Document gap9JgbekNZ72v5jDoVaOVM4Q
CORNINGHazleton
MUTAGENICITY TEST WITH T-6357
IN THE SALMONELLA - ESCHERICHL4 COLIMAMMALIAN-NECROSOME MUTATION ASSAY
FINAL REPORT
AUTHOR Timothy E.Lawlor,M.A.
PERFORMING LABORATORY CorningHazletonInc.(CHV) 9200 Leesburg Pike Vienna,Virginia22182
LABORATORY PROJECT ID CHV StudyNo.: 17387-0409
SUBMITTED TO 3M
Building220-2E-023M Center St.Paul,MN 55144-1000
STUDY COMPLETION DATE April1,1996
CHV StudyNo.: 17387-0-409
REVERSE 1 of 30
CORNINGHazleton QUALITY ASSLIUNCE STATEMIENT
STUDY TITLE:
Salmonel-lEascherichcioalMammalian-NficrosRoemveersMeutation Assay
ASSAY NO.:
17387-0-409
PROTOCOL NO.: 409,Edition4
QualityAssuranceinspectionosfthestudyand reviewof thefinalreportof theabove referenced projectwere conductedaccordingtotheStandardOperatingProceduresoftheQualityAssurance Unitand accordingtothegeneralrequirementsof theappropriateGood LaboratoryPractice regulationsF.indingsfrom theinspectionasnd finalreportreview were reportedto management and tothestudydirectoorn thefollowingdates:
In=ection/Date
FindinizRs=orted
Audi
Characterizatiofn TesterStrains- 02/14/96
02/14/96
C. Orantes
DraftReportReview -03/16/96 03/18/96
S.Ballenger
FinalReportReview -04/01/96 04/01/96
S. Ballenger
O@@ity AssuranceUnit(j CHV StudyNo.: 17387-0-409
Date'l@eleased2
C)ORNING Hazleton
STUDY COMPLIANCE AND CERTIFICATION
The studywas conducted incompliance with the Good LaboratoryPracticeregulationsas set forthby theFood and Drug Administration(FDA) inTitle21 oftheU.S. Code of Federal RegulationsPart58,issuedDecember 22, 1978, (effectivJeune 20, 1979) with any applicable amendments. There were no deviationsfrom the aforementionedregulationsorthe signed protocolthatwould affecthe integritoyfthe studyorthe interpretatioofnthe testresults.The raw datahave been reviewed by the Study Director,who certifietshatthe evaluationof the test articlaes presentedhereinrepresentsan appropriateconclusionwithin thecontextofthe study designand evaluationcriteria.
Study Director:
imothy E. 4awlor,M.A. BacterialMikagenesis Geneticand CellularToxicology
q Study Completion Date
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TABLE OF CONTENTS
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Page No. I. SUMMARY ..........................................5......... ii. STUDY INFORMATION ...................................7......... III. MATERIALS AND NIETHODS ...............................9......... IV. RESULTS AND CONCLUSIONS .............................2.3 .........
V. DATA TABLES ..........................................2.6..........
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SECTION I. SUMMARY INTRODUCTION AND CONCLUSIONS
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SUNUMLARY
A. Introduction
Attherequeosft3M,ComingHazletIonnci.nvestigTa-t6e3d5f7ormutagenic activitntyheSalmonella-Eschceorliic/hMiammalian-MicRreovseormseMeutatioAnssay. Thisassayevaluatedthetestarticlaend/oritsmetabolitefsortheirabilittyoinducereverse mutationsatthehistidinleocusinthegenome ofspecifiScalmonellatyphimuriumtestesrtrains and atthetryptophanlocusinan Escherichicaolitestesrtraibnothinthepresenceand absence ofan exogenousmetabolicactivatiosnystemofmammalian microsomalenzymes derivedfrom AroclorTm-inducerdatlive(rS9).
The dosestestedinthemutagenicityassaywere selectedbasedon theresultosfa dose rangefindinsgtudyusingtestesrtrainTsA 100 and WP2uvrA and tendosesoftestarticlreanging from 5,000to6.67gg perplate,one plateperdose,bothinthepresenceand absenceofS9 mix.
The testesrtrainussed inthemutagenicityassaywere Salmonellatyphimuriumtestesrtrains TA98, TAIOO, TA1535, TA1537, and EscherichiacolitestesrtrainWP2uvrA. Theassaywas conductedwithfivedosesoftestarticlienboththepresenceand absenceofS9 mix alongwith concurrenvtehicleand positivceontrolussingthreeplatesperdose. The dosestestedwere 5,000,3,330,1,000,333,and 100 gg perplateinboththepresenceand absenceofS9 mix.
B. Conclusions
The resultosf theSalmonella-Escherichiacoli/Mammalian-MicrosomeReverse MutationAssay indicatethatu,ndertheconditionosfthisstudy,3M's testarticlTe-,6357,did notcausea positiveincreaseinthenumber ofrevertantpserplateofany ofthetestesrtrains eitherinthepresenceor absenceofmicrosomalenzymes preparedfrom Arocloirm-inducerdat live(rS9).
