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CORNINGHazleton
MUTAGENICITY TEST ON T-6292
IN AN flVVIVO MOUSE L@ECRONUCLEUS ASSAY
FINAL REPORT
AUTHOR HemalathaMurli,Ph.D.
PERFORMING LABORATORY C-oming HazletonInc.(CHV) 9200 LeesburgPike Vienna,Virginia22182
LABORATORY PROJECT IDENTIFICATION CHV StudyNo.: 17384-0-455
SUBMITTED TO 3M
3M Center,Building220-2E-02 St.Paul,Minnesota55144-1000 STUDY COMPLETION DATE
May 2,1996
L.!J)
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QUALITY ASSURANCE STATEMENT
ProjeTcittlIen:VivoMouseMicronuclAesussay
ProjecNto.: 20996
AssayNo.: 17384
ProtocolNo.:455
EditionNo.:17
QualityAssuranceinspectionosfthestudyandreviewofthefinalreportoftheabove referenced
projectwere conductedaccordingtotheStandardOperatingProceduresoftheQualityAssurance Unitand accordingtothegeneralrequirementosftheappropriatGeood LaboratoryPractice regulationsF.indingsfromtheinspectionasnd finalreportreviewwere reportedtomanagement and tothestudydirectoorn thefollowingdates:
InsRgction/Date
FindingsRe-Ported
Auditor
WeighingofTestArticle/03/05/1996 03/05/1996
C. Smith
DilutioonfTestArticle/03/05/1996 03/05/1996
C. Smith
D mftReportReview/04/24/1996
04/25/1996
C. Orantes
FinalReportReview/05/02/1996
05/02/1996
C. Orantes
QualityAssurance t
I5atel@eleased
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STUDY COWLIANCE AND CERTTFICANON
The describedstudywas conductedincompliancewiththeGood LaboratoryPracticreegulations assetforthintheFood and Drug Administratio(nFDA) Title21 oftheU.S.Code ofFederal RegulationsPart58,issuedDecember 22,1978,(effectiJvuene20,1979)withanyapplicable amendments. Therewereno significadnetviationfsromtheaforementionerdegulationosrthe signedprotocolthatwould affectheintegritoyfthestudyortheinterpretatioofnthetestresults. Tle raw datahavebeenreviewedby theStudyDirectorw,ho certifitehsattheevaluatioonfthe testarticlaespresentedhereinrepresentasn appropriatceonclusionwithinthecontextofthe studydesignand evaluatiocnriteria.
Alltestand controlresultisnthisreportaresupportedby anexperimentadlatarecordand this recordhasbeenreviewedby theStudy Director.Allraw data,documentationr,ecordsp,rotocol and a copyofthefmalreportgeneratedasa resultofthisstudywillbe archivedinthestorage facilitioefsComing HazletonInc.foratleasotne yearfollowingsubmissionofthefinarleporto theSponsor.Aftertheone yearperiod,theSponsormay electohavetheaforementioned materialrsetaineidnthestoragefacilitioefsComing HazletonInc.foran additionapleriodof time,orsenttoa storagefacilitdyesignatebdy theSponsor.
SubmittedBy:
StudyDirector:
HemalathaMurli,Ph.D. Mammalian Cytogenetics DepartmentofGeneticand CellulaTroxicology
StudyCompletion Date
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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
InitiatiDoante
5.2
ExperimentalStartDate
5.3
ExperimentaTlerminationDate
6.0 SUPERVISORY PERSONNEL ..............................7 ........
6.1
StudyDirector
6.2
LaboratorySupervisor
7.0 OBJECTIVE ........................................7........
8.0 IVIATERIALS .......................................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
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11.0 MCRONUCLEUS STUDY ...............................I.I........ 11.1 Dose Selection 11.2 MicronucleusAssay DosingInformation
12.0 BONE MARROW I-IARVEST,SLIDE PREPARATION AND ANALYSIS ....1.2
13.0 EVALUATION CRITERIA ...............................1.3 ........ 13.1 General 13.2 Data Presentatioannd Interpretation
14.0 RESULTS AND INTERPRETATION ..........................13.........
15.0 CONCLUSION ......................................1.5........ 16.0 REFERENCES ......................................1.5........
17.0 DEVIATION FROM THE SIGNED PROTOCOL ...................1.5......
18.0 EXPERINM'NT DATA TABLES ............................1.6.........
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SUNB4ARY
MutagenicitTyeston T-6292 inan In VivoMouse NficronucleuAssay
The objectivoefthisinvivoassaywas toevaluatteheabilitoyfthetestarticlTe-,6292,toinduce micronucleiinbonemarrow polychromatiecrythrocytoefsCrl:CD-I"(ICRB)R mice.
Inthedoseselectiosntudy,thetestarticlweas solubiliziendacetone:comoil(40%:60%, v:v) and dosedby oralgavageat0,1000,1400,1800,2200,and 2600 mg/kg. Sixanimals(three males and threefemales)were assignedtoeachdosegroup.Animalswere observedforthree daysafterdosingfortoxicsignsand/ormortality.
Based on theresultosfthedoseselectiosntudy,themaximum tolerateddosewas estimatedas about2200 mg/kg. Inthemicronucleusassay,thetestarticlweas solubilizeidnacetone:comoil (40%:60%, v:v)and dosed oralgavageat550,1100,and 2200 mg/kg. Ten animals(fivemales and fivefemales)were randomly assignedtoeachdose/harvesttimegroup.Vehicleand positive controlgroups,eudianatizeadpproximatel2y4 hoursafterdosing,were includedintheassay. The animalsdosedwiththetestarticlweere euthanatizeadpproximatel2y4,48 and 72 hours afterdosingforextractioonfthebone marrow.
