Document jBzNzjpbakkm7ER0YQ76BnmZ2

CORNINGHazleton MUTAGENICITY TEST ON T-6357 IN AN IN VIVO MOUSE MICRONUCLEUS ASSAY FINAL REPORT AUTHO Hemalatha Murli,Ph.D. PERFORMING LABORATO Coming HazletonInc.(CHV) 9200 Leesburg Pike Vienna,Virginia22182 LABORATORY PROJECT IDENTIFICATION CHV Study No.: 17387-0-455 SUBMI D TO 3M 3M Center,Building220-2E-02 St.Paul,Minnesota 55144-1000 STUDY COMPLETION DATE April23, 1996 CHV Study No.: 17387-0-455 lof26 CORNINGHazleton QUALITY ASSURANCE STATEMENT ProjecTtitlel:nVivoMouseMicronucleAusssay ProjectNo.: 20996 Assay No.: 17387 ProtocolNo.: 455 EditionNo.: 17 QualityAssuranceinspectionosfthestudyand reviewof thefinalreportoftheabove referenced projectwere conductedaccordingtotheStandardOperatingProceduresoftheQualityAssurance Unitand accordingtothegeneralrequirementsof theappropriatGeood LaboratoryPractice regulationsF.indingsfrom theinspectionasnd finalreportreviewwere reportedtomanagement and tothestudydirectoorn thefollowingdates: Inspection/Date FindinizRseported Auditor Harvest/02/28/1996 02/28/1996 C. Orantes DraftReport Review/04/18,19/199604/19/1996 C. Orantes FinalReportReview/04/23/1996 04/23/1996 C. Orantes QualityAssuranceUnit Dqte"Relem/eii- CHV Study No.: 17387-0-455 2 CORNINGHazleton STUDY COMPLIANCE AND CERTIFICATION The describedstudywas conductedincompliancewiththeGood LaboratoryPracticeregulations as setforthintheFood and Drug Administratio(nFDA) Title21 oftheU.S.Code ofFederal RegulationsPart58,issuedDecember 22,1978,(effectivJeune20, 1979)withany applicable amendments. There were no significandteviationsfrom theaforementionedregulationosrthe signedprotocolthatwould affecttheintegritoyf thestudyortheinterpretatioofnthetestresults. The raw datahave been reviewedby theStudy Directorw,ho certifitehsattheevaluationof the testarticlaes presentedhereinrepresentasn appropriatceonclusionwithinthecontextofthe studydesignand evaluationcriteria. Alltestand controlresultisnthisreportaresupportedby an experimentaldatarecordand this recordhasbeen reviewedby theStudy Director.Allraw data,documentation,records,protocol and a copy of thefinal'reporgteneratedas a resultof thisstudywillbe archivedinthestorage facilitioefsComing HazletonInc.foratleastone yearfollowingsubmissionofthefinalreportto theSponsor.Aftertheone yearperiod,theSponsormay electtohave theaforementioned materialrsetainedinthestoragefacilitioefsComing HazletonInc.foran additionapleriodof time,or sentto a storagefacilitdyesignatedby theSponsor. SubmittedBy: Study Director: Hemalatha Murli,Ph.D. Mammalian Cytogenetics Departmentof Geneticand CellularToxicology -o-@ f.?3jq6 Study Completion Date CHV StudyNo.: 17387-0-455 3 CORNINGHazleton SUMMARY TABLE OF CONTENTS Page No. ................................................6.......... 1.0 SPONSOR ...........................................7......... 2.0 MATERIAL (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 ExperimentalTemiinationDate 6.0 SUPERVISORY PERSONNEL ...............................7......... 6.1 Study Director 6.2 LaboratorySupervisor 7.0 OBJECTIVE ..........................................7......... 8.0 MATERIALS .........................................8.......... 9.0 SOLUBILITY AND STABILITY: .............................8......... 10.0 DOSE SELECTION STUDY ................................9......... TRIAL I 10.1 Dose Selection 10.2 Dosing Information 10.3 Resultsand Interpretation 10.4 Conclusion CHV StudyNo.: 17387-0455 4 CORNINGHazleton TRIAL II 10.5 Dose Selection 10.6 DosingInformation 10.7 Resultsand Interpretation 10.8 Conclusion 11.0 NHCRONUCLEUSSTUDY ...............................1.3........ 11.1 Dose Selection 11.2 MicronucleusAssay DosingInformation 12.0 BONE MARROW HARVEST, SLIDE PREPARATION AND ANALYSIS ....1.5 13.0 EVALUATION CRITERIA ...............................1.5........ 