Document YjbLr1KkMa9Bv5ExDrV813QJy
CORNINGHazlcton
MUTAGENICITY TEST WITH
T-6342
IN THE SALMONELLA
- ESCHEPICHL4 COLIMAMMALIAN-MICP,(>SOMt MUTATION ASSAY
RE-1@ERSE
FINAL REPORT
AUTHOR Timothy E.Lawlor,M.A.
PERFORMING LABORATORY
Coming HazletonInc.(CHV) 9200 LeesburgPike
Vienna,Virginia22182
LABORATORY PROJECT ID CHV StudyNo.: 17073-0409
SUBM=ED TO
3M Corporation Building220-2E-02 3M Center
St.Paul,MN 55144-1000
STUDY COMPLETION DAIE December 14,1995
CHV StudyNo.: 17073-0409
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CORNINGHazleton QUALITY ASSURANCE STATEMENT
STUDY TITLE: Salmonel-lEaschericchoilai/Mammalian-MicrRoesvoemresMeutation Assay
ASSAY NO.:
17073-0-409
PROTOCOL No.: 409,Edition4,Modifiedfor3M Corporation
QualityAssuranceinspectionosfthestudyandreviewofthefinalreportoftheabove referenced projectwere conductedaccordingtotheStandardoperatingProceduresoftheQualityAssurance Unitand accordingtothegeneralrequirementosftheappropriatGeood LaboratoryPractice regulationsF.indingsfrom theinspectionasnd finalreportreviewwerereportedtomanagement and tothestudydirectoorn thefollowingdates:
Inspection/Date Plating- 09/19/95 DraftReportReview - 11/06/95
FindingsRep-Qrted 09/19/95 11/07/95
FinalReportReview - 12/14/95 12/14/95
Audito
M. Murphy
C. Orantes/ S. Ballenger C. Orantes
u@ul it@syssursaunr@acnecme@mtt
Datre-RJease-d
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STUDY COMPLIANCE AND CERTIFICATION
The study was conducted incompliance with theGood LaboratoryPracticeregulationsasset forthintheFood and Drug Administration(FDA) Title21 oftheU.S. Code ofFederal RegulationsPart58, issuedDecember 22, 1978,(effectivJeune 20, 1979) and with any applicableamendments. There were no significandteviationsfrom theaforementioned regulationosrthe signedprotocolthatwould affecttheintegritoyf the studyor theinterpretation of thetestresults.The raw datahave been reviewed by theStudy Director,who certifietshatthe evaluationofthe testarticlaes presentedhereinrepresentsan appropriateconclusionwithinthe contextof thestudy designand evaluationcriteria.
Study Director:
Timothy E. awlor,M.A. Bacterial utagenesis Genetacan- CellularToxicology
Li
Study Completion Date
CHV Study No.: 17073-0-409
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TABLE OF CONTENTS
CORNINGHazieton
Page No. 1. SUMMARY ..........................................5......... ii. STUDY INFORMATION ..................................7.......... III. MATERIALS AND METHODS ...............................9......... IV. RESULTS AND CONCLUSIONS .............................2.3.......... V. DATA TABLES .......................................26..........
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SECTION 1. SUMMARY INTRODUCTION AND CONCLUSIONS
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SUMMARY
A. Introduction
At therequesotf3M CorporatioCno,ming HazletoInnc.investigaTt-e6d342for mutageniacctiviitnytheSalmonella-Eschericcohliia/Mammalian-MicrosRoemveerseMutation Assay. This assayevaluatedthetestarticlaend/oritsmetabolitesfortheirabilittyoinduce reversemutationsatthehistidinleocusinthegenome of specifiScalmonella typhimurium tester strainasnd atthe tryptophanlocusinan Escherichiacolitesterstrainboth inthepresenceand absence of an exogenous metabolicactivationsystem of mammalian microsomal enzymes derivedfrom AroclorTm-inducedratliver(S9).
The doses testedin themutagenicityassaywere selectedbased on the resultsof a dose rangefindingstudy usingtesterstrainsTA 100 and WP2uvrA and ten doses oftestarticlreanging from 5,000 to 6.67 @Lgper plate,one plateper dose,both in thepresenceand absence of S9 mix.
ne testerstrainsused in themutagenicityassaywere Salmonella typhimurium testerstrains TA98, TAIOO, TA1535, TA1537, and EscherichiacolitesterstrainWP2uvrA. Theassaywas conducted with fivedoses oftestarticlienboththepresenceand absence of S9 mix along with concurrentvehicleand positivecontrolsusingthreeplatesper dose. The doses testedwere 5,000,3,330, 1,000,333, and 100 gg per platein both thepresenceand absence of S9 mix.
B. Conclusions
The resultosftheSalmonella- Escherichiacoli/Mammalian-Microsome Reverse Mutation Assay indicatethat,under theconditionsofthisstudy,3M Corporation'tsestarticle, T-6342, did not cause a positiveincreaseinthe number of revertantpser plateofany of thetester strainseitherinthepresenceor absenceof microsomal enzymes preparedfrom AroclofT'm-inducerdatliver(S9).