The testmaterialT,-6292,didnotinducea significainntcreasienmicronucleinbone marrow polychromatiecrythrocyteusndertheconditionosfthisassayand isconsiderendegativeinthe mouse bone marrow micronucleustest.
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MutagenicTietsytonT-629i2naninvivMoouseMicronuclAesussay 1.0 SPONSOR-3M 2.0 MATERIAL (TestArticle)
2.1 Client'IsdentificatiTo-n6:292 2.2 DateReceived:January16,1996 2.3 PhysicalDescriptionA:mber, waxy solid 2.4 GeneticsAssay No.: 17384 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 ExperimentaSltartDate: February20,1996 5.3 ExperimentaTlerminationDate:April16,1996 6.0 SUPERVISORY PERSONNEL 6.1 StudyDirectorH:emalathaMurli,Ph.D. 6.2 LaboratorySupervisorM:onica Vegarra,B.S. 7.0 OBJECTIVE The objectivoefthisinvivoassaywas toevaluatteheabilitoyfthetestarticlTe-,6292,to inducemicronucleinbonemarrow polychromatiecrythrocyteosfCrl:CD-I*(ICR)BR mice. Thisstudywas conductedusingmodificationosftheproceduressuggestedby Heddleetal.(1983).
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8.0 MATERLKLS
Adultmale and femaleniices,traiCnrl:CD-1'(ICR)BF, were purchasedfrom Charles RiverLaboratoriePso,rtage,lvflT.hishealthyr,andom bredstraiwnas selectetdo maximize geneticheterogeneitaynd atthesame timeassureaccesstoa common source. The protocolforthisstudywas approvedby theCHV-ACUC priortotheinitiatioofn dosing.
Animals were housedup tosevenpercageduringquarantinea,nd housedup tofiveper cageatrandomizationT.he temperaturaend relativheumidityweremaintainedat 726'F and 5515%, respectiveleyx,cepton February17,18,and 19,1996,forthedose selectiosntudy,when therelativheumiditywas recordedat39.1%,37.3%,and 34.5%, respectivelayn,d on February'242,4,25,and March 2,1996,fortheniicronucieuasssay, when therelativheumiditywas recordedat32.5%, 34.4%,20.8%,and 35.5%, respectivelAy.12-hourlight/12-hoduarrkcyclewas maintained.A commercialdiet (PurinOCertifieLdaboratoryPellets 5002)and waterwereavailablAed libitumfor thedurationofthestudy.The feedwas analyzedby themanufacturerforconcentrations ofspecifiehdeavy metals,aflatoxicnh,lorinatehdydrocarbonso,rganophosphatesa,nd specifiendutrientsT.he waterwas analyzedon a retrospectibvaesisforspecified microorganismsp,esticidedsu,inity, heavymetals,and halogens.Sanitizecdagingwas used forhousingtheanimals.Personnelhandlinganimalsor workingwithintheanimal facilitiweesre requiredtowear suitablperotectivgearmentsand equipment.
Animals were quarantinefdorsevendaysbeforebeingplacedon study.Animals were randomlyassignedtostudygroupsand were individualwleyighedpriortodosing.All animalsweredosed basedupon theindividuablody weights.Animalswere uniquely identifiebdy eartag.Dose ortreatmengtroupswere identifiebdy cagecard/label.
At theterminatioonfthestudyallsurvivinagnimalswereeuthanatizebdyC02hihdation followedby penetratioonfthethorax.Any extraanimalsnotusedforthestudywere usedfortraininpgurposes.
9.0 SOLUBILITY AND STABILITY:
The testarticleT,-6292,was suppliedasan amber,waxy solid.The solubilitoyfthetest articlweas evaluateidn0.5% highviscositcyarboxymethylcellulo(sCeMC), com off, andacetone:comoil(40%:60%,v:v).The vehicles0,.5% highviscositCyMC andcom oildidnotproducesuitablseuspensionosfT-6292. An acceptableemulsionwas obtainedinacetone:comoil(40%:60%,v:v)ata concentrationfabout422.3mg/ml.
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Thestabiloifthyetesmtateriuanldetrhedosincgonditioofntshiasssaiysthe responsibiliotfythesponsor.
10.0 DOSE SELECTION STUDY
10.1 Dose Selection
Dose levelsof0,1000,1400,1800,2200,and 2600 mg/kg were administerebdy oralgavageforthedoseselectiosntudy.
10.2 DosingInformation
The animalsusedinthedoseselectioanssaywere dosedon February20, 1996. The weightrangeoftheanimalsusedinthedoserangefindingassaywas 30.135.1and 21.6-26.2grams,forthemalesand females,respectivelyD.osing solutionwsere preparedjustpriortodosingand were preparedby making a 260 mg/ml stockforthehighdose(2600mg/kg).Thiswas preparedby adding 11.0ml of400/o:60%v,:vofacetone:comoil(Sigma,Lot # 2435KHXG: Duke's com oilL,ot# 5D 17 12:46)to3.1200g ofT-6292,resultinignan opaque,creamcoloredemulsionthatsettleddown toa bilayewritha finavlolume of 12.0ml. An opaque,cream-coloreedmulsionresulteudpon mixing.Dilutionosfthisstock werepreparedforthe1000,1400,1800and 2200 mg/kg doselevels.Alldosing stockswere placedon magneticstiprlatesduringthedosingprocedure.