13.1 General 13.2 Data Presentatioannd Interpretation 14.0 RESULTS AND INTERPRETATION ..........................16......... 15.0 CONCLUSION ......................................1.6 ........ 16.0 REFERENCES ......................................1.7 ........ 17.0 DEVIATIONS FROM THE SIGNED PROTOCOL ..................1.7 ...... 18.0 E32ERIMENT DATA TABLES ............................1.8......... CHV StudyNo.: 17387-0455 5 CORNINGHazleton SUNIMARY MutagenicitTyestonT-6357inanIn VivoMouse MicronucleuAsssay The objectivoefthisinvivoassaywastoevaluatteheabiliotfythetestarticlTe-,6357,toinduce micronucleiinbone marrow polychromaticerythrocyteosf Cri:CD- I2'(ICR)BR mice. In the dose selectionstudy,the testarticlweas solubilizeidndeionizedwater and dosed by oral gavage at200, 400, 610, 810,and 1010 mg/kg inTrialI and no toxicsignswere observed inany of the animals.In TrialII,animalswere dosed by oralgavage at1500,2375, 3250, 4125 and 5000 mg/kg. Six animals(threemales and threefemales)were assignedto each dose group. Animals were observedforthreedays afterdosingfortoxicsignsand/ormortality. Based on the resultsof thedose selectionstudy,the maximum toleratedose was estimatedas 2000 mg/kg. In themicronucleusassay,the testarticlweas solubilizeidndeionizedwater and dosed oralgavage at500, 1000, and 2000 mg/kg. Ten animals (fivemales and fivefemales) were randomly assignedtoeach dosefiiarvesttime group. Vehicle and positivecontrolgroups euthanatizedapproximately24 hours afterdosingwere includedinthe assay.The animals dosed with thetestarticlweere euthanatizedapproximately24, 48 and 72 hours afterdosing for extractionof the bone marrow. The testmaterial,T-6357, did not inducea significanitncreaseinmicronucleiinbone marrow polychromaticerythrocytesunder theconditionsofthisassayand isconsiderednegativeinthe mouse bone marrow micronucleustest. CHV Study No.: 17387-0455 6 CORNINGHazleton MutagenicTietsytonT-635i7naninvivMoouseMicronuclAesussay 1.0 SPONSOR:3M 2.0 MATERIAL (TestArticle) 2.1 Client'IsdentificatiTo-n6:357 2.2 DateReceived:January16,1996 2.3 PhysicalDescriptionC:lear,amber liquid 2.4 GeneticsAssay No.: 17387 3.0 TYPE OF ASSAY: In VivoMouse MicronucleusAssay 4.0 PROTOCOL NO.: 455,Edition17 5.0 STUDY DATES 5.1 InitiatiDoante:January18,1996 5.2 ExperimentalStartDate: February14,1996 5.3 ExperimentaTlerminationDate: March 22,1996 6.0 SUPERVISORY PERSONNEL 6.1 StudyDirector:HemalathaMurli,Ph.D. 6.2 LaboratorySupervisorM:onica Vegarra,B.S. 7.0 OBJECTIVE The objectivoefthisinvivoassaywas toevaluatteheabilitoyfthetestarticlTe-,6357,to inducemicronucleiinbone marrow polychromatiecrythrocyteosfCrl:CD-I*(ICR)BR mice. Thisstudywas conductedusingmodificationosftheproceduressuggestedby Heddleetal.(1983). CHV StudyNo.: 17387-0-455 7 CORNINGHazleton 8.0 MATERJALS Adult male and femalemice,strainCrl:CD-II(ICR)BR, were purchasedfrom Charles River LaboratoriesP,ortageMI. This healthy,random bred strainwas selectedto maximize geneticheterogeneityand atthesame time assureaccesstoa common source. The protocolforthisstudywas approved by the CHV-ACUC priortotheinitiatioonf dosing. Animals were housed seven per cage duringquarantine,and housed fiveat randomization.The temperatureand relativheumiditywere maintainedat726'F and 5515%, respectivelye,xcept on February 19, 1996,forthe dose selectionstudies,when therelativheumidity was recordedat3 1.1% and on the followingdatesforthe micronucleusassay:February 17, 18, 19,24, 24, and 25, 1996 when therelativehumidity was recordedat39.1%, 37.3%, 34.5%, 32.5,34.4%, and 20.8%, respectivelyA. 