Test No. 476: In Vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes [electronic resource]

2015·0.55 MB·English

by  Organisation for Economic Co-operation and Development

OECD/OCDE 476
Adopted:
28July2015
OECDGUIDELINEFORTHETESTINGOFCHEMICALS
InVitroMammalianCellGeneMutationTestsusingtheHprtandxyrtgenes
INTRODUCTION
1. TheOECDGuidelinesfortheTestingofChemicalsareperiodicallyreviewedinthelightof
scientificprogress,changingregulatoryneedsandanimalwelfare.TheoriginalTestGuideline476
(TG476)wasadoptedin1984.In1997arevisedversionwasadopted,basedonscientificprogressmadeto
thatdate.ThiscurrentrevisedversionofTG476reflectsnearlythirtyyearsofexperiencewiththistestand
alsoresultsfromthedevelopmentofaseparatenewguidelinededicatedtoinvitromammaliancellgene
mtouxtiactoiloongyt.esAtsguusiidnagntcheedtohcyummiedinnteiskiunnadseergperneep.arTaGti4o7n,6ainsdpawritllofpraovsiedreiessuocfciTnecsttaGnudiduesleifnulesguoindagnenceetitco
usersoftheseTestGuidelines.
2. Thepurposeoftheinvitromammaliancellgenemutationtestistodetectgenemutations
inducedbychemicalsubstances.Thecelllinesusedinthesetestsmeasureforwardmutationsinreporter
tgcadeheenlentdleesxsc,Xa,tHnPetsdPRpheRiTbcnyTiefm-iitugcnhtuaeahaltlnuHiyimoPntanehRnetTpeechsenottlssdelspsotd:hgeoec(treonei.lcegblto.oeucsdbstyiaiflshvfeeyetlrpryepaonanxrtisaerffnseetprsrheauricbsetsnedrtctiat-troguaoutanfiassonggtnieeshnnn,eecetHfpi(rphcgarpomttese)vspgehhe(niorntfresetif.seb.aroInrsnsdeydmalHadtlPtdolriRaatdsniTesotlfntheeeetrtsiaotXosinetPnshgReteahTnimnseudtteG(saiuHttni)pis.dreoeTrtnlthaiiilneonen)erHs,vo)PedaneRtntnhTdset
autosomallocationofthegpttransgenomayallowthedetectionofmutationsresultingfromlargedeletions
aXn-dchprososmiobslyommeito(t1i)c(2r)e(c3)o(m4b)i(n5a)(t6i)o.nnTohtedeXtPecRtTedbisyctuhrerHenPtlRyTlteessstbweicdaeulsyeuthseedHptrhtangetnheeisHlPoRcaTtedteostntfhoer
regulatorypurposes.
3. DefinitionsusedareprovidedinAnnex1.
INITIALCONSIDERATIONSANDLIMITATIONS
4. Testsconductedinvitrogenerallyrequiretheuseofanexogenoussourceofmetabolicactivation.
Theexogenousmetabolicactivationsystemdoesnotentirelymimicinvivoconditions.
5. Careshouldbetakentoavoidconditionsthatwouldleadtoartifactualpositiveresults,(i.e.
pthoessgiebnleetiicntmeartaectriioanlwoiftthhethceeltle;stsuscyshtceomn)d,intoitoncsaiunsceldudbeycdhiarnecgtesinitnerpacHtioornobsemtowlealeintyth(e7)t(e8s)t(c9)h,emiinctaerlasctainodn
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Youarefreetousethismaterialforpersonal,non-commercialpurposeswithoutseekingpriorconsent
wfrriotmtetnhepeOmEiiCsDs,ionprforvoimdetdhethOeEsCoDur.ceisdulymentioned.Anycommercialuseofthismaterialissubjectto
476 OECD/OCDE
wrietchotmhmeemneddediutmopccoymtpootonxeinctitsy(l1e0v)e(l1s1)a,sodrefeixnceedssiinvpearleavgerlaspohf1c9ytiostcooxnisciitdyer(e1d2)e.xCcyetsostiovxeifcoirtytheexHcePeRdiTngtestth.e
6. BeforeuseoftheTestGuidelineonamixtureforgeneratingdataforanintendedregulatory
pSuurcphosceo,nsiitdsehroautliodnbsearceonnsoitdneereeddewdhewthheenr,thaenrdeiifssaorweghuyl,atiotrmyaryeqpuriorveimdeentadfeoqrutaetsteinrgesouflttshefomritxhtautrep.urpose.
