Test No. 487: In Vitro Mammalian Cell Micronucleus Test [electronic resource]

2010·0.73 MB·English

by  Organisation for Economic Co-operation and Development

OECD/OCDE 487
Adopted:
22July2010
OECDGUIDELINEFORTHETESTINGOFCHEMICALS
InVitroMammalianCellMicronucleusTest
INTRODUCTION
(1.MN)inTthheeicnytviotprloamsimcroofnuicnlteeursph(aMsNevicetl)ls.assMaiycriosnaucgleeniotomxaiycitoyritgesitnaftoertfhreomdetaeccetnitornicofcmhircormonouscolmeei
tfhreagamneanpthsas(ie.es.tlaagcekionfgcaelclednitvriosmieorne.),Thoerwahssoalyedcehtercotmsotshoemaecstitvhiattyaorfeculnaasbtloegetnoicmiagnrdataenteougtehneipcolcehsemdiucrailnsg
G(1u)id(e2l)iinnecaellllsowtshatthehauvseeuonfdperrogtoonceolcselwlitdihviasnidonwidtuhroiuntgtohreaafctterinexpoploysmuerreistaottihoentienshtibsiutbosrtacnycteo.chTahliassiTnesBt
(cytoB).TheadditionofcytoBpriortothetargetedmitosisallowsfortheidentificationandselective
analysisofmicronucleusfrequencyincellsthathavecompletedonemitosisbecausesuchcellsare
binucleate(3)(4).ThisTestGuidelinealsoallowstheuseofprotocolswithoutcytokinesisblock,provided
thereisevidencethatthecellpopulationanalysedhasundergonemitosis.
2. InadditiontousingtheMNvitassaytoidentifychemicalsthatinducemicronuclei,theuseofa
cytokinesisblock,immunochemicallabellingofkinetochores,orhybridisationwithcentromeric/telomeric
probes(fluorescenceinsituhybridisation(FISH)),alsocanprovideinformationonthemechanismsof
chromosomedamageandmicronucleusformation(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16).The
labellingandhybridisationprocedurescanbeusedwhenthereisanincreaseinmicronucleusformation
andtheinvestigatorwishestodetermineiftheincreasewastheresultofclastogenicand/oraneugenic
events.
3. Micronucleirepresentdamagethathasbeentransmittedtodaughtercells,whereaschromosome
aberrationsscoredinmetaphasecellsmaynotbetransmitted.Becausemicronucleiininterphasecellscan
beassessedrelativelyobjectively,laboratorypersonnelneedonlydeterminewhetherornotthecellshave
undergonedivisionandhowmanycellscontainamicronucleus.Asaresult,thepreparationscanbescored
rheulnadtriveedlsyoqfuiccelkllsypaenrdtraenaatlmyesnits,cianncrebaesianugtotmheatpeod.weTrhiosftmhaekaesssaiyt.pFriancatlilcya,latsomsiccorroenutchloeuisamnadysairnissteeafdroomf
ilnagcgoinnvgencthiroonmaolscohmerso,motshoemrealisatbheerrpaottienotniatelsttso,dee.tge.ctOaEnCeuDplToeisdty-Giunidduecliinngea4g7e3nt(s17t)ha.tHaorweedvieffri,cultthetoMsNtvuidty
assaydoesnotallowforthedifferentiationofchemicalsinducingpolyploidyfromthoseinducing
clastogenicitywithoutspecialtechniquessuchasFISHdescribedunderparagraph2.
4. TheMNvitassayisaninvitromethodthattypicallyusesculturedhumanorrodentcells.It
providesacomprehensivebasisforinvestigatingchromosomedamagingpotentialinvitrobecauseboth
aneugensandclastogenscanbedetected.
5. TheMNvitassayisrobustandeffectiveinavarietyofcelltypes,andinthepresenceorabsence
ofcytoB.ThereareextensivedatatosupportthevalidityoftheMNvitassayusingvariousrodentcelllines
(CHO,V79,CHL/IU,andL5178Y)andhumanlymphocytes(18)(19)(20)(21)(22)(23)(24)(25)(26)
(27)(28)(29)(30)(31).Theseinclude,inparticular,theinternationalvalidationstudiesco-ordinatedby
theSocieteFran§aisedeToxicologieGenetique(SFTG)(18)(19)(20)(21)(22)andthereportsofthe
InternationalWorkshoponGenotoxicityTesting(4)(16).Theavailabledatahavealsobeenre-evaluatedin
©OECD,(2010)
Youarefreetousethismaterialforpersonal,non-commercialpurposeswithoutseekingpriorconsentfrom
ptehermOiEssCiDo,npfrroovmitdheedOthEeCDso.urceisdulymentioned.Anycommercialuseofthismaterialissubjecttowritten
487 OECD/OCDE
aweight-of-evidenceretrospectivevalidationstudybytheEuropeanCentrefortheValidationof
AasltsecrineanttiivfeicMaleltyhvoadlsid(bEyCtVhAeME)CoVfAthMeESucireonpteifaincCAodmvmiissorsyioCnom(EmCi)t,teaend(tEhSeAtCes)tm(3e2t)ho(3d3)ha(s34b)e.eTnheenduosreseodf
thehumanTK6lymphoblastoidcellline(35),HepG2cells(36)(37)andprimarySyrianHamsterEmbryo
cells(38)hasbeendescribed,althoughtheyhavenotbeenusedinvalidationstudies.
