Document 91aJMzmn8291KwwQ0NjvdYX73

CORNINGHazlcton MUTAGENICITY TEST ON T- 6294 IN AN IN VIVO MOUSE NUCRONUCLEUS FINAL REPORT AU Hemalatha Murli,Ph.D. F;j ASS MAY'L 4 1996 /\ 3m -k 0 tco@- PERFORMING LABORATORY Coming HazletonInc.(CHV) 9200 Leesburg Pike Vienna,Virginia22182 LABORATORY PR JECT IDENTIFICATION CHV StudyNo.: 17385-0-455 SUBMITTM TO 3M 3M Center,Building220-2E-02 St.Paul,MN 55144-1000 $TUDY COMPLEDON DATE May 10, 1996 CHV Study No.: 17385-0-455 1 of 24 CORNINGHazleton QUALITY ASSURANCE STATEMENT ProjecTtitleI:nVivoMouseMicronucleAusssay ProjectNo.: 20996 Assay No.: 17385 ProtocolNo.: 455 EditionNo.: 17 QualityAssuranceinspectionosf thestudyand reviewofthefmalreportoftheabove referenced projectwere conductedaccordingtotheStandardOperatingProceduresof theQualityAssurance Unitand accordingtothegeneralrequirementsof theappropriatGeood LaboratoryPractice regulationsF.indingsfrom theinspectionasnd fmal reportreviewwere reportedtomanagement and to thestudydirectoorn thefollowingdates: Inspection/Date FindingsRepQMed Auditor Dosing/03/12/1996 03/12/1996 C. Orantes Harvest/03/13/1996 03/13/1996 C. Orantes DraftReportReview/05/03,06/1996 05/06/1996 C. Orantes FinalReport Review/05/10/1996 05/10/1996 C. Orantes ----------- QualityAssuranceUnit --f)atReeleased CHV StudyNo.: 17385-0455 2 CORNINGHazleton STUDY COMPLIANCE AND CERTIFICATION The describedstudywas conducted incompliance with the Good LaboratoryPracticeregulations as setforthintheFood and Drug Administration(FDA) Title21 oftheU.S. Code of Federal RegulationsPart58, issuedDecember 22, 1978,(effectivJeune 20, 1979) withany applicable amendments. There were no significandteviationsfrom theaforementionedregulationosr the signedprotocolthatwould affecttheintegritoyfthe studyor theinterpretatiofn thetestresults. The raw datahave been reviewed by the Study Director,who certifietshatthe evaluationof the testarticlaes presentedhereinrepresentsan appropriateconclusionwithinthecontextofthe studydesignand evaluationcriteria. Alltestand controlresultsinthisreportaresupportedby an experimentaldatarecordand this recordhas been reviewed by the Study Director.All raw data,documentation,records,protocol and a copy of thefinalreportgeneratedas a resultof thisstudy willbe archivedinthe storage facilitioefsComing Hazleton Inc.foratleastone yearfollowingsubmissionofthe finalreportto theSponsor. Aftertheone yearperiod,the Sponsor may electto have theaforementioned materialsretainedinthe storagefacilitioefsComing HazletonInc.foran additionapleriodof time,or sentto a storagefacilitdyesignatedby the Sponsor. Submitted By: Study Director: Hemalatha Murli,Ph.D. Mammalian Cytogenetics Department of Geneticand CellularToxicology Study Completion Date CHV Study No.: 17385-0-455 3 CORNINGHazleton TABLE OF CONTENTS Page No. SUMMARY 6 1.0 SPONSOR ...........................................7........ 2.0 MATERIAL (TestArticle)..................................7........ 2.1 ClienfsIdentification 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 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 SELECTION STUDY ................................9........ 10.1 Dose Selection 10.2 DosingInformation 10.3 Resultsand Interpretation 10.4 Conclusion CI-RVStudyNo.: 17385-0-455 4 CORNINGHazleton 11.0 MICRONUCLEUS STUDY ................................1.1........ 11.1 Dose Selection 11.2 MicronucleusAssayDosingInformation 12.0 BONE MARROW HARVEST, SLIDE PREPARATION AND ANALYSIS ......12. 13.0 EVALUATION CRITERIA ................................1.3........ 13.1 General 13.2 Data Presentatioannd Interpretation 14.0 RESULTS AND INTERPRETATION ...........................1.3 ....... 15.0 CONCLUSION .......................................1.5........ 16.0REFERENCES .......................................1.5........ 17.0DEVIATION FROM THE SIGNED PROTOCOL .....................1.5 ....... 18.0 EXPERIMENT DATA TABLES ..............................16........ f CHV Study No.: 17385-0-455 5 CORNINGHazleton SUMMARY MutagenicitTyestonT- 6294inanInVivoMouseMicronucleuAsssay The objectivoefthisinvivoassaywas toevaluatteheabiliotfythetestarticlTe-,6294,to inducemicronucleiinbone marrow polychromaticerythrocyteosf Crl:CD-1'(ICR)BR mice. Inthe dose selectionstudy,thetestarticlweas suspended inacetone:comoil(40%:60%, v:v), and dosed by oralgavage at 1000, 2000, 3000, 4000, and 5000 mg/kg. Six animals (threemales and threefemales)were assignedtoeach dose group. Animals were observedforthreedays after dosing fortoxicsignsand/ormortality. Based on the resultsof thedose selectionstudy,themaximum toleratedose was estimatedas 4000 mg/kg. In themicronucleus assay,thetestarticlweas suspended in acetone:com oil (40%:60%, v:v)and dosed by oralgavage at 1000,2000, and 4000 mg/kg. Ten animals(five males and fivefemales)were randomly assignedto each dose/harvestime group. Vehicle and positivecontrolgroups,euthanizedapproximately24 hours afterdosing,were includedinthe assay.The animals dosed with thetestarticlweere euthanizedapproximately24, 48 and 72 hours afterdosing forextractionof thebone marrow. The testmaterial,T- 6294, did not induce a significanitncreaseinmicronucleiinbone marrow polychromaticerythrocytesunder the conditionsof thisassayand isconsiderednegativeinthe mouse bone marrow micronucleus test. CHV Study No.: 17'385-0455 6 CORNINGHazleton MutageniciTteystonT-6294inaninvivoMouseMicronucleAusssay 1.0 SPONSOR:3M 2.0 MATERIAL (TestArticle) 2.1 Client'IsdentificatioTn-:6294 2.2 Date Received: January16,1996 2.3 PhysicalDescriptionW:ax-likeainbercoloredsolid 2.4 GeneticsAssay No.: 17385 3.0 TYPE OF ASSAY: In Vivo Mouse Micronucleus Assay 4.0 PROTOCOL NO.: 455,Edition17 5.0 STUDY DATES 5.1 InitiatiDoante: January18,1996 5.2 ExperimentalStartDate: March 6, 1996 5.3 ExperimentalTerminationDate: April1,1996 6.0 SUPERVISORY PERSONNEL 6.1 StudyDirector:HemalathaMurli,Ph.D. f 6.2 LaboratorySupervisor:Monica Vegarra,B.S. 7.0 OBJECTIVE The objectivoefthisinvivoassaywas toevaluatetheabilitoyfthetestarticlTe-, 6294, toinducemicronucleinbone marrow polychromaticerythrocyteosfCrl:CD-IO(ICR)BR mice. Thisstudywas conductedusingmodificationosf theproceduressuggestedby Heddle etal.(1983). CHV StudyNo.: 17385-0455 7 CORNINGHazleton 8.0 MATERIALS Adult male and femalemice,strainCrl:CD-II(ICR) BR, were purchasedfrom Charles River LaboratoriesP,ortage,NH. This healthy,random bred strainwas selectedto maximize geneticheterogeneityand atthe same time assureaccessto a common source. The protocolforthisstudywas approved by the CHV-ACUC priortothe initiationf dosing. Animals were housed fiveper cage duringquarantine,and housed fiveatrandomization. The temperatureand relativhetuniditywere maintainedat726'F and 5515%, respectivelye,xcepton March 9, 1996,when the relativheumidity was recordedas 38.1%. A 12-hourlight1/2-hourdarkcyclewas maintained.A commercial diet(Purine CertifiedLaboratoryPelletsI # 5002) and waterwere availablea-dlibitumforthe durationofthe study.The feedwas analyzedby the manufacturerforconcentrationosf specifiedheavy metals,aflatoxinc,hlorinatedhydrocarbons,organophosphates,and specifiednutrients.The water was analyzedon a retrospectivbeasisforspecified microorganisms,pesticidesa,lkalinityh,eavy metals,and halogens. Sanitizedcagingwas used forhousingtheanimals. Personnelhandlinganimalsor working withintheanimal facilitiweesre requiredtowear suitableprotectivegarments and equipment. Animals were quarantinedforseven days beforebeing placed on study. Animals were randomly assignedto studygroups and were individuallwyeighed priortodosing. All animals were dosed based upon theindividualbody weights. Animals were uniquely identifiebdy eartag.Dose or treatmentgroupswere identifiebdy cage card/label. At theterminationofthe studyallsurvivinganimalswere euthanizedby C02 inhalation followedby penetrationofthethorax. 