PRINCIPLEOFTHETEST
HX7.PPRRTTtteesstMt)uatroearnrtegspictsetla(lnistndteXofPitchRieTenctytteiosnstt)HatppirrcotfeifecfniezecntystmoecfelatlchsteiavpiruteryisnieennsatihnteailvHoegPtuRoeT6T-Gtte,hsitwoohgruiacxnhpirntceaeu(nsTezGsy).mteThehaecitnHihvpiirbttiyt(iiionnntthhoeef
coeflTluGl,arwmheetraebaoslinsomrmaanldchealllst,swfuhritchherccoenltladiinvitshieonH.prTthu(si,ntmhuetaHnPtRcTelltsesatr)eoarbglpetto(ipnroXlPifRerTatteesitn)tehnezpyrmees,enacree
not.
8. Cellsinsuspensionormonolayerculturesareexposedtothetestchemical,bothwithandwithout
anexogenoussourceofmetabolicactivation(seeparagraph14),forasuitableperiodoftime(3-6hours),
andthensub-culturedtodeterminecytotoxicityandtoallowphenotypicexpressionpriortomutant
selection(13)(14)(15)(16). Cytotoxicityisdeterminedbyrelativesurvival(RS).i.e.,cloningefficiency
measuredimmediatelyaftertreatmentandadjustedforanycelllossduringtreatmentascomparedtothe
negativecontrol(paragraph18andAnnex2).Thetreatedculturesaremaintainedingrowthmediumfora
sufficientperiodoftime,characteristicofeachcelltype,toallownear-optimalphenotypicexpressionof
inducedmutations(typicallyaminimumof7-9days).Followingphenotypicexpression,mutantfrequency
isdeterminedbyseedingknownnumbersofcellsinmediumcontainingtheselectiveagenttodetect
mutantcolonies,andinmediumwithoutselectiveagenttodeterminethecloningefficiency(viability).
Afterasuitableincubationtime,coloniesarecounted. Mutantfrequencyiscalculatedbasedonthe
numberofmutantcoloniescorrectedbythecloningefficiencyatthetimeofmutantselection.
DESCRIPTIONOFTHEMETHOD
Preparations
Cells
9. ThecelltypesusedfortheHPRTandXPRTtestsshouldhaveademonstratedsensitivityto
chemicalmutagens,ahighcloningefficiency,astablekaryotype,andastablespontaneousmutant
frequency.ThemostcommonlyusedcellsfortheHPRTtestincludetheCHO.CHLandV79linesof
Chinesehamstercells,L5178Ymouselymphomacells,andTK6humanlymphoblastoidcells(17)(18).
CXHPOR-Tdetreistve(d19A)S(5220);cetlhlsecHoPntRaTinitnegsttchaengnpotttbreanpsegrefnoer(maenddihnavAiSn5g2thceelHlpsrbtegcaeunseedetlheetehdp)rtargeenuesehdasfobretehne
deleted.Theuseofothercelllinesshouldbejustifiedandvalidated.
10. Celllinesshouldbecheckedroutinelyforthestabilityofthemodalchromosomenumberandthe
absenceofMycoplasmacontamination(21)(22),andcellsshouldnotbeusedifcontaminatedorifthe
modalchromosomenumberhaschanged.Thenormalcellcycletimeusedinthetestinglaboratoryshould
beestablishedandshouldbeconsistentwiththepublishedcellcharacteristics.Thespontaneousmutant
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frequencyinthemastercellstockshouldalsobechecked,andthestockshouldnotbeusedifthemutant
frequencyisnotacceptable.
c1u1lturingPirnioHrAtToumseediinutmhisfotrestH,PtRheTcutelstturaensdmMayPAneefdortoXPbeRTcleteasntse(d4)o(f23p)re(-SeexeisAtinnngemxutIaTntThceellsc,leea.ngs.ebdy
bceellussceadnfboerctrhyeoptersetsearfvteerdnaonrdmtahlendotuhbalwiendgttoiumseesaasrewoartktaiinngeds.toWchkse.nThceonndeuwcltyintghathweedXwPoRrkTintegsts,torcokutcianne
c1u2l.tureofAS52cellsshoulduseconditionsthatassurethemaintenanceofthegpttransgene(19).
