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THE DEVELOPMENT SERVICES COMPANY
L5178Y TK
FINAL REPORT
MOUSE LYMPHOMA FORWARD MUTATION
WITH A CONFIRMATORY
ASSAY
WITH N-ETFOSE T-6316
ASSAY
AUTHOR
-
Maria A. Cifone,PhD
PERFORMING LABORATORY
Covance LaboratoriesInc(Covance) 9200 Leesburg Pike
Vienna,Virginia22182
LABORATORY
PROJECT IDENTIFICATION
Covance Study No.: 20785-0-431 ICH 3M Study No.: T-6316.12
SUBMITTED TO
3M CorporateToxicology 3M Center
Building220-2E-02 St.Paul,Minnesota 55133-3220
STUDY COMPLETION DATE September 05, 2000
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Covance20785-0-43I1CH 3M StudyNo.:T-6316-12
QUALITY ASSURANCE STATEMENT
L5178Y TK +/-Mouse Lymphoma Forward Mutation Assay with a ConfirmatoryAssay withN-ETFOSE T-6316
The reporthas been reviewed by the QualityAssurance Unit of Covance LaboratoriesInc.,in accordancewith the Good Laboratory Practiceregulationsas setforthintheFood and Drug Administration(FDA) Title21 of theU.S.Code of FederalRegulationsPart58,and with any applicableamendments. The followinginspectionswere conducted and the findingsreportedto the Study Directorand studydirectormanagement. Writtenstatusreportsof inspectionsand findingsareissuedto Covance management accordingtostandardoperatingprocedures.
InspectionDates
May 31, 2000 July 19-20,2000 August 30, 2000
Phase
Scoring DraftReport Review FinalReport Review
Dates Reported to Study Directorand Study
DirectorManagement
May 31, 2000
July 20, 2000
August 30,2000
Auditor
S.Ballinger P. Cdceres P. Cdceres
-::@ cl@
RepresentatiQveu,aliAtsysuranUcneit
0,/ S-/(:)o
Date
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STUDY COMPLIANCE
Covance20785-0-43I1CH 3M StudyNo.: T-6316.12
AND CERTIFICATION
The studywas conductedincompliancweiththeGood LaboratorPyracticreegulatioanssset forthin theFood and Drug Administration(FDA) Title21 of theU.S.Code of Federal RegulationsPart58, and with any applicableamendments. There were no deviationsfrom the regulationsor the signed protocolthatwould affectthe integritoyf the studyor the interpretation of thetestresults.The raw datahave been reviewed by the Study Director,who certifietshatthe evaluationof the testarticlaes presentedhereinrepresentsan appropn*ateconclusionwithinthe contextof the studydesign and evaluationcriteriaA.ll testand controlresultsin thisreportare supportedby an experimentaldata recordand thisrecordhas been reviewed by the Study
Director.
Study Director:
N@4 A. Cifone,PhD
Date
Genetic and Molecular Toxicology
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Covance20785-0-43I1CH 3M StudyNo.:T-6316.12
TA,BLE OF CONTENTS
PageNo.
ABSTRACT .........................................................................5................
STUDY INFORMATION .............................................................6................
Sponsor Test Article Assay Information Study Dates SupervisoryPersonnel OBJECTIVE ........................................................................6................
TEST SYSTEM RATIONALE ........................................................7................
MATERIALS AND METHODS .......................................................7................
Test System TestArticle ControlArticles S9 Metabolic ActivationSystem Dose RangefindingAssay Mouse Lymphoma MutagenicityAssay
DATA .............................................................................1.2...............
Data Presentation Assay Acceptance Criteria Assay EvaluationCriteria RESULTS ..........................................................................1.5...............
Test ArticleHandling Dose Rangefinding Assay Mutation Assays CONCLUSION ....................................................................1.7................
RECORDS TO BE MAINTAINED ...................................................1.7................
REFERENCES .....................................................................17................
DATA TABLES ....................................................................18................ HISTORICAL CONTROL DATA ....................................................2.9................
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Covance 20785-0-43I1CH 3M StudyNo.:T-6316.12
ABSTRACT The objectivoefthisinvitraossaywas toevaluatteheabilitoyfthetestarticlNe-,ETFOSE T-6316, toinduceforward mutationsatthethymidinekinase(TK) locusin themouse lymphoma L5178Y cellline.The testarticlweas solublein thevehicle(dimethylsulfoxidDeM,SO) at 125 mg/mL, the highestconcentrationprepared.
A dose rangefindingassaywas performed with and withoutmetabolicactivatiounsinga treatmentperiodof approximately4 hours. In addition,a dose rangefindingassaywithout metabolicactivatiounsinga treatmentperiodof approximately24 hourswas performed. The dose rangefindingassayswere initiatewdith concentrationsfrom 2.47 to 1250 @ig/mL. Inthe presenceand absence of ratliverS9 metabolicactivitwyith a 4-hourtreatmentperiod, N-ETFOSE T-6316 was noncytotoxicup to 19.7 gg/mL. Moderate cytotoxicitwyas induced from 39.3to 157 @ig/mL and higher concentrationswere excessivelycytotoxicor lethal.In the nonactivationdose rangefindingassayusinga 24-hour treatmentperiod,the testarticlienduced no cytotoxicitayt2.47 )ig/mL,weak cytotoxicitayt4.93gg/mL, and moderatelyhigh cytotoxicitayt9.85 @ig/mL. Higher concentrationswere excessivelycytotoxicor lethal.Dose levelsforthe mutation assayswere based on theseresults.
In thenonactivationmutation assaywith a treatmentperiodof approximately4 hours,sixdoses rangingfrom 25.3 to 201 gg/mL were analyzed formutant inductionand weak cytotoxicittyo high cytotoxicitwyas induced. None of the analyzed treatmentsinduced a mutant frequencythat exceeded the minimum criterifaor a positiveresponse.A confirmatoryassaywas perfon-ned.
In theconfirmatorynonactivationmutationassay,which was performed with a 24-hour treatmentperiod,seven treatmentsfrom 1.25 to 15.0 @tg/mL were analyzed and no cytotoxicittyo high cytotoxicitwyas induced. None of thetreatmentsinduced a mutant frequencythat exceeded the minimum criterifaor a positiveresponse. The testarticlweas, thereforee,valuated as negativewithoutmetabolicactivation.
In the initiamlutation assay inthe presenceof S9 metabolicactivationf,ourtreatmentsfrom 25.3 to 101 gg/mL were analyzedand weak cytotoxicittyo very high cytotoxicitwyas induced. None ofthe analyzed treatmentsin eitherassay induced a mutant frequency thatexceeded the minimum criterifaora positiveresponse.A confirmatorymutation assaywas perfon-ned.
Inthe confirmatoryactivationmutation assay,eighttreatmentsfi7om20.0 to 150 gg/mL were analyzedand weak cytotoxicittyohigh cytotoxicitwyas induced. None of thetreatments induced a mutant frequencythatexceeded the minimum criterifaora positiveresponse.The test articlweas, thereforee,valuatedas negativewith metabolicactivation.
The testarticleN,-ETFOSE T-6316, was evaluatedas negativeforinducingforward mutationsat theTK locusin L5178Y mouse lymphoma cellsunder activationand nonactivation.
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STUDY INFORMATION
Covance 20785-0-43I1CH 3M StudyNo.:T-6316-12
Sponsor 3M CorporatTeoxicology
TestArticle SponsorIdentificatiNo-nE:TFOSE T-6316 Lot 30035, 30037, 30039 Purity98.1% (August 19 sample) TM #: 1084A3 (August 19 sample) Dates Received: August 19, 1999 and June 07, 2000 Physical Description: whiteto beigesolid(August 19 sample) beigechunky solid(June07 sample) Storage Conditions: room temperature
Assay Information Type of Assay: L5178Y TK+/-Mouse LyTnphoma With A ConfirmatoryAssay ProtocolNo.: 431 ICH Edition2 Covance Study No.: 20785-0-431 ICH
Forward Mutation Assay
Study Dates
InitiationDate: May 08,2000 Experimental StartDate: May 10,2000 Experimental Termination Date: June 22,2000
Supervisory Personnel
Study Director: Maria A. Cifone,PhD Laboratory Supervisor: Kathryn Flanders,MS
OBJECTIVE
The objectiveof thisstudywas to evaluatetheabilitoyf thetestarticlteoinduce forward mutationsatthethymidinekinase(TK) locusof L5178Y mouse lymphoma cellsasassayedby colony growth inthepresenceof 5-trifluorothymidi(nTeFT). The assaydesignisbased on OECD Guideline476, updated and adopted July21, 1997,and the ICH S2B Guideline, "Genotoxicity:StandardBatteryforGenotoxicityTestingof Pharmaceuticals".