9.0 SOLUBILITY AND STABILITY: f The testarticlTe-, 6294,was suppliedas a wax-likeamber coloredsolid.The solubility ofthe testarticlweas evaluatedin2% high viscositycarboxymethylcellulos(eCMC) and thiswas not a suitablveehicle. Solubilitwyas then evaluatedinacetone:com oil (40%:60%, v:v)and a translucentc,ream-coloredemulsion was obtainedata concentrationof approximately421.75 mg/ml, which formed a bilayeraftera shortperiod of time. Shaking thisbilayerresultedinthe re-emulsificatiofnthemixture. Acetone:com oil(40%:60%, v:v)was thevehicleof choiceforthisassay.The stability of thetestmaterialunder thedosingconditionsof thisassay isthe responsibilitoyfthe sponsor. CHV Study No.: 17385-0-455 8 CORNINGHazleton 10.0 DOSE SELECTIONSTUDY 10.1 Dose Selection Dose levelsof 1000,2000,3000,4000,and 5000 mg/kg were administerebdy oralgavageforthedose selectiosntudy. 10.2 Dosing Information The animalsused inthedoseselectioanssaywere dosedon March 6,1996. The weightrangeoftheanimalsused inthedoserangefindingassaywas 26.6-34.7 and 23.1-26.7grams,forthemales andfemales,respectivelDyo.sing solutions were preparedjustpriortodosingand werepreparedby making a 500 mg/ml stockforthehighdose(5000mg/kg).Thiswas preparedby adding7.5ml of acetone(Sigma,Lot# 2435KHXG):com oil(Duke'scom oilL,ot # 5D1712:46), (40%:60%, v:v)to5.0025g ofT- 6294,resultinigna translucenttanandyellow bilayer(bottomand top)thatbecame anemulsionupon shakingwitha final volume of10.0ml. Dilutionosfthisstockwere preparedforthe1000,2000, 3000 and 4000 mg/kg doselevels.Alldosingstockswereplacedon magneticstir platesduringthedosingprocedure. Dosingwas achievedusinga 10.0ml/kgdosingvolume. Allanimalswere eight weeks and two daysoldatthetimeofdosing.An outlineofthedosingscheme is foundinthefollowingtable. DOSE GROUPS TREATMENT mF ----------------------------------------------------- T- 6294 1000 mg/kg 33 2000 mg/kg 3000 mg/kg 4000 mg/kg 33 33 33 5000 mg/kg 33 ----------------------------------------------------- Alldosesgivenwere on an acute(one-timoenly)basis.A totaolf30 animalswas used inthisassay. CHV StudyNo.: 17385-0-455 9 CORNINGHazleton 10.3 Resultsand Interpretation All animalswere examined afterdosingand dailythroughoutthedurationofthe study(threedays)fortoxiceffectasnd/ormortalitieAsl.lanimalsappeared normal immediatelyafterdosing. Approximately I hour afterdosing,allanimalsinalldose groupsappeared hypoactiveand hunched. Approximately24 hours afterdosing,allanimalsinalldosegroupsappeared hypoactiveand the5000 mg/kg dose group alsoappearedhunched. Approximately44 hoursafterdosing,allanimalsinalldosegroupsappeared hypoactiveand hunched. Some animalsinthe1000,2000,and 3000 mg/kg dose levelshad squintedeyes and othershad dyspnea. Some animalsinthe4000 and 5000 mg/kg dose levelshad dyspnea and allhad rough haircoatsO.ne female (# 6689) from the5000 mg/kg dosegroupwas founddead. Approximately74 hours afterdosing,allanimalsinalldosegroupsappeared hypoactiveand hunched. The mortalitdyataforthisassayaresummarized inthe followingtable: Summary ofMortalitieWsithin 3 Days inMice Dosed Acutelywith T- 6294 Observations Treatment 1000 mg/kg 2000 mg/kg 3000 mg/kg 4000 mg/kg 5000 mg/kg Male Female 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 1/3 10.4 Conclusion Based on theseresultst,hemaximum toleratedose was estimatedtobe 4000 mg/kg. CHVStudyNo.: 17')85-0-455 10 CORNINGHazleton 11.0 MICRONUCLEUS STUDY 11.1 Dose Selection Based on resultsfrom the dose selectionstudy,dose levelsof 1000, 2000, and 4000 mg/kg were selectedfortestinginthisstudy. 11.2 MicronucleusAssay Dosing Information The animals used inthemicronucleus assay were dosed on March 12, 1996. Cyclophosphamide (CAS # 6055-19-2;Sigma,Lot # 44H0486), thepositive control,was solubilizeidnsterildeeionizedwater(Lot# 19,preparedatCHV) and was administeredby oralgavage at80.0mg/kg. The vehiclecontrol,acetone (Sigma,Lot # 2435KHXG):com oil(Duke'scom oil,Lot # 5D1712:46), 40%:60%, v:v,was administeredconcurrentlywith the testarticlaeta volume of 10.0ml/kg. The weight range ofthe animals used inthe micronucleusassaywas 25.3- 35.6 and 21.4 - 28.4grainsforthemales and females,respectivelyT.he dosingsolutionsfortheassay were preparedby making a 400 mg/ml stockforthe high dose (4000 mg/kg). This was preparedby adding thevehicletothetest articluep toa volume of 25 mi and stirrinvgigorouslywith a spatula.A translucenttan and yellow