Document 15xzebb9zV18XjVVwXJK7MwMm
CORNINGHazleton
MUTAGENICITY TEST WITH
T-6564
IN THE SALMONELLA -ESCHEFICHIA COLI/MAMMALIAN-NECROSOME MUTATION ASSAY WITH A CONFIRMATORY ASSAY
REVERSE
FINAL REPORT
AUTHOR TimothyE.Lawlor,M.A.
PEREORMRNG LABORATORY Coming HazletonInc.(CHV) 9200 LeesburgPike Vienna,Virginia22182
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6..:
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LABORATORY PROJECT ID CHV StudyNo.: 17750-0409R
SUBMITTED TO
3M Corporation Building220-2E-023M Center
St.Paul,NIN 55144-1000
STUDY COMPLETION DATE September 13,1996
CHV StudyNo.:17750-0-409R
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CORNINGHazleton QUALITY ASSURANCE STATEMENT
STUDY TITLE: Salmonel-lEaschericchoilai/Nlammalian-MiRcervoesrosMmeuetation Assaywitha ConfirmatoryAssay
ASSAY NO.:
17750-0409R
PROTOCOL NO.: 409F,Edition4,Modifiedfor3M
QualityAssuranceinspectionosfthestudyand reviewof thefinalrepor-o.ftheabove referenced projectwere conductedaccordingtotheStandardOperatingProceduresof theQualityAssurance Unitand accordingtothegeneralrequirementosftheappropriatGeood LaboratoryPractice regulationsF.indingsfromtheinspectionasnd finalreportreviewwerereportedtomanagement
and tothestudydirectoorn thefollowingdates:
Inspection/Date
FindingsRepgrted
Auditor
ScoringPlates- 06/28/96
06/28/96
P. Conley
DraftReportReview -08/29/96 FinalReportReview - 09/13/96
08/30/96 09/13/96
C. Orantes C. Orantes
QuariAtsysu-rance Unit
DO(e Refeased
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STUDY COMPLIANCEAND CERTIFICATION
The studywas conductedincompliancewiththeGood LaboratoryPracticeregulationass set forthby theFood and Drug Administratio(nFDA) inTitle21 oftheU.S.Code ofFederal RegulationsPart58,issuedDecember 22, 1978,(effectiJvuene20,1979)withany applicable amendments. Therewere no deviationfsrom theaforementionerdegulationosr thesigned protocolthatwould affectheintegritoyfthestudyortheinterpretatioofnthetestresultsT.he raw datahave beenreviewedby theStudyDirectorw,ho certifitehsattheevaluatioonf thetest articlaespresentehdereinrepresentasn appropriatceonclusionwithinthecontextof thestudy designand evaluatiocnriteria.
StudyDirector:
Timothy E. wlor,M.A. BacteriaMlu agenesis Geneticand CellulaTroxicology
ct-13
StudyCompletionDate
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TABLE OF CONTENTS
CORNINGHazleton
Page No. I. SUMMARY ..........................................5............. ii. STUDY INFORMATION ..................................7............. III. MATERIALS AND METHODS ...............................9.............. IV. RESULTS AND CONCLUSIONS .............................24............ V. DATA TABLES ......................................2.7.............
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SECTION 1.SUMMARY INTRODUCTION AND CONCLUSIONS
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SUMMARY
A. Introduction
At therequestof 3M Corporation,Coming HazletonInc.investigateTd-6564 for mutagenic activitiyntheSalmonella-Escherichiacoli/Manirnalian-MicrosomReeverse Mutation Assay with a ConfirmatoryAssay. This assayevaluatedthetestarticlaend/orits metabolitesfortheirabilittyoinducereversemutationsatthehistidinelocusinthegenome of specificSalmonellatyphimuriumtesterstrainsand atthetryptophanlocusinan Escherichiacoli testerstrainboth inthepresenceand absence of an exogenous metabolica. ctivatiosnystem of mammalian microsomal enzymes derivedfrom Aroclofrm-inducedmt liver(S9).
The doses testedin themutagenicityassay were selectedbased on the resultsof a dose rangefindingstudyusingtesterstrainsTAIOO and WP2uvrA and tendoses of testarticlreanging from 5,000 to 6.67 gg per plate,one plateper dose,both in the presence and absence of S9 mix.
The testerstrainsused in the muta 9enicityassay were Salmonella typhimurium testerstrains TA98, TAIOO, TA1535, TA1537, and Escherichia colitesterstrainWP2uvrA. The assay was conducted with fivedoses oftestarticlienboth thepresenceand absence of S9 mix along with concurrentvehicleand positivecontrolsusingthreeplatesper dose. The dosestestedwere 5,000,3,330,1,000@3*'3-'a1n,d 100 gg per platein both the presence and absence of S9 mix. The resultsoftheinitiamlutagenicityassaywere confu-med inan independentexperiment.
B. Conclusions
The resultsoftheSalmonella-Escherichiacoli/Mammalian-Microsome Reverse Mutation Assay with a ConfirmatoryAssay indicatethat,under the conditionsofthisstudy,')M Corporation'stestarticleT,-6564, did notcause a positiveincreaseinthenumber of revertants perplateofany ofthetesterstrainseitherinthepresenceorabsence of microsomal enzymes preparedfrom Aroclor:Tm-inducerdatliver(S9).
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CORNINGHazleton SECTION 11.STUDY INFORMATION
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STUDY INFORMATION
A. Sponsor:3M Corporation
B. TestArticleT:-6564 L-13167 FC1015-X
1. Physical Description: clear colorlessliquid
2. DateReceived:05/30/96
C. Type of Assay: Salmonella-Escherichiacoli/Mammalian-Microsome Reverse MutationAssay witha ConfirmatoryAssay
I ProtocolNumber: CHV Protocol4091@,Edition4
2. CHV StudyNumber: 17750-0409R
D. StudyDates
I StudyInitiatiDoante:
06/04/96
2. ExpcrimentalStartDate:
06/12/96
3. ExperimentalTerminationDate: 08/27/96
E. StudySupervisorPyersonnel
StudyDirector:
TimothyE.Lawlor,M.A.
LaboratorySupervisor:
MichaelS.Mecchi,B.S.
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SECTION 111.MATERIALS AND METHODS
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MATERIALS AND METHODS
Theexperimentmaalterialmse,thodsandprocedureasrebasedon thosedescribebdy Ames elal(1975)andGreenandMuriel(1976).