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SECTION Il.STUDY INFORMATION
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STUDY INFORMATION
A. Sponsor: 3M
B. TestArticle:T-6357
1. Physical Description: clear amber liquid
2. Date Received: 01/16/96
C. Type of Assay: Salmonella -Escherichiacoli/Mammalian-Microsome Reverse Mutation Assay
I
ProtocolNumber: CHV Protocol409,Edition4
2. CHV Study Number: 17387-0409
D. Study Dates
1. Study InitiatioDnate:
01/30/96
2. ExperimentalStartDate:
02/04/96
3. ExperimentalTerminationDate: 02/21/96
E. Study SupervisoryPersonnel
Study Director:
Timothy E. Lawlor,M.A.
LaboratorySupervisor: Michael S.Mecchi, B.S.
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SECTION III.MATERLALS AND MIETHODS
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MATERIALS AND METHODS
Theexperimenmtaatleriamlest,hodasndproceduraersebaseodnthosdeescribbeyd Amesetal(1975a)ndGreenandMurie(l1976).
MATERIALS
A. TesterStrains
I. Salmonella typhimurium
The testerstrainsused were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535, and TA1537 as described by Ames etal (1975). "Me specificgenotypes of these strainsare shown in Table 1.
TABLEI.TES
HistidinMeutation -
hisG46 hisC3076 hisD3052
TA1535 TA1537
TA100
TA98
14GENOTYPES
AdditionaMlutations
LPS Repair R Factor
rfa uvrb
rfa uvrb
+R
Inadditiontoa mutationinthehistidinoeperon,thetestesrtraincsontaintwo additional mutationswhich enhancetheirsensitivittoysome mutageniccompounds. The rfawallmutation resultisnthelossofone oftheenzymes responsiblfeorthesynthesiosfpartofthe lipopolysaccharibdaerrietrhatformsthesurfaceofthebacteriaclellwall.The resultincgellwall deficienciyncreasepsermeabilittyocertainclasseosf chemicalssuchas thosecontaininglarge ringsystems(i.eb.enzo(a)pyrenet)hatwould otherwisebe excludedby a normalintacctellwall.
The secondmutationa, deletioonftheuvrb gene,resultisna deficienDtNA excisiornepair systemwhich greatleynhancesthesensitiviotfythesestraintsosome mutagens.Sincetheuvrb deletioenxtendsthroughthebiogene,allof thetestesrtraincsontainintghisdeletioanlsorequire thevitaminbiotinforgrowth.
StainsTA98 and TAI 00 alsocontaintheR-factoprlasmid,pKM101, which furtheirncreases thesensitiviotfythesestraintsosome mutagens.The mechanism by which thisplasmid increasessensitivittoymutagenshasbeensuggestedtobe by modifyingan existinbgacterial DNA repaiprolymerasecomplex involvedwiththemismatch-repaiprrocess.
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TestesrtraiTnAs98andTA1537arerevertferdomhistiddienpeenden(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 mutationinthetryptophanoperon,thetestesrtraicnontainas uvra DNA repair deficiencwyhich enhancesitssensitivittoysome mutageniccompounds. Thisdeficiencayllows thestraitnoshow enhancedmutabilitsyincetheuvra repairsystemwould normallyactto remove thedamaged partoftheDNA moleculeand accuratelryepairitafterwards.
TesterstraiVnvT2uvrA isrevertefdrom tryptophadnependence(auxotrophyt)otryptophan independence(prototrophbyy) basesubstitutimountagens.
3. SourceofTesterStrains
a. Salmonella typhimurium
The testerstrainsin use at CHV were received directlyfrom Dr. BruceAmes, DepartmentofBiochemistryU,niversitoyfCaliforniBae,rkeley.
b. Escherichiacoli
The testerstrainW,P2uvrA, inuse atCHV was receivedfrom the NationalCollectioonf IndustriBaalcteriaT,orreyResearchStationS,cotland(TJniteKdingdom).
4. Storageof theTesterStrains
a. FrozenPermanent Stocks
Frozenpermanentstockswere preparedby growingfresh overnighctulturesa,ddingDMSO (0-09ml/mlofculturea)nd freezinsgmallaliquots (0.5-1.m5l)at 70*C.
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b. MasterPlates
Masterplateswere preparedby streakinegachtesterstraifnrom a frozenpermanentstockontominimal agarappropriatelsyupplementedwith 1)forSalmonella typhimurium,an excessofhistidinea,nd biotina,nd fortestesrtrainTsA 98 and TA 100, ampicilli(n25gg/ml),toensurethestablmeaintenanceofthepKM101 plasmid;and 2)for Escherichicaolia,n excessoftryptophanT.esterstraimnasterplateswere storedat 5 3*C.
