Document 6R2RGw8ZkEXJ4G32DKGe5r0em
CORNINGHazleton
MUTAGENICITY TEST ON T-6564
IN AN IN VIVO MOUSE MICRONUCLEUS
ASSAY
FRNAL REPORT
Eli/,
NOV r-, 1996 ,x 3,.4 0
ico@-L'
AUTHOR Hemalatha Murli,Ph.D.
PERFORMRNG LABORATORY
Coming HazletonInc.(CHV) 9200 Leesburg Pike
Vienna,Virginia22182
LABORATORY PROJECT IDENTIFICATION CHV StudyNo.: 17750-0455
SUBMITTED TO
3M Corporation Building220-2E-02,3M Center St.Paul,Minnesota 55144-1000
STUDY COMPLETION DATE November 1,1996
CHV StudyNo.: 17750-0455
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QUALITY ASSURANCE STATEMENT
ProjecTtitleI:nVivoMouse MicronucleuAsssay
ProjectNo.:20996
Assay No.: 17750
ProtocolNo.: 455
EditionNo.: 17,Modified for3M Corporation
QualityAssurance inspectionsof thestudyand review of thefinalreportof theabove referenced projectwere conducted accordingtotheStandardOperatingProceduresofthe QualityAssurance Unit and accordingtothegeneralrequirementsof theappropriateGood LaboratoryPractice regulationsF.indingsfrom theinspectionasnd fmal reportreviewwere reportedtomanagement and tothestudydirectoron thefollowingdates:
Insl2ection/Date
FindingsR=orted
Auditor
Dosing/07/30/1996
07/30/1996
C. Orantes
Harvest/08/l/1996
08/l/1996
C. Smith
DraftReport Review/09/17,18/1996
09/18/1996
C. Orantes/C.Smith
FinalReport Review/ 11/01 1996
11/01/1996
C. Smith
(@dalitAyssurance Unit
Date Released
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STUDY CONTLLANCEAND CERUFICATION
The describedstudywas conductedincompliancewiththeGood LaboratoryPracticreegulations assetforthintheFood and Drup-Administratio(nFDA) Title21 oftheU.S.Code ofFederal RegulationPsart58,issuedDecember 22,1978,(effectiJvuene20, 1979)withany applicable amendments. Therewere no significadnetviationfsromtheaforementionerdegulationosrthe signedprotocotlhatwould affectheintegritoyfthestudyortheinterpretatioofnthetestresults.
raw datahave beenreviewedby theStudyDirectorw,ho certifietshattheevaluatioonf the testarticlaespresentedhereinrepresentasn appropriatceonclusionwithinthecontextofthe studydesignand evaluatiocnriteria.
Alltestand controrlesultisnthisreportaresupportedby an experimentaldatarecordand this recordhasbeenreviewedby theStudyDirector.Allraw data,documentationr,ecordsp,rotocol and a copy ofthefinalreportgeneratedas a resultofthisstudywillbe archivedinthestorage facilitioefsComing HazletonInc.foratleasotne yearfollowingsubmissionofthefinalreporto theSponsor.Aftertheone yearperiod,theSponsormay electtohave theaforementioned materialrsetaineidnthestoragefacilitioefsComing HazletonInc.foran additionapleriodof time,orsenttoa storagefacilitdyesignatebdy theSponsor.
SubmittedBy:
StudyDirector:
HemalathaMurli,Ph.D. MannnalianCytogenetics DepartmentofGeneticand CellulaTroxicology
Stud@ CompletionDate
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TABLE OF CONTENTS
Page No.
SUMMARY ..............................................6.............
1.0 SPONSOR ...........................................7 ............
2.0 MATERIAL (TestArticle.) .................................7 ............ 2.1 Client'Isdentification 2.2 Date Received 2.3 PhysicaDlescription 2.4 GeneticsAssay No.