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CORNINGHazleton SECTION 11.STUDY INFORMATION
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STUDY INFORMATION
A. Sponsor: 3M Corporation
B. TestArticle:T-6342
I. Physical Description: clear colorless liquid
2. Date Received: 07/21/95
C. Type of Assay: Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay
I
ProtocolNumber: CHV Protocol409,Edition4
Modifiedfor3M Corporation
2. CHV StudyNumber: 17073-0-409
D. Study Dates
I. Study InitiatioDnate:
08/28/95
2. ExperimentalStartDate:
09/12/95
3. ExperimentalTerminationDate: 09/22/95
E. Study SupervisoryPersonnel
Study Director:
Timothy E. Lawlor,M.A.
LaboratorySupervisor: Michael S.Mecchi, B.S.
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SECTION Ill.MATERIALS AND METHODS
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MATERIALS AND METHODS
The experimentmaalterialmse,thodsandprocedureasrebasedon thosedescribebdy Ames etal(1975)andGreenandMuriel(1976).
MATERIALS
A. Tester Strains
I. Salmonella typhimurium
The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TAIOO, TA1535, and TA1537 as described by Ames etal (1975). The specificgenotypes of thesestrainsare shown inTable 1.
TABLE 1. TEST
HistidineMutation
hisG46 hisC3076 hisD3052
TA1535 TA1537
TAIOO
TA98
R STRAIN GENOTYPES
AdditionalMutations
LPS Repair R Factor
rfa uvrb
rfa uvrb
+R
Inadditiontoa mutation inthe histidinoeperon,thetesterstrainscontaintwo additional mutationswhich enhance theirsensitivittyosome mutagenic compounds. The rfawall mutation resultsinthe lossof one of theenzymes responsibleforthesynthesisofpartofthe lipopolysaccharidbearrierthatforms the surfaceof thebacteriaclellwall.The resultincgellwall deficiencyincreasespermeabilityto certainclassesof chemicalssuch as thosecontaininglarge ringsystems(i.eb.enzo(a)pyrene)thatwould otherwisebe excluded by a normal intactcellwall.
The second mutation,a deletionofthe uvrb gene,resultisna deficienDtNA excisionrepair system which greatlyenhances thesensitivitoyf thesestraintsosome mutagens. Since the uvrb deletionextendsthrough thebio gene,allofthe testerstraincsontainingthisdeletionalsorequire thevitaminbiotinforgrowth.
StrainsTA98 and TA 100 alsocontainthe R-factorplasmid,pKM 101,which furtherincreases thesensitivitoyf thesestrainstosome mutagens. The mechanism by which thisplasmid increasessensitivittyo mutagens has been suggestedtobe by modifying an existingbacterial DNA repairpolymerase complex involvedwiththemismatch-repairprocess.
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TestesrtraiTnAs98andTA 1537arerevertferdomhistiddienpeenden(caeuxotropthoy) histidiniendependence(prototrophbyy) frameshifmtutagens.TA1535 isrevertebdy base substitutimountagensand TA 100 isrevertedby mutagenswhich causebothframeshiftasnd base substitutions.
2. Escherichiacoli
The testerstrainused was thetryptophanauxotroph WP2uvrA as describebdy Green and Muriel(1976).
Inadditiotnoa mutationinthetryptophaonperon,thetestesrtraicnontainsa uvra DNA repair deficiencwyhich enhancesitssensitivittoysome mutageniccompounds. Thisdeficiencayllows thestraitnoshow enhancedmutabilitsyincetheuvra repairsystemwould normallyactto remove thedamaged partoftheDNA moleculeandaccuratelryepairitafterwards.
TesterstraiWnP2uvrA isrevertefdrom tryptophadnependence(auxotrophyt)otryptophan independence(prototrophyb)y basesubstitutimountagens.
3. SourceofTesterStrains
a. Salmonella typhimurium
The testerstrainsin use atCHV were received directlyfrom Dr. BruceAmes, DepartmentofBiochemistryU,niversitoyfCaliforniBae,rkeley.
b. Escherichiacoli
The testerstrainW,P2uvrA, inuse atCHV was receivedfrom the NationalCollectioonfIndustriaBlacteriaT,orreyResearchStationS,cotland(UnitedKingdom).
4. Storageof the--T-esStterains
a. FrozenPermanentStocks
Frozenpermanentstockswere preparedby growing fresh overnighctulturesa,ddingDMSO (0.09myml ofculturea)nd freezinsgmallaliquots (0.5-1.m5l)at:5-70*C.
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b. MastePrlates
Masterplateswere preparedby streakinegachtestesrtraifnrom a frozenpermanentstockontominimalagarappropriatelsyupplementedwith 1)forSalmonella typhimurium,an excessofhistidinaen,d biotina,nd fortestesrtrainTsA 98 and TA 100, ainpicill(i2n5gg/ml),toensurethestablmeaintenanceofthepKM 101plasmid;and 2)for Escherichicaolia,n excessofftyptophanT.esterstraimnasterplatewsere storedat 5 30C.