5. Preparatioonf OvemiszhtCultures
a. Inoculation
Overnightculturefsoruseinalltestinpgrocedureswere inoculated by transferriangcolonyfrom theappropriatmeasterplatetoa flaskcontainincgulturemedium. Inoculatedflaskswere placedina shaker/incubatwohrich was programmed tobeginoperation (shaking1,25 :L25 rpm;incubation3,7 2'C) sothattheovernighctulturewsere inlogphaseor latelogphasewhen turbiditmyonitoringbegan.
b. Harvest
To ensurethatculturewsere harvestedinlatelogphase,thelength ofincubatiownas determinedby spectrophotometrmiocnitoringofculturteurbidityC.ultures were harvestedonce a predeterminedturbiditwyas reachedasdeterminedby a percent transmittanc(e%T) readingon a spectrophotometeTrh.istargeturbiditeynsuresthatcultures havereacheda densityofatleast0.5X 10'cellsperml andthattheculturehsave notovergrown. Overgrown (stationarcyu)lturemsay exhibidtecreasedsensitivittoysome mutagens.Cultures were removed from incubatiownhen thetarge%tT was reachedandwere placedat5 3"C.
6. ConfirmationofTesterStrainGenoWes
Testerstrainculturewsere checkedforthefollowingeneticmarkerson theday oftheiruseinthemutagenicitayssay:
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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cultuorfeeachstrawiansoverlaoindtoplatceosntainsienlgectmievdeiandanantibiotic sensitividtiyskcontainin1g0 gg ofcrystavliolewtas added.Sensitiviwtays demonstratedby inhibitionfbacteriaglrowthina zoneimmediatelysurroundintghedisk.
2) pKM 101 Plasn3idR-factor
The presenceofthepKM101 plasmidwas confirmedfor testesrtrainTsA98 and TAIOO by demonstratioonfresistancteoampicillinA.n aliquootfan overnightcultureofeachstraiwnas overlaiodntoplatescontaininsgelectivmeedia and an antibiotsiecnsitividtiyskcontainin1g0 gg ofampicilliwnas added.Resistancweas demonstratedby bacteriaglrowth inthezoneimmediatelysurroundingthedisk.
3) CharacteristNiucmber ofSpontaneousRevertants
The mean number ofspontaneousrevertantpserplateinthe vehiclecontroltshatarecharacteristoifctherespectivsetrainwsere demonstratedby plating 100 glaliquotosftheculturealongwiththeappropriatveehicleon selectivmeedia.
b. Escherichiacoli
1) CharacteristNiucmber ofSpontaneousRevertants
The mean number ofspontaneousrevertantpserplateinthe vehiclecontroltshatarecharacteristoifctherespectivsetrainwseredemonstratedby plating 100 plaliquotosftheWP2uvrA culturaelongwiththeappropriatveehicleon selectivmeedia.
7. TesterStrainMedi
a. CulturinBgroth
The brothusedtogrow overnightcultureosfthetesterstrainwsas Vogel-Bonnersaltsolutio(nVogeland Bomer, 1956)supplementedwith2.5% (w/v)Oxoid NutrienBtrothNo. 2 (drypowder).
b. Agar Plates
Bottom agar(25mi per15 x 100mm petridish)was VogelBonner minimalmedium E (Vogeland Bonner,1956),supplementedwith1.5% (w/v)agarand 0.2% (w/v)glucose.
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C. OverlayAgar forSelectioonfRevertants
Overlay(top)agarwas preparedwith0.7% agar(w/v)and 0.5% NaCl (w/v)and was supplementedwith10ml of 1)0.5mM histidine/biostoilnutiopner 100 rnlagarforselectioonfhistidinreevertantosr,2)0.5mM tryptophansolutiopner100ml of agarforselectioonft-yptopharnevertantsW.hen S9 mix was required2,.0ml ofthe supplementedtopagarwas usedintheoverlay.However, when S9 mix was notrequiredw,ater was addedtothesupplementedtopagar(0.5ml ofwaterper2 ml ofsupplementedtopagar)and theresultin2g.5ml ofdilutedsupplementedtopagarwas usedfortheoverlay.Thisdilution ensuredthatthefinaltopagarand amino acidsupplementconcentrationrsemainedthesame both inthepresenceand absenceof S9 mix.
B. LiverMicrosomal Ename Reactionlmxture (S9 Mix)
I. S9 Homoge ate
Livermicrosomalenzymes (S9homogenate)were purchasedfrom MolecularToxicology,Inc.A,nnapolis,NO 21401,Batch0623 (42.4mg ofproteinperml). The homogenate was preparedfrommale Sprague-Dawleyratsthathad beeninjecte(di.p.w)ith Aroclofrm1254 (200mg perml incom oil)at500 mg/kg asdescribedby Ames etal,1975.
2. S9 Mix
The S9 mix was preparedimmediatelypriortoitsuseinany experimental procedure.The S9 mix containetdhecomponentsindicateidnTableII.