bilayer(bottomand top)thatbecame an emulsion upon shakingwas obtained.Dilutionsof thisstockwere preparedfortheremaining dose levels.All dosingstockswere placedon magnetic stirplatesduring preparationand the dosingprocedure.A second group of animals(designated Secondary Dose Group) was alsoassignedtothe studyand was dosed withthe high dose of thetestarticleT.hese animalswere only used intheassay as replacementsforany which diedinthe primary dose group. Ten animals (fivemales and fivefemales)were randomly assignedto each dose/- harvesttime group. Vehicle and positivecontrolgroups,euthanized f approximately24 hours afterdosing,were includedinthe assay.The animals dosed with thetestarticlweere euthanizedapproximately24,48 and 72 hours afterdosingforextractioonf the bone marrow. An outlineofthe dosingscheme isfound inthe followingtable: CHV StudyNo.: 17385-0-455 11 CORNINGHazleton DosingScheme forMicronucleuAsssay Treatment Number ofAnimalsAssigned Primary Dose Groups Secondary Dose 24 lir 48 Hr 72 Hr Groups" M F M F M F Male Female T- 6294 100 mg/kg 200 mg/kg 55 55 55 55 55 55 - - 400 mg/kg 55 55 55 5 5 VehicleControl,40% Acetone/60% Com Oil 10.0 ml/kg 55 -- -- - - PositiveControl,Cyclophosphamide, 80.0mg/kg 5 5 - - - - The animals assignedtothe secondarydose groups were dosed and were only used to replaceanimalswhich diedinthe primarydose group atthehigh dose level.All extra animals not used as replacementswere euthanizedatthecompletion of thetrial. The age ofthe animalsatthetime of dosingwas eightweeks and one day. A totalof 120 animals was used in thisassay Volumes dosed were 10.0ml/kg based upon individualanimal weights. 12.0 BONE MARROW HARVEST, SLIDE PREPARATION AND ANALYSIS At theappropriathearvesttime,theanimalswere euthanizedwithC02, followedby penetrationof thethorax.The adheringsofttissueand epiphysesofboth femora were removed. The marrow was flushedfrom thebone and transferretdo centrifugetubes containing3 - 5 ml bovineserum (onetube foreach animal).Followingcentrifugatiotno pelletthe tissue,thesupernatantwas removed by aspiratioannd portionsof the pellet were spreadon slidesand airdried.The slideswere fixedinmethanol,and stainedin May-Gnmwald solutionfollowedby Giemsa (Schmid, 1975).The air-driesdlideswere coverslippedusing Depex* mounting medium. The slideswere coded foranalysisa,nd scoredformicronucleiand thepolychromatic erythrocyt(ePCE) to normochromaticerythrocyt(eNCE) cellratio.Standardforms were usedto recordthesedata.One thousandPCEs peranimalwere scored.The frequencyof micronucleatedcellswas expressedas percentmicronucleatedcellsbased on thetotal PCEs presentinthe scoredopticfleld.The normal frequencyofmicronucleiinthis Crl:CD-1"(ICR) BR strainisabout0.0-0.4%. CHV Study No.: 17385-0-455 12 CORNINGHazleton ThefrequenocfyPCEsversuNsCEs wasdeterminbeydscorintghenumberofPCEsand NCEs observedintheopticfieldswhilescoringthefirs1t000 erythrocytes. 13.0 EVALUATION CRITEF-IA: 13.1 General The criterifaortheidentificatioofnmicronuclewiere thoseofSchmid (1976). Micronucleiwere darklystainedand generallryound,althoughalmond and ringshapedmicronucleioccasionalloyccurred.Micronucleihad sharpborders and were generallybetween 1/20and 115thesizeof thePCE. The unitof scoring was themicronucleatedcelln,otthemicronucieust;hustheoccasionalcellwith more thanone micronucleuswas countedasone 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 analysisof thesedatawas performedusingan analysisof variance(Willer1,971)on eitheruntransformed (when variancesarehomogeneous) and ranktransformed(when variancesare heterogeneous)proportionosf cellswithmicronucleiperanimal.Iftheanalysis ofvariancewas significa(npt<0.05)a,Dunnett'ts-tes(tDunnett,1955;1964)was usedtodeterminewhich dosegroups,ifany,were significantdliyfferenftrom the negativecontrol.Analyseswere performedseparatelfyoreachharvesttimeand sexcombination.The criterifaordetermininga positivreesponseinvolveda statisticalsliygnificandtose-relateidncreaseinmicronucleatedPCES, orthe detectioonf a reproduciblaend statisticaslilgynificanptositiveresponseforat leastone doselevel.A testarticltehatinducedneithera statisticaslilgynificant dose responsenor a statisticalsliygnificanatnd reproducibleincreaseatone dose levelwas considerednegative.In eithercase,thefinaldecisionwas based on scientifjiucdgment. 