Dosingwas achievedusinga 10.0ml/kgdosingvolume. Allanimalswere eight weeks and one day oldatthetimeofdosing.An outlinoefthedosingscheme is found inthefollowingtable.
A totalof 36 animalswas usedinthisassay.
DOSE GROUPS
TREATMENT
MF
------- - - ----- -- -----------------------
T-6292
0 mg/kg
33
1000 mg/kg
33
1400 mg/kg
33
1800 mg/kg
33
2200 mg/kg
33
2600 mg/kg
33 ---
Alldosesgi-v-e-n--w-e-r-e- o-n--a-n-a-c-u--t--e(-o-n-e--timoenly)basis.
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10.3 Resultsand Interpretation
Allanimalswere examinedafterdosinganddailythroughouthedurationofthe study(thredeays)fortoxiceffectasnd/ormortalitieAsl.lanimalsappeared normal immediatelyand about4.6hoursafterdosing.
Approximately20.3hoursafterdosing,allanimalsatthe0, 1000,1400,1800 and 2600 mg/kg doselevelasppearednormal,exceptforonemale (#6524)from the 2600 mg/kg doselevelwhich appearedhypoactive.The males atthe2400 mg/kg doselevelappearedhypoactivewithsquintedeyesand chromodacryorrheaand thefemalesatthe2400 mg/kg dose levelappearedhunchedand exceptionally hypoactivewithsquintedeyesand chromodacryorrhea.
Approximately44 hoursafterdosing,theanimalsatthe0 and 1000mg/kg dose levelsappearednormal.The animalsatthe1400 and 1800mg/kg doselevels appearedhypoactive.One 2200 mg/kg doselevelfemale(#6531)was found
dead. The remaininganimalsatthe2200 and 2600 mg/kg doselevelsappeared hunchedand veryhypoactivewithdyspnea. -
Approximately68.4hoursafterdosing,theanimalsatthe0 and 1000 mg/kg dose levelsappearednormal.The animalsatthe1400and 1800mg/kg doselevels appearedhypoactivewithrough haircoatsT.he animalsatthe2200 and 2600 mg/kg dose levelsappearedhunched and hypoactivewithrough haircoats. T'hemortalitdyataforthisassayaresummarized inthefollowingtable:
Summary ofMortalitieWsithin 3 Days inMice Dosed Acutelywith T-6292
Observations
Treatment
Male
0 mg/kg
0/3
1000 mg/kg
0/3
1400 mg/kg
0/3
1800 mg/kg
0/3
2200 mg/kg
0/3
2600 mg/kg
0/3
Female 0/3 0/3 0/3 0/3 1/3 0/3
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10.4 Conclusion
Based on theseresultst,hemaximum tolerateddosewas estimatedtobe about 2200 mg/kg.
11.0 NUCRONUCLEUS STUDY
11.1 Dose Selection
Based on resultfsrom thedose selectiosntudy,doselevelsof550, 1100,and 2200 mg/kg were selectefdortestinignthisstudy.
11.2 MicronucleusAssay liosingInformation
The animalsusedinthemicronucleusassaywere dosedon March 5,1996. Cyclophosphamide(CAS # 6055-19-2;SiginaL,ot # 44HO486),thepositive controlw,as solubiliziendsterildeeionizedwater(Lot# 19,preparedatCHV) and was administerebdy oralgavageat80.0mg/kg. The vehiclecontrola, 40%:60%, v:vofacetone:comoil(Sigma,Lot# 2435KHXG and Duke'scom oilL,ot# 5D 17 12:46)w,as administerecdoncurrentlwyiththetestarticlaeta volume of 10.0ml/kg.The weightrangeoftheanimalsusedinthemicronucleus assaywas 27.2-35.2grams and 21.7-28.1grams forthemales and females, respectivelTyh.e dosingsolutionfsortheassaywerepreparedby making a 220 mg/ml stockforthehighdose(2200mg/kg).Thiswas preparedby adding 10.0ml ofvehiclteo5.5070g ofT-6292,mixingthoroughlfyorabout2 minutes and thenbringingthesuspensiontoa fmalvolume of25.0ml. A paleyellow-tan emulsionthatbecame a bilayerofpaletansolutioonvera heavy yellowsolution was obtained.Dilutionosfthisstockwerepreparedfortheremainingdoselevels. Duringformulatioannd duringthedosingprocedurea,lldosingstockswere placedon magneticstiprlates.A secondgroup ofanimals(designateSdecondary Dose Group)was alsoassignedtothestudyand was dosedwiththehighdoseof thetestarticleT.hese animalswere onlyused intheassayasreplacementsforany which diedintheprimarydosegroup.
Ten animals(fivemalesand fivefemales)wererandomlyassignedtoeachdose/harvesttimegroup.Vehicleand positivceontrolgroups,eudianafized approximatel2y4 hoursafterdosing,wereincludedintheassay.The animals dosedwiththetestarticlweere euthanatizeadpproximatel2y4,48 and 72 hours aftedrosingforextractioonfthebone marrow. An outlinoefthedosingscheme isfound inthefollowintgable:
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DosinSgchemeforMicronucleAusssay A totaolf120animalwsasusedinthiasssay
Number ofAnimalsAssigned
Treatment T-6292
2200 mg/kg
I100mg/kg
PrimaryDose Groups 24Hr 48Hr 72Hr MF MF MF
55 55 55
55 55 55
SecondaryDose
Groups' Male Female
5
5
--
550 mg/kg
55 55 55
VehicleControl,acetone:comoil,40%:60%, v:v
10.0ml/kg
5 5 --
PositivCeontrolC,yclophosphamide,80.0mg/kg 5 5
a The animalsassignedtothesecondarydose groupswere dosed and were onlyused to replaceanimalswhich diedintheprimarydosegroupatthehighdoselevel.Allextra animalsnotusedasreplacementswere eudianatizeadtthecompletionofthetrial.