TABLE II.S9 MIX COMPONENTS
H,O
IM NaH2PO4/Na2HPO4,
pH7.4
0.25M Glucose-6-phosphate
0.1OM NADP
0.825M KCI/0.2M MgCI2
S9 Homogenate
0.70ml 0.10mi 0.02ml
0.04ml 0.04ml
0.10 ml 1.00mi
C. Controls I. VehicleControls
Vehiclecontrolwsere platedforalltestesrtrainbsothinthepresenceand absenceofS9 mix. The vehiclecontrolwas platedu,singa 50 glaliquootfvehicle(equaltothe--,
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maximumaliquooft esatrticdlielutipolnateadl)o,nwgitha 100glaliquooft heappropriate testesrtraiannd a 500 tialliquootfS9 mix (when necessary)o,n selectivaegar.
2. PositivCeontrols
The combinationosfpositivceontrolsa,ctivatiocnonditioannd tester strainpslatedconcurrentlwyiththeassayareindicateidnTableIII.
IAE-Tester
5=1 S9 Mix
TA98
+
TA98
TA100
+
TAIOO
TA1535 + TA1535 -
TA1537 + TA1537 -
WP2uvrA +
WP2uvrA -
ITIVE CONTROLS
Conc
PositivCeontrol
12erplate
2-aminoanthracene
2.5jig
2-nitrofluorene
1.0gg
2-aminoanthracene
2.5 [ig
sodium azide
2.0gg
2-aminoanthracene
2.5jig
sodium azide
2.0gg
2-aminoanthracene
2.5jig
ICR-191
2.0gg
2-aminoanthracene
25.0gg
4-nitroquinoline-N-oxide1.0gg
a. SourceandGrade ofPositivCeontrolArticles
2-aminoanthracen(eCAS #613-13-8)S,igma ChemicalCo.,purity 97.5%;2-nitrofluore(nCeAS #607-57-8)A,ldrichChemicalCo.,purity98%; sodium azide (CAS #26628-22-8)S,igma ChemicalCo.,purity>98%; ICR-191(CAS #170745-0), PolyscienceIsnc.p,urity>95%; 4-nitroquinoline-N-ox(iCdAeS #56-57-5)S,igma ChemicalCo., purity>99%.
3. SteriliCtoyntrols
a. TestArticle
The most concentratetdestarticldeilutiownas checkedforsterility by platinga 50 glaliquot(thesame volume usedintheassay)on selectivaegar.
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b. S9 Mix
The S9 mix was checkedforsterilibtyyplatin0g.5ml on selective
METHODS
A. Dose RangefindingStudy
The growth inhibitoryeffect(cytotoxicityo)f the testarticleto the testsystem was determinedinorderto allow theselectionof appropriatedoses tobe testedinthemutagenicity assay.
I . Design
The dose rangefmding studywas performed using testerstrainsTA 100 and WP2uvrA both inthe presenceand absence of S9 mix. Ten doses of testarticlewere tested atone plateper dose. The testarticlweas checked forcytotoxicituyp to a maximum concentrationof 5 mg per plate.
a. Rationale
The cytotoxicitoyf thetestarticloebservedon testerstrainTA 100 isgenerallyrepresentativoef thatobserved on theothertesterstrainsand because of TA 100's comparativelyhigh number of spontaneousrevertantpser plate,gradationsofcytotoxicitcyan be readilydiscernedfrom routineexperimentalvariation.The Escherichiacolitesterstrain WP2uvrA does not possesstherfawallmutationthattheSalmonellatyphimurium strainshave and thus,a differenrtange ofcytotoxicitmyay be observed.Also,thecytotoxicitiynduced by a testarticlienthe presenceof S9 mix may vary greatlyfrom thatobservedinthe absence of S9 mix. Therefore,thiswould requirethatdifferenttestarticldeose rangesbe testedinthe mutagenicityassay based on thepresenceor absence of the S9 mix.
2. EvaluationoftheDose RangefindingSU&
Cytotoxicityisdetectableas a decreaseinthe number of revertantcolonies per plateand/orby a fl@i@ng or disappearanceofthe bacteriablackground lawn.
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3. SelectioonftheMaximum Dose fortheMutaizeniciAtsysU
a. No CytotoxicitOybserved
Sinceno cytotoxiciwtays observedinthedoserangefmdingstudy, thehighestdose oftestarticluesedinthemutagenicityassaywas thesame asthattestedinthe rangefindinsgtudy.
B. MutageniciiyAssay
1. Design
The assaywas performedusingtestesrft-aiTmA98, TAIOO, TA1535, TA1537 and WP2uvrA bothinthepresenceand absenceofS9 mix. Fivedosesoftestarticle were testedalongwiththeappropriatveehicleand positivceontrolsT.he dosesoftestarticle were selectebdased on theresultosf thedose rangefindinsgtudy.