12-hour light/12-houdrark cyclewas maintained.A commercial diet(PurinO Certified LaboratoryPellets" # 5002) and waterwere availablead libitumforthedurationofthe study. The feedwas analyzedby the manufacturerforconcentrationosf specifiehdeavy metals,aflatoxinc,hlorinatedhydrocarbons,organophosphates,and specifiednutrients. The water was analyzed on a retrospectivbeasisforspecifiedmicroorganisms,pesticides, alkalinityh,eavy metals,and halogens. Sanitizedcagingwas used forhousing the animals.Personnelhandlinganimalsor working withintheanimal facilitiweesre requiredtowear suitableprotectivegarments and equipment. Animals were quarantinedforatleastseven days beforebeing placedon study.Animals were randomly assignedtostudy groups and were individuallwyeighed priortodosing. All animalswere dosed based upon the individuablody weights.Animals were uniquely identifiebdy eartag. Dose or treatmentgroups were identifiebdy cage card/label. At theterminationof the studyallsurvivinganimalswere euthanatizedby C02 followed by penetrationof thethorax. Any extraanimalsnot used forthe studywere used for trainingpurposes. 9.0 SOLUBILITY AND STABILITY: The testarticleT,-6357, was suppliedas a clearamber liquid.The solubilitoyfthetest articlweas evaluatedindeionizedwater. A clearl,ightyellow solutionwas obtainedata concentratioonf about391.6 mg/ml. Deionizedwaterwas the vehicleofchoiceforthis assay. The stabilitoyf thetestmaterialunderthe dosingconditionsof thisassayisthe responsibilitoyfthe sponsor. CHV Study No.: 17387-0-455 8 CORNINGHazleton 10.0 DOSE SELECTION STUDY TRIAL I 10.1 Dose Selection Dose levelsof200, 400, 610,810 and 1010 mg/kg were administeredby oral gavage forthedose selectiosntudy. 10.2 Dosing Information The animalsused inthedose selectionassaywere dosed on February 14,1996. The weightrange oftheanimalsused inthedose rangefmding assaywas 28.536.6and 22.9-27.6grams,forthemales and females,respectivelyD.osing solutionwsere preparedjustpriortodosingand were preparedby making a 100 mg/ml stockforthehighdose (1000mg/kg). Thiswas preparedby adding 10.98mlof deionizedwater(Lot# 19,preparedatCHV) to 1.2081g ofT-6357, resultinigna clearpaleyellowsolutiownitha finalvolume of12.0mi. Dilutions of thisstockwere preparedfortheremainingdose levels. Dosing was achievedusinga 10.0ml/kg dosingvolume. All animalswere nine weeks oldatthetimeof dosing.An outlineofthedosingscheme isfound inthe followingtable. A totalof 30 animalswas used inthisassay. DOSE GROUPS TREATMENT MF --------------------------------------------------- T-6357 200 mg/kg 3 3 400 mg/kg 3 3 610 mg/kg 3 3 8 10 mg/kg 3 3 1010 mg/kg 3 3 ---------------------------------------------------- Alldosesgivenwere on an acute(one-timeonly)basis. CHV StudyNo.: 17387-0-455 9 CORNINGHazleton 10.3 Resultsand Interpretation Allanimalswere examinedaftedrosingand dailythroughouthedurationofthe study(thredeays)fortoxiceffectasnd/ormortalitieIsm.mediatelyfollowing dosingallanimalsappearednormaland remainedhealthyuntiltheend ofthe observatiopneriod. 10.4 Conclusion Based on theseresultst,hemaximum tolerateddose couldnotbe determined. TRIAL II 10.5 Dose Selection Dose levelsof 1500,2375,3250,4125 and 5000 mg/kg were administerebdy oral gavageforthedose selectiosntudy. 10.6 DosingInformation The animalsused inthedose selectioanssaywere dosedon February15,1996. The weightrangeoftheanimalsused inthedose rangefmding assaywas 28.536.4and 23.6-27.5grams,forthemales and females,respectivelDyo.sing solutionwserepreparedjustpriortodosingand were preparedby making a 500 mg/ml stockforthehighdose(5000mg/kg).Thiswas preparedby adding deionizedwater(Lot# 19,preparedatCHV) to7.4993g ofT-6357 up toa volume of 15.0ml,resultinigna clearpaleyellowsolutionD.ilutionosfthis stockwere preparedforthe1500,2375,3250,and 4125 mg/kg doselevels. Dosingwas achievedusinga 10.0mi/kgdosingvolume. Allanimalswere nine weeks and one day oldatthetimeofdosing.An outlinoefthedosingscheme is foundinthefollowingtable. CHV StudyNo.: 17387-0-455 10 CORNINGHazleton A totaolf30animalwsasusedinthiasssay. DOSE GROUPS TREATMENT MF ----------------------------------------------------- T-6357 1500 mg/kg 2375 mg/kg 33 33 3250 mg/kg 33 4125 mg/kg 5000 mg/kg 33 33 All dosesgi-v-e-n-w-e-r-e-o-n-a-n-a-c-u-t-e-(-o-n-e--t-im-oe-n-l-y-)-b-a-s-i-s-.