The age of theanimalsatthetimeofdosingwas eightweeks and one day.
Volumes dosed were 10.0ml/kg and were basedupon individuaalnimalweights.
12.0 BONE MARROW HARVEST, SLIDE PREPARATION AND ANALYSIS
At theappropriathearvestime,theanimalswere euthanatizewdithC02followed by penetratioonf thethoraxand theadheringsofttissueand epiphysesofbothfemorawere removed. The marrow was flushedfrom thebone and transferrteodcentrifugteubes containin3g-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-driesdlidewsere coverslippeudsingDepekO mounting medium.
The slidewsere codedforanalysisa,nd scoredformicronucleaindthepolychromatic erythrocyt(ePCE)tonormochromaticerythrocyt(eNCE) cellratio.Standardformswere usedtorecordthesedata.One thousandPCEs peranimalwere scored.Ile frequencyof micronucleatecdeuswas expressedaspercentmicronucleatecdellsbasedon thetotal PCEs presentinthescoredopticfieldT.he normalfrequencyofmicronucleinthis Crl:CD-I'(ICRB)R straiinsabout0.0-0.4%.
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ThefrequenocfyPCEsversuNsCEswasdeterminbeysdcoritnhgenumbeorfPCEsand NCEs observedintheopticfieldwshilescoringthefirs1t000erythrocytes.
13.0 EVALUATION CRITERIA:
13.1 General
The criterifaortheidentificatoifomnicronuclewiere thoseof Schmid (1976). Nficronuclewiere darklystainedand generallryound,althoughalmond and ringshapedmicronucleoiccasionalloyccurred.Micronucleihad sharpborders and were generallbyetween 1/20and 115thesizeofthePCE. The unitofscoring was themicronucleatec.edlln,otthemicronucleust;hustheoccasionaclellwith more thanone micronucleuswas countedasone micronucleatePdCE, nottwo (or more)micronucleiT.he staininpgrocedurepermittetdhedifferentiatbiyocnolor ofPCEs and NCEs (bluish-graenyd red,respectively).
13.2 Data Presentatioannd Interpretation
Dataaresummarized by sexand dosegroupsforthedifferenttimepoints. Individuaalnimaldataarealsopresented.The analysiosfthesedatawas performedusingan analysiosfvariance(Winer,1971)on eitheurntrawforined (when varianceasrehomogeneous) and ranktransformed(when variancesare heterogeneousp)roportionosf cellswithmicronuclepier animal.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 criterfioardetermininagpositivreesponseinvolveda statisticaslilgynificadnotse-relateidncreasienmicronucleatePdCES, orthe detectioonfa reproduciblaend statisticaslilgynificapnotsitivreesponseforat leastone doselevel.A testarticltehatinducedneithera statisticaslilgynificant doseresponsenor a statisticaslilgynificanatnd reproducibliencreaseatone dose levelwas considerednegative.Ineithercase,thefinaldecisionwas basedon scientifjiucdgment.
14.0 RESULTS AND INTERPRETATION:
Allanimalswereobservedimmediatelyafterdosingand periodicaltlhyroughoutthe duratioonftheassayfortoxicsymptoms and/ormortalitieAsl.lanimalsinthevehicle andpositivceontroglroups(exceptone female,# 6846,fromthe vehiclegroup,which appearedhypoactiveatabout1.4hoursafterdosing)appearednormal afterdosingand
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remainehdealtuhnytitlheapproprihaatrevetsitmesB.y abou2t0.4hourasftedrosing, female(#6846)fromthe vehiclegroupappearednormaland remainedhealthyuntilthe 24 hourharvesttime.
Approximately1.4hoursafterdosing,all550,1100 and 2200 mg/kg dose levelanimals appearedhypoactivew,ith some alsoshowingexcessivesalivation.
Approximately20.4hoursafterdosing,the550 mg/kg doselevelanimalsappeared normal.At theI100mg/kg doselevelt,he24 hourharvestmalesappearednormal and thefemalesappearedhypoactive.The 48 hourharvestanimalsappearedhypoactivewith roughhaircoatsT.he 72 hourharvestanimalsappearedhypoactivewithrough haircoats and some withdyspnea.
Approximately43.4hoursafterdosing,the550 mg/kg doselevelanimalsappeared normal.At the1100 mg/kg doselevelt,heanimalsappearedhypoactivweithrough haircoatasnd dyspnea.Some alsohad squintedeyesand were hunched. At the 2200 mg/kg doselevelt,heanimalsappearedhypoactivewithroughhaircoatsd,yspnea, squintedeyesand chromodacryorrhea.
Approximately47.5hoursaftedrosing,I female(#6916)from the2200 mg/kg doselevel and 48 hourharvestwas founddead.
Approximately68.4hoursafterdosing,the550 mg/kg doselevelanimalsappeared normal.At theI100 mg/kg doselevelo,ne female(#6920)was founddead andthe remaininganimalsappearedhypoactive.At the2200 mg/kg doselevelt,heanimals appearedhypoactivewith roughhaircoatasnd some were hunched.
