group, 72 hour females from the950 mg/kg dose group,and the positivecontrolfemales.The positivecontrol,cyclophosphamide,induced significant increasesinmicronucleatedPCEs in bothsexes ascompared tothevehiclecontrolsw,ith means and standarderrorsof 3.36% 0.97% and 4.52% 0.72% forthemales and females,respectivelyT.he datasummarized by dose group arepresentedinTable I and individualanimaldataare found inTables2 through7. Historicaclontroldataare presentedin Table 8. CHV Study No.: 17403-0455 16 CORNINGHazleton 16.0 CONCLUSION The testmaterialT,-6295,didnotinducea significainntcreasienmicronucleinbone marrow polychromatiecrythrocyteusndertheconditionosfthisassayand isconsidered negativeinthemouse micronucleusassay. 17.0 REFERENCES Dunnett,C.W.: A multiplecomparisonsprocedureforcomparingseveraltreatmentwsith a control.J.Am. StatisAts.soc.,5Q:1096-1121,1955. Dunnett,C.W.: New tablefsormultiplceomparisonswitha controlB.iometrics2,,Q:482491,1964. Heddle,J.A.,Hite,M., Kirkhmt,B.,Larsen,K.,MacGregor,J.T.N,ewell,G.W. and Salamone,M.F.: The inductioonfmicronucleaisa measureofgenotoxicity. MutationRes.,la: 61-118,1983. Schmid,W.: The micronucleutsest.MutationRes.,21:9-15,1975. Schmid,W.: The micronucleutsestforcytogenetiacnalysisC.hemical Mutagens: PrincipleasndMethods forTheirDetectionV,ol.4 (A.Hollaender, ed.).Plenum,pp.31-53,1976. Winer,B.J.:StatistiPcrailncipleisnExperimentaDlesign,McGraw-Hill,New York,Second Edition,1971. 18.0 DEVIATIONS FROM THE SIGNED PROTOCOL I. On March 10 and 16,1996,fortheseconddoserangefindinsgtudy,therelative humiditywas recordedas36.1%*and39.0%,respectivelTyh.isdidnotaffecthe animalsortheintegritoyfthestudy. 2. The actualdose levelsachievedforthedoserangefindinsgtudywere 3880 and 1630 mg/kg and not3875 and 1625 mg/kg,respectivelyT.hese dose levelsare veryclosetothedose levels(<0.05% differencem)entionedintheprotocol amendment and hencetherewas no impacton theintegritoyfthestudy. CHV StudyNo.: 17403-0455 17 CORNINGHazleton 19.0 EXPERIMENT DATA TABLES CHV StudyNo.: 17403-0-455 18 CORNINGHazleton SPONSOR: 3M MICRONUCLEUS TABLE I DATA SUMMARY TABLE TEST ARTICLE: T-6295 ASSAY: 17403 TREATMENT DOSE HARVEST TIME (HR) % MICRONUCLEATED PCEs MEAN OF 1000PER ANIMAL S.E. MALES FEMALES TOTAL CONTROLS VEFUCLE POSITIVE Water 24 hr CP 90.0mg/kg 24 hr 0.02 0.02 3.36 0.97* 0.14 0.06 4.52 0.720 0.08 0.04 3.94 0.60* RATIO PCE:NCE MEAN S.E. MALES FEMALES 0.79 0.08 0.59:k0.05 0.77 0.09 0.55 0.03** TEST ARTICLE 237.5mg/kg 24 hr 48 hr 72 hr 475.Omg&g 24 hr 49 hr 72 hr 950.0mg/kg 24 hr 48 hr 72 hr 0.06:k0.04 0.161:0.07 0.04* 0.02 0.14:k0.05 0.04 0.02 0.14 0.05 0.02 0.02 0.07 0.051 0.09 0.04 0.10 0.03 0.02zk0.02 0.12 :k0.05 0.04:L0.04 0.05 0.03' 0.08 0.04 0.02 0.02 0.03 0.031 0.02 0.02 0.08* 0.02 0.09:k0.04 0.09:k0.03 0.09 0.03 0.04 0.02 0.11 0.03 0.02 0.01 0.06 0.03 0.05 0.02 0.57:k0.11 0.52* 0.10 0.48:k0.04** 0.80:10.10 0.39:E0.110* 0.42* 0.14 0.76:b0.11 0.71 0.05 0.29:k0.06*0 0.56 0.13 0.59:k0.08 0.37* 0.0710* 0.40 0.12 0.59 0.08 0.54 0.08'** 0.44 0.112 0.17 0.05** 0.17 0.050* 1 Significantglryeaterthanthecorrcspondinvgehiclecontrolp,<0.05. ** Significantlleyssthanthecorrespondinvgehiclecontrolp,<0.05 Out offouranimals Out of threeanimals CP Cyclophosphiunide CHV StudyNo.: 17403-0-455 19 CORNINGHazleton TABLE2 MICRONUCLEUS TEST -INDIVIDUALANIMAL DATA SPONSOR: 3M TEST ARTICLE: T-6295 ASSAY NO.: 17403 TREATMENT 24 HOUR HARVEST MALE ANIMAL NUM13ER # MN RATIO PCEs/ 1000 PCEs PCE:NCE VEHICLE CONTROL POSITIVE CONTROL TEST ARTICLE Water CP 90.0mglkg 237.5mg/kg 475.0mg/kg 