Document 6R9QrvKLkvQMxerYjw3BaX0og

CORNINGHazleton MUTAGENICITY TEST ON T-6342 IN AN IN VIVO MOUSE MICRONUCLEUS ASSAY FINAL REPORT AUTHOR HemalathaMurli,Ph.D. PERFORMING L ORATORY Coming HazletonInc.(CHV) 9200 LeesburgPike Vienna,Virginia22182 LABORATORY PROJECT IDENTIFICATION CHV StudyNo.: 17073-0-455 SUBMITTED TO 3M Corporation Building220-2E-02,3M Center St.Paul,Minnesota 55144-1000 STUDY COMPLETION DATE December 14,1995 CHV StudyNo.: 17073-0-455 1 of23 CORNINGHazleton QUALITY ASSURANCE STATEMENT ProjeTcittleI:nVivoMouseMicronucleAusssay ProjectNo.: 20996 Assay No.: 17073 ProtocolNo.: 455 EditionNo.:17,Modifiedfor3M Corporation QualityAssurance inspectionosfthestudyand reviewof thefinalreportof theabove referenced projectwere conductedaccordingtotheStandardOperatingProceduresof theQualityAssurance Unit and accordingtothegeneralrequirementsof theappropriatGeood LaboratoryPractice regulationsF.indingsfrom theinspectionasnd finalreportreviewwere reportedto management and tothestudydirectoron thefollowingdates: Inspection/Date FindingsRel2orted Audito Randomizationof Animals/09/18/1995 09/18/1995 S.Ballenger DraftreportReview/10/31/1995 10/31/1995 C. Orantes FinalReport Review/ 12/14/1995 12/14/1995 C. Orantes QualityAssuranceUnit Date"ReYease-d CHV StudyNo.: 17073-0-455 2 CORNINGHazieton STUDY COMPLIANCE AND CERTIFICATION The describedstudy was conducted incompliance with theGood LaboratoryPracticeregulations as setforthinthe Food and Drug Administration(FDA), Title21 ofthe U.S. Code of Federal RegulationsPart58, issuedDecember 22, 1978,(effectivJeune 20, 1979) with any applicable amendments. There were no significandteviationsfrom theaforementionedregulationsorthe signedprotocolthatwould affecttheintegritoyf thestudyor theinterpretationf thetestresults. The raw datahave been reviewed by the Study Director,who certifietshatthe evaluationofthe testarticlaes presentedhereinrepresentsan appropriateconclusionwithin thecontextofthe studydesign and evaluationcriteria. All testand controlresultsinthisreportaresupportedby an experimentaldatarecordand this recordhas been reviewed by theStudy Director.All raw data,documentation,records,protocol and a copy of thefinalreportgeneratedasa resultofthisstudy willbe archivedinthe storage facilitioefsComing HazletonInc.foratleastone yearfollowingsubmissionof thefinalreportto theSponsor. Afterthe one yearperiod,the Sponsor may electtohave the aforementioned materialsretainedinthestoragefacilitioefsComing HazletonInc.foran additionapleriodof time,or senttoa storagefacilitdyesignatedby the Sponsor. SubmittedBy: Study Director: Hemalatha Murli,Ph.D. Mammalian Cytogenetics Department ofGeneticand CellularToxicology Study Completion Date CHV Study No.: 17073-0-455 3 CORIYING Hazleton TABLE OF CONTENTS Page No. SUNINIARY ..............................................