MATERIALS
A. TesterStrains
I
Salmonella =himurium
The testerstrainsused were theSalmonella typhimurium histidine auxotrophs TA98, TAIOO, TAIS35, and TA1537 as described by Ames etal (1975). The specificgenotypes of thesestrainsare shown in Table 1.
TABLE 1. TESTER
HistidineMutation hisG46 hisC3076 hisD"3052
TA1535 TA15'37 TAIOO
TA98
STRAIN ENOTYPES
AdditionalMutations
LPS Repair R Factor
rfa
uvrb
rfa
uvrb
+R
In additiontoa mutationinthehistidinoeperon,thetesterstrainscontaintwo additional mutationswhich enhance theirsensitivittyosome mutagenic compounds. The rfawall mutation resultsinthe lossof one oftheenzymes responsibleforthesynthesisofpartof the lipopolysaccharidbearrierthatforms the surfaceof thebacteriaclellwall. The resultinecellwall deficiencyincreasespermeabilitytocertainclassesof chemicals such asthosecontaininglarize ringsystems(i.eb.enzo(a)pyrene)thatwould otherwisebe excludedby a normal intactcellwall.
The second mutation,a deletionof theuvrb gene,resultsina deficientDNA excisionrepair system which greatlyenhances thesensitivitoyf thesestrainsto some mutagens. Sincetheuvrb deletionextendsthroughthebio gene,allofthetesterstrainscontainingthisdeletionrequirethe vitaminbiotinforgrowth.
StrainsTA98 and TA 100 alsocontainthe R-factorplasmid,pKM 101,which furtherincreases thesensitivitoyf thesestrainstosome mutagens. The mechanism by which thisplasmid increasessensitivityo mutagens has been suggestedtobe by modifying an existingbacterial DNA repairpolymerase complex involvedwith themismatch-repairprocess.
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TestesrtraiTnAs98andTAI537arerevertferdomhistiddienpeenden(caeuxotropthoy) histidiniendependenc(eprototrophbyy) fi=eshiftmutagens.TA1535 isrevertebdy base substitutimountagens and TAIOO isrevertedby mutagens which causeboth fi-ameshifatnsd base substitutimountations.
2. Lcherichia coli The testerstrainused was the tryptophanauxotroph WP2uvrA as
describebdy Greenand Muriel(1976). Inadditiotnoa mutationinthetryptophanoperon,thetestesrtraicnonta= a uvra DNA repair deficiencwyhich enhancesitssensitivittoysome mutageniccompounds. Thisdeficiencayllows thestraitnoshow enhancedmutabilitsyincetheuvra repairsystemwould normallyactto remove thedamaged partoftheDNA moleculeand accuratelryepairitafterwards.
TesterstraiWnP2uvrA isrevertefdrom tryptophandependence(auxotrophyt)otryptophan independence(prototrophbyy) basesubstitutimountagens.
3. SourceofTesterStrains a. Salmonella typhimurium The testerstrainsin use atCHV were received directlyfrom
Dr.BruceAmes, DepartmentofBiochemistryU,niversitoyfCaliforniBae,rkeley.
b. Escherichiacoli
The testerstrain,WP2uvrA, in use at CHV was receivedfrom the NationalCollectioonf IndustriBaalcteriaT,orreyResearchStationS,cotland(UnitedKingdom).
4. StorasioeftheTesterStrains
a. FrozenPermanentStocks Frozenpermanentstockswere preparedby growingfresh
overnighctulturesa,ddingDMSO (0.09ml/ml ofculturea)nd freezinsgmallaliquots (0.5-1.m5l) at:@-70*C.
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b. MasterPlates
Masterplateswere preparedby streakinegachtesterstraifnrom a frozenpermanentstockontominimalagarappropriatelsyupplementedwith 1)forSalmonella typhimurium,anexcessofhistidinaen,d biotina,nd fortestesrtrainTsA98 and TA 100, ampicilli(n25pg/ml),toensurethestablemaintenanceofthepKM101 plasmid;and 2)for Escherichicaolia,n excessoftryptophanT.esterstraimnasterplateswerestoredat 5 :@3OC.
5. PreparatioonfOvernightCultures
a. Inoculation
Overnightculturefsoruse ina testinpgrocedureswere inoculated by transferriancgolonyfrom theappropriatmeasterplatetoa flaskcontainincgulturemedium. Inoculatefdlaskwsere placedina shaker/incubatwohrich was programmed tobeginoperation (shaking1,25 25 rpm;incubation3,7 + 2*C) sothattheovernighctulturewsereinlogphaseor latelogphasewhen turbiditmyonitoringbegan.
b. Harvest
To ensurethatculturewsere harvestedinlatelogphase,thelenzth ofincubatiownas determinedby spectrophotometrmiocnitoringofculturteurbidityC.ultures were harvestedonce a predeterminedturbiditwyas reachedas determinedby a percent transmittanc(e%T) readingon a spectrophotometerT.histargeturbiditeynsuresthatcultures have reacheda densityofatleast0.5X 109cellsperml and thattheculturehsave notovergrown. Overgrown (stationarcyu)lturemsay exhibitdecreasedsensitivittoysome mutagens.Cultures were removed from incubatiownhen thetargetO/oTwas reachedand were placedat5 3'C.
6. ConflrznatiofnTesterStraiGneno=el
Testerstraicnulturewsere checkedforthefollowingeneticmarkerson theday oftheiruse inthemutagenicitayssay:
a. Salmonella typhimurium
1) rfa Wall Mutation
The presence of the rfawall mutation was confirmed by demonstratioonfthesensitiviotfytheculturteocrystavlioletA.n aliquootfan overnight
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cultuorfeachstrawiansoverlaoindtpolatceosntainsienlgectmievdeiaandanantibiotic sensitividtiyskcontainin1g0 gg ofcrystavliolewtas added.Sensitiviwtays demonstratedby inhibitionfbacteriaglrowthina zoneimmediatelysurroundingthedisk.
2) pKM 101 PlasmidR-factor
The presenceofthepKM 101 plasmidwas confirmedfor testesrtrainTsA98 and TAI 00 by demonstratioonfresistancteoampicillinA.n aliquootfan overnightcultureofeachstraiwnas overlaidontoplatescontaininsgelectivmeedia and an antibiotsiecnsitividtiyskcontainin1g0jigofampicilliwnas added.Resistancweas demonstratedby bacteriaglrowthinthezone immediatelysurroundingthedisk.