950.0mglkg 7283 0 0.66 7295 1 0.64 7292 0 0.68 7297 0 1.00 7303 0 0.99 7266 34 0.59 7274 3 0.64 7277 38 0.42 7294 29 0.53 7313 64 0.72 7265 0 0.71 7281 2 0.58 7291 0 0.91 7296 1 0.34 7321 0 0.30 7272 1 1.16 7294 2 0.56 7293 0 0.84 7307 3 0.73 7320 1 0.53 72670 0 0.53 7273 0 0.68 7297 0 1.02 7295 1 0.33 7319 0 0.26 Secondarydose groupanimal CP = Cyclophosphamide MN Micronuelcus PCE Polychromaticerythrocyte # MN PCEs = MicronucleatePdCEs NCE = Norinochromatiecrythrocyte CHV StudyNo.: 17403-0455 20 CORNINGHazleton TABLE3 MICRONUCLEUS TEST -RNDIVIDUAL ANIMAL DATA SPONSOR: 3M TEST ARTICLE: T-6295 ASSAY NO.: 17403 TREATMENT 24 HOUR HARVEST FEMALE ANIMAL NUMBER # MN RATIO PCEs/ PCE:NCE 1000 PCEs VE3UCLE CONTROL POSMVE CONTROL TEST ARTICLE Water CP 80.0mgtkg 237.5mg/kg 475.0mglkg 950.0mgtkg 7324 0 0.56 7327 2 0.73 7336 2 0.86 7350 3 0.66 7360 0 1.04 7329 61 0.54 7330 26 0.53 7334 63 0.66 7359 36 0.52 7366 40 0.49 7349 2 0.67 7355 1 0.17 7363 0 0.71 7370 1 0.43 7376 1 0.63 7337 0 0.89 7347 0 0.59 7362 2 0.58 7364 0 0.43 7377 0 0.46 7325 1 0.63 7335 0 0.86 7340 0 0.49 7361 0 0.41 7365 0 0.55 CP Cyclophosphamide MN Micronucleus PCE Polychromaticerythrocyte # MN PCEs = MicronucleatePdCEs NCE = Normochromaticcrythrocyte CHV StudyNo.: 17403-0455 21 CORNINGHazleton MICRONUCLEUS SPONSOR: 3M TEST ARTICLE: T-6295 ASSAY NO.: 17403 TREATMENT 49 HOUR HARVEST TABLE4 TEST -INDIVIDUAL ANIMAL DATA ANIMAL NUMBER # NIN RATIO PCEs/ PCE-NCE 1000 PCEs MALE TEST ARTICLE 237.5mglkg 475.0mg/kg 950.0mg/kg 7279 1 0.35 7299 4 0.45 7309 2 0.60 7312 1 0.47 7322 0 0.55 7270 0 0.65 7306 1 0.66 7317 0 0.69 7319 1 0.62 7323 0 0.92 7269 0 0.57 7271 0 0.32 7280** 2 0.71 7301 1 0.56 7314* Animalfounddead ** Secondarydosegmup animal MN Micronucleus PCE Polychromatiecrythrocyte # MN PCEs = MicronucleatePdCEs NCE = Normochromaticerythrocyte CHV StudyNo.: 17403-0-455 22 CORNINGHazleton TABLE5 MICRONUCLEUS TEST -INDIVIDUAL ANIMAL DATA SPONSOR: 3M TEST ARTICLE: T-6295 ASSAY NO.: 17403 TREATMENT 48 HOUR HARVEST. FEMALE ANIMAL NUMBER # MN RATIO PcEs/ PCE:NCE 1000 PCEs TEST ARTICLE 237.5mgtkg 475.0mglkg 950.0mg/kg 7326 1 0.79 7352 0 0.96 7353 0 0.77 7372 0 0.43 7393 0 1.04 7329 0 0.59 7341 1 0.31 7343* 7356 1 0.29 7374 0 0.30 7339 1 0.41 7358 0 0.64 73680 73690 7373 0 0.28 Animal founddead MN Micronucieus PCE Polychromaticerythrocyte # MN PCEs = MicronucleatePdCEs NCE = Noffnochromatiecrythrocyte CHV StudyNo.: 17403-0-455 23 CORNINGHazieton MICRONUCLEUS SPONSOR: 3M TEST ARTICLE: T-6295 ASSAY NO.: 17403 TREATMENT 72 HOUR HARVEST TABLE6 TEST - INDIVIDUAL ANIMAL DATA ANIMAL NUMBER # MN PCEs/ RATIO . PCE:NCE 1000 PCEs MALE TEST ARTICLE 237.5mg/kg 475.0mg/kg 950.0mgtkg 7264 1 0.32 7289 1 0.33 7299 0 0.72 7310 0 0.52 7315 0 0.07 7275 1 0.25 7278 2 0.18 7290 1 0.15 7302 3 OAO 7316 0 0.47 7269 0 0.26 7276 2 0.11 7299 0 0.31 7309 1 0.09 7311 1 0.10 MN Micronucle@s PCE Polychromatiecrythrocyte # MN PCEs - MicronucleatePdCEs NCE = Nonnochromaticerythrocyte CHV Study No.: 17403-0-455 24 CORNINGHazleton MICRONUCLEUS SPONSOR: 3M TABLE7 TEST -INDIVIDUAL ANIMAL DATA TEST ARTICLE: T-6295 ASSAY NO.: 17403 TREATNffi'NT 72 HOUR HARVEST FEMALE ANIMAL NUMBER # ?JN PCEs/ RATIO 1000 PCEs PCE:NCE TEST ARTTCLE 237.5mg/kg 415.0mg/kg 950.0mg/kg 7331 1 0.28 7348 3 0.07 7351 1 0.92 7378 1 0.40 7390 0 0.46 7339 0 0.97 7346 1 0.30 7357 2 0.38 7371 0 0.25 737S 1 0.19 7333* 0 0.10 7345 0 0.11 73670 1 0.34 7391* 0 0.11 7382 0 0.18 Secondarydose groupanimal MN Micronucieus PCE Polychromatiecrythrocyte 4 MN PCEs = MicronucientoPdCEs NCE = NorTnochromatiecrythrocyte CHV StudyNo.: 17403-0455 25 CORNINGHazleton TABLES MOUSE MICRONUCLEUS HISTORICAL CONTROL DATA 7/95THROUGH 12/95 POOLED VEFUCLE CONTROLS MN MAX AVG N POSITIVE CONTROLS Cyclophosphamide9,0.0mg/kg MIN MAX AVG N % MICRONUCLEATED PCEs PER 1000PCE MEAN OF 1000PER ANIMAL* S.E. MALES FEMALES TOTAL 0.00 0.22 0.087* 0.007 47 0.00 024 0.091* 0.008 47 0.01 0.17 0.094* 0.005 47 RATIO PCE:NCE MEAN S.E. MALES FEMALES 0.31 0.85 0.550* 0.021 47 014 1.03 0.597 0.025 47 2.00 5.68 3.692ii0.240 19 1.50 636 3.170:k0.245 19 2.41 5.38 3.426* 0.194 19 0.41 0.72 0.577* 0.020 19 0.40 0.79 0.599* 0.026 19 PCE Polychromaticrydirocyte NCE Norinochromaticryffirocyte CHV Study No.: 17403-0-455 26 CHV STUDY NO. PROTOCOL NO. 455. EDITION 17 CORNINGHazleton iN vivo mouse kncRONUCLEUS ASSAY Corning Hazleton Inc. (CHV) will conduct this study in compliance with Good Laboratory Practice (GLP) Regulations. This protocol, critical phase(s) of the work in progress and the final report will be subject to audit by Quality Assurance in accordance with SOPs at Corining Hazleton Inc. The study will be conducted by CHV at 9200 Leesburg Pike, Vienna, Virginia 22182. PART 1. SPONSOR IM RM&TION AND APPROVALS I. SPONSOR IDENTIFICATION Company Name: 319 Address: 45f-. FUJ, mfq II. TEST ARTICLE IDENTIFICATION: III. TEST ARTICLE ANALYSIS Determination of the test article stability and the test article characteristics as defined in the GLP regulations is the responsibility of the Sponsor. IV. NOTIFICATION OF REGULATORY SUBMISSION In order to comply with the GLP regulations, consulting laboratories must be notified if all or pari of a study is intended for regulatory submission. CHV maintains a master schedule of studies which fall under regulatory review. Please indicate which agency, if any, might receive the results of this study: Undetermined MAFF FDA EPA-TSCA MOHW OECD OTHER EPA-FIFR& 4/95 1 of 10 PROTOCOL NO. 455, EDITION 17 V. STUDY DATES Proposed Experimental Start Date: Proposed Experimental Termination Date: VI. APPROVAL nF STUDY PROTOCOL Study Director: Remalatha Murli, Ph.D. Sponsor's Authorized Representative: CORNINGHazleton Date: Date: 7 Z,/fs-- 4/95 2 of 10 PROTOCOL NO. 455, EDITION 17 CORNINGHaAeton PART 2 - STUDY PROTOCOL IN VIVO HOUSE HICRONUCLEUS ASSAY I. OBJECTIVE The objective of this study is to evaluate a test article for clastogenic activity and disruption of the mitotic apparatus in polychromatic erythrocyte stem cells in mouse bone marrow in vivo. II. DEFMTIONS Hicronucleus: a small chromatin body, consisting of entire chromosome(s) and/or of acentric chromosome fragment(s), which lags behind at mitotic anaphase. After telophase, these chromosome(s) and fragment(s) may not be included in the daughter nuclei, and may form single or multiple micronuclei in the cytoplasm. III. ILATIONALE The micronucleus test can serve as a rapid screen for clastogenic agents and test articles which interfere with normal mitotic cell division (Schmid, 1975; Heddle et al., 1983). Micronuclei are formed from chromosomes or chromosome fragments left behind during anaphase and can be scored during interphase because they persist (Schmid, 1975). In this assay, polychromatic erythrocytes (PCES) in the bone marrow are -scored for the presence of micranuclei. During maturation from erythroblast to erythrocyte the nucleus is extruded, while micronuclei, if present, remain in the cytoplasm. Detection of micronuclei in non-nucleated cells is thus facilitated, and time involved in searching for metaphase