5. Prel2aratioofnOvernightCultures
a. Inoculation
Overnightculturefsoruseinalltestinpgrocedureswere inoculated by transferriangcolonyfrom theappropriatmeasterplatetoa flaskcontainincgulturmeedium. Inoculatefdlaskswere placedina shaker/incubatwohrich was programmed tobeginoperation (shaking1,25 25 rpm; incubatio3n7, 2*C) sothattheovernighctulturewsereinlogphaseor latelogphasewhen turbiditmyonitoringbegan.
b. Harvest
To ensurethatculturewsere harvestedinlatelogphase,thelength ofincubatiownas determinedby spectrophotometrmiocnitoringofculturteurbidityC.ultures were harvestedonce apredeterminedturbiditwyas reachedasdeterminedby a percent transmittanc(e%T) readingon a spectrophotometeTrh.istargeturbiditeynsuresthatcultures have reacheda densityof atleast0.5X 10'cellsperml and thattheculturehsave notovergrown. Overgrown (stationarcyu)lturemsay exhibidtecreasedsensitivittoysome mutagens.Cultures were removed from incubatiownhen thetarge%tT was reachedand wereplacedat5 3OC.
6. ConfirmatioonfTesterStrainGenoilpm
Testerstraicnulturewsere checkedforthefollowinggeneticmarkerson theday of theiruse inthemutagenicitayssay:
a. Salmonella typhimurium
1) rfa Wall Mutation
The presence of the rfa wall mutation was confirmed by demonstratioonfthesensitiviotfytheculturteocrystavlioletA.n aliquootfan overnight
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cultuorfe achstrawiansoverlaoindtpolatceosntainsienlgectmievdeia ndanantibiotic sensitividtiyskcontainin1g0 pg ofcrystavliolewtas added.Sensitiviwtays demonstratedby inhibitiofnbacteriaglrowthina zoneimmediatelysurroundintghedisk.
2) pKM 101 PlasmidR-factor
The presenceofthepKM 101plasmidwas confirmedfor testesrtrainTsA98 and TAIOO by demonstratioonfresistancteoampicillinA.n aliquootfan overnightcultureofeachstraiwnas overlaiodntoplatescontaininsgelectivmeedia and an antibiotiscensitividtiyskcontaining10 4g ofaznpicillwians added. Resistancweas demonstratedby bacteriaglrowthinthezoneimmediatelysurroundingthedisk.
3) CharacteristNiucmber ofSpontaneousRevertants
T'hemean number of spontaneousrevertantpserplateinthe vehiclecontroltshatarecharacteristoifctherespectivsetrainwsere demonstratedby plating 100 glaliquotosftheculturaelongwiththeappropriatveehicleon selectivmeedia.
b. Escherichiacoli
1) CharacteristNiucmber ofSpontaneousRevertants
The mean number ofspontaneousrevertantpserplateinthe vehiclecontrolsthatarecharacteristoifctherespectivsetrainwsere demonstratedby plating 100 gl aliquotsoftheV@P2uvrA culturaelongwiththeappropriatveehicleon selectivmeedia.
7. TesterStrainMedia
a. CulturinBgroth
T'hebrothused togrow overnightcultureosfthetestesrtrainwsas Vogel-Bonnersaltsolutio(nVogeland Bonner,1956)supplementedwith2.5% (w/v)Oxoid NutrienBtrothNo. 2 (drypowder).
b. Agar Plates
Bottom agar(25ml per15x 100mm petridish)was VogelBonnerminimalmedium E (Vogeland Bonner,1956),supplementedwith1.5% (w/v)agarand 0.2% (w/v)glucose.
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C. OverlayAgar forSelectioonf Revertants
Overlay(top)agarwas preparedwith 0.7% agar(w/v)and 0.5% NaCl (w/v)and was supplementedwith 10ml of1)0.5MM histidinelbiostoilnutiopner 100 ml agarforselectioonfhistidinreevertantosr,2) 0.5mM tzyptophansolutionper 100 mi of agarforselectioonf tryptophanrevertantsW.hen S9 mix was required2,.0ml of the supplementedtop agarwas used intheoverlay.However, when S9 mix was notrequiredw,ater was added tothesupplementedtopagar(0.5ml ofwaterper2 ml of supplementedtopagar)and theresultin2g.5ml ofdilutedsupplementedtopagarwas used fortheoverlay.Thisdilution ensuredthatthefinaltop agarand amino acidsupplementconcentrationrsemainedthesame both inthepresenceand absenceof S9 mix.
B. Liver Microsomal Enzyme Reaction Mixture (S9 Mix)
I. S9 Homogenate
Livermicrosomalenzymes (S9 homogenate)were purchasedfrom MolecularToxicology,Inc.,Annapolis,MD 21401,Batch 0568 (38.8mg of proteinper ml) and Batch0569 (38.8mg of proteinperml). The homogenate was preparedfrom male SpragueDawley ratsthathad been injecte(di.p.w)ithAroclor@r1m254 (200mg perml incom oil)at 500 mg/kg asdescribedby Ames etal,1975.
2. S9 Mix
The S9 mix was preparedimmediatelypriortoitsuse inany experimental procedure.The S9 mix containedthecomponents indicateidnTable Il.