Mediaandcultureconditions
oshfo5ul%dCa0lAwp2a,pyrasonpdbreiiantmceaubicanuttliatoiunnreetdemmeupdnedrieautrmurcaeonndodifti3ino7cnu°sbCa)ttihsoahtnoucelondnsdubireteioutnshseatd(cftuohlretyumraeairnveetsagsiernloiswn,ginhcugumlitidunireflsio.egdCpeahltalmsoecs.ulpthIuterreiess
particularlyimportantthatmediaandcultureconditionsbechosentoensureoptimalgrowthofcellsduring
theexpressionperiodandoptimalcloningefficiencyforbothmutantandnon-mutantcells.
Preparationofcultures
13. Celllinesarepropagatedfromstockcultures,seededinculturemediumatadensitysuchthatthe
cellsinsuspensionsorinmonolayerswillcontinuetogrowexponentiallythroughthetreatmentand
expressionperiods(e.g.confluenceshouldbeavoidedforcellsgrowinginmonolayers).
Metabolicactivation
14. Exogenousmetabolisingsystemsshouldbeusedwhenemployingcellswhichhaveinadequate
eo1t5n.hdeorgweinsoeujsusmteitfiaebdo,liiscacacpoa-cfitayc.torT-hseupmpolsetmecnotmedmopnoslty-muisteodchsoynsdtreima,ltfhratacitsiornec(So9m)meprnedpeadrebdyfdreofamultth,eulnilveesrss
ofrodents(generallyrats)treatedwithenzyme-inducingagentssuchasAroclor1254(24)(25)(26)(27)ora
combinationofphenobarbitalandp-naphthoflavone(28)(29)(30)(31).Thelattercombinationdoesnot
conflictwiththeStockholmConventiononPersistentOrganicPollutants(32)andhasbeenshowntobeas
eatffceocntciveentarsatAirooncslorran1g2i5n4gfforromind1utcoin2g%mi(xve/dv)-fbuuntctmiaoynobxeidianscersea(s2e8d)t(o301).0%Th(ev/Sv)9ifnratchteiofnintaylpitceasltlmyeidsiuusme.d
Thechoiceofthetypeandconcentrationofexogenousmetabolicactivationsystemormetabolicinducer
employedmaybeinfluencedbytheclassofsubstancesbeingtested(33)(34)(35).
TestchemicalPreparation
Solidtestchemicalsshouldbepreparedinappropriatesolventsanddiluted,ifappropriate,prior
totreatmentofthecells(seeparagraph16).Liquidtestchemicalsmaybeaddeddirectlytothetestsystem
and/ordilutedpriortotreatmentofthetestsystem.Gaseousorvolatiletestchemicalsshouldbetestedby
appropriatemodificationstothestandardprotocols,suchastreatmentinsealedculturevessels(36)(37).
Preparationsofthetestchemicalshouldbemadejustpriortotreatmentunlessstabilitydatademonstrate
theacceptabilityofstorage.
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Testconditions
16.
Solvents
Thesolventshouldbechosentooptimizethesolubilityofthetestchemicalswithoutadversely
impactingtheconductoftheteste.g..changingcellgrowth,affectingtheintegrityofthetestchemical,
reactingwithculturevessels,impairingthemetabolicactivationsystem.Itisrecommendedthat,wherever
psooslsviebnltes,atrheefuorseexoafmapnlea,qwuaetoeursasnodlvdeinmtet(horylcuslutlufroeximdee.diGuenme)raslhloyu,ldorbgaenicconssoildveernetdsfsihrsotu.lWdenlolteesxtcaebeldish1e%d
(v/v)andaqueoussolvents(salineorwater)shouldnotexceed10%(v/v)inthefinaltreatmentmedium.If
thesolventsusedarenotwell-established(e.g.ethanoloracetone),theiruseshouldbesupportedbydata
indicatingtheircompatibilitywiththetestchemicalsandthetestsystem,andtheirlackofgenetictoxicity
attheconcentrationused.Intheabsenceofthatsupportingdata,itisimportanttoadduntreatedcontrols
(seeAnnex1)todemonstratethatnodeleteriousormutageniceffectsarcinducedbythechosensolvent.