spreads in treated cell populations is eliminated. Test articles affecting spindle-fiber function or formation as well as clastogenic agents can be detected through micronucleus induction (Schmid, 1975). IV. MATERIALS A. Animals Young adult male and female mice of the ICR strain, 810 weeks old at the time of dosing, will be purchased from Charles River Laboratories, Inc., or Harlan Sprague-Dawley, Inc. This strain has been selected to 4/95 3 of 10 PROTOCOL NO. 455, EDITION 17 CORNINGHa7leton maximize genetic heterogeneity and at the same time ensure access to a common source. B. Control Articles Cyclophosphamide (CP, 80 mg/kg; dosing volume of 10 ml/kg) will be used as the positive control article and will be administered by oral gavage. The vehicle control article will consist of the solvent or vehicle used for the test article and will be administered by the same route as, and concurrently with, the test article and in amounts equal to the maximum volumes administered to the experimental animals. The dosing volume will not exceed 20 ml/kg for oral gavage and IP administrations. The vehicles generally used in the assay are water, 0.51 aqueous carboxymethylcellulose solution, or corn oil. V. EXPERIMENTAL DESIGN A. Animal Husbandry All applicable CHV SOPs will be followed. Animals will be isolated by sex. Animals will be housed up to seven per cage during quarantine, and will be housed up to five prior to experiment initiation. Animals are housed under the following climatic conditions: temperature, 72*F * 60F; humidity, 551 t 15Z; light cycle, 12 hours light/dark. A commercial diet (Purinat Certified Laboratory Chowe 15002) and tap water will be available ad libitum. The feed is analyzed by the manufacturer for concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosp4ates, and specified nutrients. The water is analyzed biannually on a retrospective basis for specified microorganisms, pesticides, heavy metals, alkalinity, and halogens. Animals will be quarantined for at least 7 days before being placed on study. Animals will be assigned to study groups at random according to Coning Hazleton Standard Operating Procedures. Animals will be weighed prior to dosing. They will be dosed based upon the individual animal weights. Animals will be uniquely identified by ear tag. Treatment groups will be identified by cage label/card. 4/95 4 of 10 PROTOCOL NO. 455, EDITION 17 CORNINGHa2leton Sanitary cages will be used. Personnel handling animals or working within the animal facilities will be required to wear suitable protective garments and equipment. B. Dose Selection The high dose generally will be selected as 802 of the maximum tolerated dose. The high dose should produce some indication of toxicity (e.g.,death, depression of ratio of PCEs to normochromatic erythrocytes (NCEs). One-half and one-quarter of this high dose will normlly be used as the intermediateand low dose levels. respectively. Use of a high dose increases the likelihood that a weak clastogenwill be detected, and is therefore recommended. If no appropriate range finding data are available, a range finding study can be performed. The top dose tested in the dose rangefinding study will be 5000 mglkg. The dose levels tested will be issued as an amendment. DOSE RANGEFINDING STUDY The dose rangefinding study will be conducted using five treatment groups. Each of the five groups will consist of 3 male and 3 female mice. Group Designation and Treatment Regimens Group No. Number of Mice Male Female