------------------ 10.7 Resultsand Interpretation Allanimalswere examined afterdosingand dailythroughoutthedurationofthe study(thredeays)fortoxiceffectasnd/ormortalities. Immediatelyafterdosing,inthe1500mglkg dosegroup,4 animalswere hypoactivea,nd theother2 were normal.Inthe2375 mg/kg dosegroup,2 males were languidand ataxica,nd theremaininganimalswere normaland.healthy.In the3250 mg/kg dosegroup,2 males and I femalewere languidand ataxica,nd theremaininganimalswere normaland healthy.Inthe4125 mg/kg dosegroup,1 male was prostratweithlaboredbreathinga,nd theremaininganimalswere hypoactive.Inthe5000 mg/kg dosegroup,I maleand I femalewere prostrate withlaboredbreathinga,nd theremaininganimalswereslightlhyypoactive. ApproximatelyI houraftedrosing,allanimalsfromthe1500mg/kg dosegroup were normal and healthy.Inthe2375 mg/kg dosegroup,1 maleand I female were hypoactivea,ndtheremaininganimalswerenormal andhealthy.Inthe 3250 mg/kg dosegroup,I male and 2 femaleswerehypoactiveandtheremaining animalswere normaland healthy.Allanimalsfromthe4125 mg/kg dosegroup were hypoactive.Inthe5000 mg/kg dosegroup,1 male and 2 femaleswere prostratea,ndtheremaininganimalswere hypoactive. Approximately17 hoursafterdosing,allanimalsfrom the1500 mg/kg dosegroup were normal and healthy.Allanimalsfrom the2375 mg/kg werehypoactiveand hunched,and 2 males(#'s6336,6334)alsohad squintedeyeswithbloodcolored staininogn eyes.All3250 mg/kg doselevelanimalswere hypoactiveand CHV StudyNo.: 17387-0-455 11 CORNINGHazieton huncheda,nd2 males(#'s6333,6337)alsohadbloodcoloresdtaininogn eyelids. All 4125 mg/kg animalswere hypoactivewith dyspnea and blood coloredstaining on eyelids.All 5000 mg/kg animalswere hypoactivewith dyspnea;allmales also had tremors,chromodacryorrhea,and were coldtothetouch. Theircage alsohad severalblood coloredspotsin it.One female (# 6340) had urinecoloredstains, profound footsplay,and was coldtothetouch. A second female alsohad a blood coloredglazeon the eyelidsand was coldto thetouch. Approximately 42.5 hours afterdosing,allanimals from the 1500 mg/kg dose group and allfemalesfrom the 2375 mg/kg and 3250 mg/kg dose groups were normal and healthy.One 2375 mg/kg dose group male (4 6336) was found dead and the remaining males were hypoactive.One 3250 mg/kg dose group male (# 6337) was found dead and the remainingmales were hypoactive.In the 4125 mg/kg dose group,2 males (# 6332, 6338) and one female (#6355) were found dead;theremaining male were hypoactive,hunched with dyspnea;the remainingfemale was hypoactive.In the5000 mg/kg dose group,I male (# 6340) and I female(# 6360) were found dead;theremainingmales were hypoactive;theremaining females were hypoactiveand had tremors,squinted eyes and dyspnea. Approximately 66.5hours afterdosing,allanimalsfrom the 1500 mg/kg, and all survivinganimals from the 2375 mg/kg and 3250 mg/kg dose groupswere normal and healthy.All survivinganimalsinthe4125 mg/kg dose group were hypoactivewith dyspnea. In the 5000 mg/kg dose group,2 females(#'s6346, 6352) were found dead and the survivingmales were hypoactive,hunched and had dyspnea. Approximately 71.5hoursafterdosing,I male from the5000 mg/kg dose group (#6342) was found dead. Approximately 91 hoursafterdosing,I male (# 6344) from the2375 mg/kg dose group,I male from the 3250 mg/kg dose group and 1 male (#6339) from the 4125 mg/kg dose group were found dead. All remaininganimalsfrom the 1500, 2375 and 3250 mg/kg and 4125 mg/kg female dose groups were normal and healthy.The survivingmale (# 6343) from the 5000 mg/kg dose group