3.0 TYPE OF ASSAY ......................................7.............
4.0 PROTOCOL NO ........................................7............
5.0 STUDY DATES .......................................7............. 5.1 InitiationDate 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 I ...............................9............. 10.1 Dose Selection 10.2 DosingInformation 10.3 Resultsand Interpretation 10.4 Conclusion
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11.0 DOSE SELECTION STUDY Il ..............................1.1............ 11.1 Dose Selection 11.2 DosingInformation 11.3 Resultsand Interpretation 11.4 Conclusion
12.0 MICRONUCLEUS STUDY .................................1,............. 12.1 Dose Selection 12.2 MicronucleusAssay DosingInformation
13.0 BONE MARROW HARVEST, SLIDE PREPARATION AND ANALYSIS ......1.4.
14.0 EVALUATION CRITERIA .................................1.5............ 14.1 General 14.2 Data Presentatioand Interpretation
15.0 RESULTS AND INTERPRETATION ...........................16...........
16.0 CONCLUSION .......................................1.6.............
17.0 REFERENCES .......................................1.7.............
18.0 DEVIATION FROM THE SIGNED PROTOCOL .....................17........
19.0 EXPERIMENT DATA TABLES ..............................18............
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SUMMARY
MutageriiTceistotynT-656i4nanInVivMoouseMicronuclAesussay
Theobjectivoefthisinvivoassaywas toevaluatteheabilitoyfthetestarticlTe-,6564,toinduce
micronucleiinbone marrow polychromaticerythrocyteosfCrl:CD-le(ICR) BR mice.
Inthedose selectionstudy,thetestarticlweas solubilizeidndeionizedwater and dosed by oral gavage at1000, 1510,2010, 2540 and 3010 mg/kg ina firstriaalnd thendosed at1010, 1520, 2020, and 2530 mg/kg ina second vial.Sixanimals (threemales and threefemales)were assignedtoeach dose group. Animals were observedforfourdays afterdosing fortoxicsigns and/ormortality.
Based on the resultsof the dose selectionstudy,the maximum toleratedose was estimatedas about 2000 mg/kg. Inthe micronucleusassay,thetestarticlweas solubilizeidndeionizedwater and dosed by oralgavage at498,995, and 1990 mg/kg. Ten animals(fivemales and five females)were randomly assignedtoeach dose/harvestimegroup. Vehicleand positivecontrol groups,euthanizedapproximately24 hours afterdosing,were includedintheassay.The animals dosed with thetestarticlweere euthanizedapproximately24,48 and 72 hours afterdosing for extractioonf thebone n=ow.
Tle testmaterial,T-6564, didnot inducea significanitncreaseinmicronucleiinbone marro@Apolychromaticerythrocytesunder theconditionsof thisassayand isconsiderednegativeinthe mouse bone marrow micronucleustest.
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MutagenicTietsytonT-656i4nanInVivMoouseMicronuciAesussay
1.0 SPONSOR: 3M Corporation 2.0 MATERIAL (TestArticle)
2.1 Client'IsdentificatiTo-n6:564L-13167FC1015-X 2.2 DateReceived:May 30,1996 2.3 PhysicalDescriptionC:learcolorleslsiquid 2.4 GeneticsAssay No.: 17750 3.0 TYPE OF ASSAY: In Vivo Mouse MicronucleusAssay 4.0 PROTOCOL NO.: 455,Edition17,Modifiedfor'3MCorporation 5.0 STUDY DATES 5.1 InitiatiDoante: June4,1996 5.2 ExperimentalStartDate: July11,1996 5.3 ExperimentalTerminationDate: August22,1996 6.0 SUPERVISORY PERSONNEL 6.1 StudyDirector:HemalathaMurli,Ph.D. 6.2 LaboratorySupervisor:Monica Vegarra,B.S. 7.0 OBJECTIVE The objectivoefthisinvivoassaywas toevaluatteheabilitoyfthetestarticlTe-,6564,to inducemicronucleiinbone marrow polychromatiecrythrocyteosfCrl:CD-IT(ICR)BR mice. Thisstudywas conductedusingmodificationosftheproceduressuggestedby Heddleetal.(1983).
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8.0 MATERIALS
Adultmale and femalemice,straiCnrl:CD-1*(ICR)BP, were purchasedfrom Charles RiverLaboratoriePso,rtageN,fl.Thishealthyr,andom bredstraiwnas selectetdo maximize geneticheterogeneitaynd atthesame tiznaessureaccesstoa common source. The protocolforthisstudywas approvedt)@tvheCHV-IACUC priortotheinitiatioofn dosing.