Measuringcytotoxicityandchoosingexposureconcentrations
17. Whendeterminingthehighesttestchemicalconcentration,concentrationsthathavethecapability
ofproducingartifactualpositiveresponses,suchasthoseproducingexcessivecytotoxicity(seeparagraph
tp2h0ae)r,atgpirrmaeepcihopif5t)aadtsdihiootnuiloidnn,tbhteeheacvupoliHtduermdei.gmIheftdtihbeuemtaeds(tjsuecsehtepemdaircbaagylrbacupafhufse2er1si),nagomrthamerakfreikndealdchtcarhneaagntegmeeisnnttihnmeeppdHiHuoormfotsshomeoalmsaeltdiotiyauvm(osieadet
artifactualpositiveresultsandtomaintainappropriatecultureconditions.
18. Concentrationselectionisbasedoncytotoxicityandotherconsiderations(seeparagraphs20-22).
Whiletheevaluationofcytotoxicityinaninitialtestmaybeusefultobetterdefinetheconcentrationstobe
usedinthemainexperiment,aninitialtestisnotrequired.Evenifaninitialcytotoxicityevaluationis
performed,themeasurementofcytotoxicityforeachcultureisstillrequiredinthemainexperiment.
CytotoxicityshouldbeevaluatedusingRS.i.e.,cloningefficiency(CE)ofcellsplatedimmediatelyafter
treatment,adjustedbyanylossofcellsduringtreatment,basedoncellcount,ascomparedwithadjusted
cloningefficiencyinnegativecontrols(assignedasurvivalof100%)(seeAnnex2fortheformula).
19. Atleastfourtestconcentrations(notincludingthesolventandpositivecontrols)thatmeetthe
acceptabilitycriteria(appropriatecytotoxicity,numberofcells,etc.)shouldbeevaluated.Whiletheuseof
duplicateculturesisadvisable,eitherreplicateorsingletreatedculturesmaybeusedateachconcentration
tested.Theresultsobtainedintheindependentreplicateculturesatagivenconcentrationshouldbe
reportedseparatelybutcanbepooledforthedataanalysis(16).Fortestchemicalsdemonstratinglittleor
nocytotoxicity,concentrationintervalsofapproximately2to3foldwillusuallybeappropriate.Where
cytotoxicityoccurs,thetestconcentrationsselectedshouldcoverarangefromthatproducingcytotoxicity
toconcentrationsatwhichthereismoderateandlittleornocytotoxicity.Manytestchemicalsexhibitsteep
concentrationresponsecurvesandinordertocoverthewholerangeofcytotoxicityortostudythe
concentration response relationshipindetail, itmaybenecessaryto use more closely spaced
creoqnuciernetdrat(sieoenspaarnadgrmaoprhe4t3h)a.nTfhoeurusceoncoefntmroarteiontsh,anin4pcaorntciceunltarratiinonssitumaatiyonbsewphaerrteicualarrelpyeaitmpeoxrptearnitmewnhteins
usingsinglecultures.
20. Ifthemaximumconcentrationisbasedoncytotoxicity,thehighestconcentrationshouldaimto
achievebetween20and10%RS.Careshouldbetakenwheninterpretingpositiveresultsonlyfoundat
10%RSorbelow(paragraph43).
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21. Forpoorlysolubletestchemicalsthatarenotcytotoxicatconcentrationsbelowthelowest
insolubleconcentration,thehighestconcentrationanalysedshouldproduceturbidityoraprecipitatevisible
byeyeorwiththeaidofaninvertedmicroscopeattheendofthetreatmentwiththetestchemical.Evenif
ccyotnocteonxtircaittiyonopcrcoudrsucianbgovteurbtihdietyloorwewsitthinasvoilsuibblleepcroencciepnittraatteiobne,cauitseisartaidfvaicstuaablleeffteoctstemstayatreosunlltyfroonme
theprecipitate.Attheconcentrationproducingaprecipitate,careshouldbetakentoassurethatthe
precipitatedoesnotinterferewiththeconductofthetest. Thedeterminationofsolubilityintheculture
mediumpriortotheexperimentmaybeuseful.