appeared pale,hunched and hypoactive. CHV Study No.: 17387-0-455 12 CORNINGHazleton Themortaldiattyfaorthiasssaayresummariziendthefollowitnagble: SummaryofMortalitWiietshi3n Days inMiceDosedAcutelwyithT-6357 Observations Treatment 1500 mg/kg 2375 mg/kg 3250 mg/kg 4125 mg/kg 5000 mg/kg Male Female 0/3 0/3 2/3 0/3 2/3 0/3 3/3 1/3 2/3 3/3 10.8 Conclusion Based on theseresultst,hemaximum tolerateddosewas estimatedtobe 2000 mg/kg. 11.0 NUCRONUCLEUS STUDY 11.1 Dose Selection Based on resultfsrom thedose selectiosntudy,doselevelsof500, 1,000,and 2,000mg/kg were selectefdortestinignthisstudy. 11.2 MicronucleusAssay DosingInformation The animalsused inthemicronucleusassaywere dosed on February27,1996. Cyclophosphamide(CAS # 6055-19-2;Sigma,Lot # 44H0486),thepositive controlw,as solubilizeidndeionizedwater(Lot# 19,preparedatCHV) and was administerebdy oralgavageat80.0mg/kg. The vehiclecontrold,eionizedwater (Lot# 19,preparedatCHV), was administerecdoncurrentlwyiththetestarticlaet a volume of10.0 ml/kg.The weightrangeoftheanimalsusedinthemicronucleusassaywas 29.2-39.5and 23.0-32.2grwns forthemalesand females, respectivelyT.he dosingsolutionfsortheassaywere preparedby making a 200 mg/ml stockforthehighdose(2000mg/kg).Thiswas preparedby adding deionizedwaterto5.0088g ofT-6357 up toa volume of25 ml. A clearpale CHV StudyNo.: 17387-0-455 13 CORNINGHazleton yelloswolutiwoansobtainaetdaconcentraotfi2o0n0mg/mlD.ilutionftshis stockwere preparedfortheremainingdoselevels.A secondgroup ofanimals (designateSdecondaryDose Group) was alsoassignedtothestudyand was dosed atthehighdose selectedT.hese animalswere onlyused intheassayas replacementsforany which diedintheprimarydosegroup. Ten animals(fivemalesand fivefemales)were randomlyassignedtoeachdose/harvesttimegroup.Vehicleand positivceontrolgroups,euthanatized approximatel2y4 hoursafterdosing,were includedintheassay.The animals dosedwiththetestarticlweere eudmatized approximatel2y4,48 and 72 hours aftedrosingforextractionfthebone marrow. An outlinoefthedosingscheme isfound inthefollowintgable: Dosing Scheme forMicronucleusAssay A totalof120 animalswas used inthisassay Number ofAnimalsAssigned Treatment PrimaryDose Groups 24Hr 48Hr 72Hr MF MF MF SecondaryDose Groups' Male Female T-6357 500 mg/kg 1000 mg/kg 55 55 55 55 55 55 - 2000 mg/kg 55 55 55 5 5 VehicleControld,eionizedwater,10.0ml/kg 5 5 - - - - - - PositivCeontrol,Cyclophosphamide,80.0mg/kg 5 5 a The animalsassignedtothesecondarydose groupswere dosedand were onlyused to replaceanimalswhich diedintheprimarydosegroup atthehighdoselevel.Allextra: aiumalsnotused as replacementswere euthanatizeadtthecompletionofthetrial. The age of theanimalsatthetimeofdosingwas nineweeks and one day. Volumes dosedwere 10.0ml/kgandwere basedupon individuaalniinawleights. CHV StudyNo.: 17387-0-455 14 CORNINGHazleton 12.0 BONE MARROW HARVEST, SLIDE PREPARATION AND ANALYSIS At theappropriathearvesttime,theanimalswere euthanatizewdithC02 followedby penetrationofthe thoraxand the adheringsofttissueand epiphysesofboth femora were removed. The marrow was flushedfrom thebone and transferretdocentrifugetubes containing3 - 5 ml bovine serum (one tubeforeach animal).Followingcentrifugatiotno pelletthetissue,the supernatantwas removed by aspiratioannd portionsofthe pellet were spreadon slidesand airdried.The slideswere fixedin methanol,and stainedin May-Grunwald solutionfollowedby Giemsa (Schmid, 1975).The air-driesdlideswere coverslippedusing Depex4lmounting medium. The slideswere coded foranalysisa,nd scoredformicronucleiand thepolychromatic erythrocyt(ePCE) tonormochromatic erythrocyte(NCE) cellratio.Standardforms were used torecordthesedata.One thousandPCEs per animalwere scored.The frequencyof micronucleatedcellswas expressedaspercentmicronucleatedcellsbased on thetotal PCEs presentinthe scoredopticfield.The normal frequencyof micronucleiin this Crl:CD-I"(ICR)BR straiinsabout0.0-0.4%. The frequency ofPCEs versusNCEs was determined by scoringthenumber of PCEs and NCEs observed inthe opticfieldswhile scoringthe first1000 erythrocytes. 