Animalswere housedfivepercaizeduringquarantinea,nd housed fiveperca2eat randomizationT.he temperaturaend relativheumidityweremaintainedat72:k6 'F and 55 15%, respectiveleyx,cepton July31, 1996,forthedefinitivsetudy,when the humiditywas recordedas75.1%. A 12-hourlight/2l-hourdarkcyclewas maintained.A cor=ercialdiet(PurinatCertifieLdaboratorPyelletst4 5002)andwaterwere available ad libitumforthedurationofthestudy.The feedwas analyzedby themanufacturerfor concentrationosf specifiehdeavy metals,aflatoxicnh,lorinatehdydrocarbons, organophosphatesa,nd specifiendutrientsT.he waterwas analyzedon a retrospective basisforspecifiemdicroor9anisms,pesticideasl,kalinithye,avy metals,and halogens. Sanitizecdagingwas usedforhousinetheanimals.Personnelhandlin2animalsor workingwithintheanimalfacilitiweesrerequiredtowear suitablperotectivgearments and equipment.
Animalswere quarantinefdorsevendaysbeforebeingplacedon study.Animalswere randomlyassignedtostudygroupsand were individualwleyighedpriortodosing.All animalswere dosedbasedupon theindividuablody weights.Animals wereuniquely identifiebdy eartag.Dose ortreatmentgroupswereidentifiebdy cagecard/label.
At theterminatioonfthestudya survivinagnimalswere euthanizedby COI-/02 inhalatiofno,llowedby C02 inhalatioannd penetratioonfthethorax.Any extraanimals notusedforthestudywere savedfortraining.
9.0 SOLUBILITY AND STABILITY
The testarticlTe-,6564,was suppliedasa clearcolorleslsiquid.The specifigcravityof thetestarticlweas calculateidnthetestinlgaboratorays 1.22g/ml.Thisspecifigcra-,-ity was usedtopreparedosingstocksfortheassay. Deionizedwaterwas usedas thevehicle inthisassay.The detenninatioonfthetestarticlsetabiliutnyderthedosingconditionosf thisassayand thetestarticlceharacteristaiscdsefinedintheGLP regulationosfFDA (21 CFR 58.105)E,PA-TSCA (40CFR 792.105)a,nd EPA-FIFRA (40CFR 160.105)isthe responsibiliotfythesponsor.
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10.0 DOSE SELECTION STUDY I
10.1 Dose Selection
Dose levelsof 1000,1510,2010,2540 and 3010 mg/kg were administeredby oral gavage forthefirsdtose selectiosntudy.
10.2 Dosing Information
The animalsused inthefirsdtoseselectioanssaywere dosed on July11,1996. The weightrangeof theanimalsused inthedose rangefindingassaywas 30.9-35.1and 22.2-27.6grams,forthe males and females,respectivelyD.osills! solutionwsere preparedjustpriortodosingand were preparedby makiniza 301 mg/mi stockforthehighdose(3010 mg/kg ).Thiswas preparedby addiniz deioriizewdater(CHV, Lot # 19)up toa volume of 15.0ml to3.7mi of T-656.,4. resultinigna clearcolorlesssolution.Dilutionsof thisstockwere preparedfor the2540,2010, 1510 and 1000 mg/kg doselevels.
Dosing was achievedusinga 10 ml/kg dosingvolume. Allanimalswere eight weeks and threedays oldatthetime ofdosing.An outlineof thedosingscheme isfound inthefollowingtable.
Dosing Scheme forDose SelectionAssay I
Treatment T-6564 1000 mg/kg 15 10 mg/kg 2010 mg/kg 2540 mg/kg 3010 m2Ag
Male Female
3
3
3
3
3
3
3
3
A totalof '30ariimalswas used inthisassay.Alldosesgivenwere on an acute (one-timeonly)basis.
10.3 Resultsand Interpretation
All animalswere examined afterdosingand dailythroughoutthedurationof the study(fourdays)fortoxiceffectasnd/ormortalitiesT.he anirnalobservationfsor thisassayaresummarized inthefollowingtable:
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AnimalObservationfsorToxicitfyorDoseRangefindinAgssayI
Time After Dose Dosing Level (hours) (mg/kg)
Observations
=0.2
all Normal
1000 Normal
Male # 9761 and Female #s 9776 & 9788 found dead.
1510 Remaining males appeared hunched and hypoactive.
16.8
Remaining female appeared normal.*
2010, 2540, 3010
Normal
=40.2
1000, 1510, 2010, 2540
Normal
Male #s 9765 & 9767 found dead. 3010
Remaining male and allfemalesappearnormal.
1000 Normal
1510 =67.0 -
2010, 2540, 3010
1000
Male # 9766 found dead. Remaining male and female appeared normal.
Normal
Normal
-88.5
1510
2010, 2540
Remaining niale# 9774 found dead. Remaining female appeared normal.
Normal
Remaining male # 9764 appearedhypoactiveand hunched 3010
with a rough haircoat.All femalesappeared normal.