2c2o.rresponIdftnoo10prmeMci,pit2atmeg/ormLliomrit2inpgL/cmytLo.towxhiicicthyeviesroibsstehreveldo,westhte(3h8i)gh(e3s9t).tWeshtencontcheenttersattcihoenmischaolulids
notofdefinedcomposition,e.g.,substanceofunknownorvariablecomposition,complexreaction
productsorbiologicalmaterials(i.e.,ChemicalSubstancesofUnknownorVariableComposition
(UVCBs))(40),environmentalextracts,etc.,thetopconcentrationmayneedtobehigher(e.g.5mg/mL),
binetnhoeteadbsheonwceevoefrstuhfaftictiheenstecryetqotuoixriecmietny,tstomainycrdeiaffseertfhoerchounmceanntrpahtairomnacoefuetaicchalosf(t4h1e).components.Itshould
Controls
23. Concurrentnegativecontrols(seeparagraph16),consistingofsolventaloneinthetreatment
mediumandhandledinthesamewayasthetreatmentcultures,shouldbeincludedforeveryexperimental
condition.
24. Concurrentpositivecontrolsareneededtodemonstratetheabilityofthelaboratory'toidentify
mutagensundertheconditionsofthetestprotocolusedandtheeffectivenessoftheexogenousmetabolic
activationsystem,whenapplicable.ExamplesofpositivecontrolsaregiveninTable1below.Alternative
positivecontrolsubstancescanbeused,ifjustified.Becauseinvitromammaliancelltestsforgenetic
toxicityaresufficientlystandardized,testsusingtreatmentswithandwithoutexogenousmetabolic
activationmaybeconductedusingonlyapositivecontrolrequiringmetabolicactivation.Inthiscase,this
singlepositivecontrolresponsewilldemonstrateboththeactivityofthemetabolicactivationsystemand
theresponsivenessofthetestsystem.Eachpositivecontrolshouldbeusedatoneormoreconcentrations
expectedtogivereproducibleanddetectableincreasesoverbackgroundinordertodemonstratethe
sleinmsiittsisvpietycioffietdheinttehsetsTyGst(esme,eapnadratghreaprhes2p0o)n.seshouldnotbecompromisedbycytotoxicityexceedingthe
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Table1.Referencesubstancesrecommendedforassessinglaboratoryproficiencyandforselectionof
positivecontrols.
MetabolicActivation Locus ChemicalandCASNo.
condition
Absence of exogenous Hprt Ethylmethanesulfonate[CASno.62-50-0]
metabolicactivation Ethylnitrosourea[CASno.759-73-9]
4-Nitroquinoline1-oxide[CASno.56-57-51
xprt Streptonigrin[CASno.3930-19-6]
MitomycinC[CASno.50-07-7]
Presence of exogenous Hprt 3-Methylcholanthrene[CASno.56-49-5]
metabolicactivation 7,12-Dimethylbenzanthracene[CASno.57-97-6]
Benzo[a]pyrene[CASno.50-32-8]
xprt Benzo[a]pyrene[CASno.50-32-8]
PROCEDURE
Treatmentwithtestchemical
25. Proliferatingcellsaretreatedwiththetestchemicalinthepresenceandabsenceofametabolic
activationsystem.Exposureshouldbeforasuitableperiodoftime(usually3to6hoursisadequate).
t22e67s..tshoulTdhebemibnasiemdumonnutmhebesrpoonftcaenllesouussemduftoarnetacfhretqeusetn(ccyo.ntroAlagenndetrraelatgeud)idceulitsurteoattreeaatchasntdagpeasinsatghee
ssoufpfof5nitcxailenOneto6ucasenlldmsuttaoasnmttaoifnmrtaeaiqinunteaanicnsyuf1if0sicgsiepenonentrtanalunlmeyobbuesertmwouefteasnnpto5nstaiannndeeo2vu0esrxymluOct"ula6.tnutrFseor(i1na0aslolpropmnhotarasene)esoeuovsfetmnhueftoatrnesttthfe(r1ce6u)ql.uteuTnrhceeys
ttrreeaatteadtalteacsotn2c0enxtra1t0i“ocneslltsh.atIncaadudsieti9o0n%acsyutfoftiocxieinctitnyudmubreirngoftrceealtlsme(nbtut(1ne0v%erRSl)e,ssitthwaonul2dmiblelinoenc)esmsuasrtybteo
culturedduringtheexpressionperiodandplatedformutantselection(16).