The testarticlTe-,6292,inducedno significainntcreaseisnmicronucleated polychromatiecrythrocyteosverthelevelsobservedinthevehiclecontrolisn eithersex oratany oftheharvestimes.Due totoxicityt,hePCENCE ratioosfthefemalesfrom the72 hourharvestand 550,1100,and 2200 mg/kg dosegroupsweresignificantly reducedwhen compared tothevehiclecontrolfemales.The positivceontrolC,P, induced significainntcreaseisnmicmnucleatedPCEs inbothsexesascompared tothevehicle controlsw,ithmeans and standarderrorsof3.86% 0.49% and4.84%:k 0.74% forthe males and females,respectivelyT.he datasummarized by dosegrouparepresentedin TableI and individuaalnimaldataarefoundinTables2 through7. Historicaclontrol dataarepresentedinTable 8.
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15.0 CONCLUSION:
The testmaterialT,-6292,didnotinducea significainntcreasienmicronucleinbone marrow polychromatiecrythrocyteusndertheconditionosfthisassayand isconsidered negativeinthemouse micronucleusassay.
16.0 REFERENCES:
Dunnet@ C.W.: A multiplecomparisonsprocedureforcomparingseveraltreatmentwsith a control.J.Am. StatisAts.soc.,JQ:1096-1121,1955.
DunneM C.W.: New tablesformultiplceomparisonswitha controlB.iometricsM,:482491,1964.
Heddle,J.A.H,ite,M., KirkhartB,.,Larsen,K.,MacGregor,J.T.N,ewell,G.W. and Salamone,M.F.: The inductionofmicronucleiasa measureofgenotoxicity. MutationRes.,M:61-118, 1983.
Schmid,W.: -Themicronucleustest.MutationRes.,31:9-15,11975.
Schmid,W.: The micronucleutsestforcytogenetiacnalysisC.hemical Mutagens:Principleasnd Methods forTheirDetectionV,ol.4 (A.Hollaender, ed.).Plenum,pp. 31-53,1976.
Winer,B.J.:StatisticParlincipleisnExperimentalDesign,McGraw-Hill,New York,Second Edition1,971.
17.0 DEVIATION FROM THE SIGNED PROTOCOL
The followingdeviatiownas made fromthesignedprotocol.
Relativehumiditywas recordedon February17,18,and 19,1996,(forthedoseselection study)at39.1%, 37.3%,and 34.5%,respectivelayn,d on February24,24,25,and March 2,1996,(forthemicronucleusassay)w,hen therelativheumiditywas recordedat32.5%, 34.4%, 20.8%,and 35.5%,respectively.
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18.0 EXPERIMENT DATA TABLES
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SPONSOR: 3M
MICRONUCLEUS
TABLE 1 DATA SUMMARY
TABLE
TEST ARNCLE: T-6292
ASSAY: 17384
TREATMENT
CONTROLS VEtflCLE POSMVE
DOSE
HARVEST TIME (HR)
% NUCRONUCLEATED PCEs
MEAN OF 1000PER ANIMAL:K S.E.
MALES
FEMALES
TOTAL
4(r/Aocetone/ 24 hr 600/C9om Oil
CP 80.0mg&g 24 hr
0.12* 0.04 0.10 :L0.04 3.86* 0.49* 4.84 0.740
0.11 :L0.03 4.35+ OA5*
RATIO PCE:NCE
MEAN:K S.E.
MALES
FEMALES
0.51 0.08 0.40 0.04
0.72 0.08 0.51 0.04**
TEST ARNCLE
550 mg/kg 1100 mg/kg 2200 mg/kg
24 hr 48 hr 72 hr 24 hr 48 hr 72 hr 24 hr 48 hr 72 hr
0.14 * 0.06 0.10:10.00 0.12 :b0.07 0.12 :k0.04 0.14 0.04 0.10 0.05 0.1& 0.06 0.12 0.07 0.06 0.02
0.12 0.05
0.16:k0.07
0.19 :E0.04 0.06 :L0-.02 0.18 :h0.05 0.05 0.05 0.10 0.04 0.15 0.06 0.26 0.09
Significantglryeatetrhanthecorrespondinvgehiclecontrolp,<0.05. Significantlloywerthanthecorrespondinvgehiclecontrolp,<0.05.