The mortalitydata forthisassay aresummarized in the follo%%intgable:
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Summar-yofMortalitifeosrDoseRangefmdingI
Treatment T-6564 1000 mg/kg 15 10 mg/kg 2010 mg/kg 2540 mg/kg 3010 mg/kg
Male
0/3 3/3 0/3 0/3 2/@3
Female
0/3 2/') 0/3 0/3
10.4 Conclusion
Based on theseresultst,he maximum tolerateddose could not be determined.
11.0 DOSE SELECTION STUDY E
11.1 Dose Selection
Dose levelsof 1010,1520,2020 and 2530 mg/kg were administeredby oral gavage forthe second dose selectionstudy.
11.2 Dosing Information
The animalsused inthe second dose selectionassay were dosed on July 24, 1996. The weight range of the animals used in the second dose range findingassav%%-as 30.1-35.8and 2').6-28.4grams, forthe males and females,Tespecti-,-elDyo.singsolutionswere preparedjustpriortodosingand were preparedby makin2 a 253 mg/mi stockforthehigh dose (25-'1m0g/kg ). This was preparedby addine deionizedwater (CHV, Lot # 20) up toa volwne of 14.0n-dto2.9 mJ of T-6564, resultinigna clearcolorlessolution.Dilutionsofthisstockwere preparedfor the 2020, 1520 and 10 10 mg/kg dose levels.
Dosing was achievedusinga 10 ml/kg dosing volume. All animalswere eight weeks and two days oldatthetime ofdosing. An outlineof thedosing scheme is found inthe followingtable:
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DosingScheme forDose SelectioAnssay II
Treatment T-6564 1010 mg/kg 1520 mg/kg 2020 mg/kg 2530 mg/kg
Male Female
3
3
3
3
3
3
3
3
A totalof24 animalswas usedinthisassay.Alldosesgivenwere on an acute (one-timoenly)basis.
11.3 Resultsand Interpretation
Allanimalswereexamined afterdosingand dailythroughoutthedurationofthe study(thredeays)fortoxiceffectasnd/ormortalitieTsh.e animalobservations forthisassayaresummarized inthe followingtable:
Animal ObservationsforToxicityforDose RangefindingAssay 11
Time After Dose
Dosing Level
Observations
(hours) (mg/kg)
=O.i,i.i All Normal & 15.6
=17.9 1520 Female # 1417 found dead.
=21.6 1520 Male # 1405 founddead.
Z39.5 All Normal
=66.6 All Normal
=90.4 All Normal
T*hemortalitdyataforthisassavaresummarized inthefollowingtable:
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SummaryofMortaUtifeosrDoseR2ngefindiInIg
Treatment T-6564 1010 mg/kg 1520 mg/kg 2020 mg/k-g 2530 me,/kg
Male
0/3 1/-,) 0/1@ 0/3
Female
0/3 1/3 0/3 0/1)
11.4 Conclusion
Based on theseresultst,hemaximum tolerateddosewas estimatedas about 2000 mg/kg.
12.0 MICRONUCLEUS STUDY
12.1 Dose Selection
Basedon resultfsrom thedoseselectiosntudy,doselevelsof 498,995,and 1990 mg/kg were testedinthisstudy.
12.2 MicronucleusAssay DosingInformation
The animalsusedinthemicronucleusassaywere dosed on July30, 1996. Cyclophosphamide(CP),(CAS f,6055-19-2;Sigma,Lot 4 26HO473),theI>Ositive controlw,as solubilizeidnsterildeeionizedwater(Lot-419,preparedatCHV) and was administerebdy oralgavageat80.0mg/kg. The vehiclecontrol. deionizedwater(CHV, Lot 1'2r0),was administerecdoncbnently,%itthhetest articlaeta volumeof 10mukg. The weightrangeoftheanimalsusedinthe micronucleusassaywas 31.0-37.7and 22.4-28.3grams forthemales and females, respectivelTyh.e dosingsolutionfsortheassaywere preparedby makine a 199 mg/ml stockforthehighdose(1990mg/kg ).Thiswas preparedby adding deionizewdater(CHV, Lot'm20) up toa volume of30.0ml to4.9ml ofT-6564, resultinigna clearcolorlesssolutionD.ilutionosfthisstock verepreparedfor the995 and498 mg/kg doselevels.A secondgroupofanimals(designated SecondaryDose Group)was alsoassignedtothestudyand was dosedwiththe highdoseofthetestarticleT.hese animalswere onlyused intheassayas replacementfsorany which diedintheprimarydosegroup.