TABLE II.S9 Ml'
H20
IMNaH2pO4/Na2HP04, pH7.4 0.25M Glucose-6-phosphate 0.1OM NADP 0.825M KCI/0.2M MgCI2 S9 Homogenate
0.70ml 0.10ml 0.02 ml 0.04in]
0.04ml
0.10ml 1.00ml
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C. Controls
1. VehicleCon Is
Vehiclecontrolswere platedforalltestesrtrainbsoth inthepresenceand absenceof S9 mix. The vehiclecontrolwas plated,usinga 50 Illaliquotof vehicle(equaltothe maximum aliquotoftestarticldeilutiopnlated)a,longwitha 100 pl aliquotoftheappropriate testesrtraiannd a 500 Alaliquotof S9 mix (when necessary)o,n selectivaegar.
2. PositiveControls
The combinationsofpositivecontrolsa,ctivatiocnonditionand tester strainpslatedconcurrentlywith theassayareindicatedinTable111.
Tester S.M& TA98 TA98 TAIOO TAIOO TA1535 TA1535 TA1537 TA1537 WP2uvrA WP2uvrA
TAB]
S9 Mix +
+
+ + +
TIYE --ds
Conc
PositiveControl
per plate
2-aminoanthracene
2.5pg
2-nitrofluorene
1.0gg
2-aininoanthracene
2.5;ig
sodium azide
2.0gg
2-aminoanthracene
2.5gg
sodium azide
2.0 pg
2-aminoanthracene
2.5 gg
ICR- 191
2.0 gg
2-aminoanthracene
25.0 pg
4-nitroquinoline-N-oxide1.0gg
a. Sourceand Grade ofPositiveControlArticles
2-aminoanthracen(eCAS #613-13-8)S,igma Chemical Co.,purity 97.5%;2-nitrofluore(nCeAS #607-57-8)A,ldrichChemicalCo.,purity98%; sodium azide (CAS #26628-22-8),Sigma Chemical Co.,purity>98%; ICR-191 (CAS # 1707-45-0)@ PolyscienceIsnc.p,urity>95%; 4-nitroquinoline-N-ox(iCdAeS #56-57-5),Sigma Chemical Co., purity>99%.
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3. SteriliCtoyn=la a. TestArticle
The most concentratetdestarticldeilutiownas checkedforsterility by platinga 50 g] aliquot(thesame volume used intheassay)on selectivaegar.
b. S9 Mix
agar.
The S9 mix was checked forsterilibtyyplating0.5ml on selective
METHODS
A. Dose RangefindinLyStudy
The growth inhibitoryeffect(cytotoxicityo)f thetestarticleto thetestsystem was determinedinordertoallowtheselectioonfappropriatdeosestobe testedinthemutagenicity assay.
I. Design
The dose rangefindingstudywas performedusingtesterstrainTsA 100 and WP2uvrA both inthepresenceand absenceof S9 mix. Ten dosesof testarticlweere tested atone plateperdose. The testarticlweas checkedforcytotoxicituyp toa maximum concentratioonf5 mg perplate.
a. Rationale
The cytotoxicitoyfthetestarticloebservedon testerstrainTA 100 isgenerallyrepresentativoef thatobservedon theothertestesrtrainasnd becauseof TA I00's comparativelyhighnumber ofspontaneousrevertantpserplateg,radationsof cytotoxicitcyan be readilydiscernedfrom routineexperimentalvariationT.he Escherichiacolitestesrtrain WP2uvrA does notpossesstherfawallmutationthattheSalmonellatyphimuriumstrainhsave and thus,a differenrtange ofcytotoxicitmyay be observed.Also,thecytotoxicitiynducedby a testarticlienthepresenceof S9 mix may vary greatlyfrom thatobservedintheabsenceof S9 mix. Therefore,thiswould requirethatdifferenttestarticldeoserangesbe testedinthe mutagenicityassaybased on thepresenceor absenceoftheS9 mix.
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2. Evaluationofthe-Dose-RanizefindSitnugdy
Cytotoxicitiysdetectablaesa decreaseinthenumber ofrevertanctolonies perplateand/orby a thinningor disappearancoefthebacteriablackgroundlawn.
3. aelectioonftheMaximum Dose fortheMutaizeniciAtsysU
a. No CytotoxicitOybserved
Sinceno cytotoxiciwtays observedinthedoserangefindinsgtudy, thehighestdose of testarticluesedinthemutagenicitayssaywas thesame asthattestedinthe rangefindinsgtudy.
B. MutageniciijAssay
I. Design
The assaywas performedusingtestesrtrainTsA98, TA 100,TA 1535, TA1537, and WP2uvrA bothinthepresenceand absenceofS9 mix. Fivedosesoftestarticle weretestedalongwiththeappropriatveehicleand positivceontrolsT.he dosesoftestarticle were selectedbasedon theresultosf thedoserangefindinsgtudy.
2. Frequencyand Routeofadministration
The testesrtrainwsere exposedtothetestarticlveiatheplate incorporatiomnethodologyoriginalldyescribedby Ames etal(1975)and Maron and Ames (1983).Thismethodologyhasbeenshown todetecta widerangeofclasseosfchemical mutagens.Intheplateincorporatiomnethodology,thetestarticlteh,etestesrtraiannd theS9 mix (whereappropriatew)ere combinedinmoltenagarwhich was overlaiodntoa minimalagar plate.Followingincubatioant37 2*C for48 8 hr,revertanctoloniewserecounted.All dosesofthetestarticlet,hevehiclecontrolasnd thepositivceontrolwsereplatedintriplicate.