Route Duration (Days) 1 3 3 PO 3 2 3 3 PO 3 3 3 3 PO 3 4 3 3 PO 3 5 3 3 PO 3 4/95 5 of 10 PROTOCOL NO. 455, EDITION 17 COFMINGHazleton The route of administration will be oral gavage. In the event that test article characteristics preclude oral gavage. IP injection will be employed. These routes of administration have been selected because they are the most common routes of administration for this test procedure. The dosing volume will not exceed 20 ml/kg for oral gavage and IP administrations. Other routes of administration that may be used are intravenous, intramuscular, sub-cutaneous administrations or by feed. The test material will generally be solubilized in one of the following solvents: water, 0.9X saline, 0.51 aqueous carboxymethylcellulose solution, or com oil. All animals will be dosed based upon individual body weights. Dose levels will be assigned by a protocol amendment. Body weights will be taken prior to dosing. Dosing formulation will be prepared just prior to dosing. Dosing solutions will be prepared and held at ambient temperatures until dosing (0-2 hours). All animals will be euthanized 3 days after receiving a single dose. The animals will be observed daily for toxic signs and mortality for the duration of the study. Animals will be euthanized by C02 inhalation followed by penetration of the thorax. The daily observations of toxic symptoms and/or mortalities data will be used to estimte* the Maximum Tolerated Dose (MTD). Doses will then be assigned for the subsequent cytogenetics assay. MICRONUCLEUS STUDY C. Dosing Schedule and Route of Administration Normlly an acute dosing regimen (single administration) will be used (see Table below). Harvest will be approxim4tely 24, 48, 72 hours after administration of the test article, and at approximately 24 hours after administration of the control articles. A total of 110 animals will be used. Equal numbers of males and females will be used at each treatment group. An additional group of animals consisting of 3-10 males and 3-10 females may be dosed as a secondary dose group with the high dose of the test material. This group will be dosed if toxicity is expected at the high dose and the animals in this group will only be used as replacements for any which die prior to euthanasia. The use of the secondary dose group will be determined by the study director. Freshly prepared solutions will be 4/95 6 ot 10 PROTOCOL'NO. 455, EDITION 17 CORNINGHazleton employed. The animals will be observed daily for toxic signs and mortality. NUMBER OF ANIMALS USED FOR HICRONUCLEUS ASSAY Group No. Treatment Harvest Times After Treatment (Males and Females) 24 Hours 48 Hours 72 Hours Total 1 Positive Control 5+5 2 Vehicle Control 5+5 ----- ----- 5+ 5 5+ 5 3 Low Dose 5+5 5+ 5 5+ 5 15 + 15 4 Medium Dose 5+5 5+ 5 5+ 5 15 + 15 5 High Dose 5+5 5+5 5+ 5 15 + 15 TOTAL 25 + 25 15 + 15 15 + 15 55 + 55 The route of administration will be oral gavage. In the event that test article characteristics preclude oral gavage, IP injection will be employed. The dosing volume will not exceed 20 ml/kg. These routes of administration have been selected because they are the most common routes of administration for this test procedure. Other routes of administration that may be used are intravenous, intramuscular, sub-cutaneous administrations or by feed. D. Extraction of Bone Marrow Euthanasia will be with C02, followed by penetration of the thorax, and hind limb bones will be removed for marrow extraction. The marrow will be flushed from the bone and transferred to centrifuge tubes containing 3-5 ml bovine serum (one tube for each animal). E. Preparation of Slides Following centrifugation to pellet the tissue, the supernatant will be removed by aspiration and portions of the pellet will be spread on slides and air-dried. The slides will then be fixed in methanol, stained in May-Grunwald Solution and Giemsa, and protected by mounting with coverslips. For control of bias, all slides are coded for analysis. 