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 InitiatioDnate 5.2 ExperimentalStartDate 5.3 ExperimentaTlerminationDate 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 SELECTION STUDY ...............................8........... 10.1 Dose Selection 10.2 DosingInformation 10.3 Resultsand Interpretation 10.4 Conclusion CHV StudyNo.: 17073-0-455 4 11.0 12.0 13.0 14.0 15.0 16.0 17.0 CORNINGHazleton MICRONUCLEUS STUDY ...............................1.0 .......... 11.1 Dose Selection 11.2 MicronucleusAssayDosingInformation BONE MARROW HARVEST, SLIDE PREPARATION AND ANALYSIS ....1.1 EVALUATION CRITERIA ...............................1.2 .......... 13.1 General 13.2 Data Presentatioannd Interpretation RESULTS AND INTERPRETATION .........................1.3.......... CONCLUSION ......................................13........... REFERENCES ......................................1.4 .......... EXPERIMENT DATA TABLES ............................1.5........... CHV StudNyo.:17073-0-455 5 SUN*AARY CORNINGHazleton MutagenicityTeston T-6342 inan In VivoMouse Mcronucleus Assay The objectivoefthisinvivoassaywas toevaluatteheabilitoyfthetestarticlTe-,6342,toinduce micronucleinbone marrow polychromaticerythrocyteosfCrl:CD-II(ICR)BR mice. In thedoseselectiosntudy,thetestarticlweas solubilizeidndeionizedwaterand dosed by oral gavageat500,l625,2750,3875,and 5000mg/kg. Sixanimals(threemalesand threefemales) were assignedto each dose group. Animals were observedforthreedays afterdosingfortoxic signsand/ormortality. Based on theresultosfthedose selectiosntudy,themaximum toleratedose was estimatedas >5000 mg/kg. In themicronucieusassay,thetestarticlweas solubilizeidndeionizedwater and dosedby oralgavage at1250,2500,and 5000 mg/kg. Ten animals(fivemales and fivefemales) were randomly assignedto eachdose/harvesttimegroup.Vehicleand positivecontrolgroups euthanatizeadpproximately24 hoursafterdosingwere includedintheassay.The animalsdosed withthetestarticlweere euthanatizeadpproximately24,48 and 72 hours afterdosingfor extractioonfthebone marrow. The testmaterialT,-6342, didnotinducea significanitncreaseinmicronucleiinbone marrow polychromaticerythrocyteusndertheconditionsof thisassayand isconsiderednegativeinthe mouse bone marrow micronucleustest. CHV StudyNo.: 17073-0-455 6 CORNINGHazleton MutagenicTietsyotnT-634i2naninvivMoouseMicronuciAesussay 1.0 SPONSOR: 3M Corporation 2.0 NIATERIAL (TestArticle) 2.1 Client'IsdentificatiTo-n6:342 2.2 DateReceived:July21,1995 2.3 PhysicalDescriptionC:lear,colorleslsiquid 2.4 GeneticsAssay No.: 17073 3.0 TYPE OF ASSAY: In VivoMouse MicronucleusAssay 4.0 PROTOCOL NO.: 455,Edition17,Modifiedfor3M Corporation 5.0 STUDY DATES 5.1 InitiatiDoante: August 15,1995 5.2 ExperimentalStartDate:August 29,1995 5.3 ExperimentalTerminationDate: October16,1995 6.0 SUPERVISORY PERSONNEL 6.1 StudyDirectorH:emalathaMurli,Ph.D. 6.2 LaboratorySupervisorM:onica Vegarra,B.S. 7.0 OBJECTIVE The objectivoefthisinvivoassaywas toevaluatteheabilitoyfthetestarticlTe-,6342,to inducemicronucleiinbone marrow polychromatiecrythrocyteosfCrl:CD-I*(ICR)BR mice. Thisstudywas conductedusingmodificationosftheproceduressuggestedby Heddleetal.