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Covance 20785-0-43I1CH 3M StudyNo.:T-6316.12
TEST SYSTEM RATIONALE
Thyinidinkeinase(TK)isanenzyme thatallowscelltsosalvagtehymidinferomtheculture medium forDNA synthesis.IfthethyrnidineanalogTFT isincludedinthegrowth medium, the analogwillbe phosphorylatedviatheTK pathway and willcausecelldeathby inhibitinDgNA synthesis.Cellswhich areheterozygousattheTK locus(TK+'-)may undergo a single-step forward mutation tothe TK-/-genotype inwhich littloerno TK activitryemains. Such mutants areviableinnormal culturemedium because de novo DNA synthesisdoes not requireexogenous thyrnidine.TK-/-mutants,however, are resistanttoTFT because they cannot incorporatethis toxicanalog ofthyrnidineintoDNA. Cellswhich grow to form coloniesinthepresenceof TFT arethereforeassumed tohave mutated, eitherspontaneouslyor induced by the testarticlea,tthe
TK-1-locus.
MATERIALS
AND METHODS
Test System Test Cells. The mouse lymphoma L5178Y cellline,heterozygous atthe TK locus and designatedas clone3.7.2C,was used forthisassay. Stock cultureswere obtainedfrom Dr. Donald Cliveand arestoredin liquidnitrogen.Laboratory culturesforthemutation assays were maintainedin logarithmicgrowth by serialsubculturingforup to4 months and were then replacedby cellsfrom the frozenstock. Cultureswere grown in a shakerincubatorat approximately37'C. A continuouscelllogwas kept to recordgrowth,doublingtimes,and subcultureoperations.To reduce the frequencyof spontaneous TK-/-mutants priorto use inthe mutation assay,cellcultureswere exposed to conditionsthatselectedagainsttheTK-/-phenotype and then returnedtonormal growth medium for3 to 8 days.
Media and Cell Culture Conditions. The medium used forthisstudywas RPMI 1640 (Amacher etal.,1980;Cliveetal.,1987) supplemented with horse serum (10% by volume), PluronicoF68, L-glutamine,sodium pyruvate,penicilliannd streptomycin.Treatment medium was Fischer'smedium with the same medium supplements used inthe culturemedium except thatthehorse serum concentrationwas reduced to 5% by volume. Cloning medium consistedof the R-PMI 1640 culturemedium with up to 20% horseserum, withoutPluronicoF68 and with the additionof 0.24% BBL(& agarto achieve a semisolidstate.Selectionmedium was cloning medium thatcontained3 @ig/mLof TFT (Cliveetal.,1987).
Test for Mycoplasma Contamination. Mycoplasma testingwas performed by a commercial laboratoryon stockculturespriortopreparingstocksforfreezing.Stock culturesforthe mutation assayswere maintained up to4 months afterthawing a sample from a frozenstock. Mycoplasma testingwas alsoperformed on culturesthawed foruse inassayspriorto discarding thestock.This ven*fiedthattherewere no mycoplasma during thelifeof theculture.Both direct culturingmethods and the indirectHoechst stainingmethod were used.
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Covance20785-0-43I1CH 3M StudyNo.: T-6316.12
KaryotypeStabilityK.aryotypestabiliatsym,easuredby mean chromosomalnumber,was routinelyperfon-nedon stockculturesused forassays.Mean chromosomal number was determined priorto discardingthe stockto ensurethatchanges inthe culturehad not occurred. Karyotype analysis,includingbanding, was performed on cellsafterpreparationof a freshftozen stock culture.
Test Article The sponsor was responsibleforthe determinationof the testarticlestabilitaynd the testarticle characteristicas definedin the GLP regulations.
Control Articles Vehicle Control Article.The vehicleof choiceforthe testarticlweas DMSO (Sigma Chemical Co., Lot No. 99HOO20, CAS #67-68-5). Concurrentvehiclecontrolswere performed foreach portionofthe assayby exposing thecellstoDMSO inculturemedium. Intheactivatiopnortion of the assays,the vehiclecontrolswere alsoexposed tothe S9 metabolic activatiomnix. Single vehiclecontrolcultureswere initiateidn thedose rangefindingassays and threevehiclecontrol cultureswere initiateidn themutation assays.
PositiveControl Articles.The positivecontrolarticlelsistedbelow were chosen because of the largedatabase availableand because both chemicalsinduce small and largecolonies(Clive etal.,1987,Young etal.,199 1).
Methyl methanesulfonate(MMS) isa directactingmutagen thatishighlymutagenic to L5178Y TK+@-cells.MMS (AldrichChemical Co.,Lot No. 08109BU, CAS # 66 27-3)was used in duplicateat 13 @tg/niJf-orthe nonactivationassay with a 4-hour treatmentperiod,and in duplicateata concentrationof 6.5 ptg/mL forthe nonactivationassaywith a 24-hour treatment period.
Methylcholanthrene(MCA) requiresmetabolicactivatiobny microsomal enzymes to become mutagenic to L5178Y TK+/-cells.MCA (Sigma Chemical Co.,Lot No. 77H2515, CAS # 56-49-5)was used at2 @ig/mL and 4 @tg/mL as a positivecontrolforassaysperformed with S9 metabolicactivation.
S9 Metabolic Activation System The invitrometabolicactivatiosnystem was comprised of ratliverenzymes (S9 fractiona)nd an energyproducing system (CORE) comprised of nicotinamideadenine dinucleotidpehosphate (NADP, sodium salt)and isocitrateT.he S9 enzymes were preparedcommercially(Molecular Toxicology,Inc.,Boone, NC) and were testedforsterilitaynd enzyme activityT.he enzymes were obtainedfrom the9000 x g supernatant(S9)of liverhomogenate from male Sprague Dawley ratstreatedwith Aroclor 1254,which inducesmixed functionoxidaseenzymes capable of transformingchemicalstomore activeforms. Rats were treatedonce with 500 mg/kg of Aroclor 1254,and the S9 was prepared approximately5 days later.The S9 and reactionmixture (CORE) were retainedfrozenatabout -80'C untilused.
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Covance20785-0-43I1CH 3M StudyNo.: T-6316.12
S9 MetabolicActivatioSnystem
Component
FinalConcentrationin Cultures
-NADP (sodiumsalt)
3 mM
Isocitrate
15 mM
S9 homogenate
-10 @tL/mL
Dose Rangefinding Assay A preliminarydose rangefindingcytotoxicityexperiment was performed with a treatmentperiod of approximately4 hours with and withoutS9 activations,incesubstantiaslhiftsintoxicityoften occur forthetwo testconditions.In addition,a nonactivationassaywith a treatmentperiodof approximately24 hours was performed. A wide range of testarticlceoncentrationswas tested forcytotoxicitsytartinagt 1250 @ig/mL,and followedby nine lower concentrationsprepared in 2-folddilutionsteps.Afteran exposuretime of approximately4 hours or approximately 24 hours at35-38'C in an orbitalshakerat80 10 orbitsper minute,the cellswere washed twice with culturemedium and resuspendedin culturemedium. The cellsreceivingthe24-hour treatmentwere counted and subcultured,as necessary.Following theterminationof the treatmentperiods,the cellswere incubatedovernightusingproceduresidenticatlo thosefor mutation experiments.A cellcountwas determinedafterthe growth periodtomeasure the reductionincellgrowth relativteo theconcurrentvehiclecontrolcellcultures.The preliminary dose rangefindinginformationwas then used to selectdose levelsforthe mutation assay.
Test articleconcentrationsare chosen to cover a toxicityrange from 10% to 20% survivaltono apparen- teffecton growth compared tothevehiclecontrol.Iflittloer no toxicitiysobserved and solubilitiysmaintained,themutationexperiment isinitiatewdith a maximum concentrationof 5 mg/mL or 10 mM (which everislowest).Ifprecipitatioonf thetestarticloeccursin the culturemedium, themaximum applieddose isatleasttwicethe solubilitlyimitin culture medium. In thisassay,thehigh dose limitedby cytotoxicity.
Mouse Lymphoma Mutagenicity Assay The assayprocedureused was based on thatreportedby Clive and Spector (1975),Cliveetal. (1979),Amacher etal.(1980)and Cliveetal.(1987). Although tenor elevendoseswere selectedto initiataemutation experiment,the objectivewas to carryatleastfourdoses through theentireassay.This procedurecompensated fornormal variationisn cellulatroxicityand helped toensure thechoice of atleastfourdoses over a wide cytotoxicitryange.