4/95 7 of 10 PROTOCOL NO. 455, EDITION 17 CORNINGHazleton F. Scoring the Slides An attempt will be made to score one-thousand PCEs per animal. The frequency of micronucleated cells will be expressed as percent micronucleated cells based on the number of PCEs analyzed. The normal background frequency of micronuclei in the ICR mouse strain is around 0.0-0.4Z. The frequency of PCEs versus mature erythrocytes (NCES) will be determined by scoring the number of PCEs and NCEs observed in the optic fields while scoring the first 1000 erythrocytes on the slide. V3:. DATA The criteria for the identification of micronuclei are those of Schmid (1976). Hicronuclei are darkly stained and generally round, although almond and ring-shaped micronuclei occasionally occur. Micronuclei have sharp borders and are generally between 1/20 and 1/5 the size of The PCE. The unit of scoring is the micronucleated cell, not the micronucleus; thus the occasional cell with more than one micronucleus is counted as one micronucleated PCE, not two (or more) micronuclei. The staining procedure permits the differentiation by color of polychromatic and normochromatic erythrocytes (bluish-grey and red, respectively). Data Presentation The data reported will include the number of PCEs scored, the number of micronucleated PCES, the percentage of micronucleated PCES, and the ratio of polychromatic to normochromatic erythrocytes for each experimental animal. Evaluation Criteria The criteria for a positive response is a statistically significant dose-related increase in micronucleated PCES, or the detection of a reproducible and statistically significa'ntpositive response for at least one dose level. A test article that induces neither a statistically significant dose response nor a statistically significant and reproducible increase at one dose level is considered negative. In either case, the final decision is based upon scientific judgement. 4/95 8 of 10 PROTOCOL NO. 455, EDITION 17 CORNINGHazieton VII. TEST INTERPRETATION The analysis of this data will be performed using an analysis of variance (Winer, 1971) on either untransfomed (when variances are homogeneous) or rank transformed (when variances are heterogeneous) proportions of cells with micronuclei per animal. If the-aixalysisof variance is significant (p<0.05), a Dunnett's t-test (Dunnett, 1955; 1964) will be used to determine which dose groups, if any, are significantly different from the negative control. Analyses will be performed separately for each harvest time and sex combination. VIII. REFERENCES Dunnett, C.W.: A multiple comparisons procedure for comparing several treatments with a control. J. Am. Statist. Assoc., 10:1096-1121. 