(1983). CHV StudyNo.: 17073-0-455 7 CORNINGHazleton 8.0 MATERIALS Adultmale and femalemice,straiCnrl:CD-I'(ICR)Bl@,were purchasedftom Charles River LaboratoriesP,ortageNU.. This healthy,random bredstrainwas selectedto maximize geneticheterogeneitaynd atthesame time assureaccesstoa common source. The protocolforthisstudywas approvedby theCHV-ACUC priortotheinitiatiofn dosing. Animals were housed up tosevenpercage duringquarantinea,nd housed up to fiveper cageatrandomization.The temperatureand relativheumiditywere maintainedat 726'F and 5515%, respectivelyA.12-hourlight/12-houdrarkcyclewas maintained. A commercialdiet(PurinO CertifieLdaboratoryPellet0s# 5002)and waterwere availablaed libitumforthedurationofthestudy.The feedwas analyzedby the manufacturerforconcentrationosf specifiehdeavy metals,aflatoxinc,hlorinated hydrocarbons,organophosphatesa,nd specifiendutrientsT.he waterwas analyzedon a retrospectivbeasisforspecifiemdicroorganisms,pesticidesa,lkalinithye,avy metals,and halogens.Sanitizedcagingwas usedforhousingtheanimals.Personnelhandling animalsor working withintheanimalfacilitiweesre requiredto wear suitablperotective garmentsand equipment. Animals were quarantinedforseven beforebeingplacedon study.Animals were randomly assignedto studygroupsand were individuallwyeighed priortodosing.All animalswere dosed basedupon theindividuablody weights.Animals were uniquely identifiebdy eartag.Dose or treatmentgroupswere identifiebdy cage card/label. At theterminatioonf thestudyallsurvivinganimalswere euthanatizebdyC02 inhalationf,ollowedby penetratioonfthethorax.Any extraanimalsnotused forthe studywere euthanatizebdyC02inhalation,followedby penetratioonf thethorax. 9.0 SOLUBILITY AND STABILITY: The testarticleT,-6342,was suppliedas a clearcolorleslsiquid.The vehicleselectedfor thisassaywas deionizedwater.The stabiliotfythetestmaterialunder thedosing conditionsof thisassayistheresponsibiliotfythesponsor. 10.0 DOSE SELECTION STUDY 10.1 Dose Selection Dose levelsof 500, 1625,2750, 3875,and 5000 mg/kg were administeredby oral gavage forthedose selectionstudy. CHV StudyNo.: 17073-0-455 8 CORNINGHazleton 10.2 DosingInformation The animalsusedinthedose selectioanssaywere dosedon August 29, 1995. The weightrangeoftheanimalsusedinthedoserangefindingassaywas 26.5-33.5 and 21.1-25.1grams,forthemalesand femalesr,espectivelyD.osing solutions were preparedjustpriortodosingand werepreparedby making a 500 mg/ml stockforthehighdose(5000mg/kg).Thiswas preparedby adding6.6ml of deionizedwater(Lot# 17,preparedatCHV) to6.0007g ofT-6342,resultinigna