InitialNonactivation Assay. Logarithmicallygrowing laboratorystockcultureswere seeded intoa seriesof tubesat6 x 106 cellspertube. The cellswere pelletedby centrifugationt,he culturemedium removed, and the cellsresuspended to a finalvolume of 10 mL of treatment medium. The tubeswere placedin an orbitalshaker incubatorat35-38'C and rotatedat 80 10 orbitsperminute. Afteran exposure periodof approximately4 hours,thecellswere
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Covance 20785-0-43I1CH 3M StudyNo.: T-6316.12
centrifugaenddthetreatmenmtedium removed.The cellwserethenwashed,resuspendeidn 20 mL of culturemedium and returnedto theorbitalshakerincubator.
The assayconditionsconsistedof vehiclecontrolsintriplicattew,o positivecontrolsand eleven differentestarticledose levelsusingone cultureper dose level.Treatedculturesmay be eliminatedduring the expressionperiodas long as fourdose levelsare leftforanalysisofmutant induction.
The appearance of the treatedcultureswas recordedboth atthetime of treatmentand afterthe treatmentperiod. When thepH indicatorinthemedium had a colorchange, thepH was measured usingpH indicatorstrips.
A standardexpressionperiodof 2 days was used to allow formutant recovery,growth and
expressionof the TK-/-phenotype. Celldensitieswere determinedon Day I and were adjustedto
3 x 105 cells/mL in 20 mL of growth medium. Ifthecellsina culturefailedtomultiplyto a
105
densityof 4 x
cells/mL on the firsdtay aftertreatment,the culturewas
not subcultured.On
Day 2,cellcounts were againdetermined,and appropriatecultureswere selectedforcloningand
mutant selection.
Six doses were selectedformutant analysis.Ifpossible,doses were selectedto includenontoxic to highlytoxictreatmentconditions.Although relativgerowth givesan indicatioonf whether a testarticlieshighlyor weakly cytotoxics,pecificrangesforweak, moderate,highlytoxicand excessivelytoxicarenot specified.Severalfactorsgo intothecalculationand theStudy Director makes a decisionconcerningthelevelof cytotoxicittyhathas occurred.Cultureswith cell densitieslessthan approximately3 x 105 cells/mLwere not consideredforselection.
A totalof 3 x 106 cellsfrom each selectedtubewas suspended in selectionmedium in softagar torecoverTFT-resistantmutants. This sample was distributeidntothree100 mm dishes.The absoluteselectioncloningefficiencywas determinedby seedingthreedisheswith a totalof approximately600 cellsin agar cloningmedium. All disheswere placed inan incubatorat35-
38'C with 4-61/@C02:95% humidifiedair.After 13 days intheincubator,thecolonieswere countedwith theLoatsAssociates,Inc.(LAI) High ResolutionColony Counter(HRCC) System forthe Mouse Lymphoma Assay, Version 1.05bO4c.
The mutant frequency was calculatedas the ratioof the totalnumber of mutant coloniesfound in each setof threemutant selectiondishesto the totalnumber of cellsseeded,adjustedby the absoluteselectioncloningefficiency.Ifone dishin eithersetwas lostdue tocontaminationor othercause,the colony count of the missing dishwas determinedby a proportionequationbased upon the weights of the threedishesof the setand the colony countsin the two acceptable dishes.Ifa lostplatewas not availableforweighing,thecolony count of thelostplatewas determined from the average of the two remaining acceptableplates.
The measurement of the cytotoxicitoyf each treatmentwas the relativesuspensiongrowth of the cellsover the2-day expressionperiod(forthe4-hourtreatment)or therelativseuspension growth over the3-day treatmentand expressio-nperiod(forthe24-hour treatment)multipliedby
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Covance20785-0-43I1CH 3M StudyNo.:T-6316.12
therelaticvleoningefficienactythetimeofselectioAnl.thoughnotstrictalmyeasureofcell sur-vivatlh,isparameter(calledrelativgerowth or relativteotalgrowth,RTG) providesa measure of the effectivenesosf treatmentand was used as the basisforselectingdoses forany necessary subsequent trials.
Conf-irmatory Nonactivation Assay. The confirmatoryassayprocedure was similartothe assay using a treatmentperiodof approximately4 hours exceptthattheexposure time was increasedto approximately24 hours. The assay was conducted using resultsfrom the dose rangefindingassaywith a treatmentperiodof approximately24 hours. Ten treatmentswere initiateda,nd seven were analyzed formutant induction.
The cellswere obtainedfrom logarithmicallgyrowing laboratorystockculturesand were seeded intoa seriesof tubes at6 x 106 cellsper tube. The cellswere pelletedby centrifugationt,he culturemedium removed, and the cellsresuspended in a finalvolume of 20 mL of treatment medium. The dosed tubeswere closed,vortexed and placedin an orbitalshakerincubatorat3538'C at80 10 orbitsper minute. Aftera treatmentperiodof approximately24 hours,thecells were centrifugedand the treatmentmedium removed. The cellswere thenwashed, and resuspendedin 20 mL of growth medium at3 x 105 cellsper mL. The cultureswere then returnedtothe orbitalshaker.
A standardexpressionperiodof 2 days followingterminationof treatmentwas used to allow recovery,growth and expressionofthe TK-/-phenotype. Celldensitieswere determinedthe day aftertreatmenttermination,and each culturewas adjustedto3 x 105 cells/mLin 20 mL of growth medium. Ifthecellsina culturefailedtomultiplyto a densityof 4 x 105/mL on thefirst day aftertreatmenttermination,the culturewas returnedtothe incubatorwithoutbeing subcultured.The next day cellcounts againwere determined,and appropriatecultureswere selectedforcloningand mutant selection.Cultureswere counted 12 days aftercloning.
InitialActivationAssay. The activationassaywas identicatlo the nonactivationassayexcept fortheadditionof theS9 fractionofratliverhomogenate and necessarycofactors(CORE) duringthetreatmentperiodof approximately4 hours. The treatmentmedium includedthisS9 activatiomnix, which was preparedinunediatelypriortouse and kept chilled.Each batchof S9 was testedforactivatioonf theMCA positivecontrolarticlbeeforeitwas used forthe mutagenicityassay.The S9 homogenate was commercially prepared(MolecularToxicology, Inc.,Boone, N.C.,Lot No. 955) and consistedof the 9000 x g supernatantfrom the homogenized liversof Aroclor 1254-inducedadultmale Sprague Dawley rats.Cultureswere counted 13 days aftercloning.Eleven treatmentswere initiateda,nd fourwere cloned formutant analysis.
Confirmatorv ActivationAssay. The proceduresused in theconfirmatoryactivatioanssay were identicatlo theinitiaalctivatioanssay.Eleven treatmentswere initiateadn,d eightwere cloned formutant analysis.Cultureswere counted 13 days aftercloning.
SizingAnalysis. The L5178Y TK +/-mutation assay produces a bimodal distributioonf largeand smallmutant colonies.The originof thebimodal distributioonf mutant colonysizesis
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Covance 20785-0-43I1CH 3M StudyNo.: T-6316.12
consideretdoreflectthetypesofgenetidcamage,withthelargecoloniedserivedfromcellwsith intragenicmutations thataffectonly theTK gene and the smallcoloniestheresultof larger mutationsthataffectcellgrowth as well asthe TK gene (Hozier etal.,1985).
Both thesmalland largecolonieswere quantifiedforallcultures.A bimodal curve isgenerated and small and largecoloniesare quantitatedby the areasunder the curves. The largecolonies presumably an'sefrom pointmutationsand the smallcoloniesfrom chromosome changes. The LAI Automated Colony Counter was used. This countersizesallanalyzedcultures.
DATA
Data Presentation The historicaclontroldataarepresentedafterthedatatables.This reportincludesthefollowing information:
Dose Rangefluding Data. These dataincluded,foreach treatmentconditiont,hedailycell densitiesofthe individualculturesand the celldensitiesrelativetothe vehiclecontrols.
Cell Growth Characteristics.This includedthe number of cellsseeded forthe analysisof
mutant frequencies,the phenotypic expressiontime, and the selectiveagentand concentration.