3) CharacteristNiucmber of SpontaneousRevertants
The mean number of spontaneousrevertantpserplateinthe vehiclecontroltshatarecharacteristoifctherespectivsetrainwsere demonstratedby plating 100 glaliquotosftheculturaelongwiththeappropriatveehicleon selectivmeedia.
b. Escherichiacoli
I CharacteristNiucmber of SpontaneousRevertants
The mean number of spontaneousrevertantpserplateinthe vehiclecontroltshatarecharacteristoifctherespectivsetrainwsere demonstratedby plating100 plaliquotosftheWP2uvrA culturaelongwiththeappropriatveehicleon selectivmeia.
7. TesterStraiMnedia
a. CulturinBgroth
The brothusedtogrow overnightcultureosf thetesterstrainwsas Vogel-Bonnersaltsolutio(nVogeland Bonner,1956)supplementedwith2.5% (w/v)Oxoid NutrienBtrothNo. 2 (drypowder).
b. Agar Plates
Bottom agar(25mi per 15x 100 mm petridish)was VogelBonner minimalmedium 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 preparedwith0.7% agar(w/v)and 0.5% NaCl (w/v)andwas supplementedwith10ml of1)0.5 mM histidinelbiostoilnutiopner 100 ml agarforselectioonfhistidinreevertantosr,2)0.5mM tryptophasnolutiopner100 ml of agarforselectioonftryptopharnevertantsW.hen S9 mix was required2,.0ml ofthe supplementedtopagarwas used intheoverlay.However, when S9 mix was notrequiredw,ater was addedtothesupplementedtopagar(0.5ml ofwaterper2 ml ofsupplementedtopagar)and theresultin2g.5ml ofdilutedsupplementedtopagarwas usedfortheoverlay.Thisdilution ensuredthatthefinaltopagarand amino acidsupplementconcentrationrse.mainedthesame both inthepresenceand absenceofS9 mix.
B. LiverMicrosomal Enzyme Reaction Mixture (S9 Mix)
I. S9 Homogenate
Livermicrosomalenzymes (S9 homogenate)werepurchasedfrom MolecularToxicologyI,nc.A,nnapolisl,@M 21401,Batch0642 (39.5mg ofproteipnerml) and Batch0646 (43.4mg ofproteinperml).The homogenate was preparedfrom male SpragueDawley ratsthathadbeen injecte(di.p.w)ithAroclorTm1254(200mg perml incom oil)at 500 mg/kg asdescribebdy Ames etal,1975.
2. S9 Mix
The S9 mix was preparedimmediatelypriortoitsuseinany experimental procedure.The S9 mix containedthecomponents indicateidnTableII.
TABLE
!OMPONENTS
H20
IM NaH2Po4Na2HP04, pH 7.4 0.25M Glucose-6-phosphate 0.1OM NADP 0.825M KCUO.2MMgCI2 S9 Homogenate
0.70ml 0.10ml 0.02ml 0.04ml 0.04ml 0.10 mi 1.00ml
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C. Controls
1. VehicleControls
Vehiclecontrolwsere platedforalltestesrtrainbsothinthepresenceand absenceofS9 mix. The vehiclecontrolwas platedu,singa 50 glaliquotofvehicle(equaltothe maximum aliquootftestarticldeilutiopnlated)a,longwitha 100 plaliquootftheappropriate testesrtraiannd a 500 glaliquootfS9 mix (when necessary)o,n selectivaeizar.
2. PositivCeontrols
The combinationsofpositivceontrolsa,ctivatiocnonditioannd tester strainpslatedconcurrentlwyiththeassayareindicateidnTableIII.
TABLE 111.POSITIVE CONTROLS
Tester
Conc
Stmin S9 Mix PositivCeontrol
12er12late
TA98
+
2-aminoanthracene
2.5jig
TA98
2-nitrofluorene
1.0gg
TAIOO
+
2-aminoanthracene
2.5;ig
TAIOO
sodium azide
2.0gg
TA1535
+
2-aminoanthracene
2.5gg
TA1535 -
sodium azide
2.0 ;ig
TA1537 +
2-aminoanthracene
2.5gg
TA1537 -
ICR-191
2.0gg
WP2uvrA +
2-aminoanthracene
25.0jig
WP2uvrA -
4-nitroquinoline-N-oxid1e.0gg
a. Sourceand Grade ofPositivCeontrolArticles
2-aininoanthrace(nCeAS #61')-13-8)S,igma ChemicalCo., purity2t97.5%;2-nitrofluore(nCeAS #607-57-8)A,ldrichChemicalCo.,purity98%: sodium azide(CAS 426628-22-8)S,igma ChemicalCo.,purity>98%; ICR-191 (CAS #1707-45-0). PolyscienceIsnc.p,urity>95% and Sigma ChemicalCo.,purity98%; 4-nitroquinoline-N-o\lde (CAS #56-57-5)S,igma ChemicalCo.,purit>y99%.
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SterilCiotnvtrols
a. TestArticle
The most concentratetdestarticldeilutiownas checkedforsterility by platinga 50 glaliquot(thesame volume usedintheassay)on selectivaegar.
b. S9 Mix
agar.
ne S9 mix was checkedforsterilibtyyplatin0g.5ml on selective
METHODS
A. Dose RangefindingStudy
The growthinhibitoreyffec(tcytotoxicitoyf)thetestarticlteothetestsystemwas determinedinordertoallowtheselectioonfappropriatdeosestobe testedinthemutagenicity assay.
I. Design
The doserangefmdingstudywas performedusingtesterstrainTsA 100 and WP2uvrA bothinthepresenceand absenceof S9 mix. Ten dosesoftestarticlweere tested atone plateperdose.The testarticlweas checkedforcytotoxiciutpy toa maximum concentratioonf5 mg perplate.
a. Rationale
The cytotoxicitoyfthetestarticloebservedon testesrtraiTnA 100 isgenerallryepresentatiovfethatobservedon theothertestesrtrainasnd becauseofTA I00's comparativelhyighnumber ofspontaneousrevertantpserplateg,radationosfcytotoxicitcyan be readildyiscernedfromroutinexperimentavlariationT.he Escherichiacolitestesrtrain WP2uvrA doesnotpossesstherfawallmutationthattheSalmonellatyphimuriumstrainhsave and thus,a differenrtangeofcytotoxicimtayy be observed.Also,thecytotoxiciitnyducedby a testarticlienthepresenceofS9 mix may varygreatlfyrom thatobservedintheabsenceof S9 mix. Thereforet,hiswould requirethatdifferenttestarticldeoserangesbetestedinthe mutagenicityassaybasedon thepresenceor absenceof theS9 mix.