14.0 RESULTS AND INTERPRETATION: All animalswere observedimmediatelyafterdosingand periodicalltyhroughoutthe durationof theassayfortoxicsymptoms and/ormortalitiesA.llanimalsinthevehicle and positivceontrolgroupsappearednormal afterdosingand remainedhealthyuntilthe appropriateharvesttimes. CHV StudyNo.: 17385-0455 13 CORNINGHazleton AlltesatrticdloesegdroupasppearneodrmailmmediataefltyedrosingA.pproximately one hourafterdosing,allanimalsatalldoselevelsappearedhypoactive. Approximately21.5hoursafterdosing,allanimalsinthe1000mg/kg dosegroup appearedslightlhyypoactivew,ithsome showingsignsofdyspnea.Allanimalsinthe 2000 mg/kg dosegroup appearedhypoactivew,ithmostshowing signsofdyspnea,and some femalesalsohad roughhaircoatsand lacriminatioAnl.lanimalsinthe4000 mg/kg dose groupappearedhypoactive,withmost showing signsofdyspnea,and most females alsohad ungroomed haircoatsand excessivelacriminationT.hreefemales(#'s72437,2 hourharvestgroup;7133,7214,secondarydosegroup)from the4000 mg/kg dosegroup were found dead. Approximately45.5hoursafterdosing,allanimalsinthe1000mg/kg dosegroup appearedslightlhyypoactivew,ithsome havingroughhaircoats.Allanimalsinthe 2000 mg/kg dosegroup appearedslightlhyypoactivea,nd some femalesalsowere ungroomed andhunched. One female(#7209)fromthe2000 mg/kg doseand 72 hour harvestgroupwas found dead. Allanimalsinthe4000 mg/kg dosegroupappeared slightlhyypoactivew,ithseveralshowing signsofdyspnea,and some femalesalsowere hunched andhad ungroomed haircoats. Approximately69.5hoursafterdosing,allanimalsinalldose groupsappearednormal, exceptforone female(#7192)from the1000 mg/kg dosegroupappearedhypoactive, coldto touch,had dyspnea,and was hunched. The testarticlTe-. 6294,inducedno significainntcreaseisnmicronucleated polychromaticerythrocyteosverthelevelsobservedinthevehiclecontrolisneithesrex oratany oftheharvestimes.The PCE/NCE ratioisnthemalesfromthepositivceontrol group,24 and 48 hourmales from the 1000 and 2000 mg/kg dose groups,and 48 hour males fromthe4000 mg/kg dosegroupwere significanthliygherthanthecorresponding vehiclecontromlales.The positivceontrolC,P, inducedsignificainntcreaseisnmicrof nucleatePdCEs inbothsexesascomparedtothevehiclecontrolsw,ithmeans and standarderrorsof2.42% 0.12% and 5.14% 0.65% forthemalesand females,respectivelyT.he datasummarizedby dosegrouparepresenteidnTableI and individual animaldataarefoundinTables2 through7. Historicaclontroldataarepresentedin Table 8. CHV StudyNo.: 17385-0-455 14 CORNINGHazleton 15.0 CONCLUSION: The testmaterialT,- 6294,didnotinducea significainntcreasienmicronucleinbone marrow polychromatiecrythrocyteusndertheconditionosfthisassayand isconsidered negativeinthemouse micronucleusassay. 16.0 REFERENCES: Dunnett,C.W.: A multiplecomparisonsprocedureforcomparingseveratlreatmentwsith a controlJ..Am. StatisAts.soc.,U: 1096-1121,1955. Dunnett,C.W.: New tablesformultiplecomparisonswitha controlB.iometrics2,0:482491,1964. Heddle,J.A.,Hite,M., KirkhartB,.,Larsen,K.,MacGregor,J.T.N,ewell,G.W. and Salamone,M.F.:The inductioonfmicronucleaisa measureofgenotoxicity. MutationRes.,123:61-118,1983. Schmid,W.: The micronucleutsest.MutationRes.,31:9-15,1975. Schmid,W.: The micronucleutsestforcytogenetiacnalysisC.hemical Mutagens:Principleasnd Methods forTheirDetectionV,ol.4 (A.Hollaender, ed.).Plenum,pp.31-53,1976. Winer,B.J.:StatisticParlincipleisnExperimenW Design,McGraw-Hill,New York, Second Edition1,971. 