Phenotypicexpressiontimeandmeasuringmutantfrequency
Afterthetreatmentperiod,cellsareculturedtoallowexpressionofthemutantphenotype.A
minimumof7to9daysgenerallyissufficienttoallownearoptimalphenotypicexpressionofnewly
inducedHprtandxprtmutants(42)(43).Duringthisperiod,cellsareregularlysub-culturedtomaintain
theminexponentialgrowth.Afterphenotypicexpression,cellsarere-platedinmediumwithandwithout
selectiveagent(6-thioguanine)forthedeterminationofthenumberofmutantsandcloningefficiencyat
thetimeofselection,respectively.Thisplatingcanbeaccomplishedusingdishesformonolayercultures
ormicrowellplatesforcellsinsuspension.Formutantselection,cellsshouldbeplatedatadensityto
assureoptimummutantrecovery(i.e.,avoidmetaboliccooperation)(16). Platesareincubatedforan
appropriatelengthoftimeforoptimumcolonygrowth(e.g.,7-12days)andcoloniescounted. Mutant
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frequencyiscalculatedbasedonthenumberofmutantcoloniescorrectedbythecloningefficiencyatthe
timeofmutantselection(seeAnnex2forformulas).
Proficiencyofthelaboratory
28. Inordertoestablishsufficientexperiencewiththetestpriortousingitforroutinetesting,the
laboratoryshouldhaveperformedaseriesofexperimentswithreferencepositivesubstancesactingvia
differentmechanisms(atleastoneactivewithandoneactivewithoutmetabolicactivationselectedfrom
thesubstanceslistedinTabic1)andvariousnegativecontrols(usingvarioussolvents/vehicles).These
positiveandnegativecontrolresponsesshouldbeconsistentwiththeliterature.Thisisnotapplicableto
laboratoriesthathaveexperience,i.e.thathaveanhistoricaldatabaseavailableasdefinedinparagraphs30
to33.
29. Aselectionofpositivecontrolsubstances(seeTable1inparagraph25)shouldbeinvestigatedin
theabsenceandinthepresenceofmetabolicactivation,inordertodemonstrateproficiencytodetect
mutagenicsubstances,todeterminetheeffectivenessofthemetabolicactivationsystemandtodemonstrate
tsheelecatpipornoparnidatoefnetshseosfcotrhiengceplrlocgerdouwrtesh.cAondriatnigoensofducroinncgenttrreaattimoennst,ofpthheenosteylpeicctedexspurbesstsainocnesansdhomuultdabnet
chosensoastogivereproducibleandconcentration-relatedincreasesabovethebackgroundinorderto
demonstratethesensitivityanddynamicrangeofthetestsystem.
Historicalcontroldata
30. Thelaboratoryshouldestablish:
--AAhhiissttoorriiccaallpnoesgiattiivveec(ounnttrroelatreadn,gseolavnedntd)isctornitbrutoilorn,angeanddistribution.
31. Whenfirstacquiringdataforanhistoricalnegativecontroldistribution,concurrentnegative
controlsshouldbeconsistentwithpublishedcontroldata(21).Asmoreexperimentaldataareaddedtothe
controldistribution,concurrentnegativecontrolsshouldideallybewithinthe95%controllimitsofthat
distribution(16)(44)(45).
32. Thelaboratory’shistoricalnegativecontroldatabaseshouldinitiallybebuiltwithaminimumof
10experimentsbutwouldpreferablyconsistofatleast20experimentsconductedundercomparable
experimentalconditions.Laboratoriesshouldusequalitycontrolmethods,suchascontrolcharts(e.g.C-
chartsorX-barcharts(46)),toidentifyhowvariabletheirpositiveandnegativecontroldataare,andto
showthatthemethodologyis'undercontrol'intheirlaboratory(45).Furtherrecommendationsonhowto
buildandusethehistoricaldata(i.e.criteriaforinclusionandexclusionofdatainhistoricaldataandthe
acceptabilitycriteriaforagivenexperiment)canbefoundintheliterature(44).