13.0 EVALUATION CRITERIA: 13.1 General The criterifaortheidentificatioofnmicronucleiwere thoseof Schmid (1976). Micronucleiwere darklystainedand generallyround,althoughalmond and ringshapedmicronucleioccasionallyoccurred.Micronucleihad sharpborders and were generallybetween 1/20and 115thesizeof thePCE. The unitof scoring was themicronucleatedcell,not themicronucieus;thustheoccasionalcellwith more thanone micronucleuswas counted asone micronucleatedPCE, nottwo (or more) micronuclei.The stainingprocedurepermittedthe differentiatiboyncolor of PCEs and NCEs (bluish-greaynd red,respectively). 13.2 Data Presentationand Interpretation Data aresummarized by sex and dose groups forthe differentimepoints. Individualanimal dataarealsopresented.The analysisof thesedatawas performed usingan analysisofvariance(Winer, 197 1)on eitheruntransformed (when variancesarehomogeneous) and rank transformed(when variancesare CHV Study No.: 17'387-0-455 15 CORNINGHazleton heterogeneporuosp)ortioofncsellwsitmhicronucpleerainimalI.ftheanalysis ofvariancweas significa(npt<0.05)a,Dunnetfst-tes(tDunnett,1955;1964)was usedtodeterminewhichdosegroups,ifany,were significantdliyfferenftrom the negativecontrol.Analyseswere performedseparatelfyoreachharvestimeand sexcombination.The criterfioardetermininag positivreesponseinvolveda statisticaslilgynificadnotse-relateidncreaseinmicronucleatePdCES, orthe detectioonfa reproduciblaend statisticaslilgynificapnotsitivreesponseforat leastone doselevel.A testarticltehatinducedneithearstatisticaslilgynificant dose responsenor a statisticaslilgynificanatnd reproducibliencreaseatone dose levelwas considerednegative.Ineithercase,thefinaldecisionwas basedon scientifjiucdgment. 14.0 RESULTS AND INTERPRETATION: Allanimalswere observedimmediatelyaftedrosingand periodicaltlhyroughouthe duratioonftheassayfortoxicsymptoms and/ormortalitieAsl.lanimalsinthevehicle andpositivceontrolgroupsappearednormal afterdosingandremainedhealthyuntilthe appropriathearvesttimes.Alltestarticldeosed groupsappearednormalimmediately afterdosingand remainedhealthyuntiltheappropriathearvesttimes,exceptforone 72 hourmale atthe1000 mg/kg doselevel(#6562),which was founddeadabout72 hours postdosing. The testarticlTe-,6357,inducedno significanitncreasesinmicronucleated polychromaticerythrocyteosverthelevelsobservedinthevehiclecontrolisneithersex oratany oftheharvestimes.Due totoxicitya,significarnetductiownas observedinthe PCE/NCE ratioosfthemalesfrom the500,1000,and 2000 mg/kg dosegroupsatthe72 hourharvestime.The positivceontrolC,P, inducedsignificainntcreaseisnmicronucleatedPCEs inboth sexesascompared tothevehiclecontrolsw,ithmeans and standarderrorsof 7.98% 1.30% and 4.16% 0.92% forthemalesand females,respectivelyT.he datasummarizedby dosegrouparepresenteidnTableI and individual animaldataarefound inTables2 through7. Historicaclontroldataarepresentedin Table 8. 15.0 CONCLUSION: The testmaterialT,-6357,didnotinducea significainntcreasienmicronucleinbone marrow polychromatiecrythrocyteusndertheconditionosfthisassayand isconsidered negativeinthemouse micronucleusassay. CHV StudyNo.: 17387-045 5 16 CORNINGHazleton 16.0 REFERENCES: Dunnett,C.W.: A multiplecomparisonsprocedureforcomparing severaltreatmentwsith a control.J.Am. StatistA.ssoc.,5-Q:1096-11211,955. Dunnett,C.W.: New tablesformultiplecomparisonswitha control.BiometricsM,:482491,1964. Heddle,J.A.,Hite,M., KirkhartB,.,Larsen,K.,MacGregor, J.T.N,ewell,G.W. and Salamone,M.F.: The inductionof micronucleias a measure of genotoxicity. MutationRes.,LU:61-118, 1983. Schmid, W.: The micronucleustest.MutationRes.,21:9-15,1975. Schmid,W.: The micronucleustestforcytogenetiacnalysisC.hemical Mutagens: Principleasnd Methods forTheirDetectionV,ol.4 (A.Hollaender, ed.).Plenum, pp.31-53,1976. Winer,B.J.:StatisticParlincipleisnExperimentalDesign,McGraw-Hill,New York, Second Edition,1971. 