0.13 :k0.04
0.13:k0.03
0.15 0.04 0.09 0.02 0.16 0.03 0.09 0.04 0.14 0.04 0.13 0.05 0.16 0.05
0.56 0.05 0.78 :k0.11 0.61 0.12 OA7 * 0.03 0.70 :k0.06 0.63 :1:0.07 0.52 0.04 0.92 0.11 0.57 0.05
1
CP - Cyclophosphamide
0.75 0.08 0.62:10.13 0.48 :L0.04*0 0.73 0.13 0.64 0.08 0.44 0.04** 0.60 0.11 0.56 0.07 0.30 0.020*
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NUCRONUCLEUS
TABLE2 TEST -INDIVIDUAL ANIMAL DATA
SPONSOR. 3M
TEST ARTICLE: T-6292
ASSAY NO.: 17384 TREATNM'NT
24 HOUR HARVEST
MALE
ANIMAL NUMBER
NO. MN PCEs (1000)
RATIO PCE.NCE
VEHICLE CONTROL
40% Acetone/600C/o.m Oil
6946
2
0.64
6960
2
0.26
6870
1
0.37
6892
1
0.72
69%
0
0.56
POSITIVE CONTROL
CP 80.0mg/kg
6955
43
0.34
6867
42
0.43
6868
51
0.49
6986
35
0.45
6891
22
017
TEST ARTICLE
550 mg/kg
6859
2
0.63
6962
2
0.38
6872
3
0.52
6893
0
0.63
6993
0
0.64
1100 mg/kg
6961
1
0.56
6863
1
0.43
6873
2
0.37
6879
0
0.48
6889
2
0.49
2200 mg/kg
6856
1
0.57
6865
1
0.47
6871
2
0.38
6874
4
0.62
6898
1
0.54
CP Cyclophosphunide MN = Micronucieus PCE - Polychromaticcryduwyte # MN PCEs - MicronucleatePdCEs NCE = Normochromaticerythrocyte
CHV StudyNo.: 17384-0-455
18
CORNINGHazleton
MICRONUCLEUS
TABLE3 TEST -INDIVIDUAL ANIMAL DATA
SPONSOR.- 3M
TEST ARNCLE: T-6292
ASSAY NO.: 17384 TREATMENT
24 HOUR HARVEST
FEMALE
NO. MN ANINIAL PCEs NUMBER (1000)
RATIO PCE.NCE
VE19CLE CONTROL
400/.Acetone/600C/o.m Oil
6900
2
6902
1
6923
0
6929
2
6953
0
POSrfM CONTROL
CP 80.0mglkg
6897
32
6898
56
6906
34
6926
72
6945
48
TEST ARNCLE
550 mg&g
6908
3
6909
1
6930
1
6941
1
6951
0
I100 mg/kg
6907
1
6921
1
6929
0
6935
1
6949
0
2200 mgtkg
6904
2
6914
0
6917
1
6942
2
6952
0
0.65 0.69 1.01 0.59 0.64
0.54 0.60 0.41 0.42 0.57
0.53
0.69
0.86 0.68 1.00
0.37
1.00
0.94 0.45 0.89
0.72 0.75 0.26 OAI 0.87
CP Cyclophosphamide MN Micronucleus PCE Polychromatiecrythrocyte # MN PCEs = MicronuclcatePdCEs NCE - Normochromaticcridwocyte
CHV StudyNo.: 17384-0-455
19
CORNINGHazleton
MICRONUCLEUS
TABLE4 TEST -INDIVIDUAL ANIMAL DATA
SPONSOR. 3M
TEST ARNCLE: T-6292
ASSAY NO.: 17384 TREATMENT
48 HOUR HARVEST
MALE
ANIMAL NUMBER
NO. MN PCEs (1000)
RANO PCE:NCE
TEST ART3CLE
550 mglkg 1100 mg/kg 2200 mgtkg
6939
1
1.10
6949
1
0.91
6966
1
0.82
6876
1
0.52
6897
1
0.54
6947
2
0.79
6869
2
0.63
6891
1
0.71
6884
0
0.52
6892
2
0.97
6944
3
0.75
6853
3
0.92
6977
0
1.19
6890
0
0.69
6895
0
0.58
MN Micronucieus PCE Polychromaticrythrocyto
# MN PCEs = MicronucleatePdCEs NCE = Normochromaticerfthrocyte
CHV StudyNo.: 17384-0455
20
CORNINGHazleton
MICRONUCLEUS SPONSOR. 3M
TABLE5 TEST -INDIVIDUAL ANIMAL DATA
T'EST ARTICLE: T-6292
ASSAY NO.: 17384 TREATMENT
48 HOUR HARVEST
FEMALE
ANIMAL NLTMBER
NO. MN PCEs (1000)
RATIO PCE-NCE
TEST ARTICLE
550 mg/kg I100 mgtkg 2200 mg/kg
6910
3
0.43
6925
0
0.87
6927
3
039
6939
2
0.40
6943
0
1.01
6911
2
0.61
6931
0
0.93
6934
2
0.43
6946
2
0.65
6955
3
0.55
6901
3
0.61
6916*
6932
2
0.44
6937
0
0.75
6950
1
OA3
Animal found dead MN - Micronucieus PCE - Polychromatiecryduvcyte # MN PCEs - MicronucleatePdCEs NCE = Normochromaticerythrocyte
CHV StudyNo.: 17384-0-455
21
CORNINGHazleton
MICRONUCLEUS
TABLE6 TEST -RNDIVIDUAL ANIMAL DATA
SPONSOP, 3M
TEST ARTICLE: T-6292
ASSAY NO.: 17394 TREATNffi'NT
72 HOUR HARVEST
MALE
ANIMAL NUMBER
NO. MN PCEs (1000)
RATIO PCE:NCE
T*ESTARTICLE
550 mglkg
6939
0
1.00
6958
3
0.24
6879
0
0.61
6980
3
0.61
6994
0
0.59
1100 mg/kg
6937
0
0.62
6941
0
0.94
6943
1
0.53
6851
1
0.71
6964
3
0.45
2200 mgtkg
6940
1
0.72
6945
1
0.59
6949
0
0.60
6957
1
0.41
6895
0
0.55
MN Micronucieus PCE Polychromatiecrythrocyte # MN PCEs = MicronucleatePdCEs NCE = Normochromaticerythrocyte
CHV StudyNo.: 17384-0455
22
CORNINGHazleton
NUCRONUCLEUS
TABLE7 TEST -INDIVIDUAL ANIMAL DATA
SPONSOK- 3M
TEST ARTICLE: T-6292
ASSAY NO.: 17394 TREATh4ENT
72 HOUR ELikRVEST
FEMALE
ANIMAL NUMBER
NO. MN RANO PCEs PCE-NCE (1000)
TEST AR71CLE
550 mg/kg
6903
2
0.58
6915
1
0.56
6940
3
0.38
6949
1
0.43
6954
2
0.46
1100 mglkg
6920*
6922
2
0.32
6938
0
0.53
6944
0
0.44
6947
0
0.45
2200 mg/kg
6905
2
0.24
6918
1
0.37
6919
2
0.26
6924
2
0.34
6956
6
0.31
Anitnalfounddead MN Micronucleus PCE Polychromatiecrythrocyte # MN PCEs - MicronucleatePdCEs
NCE = Nonnochromaticerythrocyte
CHV StudyNo.: 17384-0-455
23
CORNINGHazleton
TABLES
MOUSE NUCRONUCLEUS HISTORICAL CONTROL DATA 7/95THROUGH 12/95
POOLED VEIECLE CONTROLS MIN MAX AVG N
% MICRONUCLEATED PCEs PER 1000PCE
MEAN OF 1000PER ANIMAL S.E.