In additiond,ailycelldensitieisntheindividualculturescarriedthroughtheexpressionperiod
and cloningwere included.The suspensiongrowth forvehicleand positivecontrolsf,orthe 4-hourtreatmentstudiescalculatedas (Day I celldensity/3x 105) X (Day 2 celldensity/3x 105
or Day I densityifnot splitback) describesthegrowth of the controlculturesduringthe
expressionperiod.For thenonactivationassaywith a 24-hour treatmentperiod,the suspension
growth forvehicleand positivecontrolsc,alculatedas [Treatmenttermination(Day 1)cell
density/3x
10']x
[Day
2
celldensity/3x
05
1
or Day
I densityifnot splitback] x [Day 3 cell
density/3x 105 or Day 2 densityifnot splitback],describesthe growth of the controlcultures
duringthe expressionperiod.For the test-artictlreeatedcultures,therelativesuspensiongrowths
were includedand calculatedas above, and were expressed as percentagesof the average vehicle
controlsuspensiongrowth. The relativceloningefficiencieosf testarticle-treatceudltureswere
expressedaspercentagesof the average vehiclecontrolcloningefficiency.The expressionof
RTG was theparameter thatwas used to assesstreatmentcytotoxicityand was obtainedby
multiplyingtherelativesuspensiongrowth by the relativceloning efficiency/100.
Mutant SelectionData. The mutant selectiondataincludesthe totalmutant colonies(i.et.otal number of mutant colony countsobtainedfrom 3 x 106 CellS sampled ftom one culture,seeded intoselectivmeedium and dividedamong threeculturedishes)and thetotalviablecolonies
(i.et.hetotalnumber of colony countsobtainedfrom 600 cellssampled from one culture,seeded
intononselectivemedium and dividedamong threeculturedishes).These were used tocalculate
themutant frequencyforeach culture.The ratioof cellsseeded formutant selectiotno cells seeded forcloningefficiencywas 0.5 x 104 . Therefore,the mutant frequencywas: (totamlutant
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Covance20785-0-43I1CH 3M StudyNo.:T-6316.12 4 colonies/totavliablecolonies)x 2 x 104. Mutant frequencywas given inunitsof 10-6. Also includedinthissectionwas the cloningefficiencyof vehicleand positivecontrolscalculatedas (totalnumber of viablecolonies/600x) 100%.
Assay Acceptance Criteria An assaywas consideredacceptableforevaluationof thetestresultsonlyifallofthecriteria given below were satisfiedA.ctivationornonactivationassayswere repeatedindependently,as needed, to satisfyacceptancecriteriaT.he Study Directorreviewed each assayin accordance with the followingcriteria:
Acceptable Controls. The averageabsolutecloningefficiencoyf thevehiclecontrolsshouldbe between 60% and 130%. A valuegreaterthan 100% ispossiblebecause oferrorsincellcounts (usually 10%) and variationisn celldivisionduringunavoidabledelaysbetween countingand cloningof many cultures.
A minimum acceptablevalueforthe average suspensiongrowth of the vehiclecontrolsfor 2 days isan 8.0-foldincreaseover theoriginalcellconcentrations.Lower valuesrenderan assay unacceptablebecause of poor cellgrowth.
The background mutant frequency(averageof vehiclecontrolvalues)iscalculatesdeparatelyfor concurrentactivationand nonactivationassays,even though the same populationof cellsisused foreach condition.For both conditions,the normal range of background frequenciesforassays performed with differenctellstocksis30 x 10-6 to 120 x 10-6 . Assays with backgrounds outside thisrange arenot necessarilyinvalidbut willbe evaluatedwith caution.
At leastone of thepositivecontrolculturesineach trialshould induce a mutant frequencyof at least200 x 10-6 . A trialmay be acceptablewithouta positivecontrol(due tocontaminationor technicalerror)only ifthetestarticliespositive.
Acceptable High Dose. For testarticlewsith weak or no mutagenic activitya,n assayshould includeappliedconcentrationsthatreduce the RTG to 10% or 20% of theaveragevehicle controlor reachthemaximum appliedconcentrationsgiven intheevaluationcriteriaR.TG representsa calculatioonf survivalthatisbased on both relativesuspensiongrowth duringthe expressionperiodand relativecloningefficiencyatthe time ofplating.Because mutant frequenciesincreaseas a functionof lethalitya,n attemptto obtaintreatmentsin therange of 10% to 20% RTG must be made inordertoconsiderthe assayas conclusive.This requirement was waived ifthe concentrationof the highestassayeddose was atleast75% of a higher, excessivelycytotoxicdose levelor ifthehighestdose was atleasttwicethesolubilitlyimitof the testarticlien culturemedium. There isno maximum toxicityrequirementfortestarticlewshich clearlyshow mutagenic activity.
Acceptable Number of Doses. Mutant frequencywillbe consideredacceptableforevaluation only iftherelativecloningefficienciys 10% or greaterand thetotalnumber ofviableclones exceeds about60. These limitsavoidproblems with thestatisticdailstributioonf scorable coloniesamong dishes.
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Covance20785-0-43I1CH 3M StudyNo.: T-6316.12
Mutantfrequenciefsoratleasftourdifferetnetstarticdloeselevelasrenon-naldleyten-niniend each assay;a minimum of four analyzed culturesisconsiderednecessaryto accepta singleassay forevaluationof the testarticle.
Mutant frequenciesare normally derivedfrom setsof threedishesforboth mutant colony count and viablecolony count. In orderto allow forlossesdue tocontaminationor otherreasons,an acceptablemutant frequency can be calculatedfrom a minimum of two dishesper set.
Assay Evaluation Criteria The testarticlewas evaluatedaspositive,negative,or equivocalinthisassay.A positive evaluationindicatesthatthe testarticliesa mutagen (inducesgene/chromosomal mutations)in thistestsystem. A negativeevaluationindicatesthetestarticliesno=utagenic inthistest system and causes no responsesthatcan be interpretedaspositive.However, thereare substancesforwhich theresultsremain questionable;any testarticlien thiscategorywillbe evaluatedas equivocal inthistestsystem.
Evaluation of a PositiveResponse. The testarticliesevaluatedas positiveifdose-dependent increasesof 2-foldor greaterinmutant frequencyare obtainedover the concurrentbackground mutant frequency.The background mutant frequencyisdefinedas the averagemutant frequency of thevehiclecontrolcultures.The 2-foldor greaterincreaseisbased on extensiveexperience which indicatessuch responsesarerepeatablein additionaltrials.Itisdesirabletoobtainthis relationshifporatleastthreedoses,but thisgoaldepends on the dose stepschosen forthe assay and toxicityatwhich mutagenic activityappears.
The dose-dependentrequirementiswaived ifa largeincreaseinmutant frequency(4-foldor higher)isobtainedfora singledose ator near thehighesttestabletoxicity.However, forthetest articlteobe evaluatedaspositive,any increasesmust be repeatableina second trial.
Evaluation of a Negative Response. The testarticlweas evaluatedasnegativeifa 2-fold increaseinmutant frequencywas not observed for(1)a range of doses thatextendedtotoxicities causing 10% to20% RTG, or (2)forrelativelnyontoxictestarticlesa,range of doses extending tothemaximum concentratioonf 5 mg/mL or 10 mM (whicheverislower),or (3)a rangeof dosesthatextended toa levelapproximatelytwice thesolubilitlyimitin culturemedium, or (4)theincrease(sa)renot repeatablein a confirmatorytrial.
Evaluationof an Equivocal Response. The testarticlweas evaluatedas equivocalin thistest system iftherewas no consistentevidence foreithera positiveor negativeevaluation.
Other Considerations. Treatments with testarticletshatreduced the RTG tolessthan 10% may occurin an assay,buttheresultsobtainedatthishich toxicitywere consideredbiologically irrelevanatnd were not used to evaluatethe testarticle.
For some testarticlest,hecorrelatiobnetween toxicityand appliedconcentrationispoor. The interactioonf thetestarticlweith thecellsisnot always repeatableor controllebdy
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Covance20785-0-43I1CH 3M StudyNo.: T-6316.12
concentratioInn.thiscaset,heinducedtoxicitaysmeasuredby theRTG isa bettemreasureofa dose relatedresponse than appliedconcentration.Therefore,indeterminingifa doserelationshiipspresent,eithertestarticlceoncentrationor induced toxicity(RTG) may be used.
This presentationof theevaluationprocessmay not encompass alltestsituationsa,nd the Study Directormay have used othercriteritao arriveatan evaluation.The reportprovidesthe reasoninginvolved when departuresfrom the above descriptionoccur.
RESULTS
Test ArticleHandling The testarticleformed unworkable,non-homogeneous suspensionsinwater atconcentrationsof 498, 300, 100 and 50.0 mg/mL. In DMSO, thetestarticlfeormed a solutionat500 mg/mL. For thisreason,DMSO (CAS # 67-68-5,Sigma Chemical Co.,Lot No. 99HOO20) was used asthe vehicle.