17.0 DEVIATION FROM THE SIGNED PROTOCOL Due tounknown reasonso,n March 9,1996,therelativheumiditywas recordedas 38.1%. Thishad no impacton theanimalsortheintegritoyfthestudy. CHV StudyNo.: 17385-0-455 15 CORNINGHazleton 18.0 EXPEPIMENT DATA TABLES f CHV StudyNo.: 17385-0-455 16 CORNINGHazleton TABLE I SPONSOR: 3M TEST ARTICLE: T-6294 N,NCRONUCLEUS DATA SUMMARY TABLE ASSAY: 17385 TREATMENT DOSE HARVEST TIMIE (HR) CONTROLS VEFUCLE 400/.Acetone/ 24 hr 600/.Com oil POSITIVE CP 80.0mg/kg 24 hr % MICRONUCLEATED PCEs MEAN OF 1000PER ANIMAL + S.E. MALES FEMALES TOTAL 0.12:k0.06 0.02:L0.02 0.07:k0.03 2.42* 0.12* 5.14 0.65* 3.78 0.55* RATIO PCE:NCE M[EAN:E S.E. MALES FEMALES 0.59 0.07 0.74:L0.10 0.89 0.07* 0.91 0.05 TEST ARTICLE 1000mg/kg 24 hr 48 hr 72 hr 2000mgtkg 24 hr 48 hr 72 hr 4000 mg/kg 24 hr 48 hr 72 hr 0.08-+0.06 0.10 0.0.4 0.14:k 0.04 0.02 +0.02 0.12 0.04 0.20 0.07 0.14 0.07 0.04 0.02 0.10:b0.00 0.04 0.02 0.12 0.06 0.30 0.29 0.06 0.06 0.04 0.02 0.031:0.03 0.12 0.06 0.08 0.06 0.04+0.02 Sigpificantglryeatetrhanthecorrespondinvgehiclecontrolp,<0.05. 0.06 0.03 0.11 0.03 0.22:k0.13 0.04:k0.03 0.08 0.02 0.12 0.05 0.13 0.04 0.06 0.03 0.07 0.02 1.05 0.030 0.92 0.07* 0.54:L 0.08 1.02 0.05* 0.89 0.040 0.48 0.09 0.67 0.07 0.93 0.04* 0.48 0.07 0.84 0.03 1.00:k0.03 0.58:L0.05 0.82:L0.09 1.00 0.06 0.58 0.08 0.76 0.05 0.80 0.07 0.55 0.03 1 CP = Cyclophosphamide CHV StudyNo.: 17385-0-455 17 CORNINGHazleton MICRONUCLEUS SPONSOR: 3M TEST ARTICLE: T-6294 ASSAY NO.: 17385 TREATMENT 24 HOUR HARVEST TABLE2 TEST - INDIVIDUAL ANIMAL DATA ANIMAL NUMBER # MN RATIO PCEs/ PCE-NCE 1000 PCEs MALE VEHICLE CONTROL POSITIVE CONTROL TEST ARTICLE 40% Acetone/600/.Com oil 7128 0 7130 0 7167 3 7169 2 7179 1 CP 80.0 m&g 7132 25 7134 26 7139 27 7153 22 7155 21 1000 mglkg 7129 0 7149 3 7159 1 7165 0 7176 0 2000 mgtkg 7136 0 7162 1 7166 0 7170 0 7180 0 4000 mgtkg 7142 0 7150 1 7157 0 7163 2 7171 4 0.75 0.68 0.32 0.60 0.62 0.95 0.74 1.11 0.89 0.78 1.08 0.99 1.08 0.97 1.11 0.89 1.06 1.10 1.12 0.91 0.67 0.59 0.73 0.47 0.87 CP Cyclophosphamide ,N4N Micronucieus PCE Polychromatic erythrocyte # MN PCEs = Micronucleated PCEs NCE = Norrnochromatic erythrocyte CHV StudyNo.: 17385-0-455 18 CORNINGHazleton TABLE3 MICRONUCLEUS TEST - INDIVIDUAL ANIMAL DATA SPONSOR: 3M TEST ARTICLE: T-6294 ASSAY NO.: 17385 TREATMENT 24 HOLRR HARVEST FEMALE ANIMAL NUMBER # MN PCEst RATIO 1000 PCEs PCE-NCE VE19CLE CONTROL POSITIVE CONTROL TEST ARNCLE 40% Acetone/60%Com oil 7190 0 7194 0 7200 1 7215 0 7222 0 CP 80.0mgfkg 7196 52 7201 56 7207 36 7208 40 7211 73 1000 mg/kg 7189 1 7193 1 7199 0 7237 0 7242 0 2000 mgtkg 7204 0 7216 0 7225 0 7233 3 7241 0 4000 mgfkg 7206 1 7221 0 7229 2 7232 0 7235 3 0.96 0.63 0.79 0.90 0.41 0.98 1.04 0.84 0.75 0.92 0.80 0.94 0.85 0.78 0.86 0.97 1.01 0.51 0.80 0.79 0.95 0.71 0.76 0.71 0.66 CP Cyclophosphamide MN Micronucieus PCE Polychromaticerythrocyte # MN PCEs = MicronucleatedPCEs NCE = Norinochromaticerythrocyte CHV StudyNo.: 17385-0-455 19 CORNINGHazleton MICRONUCLEUS SPONSOR: 3M TEST ARTICLE: T-6294 ASSAY NO.: 17385 TREATMENT 48 HOUR HARVEST TEST ARTICLE TABLE4 TEST - INDIVIDUAL ANIMAL DATA MALE 1000 mgtkg 2000 mgtkg 4000 mgtkg ANlN4AL NUMBER # MN PCEs/ 1000 PCEs RATIO PCE:NCE 7131 2 0.83 7135 0 0.89 7158 2 0.81 7177 1 0.90 7178 0 1.18 7137 0 0.89 7145 2 0.96 7151 1 0.73 7172 1 0.89 7182 2 0.99 7127 0 0.93 7140 0 0.81 7141 1 0.85 7144 1 1.02 7160 0 1.03 MN Micronucleus PCE Polychromaticerythrocyte # MN PCEs = MicronucleatedPCEs NCE = Normochromatic erythrocyte CHV StudyNo.: 17385-0-455 20 CORNINGHazleton MICRONUCLEUS TABLE5 TEST -INDIVIDUAL ANIMAL DATA SPONSOR: 3M TEST ARTICLE: T-6294 ASSAY NO.: 17385 TREATMENT 48 HOUR HARVEST FEMALE ANIMAL NUMBER # MN RATIO PCEs/ PCE:NCE 1000 PCEs TEST ARTICLE 1000 mgtkg 2000 mgtkg 4000 mg/kg 7186 3 0.89 7212 2 1.10 7217 0 1.04 7228 0 0.98 7236 1 0.98 7197 0 1.04 7205 1 1.18 7226 1 0.94 7227 0 0.81 7245 0 1.02 7188 1 0.63 7210 0 0.85 7219 3 1.03 7220 0 0.86 7239 0 0.66 NfN Micronucleus PCE Polychromaticcrarocyte # MN PCEs = MicronucleatePdCEs NCE = Nonnochromaticcrythrocyte CHV StudyNo.: 17385-0455 21 CORNINGHazleton MICRONUCLEUS SPONSOR: 3M TEST ARTICLE: T-6294 ASSAY NO.: 17385 TREATMENT 72 HOUR HARVEST TEST ARUCLE TABLE6 TEST -INDIVIDUAL ANIMAL DATA MALE 1000 mglkg 2000 mgtkg 4000 mgtkg ANIMAL NUMBER # NIN RATIO PCEs/ PCE:NCE 1000 PCEs 7143 0 0.34 7146 1 0.65 7148 2 0.79 7154 2 0.49 7183 2 0.42 7138 0 0.74 7147 1 0.47 7156 4 0.64 7175 2 0.30 7184 3 0.26 7126 1 0.72 7152 1 0.47 7164 1 0.49 7181 1 0.34 7185 1 0.38 MN Micronucieus PCE Polychromatiecrythrocyte # MN PCEs = MicronucleatedPCEs NCE = Normochromaticerythrocyte CHVStudyNo.: 17385-0-455 22 CORNINGHazleton TABLE7 MICRONUCLEUS TEST - INDIVIDUAL ANIMAL DATA SPONSOR. 