2. Frequencyand RouteofAdministration
The testesru-aimwere exposedtothetestarticlveiatheplate incorporatiomnethodology originalldyescribedby Ames etal(1975)and Maron and Ames (1983).Thismethodologyhasbeen shown todetecta wide rangeofclasseosfchemical mutagens.Intheplateincorporatiomnethodology,thetestarticlteh,etestesrtraiannd theS9 mix (whereappropriatew)ere combined inmoltenagarwhich was overlaiodntoa minimalagar plate.Followingincubatioant37 2*C for48 8 hr,revertanctoloniewserecounted.All dosesofthetestarticlet,hevehiclecontrolasnd thepositivceontrolwsere platedintriplicate.
C. PlatingProcedures
These procedureswereusedinboththedoserangefindinsgtudyandthe mutagenicitayssay.
Each platewas labeledwitha codewhich identifietdhetestarticltee,stphase,testesrtrain, activatiocnonditioannd dose.The S9 mix and dilutionosfthetestarticlweereprepared immediatelypriortotheiruse.
When S9 mix was notrequired1,00 gloftestesrtraiannd 50 glofvehicleortestarticldeose was added to2.5ml of moltenselectivteopagar(maintainedat45 2*C). When S9 mix was required5,00 glofS9 mix, 100 gloftestesrtraiannd 50 glofvehicleortestarticldeosewas added to2.0ml ofmolten selectivteopagar.Aftertherequiredcomponentshad beenadded,the
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mixturweasvortexaenddoverlaoindtothesurfaocfe25mlofminimablottoamgacrontainiend a 15x 100 mm petridish.Aftertheoverlayhad solidifietdh,eplatewsere inverteadnd incubatefdor48 8 hrat37 :b2*C. Positivceontroalrticlewsere platedusinga 50 glplating aliquot.
D. ScoringthePlates
Plateswhich were notevaluatedimmediatelyfollowingtheincubatiopneriod wereheldat5 3*C untilsuchtimethatcolonycountingand bacteriablackgroundlawn evaluatiocnouldtakeplace.
I. BacteriaBlackgroundLawn Evaluation
The conditionofthebacteriablackgroundlawn was evaluatedfor evidenceofcytotoxiciatnydtestarticlperecipitatEev.idenceofcytotoxiciwtays scoredrelative tothevehiclecontrolplateandwas recordedalongwiththerevertanctountsforallplatesatthat doseon thedatatablesusingthecode systempresentedattheend oftheMaterialsand Methods Section.
2. CountingRevertantColonies
The number ofrevertanctoloniesperplateforthevehiclecontrolasnd all platescontainintgestarticlweere countedmanually.The number ofrevertanctoloniesperplate forthepositivceontrolwsere countedby automatedcolonycounter.
E. AnalysisofData
For allreplicatpelatingst,hemean revertantpserplateand thestandarddeviation were calculatedT.he resultosfthesecalculatioanrsepresentedintabularformintheData TablesSectionof thisreport.
EVALUATION OF TEST RESULTS
Beforeassaydatawere evaluatedt,hecriterifaora validassayhad tobe met.
A. CriteriaFor A Valid AlLay-
The followingcriteriwaere usedtodeterminea validassay:
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1. TesteSrtraIintegriStayl:moneltlyaphimurium a. rfaWallMutation
To demonstrtahteperesenocfetherfawalmlutatiotne,stsetrrain cultureesxhibitesdensitivittoycrystavliolet.
b. pKM 101 Plasmid
To demonstratethepresenceoftheR-factorplasmid,pKM 101, cultureosftestesrtrainTsA98 and TAIOO exhibiterdesistancteoampicillin.
C. CharacteristNiucmber ofSpontaneousRevertants
To demonstratetherequirementforhistidinteh,etestesrtrain
culturesexhibiteda characteristniucmber ofspontaneousrevertantpserplatewhen platedalong
withthevehicleunderselectivceonditionsT.he acceptablreangesforthevehiclecontrolswere
as follows:
TA98
8 - 60
TA100
60 - 240
TA1535
4 - 45
TA1537
2 - 25
2. TesterStrainInte2Tity: Escherichiacoli
a. CharacteristNiucmber of SpontaneousRevertants
To demonstratetherequirementfortryptophant,hetestesrtrain culturexhibitead characterisntuimcber ofspontaneousrevertantpserplatewhen platedalong withthevehicleunderselectivceonditionsT.he acceptablreangefortheWP2uvrA vehicle controlswas 5 to40 revertantpserplate.
3. TesterStrainCultureDensity
To demonstratethatappropriatneumbers ofbacteriareplatedt,hedensity oftestesrtraicnulturewsere greaterthanorequalto0.5x 10'bacteripaerml and/orhad reached a targetlevelofturbiditdyemonstratedtoproduceculturewsitha densitygreaterthanorequalto 0.5x IC@bacteriapermi.
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4. PositivCeontrolValues
a. PositivCeontrolValuesintheAbsence ofS9 Mix
To demonstratethatthetestesrtrainwsere capableofidentifyinag mutagen,themean valueofa positivceontrolfora respectivteestesrtraienxhibiteadtleasta 3-foldincreaseoverthemean valueofthevehiclecontrolforthatstrain.
b. PositivCeontrolValuesinthePresenceofS9 Mix (S9Mix Integrity)
To demonstratethattheS9 mix was capableofmetabolizinag promutagentoitsmutagenicform(s)t,hemean valueofthepositivceontroflora respective testesrtraininthepresenceoftheS9 mix exhibiteadtleasta 3-foldincreasoeverthemean value ofthevehiclecontrolforthatstrain.