N-ETFOSE T-6316 was solublein thevehicle(DMSO) at125 mg/mL, thehighestconcentration prepared foruse inthe assay. The stockused fordosing the highestconcentrationof thetest articlewas prepared inDMSO at I00-times the highestdesiredtreatmentconcentration.Further stockswere preparedby performing serialdilutionsinthevehicle.Dosing was initiatebdy performing 1:100 dilutionosf thestocksintomedium containingthecells.Alldosingwas performed with freshlyprepared testarticlsetocks.
Dose Rangefinding Assay The testarticleN,-ETFOSE T-6316, was testedin a preliminarydose rangefindingassaywith a treatmentperiodof approximately4 hours both with and without S9 metabolicactivatioannd a preliminarydose rangefindingnonactivationassaywith a treatmentperiodof approximately 24 hours. Ten dose levelswere used in each case thatranged from 2.47 to 1250 @ig/mL;a vehiclecontrolwas includedunder each condition.
In thepresence and absence of ratliverS9 metabolic activitywith a 4-hour treatmentperiod (Table 1),N-ETFOSE T-6316 was noncytotoxicup to 19.7gg/mL. Moderate cytotoxicitwyas induced from 39.3 to 157 gg/mL and higherconcentrationswere excessivelycytotoxicor lethal. In thenonactivationdose rangefindingassayusing a 24-hour treatmentperiod(Table2),thetest articlienduced no cytotoxicitayt2.47 gg/mL, weak cytotoxicitayt4.93 @ig/mL,and moderately high cytotoxicitayt9.85 gg/mL. Higher concentrationswere excessivelycytotoxicor lethal. Dose levelsforthe mutation assayswere based on theseresults.
Mutation Assays InitialNonactivation Mutation Assav. Eleven concentrationsofN-ETFOSE T-6316 at25.3, 50.5,75.5,101, 151,201, 251, 301, 401, 501 and 601 gg/mL were initiatedT.reatmentsatand above 251 gg/mL were terminatedbecause of excessivecytotoxicityT.he remainingsix treatmentsinduced weak cytotoxicittyo high cytotoxicit(y67.6% to 14.4% relativgeowths). In
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Covance 20785-0-43I1CH 3M StudyNo.: T-6316.12
orderfora treatmenttobe evaluatedas mutagenic inthe initianlonactivationassay,a mutant
X
frequency greaterthan 127.1
10-6
was
required.This thresholdvaluewas
equal to twicethe
average mutant frequencyof theconcurrentvehiclecontrols(background mutant frequency).
None of theanalyzedtreatmentinduced thislevelof mutant action(Table3). Sizingdatais
shown inTable 4. A confin-natorayssaywas initiated.
Conrirmatory NonactivationMutation Assay. In the confirmatorynonactivationassay,the treatmentperiodwas approximately24 hours and resultsareshown in Table 5. Ten treatments at 1.25,2.50,5.00,7.50,10.0,12.5,15.0,20.0,30.0 and 40.0 gg/mL were initiatedT.reatments at20.0,30.0 and 40.0 ptg/mL were terminatedbecause of excessivecytotoxicity.The remaining seven treatmentswere selectedformutant analysisand induced no cytotoxicittyo high cytotoxicit(y94.2% to 18.7% relativgerowths).None of theanalyzedtreatmentsinduceda mutant frequencythatexceeded the minimum criterionof 111.2x 10-6.The testarticlies, thereforec,onsiderednegativewithoutmetabolicactivationS.izingdata forthe confirmatory
assay isshown inTable 6.
InitialActivation Mutation Assay. Eleven treatmentsat25.3,50.5,75.5,101, 151, 201,251, 301, 401, 501 and 601 @ig/mL were initiateda,nd treatmentsatand above 151 gg/mL were ten-ninatebdecause of excessivecytotoxicityT.he remaining four doses were cloned formutant analysisand inducedmoderate tovery high cytotoxicit(y60.0% to 9.4% relativgerowths).The minimum criteriofnora positiveresponseinthistrialwas 156.7x 10-6 . None of the analyzed treatmentsinduced thislevelof mutant action(Table7). A confirmatoryassaywas perfon-ned.
Sizingdataforthe initiaalssayisshown in Table 8.
Confirmatory ActivationMutation Assay. In the confirmatory.4.ssawyith metabolic activation (Table 9),eleventreatmentsat5.00,10.0,20.0,30.0,40.0,50.0,60.0,80.0,100, 150 and 200 @ig/mL were initiatedT.reatments at 5.00 and 10.0 @ig/mL were terminatedbecause there were sufficienhtigherdoses availablefor analysisand the 200 @tg/mL treatmentwas terminated because of excessivecytotoxicityT.he remaining eightdosesinduced weak cytotoxicittyohigh cytotoxicit(y70.4% to 15.4% relativgerowths).None of theanalyzedtreatmentsinduceda mutant frequencythatexceededthe minimum criterioaf 122.2x 10-6 . The testarticlweas evaluatedasnegativewith metabolicactivatioinn thisassay.Sizingdata fortheconfirmatory
assay isshown in Table 10.
Control Values. The average cloning efficienciefsorthe vehiclecontrolswere 88.6% and 83.4% without activatioannd 84.1% and 77.3% with S9 metabolicactivationw,hich demonstrated acceptablecloningconditionsforthe assays.The positivecontrolculturesN,IMS (nonactivationa)nd MCA (activationi)nduced largeincreasesin mutant frequencythatwere greatlyin excessof theminimum criteria.
SizingAnalysis. The L5178Y TK+/- mutation assayproduces a bimodal distributioonf largeand small mutant colonies.The originof thebimodal distributioonf mutant colony sizesis consideredtoreflecthetypesof geneticdamage, with thelargecoloniesderivedfrom cellswith intragenicmutationsthataffectonly theTK gene and thesmallcoloniestheresultof larger mutationsthataffectcellgrowth as well as theTK gene. Colony sizingwas performed on all
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Covance 20785-0-43I1CH 3M StudyNo.:T-6316.12 culture(sTable4s,6,8 and10).Mutantcoloniefsromalltheculturesshowedtheexpected bimodal distributioannd mutant coloniesfrom MMS and MCA treatedculturesshowed both small and largecolonies.
CONCLUSION The testarticleN,-ETFOSE T-6316, was evaluatedas negativefor inducingforward mutations at theTK locusinL5178Y mouse lymphoma cellsunder activatioannd nonactivatiocnonditions used in thisstudy. Treatmentsup to the testinglimitforthisassay were analyzed.
RECORDS TO BE MAINTAINED All raw data,documentation,records,protocoland a copy of the finalreportgeneratedas a result of thisstudywillbe archivedinthe storagefacilitioefs Covance-Vienna, foratleast1 year followingsubmissionof thefinalreportto theSponsor. Afterthe 1-yearperiod,theSponsor may electtohave the aforementionedmaterialsretainedinthe storagefacilitioefsCovanceVienna foran additionalperiodof time,or sentto a storagefacilitdyesignatedby the Sponsor.
REFERENCES Amacher, D.E.,PailletS,.C.,Turner, G.N.,Ray, V.A., and Salsburg,D.S. (1980) Pointmutations atthethymidinekinaselocusin L5178Y mouse lymphoma cellsIl.Test validatioannd interpretationM.utation Research,72:447-474.
Clive,D. and Spector,J.F.S.(1975) Laboratoryprocedureforassessingspecificlocusmutations attheTK locusinculturedL5178Y mouse lymphoma cells.Mutation Research,31:17-29.
Clive,D.,Johnson,K.O., Spector,J.F.S.B,atson,A.G., and Brown, M.M.M. (1979)Validation and characterizatioonf the L5178Y TK+/-mouse lymphoma mutagen assaysystem. Mutation Research, 59:61-108.
Clive,D.,Caspary,W., Kirby,P.E.,Krehl,R.,Moore, M., Mayo, J.,and Oberly,T.J.(1987) Guide forperforming themouse lyinphoma assayformammalian cellmutagenicity.Mutation Research, 189:143-156.
Hozier,J.,Sawyer,J.,Clive,D., and Moore, M.M. (1985)Chromosome 11 aberrationisnsmall colony L5178Y TK-@-mutants earlyintheirclonalhistory.Mutation Research,147:237-242.
Young, R.,OveisitorkF,.,Harrington-Brock,K., Schalkowsky, S.,Moore, M., and Myhr, B. (1991) Quantativesizeanalysisof L5178 TK+l-mutant coloniesinsoftagar;an interlaboratory comparison. Environ.Molec. Mutagenesis,17,(Suppl.19),79.