clearc,olorlesssolutio.n Dilutionosfthisstockwerepreparedfortheotherdose levels. Dosing was achievedusinga 10mi/kgdosingvolume. Allanimalswere eight weeks and one day oldatthetimeofdosing.An outlineofthedosingscheme is foundinthefollowintgable. A totalof 30 animalswas usedinthisassay. DOSE GROUPS TREATMENT mF ----------------------------------------------------- T-6342 500 mg/kg 1625mg/kg 33 33 2750 mg/kg 33 3875 mg/kg 5000 mg/kg 33 33 Alldosesgi-v-e-n-w-e-r-e-o-n-a-n-a-c-u-t-e-(-o-n-e--t-i-m-oe-nl-y-)-b-a-s-i-s-.------------------- 10.3 Resultsand Interpretation Allanimalswere examinedafterdosingand dailythroughouthedurationofthe study(threedays)fortoxiceffectasnd/ormortalitieAsl.lanimalsappeared normal immediatelyafterdosingand remainedhealthyuntiltheend ofthe observatiopneriod.The mortalitdyataforthisassayaresummarized inthe followingtable: CHV StudyNo.: 17073-0455 9 CORNINGHazleton Summary ofMortalitiWeisthin3Days inMiceDosedAcutelwyithT-6342 Obsgryations Treatment 500 mg/kg 1625 mg/kg 2750 mg/kg 3875 mg/kg 5000 mg/kg mair, 0/3 0/3 0/3 0/3 0/3 Female 0/3 0/3 0/3 0/3 0/3 10.4 Conclusion Based on theseresultst,hemaximum toleratedose was estimatedtobe >5000 mg/kg. 11.0 MICRONUCLEUS STUDY 11.1 Dose Selection Based on resultfsrom thedose selectiosntudy,dose levelsof 1250,2500,and 5000 mg/kg were selectedforthisstudy. 11.2 MicronucleusAssay Dosing Information The animalsused inthemicronucteusassaywere dosed on September 19,1995. Cyclophosphamide(Sigma,Lot # 43HO269, CAS # 6055-19-2),thepositive controlw,as solubilizeidnsterildeeionizedwater(Lot# 17,preparedatCHV) and was administeredby oralgavage at80 mg/kg. The vehiclecontrold,eionized water(Lot# 17,preparedatCHV), was administerecdoncurrentlwyiththetest articlaeta volume of 10 mukg. The weightrangeoftheanimalsusedinthe micronucieusassaywas 28.7- 37.6 g and 21.1- 25.6g grams forthemales and females,respectivelyT.he dosingsolutionfsortheassaywere preparedby making a 500 mg/ml stockforthehighdose(5000mg/kg). Thiswas preparedby addingdeionizedwaterto 11.0008g of T-6342 up to a volume of 22.0mi. A clear,colorlessolutionwas obtained.Dilutionsofthisstockwere preparedfor theremainingdose levels. CHV StudyNo.: 17073-0455 10 CORNINGHazieton Tenanimal(sfivmealesandfivfeemalewse)rerandomlayssignteodeachdose/harvesttimegroup. Vehicleand positivceontrolgroups,euthanatized approximately24 hoursafterdosing,were includedintheassay.The animals dosed with thetestarticlweere euthanatizeadpproximately24,48 and 72 hours afterdosingforextractioonfthebone marrow. An outlineof thedosingscheme isfound inthefollowingtable: Dosing Scheme forMicronucieusAssay A totalof 110 animalswas used in thisassay Number of Animals Assigned Treatment T-63T21250 mg/kg 24 Hr 48 Hr 72Hr MF MF MF 55 55 55 2500 mg/kg 5000 mg/kg 55 55 55 55 55 55 VehicleControl,deionizedwater 10 ml/kg 5 5 -- PositiveControl,Cyclophosphamide,80 mg/kg 5 5 The age of theanimalsatthetimeof dosingwas eightweeks and one day. Volume dosed was 10 ml/kg and was based upon individuaalnimalweight. 