An acceptablpeositivceontrolinthepresenceofS9 fora specifisctraiwnas evaluatedashaving demonstratedboththeintegritoyftheS9 mix andtheabilitoyfthetestesrtr@ todetecta mutagen.
5. czotoxicity
A minimum ofthreenon-toxicdoseswere requiredtoevaluateassaydata.
B. CriteriaFor A PositiveResponse
Once thecriterifaora validassayhad beenmet,respongesobservedintheassay were evaluatedasfollows:
I TesterStrainsTA98 TA 100. and WP2uvrA
For a testarticlteobe consideredpositivei,thad toproduceatleasta 2-foldincreasein themean revertantpserplateofatleastone ofthesetestesrtrainosverthe mean revertantpserplateoftheappropriatveehiclecontrol.Thisincreasienthemean number of revertantpserplatehad tobe accompaniedby a doseresponsetoincreasincgoncentrationosfthe testarticle.
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2. TesterStrainsTA 1535 and TA 1537
Fora testarticlteobe consideredpositivei,thad toproduceatleasta 3-foldincreaseinthemean revertantpserplateofatleastone ofthesetestesrtrainosverthe mean revertantpserplateoftheappropriatveehiclecontrol.Thisincreasienthemean number of revertantpser platehad to be accompaniedby a dose responsetoincreasincgoncentrationosfthe testarticle.
RECORDS TO BE MAINTAINED
Allraw data,documentationr,ecords,theprotocola,nd thefinalreportgeneratedasa resulotf thisstudywillbe archivedinthestoragefacilitioefsComing HazletonInc.foratleasotne year followinsgubmissionofthefinalreportotheSponsor.Aftertheone yearperiodt,heSponsor may electohavetheaforementionedmaterialrsetainedinthestoragefacilitioefsComing HazletonInc.foran additionapleriodoftimeorsenttoa storagefacilitdyesignatebdy the Sponsor.
REFERENCES
Ames, B.N.,J.McCann, and E. Yamasaki. Methods fordetectingcarcinogensand mutagens withtheSalmonella/Mammalian-MicrosomeMutagenicitTyest.MutationResearch 21:347-364(1975).
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 testinugsingtrp'reversioinnEscherichicaoli. MutationResearch31:3-32 (1976).
Maron,D.M., andB. Ames. RevisedMethods fortheSalmonellaMutagenicitTyest. MutationResearch112:173-215(1983).
Vogel,H.J.,and D.M. Bonner. AcetylomithinasoefE.coli:Partiaplurificatiaonnd some propertiesJ.Biol.Chem. 2U:97-106 (1956).
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BACTERIAL BACKGROUND LAWN EVALUATION CODE
Theconditiooftnhebackgroubnadcterliaawlnisevaluatbeodthmacroscopicaanldly microscopical(luysinga dissectinmgicroscope)forindicatioonfscytotoxiciatnydtestarticle precipitaatsefollows:
CODE DEFINITION CHARACTERISTICS OF BACKGROUND LAWN
I Normal
A healthymicrocolonylawn.
2 Slightly Reduced
A noticeabldeiin@ngofthemicrocolonylawnand an increaseinthesizeofthemicrocoloniecsompared tothevehicle controlplate.
3 Moderately Reduced
A marked ffiinninogfthemicrocolonylawn and an increaseinthesizeofthemicrocoloniecsompared tothevehicle controlplate.
4 Extremely Reduced
An extremethinningofthemicrocolonylawn andan increaseinthesizeof themicrocoloniecsompared to thevehicle controlplate.
5 Absent
A completelackofany microcolonylawn.
6 Obscuredby Precipitate
The backgroundbacteriallawn cannotbe accurately evaluatedue to microscopicand/ormacroscopictestarticle precipitate.
Evidenceofmacroscopictestarticlperecipitaotne theplatesisrecordedby additionofthe followingprecipitactoedetothecodenumber usedtoevaluatetheconditionofthebackground bacteriallawn.
sp Slight Precipitate
Noticeablemacroscopicprecipitaotne theplate, however,theprecipitadtoeesnot influencaeutomatedcountingofthe plate.
mp Moderate Precipitate
The amount ofmacroscopicprecipitaotne theplate would interferweithautomatedcountingt,husrequirintgheplateto be hand counted.
hp Heavy Precipitate
The largeamount ofmacroscopicprecipitatoen the platemakes therequirehdand countingdifficult.
Example:4mp would indicatae plateobservedtohave an extremelyreducedbackgroundlawn which hadtobe countedmanuallydue tothemarked amount ofmacroscopictestarticle precipitate.