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Covance 20785-0-431ICH 3M Study No.: T-6316.12
DATA TABLES
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Covance 20785-0-431 ICH 3M Study No.: T-6316.12
TABLE 1:CYTOTOXICITY ASSAY WITH N-ETFOSE T-6316(4-HOUR)
TEST ARTICLE: N-ETFOSET-6316
STUDY NUMBER: 20785-0-431ICH
TEST DATE: 05/10/2000
VEHICLE: DMSO
COMMENTS ON TREATMENT: -4-HourTreatmentPeriod
APPLIED CONCENTRATION
ilg/mL VCC 2.47 4.93 9.85 19.7 39.3 78.5 157 313 625 1250
WITHOUT S9 ACTIVATION
CELL DENSITY/mL
% VEHICLE
(Xlol)a
CONTROL'
10.0
100.0
9.2
92.0
10.8
108.0
11.8
118.0
9.1
91.0
4.7
47.0
3.3
33.0
3.5
35.0
0.4
4.0
0.0
0.0
0.0
0.0
WITH S9 ACTIVATION
CELL DENSITY/mL % VEHICLE
(X IOI)a
CONTROL'
10.4
100.0
10.8
103.8
9.4
90.4
11.7
112.5
9.2
88.5
5.1
49.0
2.8
26.9
---2.4
23.1
0.3
2.9
0.0
0.0
0.0
0.0
aCelldensitydeterminedby hemocytometer brelativteovehiclecontrolcelldensityforalltreatments cVC =Vehiclecontrol1,% DMSO
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Covance 20785-0-43I1CH 3M StudyNo.: T-6316-12
TABLE 2:CYTOTOXICITY ASSAY WITH N-ETFOSE T-6316 WITHOUT S9 ACTIVATION (24-HOUR)
TEST ARTICLE: N-ETFOSE T-6316
STUDY NUMBER: 20785-0-431 ICH
TEST DATE: 05/10/00
VEHICLE: DMSO COMMENTS ON TREATMENT:
-24-Hour TreatmentPeriod
APPLIED CONCENTRATION
mg/mL
VCC 2.47 4.93 9.85 19.7 39.3 78.5 157 313 625 1250
CELL DENSITY/ML
(Xlol)a
DAY1 11.0 12.1 7.2 5.7 0.3* 0.0* 0.0* NTC NTC NTC NTC
DAY2 7.0 6.4 7.3 3.3 1.0 0.0 0.0 NTC NTC NTC NTC
'Celldensitydetermined by hemocytometer brelativetovehiclecontrolcelldensityforalltreatments ':VC = Vehicle control,1% DMSO NTC = Not counted,cytotoxic *Not subcultured
% VEHICLE CONTROL'
100.0 100.6 68.3 24.4
3.9 0.0 0.0 ---------
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Covance 20785-0-431 ICH 3M Study No.: T-6316.12
TABLE 3:INITIAL MUTATION ASSAY WITHOUT ACTIVATION
A. TEST ARTICLE: N-ETFOSE T-6316 B. GENETICS ASSAY NO.: 20785-0-431ICH C. VEHICLE: DMSO D. SELECTIVE AGENT: TFT 3.0pg/mL
E. TR-EATMENT DATE: OS/16/2000 F. CELLS ANALYZED: 3x106
G. TREATMENT PERIOD: -4 hours
H. EXPRESSION PERIOD: 2 days
TestCondition
DailyCell Cumulative Total Total Cloning
Counts(Cell/mL, RSGA
Mutant Viable Efficienc@b
IOE5 Units)
ColoniesColonies
Day I Day 2
NonactivationControls'
AVG
AVG
vc
vc
VehicleControl VehicleControl VehicleControl
10.0 14.2 15.8
188
501 83.5
12.2 11.5 15.6
157' 544 90.7
11.7 12.2 15.9 15.7 160h
549 91.5 88.6
Relative Mutant
Growth Frequency (IOE-6 UnitS)d
94.5
74.9
101.5
57.5
104.1
58.2
,MMS 13 gg/mL MMS 13 pg/mL
6.9 12.4 9.5 8.2 12.0 10.9
389' 395
271 45.1 225' 37.5
30.8
287.9'
29.4
350.8'
TestCompound pg/mL
Relativeto Vehicle Control
Relativeto Vehicle Control
25.3
7.8 14.2
78.2
183
459
86.4
67.6
79.8
50.5
4.6 10.6
34.4
170h
483
91.0
31.3
70.2
75.5
2.89 9.7
20.5
142
469
88.3
18.1
60.5
101
1.39 8.2
17.4
171
465
87.5
15.2
73.7
151
2.19 8.5
18.0
145
485
91.4
16.4
59.8
201
2.09 8.0
16.9
173
451
84.8
14.4
77.0
'RSG (Day I Count/3) (Day 2 Count)/3(orDay I Count ifnotsubcultured)
bc]oningEfficiency= TotalViableColony Count/Number of CellsSeeded * 100
crelativGerowth = (RelativSeuspensionGrowth * RelativeCloning Efficiency)100
dmutant Frequency = (TotalMutant Colonies/TotaVliableColonies)* 2x IOE-4
Decimal ismoved to expressthefrequencyinunitsof IOE-6 'VehicleControl= I% DMSO
PositiveControl:MMS = Methyl methanesulfonate fmutagenic.Exceeds Minimum Criterioonf 127.1X IOE-6
gnot Subcultured hOne platecontaminated.Totalcountscalculatebdy usinga weight proportion
Covance 20785-0-431 ICH 3M Study No.: T-6316.12
TABLE 4: SIZING DATA FOR INITIAL MUTATION ASSAY WITHOUT ACTIVATION
A. TEST ARTICLE: N-ETFOSET-6316
B. GENETICS ASSAY NO.: 20785-0-431 ICH
C. VEHICLE: DMSO D. SELECTIVE AGENT: E. TREATMENT DATE:
TFT 3.0pg/mL 05/16/2000
Test Condition VehicleControl'
Conc.