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Tenanimal(sfivmealeasndfivfeemalewse)rerandomlayssignteodeachdose/harvestimegroup.Vehicleand positivceontrolgroups,euthanized approximatel2y4 hoursafterdosing,were includedintheassay.The animals dosedwiththetestarticlweere euthanizedapproximately24,48 and 72 hours afterdosingforextractioonfthebone marrow. An outlineofthedosingscheme
isfoundinthefollowintgable:
DosingScheme forMicronucleusAssay
T-6564
Treatment 498 mg/kg
Number ofAnimalsAssigned
PrimaryDose Groups SecondaryDose
24 Hr 48 lir 72 Hr
Group '
MF MF MF
MF
55 55 55
995 mg/kg
55 55 55
1990 mg/kg
55 55 55
10 10
VehicleControl.deionizedwater,10 ml/kg
55 -- --
--
PositivCeontrolC,vclophosphamide,80.0mg/kg 5 5
The animalsassignedtothesecondarydosegroupswere dosedand wereonlyusedto replaceanimalswhich diedintheprimarydosegroupatthehighdoselevel.Allextra animalsnotused asreplacementswere euthanizedatthecompletionoftheVW.
A totalof 130animalswas usedinthisassay.The age oftheanimalsatthetimeof dosingwas eightweeks and one day.
Volumes dosedwere 10mukg and were basedupon individuaalnimalweights.
l').OBONE MARROW HARVEST, SLIDE PREPARATION AND ANALYSIS
At theappropriathearvestime,theanimalswereeuthanizebdy C02/02 inhalation. followedby C02 inhalatioandpenetrationfthethorax.The adheringsofttissuaend epiphysesofbothfemorawereremoved. The marrow was flushedfrom theboneand =mferred tocentifugetubescontainin3g -5 ml bovineserum (onetubeforeach animal).Followingcentifugatiotnopelletthetissuet,hesupernatanwtas removed by aspiratioannd portionosfthepellewtere spreadon slidesand airdried.The slideswere fixedinmethanol,and staineidnMay-Grunwald solutiofnollowedby Giemsa (Schmid, 1975).The air-driesdlidewsere coverslipped.
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Theslidweesrecodefdoranalysainsd,scorefdormicronucalnedithepolychromatic erythrocyt(ePCE)tonormochromaticerythrocyt(eNCE) cellratioS.tandardformswere usedtorecordthesedata.One thousandPCEs peranimalwere scored.The frequencvof micronucleatedcellswas expressedaspercentmicronucleatedcellsbased on thetotal PCEs presentinthescoredopticfield.The normalfrequencyofmicronucieilnthis Crl:CD-I'O(ICRB)R straiinsabout0.0-0.4%.
The frequencyofPCEs versusNCEs was determinedby scoringthenumber of PCEs and NCEs observedintheopticfieldwshilescoringthefirs1t000 erythrocytes.
14.0 EVALUATION CRITERIA
14.1 General
The criterfioartheidentificatioofmnicronuclewiere thoseofSchmid (1976). Micronucleiwere darklystainedand generallryound,althoughalmond and ringshapedmicronucleoiccasionalloyccurred.Micronucleihad sharpborders and were generallbyetween 1/20and 115thesizeof thePCE. The unitof scoring was themicronucleatecdelln,otthemicronucleust;hustheoccasionaclellwith more thanone micronucleuswas countedasone micronucleatePdCE, nottwo (or more)micronucleiT.he staininpgrocedurepermittetdhedifferentiatbiyoncolor ofPCEs and NCEs (bluish-graenyd red,respectively).
14.2 Data Presentatioannd Interpretation
Data aresummarizedby sexand dosegroupsforthedifferenttimepoints. Individuaalnimaldataarealsopresented.The analysisofthesedatawas performedusingan analysiosfvariance(Winer,1971)on eitheruntransformed (when variancesarehomogeneous)and ranktransformed(when variancesare heterogeneousp)roportionosf cellwsithmicronuclepieranimal.Iftheanalysis ofvariancweas significa(npt<0.05)a,Dunnett'ts-tes(tDunnett,1955;1964)was usedtodeterminewhich dosegroups,ifany,were significantdliyfferenftromthe negativecontrol.Analyseswere performedseparatelfyoreach harvesttimeand sexcombination.The criterifaordetermininga positivreesponseinvolveda statisticaslilgynificadnotse-relateidncreaseinmicronucleatePdCES, or the detectioonfa reproduciblaend statisticaslilgynificapnotsitivreesponseforat leastone doselevel.A testarticltehatinducedneithera statisticaslilgynificant dose responsenora statisticaslilgynificaanntd reproducibliencreaseatone dose levelwas considerednegative.In eithercase,thefinaldecisionwas basedon scientifjiucdgment.