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2. EvaluatioonftheDose RangefindingStudy
Cytotoxicitiysdetectablaes a decreaseinthenumber of revertanctolonies perplateand/orby a thinningordisappearancoef thebacteriablackgroundlawn.
3. Selectionof theMaximum Dose fortheMutaizenicitAv,s_av
a. No CytotoxicitOybserved
Sinceno cytotoxiciwtays observedinthedoserangefindinsgtudy, thehighestdoseleveloftestarticluesed inthemutagenicitayssaywas the same asthattestedin therangefindinsgtudy.
B. MutagenicityAssa3:
1 . Desi Lyn
The assay was performed using testerstains TA9 8, TA 100, TA 15'@5, TA15-')7,and WP2uvrA both inthe presence and absence of S9 mix. Five doses of testarticle were testedalongwiththeappropriatveehicleand positivceontrolsT.he dosesoftestarticle were selectedbasedon theresultosfthedoserangefmdingstudy.The resultosftheinitial mutagenicitayssaywere confirmedinan independentexperiment.
2. Freguencvand RouteofAdministration
The testesrtrainwsere exposedtothetestarticlveiatheplate incorporatiomnethodologyoriginalldyescribedby Ames etal (1975)and Maron and Ames (1983).Thismethodologyhas beenshown todetecta wide rangeofclassesofchemical mutagens. Intheplateincorporatiomnethodology,thetestarticlteh,etesterstraiannd theS9 mix (whereappropriatew)erecombined inmoltenagarwhich was overlaiodntoa minimalaear plate.Followingincubatioant37:E2*C for48 * 8 hr,revertanctoloniewsere counted.All dosesofthetestarticlet,hevehiclecontrolasnd thepositivceontrolwsereplatedintriplicate.
C. PlatineProcedures
These procedureswere used inboththedoserangefindinsgtudyand the mutagenicityassay.
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Each platewas labeledwitha codewhich identifietdhetestarticltee,stphase,testesrtrain, activatiocnonditioannd doselevel.'Me S9 mix and dilutionosfthetestarticlweereprepared immediatelypriortotheiruse.
When S9 n-Liwxas notrequired1,00 gloftestesrtraiannd 50 piofvehicleortestarticldeose was addedto2.5mi ofmoltenselectivteopagar(maintaineadt45 2*C). )klenS9 mix was required5,00 glofS9 mix,100 gloftestesrtraiannd 50 glofvehicleortestarticldeosewas addedto2.0ml ofmoltenselectivteopagar.Aftertherequiredcomponentshad been added,the mixturewas vortexedand overlaiodntothesurfaceof25 ml ofminimalbottomagarcontainedin a 15x 100 mm petridish.Aftertheoverlayhad solidifietdh,eplateswereinvertedand incubatedfor48 8 hrat37 2*C. Positivceontrolarticlewsereplatedusinga 50 glplating aliquot.
D. ScoringthePlates
Plateswhich were notevaluatedimmediatelyfollowingtheincubatiopneriod were heldat5 3 *C untilsuchtimethatcolonycountingand bacteriablackgroundlawn evaluatiocnouldtakeplace.
I. BacteriaB]ack=und Lawn Evaluation
The conditioonfthebacteriablackgroundlawnwas evaluatedfor evidenceofcytotoxiciatnyd testarticlperecipitatEev.idenceofcytotoxiciwtays scoredrelative tothevehiclecontrolplateand was recordedalongwiththerevertanctountsforallplatesatthat doselevelon thedatatablesusingthecode systempresentedattheend oftheMaterialsand Methods Section.
2. CountingRevertantColonies
The number ofrevertanctoloniepserplateforthevehiclecontrolasnd all platescontainintgestarticlweerecountedmanually.The number ofrevertanctoloniesperplate forthepositivceontrolwsere countedby automatedcolonycounterwiththeexceptionofthe positivceontrolfsortestesrtraiWnP2uvrA intheabsenceofS9 mix,inExperiment17750-B1, which were countedmanually.
E. AnalysisofData
Forallreplicatpelatingst,hemean revertantpserplateand thestandarddeviation were calculatedT.he resultosfthesecalculationasrepresentedintabularform intheData TablesSectionof thisreport.
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EVALUATION OF TEST RESULTS
Beforaessadyatawereevaluattehde,critefroiraavaliadssayhadtobemet.
A. CriteriaFor A ValidAssav
The followingcriteriwaere used todeterminea validassay:
I TesterStrainIntegdiy: Salmonella typhimurium
a. rfaWall Mutation
To demonstrate the presenceof the rfawall mutation,testerstrain culturesexhibitesdensitivitoycrystavliolet.
b. pKM 101 Plasmid
To demonstratethepresenceof theR-factorplasmid,pK-M 101, cultureosf testesrtrainTsA98 and TAIOO exhibitedresistancteoampicillin.
C. CharacteristNiucmber of SpontaneousRevertants
To demonstratetherequirementforhistidinet,hetestesrtrain cultureesxhibiteda characteristniucmber of spontaneousrevertantpserplatewhen platedalong withthevehicleunderselectivceonditions.The acceptablerangesforthemean vehiclecontrols were as follows:
TA98 TAIOO TAIS35 TA 15-7')
8 - 60 60 - 240
4 - 45 2 - 25
2. TesterStrainInteyfit:yEscherichiacoli
a. CharacteristNiucmber of SpontaneousRevertants
To demonstratetherequirementfortryptophant,hetestesrtrain cultureexhibiteda characteristniucmber of spontaneousrevertantpser platewhen platedalong
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%&ithevehiculnedesrelectciovneditioTnhse.acceptarbalnegfeortheWP2uvrAmeanvehicle controlwsas 5 to40 revertantpserplate.