3M TEST ARTICLE: T-6294 ASSAY NO.: 17385 TREATMENT 72 HOUR HARVEST FEMALE ANIMAL NUMBER # MN PCEst 1000 PCEs RATIO PCE:NCE TEST ARNCLE 1000 mgtkg 2000 mg/kg 4000 mgtkg 7187 0 0.56 7192 14 0.73 7195 0 0.47 7213 1 0.64 7223 0 0.52 7191 0 0.58 7199 0 0.39 7209* 7219 0 0.59 7230 1 0.77 7202 1 0.62 7203 0 0.56 7224 0 0.46 7231 0 0.51 7234 1 0.59 Animal found dead &fN Micronucieus PCE Polychromaticerythrocyte # MN PCEs = MicronucleatedPCEs NCE Normochromatic erythrocyte CHV StudyNo.: 17385-0455 23 CORNINGHazleton TABLE8 MOUSE MICRONUCLEUS HISTORICAL CONTROL DATA 7/95 THROUGH 12/95 POOLED VEI-HCLE CONTROLS MIN MAX AVG N % MICRONUCLEATED PCEs PER 1000 PCE MEAN OF 1000 PER ANIMAL * S.E. MALES FEMALES TOTAL 0.00 0.22 0.087:k0.007 47 0.00 0.24 0.081:Lo.oog 47 0.01 0.17 0.094* 0.005 47 POSITIVE CONTROLS Cyclophosphwnide,90.0mgtkg NffN MAX AVG N 2.00 5.68 3.692* 0.240 19 PCE Polychromaticerythrocyte NCE Norinochromaticerythrocyte 1.50 6.36 3.170:k0.245 19 2.41 5.38 3.426 :k0.184 19 RATIO PCE:NCE MEAN * S.E. N4ALES FEMALES 0.31 0.85 0.550:k0.021 47 0.24 1.03 0.587A:0.025 47 OAI 0.72 0.577* 0.020 19 0.40 0.79 0.588:E0.026 19 CHV Study No.: 17385-0455 24 CHV STUDY NO. PROTOCOL NO. 455. EDITION 17 CORNINGHazleton IN VIVO MOUSE HICRONUCLEUS 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 I. SPONSOR IDENTIFICATION Company Name: 3A4 Address: @5f - INFORMATION AND APPROVALS 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 t In order to comply with the GLP regulations, consulting laboratories must be notified if all or part 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 IL---i MAFF IL---J MOHW FDA 'L--J EPA-TSCA rr@@= rr-,==9 LL===='J OECD IL-@] OTHER IL----i EPA-FIFRAL 4/95 1 of 10 PROTOCOL NO. 455, EDITIOII 17 V- STUDY ]DATES Proposed Experimental Start Date: Proposed Experimental Termination Date: VI- APPROVAL OF STUDY PROTOCOL Study Director: Hemalatha Murli, Ph.D. Sponsor's Authorized Representative: CORNINGHa7leton Date.- Date: f 4/95 2 of 10 PROTOCOL NO. 455, EDITION 17 CORNINGHazleton PART 2 - STUDY PROTOCOL 1. OBJECTM IN VIVO MOUSE HICRONUCLEUS ASSAY 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. DEFINITIONS Hicronucleust a small chromatin body, consistingof 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. RATIONALE 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 micronuclei. 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, 0.5Z aqueous carboxymethylcellulose solution, or corn oil. V. EXPERIHENTAL 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 ---iveprior to experiment initiation. Animals are housed under the following climatic conditions: temperature, 72*F 6'F; humidity, 55Z 151; light cycle, 12 hours light/dark. A commercial diet (Purir.at 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 hydroc;rbons, organophosphates, and specified nutrients. The water is analyzed biannually on a retrospective basis for specified microorganisms, pestic'-des, heavy metals, alkalinity, and halogens. A-i;-zralwsill be quarantined for at least 7 days before being placed on study. Aniir.alswill be assigned to study groups at random accord;-ng to Coning Hazleton Standard Operating Procedures. Animals will be weighed prior to dosing. They w4-ll be dosed based upon the individual animal weights. Animals will be uniquely identified by ear tag. Treatment groups will be identified by cage labellcard. 4/95 4 of 10 PROTOCOL NO. 455, EDITION 17 CORNINGHazleton Sanitary cages will be used. Personnel handling animals or working within the animal facilitieswill 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 normally be used as the intermediateand low dose levels, respectively. Uae 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 mg/kg. 