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15.0 RESULTSAND RNTEPPRETATION
Allanimalswereobservedimmediatelyafterdosingand periodicaltlhyroughouthe durationoftheassayfortoxicsymptoms and/ormortalitieAsl.lanimalsinthevehicle and positivceontrolgroupsappearednormal afterdosingand remainedhealthyuntilthe appropriathear-vesttimes.Alltestarticldeosedgroupsappearednormal inunediately afterdosing.Approximately22 hoursaftedrosing,one male (#1605)fromthesecondary dose groupwas founddead.Allotheranimalsappearednormal atthistimeand remained healthyuntiltheappropriathearvestimes.
The testarticlTe-,6564,inducedno significainntcreaseisnmicronucleated polychromaticerythrocyteosverthelevelosbservedinthevehiclecontrolisneithesrex oratany oftheharvestimes.The positivceontrolC,yclophosphamide,induced significanitncreaseisnmicronucleatePdCEs inbothsexesas compared tothevehicle controlsw,ithmeans and standarderrorsof 3.86% 0.55% and 3.58% 0.79% forthe malesand females,respectivelyT.he datasummarizedby dosegroup arepresentedin Table I and individuaalnimaldataarefoundinTables2 through7. Historicaclontrol dataarepresentedinTable 8.
16.0 CONCLUSION
The testmaterialT,-6564,didnotinducea significainntcreasienmicronucleinbone marrow polychromaticerythrocyteusndertheconditionosfthisassayand isconsidered negativeinthemouse micronucieusassay.
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17.0 REFERENCES
Dunnet@ C.W.: A multiplecomparisonsprocedureforcomparingseveraltreatmentswith a control.J.Am. StatisAts.soc.,JQ:1096-1121,1955.
Dunnett,C.W.: New tablesformultiplceomparisonswitha controlB.iometrics2,@: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.,M:61-118, 1983.
Schmid,W.: The micronucieustest.MutationRes.,2J:9-15,1975.
Schmid,W.: The micronucleustestforcytogenetiacnalysisC.hemical Mutagens:Principleasnd Methods forTheirDetectionV,ol.4 (A.Hollaender, ed.).Plenum,pp.31-53,1976.
Winer,B.J.:StatistiPcrailncipleisnExperimentaDlesim McGraw-Hill,New York, Second Edition1,971.
18.0 DEVIATIONS FROM THE SIGNED PROTOCOL
I. Inbothtrialosfthedoserangefmdingstudiesa,nimalswere observedforfour daysand notthreedavsafterdosing.Thishad no impacton studyintegrity.
2. Due toa technicaolversightm,ore than1000 erythrocytewsere analyzedfor animal#'s1576and 1628. Thishad no impacton theintegritoyfthestudy.
3. On July31,1996,forthedefinitivsetudy,thehumiditywas recordedas 75.1 Thishad no impacton theintegritoyfthestudy.
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i i
19.0 EX]PERIMENT DATA TABLES
i 2
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TABLE I
SPONSOR: 3M Corporation MICRONUCLEUS DATA SUMMARY TABLE
TEST ARTICLE: T-6564
ASSAY: 17750
TREATMENT DOSE
HARVEST TIME (HR)
% MICRONUCLEATED PCEs
MEAN OF 1000PER ANIMAL * S.E.
MALES
FEMALES
TOTAL
CONTROLS VEtflCLE POSMVE
Water
24 hr 0.08* 0.06 0.08* 0.04 0.08* 0.03
CP 80.0mg&g 24 hr 3.86* 0.55* 3.58* 0.790 3.72* 0.460
RATIO PCE:NCE
MEAN:L S.E.