3. TesterStrainCultureDenaiI3L
To demonstratethatappropriatneumbers of bacteriareplatedt,hedensity oftestesrtraicnulturewsere greatetrhanorequalto 0.5x 109bacteripaerml and/orhad reached a targetlevelofturbiditdyemonstratedtoproduceculturewsitha densitygreaterthanor equalto 0.5x 109bacteripaerml.
4. PositivCeontrolValues
a. PositivCeontrolValuesintheAbsenceofS9 Mix
To demonstratethatthetestesrtrainwsere capableof identifyinag mutagen,themean valueofa positivceontrolfora respectivteestesrtrainexhibiteadtleasta 3-foldincreasoeverthemean valueofthevehiclecontrolforthatstrain.
b. PositivCeontrolValuesinthePresenceofS9 Mix (S9Mix Integrity)
To demonstratethattheS9 mix was capableofmetabolizinga promutagentoitsmutagenicform(s)t,hemean valueofthepositivceontrolfora respective testesrtraiinnthepresenceoftheS9 mix exhibiteadtleasta 3-foldincreaseoverthemean value of thevehiclecontrolforthatstrain.
An acceptablpeositivceontrolinthepresenceof S9 mix fora specifisctrainwas evaluatedas havingdemonstratebdoththeintegritoyftheS9 mix and theabilitoyfthetestesrtraitnodetecta mutagen.
5. Cvtotoxiciiy
A minimum ofthreenon-toxicdoseswere requiredtoevaluateassaydata.
B. CriteriaFor A PositivReeslionse
Once thecriterifaora validassayhad been met,responsesobservedintheassay were evaluatedasfollows:
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1. TesterStrainTsA98. TA 100,and WP2uvrA
For a testarticlteobe consideredpositivei,thad toproduceatleasta 2-foldincreaseinthemean revertantpserplateofatleastone of thesetestesrtrainosverthe mean revertantpserplateoftheappropriatveehiclecontrolT.hisincreasienthemean number of revertantpserplatehad tobe accompanied by a dose responseto increasincgoncentrationosfthe testarticle.
2. TesterStrainsTA 1535 and TAI 537
Fora testarticlteobe consideredpositivei,thad toproduceatleasta 3-foldincreaseinthemean revertantpserplateofatleastone of thesetestesrtrainosverthe mean revertantpserplateoftheappropriatveehiclecontrolT.hisincreasienthemean number of revertantpserplatehad tobe accompaniedby a doseresponseto increasincgoncentrationosf the testarticle.
RECORDS TO BE MMNTAINED
Allraw data,documentationr,ecordst,heprotocola,nd thefinalreportgeneratedas a resultof thisstudywillbe archivedinthestoragefacilitioefsCorningHazletonInc.foratleastone year followingsubmissionofthefinalreportotheSponsor.Aftertheone yearperiod,theSponsor may electohavetheaforementionemdaterialrsetaineidnthestoragefacilitioefsComing HazletonInc.foranadditionapleriodoftimeorsenttoa storagefacilitdyesignatebdy the Sponsor.
REFERENCES
Ames, B.N.,J.McCann, and E.Yamasaki. Methods fordetectincgarcinogensand mutagens withtheSalmonella/Mammalian-MicrosomMeutagenicitTyest.MutationResearch 21:347-364(1975).
Brusick,D.J.,V.F.Simmon, H.S.Rosenkranz,V.A. Ray,and R.S.Stafford. An evaluatioonftheEscherichiacoliWP2 and WP2uvrA reversemutationassay. MutationResearch76:169-190(1980).
Green,M.H.L. and W.J.Muriel.Mutagen testinugsingtrp'reversioinnEscherichicaoli. MutationResearch31:3-32.(1976).
CHV StudyNo.: 17750-0-409R
21
CORNINGHazieton
MaronD,.M.a,ndB.Ames.RevisemdethodfsortheSalmonelMluatagenicTietsyt. MutationResearchJU: 173-215(1983).
Vogel,H.J.,and D.M. Bonner.AcetylomithinasoefE coli:Partiaplurificatiaonnd some propertieJs.Biol.Chem. M:97-106 (1956).
CHV StudyNo.: 17750-0-409R
22
CORNINGHazleton
BACTERIAL BACKGROUND LAWN EVALU
Theconditoifotnhebackgroubnadcterliaawlnisevaluatbeodthmacroscopicaanldly Microscopical(luysinga dissectinmgicroscope)forindicationosfcytotox-:-itaynd testarticle precipitaatsefollows:
CODE DEFMTION CHARACTERISTICS OF BACKGROUND LA)XRN
I Normal
A healthymicrocolonylawn.
2
Slightly A noticeabldeift@g ofthemicrocolonylawn andan
Reduced
increaseinthesizeofthemicrocoloniecsompared tothevehicle
controlplate.
3
Moderately A marked ddnningofthemicrocolonylawn and an
Reduced
increaseinthesizeof themicrocoloniecsompared tothevehicle
controlplate.
4
Extremely An extremethinningofthemicrocolonylawn and an
Reduced
increaseinthesizeof themicrocoloniecsompared to thevehicle
controlplate.
5 Absent
A completelackofany microcolonylawn.
6
Obscuredby The backgroundbacteriallawn cannotbe accurately
Precipitate evaluatedue tomicroscopicand/ormacroscopictestarticlperecipitate.
Evidenceofmacroscopictestarticlperecipitaotne theplatesisrecordedby additioonfthe followingprecipitactoedetothecodenumber usedtoevaluatteheconditionofthebackground bacteriallawn.
sp Slight
Noticeablemacroscopicprecipitatoen theplate,
Precipitate however,theprecipitadtoees notinfluencaeutomatedcountingof the
plate.
mp Moderate The amount of macroscopicprecipitatoen theplate Precipitate would interferweithautomatedcounting,thusrequirintgheplatetobe
hand counted.
hp Heavy
The largeamount ofmacroscopicprecipitaotne the
Precipitate platemakes therequirehdand countingdifficult.
Example:4mp would indicataeplateobservedtohave an extremelyreducedbackgroundlawn which had tobecountedmanuallydue tothemarked amount ofmacroscopictestarticle precipitate.