CHV StudyNo.: 17387-0-409
22
CORNINGHazleton
SECTION IV. RESULTS AND CONCLUSIONS
CHV Study No.: 17387-0-409
- 23
CORNINGHazleton
RESULTS
A. TestArticlHeandlinLy
The testarticlTe-,6357,was storeadtroomtemperatureD.eionizewdater(CHV lots336 and 337) was used as thevehicle.At 100 mg per ml, which was the most concentrated stockdilutionprepared,thetestarticlfeormed a clearcolorlessolution.The testarticle remained a solutionin allsucceedingdilutionpsreparedforthe mutagenicityassay.
B. Dose Rangefinding Study
Doses tobe testedinthe mutagenicityassaywere selectedbased on theresultsof thedose rangefindingstudy conducted on the testarticluesingtesterstrainsTA 100 and WP2uvrA inboth the presenceand absence of S9 mix (oneplateper dose). Ten doses oftest articlef,rom 5,000 to 6.67 Rg per plate,were testedand theresultsarepresentedinTables 1 and 2. These datawere generatedinExperiment 17387-Al. No cytotoxicitwyas observedineither thepresence or absence of S9 mix as evidencedby a normal background lawn and no decreasein thenumber of revertantpser plate.
C. Mutagenicity Assay
The mutagenicityassay resultsforT-6357 arepresentedinTables 3 and 4. These datawere generatedin Experiment 17387-Bl. The dataarepresentedas mean revertantpser plate standarddeviationforeach treatmentand controlgroup (Table4 ) and as individuapllate counts(Table3).
The resultsof the dose rangefindingstudywere used to selectfivedoses tobe testedinthe mutagi!nicitayssay.The doses testedwere 5,000,3,330,1,000,333,and 100 pg per plateinboth thepresence and absence of S9 mix.
In Experiment 17387-B 1 (Tables3 and 4),alldatawere acceptableand no positiveincreasesin thenumber ofrevertantsper platewere observed with any ofthe testerstrainseitherinthe presenceor absence of S9 mix.
Allcriterifaora validstudywere met.
CHV Study No.: 17'-8'7l-0-409
24
CORNINGHazleton
CONCLUSIONS
TheresulotftsheSalmonel-lEaschericchoilai/Mammalian-MicRreovseormsMeeutation Assay indicattehatu,ndertheconditionosfthisstudy,3M'stestarticlTe-,6357,didnotcausea positiviencreaseinthenumber of revertantpserplateofany ofthetestesrtrainesitheirnthe presenceorabsenceofmicrosomalenzymes preparedfrom AroclorTm-inducerdatliver(S9).
CHV StudyNo.: 17387-0-409
25
CORNINGHazleton SECTION V. DATA TABLES
CHV StudyNo.: 17387-0-409
26
CORNINGHazlcton
TABLE I DOSE RANGEFINDING TEST ARTICLE ID: T-6357 EXPERIMENT ID: 17387-Al VEHICLE: Deionized water
STUDY
DATE PLATED: DATE COUNTED:
04-Feb-96 07-Feb-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
WITH
TA100 REVERTANTS S9
BACKGROUND LAWN
EVALUATION*
PER PLATE
WITHOUT S9
REVERTANTS
BACKGROUND
PER
LAWN
PLATE
EVALUATION*
97
1
102
1
125
1
87
1
112
1
108
1
103
1
82
1
125
1
136
1
114
1
135
1
85
1
115
1
87
1
88
1
98
1
102
1
86
1
93
1
120
1
105
1
Background Lawn Evaluation 1 - normal 4 - extremely reduced op - slight precipitate
Cod*m:
2 - slightly reduced 5 - absent up - moderate precipitate
(requir*a hand count)
3 - moderately reduced 6 - obscured by precipitate hp - heavy precipitate
(requires hand count)
CHV Study No.: 17387-0-409
27
CORNINGHazleton
TABLE 2 DOSE RANGEFINDING TEST ARTICLE ID: T-6357 EXPERIMENT ID: 17387-Al VEHICLE: Deionized water
STUDY
DATE PLATED: DATE COUNTED:
04-Feb-96 07-Feb-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*
14
1
11
1
13
1
26
1
24
1
21
1
16
1
20
1
11
1
21
1
19
1
16
1
22
1
19
1
13
1
13
1
20
1
22
1
13
1
11
1
18
1
13
1
Background Lavn Zvaluation I - normal
4 - extremely
reduced