6. DefinitionsusedareprovidedinAnnex1.
INITIALCONSIDERATIONS
7. Testsconductedinvitrogenerallyrequiretheuseofanexogenoussourceofmetabolicactivation
unlessthecellsaremetabolicallycompetentwithrespecttothesubstancesbeingtested.Theexogenous
metabolicactivationsystemdoesnotentirelymimicinvivoconditions.Careshouldalsobetakentoavoid
conditionsthatwouldleadtoartifactualpositiveresultswhichdonotreflectintrinsicmutagenicity,and
mayarisefromsuchfactorsasmarkedchangesinpHorosmolality,orbyhighlevelsofcytotoxicity(39)
(40)(41).IfthetestchemicalcausesachangeinthepHofthemediumatthetimeofaddition,thepH
shouldbeadjusted,preferablybybufferingthestocksolutionsothatallthevolumesatalltest
concentrations,andforallcontrols,remainthesame.
8. Toanalysetheinductionofmicronuclei,itisessentialthatmitosishasoccurredinbothtreated
anduntreatedcultures.Themostinformativestageforscoringmicronucleiisincellsthathavecompleted
onemitosisduringoraftertreatmentwiththetestsubstance.
PRINCIPLEOFTHETEST
9. Cellculturesofhumanormammalianoriginareexposedtothetestsubstancebothwithand
withoutanexogenoussourceofmetabolicactivationunlesscellswithanadequatemetabolizingcapability
areused.Concurrentsolvent/vehicleandpositivecontrolsareincludedinalltests.
10. Duringorafterexposuretothetestsubstance,thecellsaregrownforaperiodsufficienttoallow
chromosomeorspindledamagetoleadtotheformationofmicronucleiininterphasecells.Forinductionof
aneuploidy,thetestsubstanceshouldordinarilybepresentduringmitosis.Harvestedandstained
interphasecellsareanalysedforthepresenceofmicronuclei.Ideally,micronucleishouldonlybescoredin
thosecellsthathavecompletedmitosisduringexposuretothetestsubstanceorduringthepost-exposure
period,ifoneisused.Inculturesthathavebeentreatedwithacytokinesisblocker,thisisachievedby
scoringonlybinucleatecells.Intheabsenceofacytokinesisblocker,itisimportanttodemonstratethatthe
cellsanalysedarelikelytohaveundergonecelldivisionduringorafterexposuretothetestsubstance.For
allprotocols,itisimportanttodemonstratethatcellproliferationhasoccurredinboththecontroland
treatedcultures,andtheextentoftestsubstance-inducedcytotoxicityorcytostasisshouldbeassessedin
thecultures(orinparallelcultures)thatarescoredformicronuclei.
DESCRIPTIONOFTHEMETHOD
Preparations
c1e1l.llinesCsuulcthuraesdCpHrOim,arVy79h,umCaHnL/pIeUri,phaenrdalLb5l1o7o8dYlycmepllhsocmyatyesbe(5)us(e1d9)((1482))((1493))(a2n0d)a(2n1u)m(b2e2)ro(f25r)od(e2n6)t
(27)(28)(30).Theuseofothercelllinesandtypesshouldbejustifiedbasedontheirdemonstrated
performanceintheassay,asdescribedintheAcceptabilityCriteriasection.Becausethebackground
frequencyofmicronucleiwillinfluencethesensitivityoftheassay,itisrecommendedthatcelltypeswith
alow,stablebackgroundfrequencyofmicronucleusformationbeused.
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12. Humanperipheralbloodlymphocytesshouldbeobtainedfromyoung(approximately18-35
yearsofage),healthy,non-smokingindividualswithnoknownrecentexposurestogenotoxicchemicalsor
radiation.Ifcellsfrommorethanonedonorarepooledforuse,thenumberofdonorsshouldbespecified.
Themicronucleusfrequencyincreaseswithageandthistrendismoremarkedinfemalesthaninmales
(44)andthisshouldbetakenintoaccountintheselectionofdonorcellsforpooling.
Mediaandcultureconditions
t1e3.mperaturAep,praonpdrihautmeidciutlyt)ureshmouelddiubmeuasneddifnocrubmaatiinotnaicnoinndgitciuolntusre(sc.ulEtsutraeblviesshseelds,celCl0l2inecsonacenndtrsattriaoinn,s
shouldbecheckedroutinelyforthestabilityofthemodalchromosomenumberandtheabsenceof
mycoplasmacontamination,andshouldnotbeusedifcontaminatedorifthemodalchromosomenumber
haschanged.Thenormalcellcycletimeforthecultureconditionsusedinthetestinglaboratoryshouldbe
known.Ifthecytokinesis-blockmethodisusedthentheconcentrationofthecytokinesisinhibitorshould
beoptimisedfortheparticularcelltypeandshouldbeshowntoproduceagoodyieldofbinucleatecells
forscoring.