1955. Dunnett, C.W.: New tables for multiple comparisons with a control. Biometrics, 20:482-491, 1964. Heddle, J.A., Hite, M., Kirkhart, B.. Larsen, K., MacGregor, J.T., Newell, G.W. and Salamone, M.F.: The induction of micronuclei as a measure of genotoxicity. Mutation Res., 123:61-118, 1983. Schmid, W.: The micronucleus test. Mutation Res. , 31:9-15, 1975. Schmid, W.: The micronucleus test for cytogenetic analysis. In, Chemical Mutagens: Principles and Methods for Their Detection, Vol. 4 (A. Hollaender, ed.). Plenum, pp. 31-53, 1976. Winer, B.J.: Statistical Principles in Experimental Design, McGraw-Hill, New York, Second Edition, 1971. IX. REPORT FORM&T CHV employs a standard report format for each assay design. The final report will provide the following information. 0 Sponsor identification. 0 Quality Assurance statement. 0 Statement of GLP Compliance. 9 Signature of study director. 0 Test article identification and CHV Study Number. A physical description of the test article and date of receipt will be included in this section. 0 Type of assay and protocol number. 0 Dates of study initiation and completion. 0 Study director and senior technician. 0 Methods. 4195 9 of 10 PROTOCOL NO. 455, EDITION 17 CORNINGHazleton 0 Evaluation criteria. 0 Interpretationof results. 0 Conclusions. 9 References. 0 Test results presented in tabular form. X. CHANGES OR R M SIONS Any changes or revisions of this approved protocol will be documented, signed by the Study Director, dated, and maintained with this protocol. XI. AN3MAL CARE AND USE STATEMENT In the opinion of the Study Director, no alternative testing methods are appropriate. the study does not duplicate any previous work with this material, and the number and species selected are appropriate. This protocol will be reviewed by the CHV-IACUC for compliance with regulatory guidelines concerningthe care and use of animals. If not in compliance, a modificationwill be required. Any changes or revisions of this approved protocol will be sent to the CHV-IACUC for their review. XII. RECORDS TO BE MAINTAIM All raw data, documentation, records, protocols, and the final report generated as a result of this study will be archived in the storage facilities of Coning Hazleton Inc. for at least one year following submission of the final report to the sponsor. After the one year period, the sponsor may elect to have the aforementioned materials retained in the storage facilitiesof Corning Hazleton Inc. for an additional period of time or sent to a storage facility designatedby the sponsor. 4195 10 of 10 AMENDNffiNT TO THE STUDY PROTOCOL STUDY TITLE: IN VIVO MOUSE MICRONUCLEUS ASSAY PROTOCOL NO.: 455,Edition17 STUDY NO.: 17403-0-455 PageIofI Amendment #I Section2,PartV.B. Based on thesolubilityestt,hetestarticlweillbe suspendedinwater forthestudy.Inthedoseselectiosntudy,doselevelsof 500,1625, 2750, 3875, and 5000 mg/kg willbe tested. STUDY DIRECTOR t)n'4U' @L'.4AI^' Hemalatha Murli,Ph.D. Mammalian Cytogenetics Departmentof Geneticand CellulaTroxicology :@ / c /g@ Date AMENDMENT TO THE STUDY PROTOCOL STUDY TITLE: PROTOCOL NO.: flvVIVO MOUSE 455,Edition17 MCRONUCLEUS ASSAY STUDY NO.: 17403-0-455 Page Iof I Amendment 42 Section2,PartV.B. The doseselectiosntudywillbe repeatetdestindgoselevelosf700,1000, and 1300mg/kg. STUDY DIRECTOR Hemalatha Murli, Ph.D. Mammalian Cytogenetics Department of Geneticand CellularToxicology 3115-lq 6 Date AMENDMENT TO THE STUDY PROTOCOL STUDY TITLE: IN VIVO MOUSE MICRONUCLEUS ASSAY PROTOCOL NO.: 455,Edition17 STUDY NO.: 17403-0-455 Page 1of I Amendment #3 Section2,PartV.B. Basedon theresultosfthedoseselectiosntudy,themouse micronucleus assaywillbe conductedtestindgoselevelosf237.5,475,and950 mg/kg. A secondarydosegroupwillbe used. STUDY DIRECTOR A Hemalatha Murli, Ph.D. Mammalian Cytogenetics Department of Geneticand CellularToxicology j q4 D@t