C. PlatingProcedures
These procedureswere usedinboththedose rangefindinsgtudyand the mutagenicitayssay.
Each platewas labeledwitha code which identifietdhetestarticlet,estphase,testesrtrain, activatiocnonditionand dose.The S9 mix and dilutionosfthetestarticlweere prepared immediatelypriortotheiurse.
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When S9 mix was notrequire1d0,0Aloftestesrtraiannd50 Alofvehicloertesatrticdloese was added to2.5 ml ofmolten selectivteop agar(maintainedat45 2 OC). When S9 mix was required,500 pLIof S9 mix, 100 gloftesterstrainand 50 ;ilof vehicleor testarticledose was added to2.0 ml of molten selectivteop agar. Aftertherequiredcomponents had been added, the mixturewas vortexedand overlaidontothe surfaceof 25 ml ofminimal bottom agarcontainedin a 15 x 100 mm petridish.Aftertheoverlayhad solidifietdh,eplateswere invertedand incubatedfor48 8 hrat37 2 *C. Positivecontrolarticlewsere platedusinga 50 glplating aliquot.
D. Scoring the Plates
Plateswhich were not evaluatedimmediatelyfollowingtheincubationperiod were heldat5 3 *C untilsuch time thatcolony countingand bacteriablackground lawn evaluationcould takeplace.
I. BacterialBackeround Lawn Evaluation
The conditionofthe bacteriablackground lawn was evaluatedfor evidenceof cytotoxicitaynd testarticlperecipitateE.vidence ofcytotoxicitwyas scoredrelative tothe vehiclecontrolplateand was recordedalong withtherevertanctounts forallplatesatthat dose on the datatablesusing thecode system presentedatthe end of the Materialsand Methods Section.
2. -CountiniRzevertantColonies
The number ofrevertanctoloniesper plateforthe vehiclecontrolsand all platescontainingtestarticlweere countedmanually. The number of revertantcoloniesperplate forthe positivecontrolswere countedby automated colonycounter.
E. Analysis-ofData
For allreplicateplatingst,he mean revertantpser plateand the standarddeviation were calculated.The resultsofthesecalculationasrepresentedintabularform intheData TablesSectionof thisreport.
EVALUATION
OF TEST RESULTS
Before assaydatawere evaluated,thecriterifaora validassayhad tobe met.
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A. CriterFioarA ValidAssay
Thefollowicnrgiterwieareusedtodetermianevaliadssay:
I TesterStrainInteg[ity:Salmonella typhimurium
a. rfaWall Mutation
To demonstrate thepresence of therfawall mutation,testerstrain cultureesxhibitedsensitivittoycrystavliolet.
b. PKMIOI Plasmid
To demonstratethepresenceof theR-factorplasmid,pKM 101, cultureosftesterstrainTsA98 and TA 100 exhibitedresistancteo ampicillin.
C. CharacteristNiucmber of SpontaneousRevertants
To demonstratetherequirementforhistidinet,hetestesrtrain
culturesexhibiteda characteristniucmber ofspontaneousrevertantpserplatewhen platedalong
withthevehicleunder selectivceonditionsT.he acceptablerangesforthevehiclecontrolswere
as follows:
TA98
8 - 60
TAIOO
60 - 240
TA1535
4 - 45
TA1537
2 - 25
2. TesterStrainIntegrity:Escherichiacoli
a. CharacteristNiucmber of SpontaneousRevertants
To demonstratetherequirementfortryptophan,thetesterstrain cultureexhibiteda characteristniucmber of spontaneousrevertantpserplatewhen platedalong withthevehicleunder selectivceonditions.The acceptablerangeforWP2uvrA was 5 to 40 revertantpserplate.
3. JesterStrainCultureDenjily
To demonstratethatappropriatneumbers of bacteriareplated,thedensity oftesterstraincultureswere greaterthanor equalto0.5x 10"bacteripaerml and/orhad reached
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atargelteveolfturbididteymonstratteopdroducceulturweisthadensitgyreattehranorequatlo 0.5x 10'bacteripaer mi.
4. PositiveControlValues
a. PositiveControlValuesintheAbsence of S9 Mix
To demonstratethatthetestesrtrainwsere capableof identifyinag mutagen,themean valueof a positivecontrolfora respectivteestesrtrainexhibitedatleasta 3-foldincreaseover themean valueofthevehiclecontrolforthatstrain.
b. PositiveControlValues inthePresenceof S9 Mix (S9Mix Integrity)
To demonstratethattheS9 mix was capableof metabolizinga promutagen to itsmutagenicform(s),themean valueof thepositivceontrolfora respective testerstraininthepresenceof theS9 mix exhibitedatleasta 3-foldincreaseover themean value ofthevehiclecontrolforthatstrain.
An acceptablepositivecontrolinthepresenceof S9 fora specifisctrainwas evaluatedas having demonstratedboththeintegritoyftheS9 mix and theabilitoyfthetestesrtrainto detecta mutagen.
2. czotoxiciiy
A minimum ofthreenon-toxicdoseswere requiredto evaluateassaydata.