33. Negativecontroldatashouldconsistofmutantfrequenciesfromsingleorpreferablyreplicate
culturesasdescribedinparagraph23.Concurrentnegativecontrolsshouldideallybewithinthe95%
Wcohnetrroelcloinmictusrroefntthneegdaitsitvreibuctoinotnroolfdtahtealfaablolraotuotrsyi’dsehtihseto9ri5ca%lcnoengtartoilvelicmointtrtohleydamtaaybasbee(a1c6c)ep(t4a4b)le(4f5o).r
inclusioninthehistoricalcontroldistributionaslongasthesedataarenotextremeoutliersandthereis
evidencethatthetestsystemis'undercontrol'(seeabove)andthereisevidenceofnotechnicalorhuman
failure.
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Anychangestotheexperimentalprotocolshouldbeconsideredintermsoftheirconsistencywith
thelaboratory’sexistinghistoricalcontroldatabases.Anymajorinconsistenciesshouldresultinthe
establishmentofanewhistoricalcontroldatabase.
DATAANDREPORTING
Presentationoftheresults
35. Thepresentationofresultsshouldincludeallofthedataneededtocalculatecytotoxicity
(expressedasRS).Thedata,forbothtreatedandcontrolcultures,shouldincludethenumberofcellsatthe
endoftreatment,thenumberofcellsplatedimmediatelyfollowingtreatment,andthecolonycounts(or
numberofwellswithoutcoloniesforthemicrowellmethod).RSforeachcultureshouldbeexpressedasa
percentagerelativetotheconcurrentsolventcontrol(refertoAnnex1fordefinitions).
36. Thepresentationofresultsshouldalsoincludeallofthedataneededtocalculatethemutant
frequency.Dataforbothtreatedandcontrolcultures,shouldinclude:(1)thenumberofcellsplatedwith
andwithoutselectiveagent(atthetimethecellsareplatedformutantselection),and(2)thenumberof
coloniescounted(orthenumberofwellswithoutcoloniesforthemicrowellmethod)fromtheplateswith
andwithoutselectiveagent. Mutantfrequencyiscalculatedbasedonthenumberofmutantcolonies(in
t3h8e.plateswithselectiveagent)correctedbythecloningefficiency(fromtheplateswithoutselective
agent).Themutantfrequencyshouldbeexpressedasthenumberofmutantcellspermillionviablecells
(refertoAnnex1fordefinitions).
37. Individualculturedatashouldbeprovided.Additionally,alldatashouldbesummarisedin
tabularform.
AcceptabilityCriteria
Acceptanceofatestisbasedonthefollowingcriteria:
-Theconcurrentnegativecontrolisconsideredacceptableforadditiontothelaboratoryhistorical
39. negativecontroldatabaseasdescribedinparagraph33.
-Concurrentpositivecontrols(seeparagraph24)shouldinduceresponsesthatarecompatiblewith
thosegeneratedinthehistoricalpositivecontroldatabaseandproduceastatisticallysignificant
increasecomparedwiththeconcurrentnegativecontrol.
-Twoexperimentalconditions(i.e.,withandwithoutmetabolicactivation)weretestedunlessone
resultedinpositiveresults(seeparagraph25).
-Adequatenumberofcellsandconcentrationsareanalysable(paragraphs25,26and19).
-Thecriteriafortheselectionoftopconcentrationareconsistentwiththosedescribedin
paragraphs20,21and22.
Evaluationandinterpretationofresults
Providingthatallacceptabilitycriteriaarefulfilled,atestchemicalisconsideredtobeclearly
positiveif,inanyoftheexperimentalconditionsexamined:
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a)atleastoneofthetestconcentrationsexhibitsastatisticallysignificantincreasecomparedwith
theconcurrentnegativecontrol,
b)theincreaseisconcentration-relatedwhenevaluatedwithanappropriatetrendtest,
c)anyoftheresultsareoutsidethedistributionofthehistoricalnegativecontroldata(e.g.Poisson-
based95%controllimit;seeparagraph33).
Whenallofthesecriteriaaremet,thetestchemicalisthenconsideredabletoinducegene
mutationsinculturedmammaliancellsinthistestsystem. Recommendationsforthemost
appropriatestatisticalmethodscanbefoundintheliterature(45)(47).