op - slight precipitate
Codes: 2 - slightly 5 - absent
up - moderate (requires
reduced
precipitate hand count)
3 - moderately
reduced
6 - obscured
by precipitate
hp - heavy precipitate
(requires
hand count)
CHV Study No.: 17387-0-409
28
CORNINGHa7leton
TABLE 3 MUTAGENICITY ASSAY
INDIVIDUAL PLATE
RESULTS COUNTS
TEST ARTICLE ID: T-6357
EXPERIMENT ID: 17387-Bl
DATE PLATED: 14-Feb-96
VEHICLE: Deionized water
DATE COUNTED: 21-Feb-96
PLATING ALIQUOT: 50 pi
XEMTkaS
PER PLATE
DOSE/PLATE
MICROSOMES: Rat Liver VEHICLE CONTROL
TA98-
123
29 36 23
TA100 ----
TAIS33
1 23
1 23
130 140 131
10
9
9
TA1537
12 3
4
7
a
TEST ARTICLE
100 jig
333 pg 1000 ps
3330 pg
5000 pg
30 32 24
26 37 16
15 16 17
26 27 is
19 24 24
135 143 149
113 99 127
122 119 138
134 146 132
139 129 117
12 14 12
12 16 14
11 23 11
12 16 14
10 11 14
10
9
a
3 10 9
la
3
7
5 10
2
44 9
POSITIVE CONTROL
1086 1106 1231
1400 1443 1450
227 206 348
134 101 130
BACKGROUND
TAWN'
VP2uvrA 123
11 17
6
1
25
7 14
1
13 13 14 1
14 11 13
1
10 20 19
1
16 15 21 1
393 344 372
1
KICROSOMES: None VEHICLE CONTROL TEST ARTICLE
POSITIVE CONTROL
100 mg 333 VS 1000 pg 3330 pg 5000 pg
20 17
8
11 12
9
15 12 11
14 18 12
6 17
9
9 20 11
158 151 162
84 114
79
93 106
84
91 90 so
101
77
98
77 85 98
84 93 86
726 734 779
5 12 11
14 12
9
8
a 10
7 11 13
13 16 10
13 15 10
646 599 701
6
4
a
13 14 12
1
2
4
5
7
9
5
3 11
6
3
5
4
4
7
5
20 10 14 1
12
6
12
1
14 11
9
2
14 12 13 1
17 13
7
1
463 553 742 124 153 119 1
TA98
TA100
TA1535 TA1537 WP2uvrA
2-aminoanthracen* 2-aminoanthracene 2-amino&nthracono 2-aminoanthracens 2-aminoanthracens
2.5 pg/plate 2.5 pg/plate 2.5 pg/plate 2.5 pg/platte 25.0 pg/plate
... TA98 TA100 TA1535 TA1537 WP2uvrA
2-nitrofluorens sodium azido sodium aside ICR-191 4-nitroquinaline-N-oxide
1.0 pe/plate 2.0 pg/plate 2.0 pg/plate 2.0 pg/plate 1.0 pg/plate
Background Lawn Evaluation 1 - normal 4 - &xtrously reduced
ap - 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.: 17387-0-409
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CORNINGHazleton
TABLE 4 NUTAGENICITY ASSAY
SUMMARY
RESULTS
TEST ARTICLE ID: T-6357
EXPERIMENT ID: 17387-Bl
DATE PLATED: 14-Feb-96
VEHICLE: Deionized water
DATE COUNTED: 21-Feb-96
PLATING ALIQUOT: 50 pl
DOSZ/PLkTR
KICROSOMES: Rat Liver VEHICLE CONTROL
TA98
ME"
S.D.
29
7
TEST ARTICLE
100 pg
333 pg 1000 ps
3330 pg
5000 pg
29 4
26 11
16
1
24 5
22
3
MEM REVERTANTS PER PLATE WITH STANDARD DEVIATION
TAIOO MEAN S.D.
TAIS35
MEAN S.D.
TA1537
MF"
S.D.
134
6
142 7
113
14
126
10
137 8
128
11
9
1
13 1
14
2
is
7
14 2
12
2
6
2
91
7
4
7
4
64
6
3
POSITIVE CONTROL
1141
79
1431
27
260 77
122
18
MIGROSOMES: None VEHICLE CONTROL TEST ARTICLE
POSITIVE CONTROL
15
6
100 ps 333 pg 1000 PS 3330 pg 3000 pg
11
2
13
2
15
3
11
6
13
6
157
6
92
19
94 11
87
6
92 13
87 11
as
5
746
29
9
4
12
3
9
1
10
3
13
3
13
3
649
51
6
2
4
2
7
2
7
4
4
1
5
2
586 142
WP2uvrA
M&M
S.D.
BACKGROUND
TAW'
11
6
1
12 4
1
13
1
1
13
2
1
16
6
1
17
3
1
370
25
1
13
1
1
is
5
1
10
3
1
12
3
1
13
1
1
12
5
1
132
is
I
TA98 TA100 TA1535 TA1537 WP2uvrA
2-amincanthraceno 2-aminoanthracens 2-aninoanthracens 2-amincanthracens 2-"incanthracens
2.5 pg/plate 2.5 pg/plate 2.5 pg/plate 2.5 jig/plate 25.0 pg/plate
... TA98 TA100 TA1535 TA1537 WP2uvrA
2-nitrofluerens sodium aside sodium aside ICR-191 4-nitraquinaline-N-axide
1.0 pg/plate 2.0 pg/plate 2.0 pg/plate 2.0 VS/plate 1.0 pg/plate
Background Lavn Evaluation I - normal 4 - extremely 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.: 17387-0409
30