CHV StudyNo.: 17750-0-409R
23
CORNINGHazleton
SECTION TV.RESULTS AND CONCLUSIONS
CHV StudyNo.: 17750-0-409R
24
CORNINGHazieton
RESULTS
A. TestArticlHeandlilLg
The testarticlTe-,6564,was storedatroom temperatureD.eionizedwater (CHV Lots347,349,and 352)was usedasthevehicle.At 100 mg perml,which was themost concentratesdtockdilutiopnreparedt,hetestarticlfeormeda clearc,olorlesssolutionT.he test articlreemaineda solutioinnallsucceedingdilutionpsreparedforthemutagenicitayssay.
B. Dose Ran-.efindinSat.udv
Doses tobe testedinthemutagenicityassaywere selectebdased on theresultosf thedoserangefindinsgtudyconductedon thetestarticluesingtesterstrainTsA 100 and WP2uvrA inboththepresenceand absenceof S9 mix (oneplateperdose).Ten dosesoftest articlfer,om 5,000to6.67jigperplate,were testedand theresultasrepresentedinTablesI and 2. These datawere generateidnExperiment17750-Al.No cytotoxiciwtays observedineither thepresenceorabsenceof S9 mix as evidencedby a normal backgroundlawn and no decreasein thenumber of revertantpserplate.
C. MutageniciLvAssav
The mutagenicitayssayresultfsorT-6564 arepresenteidnTables3 through7. These dataweregeneratedinExperiments17750-BI,17750-Cl,and 17750-DI.The dataare presentedas mean revertantpserplate::sEtandarddeviationforeach treatmentand control group(Tables4,6,and 7)and asindividuapllatecounts(Tables3,5,and 7).
The resultosfthedoserangefindinsgtudywere usedto selectfivedosestobe testedinthe mutagenicitayssay.The dosestestedwere 5,000,3,330,1,000,333,and 100 gg perplateinboth thepresenceand absenceof S9 mix.
Intheinitimaultagenicitayssay,Experiment17750-BI (Tables3 and4),and intheconf=atory assay,Experiment17750-CI (Tables5 and 6),alldatawere acceptablaend no positiviencreases inthenumber ofrevertantpserplatewere observedwithany of thetesterstrainesitherinthe presenceorabsenceofS9 mix. InExperiment17750-C1,a non-doseresponsive5.0-fold increaswehich didnotmeet thecriterifaora positiveresponsewas observedwithtestesrtrain TA1537 intheabsenceofS9 mix. For thisreasontestesrtraiTnA1537 was retesteidnthe absenceofS9 mix inExperiment17750-DI.
InExperiment17750-DI(Table7),a datawereacceptablaend and no positiviencreaseisnthe number oerevertantpserplatewereobservedwithtestesrtraiTnA1537 inthe absenceof S9 mix.
Allcriterifaora validstudywere met.
CHV StudyNo.: 17750-0-409R
25
CORNINGHazleton
CONCLUSIONS The resultosftheSalmonell-aEscherichicaoli/Mamrnalian-MicrosoRmeeverseMutation Assay witha Confir7natorAyssay indicattehat,undertheconditionosfthisstudy,3M CorporatioWstestarticlTe-,6564,didnotcausea positiviencreaseinthenumber of revertants perplateofany ofthetestesrtrainesitherinthepresenceor absenceofmicrosomalenzymes preparedfromAroclorTm-inducerdatliver(S9).
CHV StudyNo.: 17750-0-409R
26
CORNINGHazleton SECTION V. DATA TABLES
CHV StudyNo.: 17750-0409R
27
CORNINGHazleton
TABLE I DOSE RANGEEINDING TEST ARTICLE ID: T-6564 EXPERIMENT ID: 17750-Al VEHICLE: Deionized vater
STUDY
DATE PLATED: DATE COUNTED:
12-Jun-96 18-Jun-96
jig/PLATE
0.00 (Vehicle) (50 pl)
Test Article 6.67
10.0 33.3 66.7 100 333 667 1000 3330 5000
REVERTANTS PER
PLATE
WITH
TAIOO REVERTANTS S9
BACKGROUND LAWN
EVALUATION*
PER PLATE
WITHOUT S9
REVERTANTS
BACKGROUND
PER
LAWN
PLATE
EVALUATION*
85
1
78
1
106
1
128
1
120
1
122
1
113
1
126
1
121
1
110
1
112
1
118
1
91
1
96
1
78
1
105
1
84
1
113
1
96
1
86
1
81
1
99
1
background
La@ Eval@tion
I :or@l
4
xzre.ely
reduced
up slight precipitate
Codes: 2 - slightly 5 - absent
up - moderate (requires
reduced
precipitate hand count)
3 - moderately
reduced
6 - obscured
by precipitate
hp - hea@y precipitate
(requires
hand count)
CHV Study No.: 17750-0-409R
28
TEST ARTICLE ID: T-6564 EXPERIMENT ID: 17750-Al VEHICLE: Deicnized water
CORNINGHazieton
TABLE 2 DOSE RANGEFINDING
STUDY
DATE PLATED: DATE COUNTED:
12-Jun-96 18-Jun-96
pg/PLATE
0.00 (Vehicle) (50 pl)
Test Article 6.67
10.0 33.3 66.7 100 333 667 1000 3330 5000
REVERTANTS PER
PLATE
WP2uvrA REVERTANTS WITH S9
BACKGROUND LAWN
EVALUATION*
PER PLATE
WITHOUT S9
REVERTANTS
BACKGROUND
PER
LAWN
PLATE
EVALUATION*
10
1
12
1
13
1
12
1
16
1
9
1
15
1
16
1
10
1
9
1
11
1
10
1
14
1
11
1
16
1
22
1
10
1
8
1
20
1
14
1
6
1
5
1
Background
Lawn Z@al"tion
1 - normal 4 - extremely
reduced
op - slight precipitate
r-odea-. 2 - slightly 5 - absent
op - moderate (requires
reduced
precipitate hand count)
3 - moderately
reduced
6 - obscured by precipitate
hp - heavy precipitate
(requires hand count)
CHV Study No.: 17750-0-409R
29
CORNINGHazleton
TABLE 3 MUTAGENICITY ASSAY
INDIVIDUAL PLATE
RESULTS COUNTS
TEST ARTICLE ID: T-6564
EXPERIMENT ID: 17750-Bl DATE PLATED: 20-Jun-96
DATE COUNTED: 28-Jun-96
VEHICLE: Deionized wator PLATING ALIQUOT: 50 pl
DOSE/?LATE
HICROSOMES: Rat Liver VEHICLE CONTROL
TEST ARTICLE
100 ps 333 pg 1000 ps 3330 pg 5000 06
POSITIVE CONTROL
TA9$
1
2
3
25 22 13
21 29 is 16 27 25 27 16 29 25 27 19 23 25 25
860 852 970
REVERTANTS PER PLATE
TA100
1
2
3
116 106 203
95 109 102 101 104 of 206 100 101 114 110 123 111 99 109
936 $92 610
TAIS35
1
2
3
15 17
9
13 13 10 a 11 is
10 12 15 is 10 13 22 12 10
141 234 124
TAIS37
1
2
3
9 10
5
10 11
5
5
6
2
a
5 12
12
a 10
7
7
5
240 202 190
VP2u@rA
1
2
BACKGROUND laim.