40. Providingthatallacceptabilitycriteriaarcfulfilled,atestchemicalisconsideredclearlynegative
if,inallexperimentalconditionsexamined:
a)noneofthetestconcentrationsexhibitsastatisticallysignificantincreasecomparedwiththe
concurrentnegativecontrol,
b)thereisnoconcentration-relatedincreasewhenevaluatedwithanappropriatetrendtest,
c)allresultsareinsidethedistributionofthehistoricalnegativecontroldata(e.g.Poisson-based
95%controllimit;seeparagraph33).
Thetestchemicalisthenconsideredunabletoinducegenemutationsinculturedmammaliancells
inthistestsystem.
43.
41. Thereisnorequirementforverificationofaclearlypositiveornegativeresponse.
42. Incaseswhentheresponseisneitherclearlynegativenorclearlypositiveasdescribedabove,or
ji4nu4.dogredmerenttoaassnids/toirnefusrttahbelrishiinnvgestthiegabtiioolnosg.icalPerrelfeovramnicnegofaarreepseulatt,tehxepedraitamesnhtouplodsbseibelvyaluusaitnegdbmyodeixfpieerdt
experimentalconditions(e.g.concentrationspacing,othermetabolicactivationconditions [i.e.S9
concentrationorS9origin])couldbeuseful.
Inrarecases,evenafterfurtherinvestigations,thedatasetwillprecludemakingaconclusion
ofpositiveornegativeresults.Thereforethetestchemicalresponseshouldbeconcludedtobeequivocal
(interpretedasequallylikelytobepositiveornegative).
Testreport
Thetestreportshouldincludethefollowinginformation:
Testchemical:
source,lotnumber,limitdateforuse,ifavailable;
stabilityofthetestchemicalitself,ifknown;
solubilityandstabilityofthetestchemicalinsolvent,ifknown;
measurementofpH,osmolality’andprecipitateintheculturemediumtowhichthetest
chemicalwasadded,asappropriate.
Mono-constituentsubstance:
physicalappearance,watersolubility,andadditionalrelevantphysicochemicalproperties;
©OECD,(2015) 9
476 OECD/OCDE
chemicalidentification,suchasIUPACorCASname,CASnumber,SMILESorInChlcode,
structuralformula,purity,chemicalidentityofimpuritiesasappropriateandpractically
feasible,etc.
Multi-constituentsubstance,UVBCsandmixtures:
characterisedasfaraspossiblebychemicalidentity(seeabove),quantitativeoccurrenceand
relevantphysicochemicalpropertiesoftheconstituents.
Solvent:
justificationforchoiceofsolvent;
percentageofsolventinthefinalculturemedium.
Cells:
ForLaboratorymastercultures:
- type,sourceofcelllines;
numberofpassages,ifavailable,andhistoryinthelaboratory;
karyotypefeaturesand/ormodalnumberofchromosomes;
methodsformaintenanceofcellcultures;
absenceofmycoplasma;
celldoublingtimes.
Testconditions:
rationaleforselectionofconcentrationsandnumberofculturesincluding,e.g.cytotoxicitydata
andsolubilitylimitations;
occroonmmcpgeons/timrtaLitoionornomfomfMetdeoisfta,ccuhClet0muir2cecaomlnecedexinputrrmea)st;isoend,ahsufmiindailtcyolnecveenlt;rationintheculturemedium(e.g.pg
concentration(and/orvolume)ofsolventandtestchemicaladdedintheculturemedium;
incubationtemperature;
incubationtime;
durationoftreatment;
celldensityduringtreatment;
- typeandcompositionofmetabolicactivationsystem(sourceofS9,methodofpreparationof
tineS9mix,theconcentrationorvolumeofS9mixandS9intinefinalculturemedium,quality
controlsofS9);
positiveandnegativecontrolsubstances,finalconcentrationsforeachconditionoftreatment;
lengthofexpressionperiod(includingnumberofcellsseeded,andsubculturesandfeeding
schedules,ifappropriate);
identityoftheselectiveagentanditsconcentration;
criteriaforacceptabilityoftests;
methodsusedtoenumeratenumbersofviableandmutantcells;
methodsusedforthemeasurementsofcytotoxicity;
anysupplementaryinformationrelevanttocytotoxicity'andmethodused;
durationofincubationtimesafterplating;
criteriaforconsideringstudiesaspositive,negativeorequivocal;
methodsusedtodeterminepH.osmolalityandprecipitation.
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