Preparationofcultures
14. Establishedcelllinesandstrains:cellsarepropagatedfromstockcultures,seededinculture
mediumatadensitysuchthatthecultureswillnotreachconfluencyinmonolayers,andsuspension
cultureswillnotreachexcessivedensitybeforethetimeofharvest,andincubatedat37°C.
l15y.mphocytLeysm,pahroeccyutletsu:redwihnotlheepbrleosoedncetroefataedmitwoigtehnea.ng.panhtyit-ochoaaegmualgangtlut(ien.ig.n(hPepHaAr)in)p,riorortoseexppaorsautreed
tothetestsubstanceandcytoB.
Metabolicactivation
16. Exogenousmetabolisingsystemsshouldbeusedwhenusingcellswithinadequateendogenous
metaboliccapacity. Themostcommonlyusedsystemisaco-factor-supplementedpost-mitochondrial
fraction(S9)preparedfromtheliversofrodentstreatedwithenzyme-inducingagentssuchasAroclor1254
(45)(46)oracombinationofphenobarbitoneandp-naphthoflavone(46)(47)(48)(49).Thelatter
combinationdoesnotconflictwiththeStockholmConventiononPersistentOrganicPollutants(50)and
hasbeenshowntobeaseffectiveasAroclor1254forinducingmixed-functionoxidases(46)(47)(48)
(49).TheS9fractiontypicallyisusedatconcentrationsrangingfrom1-10%(v/v)inthefinaltestmedium.
Theconditionofametabolicactivationsystemmaydependupontheclassofchemicalbeingtestedandin
somecasesitmaybeappropriatetoutilisemorethanoneS9concentration.
17. Geneticallyengineeredcelllinesexpressingspecifichumanorrodentactivatingenzymesmay
eliminatetheneedforanexogenousmetabolicactivationsystem,andmaybeusedasthetestcells.Insuch
casesthechoiceofthecelllinesusedshouldbescientificallyjustified,e.g.byrelevanceofthemixed
functionoxidasesforthemetabolismofthetestsubstance(51),andtheirresponsivenesstoknown
clastogensandaneugens(seeseparatesectiononAcceptabilityCriteria).Itshouldberecognizedthatthe
substancebeingtestedmaynotbemetabolisedbytheexpressedmixedfunctionoxidase(s);inthiscase,the
negativeresultswouldnotindicatethatthesubstancecannotinducemicronuclei.
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Testsubstancepreparation
18. Solidtestsubstancesshouldbedissolvedinappropriatesolventsorvehiclesanddiluted,if
appropriate,priortotreatmentofthecells.Liquidtestsubstancesmaybeaddeddirectlytothetestsystems
and/ordilutedpriortotreatment.Gasesorvolatilesubstances shouldbetestedbyappropriate
modificationstothestandardprotocols,suchastreatmentinsealedvessels(52)(53).Freshpreparationsof
thetestsubstanceshouldbeusedunlessstabilitydatademonstratetheacceptabilityofstorage.
TestConditions
Solvents/vehicles
19. Thesolvent/vehicleshouldnotreactwiththetestsubstance,orbeincompatiblewiththesurvival
ofthecellsorwiththemaintenanceofS9activityattheconcentrationused.Ifotherthanwellestablished
ssoulpvpeonrtt/evdehbiycldeasta(ei.ngd.icwaattienrg,thceeilrlcocmupltautriebilmietdyiwuimt,htdhiemetetshtyslubssutlafnocxeidaen)datrheeirusleadc,kotfhegierneutsiectsohxoiuciltdy.bIet
isrecommendedthat,whereverpossible,theuseofanaqueoussolvent/vehicleshouldbeconsideredfirst.
UseofcytoBasacytokinesisblocker
20. OneofthemostimportantconsiderationsintheperformanceoftheMNvitassayisensuringthat
thecellsbeingscoredhavecompletedmitosisduringthetreatmentorthepost-treatmentincubationperiod,
ifoneisused.CytoBistheagentthathasbeenmostwidelyusedtoblockcytokinesisbecauseitinhibits
actinassembly,andthuspreventsseparationofdaughtercellsaftermitosis,leadingtotheformationof
binucleatedcells(5)(54)(55).Micronucleusscoring,therefore,canbelimitedtocellsthathavegone
throughmitosisduringoraftertreatment.Theeffectofthetestsubstanceoncellproliferationkineticscan
bemeasuredsimultaneously.CytoBshouldbeusedofasacytokinesisblockerwhenhumanlymphocytes
areusedbecausecellcycletimeswillbevariablewithinculturesandamongdonorsandbecausenotall
lymphocyteswillrespondtoPHA.Othermethodshavebeenusedwhentestingcelllinestodetermineif
thecellsbeingscoredhavedivided;theseareaddressedbelow(seeParagraph26).