MALES
FEMALES
0.53 0.08 0.61 0.05
0.73t 0.07 0.96* 0.17
TEST ARTICLE
498 mg/kg 24 hr 48 hr 72 hr
995 me&-9 24 hr 48 hr 72 hr
1990mg/kg 24 hr 48 hr 72 hr
0.20* 0.06 0.02 0.02 0.09 0.06 0.12 0.06 0.12 0.04 0.10* 0.08 0.30* 0.13 0.00* 0.00 0.08* 0.06
0.06* 0.06 0.08* 0.04 0.12* 0.07 0.18* 0.16 0.06 * 0.04 0.10* 0.06 0.09 * 0.06 0.04* 0.02 0.12 :k0.04
0.13* 0.05 0.05* 0.02 0.10* 0.04 0.15* 0.09 0.09 * 0.03 0.10* 0.05 0.19* 0.08 0.02* 0.01 0.10* 0.03
0.71 0.08 0.59* 0.05 0.52 * 0.08 0.69 .i0.12 0.63 0.03 0.45 0.08 0.69 0.07 0.60 0.06 0.49 :L 0.11
0.92 * 0.05 0.53* 0.04 1.12 * 0.43 1.03 * 0.13 0.54 * 0.06 0.95 0.21 0.75 0.07 0,56 0.07 0.78 Oll
Significantglrveaterthanthecorrespondinvgehiclecontrolp,<0.05. CP - Cvclophosphamide PCE = Polvchromatiecrvthrocyte NCE = NorTnochromaticerythrocyte
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TABLE2
MICRONUCLEUS TEST - INDIVIDUAL ANIMAL DATA
SPONSOR: 3M Corporation
TEST ARTICLE: T-6564
ASSAY NO.: 17750 TREATMENT
24 HOUR HARVEST
MALE
ANIMAL NUMBER
# MN
RATIO
PCEs/ PCE:NCE
1000 PCEs
VEMCLE CONTROL POSMVE CONTROL
TEST ARNCLE
Water CP 90.0mg/kg
498 mgtkg 995 mglkg 1990 mg/kg
1557
1
1574
0
1576
0
1585
3
1614
0
1564
46
1566
32
1569
25
1607
56
1612
34
1562
3
1570
1
issi
3
1586
3
1587
0
1568
1
1571
0
591
3
11598
2
1602
0
1558
2
1567
0
1580
8
1594
3
1611
2
0.52 0.79* 0.62 0.39 0.35
0.62 0.49 0.77 0.64 0.53
0.73 0.73 0.97 0.61 0.48
0.69 0.64
04 0I.-80 0.29
0.72 0.61 0.96 0.55 0.63
CP Cyclophosphamide PCE - Polychromaticcrarocyte # NM PCEs - MicronucleatePdCEs NCE = Normochromaticerythrocyte
1059 erythrocytesscored
CHV StudyNo.: 17750-0-455
20
CORNINGHazleton
TABLE3
MICRONUCLEUS TEST -INDIVIDUAL ANIMAL DATA
SPONSOR: 3M Corporation
TEST ARTICLE: T-6564
ASSAY NO.: 17750 TREATMENT
24 HOUR HARVEST
FEMALE
ANIMAL NLTMBER
# MN RATIO PCEs/ PCE:NCE 1000 PCEs
VEICCLE CONTROL POSITIVE CONTROL TEST ARTICLE
Wamt CP 90.0mg/kg
499 mglkg 995 m&lkg 1990 mg/kg
1669
1
0.65
1671
2
0.81
1678
0
0.82
1679
1
0.89
1683
0
0.50
1641
51
0.72
1653
33
1.06
1663
56
0.57
1664
25
0.89
1667
14
1.56
1627
0
0.97
1630
0
1-00
1635
0
0.74
1644
3
0.90
1652
0
1.01
1633
0
0.78
1637
0
0.77
1660
0
0.96
1666
8
1.26
1672
1
1.39
1643
0
0.89
1650
0
0.67
1668
1
0.88
1681
3
0.53
1682
0
0.78
CP Cyclophosphamide PCE - Polychromaticrythrocvtc # MN PCF-s- MicronucleatePdCEs NCE - Nomochromatic erythrocytc
CHV StudyNo.: 17750-0-455
21
CORNINGHazleton
TABLE4
MICRONUCLEUS TEST - INDIVIDUAL ANIMAL DATA
SPONSOR: 3M Corporation
TEST ARTICLE: T-6564
ASSAY NO.: 17750 TREATMENT
48 HOUR HARVESR
MALE
ANIMAL NUMBER
# MN RATIO PCEs/ PCE:NCE 1000 PCEs
TEST ARNCLE
499 mglkg 995 mwkg 1990
1563
0