12.0 BONE MARROW HARVEST, SLIDE PREPARATION AND ANALYSIS At theappropriathearvestime,theanimalswereeuthanatizewdithC02 followedby penetratioonf thethoraxand theadheringsofttissueand epiphysesof bothfemora were removed. The marrow was flushedfrom thebone and transferretdo centrifugteubes containing3 -5 ml bovineserum (onetubeforeachanimal).Followingcentrifugatitoon' pellethetissuet,hesupernatanwtas removed by aspiratioannd portionsofthepellet were spreadon slidesand airdried.The slideswere fixedinmethanol,and stainedin May-Grunwald solutiofnollowedby Giemsa (Schmid,1975).The air-driesdlideswere coverslippeudsingDepex* mounting medium. The slideswere coded foranalysisa,nd scoredformicronucleiand thepolychromatic erythrocyt(ePCE) tonormochromaticerythrocyt(eNCE) cellratio.Standardformswere CHV StudyNo.: 17073-0-455 11 CORNINGHazleton usedtorecortdhesdeata.OnethousanPdCEsperanimawlerescoredT.hefrequenocfy micronucleatedcellswas expressedaspercentmicronucleatedcellsbased on thetotal PCEs presentinthescoredopticfield.The normal frequencyof micronucleiinthis Crl:CD-I'(ICR)BR straiinsabout0.0-0.41/o. The frequencyof PCEs versusNCEs was determinedby scoringthenwnber of PCEs and NCEs observedintheopticfieldwshilescoringthefirs1t000 erythrocytes. 13.0 EVALUATION CRITERIA: 13.1 General The criterifaortheidentificatioofnmicronucleiwere thoseof Schmid (1976). Micronucleiwere darklystainedand generallryound,althoughalmond and ringshapedmicronucleioccasionalloyccurred.Micronucleihad sharpborders and were generallbyetween 1/20and 1/5thesizeofthePCE. The unitof scoring was themicronucleatecdelln,otthemicronucleust;hustheoccasionalcellwith more thanone micronucleuswas countedas one micronucleatedPCE, nottwo (or more) micronuclei.The staininpgrocedurepermittedthedifferentiatiboyncolor of PCEs and NCEs (bluish-greaynd red,respectively). 13.2 Data Presentatioannd Interpretation Data aresummarized by sexand dose groupsforthedifferenttime points. Individualanimaldataarealsopresented.The analysisofthesedatawas performedusingan analysiosf variance(Winer,1971)on eitheruntransformed (when variancesarehomogeneous) and ranktransformed(when variancesare heterogeneousp)roportionosfcellswith micronucleiperanimal.Iftheanalysis ofvariancewas significa(npt<0.05)a, Dunnett'ts-tes(tDunnett,1955;1964)was used todeterminewhich dose groups,ifany,were significantdliyfferenftrom the negativecontrol.Analyseswere performedseparatelfyoreach harvesttime and sex combination.The criterifaordetermininga positiveresponseinvolveda statisticalsliygnificandtose-relateidncreaseinmicronucleatedPCES, or the detectioonf a reproduciblaend statisticaslilgynificanptositivreesponseforat leastone dose level.A