B. CriteriaFor A PositiveResponse
Once thecriterifaora validassayhad been met,responsesobservedintheassay were evaluatedas follows:
I. TesterStrainsTA98.TA 100.and WP2uvrA
For a testarticlteo be consideredpositivei,thad toproduceatleasta 2-foldincreasein themean revertantpserplateofatleastone of thesetesterstrainosverthe mean revertantpserplateoftheappropriatveehiclecontrol.Thisincreaseinthemean number of revertantpserplatehad tobe accompanied by a dose responsetoincreasingconcentrationosf the testarticle.
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2.
1TAI537
For a testarticlteobe consideredpositivei,thad toproduceatleasta 3-foldincreaseinthemean revertantpserplateofatleastone ofthesetestesrt-aimoverthe mean revertantpserplateoftheappropriatveehiclecontrol.Thisincreasienthemean number of revertantpserplatehad tobe accompaniedby a dose responsetoincreasincgoncentrationosfthe testarticle.
RECORDS TO BE MAINTAINED
Allraw data,documentationr,ecordst,heprotocola,nd thefinalreportgeneratedasa resulotf thisstudywillbe archivedinthestoragefacilitioefsComing HazletonInc.,foratleastone year followingsubmissionofthefinalreportotheSponsor.Aftertheone yearperiod,theSponsor may electohavetheaforementionemdaterialrsetaineidnthestoragefacilitioefsComing HazletonInc.,foran additionapleriodoftimeorsenttoa storagefacilitdyesignatebdy the Sponsor.
REFERENCES
Ames, B.N.,J.McCann, and E.Yamasaki. Methods fordetectincgarcinogenasnd mutagens withtheSalmonella/Mammalian-MicrosomMeutagenicitTyest.MutationResearch 3-L:347-36(41975).
BrusickD,.J.,V. F.Simmon, H. S. Rosenkranz,V. A. Ray,andR. S.Stafford. An evaluatioonftheEscherichicaoliWP2 and WP2uvrA reversemutationassay. MutationResearch76:169-190(1980).
Green,M.H.L. and W. J.Muriel.Mutagen testinugsingirp'reversioinnEscherichicaoli. MutationResearch31:3-32 (1976).
Maron, D.M.,and B. Ames. RevisedMethods fortheSalmonellaMutagenicitTyest. MutationResearchM: 173-215(1983).
Vogel,H.J.,andD.M. Bonner.AcetylomithinaosfeE. coli:Partiaplurificatiaonnd some propertieJs.Biol.Chem. M:97-106 (1956).
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BACTF,RIALBACKGRO
EVALUATION CODE
Theconditiooftnhebackgroubnadcterliaawlnisevaluatbeodthmacroscopicaanldly microscopical(luysinga dissectinmgicroscope)forindicatioonfscytotoxiciatnyd testarticle precipitaatsefollows:
CODE
DEFMTION
CHARACTERISTICS OF BACKGROUND LAWN
I
Normal
A healthymicrocolonylawn.
2
Slightly
A noticeabltehinninogfthemicrocolonylawn and an
Reduced
increaseinthesizeofthemicrocoloniecsompared tothe
vehiclecontropllate.
3
Moderately
A marked thinninogfthemicrocolonylawn and an
Reduced
increaseinthesizeofthemicrocoloniecsompared tothe
vehiclecontrolplate.
4
Extremely
An extremethinningofthemicrocolonylawn and an
Reduced
increaseinthesizeofthemicrocoloniecsompared tothe
vehiclecontrolplate.
5
Absent
A completelackofanymicrocolonylawn.
6
Obscured by
The backgroundbacterial]awn cannotbe accurately
Precipitate
evaluatedue tomicroscopicand/ormacroscopictest
articlperecipitate.
Evidenceofmacroscopictestarticlperecipitaotne theplatesisrecordedby additionofthe followingprecipitatceodetothecodenumber usedtoevaluatteheconditioonfthebackground bacterial]awn.
SP
Slight
Noticeablemacroscopicprecipitaotne theplate,
Precipitate
however,theprecipitadtoees notinfluenceautomated
countingoftheplate.
MP
Moderate
The amount ofmacroscopicprecipitaotne theplate
Precipitate
would interferweithautomatedcountingt,husrequirintghe
platetobe hand counted.
hp
Heavy
The largeamount ofmacroscopicprecipitatoen the
Precipitate
platemakes therequiredhand countingdifficult.
Example:4mp would indicataeplateobservedtohavean extremelyreducedbackgroundlawn which had tobe countedmanuallydue tothemarked amount ofmacroscopictestarticle precipitate.
CHV StudyNo.: 17073-0-409
22
CORNINGHazleton
SECTION IV.RESULTS AND CONCLUSIONS
CHV StudyNo.: 17073-0409
23
CORNINGHazieton
RESULTS
A. TestArticHlaendha
ThetesatrticTl-e6,342w,asstoraetdroomtemperatuDrei.onizweadte(rCHV Lot326)was usedasthevehicle.At 100mg perml,which was themost concentratesdtock dilutiopnreparedt,hetestarticlfeormeda clearc,olorlesssolutionT.he testarticlreemaineda solutioinnallsucceedingdilutionpsreparedforthemutagenicitayssay.