3
is 12 12
1
12 10 19
1
20 24
9
1
14 14 14
2
11 16
9
1
13 13
5
1
314 279 270
1
MICROSOMES: Non* VEHICLE CONTROL @MST ARTICLE
POSITIVE CONTROL
12 12 12
91 74 as
14 10
6
5
4
7
is 14 10
1
100 Pt 333 pg 1000 pg 3330 V& 5000 pg
14 10 13
13 11
4
16 11 12
is 10
2
11 11 13
$1 104 33 101 95 86
97 98 92 lag 93 75 106 log 204
11
9
5
10 13
5
11 10
7
12 10
6
16 14 12
6
7
1
6
6
6
6
6
5
4
5
1
9.. 5
4
a
7 13
1
12 12 11
1
a 12
9
1
12
a 11
1
13 13
9
2
105 99 46
470 419 460
460 427 400 1233 913 1110
235 102 85
2
TA98 TAIOD TAIS35 TAIS37 WP2uvrA
2-&ainoonthracene 2-ami@nthr&cane 2-anineanthtscono 2-aminoanthraceno 2-auinoanthracen*
2.3 ps/plate 2.5 pg/plato 2.5 VS/plote 2.5 pg/plate 25.0 pg/plato
TA98 TAIOO TAIS35 TAIS37 W?2uvrA
2-nitrofluarene sodium azide *odium azide ICR-191 4-nitraquincline-N-axide
1.0 pa/plate 2.0 pg/plato 2.0 pg/l)late 2.0 mg/plate 1.0 vg/plate
background Lawn Evaluation I - normal 4 - extr@ely reduced
up - slight precipitate
Codoxi 2 - slightly reduced 5 - absent
up - moderate precipitate (r*quiran hand count)
3 - moderately reduced 6 - obscured by precipitate bp - heavy precipitate
(requires hand count)
CHV StudyNo.: 17750-0-409R
30
CORNINGHazleton
TABLE 4 MUTAGENICITY ASSAY
SUMMARY
RESULTS
TEST ARTICLE ID: T-6564 EXPERIMENT ID: 17750-Bl DATE PLATED: 20-Jun-96 DATE COUNTED: 28-Jun-96
VEHICLE: Deicnized vater PLATING ALIQUOT: 50 pl
DOSE/PLATE-
HICROSOMES: Rat Li@er VEHICLE CONTROL
- TA98
MEAN
S.D.
20
6
TEST ARTICLE
100 pg 333 pg 1000 PS 3330 pg 5000 mg
23
6
23
6
24
7
20
4
24
1
POSITIVE CONTROL
$94 66
HEM RMRTAMS
PER PLATE WM STANDARD DEVIATION
0
HEM
S.D.
MEAN
S.D.
KLAN
S. ID.
109
5
99 12
100
4,
102
3
126
8
108
9
14
4
12
2
11
4
12
3
14
4
12
1
a
3
9
3
4
2
a
4
10
2
6
1
$79 64
133
9
211 26
BACXGROVND
KLAN S.D.
13
2
is
5
1
is
a
I
14
0
1
12
4
1
10
5
1
288 23
1
MICROSOMZS: None VEKI= CONTROL @FEST ARTICLE
POSITIVE CONTROL
100 ps 333 VS 2000 PS 3330 pg 5000 pit
12
0
22
2
9
5
13
3
9
7
12
1
97 10
83
9
89 13
94
8
96
3
92 17
206
2
450 27
10
4
8
3
9
4
9
2
9
3
14
2
429 30
5
2
5
3
6
0
6
1
3
2
6
3
1087 159
13
3
1
9
3
1
12
2
1
10
2
1
10
2
1
12
2
1
107 25
1
TA95 TAIOO TAIS35 TAIS37 W?2uvrA
2-amineanthracens 2-aminoanthr&cene 2-"inoanthracene 1-"inoanthratens 2-"inoanthrae.ne
2.5 pg/plate 2.5 pit/plate 2.5 pg/plate 2.5 VS/plate 25.0 mg/p2ate
TA9$ TAIOO TA1535 TA1537 W?2u@rA
2-nitrafluorens sodium azide sodium aside ICR-191 4-nitroquinolino-N-axide
1.0 vg/plata 2.0 pg/plate 2.0 pg/plate 2.0 mg/plat* 1.0 vg/plat*
Sackground Lo@ tvaluation 1 - normal 4 - extremely reduced
ap - slight precipitate
Codeez 2 - slightly reduced 3 - absent
up - moderate precipitate (requires hand count)
3 - moderately reduced 6 - obscured by procipi-tat. hp - heavy precipitate
(requires hand count)
CHV StudyNo.: 17750-0-409R
31
CORNINGHazleton
TABLE 5 MUTAGENICITY ASSAY
INDIVIDUAL PLATE
RESULTS COUNTS
TEST ARTICLE ID: T-6564
EXPERIMENT ID: 17750-Cl DATE PLATED: 25-Jul-96
DATE COUNTED: 30-Jul-96. 31-Jul-96
VEHICLE: Deionized vater PLATING ALIQUOT: 50 pl
DOSE/?LATE
MICROSOMES: Rat Liver VEHICLE CONTROL
TEST ARTICLE
100 ps 333 Mg 1000 ps 3330 VS 5000 ma
POSITIVE CONTROL
TA
1
2
3
25
25
18
21
21
28
27
33
25
33
26
29
24
30
27
25
33
25
769 624 815
RZVUTAMS rU PLATE
1
2
3
94
76 103
89 99
95
102 105
76
$1 115 109
106
96 124
107
87
ag
949 1208 1066
1
2
3
17
14 17
a
11
9
16 16
9
22
10
8
9
18
12
a
6 17
146 196 136
1
2
3
4
7
6
12
4 11
8
6 19
7
7
10
0
4
4
a
7
6
89 129
91
"CKGROUND lAw.