21. TheappropriateconcentrationofcytoBshouldbedeterminedbythelaboratoryforeachcelltype
toachievetheoptimalfrequencyofbinucleatedcellsinthesolvent/vehiclecontrolcultures.The
appropriateconcentrationofcytoBisusuallybetween3and6pg/ml.
Measuringcellproliferationandcytotoxicityandchoosingexposureconcentrations
22. Whendeterminingthehighesttestsubstanceconcentrationtobetested,concentrationsthathave
thecapabilityofproducingartifactualpositiveresponses,suchasthoseproducingexcessivecytotoxicity,
precipitationintheculturemedium,andmarkedchangesinpHorosmolality(39)(40)(41),shouldbe
avoided.
23. Measurementsofcellproliferationaremadetoassurethatthetreatedcellshaveundergone
mitosisduringtheassayandthatthetreatmentsareconductedatappropriatelevelsofcytotoxicity(see
Paragraph29).Cytotoxicityshouldbedeterminedwithandwithoutmetabolicactivationincellsthatdo
notrequiremetabolicactivationusingtherelativeincreaseincellcounts(RICC)orrelativepopulation
doubling(RPD)(seeAnnex2forformulas)unlesscytoBisused.WhencytoBisused,cytotoxicitycanbe
determinedusingthereplicationindex(RI)(seeAnnex2forformula).
24. TreatmentofcultureswithcytoB,andmeasurementoftherelativefrequenciesofmononucleate,
binucleate,andmulti-nucleatecellsintheculture,providesanaccuratemethodofquantifyingtheeffecton
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cellproliferationandthecytotoxicorcytostaticactivityofatreatment(5),andensuresthatonlycellsthat
dividedduringoraftertreatmentarescored.
25. InstudieswithcytoB,cytostasis/cytotoxicitycanbequantifiedfromthecytokinesis-block
p(rsoeleifAenranteixon2infdoerxf(oCrBmPulIa)s)(.5)W(h26e)n(5c6y)tooBrmisayusbeeddteoriavsesdesfsrocemllthperoRlIifferraotmioant,laeasCtB5P0I0ocrelRlsIpsehrocuulldtubree
determinedfromatleast500cellsperculture.Thesemeasurementsamongotherscanbeusedtoestimate
cytotoxicitybycomparingvaluesinthetreatedandcontrolcultures.Assessmentofothermarkersof
cytotoxicity(e.gconfluency,cellnumber,apoptosis,necrosis,metaphasecounting)canprovideuseful
information.
26. InstudieswithoutcytoB,itisnecessarytodemonstratethatthecellsscoredintheculturehave
undergonedivisionduringorfollowingtreatmentwiththetestsubstance,otherwisefalsenegative
responsesmaybeproduced.Methodsthathavebeenusedforensuringthatdividedcellsarebeingscored
includeincorporationandsubsequentdetectionofbromodeoxyuridine(BrdU)toidentifycellsthathave
replicated(57),theformationofcloneswhencellsfrompermanentcelllinesaretreatedandscoredinsitu
onamicroscopeslide(ProliferationIndex(PI))(25)(26)(27)(28),orthemeasurementofRelative
PopulationDoubling(RPD)orRelativeIncreaseinCellCount(RICC)orotherprovenmethods(16)(56)
(58)(59)(seeAnnex2forformulas).Assessmentofothermarkersforcytotoxicityorcytostasis(e.g.
confluency,cellnumber,apoptosis,necrosis,metaphasecounting)canprovideusefulinformation.
27. Atleastthreeanalysabletestconcentrationsshouldbeevaluated.Inordertoachievethis,itmay
benecessarytoperformtheexperimentusingalargernumberofcloselyspacedconcentrationsandanalyse
micronucleusformationinthoseconcentrationsprovidingtheappropriaterangeofcytotoxicities.An
alternativestrategyistoperformapreliminarycytotoxicitytesttonarrowtherangeforthedefinitivetest.
28. Thehighestconcentrationshouldaimtoproduce55±5%cytotoxicity.Higherlevelsmayinduce
ccohnrcoemntorsaotmioensdasemlaegcetedasshaoulsdeccoonvdaerryaerfafencgteoffrocmyttohtaotxipcriotdyuc(6i0n)g.5W5he+re5%cyctyottootxoixciictiyty,occtoursli,ttlteheortensot
cytotoxicity.
29. Ifnocytotoxicityorprecipitateisobserved,thehighesttestconcentrationshouldcorrespondto
0.01M,5mg/mLor5pl/mL,whicheveristhelowest.Theconcentrationsselectedforanalysisshould,in
general,beseparatedbyaspacingofnomoreVtlOan Fortestsubstancesthatexhibitasteep
concentration-responsecurve,itmaybenecessarytomorecloselyspacethetestsubstanceconcentrations
sothatculturesinthemoderateandlowtoxicityrangesalsowillbescored.