testarticltehatinducedneithera statisticaslilgynificant dose responsenora statisticaslilgynificanatnd reproducibliencreaseatone dose levelwas considerednegative.Ineithercase,thefinaldecisionwas based on scientifjiucdgment. CHV StudyNo.: 17073-0455 12 CORNINGHazleton 14.0 RESULTS AND INTERPRETATION: Allanimalswere observedimmediatelyafterdosingand periodicalltyhroughoutthe durationof theassayfortoxicsymptoms and/ormortalitiesA.llanimalsinthevehicle and positivecontrolgroupsappearednormal afterdosingand remained healthyuntilthe appropriathearvesttimes.Allanimalsfrom the1250 and 2500 mg/kg dosegroups appearednormal inunediatelayfterdosingand remainedhealthyuntiltheappropriate harvesttimes. Immediatelyafterdosing,allanimalsinthe5000 mg/kg dose group appearednormal. Approximately24 hoursafterdosing,2 males (#'s2712,48 hour harvestgroup;2714,72 hour harvestgroup)from the5000 mg/kg dose group were found dead. One male (#2718,72 hour harvestgroup)appearedhunched and weak with squintedeyes.Allother animalsappearednormal atthistime. Approximately48 hoursafterdosing,Imale (#2718,72 hour harvestgroup)and I female(#2780,72 hourharvestgroup)from the5000 mg/kg dose group were found dead. Allotheranimalsappearednormal atthistimeand remainedhealthyuntiltheappropriate harvesttimes. The testarticleT,-6342,inducedno significanitncreaseisnmicronucleated polychromaticerythrocyteosver thelevelsobservedinthe vehiclecontrolsineithersex or atany of theharvesttimes.Due totoxicityt,hePCE/NCE ratiosof themales and femalesfrom the5000 mg/kg dosegroup atthe72 hour harvestgroup were significantly lowerthanthevehiclecontrolanimals.The positivceontrolC,P, inducedsignificant increasesinmicronucleatedPCEs inboth sexesas compared tothevehiclecontrolsw,ith means and standarderrorsof 5.44% 0.37% and 2.50% 0.33% forthemales and females,respectivelyT.he datasummarized by dose group arepresentedinTable I and individuaalnimal dataarefound inTables2 through7. Historicaclontroldataare presentedinTable 8. 15.0 CONCLUSION: The testmaterialT,-6342,didnotinducea significanitncreaseinmicronucleiinbone marrow polychromaticerythrocyteusndertheconditionsofthisassayand isconsidered negativeinthemouse micronucleusassay. CHV StudyNo.: 17073-0-455 13 CORNINGHazleton 16.0 REFERENCES: DunneM C.W.: A multiplecomparisonsprocedureforcomparing severaltreatmentswith a control.J.Am. StatistA.ssoc.,IQ:1096-1121,1955. Dunnett,C.W.: New tablesformultiplecomparisonswith a control.Biometrics,20:482491,1964. Heddle,J.A.,Hite,M., Kirkhart,B.,Larsen,K.,MacGregor, J.T.,Newell,G.W. and Salamone, M.F.: The inductionofmicronucleias a measure ofgenotoxicity. Mutation Res.,123:61-118,1983. Schmid, W.: The micronucleustest.Mutation Res.,U:9-15, 1975. Schmid, W.: The micronucleustestforcytogenetiacnalysis.Chemical Mutagens: Principleasnd Methods forTheirDetection,Vol.4 (A. Hollaender, ed.).Plenum, pp. 