17.0 DEVIATIONS FROM THE SIGNED PROTOCOL The followingdeviationwsere made from thesignedprotocol. I . On February19,1996,forthedose selectiosntudiest,herelativheumiditywas recordedat3 1.1%. For themicronucleusassay:on February 17,18,19,24,24, and 25, 1996,therelativheumiditywas recordedat39.1%, 37.3%, 34.5%, 32.5%, 34.4%, and 20.8%, respectivelyT.hisdidnot affectheanimalsand thereWas no impacton theintegritoyf thisstudy. 2. Due toa technicaelrrorinthepreparatioonf dosingstocksforTrialIof thedose selectiosntudy,theactualdose levelsused were 610,8 10,and 1010 mg/ml. The differencies<2% and had no impacton theintegritoyf thisstudy. CHV StudyNo.: 17387-0-455 17 CORNINGHazleton 18.0 EXPERIMENT DATA TABLES CHV StudyNo.: 17387-0-455 -- 18 - I CORNINGHazleton SPONSOR: 3M MICRONUCLEUS TABLE 1 DATA SUMMARY TABLE TEST ARTICLE: T-6357 ASSAY: 17387 TREATMENT DOSE HARVEST TIME (HR) % HOCRONUCLEATED PCEs MEAN OF 1000PER ANDAAL S.E. MALES FEMALES TOTAL CONTROLS VEFUCLE POSITIVE Water CP 80.0mgtkg 24 hr 24 hr 0.02;k0.02 7.98 :h1.30* 0.04:10.02 4.16 0.920 0.03 0.02 6.07* 0.98* RATIO PCE:NCE MEAN S.E. MALES FEMALES 0.67 0.05 0.78 0.08 0.71 0.08 0.71 0.05 TEST ARTICLE 500 mglkg 1000mg/kg 2000 mglkg 24 hr 48 hr 72 hr 24 hr 48 hr 72 hr 24 hr 48 hr 72 hr 0.02 0.02 0.04 0.04 0.00:k0.00 0.10 0.03 0.10 0.05 0.03 0.03 0.06 0.06 0.02 0.02 0.02 :h0.02 0.10 0.03 0.02 0.02 0.02 :k0.02 0.04 :10.02 0.04 0.02 0.00 0.00 0.10 0.04 0.04 :k0.02 0.04 :k0.02 0.06 0.02 0.03:10.02 0.01 0.01 0.07 0.02 0.07 0.03 0.01 :k0.01 0.08 0.04 0.03 0.02 0.03 0.02 0.72 0.09 0.54 0.12 0.73 0.02 0.62 0.03 0.43 0.06** 0.63 0.05 0.52 0.03 0.77 :k0.04 0.65 0.06 0.56 :k0.03 0.29 0.04** 0.58 0.06 0.61 0.07 0.70 0.07 0.67 0.04 0.69* 0.04 0.36 0.03** 0.53 0.04 Significantlgyreaterthan thecorrespondingvehiclecontrol,p<0.05. Significantllyower than thecorrespondingvehiclecontrolp,<0.05. CP Cyclophosphamidc CHV StudyNo.: 17387-0455 19 CORNINGHazleton MICRONUCLEUS TABLE2 TEST - INDIVIDUAL ANIMAL DATA SPONSOR: 3M TEST ARTICLE: T-6357 ASSAY NO.: 17387 TREATMENT 24 HOUR HARVEST MALE ANIMAL NUMBER NO.MN PCEs (1000) RATIO PCE:NCE VEFUCI.E CONTROL Water POSMVE CONTROL CP 80.0 mgtkg TEST ARTICLE 500 mgtkg 1000 -g/kg 2000 mglkg 6556 0 0.47 6560 0 0.65 6599 1 0.72 6595 0 0.71 6605 0 0.78 6548 125 0.58 6553 63 1.06 6573 90 0.63 6579 71 0.78 6580 50 0.83 6551 0 1.01 6581 0 0.54 6585 1 0.86 6597 0 0.57 6598 0 0.65 6550 0 0.72 6561 1 0.66 6569 1 0.56 6572 1 0.72 6574 2 0.48 6547 0 0.78 6549 3 0.63 6597 0 0.55 6599 0 0.40 6602 0 0.70 CP Cyclophosphamide MN Micronucieus PCE Polychromaticerythrocyte # KN PCEs = MicronucleatedPCEs NCE = Normochromatic erythrocyte CHV StudyNo.: 17387-0455 20 CORNINGHazleton MICRONUCLEUS TABLE3 TEST - INDIVIDUAL ANIMAL DATA SPONSOR: 3M TEST ARTICLE: T-6357 ASSAY NO.: 173 87 TREATMENT 24 HOUR HARVEST FEMALE ANIMAL NUMBER NO.MN PCEs (1000) RATIO PCE:NCE VEFUCLE CONTROL Water POSINVE CONTROL CP 80.0 mg/kg TEST ARNCLE 500 mg/kg 1000 mgfkg 2000 mglkg 6612 0 6617 0 6625 1 6637 1 6647 0 6616 27 6633 29 6641 24 6656 64 6658 64 6607 2 6610 1 6626 1 6642 1 6646 0 6611 1 6615 0 6634 1 6649 0 6666 0 6621 1 6627 0 6644 0 6653 2 6665 2 0.57 0.96 0.75 0.75 0.52 0.81 0.75 0.78 0.69 0.53 0.73 0.79 0.60 0.72 0.79 0.76 0.92 0.65 0.78 0.75 0.54 0.76 0.88 0.56 0.77 CP Cyclophospharnide MN MicTonucieus PCE Polychromaticerythrocyte # MN PCEs = MicronucleatedPCES NCE = Normochromatic erythrocyte CHV StudyNo.: 17387-0-455 21 CORNINGHazleton MICRONUCLEUS TABLE4 TEST -INDIVIDUAL ANIMAL DATA SPONSOR: 3M TEST ARTICLE: T-6357 