B. Dose RangefindingStudy
Doses tobe testedinthemutagenicitayssaywereselectebdasedon theresultosf thedoserangefindinsgtudyconductedon thetestarticluesingtestesrtrainTsA 100 and WP2uvrA inboththepresenceand absenceofS9 mix (oneplateperdose).Ten dosesoftest articlfer,om 5,000to6.67gg perplatew,ere testedand theresultasrepresentedinTablesI and 2. These datawere generatedinExperiment17073-AI.No cytotoxicitwyas observedineither thepresenceorabsenceof S9 mix as evidencedby anormal backgroundlawn and no decreasein thenumber ofrevertantpserplate.
C. Mutageniciiy Assay
The mutagenicityassayresultfsorT-6342 arepresentedinTables3 and 4. These datawere generatedinExperiment17073-B1. The dataarepresentedasmean revertantpser plate standardeviatiofnoreachtreatmenatnd controglroup(Table4 )and asindividuapllate counts(Table3).
The resultosf thedose rangefindinsgtudywere usedtoselecftivedosestobe testedinthe mutagenicitayssay.The dosestestedwere 5,000,3,330,1,000,333,and 100 gg perplateinboth thepresenceand absenceof S9 mix.
InExperiment17073-BI (Tables3 and 4),alldatawere acceptablaend no positiviencreaseisn thenumber ofrevertantpserplatewere observedwithany ofthetestesrtrainesitheirnthe presenceor absenceof S9 mix.
Allcriterfioara validstudyweremet.
CHV StudyNo.: 17073-0-409
24
CORNINGHazieton
CONCLUSIONS
T'heresultosftheSalmonella-Eschericchoilai/Mammalian-MicrosoRmeverseMutationAssay indicattehatu,ndertheconditionosfthisstudy,3M CorporatioetsestarticlTe-,6342,didnot causea positiviencreaseinthenumber of revertantpserplateofany ofthetestesrtrainesitheirn thepresenceorabsenceofmicrosomalenzymes preparedfromAroclorTm-inducerdatliver(S9).
CHV StudyNo.: 17073-0-409
25
CORNINGHazleton
SECTION V. DATA TABLES
CHV Study No.: 17073-0-409
26
CORNINGHazieton
TABLE I DOSE RANGEPINDING TEST ARTICLE ID: T-6342 EXPERIMENT ID: 17073-Al VEHICLE: Deionized water
STUDY
DATE PLATED: DATE COUNTED:
12-Sep-95 15-Sep-95
pg/PLATE
REVERTANTS PER
PLATE
WITH
TAIOO REVERTANTS S9
BACKGROUND LAWN
EVALUATION*
PER PLATE
WITHOUT S9
REVERTANTS
BACKGROUND
PER
LAWN
PLATE
EVALUATION*
0.00 (Vehicle)
(50 pi)
127
1
Test Article 6.67
103
1
10.0
100
1
33.3
85
1
66.7
120
1
100
112
1
333
138
667
107
1
1000
119
1
3330
ill
1
5000
118
1
93
1
87
1
81
1
77
1
105
1
98
1
113
1
97
1
79
1
107
1
92
1
Background Lavn Evaluation I - nor-%al 4 - extremely reduced
op - &light precipitate
Codes: 25up -
slightly reduced absent moderate precipitate (requires hand count)
3 - moderately reduced 6 - obscured by precipitate hp - heavy precipitate
(requires hand count)
CHV Study No.: 17073-0-409
27
CORNINGHazieton
TABLE 2 DOSE RANGEPINDING TEST ARTICLE ID: T-6342 EXPERIMENT ID: 17073-Al VEHICLE: Deicnized water
STUDY
DATE PLATED: DATE COUNTED:
12-Sep-95 15-Sep-95
pg/PLATE
REVERTANTS PER
PLATE
WP2uvrA REVERTANTS WITH S9
BACKGROUND LAWN
EVALUATION*
PER PLATE
WITHOUT S9
REVERTANTS
BACKGROUND
PER
LAWN
PLATE
EVALUATION*
0.00 (Vehicle)
(50 pl)
16
1
Test Article 6.67
24
1
10.0
19
1
33.3
11
1
66.7
12
1
100
23
1
333
20
1
667
8
1
1000
14
1
3330
20
1
5000
18
1
20
1
11
1
16
1
11
1
12
1
24
1
23
1
15
1
19
1
21
1
Background Lawn Evaluation I nor@al 4 extremely reduced op slight precipitate
Codes: 2 5
op
- slightly reduced - absent - moderate precipitate
(requires hand count)
3 - moderately reduced 6 - obscured by precipitate hp - heavy precipitate
(requires hand count)
CHV Study No.: 17073-0409
28
CORNINGHazieton