31-53, 1976. Winer,B.J.:StatisticParlincipleisn ExperimentalDesign,McGraw-Hill, New York, Second Edition,1971. CHV Study No.: 17073-0455 14 CORNINGHazleton 17.0 EXPERIMENT DATA TABLES CHV Study No.: 17073-0-455 15 CORNING Hazleton SPONSOR: 3M Corporation TEST ARTICLB:T-6342 ASSAY:17073 TREATMENT CONTROLS VEHICLE POS:ETIVE-CP HARVEST DOSE TIME (HR) Water 80.0 mg/kg 24 hr 24 hr TABLE 1 MICRONUCLEUS DATA SUMKARY TABLE MEAN MALES % MICRONUCLEATED PCEs OF 1000 PER ANIMAL :i S.E. FEMALES TOTAL 0.14 0.02 5.44 0.37* 0.02 :t0.02 2.50 0.33, 0.08 3.97 0.02 0.54* RATIO PCE:NCE MEAN :t S.E. MALES FEMALES 0.51 0.06 0.64 :t 0.07 0.69 0.11 0.63 0.05 TEST ARTICLE 1250 mg/kg 2500 mg/kg 5000 mg/kg 24 hr 48 hr 72 hr 24 hr 48 hr 72 hr 24 hr 48 hr 72 hr 0.28 0.05 0.00 0.00 0.06 0.02 0.12 0.04 0.02 0.02 0.20 0.07 0.06 0.04 0.15 0.09 0.17 0.03 0.00 :t 0.00 0.02 :t0.02 0.02 :t0.02 0.04 -t0.02 0.02 0.02 0.06 0.04 0.10 0.04 0.06 0.02 0.03 0.03 0.14 0.05 0.01 0.01 0.04 0.02 0.08 0.02 0.02 :t0.01 0.13 :t0.04 0.08 0.03 0.10 t 0.04 0.09 0.03 0.69 0.09 0.68 0.08 0.36 0.08 0.69 0.07 0.79 0.07 0.47 0.07 0.80 0.08 0.56 0.10 0.17 :t 0.04* 0.75 0.17 0.74 0.06 0.59 :t 0.06 0.64 0.05 0.65 0.01 0.64 0.07 0.79 0.09 0.63 0.06 0.24 0.05* *Significantly different from the corresponding vehicle control. p<0.05. CHV StudyNo.: 17073-0455 16 CORNINGHazleton MICRONUCLEUS TABLE 2 TEST - INDIVIDUAL ANIMAL DATA SPONSOR:3M Corporation TEST ARTICLE:T-6342 ASSAY NO.:17073 TREATMENT ANIMAL NUMBER NO.MN PCEs (1000) RATIO PCE:NCE 24 HOUR HARVEST MALE VEHICLE CONTROL Water POSITIVE CONTROL CP 80.0 mg/kg TEST ARTICLE 1250 mg/kg 2500 mg/kg 5000 mg/kg 2682 1 2689 2 2694 1 2720 2 2721 1 2696 46 2698 61 2715 52 2723 65 2728 48 2683 4 2691 1 2697 3 2703 3 2717 3 2679 2 2685 1 2693 0 2702 2 2724 1 2676 0 2684 0 2709 1 2713 0 2727 2 0.32 0.54 0.48 0.66 0.52 0.79 0.80 0.53 0.58 0.49 0.67 0.78 0.98 0.45 0.59 0.74 0.47 0.90 0.72 0.60 0.51 0.90 0.81 0.98 0.78 MN = Micronucleus PCE = Polychromatic erythrocyte NO.MN PCES - Micronucleated PCEs NCE-Normochromatic erythrocyte CHV Study No.: 17073-0-455 17 CORNINGHazleton MICRONUCLEUS TABLE 3 TEST - INDIVIDUAL SPONSOR:3M Corporation ANIMAL DATA TEST ARTICLE:T-6342 ASSAY NO.:17073 TREATMENT 24 HOUR HARVEST FEMALE ANIMAL NUMBER NO.MN FCES (1000) RATIO PCE:NCE VEHICLE CONTROL Water POSITIVE CONTROL CP 80.0 mg/kg TEST ARTICLE 1250 mg/kg 2500 mg/kg 5000 mg/kg 2741 0 2744 1 2773 0 2776 0 2779 0 2734 31 2746 17 2749 29 2760 17 2783 31 2735 0 2742 0 2750 0 2771 0 2781 0 2732 0 2733 0 2738 0 2755 1 2772 1 2743 0 2757 0 2761 2 2763 2 2768 1 MN = Micronucleus PCE - Polychromatic erythrocyte NO.MN PCEs - Micronucleated PCEs NCE=Normochromatic erythrocyte 0.76 0.76 0.70 0.27 0.94 0.78 0.71 0.62 0.54 0.48 1.03 1.25 0.69 0.39 0.39 0.56 0.64 0.81 0.54 0.67 0.92 0.56 0.83 1.04 0.61 CHV StudyNo.: 17073-0-455 18 CORNINGHazieton MICRONUCLEUS TABLE 4 TEST - INDIVIDUAL SPONSOR:3M Corporation ANIMAL DATA TEST ARTICLE:T-6342 ASSAY NO.:17073 TREATMENT 48 HOUR HARVEST MALE ANIMAL NUMBER NO.MN PCEs (1000) RATIO PCE:NCE TEST ARTICLE 1250 mg/kg 2500 mg/kg 5000 mg/kg 2680 0 2681 0 2695 0 2705 0 2710 0 2675 1 2688 0 2707 0 2726 0 2729 0 2677 1 2699 1 2700 4 2708 0 2712* *Animals found dead MN - Micronucleus PCE = Polychromatic erythrocyte NO.MN PCEs - Micronucleated PCEs NCE=Normochromatic erythrocyte 0.44 0.54 0.77 0.77 0.89 0.52 0.81 0.96 0.82 0.83 0.32 0.64 0.47 0.79 CHV StudyNo.: 17073-0-455 19 CORNINGHazieton MICRONUCLEUS TABLE 5 TEST - INDIVIDUAL ANIMAL DATA SPONSOR:3M Corporation TEST ARTICLE:T-6342 ASSAY NO.:17073 TREATMENT 48 HOUR HARVEST FEMALE ANIMAL NU.MN RATIO NUMBE,, (PiCoEsoo) 'CE:NCE TEST ARTICLE 1250 mg/kg 2500 mg/kg 5000 mg/kg 273o 0 2736 0 2747 1 2754 0 2775 0 2737 0 2745 1 2758 0 2759 0 2762 0 2739 1 2753 0 2756 1 2767 1 2770 0 MN - Micronucleus PCE @ PolychromatiC erythrocyte NO.MN PCEs - Micronucleated PCEs NCE=Normochromatic erythrocyte 0.94 0.62 0.74 0.75 0.64 0.67 0.69 0.62 0.65 0.63 0.43 0.78 0.68 0.67 0.61 CHV StudyNo.: 17073-0-455 20 CORNINGHazleton MICRONUCLEUS TABLE 6 TEST - INDIVIDUAL ANIMAL DATA SPONSOR:3M Corporation TEST ARTICLE:T-6342 ASSAY NO.:17073 TREATMENT ANIMAL NUMBER NO.MN RATIO PCEs (1000) pCE:NCE 72 HOUR HARVEST MALE TEST ARTICLE 1250 mg/kg 2500 mg/kg 5000 mg/kg 2678 0 2687 1 2716 1 2719 1 2722 0 2692 0 2701 3 2704 4 2711 2 2725 1 2686 1 2690 2 2706 2 2714* 2718* *Animals found dead MN - Micronucleus PCE - Polychromatic erythrocyte NO.MN PCEs - Micronucleated PCEs NCE=Normochromatic erythrocyte 0.26 0.30 0.17 0.51 0.56 0.28 0.37 0.52 0.49 0.70 0.11 0.16 0.26 CHV StudyNo.: 17073-0455 21 CORNINGHazleton MICRONUCLEUS TABLE 7 TEST - INDIVIDUAL ANIMAL DATA SPONSOR:3M Corporation TEST ARTICLE:T-6342 ASSAY NO.:17073 TREATMENT ANIMAL NUMBER NU.MN PCES (1000) RATIO PCE:NCE 72 HOUR HARVEST FFAALE TEST ARTICLE 1250 mg/kg 2500 mg/kg 5000 mg/kg 2740 0 2751 0 2766 1 2769 0 2778 0 2748 1 2752 0 2764 0 2774 0 2777 2 2731 0 2765 0 2780* 2782 1 2784 0 0.68 0.48 0.68 0.40 0.71 0.68 0.45 0.54 0.85 0.66 0.24 0.38 0.18 *Animals found dead MN = Micronucleus PCE - Polychromatic erythrocyte NO.MN PCEs - Micronucleated PCEs NCE-Normochromatic erythrocyte CHV StudyNo.: 17073-0-455 22 CORNINGHazleton MOUSE MICRONUCLEUS TABLE 8 HISTORICAL CONTROL DATA 2/95 THROUGH 9/95 FOOLED VEHICLE CONTROL MIN MAX AVG N % MICRONUCLEATED PCEs per KEAN OF 1000 PER ANIMAL MALES FEMALES 1000 PCE t S.E. TOTAL 0.00 0.26 0.08 * 0.06 49 0.00 0.16 0.06 t 0.04 49 0.01 0.18 0.07 t 0.04 49 RATIO MP" MALES PCE:NCE :t S. E. FEMALES 0.32 0.82 0.36 t 0.13 49 0.34 1.03 0.61 t 0.14 49 POSITIVE CONTROL Cyclophosphamide. 80 mg/kg MIN KAX AVG N 1.72 5.44 3.25 :t0.94 22 1.50 6.36 3.00 t 1.05 22 1.81 0.44 5.38 0.72 3.13 t 0.81 0.58 * 0.09 22 1 22 0.44 0.81 0.62 t 0.11 22 CHV StudyNo.: 17073-0-455 23