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 CORNINGHa7leton 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, 0.91 saline, 0.5Z aqueous carboxymethylcellulose solution, or corn 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 estimate the Maximum Tolerated Dose (MTD). Doses will then be assigned for the subsequent cytogenetics assay. MICROMUCLEUS STUDY C. Dosing Schedule and Route of Administration Normally an acute dosing regimen (single administration) will be used (see Table below). Harvest will be approximately 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 ani-mals 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 of 10 PROTOCOL NO. 455, EDITION 17 CORNINGHzzleton employed. The animals will be observed toxic signs and mortality. daily for NUMBER OF ANIMALS USED FOR M:ICRONUCLEUS ASSAY Group No. Treatment Harvest Times After Treatment (Males and Females) 24 Hours 48 Hours 72 Hours 1 Positive Control 5+5 2 Vehicle Control 5+5 ----- ----- 3 Low Dose 5+5 5+5 5+5 4 Medium Dose 5+5 5+5 5+ 5 5 High Dose 5+ 5 5 +5 5+ 5 Total 5+ 5 5+ 5 15 + 15 15 + 15 15 + 15 TOTAL 25 + Z5 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 mlfkg. 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 CORNINGHa7leton 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. Vi. DATA The criteria for the identification of micronuclei are those of Schmid (1976). Micronuclei 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 micranucleus 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 significant positive 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 CORNINGHa7leton VII. TEST INTERPRETATION The analysis of this data will be performed using an analysis of variance (Winer, 1971) on either untransformed (when variances are homogeneous) or rank transformed (when variances are heterogeneous) proportions of cells with micronuclei per animal. If the analysis of 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.. 50:1096-1121, 1955. Dunnett, C.W.: New tables for multiple comparisons with a control. Biometrics, 20:482-491, 1964. Heddle, J.A., Hite, M., Yirkhart, 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 Re.s., 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 FORMAT CHV employs a standard report format for each assay design. The final report will provide the following informtion. 0 Sponsor identification. 0 Quality Assurance statement. 0 Statement of GLP Compliance. 0 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 Interpretation of results. 9 Conclusions. 0 References. 0 Test results presented in tabular form. X. CHMGES OR REVISIONS Any changes or revisions of this approved protocol will be documented, signed by the Study Director. dated, and maintained with this protocol. XI. ANIMAL 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 concerning the care and use of animals. If not in compliance, a modification will be required. Any changes or revisions of this approved protocol will be sent to the CHV-TACUC for their review. XII. RECORDS TO BE MAINTAINED 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 facilities of Corning Hazleton Inc. for an additional period of time or sent to a storage facility designated by the sponsor. 4/95 10 of 10 AMENDN4ENT TO THE STUDY PROTOCOL STUDY TITLE: IN VIVO MOUSE MICRONUCLEUS ASSAY PROTOCOL NO.: 455,Edition17 STUDY NO.: 17385-0-455 Page I of I Amendment #I Section2,PartV.B. The SponsorhasLD50 datainratsof2459 mg/kg inmales and 1580 mg/kg infemales.Based on thisinformationt,hedoseselectiosntudywillbe conductedtestindgosegroupsof 1000,2000,3000,4000,and 5000 mg/kg. The testarticlweillbe solubilizeidnacetone/comoil mixture. STUDY DIRECTOR HemalathaMurli,Ph.D. Date Mammalian Cytogenetics DepartmentofGeneticand CellulaTroxicology AMENDMENT TO THE STUDY PROTOCOL STUDY TITLE: IN VIVO MOUSE MICRONUCLEUS ASSAY PROTOCOL NO.: 455,Edition17 STUDY NO.: 17385-0-455 Page I of I Amendment #2 Section2,PartV.C Based on theresultosfthedoseselectiosntudy,doselevelsof 1000,2000, and 4000 mg/kg willbe testedinthemouse micronucleusassay.A secondarydosegroupwillalsobe used. STUDY DIRECTOR HemalathaMurli,Ph.D. Mammalian Cytogenetics DepartmentofGeneticand CellulaTroxicology -3)t)iqb Date