TEST ARTICLE ID: T-6342 EXPERIMENT ID: 17073-Bl DATE PLATED: 19-Sep-95 DATE COUNTED: 22-Sep-95
TABLE 3 MUTAGENICITY ASSAY
INDIVIDUAL PLATE
RESULTS COUNTS
VEHICLE: Deionizod vater PLATING ALIQUOT: 50 lil
REVERTANTS PER PLATE
DOSE/ZLATE
MICROSOMES: Rat Liver VEHICLE CONTROL
TA90
1
2
3
11 25 24
-TAIOO
1
2
3
127 113 128
TA153S
1
2
3
13 15
7
TA1537
1
2
3
la
9
7
TEST ARTICLE POSITIVE CONTROL
100 jig 333 pg 1000 PS 3330 pg 5000 ps
30 36 38 30 27 28 29 34 26 38 31 34 23 19 is
848 995 810
109 115 121 114 III 114 128 123 119 108
112 111 ill IIB 126
16 13 11
5 21 14
13 17
9
13 14 11
15 10 12
926 953 999
110 108 122
6 12
8
7
5 10
10
3
9
10 11
5
10
7
6
107 122 108
WP2uvrA
1
2
BACXGROUND LAW'
3
20 19 19
1
30 14 17
1
IS 21 15
1
12 14 13
1
15 16 16
1
20 29 24
1
201 271 163
1
MICROSOMES: None VEHICLE CONTROL TEST ARTICLE
POSITIVE CONTROL
100 pg 333 pg 1000 ps 3330 pg 5000 ps
14 12 13
19 19 10
11 21 17
12 19 14
10 16 15
20 17
9
129 154 156
90 86 86
97 $a 107 103 107 100 104 91 97 82 97 98 92 72 48
841 817 861
7
5
6
5
8
5
13 13 19
6
9 17
6
6
9
9 16
9
582 599 556
7
a
1
3
7
7
10
7 23
5 10
4
4
6
4
12
9
6
202 224 218
10 15 is
1
26 11 15
1
18 15 17
1
27 18 24
1
9 22 19
1
10 16 17
1
39 45 76
2
TA98 TAIOO TAIS35 TAIS37 WP2uvrA
2-aminoanthrac*ne 2-"Inoanthracens 2-aminoanthcacens 2-aminaenthracens 2-aminoanthr&4cono
2.5 2.5 2.5 2.3 25.0
VS/plate pg/p2ate pg/plate Mg/plato pg/plate
... TA98 TAIOO TA1335 TA1337 WP2uvrA
2-nitrefluarone sodium aside sodium aside ZC]t-191 4-nitroquinaline-N-oxide
1.0 pa/plate 2.0 pg/p2at* 2.0 pg/plate 2.0 pa/plate 1.0 Mg/plate
Background Lawn Evaluation I - normal 4 - extremely reduced
op I slight precipitate
Codes: 2 - slightly reduced 5 - absent
op - moderate precipitate (requires hand count)
3 - moderately reduced 6 - oucured by precipitate hp - heavy precipitate
(requires hand count)
CHV StudyNo.: 17073-0-409
29
CORNINGHazleton
TABLE 4 MUTAGENICITY ASSAY
SUMMARY
RESULTS
TEST ARTICLE ID: T-6342
F"ERIMENT ID: 17073-BI
DATE PLATED: 19-Sep-95
VEHICLE: Dsionized water
DATE COUNTED: 22-Sep-95
PLATING ALIQUOT: 50 pl
MM REVERTANTS PER PLATS WITH STANDARD DEVIATION
DOSE/Pbm
MICROSOMES: Rat Liver VEHICLE CONTROL
TA98
MEAN
S.D.
20
a
TA100
MEAN
S.D.
117
2
Tkl535
MEAN
S.D.
12
4
TA1537
MEAN
S.D.
9
2
TEST ARTICLE
100 ps 333 pg 1000 ps 3330 yg 5000 ps
35
4
28
2
30
4h
34
4
20 3
112
3
Ila
4
114
4
123
5
Ila 9
13
3
11
3
13
4
13
2
12 3
9
3
7
3
7
4
9
3
a2
POSITIVE CONTROL
864 99
923 32
113
8
112
a
HICROSOMES: None VEHICLE CONTROL TEST ARTICLE
POSITIVS CONTROL
100 og 333 pg 1000 P& 3330 pg 5000 ps
13
1
16
5
16
5
15
4
14
3
is
6
146 15
87
2
97 10
203
4
97
7
92
9
84 11
$40
22
6
1
6
2
15
3
11
5
7
2
11
4
579
22
5
4
6
2
10
3
6
3
5
1
9
3
215
11
WP2uvrA
MEAN
S.D.
BACKGROUND LAWN'
19
1
1
20
9
1
17
3
1
13
1
1
16
1
1
24 5
1
178 20
1
14
4
1
17
a
1
17
2
1
23
3
1
17
7
1
14
4
1
53 20
1
TA90 TAIOO TAIS33 TAIS37 WP2uvrA
2-amincenthracene 2-aminaenthracene 2-sainoanthrac*ne 2-aminoonthracene 2-aminoanthracens
2.5 2.5 2.5 2.5 23.0
pS/plate pg/plate pt/plate p$/plate p&/plate
... TA98 TAIOO TA2533 TA1337 WP2uvrA
2-nitrofluorens sodium aside sodium azide ICR-191 4-nitroquinalina-M-axido
1.0 ps/plato 2.0 pa./plate 2.0 pg/plate 2.0 pa/plate 1.0 pg/plate
Background Lawn Evaluation 1 - normal
4 m extremely reduced ap m slight precipitate
Codes: 2 - slightly reduced 5 - absent
up - moderate precipitate (requires hand count)
3 - moderately reduced 6 m obscured by precipitate hp - heavy precipitate
(requires hand count)
CHV StudyNo.: 17073-0-409
30