0.60
1582
0
0.75
1593
1
0.43
1603
0
0.54
1619
0
0.62
1572
1
0.67
1599
1
0.66
1596
2
0.52
1604
2
0.66
1615
0
0.62
1577
0
0.75
1579
0
0.68
1583
0
0.60
1595
0
0.41
1606
0
0.53
PCE Poivchromatiecrvthrocyte MN PCEs = Micronucleatcd PCEs
NCE Norrnochromatic crythroc.vtc
CHV StudyNo.: 17750-0-455
22
CORNINGHazieton
TABLE5
MICRONUCLEUS TEST - INDIVIDUAL ANIMAL DATA
SPONSOR: 3M Corporation
TEST ARTICLE: T-6564
ASSAY NO.: 17750 TREATMENT
48 HOUR HARVEST
FEMALE
ANIMAL NUMBER
# NfN RATIO
PCEs/ PCE:NCE 1000 PCEs
TEST ARTICLE
498 mgtkg "S mlag 1990 mfag
1628
2
1636
1
1639
0
1654
1
1662
0
1621
1
1649
0
1651
0
1657
0
1665
2
1626
0
632
1
11634
0
1639
1
1677
0
0.58* 0.66 0.39 0.49 0.54
0.36 0.4.4 0.57 0.71 0.59
0.40 0.64 0.49 0.77 0.51
PCE Poivchromaticerythrocyte # MN PCEs - MicronucleatePdCEs NCE = Normochromaticerythrocyte
1005 erythrocytesscored
CHV StudyNo.: 17750-0455
23
CORNINGHazleton
TABLE6
MICRONUCLEUS TEST - RNDIVIDUAL ANIMAL DATA
SPONSOR: 3M Corpomtion
TEST ARTICLE: T-6564
ASSAY NO.: 17750 TREATMENT
72 HOUR HARVEST
MALE
ANIMAL NUMBER
# NIN RATIO PCEst PCE:NCE 1000 PCE4
TEST ARTICLE
498 mg/kg 995 mg/kg 1990 m&lkg
1561
0
0.65
565
0
0'74
11599
0
0.44
1600
3
0.34
1609
1
0.42
1555
1
0.49
1556
0
0.17
1559
0
0.59
1590
4
0.45
1597
0
0.57
1554
3
0.21
1560
1
0.78
1573
0
0.63
1613
0
0.24
1617
0
0.59
PCE Polychromatiecrythrocyte # MN PCEs = MicronucleatePdCEs NCE - Normochromaticerythrocyte
CHV StudyNo.: 17750-0455
24
CORNINGHazleton
TABLE7
MICRONUCLELJS TEST - INDIVIDUAL ANIMAL DATA
SPONSOR: 3M Corpomtion
TEST ARTICLE: T-6564
ASSAY NO.: 17750 TREATNIENT
72HOUR HARVEST
FEMALE
ANIMAL NUMBER
# MN RATIO PCEst 1000 PCEs PCE:NCE
TEST ARTICLE
498 mglkg 995 MBAs 1990 mg/kg
1619
0
0.60
1620
1
0.73
1624
0
0.45
1656
4
2.82
1680
1
0.99
1645
2
1.60
1648
3
0.79
1658
0
0.66
1674
0
0-33
1675
0
0.89
1622
1
0.67
1625
2
0.73
1629
0
0.34
1640
2
1.59
1642
1
0.56
PCE Polychromatiecrythrocyte 4 MN PCEs = MicronucieatePdCEs NCE Norrnochromaticerythrocyte
CHV StudyNo.: 17750-0455
25
CORNINGHazieton
TABLES
MOUSE NUCRONUCLEUS HISTORICAL CONTROL DATA 7195THROUGH 12/95
POOLED VEIECLE CONTROLS MrN MAX AVG N
% MICRONUCLF.ATED PCEs PER 1000 PCE
MEAN Of 1000 PER ANIMAL * S.E.
MALES
FEMALES
TOTAL
0.00 0.22 0.097:L0.007 47
0.00 014 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 0.350* 0.021 47
014 1.03 0.587* 0.025 47
POSITIVE CONTROLS Cyclophosphwnide9,0.0mgfkg
MrN MAX AVG
N
2.00 5.69 3.692* 0140 19
1.50 6.36 3.170* 0145 19
2.41 5.38 3.426* 0.184 19
0.41 0.72 0.577* 0.020 19
0.40 0.79 0.598* 0.026 19
PCE = Polychromaticerythrocyte NCE = Normochromaticcrarocyte N Number ofharvests
CHV StudyNo.: 17750-0-455
26