1
2
3
12
14
18
1
14
16
10
1
10
is
9
1
24
13
14
1
24
16
13
1
21
14
20
1
131
90 log
I
MICROSOMES:
None
VEHICLE CONTROL
TEST ARTICLE
POSITIVE CONTROL
23
19
15
79
ag
70
16
9
19
1
4
2
13
16
23
1
100 PS 333 pg 1000 ps 3330 pg 3000 pg
16
17
17
21
a
16
20
9
a
21
is 11
10
17
17
71 105
76
74
99
73
74
71
Is
78
75
78
99
80
77
16
17
17
14
9
6
9
9
8
25
14
10
16
13
16
6
5
4
6
6
a
1
6
7
16
10
3
8-- 7
2
15
10
10
1
is Is
29
1
6
13
19
1
23
9
6
1
9
17
10
1
207 208 172
548 397 395
644 619 663
462 477 463
187 207 160
1
TA9@ TAIOO TAIS35 TAIS37 W?2uvrA
2-sainoan threcons 2-@ineanthr' acess 2-mdneanthraccus 2-asinoanthracene 2-amineanthracene
2.5 pg/plate 2.3 pg/plato
2.5 2.5 25.0
VS/plate ps/plate pg/plate
... TA98 TAIOO TAIS35 TA1537 WP2uvrA
2-nltrcfluorene
sodium sodium ICK-191
azide &tide
4-nitroquinoline-M-exide
1.0 ma/plate 2.0 pg/plate 2.0 VS/plato 2.0 MS/plate 1.0 pa/ilato
Background Lava Evaluation
normal
extremely reduced
op
slight precipitate
Codes: 2 - slightly
reduced
5 - absent op - moderate
precipitate
(requires lumd count)
3 - moderately reduced 6 - obscured by precipitate hp - heavy precipitate
(requires hand count)
CHV StudyNo.: 17750-0-409R
32
CORNINGHazleton
TABLE 6 MUTAGENICITY ASSAY
SUMMARY
RESULTS
TEST ARTICLE ID: T-6564 EXPERIMENT ID: 17750-Cl DATE PLATED: 25-Jul-96 DATE COUNTED: 30-Jul-96. 31-Jul-96
VEHICLE: Daianized water PLATING ALIQUOT: 50 pl
MEAN MQMTANTS
PER PLATE Wn2 STANDARD DEVIATION
DOSE/FLA
MICROSOMLS. JL&t Liver VEHICLE CONTROL
MEAN
S.D.
23
4
TEST ARTICLE
100 PS
23
4
333 jig
29
4
1000 PS
29
4
3330 pg
27
3
5000 PS
28
5
POSITIVE CONTROL
736 100
MEAN S.D.
91 14
94
5
94 26
103 15
203
9
94 11
1041 82
PMAN
S.D.
16
2
9
2
14
4
10
2
13
5
10
6
160 32
XLAN S.D.
6
2
9
4
11
7
a
2
5
2
a
I
103 23
RACKGROVND LAWN'
MEAN S.D.
is
3
1
13
3
1
11
3
1
27
6
1
is
6
1
is
4
1
116 31
1
KICROSOMES: None VEHICLE CONTROL
is 3
79 10
is
5
2
2
17
3
1
TEST ARTICLE
100 ps 333 VS 1000 ps 3330 pg 5000 ps
17
1
15
7
12
7
16
5
15
4
64 is
79
9
78
9
77
2
as 12
17
1
20
3
9
1
13
3
15
2
5
1
7
1
5
3
10
7
5
4
12
3
1
16
2
1
13
7
1
9
4
2
12
4
1
POSITIVE CONTROL
196 21
580 24
642 22
467
8
IBS 24
1
TA9$ TAIOO TA1535 TAI$37 WP2uvrA
2-aminoanthraceno 2-saineanthracene 2-@ineanthracens 2-mineanthracens 2-minoanthracene
2.5 Mg/plate 2.5 pg/plate 2.5 p$/plate 2.5 Mg/plate 25.0 pg/plate
... TAIS TAICO TAIS35 TAIS37 WP2uvrA
2-nitrofluerene sodium amid* sodium aside ICR-191 4-nitroquinaline-N-exide
2.0 pa/plnte 2.0 mg/plste 2.0 pg/plate 2.0 mg/p2sto 1.0 pglpi&te
background Lavn Evaluation I - normal 4 - extra@ly reduced
op - slight precipitate
Codes: 2 - slightly reduced 5 - absent
up moderate precipitate (requires hand count)
3 - moderately reduced 6 - obscured by precipitate hp - heavy precipitate
(requires hand count)
CHV StudyNo.: 17750-0-409R
33
CORNINGHazleton
TABLE 7 MUTAGENICITY ASSAY INDIVIDUAL PLATE COUNTS
RESULTS AND SUMKARY
TEST ARTICLE ID: T-6564
EXPERIMPM ID: 17750-Dl DATE PLATED: 25-Aug-96
DATE COUNTED- 27-Aug-96
VEHICLE: Deionized water PLATING ALIQUOT: 50 pl
MICROSOMES: No" VEHICLE CONTROL TEST ARTICLE
POSITIVE CONTROL
100 333 1000 ps 3330 VS 5000 PS
REVERTANTS PER PLATE
2
2
3
10
2
9
9
6
5
9
5
4
5
6
a
6
a
5
3
1
9
128 63 60
MZAN REVERTANTS PER PLATE WXTB STANDARD DEVIATION
NEAR S.D.
7
4
7
2
6
3
6
2
6
2
4
4
$4 38
BACKGROUND IAW'
1
1 1 1 1 1
1
TA1537 ICR-291
2.0 pg/plate
background Lawn Evaluation I - normal 4 - extremely reduced ap - slight precipitate
Codes: 2 - slightly reduced 5 - absent up - moderate precipitate (requires hand count)
3 - so4orately reduced 6 - al@ecured by precipitate hp - heavy precipitate
(requires hand count)
CHV StudyNo.: 17750-0409R
34