30. Whensolubilityisalimitingfactor,themaximumconcentration,ifnotlimitedbycytotoxicity,
shouldbethelowestconcentrationatwhichminimalprecipitateisvisibleincultures,providedthereisno
interferencewithscoring.Evaluationofprecipitationshouldbedonebymethodssuchaslightmicroscopy,
notingprecipitatethatpersists,orappearsduringculture(bytheendoftreatment).
Controls
31. Concurrentpositiveandsolvent/vehiclecontrolsbothwithandwithoutmetabolicactivation
shouldbeincludedineachexperiment.
32. Positivecontrolsareneededtodemonstratetheabilityofthecellsused,andthetestprotocol,to
identifyclastogensandaneugens,andtoaffirmthemetaboliccapabilityoftheS9preparation.Thepositive
controlsshouldemployknowninducersofmicronucleusformationatconcentrationsexpectedtogive
small,butreproducibleincreasesoverbackground,anddemonstratethesensitivityofthetestsystem.
5 ©OECD,(2010)
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487 OECD/OCDE
Positivecontrolconcentrationsshouldbechosensothattheeffectsareclearbutdonotimmediatelyreveal
theidentityofthecodedslidestothereader.
b33e.usedtAocdleamsotnosgternattehatbortehquitrheesmmeettaabboolliiccaccotmipvaettieonnc(ee.ga.ndcyctlheopahboislpithyamoifdet;hebetnezsot[as]ypsytreemnet)osdheotuelcdt
clastogens.Otherpositivecontrolsubstancesmaybeusedifjustified.Becausesomepositivecontrolsthat
needmetabolicactivationmaybeactivewithoutexogenousmetabolicactivationundercertaintreatment
conditionsorincertaincelllines,theneedformetabolicactivation,andtheactivityoftheS9preparation,
shouldbetestedintheselectedcelllineandattheselectedconcentrations.
34. Atthepresenttime,noaneugensareknownthatrequiremetabolicactivationfortheirgenotoxic
activity(16).Currentlyacceptedpositivecontrolsforaneugenicactivityare,forexample,colchicineand
vinblastine.Othersubstancesmaybeusediftheyinducemicronucleisolely,orprimarily,through
aneugenicactivity.Toavoidtheneedfortwopositivecontrols(forclastogenicityandaneugenicity)
withoutmetabolicactivation,theaneugenicitycontrolcanserveasthepositivecontrolwithoutS9,andthe
clastogenicitycontrolcanbeusedtotesttheadequacyofthemetabolicactivationsystemused.Positive
controlsforbothclastogenicityandaneugenicityshouldbeusedincellsthatdonotrequireS9.Suggested
positivecontrolchemicalsareincludedinAnnex3.
35. Theuseofchemicalclass-relatedpositivecontrolchemicalsmaybeconsidered,whensuitable
substancesareavailable.Allpositivecontrolsubstancesusedshouldbeappropriateforthecelltypeand
activationconditions.
36. Solvent/vehiclecontrolsshouldbeincludedforeveryharvesttime.Inaddition,untreated
negativecontrols(lackingsolvent/vehicle)shouldalsobeusedunlesstherearepublishedorlaboratory
historicalcontroldatademonstratingthatnogenotoxicorotherdeleteriouseffectsareinducedbythe
chosensolventattheconcentrationsused.
PROCEDURE
TreatmentSchedule
37. Inordertomaximisetheprobabilityofdetectingananeugenorclastogenactingataspecific
stageinthecellcycle,itisimportantthatsufficientnumbersofcellsaretreatedwiththetestsubstance
duringallstagesoftheircellcycles.Thetreatmentscheduleforcelllinesandprimarycellculturesmay,
therefore,differsomewhatfromthatforlymphocyteswhichrequiremitogenicstimulationtobegintheir
cellcycleandtheseareconsideredinParagraphs41-43(16).
38. Theoreticalconsiderations,togetherwithpublisheddata(18)indicatethatmostaneugensand
clastogenswillbedetectedbyashorttermtreatmentperiodof3to6hrsinthepresenceandabsenceofS9,
fsafoatlmelproltwehededbabetgyainrtneiimmnoegvaeoqlruaiotvfatlhteehneetnttdeostoafbstourubetsatta1m.ne5cnet—a(2Sn.ed0etaTiamgberlsoewtt1h)he.Snpaoemrripmoladilno(gfi.oe1r.u5rnet-croev2ae.tr0eydc)teliclmeelcslycmclyaecysleb(e6l)e.enxgCtteehlnledsietdahreierf
itisknownorsuspectedthatthetestsubstanceaffectsthecellcyclingtime(e.g.whentestingnucleoside
analogues).