ASSAY NO.: 17387 TREATMENT 48HOUR HARVEgr MALE ANIMAL NUMBER NO.MN RATIO PCEs PCE:NCE (1000) TEST ARTICLE 500 mgtkg 1000 mg/kg 2000 -gtkg 6554 0 0.77 6557 0 0.51 6569 0 0.72 6583 0 0.60 6594 2 0.12 6566 0 0.58 6570 1 0.49 6582 3 0.77 6601 1 0.91 6603 0 0.61 6552 1 0.78 6584 0 0.64 6591 0 0.66 65% 0 0.56 6604 0 0.71 MN Micronucleus PCE Polychromaticerythrocyte # MN PCEs = MicronucleatePdCEs NCE = Normochromaticerythrocyte CHV StudyNo.: 17387-0-455 22 CORNINGHazleton MICRONUCLEUS TABLE5 TEST - INDIVIDUAL ANIMAL DATA SPONSOR: 3M TEST ARTICLE: T-6357 ASSAY NO.: 17387 TREATMENT 48 HOUR HARVEST FEMALE ANI NO MN RATIO PCEs NU ER PCE-NCE (1000) TEST ARTICLE 500 mgtkg 1000 mg/kg 2000 mg/kg 6613 0 0.60 6629 1 0.72 6648 0 0.56 6651 0 0.61 6661 0 0.61 6638 0 0.55 6639 1 0.47 6640 0 0.53 6645 0 0.59 6652 1 0.64 6608 1 0.78 6620 0 0.68 6624 1 0.78 6629 0 0.59 6664 0 0.63 NM Micronucleus PCE Polychromaticerythrocyte # MN PCEs = MicronucleatedPCEs NCE = NorTnochromaticerythrocyte CHV StudyNo.: 17387-0-455 23 CORNINGHazleton MICRONUCLEUS TABLE6 TEST - INDIVIDUAL ANIMAL DATA SPONSOR: 3M TEST ARTICLE: T-6357 ASSAY NO.: 17387 TREATMENT 72 HOUR HARVEST MALE ANIMAL NUMBER NO.MN PrEs (1000) RATIO PCE:NCE TEST ARTICLE 500 -glkg 1000 mglkg 2000 mgtkg 6563 0 0.35 6571 0 0.30 6577 0 0.51 6596 0 0.39 6606 0 0.61 6558 1 0.27 6559 0 0.19 6562* 6565 0 0.31 6588 0 0.40 6564 0 0.39 6567 0 0.37 6576 1 0.46 6578 0 0.35 6592 0 0.24 Animal found dead MN Micronucleus PCE Polychromaticerythrocyte # MN PCEs = MicronucleatedPCEs NCE = Normochromatic erythrocyte CHV StudyNo.: 17387-0-455 24 CORNINGHazleton MICRONUCLEUS TABLE7 TEST - INDIVIDUAL ANIMAL DATA SPONSOR:3M TEST ARTICLE: T-6357 ASSAY NO.: 17387 TREATMENT 72 HOUR HARVEST FEMALE ANIMAL NUMBER NO.MN PCEs (1000) RATIO PCE:NCE TEST ARTTCLE Soo mg/kg 1000 mglkg 2000 mg/kg 66n 0 0.56 6631 0 0.58 6636 0 0.53 6657 0 0.45 6659 1 0.46 6609 0 0.76 6614 0 0.64 6618 0 0.59 6619 0 0.51 6643 0 0.41 6623 1 0.66 6635 0 0.41 6654 0 0.54 6655 1 0.57 6662 0 0.45 MN Micronucieus PCE Polychromaticerythrocyte # MN PCEs = MicronucleatedPCEs NCE = Normochromatic erythrocyte - CHV StudyNo.: 17387-0455 25 CORNINGHazleton TABLE8 MOUSE MICRONUCLEUS HISTORICAL CONTROL DATA 7/95THROUGH 12/95 POOLED VEMCLE CONTROLS MIN MAX AVG N % MICRONUCLEATED PCEs PER 1000 PCE MEAN OF 1000 PER ANIMAL S.E. MALES FEMALES TOTAL 0.00 0.22 0.097 0.007 47 0.00 0.24 0.081 0.008 47 0.01 0.17 0.084 0.005 47 RATIO PCE:NCE MEAN S.E. MALES FEMALES 0.31 0.85 0.5500.021 47 0.24 1.03 0.587.+0.025 47 POSrrIVE CONTROLS Cyclophosphamide8,0.0mgtkg MIN MAX AVG N 2.00 5.68 3.692:k0.240 19 PCE Polychromatiecrythrocyte NCE Normochromaticerythrocyte 1.50 6.36 3.170 0.245 19 2.41 0.41 5.38 0.72 3.426:k0.194 0.577 0.020 19 1 19 0.40 0.79 0.58&:L0.026 19 CHV StudyNo.: 17387-0455 26 Corning li@iziL,.lt,oi,c-.i @i2t)(I).ecs)ii:r!4 V;,o!ir.3. '7()3 221 @4'.-1 April19, 1996 Steven C. Gordon, Ph.D.,DABT 3M Medical Department Building220-2E-02,3M Center St.Paul,NW 55144-1000 CORNING Hazleton RE: DRAFT REPORT AND PROTOCOL AMENDMENTS InVivo MicronucleusAssay ProtocolNo.: 455CO, Ed. No.: 4,Modified forSponsor GeneticsAssay No.: 17387,17387-1 TestMaterial:T-6357 Dear Dr. Gordon: Enclosed pleasefindtwo (2)copiesof theabove referencedreport.The reportincludesan unsigned QualityAssurance Statementand Compliance and CertificatioSntatement,which will be signed upon issuanceof theFinalReport. The finalreportwillbe issuedafteryour review and notificatiotno us. Pleasecontactthe undersignedwith any questions,comments, or necessaryrevisionsatyour earliesctonvenience. The reportwillbe finalizeadfterI year ifno notificatioinsreceived. Thank you forgivingus thisopportunitytowork with you. Sincerely, CORNING Hazleton Hema Murli,Ph.D. Mammlian Cytogenetics Department of Genetic and CellularToxicology HM/paj enclosures