MALES
FEMALES
TOTAL
0.00 0.22 0.087 0.007 47
0.00 0.24 0.081 0.008 47
0.01 0.17 0.094* 0.005 47
RATIO PCE:NCE
MEAN * S.E.
MALES
FEMALES
0.31 0.95 0.550 0.021 47
0.24 1.03 0.587* 0.025 47
POSMVE CONTROLS Cyclophosphamicl8e0,.0mgtkg
MN
MAX AVG N
2.00 5.68 3.692* 0.240 19
PCE = Polychromaticerythrocyte 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 0.588:k0.026 19
CHV StudyNo.: 17384-0-455
24
CHV STUDY NO. PROTOCOL NO. 455, EDITION 17
CORNING Hizleton
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 INFORMATION AND APPROVALS
I. SPONSOR IDENTIFICATION
Company Name: Address:
314 loaad,
/niv/
II. TEST ARTICLE IDENTIFICATION:
2. ?2,
III. TEST ARTICLE ANALYSIS
Determination of the test article stability and the test article characteristics as defined in the GLP regulations is the responsibility of the Sponsor.
IV. NOTIFICATION OF REGULATORY SUBMISSION
In order to comply with the GLP regulations, consulting laboratories must be notified if all or 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 MAFF
IL----i FDA MOHW
IL----J EPA-TSCA
OECD
OTHER
IL---i EPA-FIFRA
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PROTOCOL NO. 455, EDITION 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:
CORNINGHazleton
Date: Date;
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PART 2 - STUDY PROTOCOL
IN VIVO HOUSE HICRONUCLEUS ASSAY
I. OBJECTIVE
The objective of this study is to evaluate a test article for clastogenic activity and disruption of the mitotic apparatus in polychromatic erythrocyte stem cells in mouse bone marrow in vivo.
II. DEFINITIONS,
Hicronucleus:a smallchromatinbody,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. M&TERIALS
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
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PROTOCOL NO. 455, EDITION 17
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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 five prior to experiment initiation. Animals
are housed under the following climatic conditions: temperature, 720F 6'F; humidity, 55Z 15Z; light
cycle, 12 hours light/dark. A commercial diet
(Purinat Certified Laboratory Chow* 15002) and tap water will be available ad libitum. The feed is
analyzed by the manufacturer for concentrations of
specified heavy metals, aflatoxin, chlorinated
hydrocarbons, organophosphates,
and specified
nutrients. The water is analyzed biannually on a
retrospective basis for specified microorganisms,
pesticides, heavy metals, alkalinity, and halogens. Animals will be quarantined for at least 7 days before
being placed on study.
Animals will be assigned to study groups at random
according to Coning Hazleton Standard Operating
Procedures.
Animals will be weighed prior to dosing.
They will be dosed based upon the individual animal
weights.
Animals will be uniquely identified by ear
tag. Treatment groups will be identified by cage
label/card.
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Sanitary cages will be used. Personnel handling animals or working within the animal facilities will be required to wear suitable protective garments and equipment.
B.
Dose Selection
The high dose generally will be selected as 802 of the maximum tolerated dose. The high dose should produce some indication of toxicity (e.g., death, depression of ratio of PCEs to normochromatic erythrocytes (NCEs). One-half and one-quarter of this high dose will normally be used as the intermediate and low dose levels, respectively. Use of a high dose increases the likelihood that a weak clastogen will be detected, and is therefore recommended.
If no appropriate range finding finding study can be performed. rangefinding study will be 5000 will be issued as an amendment.
data are available, a range The top dose tested in the dose
mg/kg. The dose levels tested
DOSE F.ANGEFINDING 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
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PROTOCOL NO. 455, EDITION 17
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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, 0.9Z 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.
MICRONUCLEUS
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 animals consisting of 3-10 males and 3-10 females may be dosed as a secondary dose group with the high dose of the test material. This group will be dosed if toxicity is expected at the high dose and the animals in this group will only be used as replacements for any which die prior to euthanasia. The use of the secondary dose group will be determined by the study director. Freshly prepared solutions will be
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employed. The animals will be observed daily for toxic signs and mortality.
NUMBER OF ANIMALS USED FOR HICRONUCLEUS ASSAY
Group No. Treatment
Harvest Times After Treatment (Hales 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 + 25
15 + 15
15 + 15 55 + 55
The route of administration will be oral gavage. In the event that test article characteristics preclude oral gavage, IP injection will be employed. The dosing volume will not exceed 20 ml/kg. These routes
of administration have been selected because they are the most common routes of administration for this test procedure. Other routes of administration that may be used are intravenous, intramuscular, sub-cutaneous administrations or by feed.