39. BecauseofthepotentialcytotoxicityofS9preparationsforculturedmammaliancells,an
extendedexposuretreatmentof1.5-2.0normalcellcyclesisusedonlyintheabsenceofS9.Inthe
extendedtreatment,optionsareofferedtoallowtreatmentofthecellswiththetestchemicalintheabsence
orpresenceofcytoB.Theseoptionsaddresssituationswheretheremaybeconcernregardingpossible
interactionsbetweenthetestsubstanceandcytoB.
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40.
schedulesTmhaeysbueggmeosdtiefdiecdeldlepterenadtimnegntonscthheedsutlaebsiliatryeorprreesaecntitveidtyinofTtahbeletest1.suTbhsetsaencegeonrertahleptarretaitcmuelnatr
growthcharacteristicsofthecellsbeingused.Alltreatmentsshouldcommenceandendwhilethecellsare
growingexponentially.Theseschedulesarepresentedinmoredetailsinparagraphs41-47following.
Table1.CelltreatmentandharvesttimesfortheMNvitassay
Lymphocytes,primarycells +S9 Treatfor3-6hrsinthepresenceofS9;
andcelllinestreatedwith removetheS9andtreatmentmedium;
cytoB addfreshmediumandcytoB
harvest1.5—2.0normalcellcycleslater.
-S9 Treatfor3-6hrs;
Short removethetreatmentmedium;
exposure addfreshmediumandcytoB;
harvest1.5—2.0normalcellcycleslater.
-S9 OptionA:Treatfor1.5-2normalcellcyclesinthepresence
Extended ofcytoB;
exposure harvestattheendoftheexposureperiod.
OptionB:Treatfor1.5-2.0normalcellcvcles;
removethetestsubstance;
addfreshmediumandcytoB;
harvest1.5-2.0normalcellcycleslater.
CelllinestreatedwithoutcvtoB
(IdenticaltothetreatmentschedulesoutlinedabovewiththeexceptionthatnocytoBisadded)
Lymphocytes,primarycells,andcelllineswithcytoB
4418.hrsaftFerorPlHyAmphstoicmyutleast,iotnh,ewmhoesntecfyfcilceiesnytnacphprornoiascahtiiosntowisltlarthatvheeedxipsoapspueraeretodt(h5e).teIsnttshuebsitniatnicaelaasts4a4y-,
cmeelldsiuarmeitsrreaetmeodvfeodra3ndtore6plharcsedwiwtihththferetsehstmseudbisutamncceonitnaitnhiengabcsyetnocB,eaannddtphreesceelnlcsearoefhSa9r.veTshteedt1r.e5at—me2n.t0
normalcellcycleslater.
42. Ifbothinitialtestsoftheshort(3-6hrs)treatmentarenegativeorequivocal,asubsequent,
eacxcteepntdaebdle.exHpoowseuvreert,reItatmmiegnhttwbiethmoourteSa9ppirsopursieadt.eTtowofoltlroewatOmpetnitonopAtiofnosrsatriemualvaatieldabllyemapnhodcyarteeseqwuhaelrley
exponentialgrowthmaybedecliningat96hrsfollowingstimulation.Also,culturesofcellsshouldnot
havereachedconfluencebythefinalsamplingtimeinOptionB.
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• hOaprtvieosnteAd:atTthheeecenldlsofartehettrreeaattedmewnittthimteh.etestsubstancefor1.5-2.0normalcellcycles,and
• tOrpetaitomnenBt:mTehdeiucmellissraeremotvreeadteadndwirtehpltahceedtewsittshufbrsetsahncmeedfiorum1,.5an-d2t.h0encoelrlmsalarecehlalrcvyecslteesd.aTftheer
additional1.5-2.0normalcellcycles.
4n3o.tnecessParriymtaorystciemlullsaatendthceelmllwiintehsPshHoAuldfobre44tr-e4a8tehdrsi.naCeslilmsiloatrhemranthnaenrltyomlpyhmopchyotceystsehsoeuxlcdebpettehxaptoisteids
suchthatatthetimeofstudytermination,thecellsarestillinlog-phasegrowth.
CelllineswithoutcytoB
44. Cellsshouldbetreatedfor3-6hrsinthepresenceandabsenceofS9.Thetreatmentmediumis
removedandreplacedwithfreshmedium,andthecellsareharvested1.5-2.0normalcellcycleslater.
45. Ifbothinitialtestsoftheshort(3-6hrs)treatmentarenegativeorequivocal,asubsequent,
extendedexposuretreatment(withoutS9)isused.Twotreatmentoptionsareavailable,bothofwhichare
equallyacceptable:
• hOaprtvieosnteAd:atTthheeecenldlsofartehettrreeaatetdmewnittthimteh.etestsubstancefor1.5-2.0normalcellcycles,and
• tOrpetaitomnenBt:mTehdeiucmellissraeremotvreeadteadndwirtehpltahceedtewsittshufbrsetsahncmeedfiorum1,.5an-d2t.h0encoelrlmsalarecehlalrcvyecslteesd.aTftheer
additional1.5-2.0normalcellcycles.