I% 1% 1%
Cum. RSG Day I Day 2
88.5 108.0 103.5
100.2 99.0 100.7
Cloning EfficienCyb
Abs % Rel %
83.5 90.7 91.5
94.3 102.5 103.3
Relative Growth'
94.5 101.5 104.1
Mutant Frequency
(X I0-6)d
Total Small Large
74.9 57.5 58.2
42.3 43.0 34.8
32.7 14.5 23.4
MMSF (pg/mL)
13
61.1 60.4
45.1 50.9
30.8
287.9 214.1 73.8
13
72.6 69.5
37.5 42.4
29.4
350.8 169.6 181.2
Test Article(@Lg/mL)
25.3 50.5 75.5 101
69.0 40.7 24.8 11.5
78.2 34.4 20.5 17.4
76.6 80.6 78.2 77.5
86.4 91.0 88.3 87.5
67.6 31.3 18.1 15.2
79.8 70.2 60.5 73.7
40.9 42.8 34.0 44.6
151
18.6 18.0
80.9 91.4
201
17.7 16.9
75.1 84.8
16.4
59.8 35.1
14.4
77.0 47.9
'Cum. RSG = Cumulative Suspension Growlh Relativetothe Average Vehicle ControlSuspension Growth
39.0 27.4 26.5 29.1 24.7 29.1
bcloning Efficiency= Total ViableColony Count/Number of CellsSeeded * 100
'RelativeGrowth = (RelativeSuspensionGrowth * RelativeCloning Efficiency/) 100
dmutant Frequency = (TotalMutant Colonies/TotalViable Colonies) * 2x IOE-4
Decimal ismoved to expressthe frequencyinunitsof IOE-6 Expressed as Total Mutant Frequency,Small Colony Mutant Frequency and Large Colony Mutant Frequency
'VehicleControl= DMSO
fpositiveControl:MMS = Methyl methanesulfonate
Colony Counts increasedby 9.099% tocompensate forareaof dish not scanned
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Covance 20785-0-431 ICH 3M Study No.: T-6316.12
TABLE 5: CONFIRNIATORY
A. TEST ARTICLE: N-ETFOSET-6316
B. GENETICS ASSAY NO.: 20785-0-431 ICH C. VEHICLE: DMSO D. SELECTIVE AGENT: TFT 3.0 pg/mL
MUTATION
ASSAY WITHOUT ACTIVATION
E. TREATMENT DATE: 06/07/2000 F. CELLS ANALYZED: 3x]06 G. TREATMENT PERIOD: -24 hours H. EXPRESSION PERIOD: 2 days
Test Condition
Daily CellCounts Cumulative (Cell/mL,IOE5 Units) RSGA
Day I Day 2 Day 3
NonactivationControls'
AVG
vc
Total Total Mutant Viable Colonies Colonies
Cloning Efficiencby
AVG vc
Relative Growth
(%)C
Mutant
Frequency (IOE-6
UnitS)d
Vehicle Control Vehicle Control Vehicle Control
MMS 6.5 @Lg/mL MMS 6.5gg/mL
Test Compound pg/mL
12.0 7.9 12.0 43.1
133
14.3 6.2 11.4 37.4
154
14.7 6.1 16.4 54.5 44.7 128
9.5 4.9 6.8 11.7
571
9.6 4.5 5.2
8.3
574
Relativeto Vehicle Control
464
77.3
87.4
597
99.5
99.9
441
73.5 83.4 107.4
211
35.1
11.0
233
38.9
8.7
Relativeto Vehicle Control
57.4 51.6 57.9
542.0' 491.6'
1.25
10.0 7.5 10.6
65.9
173
568
113.6
74.9
61.0
2.50
9.3 6.6 16.2
82.4
136
463
92.4
76.2
59.0
5.00
12.1 9.1 12.2
111.4
158
423
84.6
94.2
74.7
7.50
9.4 6.5 15.7
79.5
147
433
86.6
68.8
68.0
10.0
9.6 7.0 10.8
60.2
148
552
110.3
66.4
53.8
12.5
6.6 5.9 12.2
39.4
133
424
84.8
33.4
62.7
'RSG
15.0
6.3 3.99 13.5
21.2
169
442
88.3
18.7
76.5
[Treatmenttermination(Day 1)celldensity/3x1051x [Day 2 celldensity/3xl05 orDay I densityifnot splitback] x
[Day 3 celldensity/3xlO5or Day 2 densityifnot splitback]
bcloning Efficiency= TotalViableColony CountfNumber of CellsSeeded * 100
rrelativeGrowth = (RelativeSuspensionGrowth * RelativeCloning Efficiency) 100
dmutant Frequency = (TotalMutant Colonies/TotalViable Colonies) 2x IOE-4
Decimal ismoved to expressthefrequency inunitsof IOE-6 'VehicleControl= I% DMSO
PositiveControl:MMS = Methyl methanesulfonate fmutagenic.Exceeds Minimum Cfiterionof 111.2X IOE-6
gnot subcultured
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Covance 20785-0-431ICH 3M Study No.: T-6316.12
TABLE 6: SIZING DATA FOR CONFIRMATORY
A. TEST ARTICLE: N-ETFOSET-6316
B. GENETICS ASSAY NO.: 20785-0-431 ICH C. VEHICLE: DMSO D. SELECTIVE AGENT: TFT 3.0pg/mL E. TREATMENT DATE: 06/07/2000
Cum. RSG
Test Condition
VehicleControl'
Conc.
1% 1% 1%
Day I Day 2 Day 3
87.8 104.6 107.6
104.1 97.4 98.5
94.3 83.8 121.9
MUTATION ASSAY WITHOUT ACTIVATION
Cloning
Efficiency,
s%
Re]%
77.3 99.5 73.5
92.7 119.3 88.1
Relative Growthc
(%)
87.4 99.9 107.4
Mutan(txFlrequency
o-6)d
Total Small Large
57.4 51.6 57.9
24.5 24.5 32.2
32.9 27.1 25.7
MMS'(pg/mL)
6.5
69.5 51.1 26.2
35.1 42.1
6.5
70.2 47.4 18.6
38.9 46.7
11.0
542.0 336.8 205.2
8.7
491.6 316.8 174.8
Test Article(@ig/mL)
1.25
2.50 5.00
7.50 10.0
12.5
15.0
73.2
68.0 88.5
68.8 70.2
48.3
46.1
82.4
67.4 120.9
67.1 73.8
42.8
20.8
65.9
82.4 111.4
79.5 60.2
39.4
21.2
94.7
77.1 70.6
72.2 92.0
70.7
73.6
acum. RSG = Cumulative Suspension Growth Relative to the Average
113.6
92.4 84.6
86.6 110.3
84.8
88.3
74.9
76.2 94.2
68.8 66.4
33.4
18.7
61.0
59.0 74.7
68.0 53.8
62.7
76.5
Vehicle Control SuspensionGrowth
29.9
27.8 37.1
34.3 31.2
35.0
39.5
bcloning Efficiency = Total Viable Colony CountfNumber of Cells Seeded * 100
'Relative Growth = (Relative Suspension Growth * Relative Cloning Efficiency) / 100 dM utant Frequency = (Total Mutant Colonies/Total Viable Colonies) * 2x IOE-4
Decimal is moved to express the frequency in units of IOE-6 Expressed as Total Mutant Frequency, Small Colony Mutant Frequency
and Large Colony
Mutant
Frequency
'Vehicle Control = DMSO fpositiveControl: MMS = Methyl methanesulfonate Colony Counts increased by 9.099% to compensate
for area of dish not scanned
31.1
31.1 37.6
33.8 22.5
27.8
37.0
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Covance 20785-0-431ICH 3M Study No.: T-6316.12
TABLE 7: INITIAL MUTATION
A.TEST ARTICLE: N-ETFOSET-6316
B. GENETICS ASSAY NO.: 20785-0-431ICH C. VEHICLE: DMSO D. SELECTIVE AGENT: TFT 3.0pg/mL
ASSAY WITH ACTIVATION
E. TREATMENT DATE: 05/16/2000 F.CELLS ANALYZED: 3x106 G. TREATMENT PERIOD: -4 hours H. EXPRESSION PERIOD: 2 days
TestCondition
DailyCell Cumulative Counts(Cell/mL, RSGA
IOE5 Units)
Total Total Mutant Viable Colonies Colonies
Day I Day 2
S9-ActivatioCnontrols'
AVG
S9 Batch Number: 955
vc
Cloning Efficiencyb
Relative Growth
(%)C
Mutant Frequency
(IOE-6 Un its)d
AVG vc
VehicleControl
12.7 17.3 24.4
170h
437
72.9
112.5
77.9
VehicleControl
11.1 15.0 18.5
201
503
83.8
98.0
79.8
VehicleControl
10.8 11.3 13.6 18.8 221
573
95.5 84.1
81.8
77.3
MCA 2 pg/mL MCA 4 pg/mL
7.6 15.1 12.8 6.2 15.6 10.7
605
381
63.5
607
391
65.1
51.1
318.1'
44.2
310.6'
Test Compound pg/mL
Relativeto Vehicle Control
Relativeto VehicleControl
25.3
8.8 11.9
61.8
167
490
97.1
50.5
3.19 13.0
23.0
149
476
94.3
75.5
1.69 7.5
13.3
178
490
97.1
101
0.59 6.2
11.0
167
433
85.9
'RSG = (Day I Count/3) (Day 2 Count)/3(orDay I Count ifnot subcultured)
bC loningEfficiency= TotalViableColony Count/Number of CellsSeeded * 100
'RelativGerowth = (RelativeSuspensionGrowth * RelativeCloningEfficiency)100
60.0
68.2
21.7
62.8
12.9
72.6
9.4
77.1
dmutantFrequency= (TotalMutant Colonies/TotaVliableColonies)* 2x]OE-4
Decimal ismoved toexpressthefrequencyinunitsof IOE-6 'VehicleControl= I% DMSO
PositiveControl:MCA = Methylcholanthrene fmutagenic.Exceeds Minimum Criterioonf 156.7X IOE-6
gnot subcultured hone platecontaminated.Totalcountscalculatebdy usinga weightproportion
-25-
Covance 20785-0-431ICH 3M Study No.: T-6316.12
TABLE 8: SIZING DATA FOR INITIAL MUTATION ASSAY WITH ACTIVATION
A. TEST ARTICLE: N-ETFOSE T-6316
B. GENETICS ASSAY NO.: 20785-0-431 ICH
C. VEHICLE: DMSO D. SELECTIVE AGENT: TFT 3.0pg/mL E. TREATMENT DATE: 05/16/2000
Cum. RSG (%)'
Test Condition Vehicle Control'
Conc.