D.
Extraction of Bone Marrow
Euthanasia will be with C02, followed by penetration of the thorax, and hind limb bones will be removed for marrow extraction. The marrow will be flushed from the bone and transferred to centrifuge tubes containing 3-5 ml bovine serum (one tube for each animal).
E.
Preparation of Slides
Following centrifugation to pellet the tissue, the supernatant will be removed by aspiration and portions of the pellet will be spread on slides and air-dried.
The slides will then be fixed in methanol, stained in Hay-Grunwald Solution and Giemsa, and protected by mounting with coverslips. For control of bias, all slides are coded for analysis.
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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 an 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. Hicronuclei have sharp borders and are generally between 1/20 and 1/5 the size of The PCE. The unit of scoring is the micronucleated cell, not the micronucleus; thus the occasional cell with more than one micronucleus is counted as one micronucleated PCE, not two (or more) micronuclei.
The staining procedure permits the differentiation by color of polychromatic and normochromatic erythrocytes (bluish-grey and red, respectively).
Data Presentation
The data reported will include the number of PCEs scored, the number of micronucleated PCES, the percentage of micronucleated PCES, and the ratio of polychromatic to normochromatic erythrocytes for each experimental animal.
Evaluation Criteria
The criteria for a positive response is a statistically significant dose-related increase in micronucleated PCES, or the detection of a reproducible and statistically 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.
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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., Kirkhart, B., Larsen, K., MacGregor, J.T., Newell, G.W. and Salamone, M.F.: The induction of micronuclei as a measure of genotoxicity. Mutation Res., 123:61-118, 1983.
Schmid, W.: The micronucleus test. Mutation Res., 31:9-15, 1975.
Schmid, W.: The micronucleus test for cytogenetic analysis. In, Chemical Mutagens: Principles and Methods for Their Detection, Vol. 4 (A. Hollaender, ed.). Plenum, pp. 31-53, 1976.
Winer, B.J.: Statistical Principles in Experimental Design, McGraw-Hill, New York, Second Edition, 1971.
IX. REPORT FORMAT
CHV employs a standard report formt for each assay design. The final report will provide the following information.
Sponsor identification.
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,@articleand 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.
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PROTOCOL NO. 455. EDITION 17
CORNING- Ha7leton
0
Evaluation criteria.
0
Interpretation of results.
0
Conclusions.
9
References.
0
Test results presented in tabular form.
X.
CHANGES 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 STATEZ-FENT
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-T-ACUC 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.
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Corni)-1i-.g7azl,-ton Inc. 9'@00Leesburg ]like Vienna, \li.@,,inia 7,,3'093.54(@O 7('),@;.i5.(,947Fax
April25, 1996
StevenC.Gordon,Ph.D.D,ABT 3M MedicalDepartment Building220-2E-02,3M Center St.Paul,MN 55144-1000
CORNINGHazleton
RE: DRAFT REPORT AND PROTOCOL AMENDMENTS
InVivo NficronucleuAsssay ProtocolNo.: 455CO, Ed.No.:4,ModifiedforSponsor GeneticsAssayNo.: 17384,17384-1 TestMaterial:T-6292
Dear Dr.Gordon:
Enclosedpleasefindtwo (2)copiesoftheabovereferencerdeport.The reportincludeasn unsignedQualityAssuranceStatementand Compliance and CertificatiSotnatementw,hich will be signedupon issuanceoftheFinalReport.
Alsoenclosedisa copyofprotocolamendments.
The finalreportwillbe issuedafteyrourreviewand notificatitoonus.Pleasecontacthe undersignedwithany questionsc,omments, ornecessaryrevisionastyour earliesctonvenience. The reportwillbe fmalizedafterI yearifno notificatiiosnreceived.
Thank you forgivingus thisopportunittyowork withyou.
Sincerely,
CORNING Hazleton
/JU*tO, 14u@ Hema Murli,Ph.D. Mammlian Cytogenetics Departmentof Geneticand CellulaTroxicology
HM/paj enclosures
AMENDMENT
TO THE STUDY PROTOCOL
STUDY TITLE: IN VIVO MOUSE WCRONUCLEUS ASSAY
PROTOCOL NO.: 455,E1dition17
STUDYNO.:
17384-0-455
Page I of I
Amendment #I
Section2,PartV.B.
The SponsorhasLD50 datainratsof1463 mg/kg solubilizeidn acetone/comoilmixture.Based on thisinformationt,hedoseselection studywillbe conductedwithacetone/comoilpreparationtsestindgose
groupsof0,1000,1400,1800,2200,and 2600 mg/kg.
STUDY DIRECTOR
)iU@ A a Lk@-a @b HemalathaMurli,Ph.D. Mammalian Cytogenetics DepartmentofGeneticand CellulaTroxicology
Iq@
Date
AMENDMENT TO THE STUDY PROTOCOL
STUDY TITLE: flVVIVO MOUSE MICRONUCLEUS ASSAY
PROTOCOL NO.: 455,Edition17
STUDY NO.:
17384-0-455
Page I of I
Amendment #2
Section2,PartV.C. Based on theresultosf theresultosf theselectiosntudy,themouse micronucleusassaywillbe conductedtestindgose groupsof 550, 1100, and 2200 mg/kg. A secondarydosegroup willbe used.
STUDY DIRECTOR
a.,
HemalathMaurliP,h.D. Mammalian Cytogenetics Departmentof Geneticand CellulaTroxicology
@).24'116
Date