46. Inmonolayers,mitoticcells(identifiableasbeingroundanddetachingfromthesurface)maybe
presentattheendofthe3-6hrtreatment.Becausethesemitoticcellsareeasilydetached,theycanbelost
whenthemediumcontainingthetestsubstanceisremoved.Careshouldbetakentocollectthesewhen
culturesarewashed,andtoreturnthemtothecultures,toavoidlosingcellsthatareinmitosis,andatrisk
formicronuclei,atthetimeofharvest.
Numberofcultures
47. Duplicate cultures shouldbe usedforeach test substance concentration andforthe
vehicle/solventandnegativecontrolcultures.Whereminimalvariationbetweenduplicateculturescanbe
demonstratedfromhistoricallaboratorydata,itmaybeacceptableforsingleculturestobeused.Ifsingle
culturesareused,itisrecommendedthatanincreasednumberofconcentrationsbeanalysed.
Cellharvestandslidepreparation
48. Eachcultureisharvestedandprocessedseparately.Cellpreparationmayinvolvehypotonic
treatment,butthisstepisnotnecessaryifadequatecellspreadingisotherwiseachieved.Different
techniquescanbeusedinslidepreparationprovidedthathigh-qualitycellpreparationsforscoringare
obtained.Cellcytoplasmshouldberetainedtoallowthedetectionofmicronucleiand(inthecytokinesis-
blockmethod)reliableidentificationofbinucleatecells.
4d(96y.2e)s)(c5a9n).TelhTiehmeisnluaistdeeessocofamaneDboeNfAtshteasipanreetcdiiffuaicsctisnsgtaasivsnaorc(iieao.tuges.damcweritithdhoidnuess,ionrsgaunacghneoa(sn6-1G)DiNeomArsHsapoeeoccrihffsilctuso3tra3ei2snc5.e8nAtnptlDiu-sNkiApnyerstoponeccihinof-riYec
©OCDE,(2010)
OECD/OCDE 487
aspnetciibfoidciesp,rimFeIrSs,HtwoigtehthperanwcietnhtraopmperroipcriDatNeADNprAobecso,untoerrsptraiinmiendg,incasnitbuelaubseeldlintgowidietnhtipfyantcheentcroonmteernet-s
(chromosome/chromosomalfragment)ofmicronucleiifmechanisticinformationoftheirformationisof
interest(15)(16).Othermethodsfordifferentiationbetweenclastogensandaneugensmaybeusedifthey
havebeenshowntobeeffective.
Analysis
50. Allslides,includingthoseofthesolvent/vehicleandthecontrols,shouldbeindependentlycoded
beforethemicroscopicanalysis.Alternatively,codedsamplescanbeanalysedusingavalidated,
automatedflowcytometricorimageanalysissystem.
51. IncytoB-treatedcultures,micronucleusfrequenciesshouldbeanalysedinatleast2000
binucleatedcellsperconcentration(atleast 1000binucleatedcellsperculture;twoculturesper
concentration).Ifsingleculturesareused,atleast2000binucleatedcellsperconcentrationshouldbe
scoredfromthatculture.Ifsubstantiallyfewerthan1000binucleatecellsperculture,or2000ifasingle
cultureisused,areavailableforscoringateachconcentration,andifasignificantincreaseinmicronuclei
isnotdetected,thetestshouldberepeatedusingmorecells,oratlesstoxicconcentrations,whicheveris
appropriate.Careshouldbetakennottoscorebinucleatecellswithirregularshapesorwherethetwo
nucleidiffergreatlyinsize;neithershouldbinucleatecellsbeconfusedwithpoorlyspreadmulti-nucleate
cells.Cellscontainingmorethantwomainnucleishouldnotbeanalysedformicronuclei,asthebaseline
micronucleusfrequencymaybehigherinthesecells(63)(64)Scoringofmononucleatecellsisacceptable
ifthetestsubstanceisshowntointerferewithcytoBactivity.
52. IncelllinesassayedwithoutcytoBtreatment,micronucleishouldbescoredinatleast2000cells
perconcentration(atleast1000cellsperculture;twoculturesperconcentration).Whereonlyoneculture
perconcentrationisused,atleast2000cellsshouldbescoredfromthatculture.
53. WhencytoBisused,aCBPIoranRIshouldbedeterminedtoassesscellproliferation(see
Annex2)usingatleast500cellsperculture.WhentreatmentsareperformedintheabsenceofcytoB,itis
essentialtoprovideevidencethatthecellsbeingscoredhaveproliferated,asdiscussedinParagraphs24-
27.