1% 1% I%
Day I Day 2
110.1 96.2 93.6
129.7 98.3 72.0
Cloning EfficienCyb Abs % Rel %
72.9 83.8 95.5
86.7 99.7 113.6
Relative Growth'
(%)
112.5 98.0 81.8
Mutant Frequency
(X 10-6)1
Total Small Large
77.9 79.8 77.3
35.3 49.9 46.9
42.6 29.9 30.5
MCA'(@ig/mL)
2
65.9 67.7
63.5 75.5
51.1
318.1 150.1 167.9
4
53.8 57.1
65.1 77.4
44.2
310.6 166.5 144.1
TestArticle(pg/mL)
25.3 50.5 75.5 101
76.3 26.9 13.9 4.3
61.8 23.0 13.3 11.0
81.6 97.1 79.3 94.3 81.6 97.1 72.2 85.9
60.0 21.7 12.9 9.4
68.2 62.8 72.6 77.1
38.8 38.5 42.8 55.4
29.4 24.3 29.8 21.7
'Cum. RSG = Cumulative Suspension Growth Relativeto theAverage VehicleControl Suspension Growth
bcloning Efficiency= TotalViable Colony Count/Number of CellsSeeded * 100
'RelativeGrowth = (Relafl'vSeu'spension Growth * RelativeCloning Efficiency/) 100
dmutant Frequency = (TotalMutant Colonies@'rotaVliable Colonies) * 2x IOE-4
Decimal ismoved toexpressthefrequency inunitsof IOE-6
Expressed as Total Mutant Frequency, Small Colony Mutant Frequency and Large Colony Mutant Frequency
'VehicleControl= DMSO
fpositivCeontrol:MCA = Methylcholanthrene
Colony Counts increasedby 9.099% to compensate forareaof dish not scanned
-26-
TABLE 9: CONFIRMATORY
A. TEST ARTICLE: N-ETFOSET-6316
B. GENETICS ASSAY NO.: 20785-0-431ICH C. VEHICLE: DMSO D. SELECTIVE AGENT: TFT 3.0pg/mL
Covance 20785-0-431ICH 3M Study No.: T-6316.12
MUTATION ASSAY WITH ACTIVATION
E. TREATMENT DATE: 06/07/2000 F. CELLS ANALYZED: 3x 106 G. TREATMENT PERIOD: -4 hours H. EXPRESSION PERIOD: 2 days
TestCondition
DailyCell Cumulative Total Total
Counts(Cell/mL, RSG a
Mutant Viable
IOE5 Units)
Colonies Colonies
Cloning Efficiencb y
Day I Day 2
S9-ActivatioCnontrols'
AVG
AVG
S9 Batch Number: 955
vc
vc
Relative Growth
(%)C
Mutant Frequency
(IOE-6 Un its)d
VehicleControl VehicleControl VehicleControl
MCA 2 @Lg/niL MCA 4 @Lg/niL
9.4 16.9 17.7
128
9.1 15.9 16.1
149
14.3 13.9 22.1 18.6 147
7.3 13.7 11.1
472
9.1 12.6 12.7
412
479 79.8
97.9
461
76.9
85.9
452 75.3 77.3 115.5
443
73.8
57.0
405
67.5
59.7
53.3 64.8 65.2
213.3' 203.8'
TestCompound pg/mL
Relativeto Vehicle Control
Relativeto Vehicle Control
20.0 30.0 40.0 50.0 60.0 80.0 100 150
8.0 19.4 7.5 12.0 5.5 17.0 5.2 14.2 3.49 14.4 2.19 13.9 2.29 10.5
2.89 7.5
92.7 53.8 55.8 44.1 25.8 24.9 18.8 13.4
131
352
132
525
128
433
122
445
100
444
122
453
135
464
153
532
75.9 113.1 93.3 95.9 95.7 97.6 99.9 114.7
70.4
74.3
60.8
50.3
52.1
58.9
42.3
54.9
24.7
45.2
24.3
54.0
18.8
58.4
15.4
57.4
aRSG (Day ICount/3) (Day 2 Count)/3(orDay ICount ifnotsubcultured)
bcloningEfficiency= TotalViableColony Count/Number ofCellsSeeded * 100
'RelativGerowth = (RelativeSuspensionGrowth * RelativeCloningEfficiency)100
dMUtantFrequency= (TotalMutant Colonies/TotaVliableColonies) 2x IOE-4
Decimal ismoved to expressthefrequencyinunitsof IOE-6 'VehicleControl= I% DMSO
PositiveControl:MCA = M ethylcholanthrene rmutagenic.Exceeds Minimum Criterioonf 122.2X IOE-6
gnot Subcultured
-27-
Covance 20785-0-431ICH 3M Study No.: T-6316.12
TABLE 10: SIZING DATA FOR CONFIRMATORY
MUTATION
ASSAY WITH ACTIVATION
A. TEST ARTICLE: N-ETFOSE T-6316 B. GENETICS ASSAY NO.: 20785-0-431 ICH C. VEHICLE: DMSO D. SELECTIVE AGENT: TFT 3.0@ig/mL E. TREATMENT DATE: 06/07/2000
Test Condition Vehicle Control'
Conc.
I% 1% 1%
Cum. RSG Day I Day 2
86.0 83.2 130.8
94.9 86.4 118.7
Cloning Efficiency' Abs % Rel %
79.8 76.9 75.3
103.2 99.5 97.3
Relative Growth'
(%)
97.9 85.9 115.5
Mutant Frequency
(X I0-I)d
Total Small Large
53.3 23,7 64.8 28.4 65.2 26.6
29.6 36,4 38.6
MCA, (pg/ML)
2
66.8 59.7
73.8 95.5
57.0
213.3 86.2 127.1
4
83.2 68.5
67.5 87.2
59.7
203.8 97.0 106.7
TestArticle(pg/niL) 20.0 30.0 40.0 50.0 60.0 80.0 100 150
73.2 68.6 50.3 47.6 31.1 19.2 20.1 25.6
92.7 53.8 55.8 44.1 25.8 24.9 18.8
13.4
58.7 87.5 72.2 74.2 74.0 75.5 77.3 88.7
75.9 113.1 93.3 95.9 95.7 97.6 99.9 114.7
70.4 60.8 52.1 42.3 24.7 24.3 18.8 15.4
74.3 50.3 58.9 54.9 45.2 54.0 58.4
57.4
32.8 21.6 29.7 29.4 23.6 28.4 30.1
29.5
41.5 28.7 29.2 25.5 21.6 25.5 28.2
27.9
'Cum. RSG = Cumulative Suspension Growth Relativeto theAverage Vehicle ControlSuspension Growth
bcloning Efficiency= TotalViable Colony Count/Number of CellsSeeded * 100
'RelativeGrowth = (RelativeSuspensionGrowth * RelativeCloning Efficiency/)100
dmutant Frequency = (TotalMutant Colonies/TotalViable Colonies)* 2x IOE-4
Decimal ismoved toexpressthe frequencyinunitsof IOE-6 Expressed asTotal Mutant Frequency,Small Colony Mutant Frequency and Large Colony Mutant Frequency
'VehicleControl= DMSO
fpositiveControl:MCA = M ethylcholanthrene
Colony Counts increasedby 9.099% tocompensate forareaof dish not scanned
-28-
Covance 20785-0-43I1CH 3M StudyNo.: T-6316.12
HISTORICAL CONTROL DATA
NonactivatiSotnudies
Pooled negativeand vehiclecontrolmutant frequencies
Mean ( SD)
53.0 22.0x 10-6
Range
20.5 to 114.8 x 10
Number of experiments
52
Number of controls
156
Positivecontrolmutant frequencies(6.5@ig/mL methyl methanesulfonate)
Mean ( SD)
272.7 135.7x 10-6
Range
115.9to632.lx 10-6
Number of experiments
51
Number of controls
51
Positivecontrolmutant frequencies(13 @ig/mL methyl methanesulfonate)
Mean ( SD)
483.9 315.2x 10'
Range
176.1 to 1996.4 x 10-6
Number of experiments
52
Number of controls
52
ActivationStudies
Pooled negativeand vehiclecontrolmutant .frequencies
Mean (:ESD)
65.3 27 1 x 10-6
Range
27.6 to 150.3 x 10-6
Number of experiments
54
Number of controls
162
Positivecontrolmutant frequencies(2.0@ig/mL 3-methylcholanthrene)
Mean ( SD)
454.5 166.2x 10-6
Range
204.8 to 787.8 x 10-6
Number of experiments
54
Number of controls
54
Positivecontrolmutant frequencies(4.0gg/mL 3-methylcholanthrene)
Mean ( SD)
567.0 248.3 x 10'
Range
218.9 to 1414.9 x 10-6
Number of experiments
54
Number of controls
54
Because some experiments containedmultiplecontrols,the number of independentcontrol culturesexceeded the number of experiments.
-29-