Acceptabilitycriteria
54. AlaboratoryproposingtousetheMNvitassaydescribedinthisTestGuidelineshould
demonstrateitsabilitytoreliablyandaccuratelydetectsubstancesofknownaneugenicandclastogenic
activity,withandwithoutmetabolicactivation,aswellasknownnegativesubstances,usingthereference
substancesinAnnex3.Asevidenceofitsabilitytoperformthistestmethodcorrectly,thelaboratory
shouldprovideevidencethatthecellsbeingscoredformicronucleusformationhavecompletedone
nucleardivisionifthetestisperformedwithouttheuseofcytoB.
55. ThechemicalsinAnnex3arerecommendedforuseasreferencechemicals.Substituteor
additionalchemicalscanbeincludediftheiractivityisknownandiftheyinducemicronucleibythesame
mechanismsofaction,andiftheyareshowntoberelevanttothechemicalsthatwillbetestedusingthe
MNvitprocedure.Justificationcouldincludeavalidationstudyemployingabroadvarietyofsubstancesor
focusedonanarrowerspectrumbasedonthechemicalclassofthetestsubstanceorthemechanismof
damagebeingstudied.
56. Solvent/vehiclecontrolanduntreatedculturesshouldgivereproduciblylowandconsistent
micronucleifrequencies(typically5-25micronuclei/1000cellsforthecelltypesidentifiedinparagraph
9 ©OECD,(2010)
487 OECD/OCDE
11).Othercelltypesmayhavedifferentrangesofresponseswhichshouldbedeterminedwhenvalidating
themforuseintheMNvitassay.Datafromnegative,solvent,andpositivecontrolsshouldbeusedto
establishhistoricalcontrolranges.Thesevaluesshouldbeusedindecidingtheadequacyoftheconcurrent
negative/positivecontrolsforanexperiment
5o7f.anewcIefllmitnypoer)cahraenpgreosptoosethdefporrotthoecoalss(aey.,g.tuhseenotfheaeuftfoemcattievednienssstoefadthoefcmhaannugaelsshcoourlidngbteedcehnmioqnusetsr;atuesed
beforethemodifiedprotocolcanbeconsideredacceptableforuse.Demonstrationofeffectivenessincludes
demonstrationthatthemajormechanismsofchromosomebreakageandgainorlosscanbedetected,and
thatappropriatepositiveandnegativeresultscanbeachievedfortheclassoftheindividualsubstance,or
thebroadrangeofsubstances,tobetested.
DATAANDREPORTING
Treatmentofresults
58. Ifthecytokinesis-blocktechniqueisused,onlythefrequenciesofbinucleatecellswith
micronuclei(independentofthenumberofmicronucleipercell)areusedintheevaluationofmicronucleus
induction.Scoringofthenumbersofcellswithone,two,ormoremicronucleicouldprovideuseful
information,butisnotmandatory.
59. Concurrentmeasuresofcytotoxicityand/orcytostasisforalltreatedandsolvent/vehiclecontrol
culturesshouldbedetermined(58).TheCBP1ortheRishouldbecalculatedforalltreatedandcontrol
ccuylttouBr,esthaesRmPeaDsuorretmheenRtIsCoCfcoerllPIcyschloeulddelbaeyuwsheedn(steheeAcyntnoekxin2e)s.is-blockmethodisused.Intheabsenceof
60. Individualculturedatashouldbeprovided.Additionally,alldatashouldbesummarisedin
tabularform.
61. ChemicalsthatinducemicronucleiintheMNvitassaymaydosobecausetheyinduce
chromosomebreakagechromosomeloss,oracombinationofthetwo.Furtheranalysisusinganti-
kinetochoreantibodies,centromerespecificinsituprobes,orothermethodsmaybeusedtodetermine
whetherthemechanismofmicronucleusinductionisduetoclastogenicand/oraneugenicactivity.
Evaluationandinterpretationofresults
62. Thereisnorequirementforverificationbyadditionaltestingofaclearpositiveornegative
response.Equivocalresultsmaybeclarifiedbyanalysisofanother1000cellsfromalltheculturestoavoid
lossofblinding.Ifthisapproachdoesnotresolvetheresult,furthertestingshouldbeperformed.
Modificationofstudyparametersoveranextendedornarrowedrangeofconditions,asappropriate,should
beconsideredinfollow-upexperiments.Studyparametersthatmightbemodifiedincludethetest
concentrationspacing,thetimingoftreatmentandcellharvest,and/orthemetabolicactivationconditions.
63. Thereareseveralcriteriafordeterminingapositiveresult,suchasaconcentration-related
increaseorastatisticallysignificantincreaseinthenumberofcellscontainingmicronuclei.Thebiological
relevanceoftheresultsshouldbeconsideredfirst.Considerationofwhethertheobservedvaluesarewithin
oroutsideofthehistoricalcontrolrangecanprovideguidancewhenevaluatingthebiologicalsignificance
oftheresponse.Appropriatestatisticalmethodsmaybeusedasanaidinevaluatingthetestresults(65).
However,theresultsofstatisticaltestingshouldbeassessedwithrespecttodose-responserelationship.
Reproducibilityandhistoricaldatashouldalsobetakenintoconsideration.”
©OCDE,(2010) 10