OECD TG492眼刺激性体外测试

OECD/OCDE492Adopted:9October20171©OECD,(2017)Youarefreetousethismaterialsubjecttothetermsandconditionsavailableathttp://www.oecd.org/termsandconditions/.ThisGuidelinewasadoptedbytheOECDCouncilbywrittenprocedureon9October2017[C(2017)97].OECDGUIDELINEFORTHETESTINGOFCHEMICALSReconstructedhumanCornea-likeEpithelium(RhCE)testmethodforidentifyingchemicalsnotrequiringclassificationandlabellingforeyeirritationorseriouseyedamageINTRODUCTION1.Seriouseyedamagereferstotheproductionoftissuedamageintheeye,orseriousphysicaldecayofvision,followingapplicationofatestchemicaltotheanteriorsurfaceoftheeye,whichisnotfullyreversiblewithin21daysofapplication,asdefinedbytheUnitedNationsGloballyHarmonizedSystemofClassificationandLabellingofChemicals(UNGHS)(1).AlsoaccordingtoUNGHS,eyeirritationreferstotheproductionofchangesintheeyefollowingtheapplicationofatestchemicaltotheanteriorsurfaceoftheeye,whicharefullyreversiblewithin21daysofapplication.TestchemicalsinducingseriouseyedamageareclassifiedasUNGHSCategory1,whilethoseinducingeyeirritationareclassifiedasUNGHSCategory2.TestchemicalsnotclassifiedforeyeirritationorseriouseyedamagearedefinedasthosethatdonotmeettherequirementsforclassificationasUNGHSCategory1or2(2Aor2B)i.e.,theyarereferredtoasUNGHSNoCategory.2.Theassessmentofseriouseyedamage/eyeirritationhastypicallyinvolvedtheuseoflaboratoryanimals(OECDTestGuideline(TG)405;adoptedin1981andrevisedin1987,2002,2012and2017)(2).ThechoiceofthemostappropriatetestmethodandtheuseofthisTestGuidelineshouldbeseeninthecontextoftheOECDGuidanceDocumentonanIntegratedApproachesonTestingandAssessmentforSeriousEyeDamageandEyeirritation(39).3.ThisTestGuidelinedescribesaninvitroprocedureallowingtheidentificationofchemicals(substancesandmixtures)notrequiringclassificationandlabellingforeyeirritationorseriouseyedamageinaccordancewithUNGHS.Itmakesuseofreconstructedhumancornea-likeepithelium(RhCE)whichcloselymimicsthehistological,morphological,biochemicalandphysiologicalpropertiesofthehumancornealepithelium.Fourotherinvitrotestmethodshavebeenvalidated,consideredscientificallyvalidandadoptedasOECDTestGuidelines(TGs)437(3),438(4),460(5)and491(6)toaddressthehumanhealthendpointseriouseyedamage/eyeirritation.492OECD/OCDE2©OECD,(2017)4.TwovalidatedtestmethodsusingcommerciallyavailableRhCEmodelsareincludedinthisTestGuideline.Validationstudiesforassessingeyeirritation/seriouseyedamagehavebeenconducted(7)(8)(9)(10)(11)(12)(13)usingtheEpiOcular™EyeIrritationTest(EIT)andtheSkinEthic™HumanCornealEpithelium(HCE)EyeIrritationTest(EIT).EachofthesemethodsmakesuseofcommerciallyavailableRhCEtissueconstructsastestsystem,whicharereferredtointhefollowingtextastheValidatedReferenceMethods–VRM1andVRM2,respectively.Fromthesevalidationstudiesandtheirindependentpeerreview(9)(12)itwasconcludedthattheEpiOcular™EITandSkinEthic™HCEEITareabletocorrectlyidentifychemicals(bothsubstancesandmixtures)notrequiringclassificationandlabellingforeyeirritationorseriouseyedamageaccordingtoUNGHS(1),andthetestmethodswererecommendedasscientificallyvalidforthatpurpose(13).5.Itiscurrentlygenerallyacceptedthat,intheforeseeablefuture,nosingleinvitrotestmethodwillbeabletofullyreplacetheinvivoDraizeeyetest(2)(14)topredictacrossthefullrangeofseriouseyedamage/eyeirritationresponsesfordifferentchemicalclasses.However,strategiccombinationsofseveralalternativetestmethodswithin(tiered)testingstrategiessuchastheBottom-Up/Top-DownapproachmaybeabletofullyreplacetheDraizeeyetest(15).TheBottom-Upapproach(15)isdesignedtobeusedwhen,basedonexistinginformation,achemicalisexpectednottocausesufficienteyeirritationtorequireaclassification,whiletheTop-Downapproach(15)isdesignedtobeusedwhen,basedonexistinginformation,achemicalisexpectedtocauseseriouseyedamage.TheEpiOcular™EITandSkinEthic™HCEEITarerecommendedtoidentifychemicalsthatdonotrequireclassificationforeyeirritationorseriouseyedamageaccordingtoUNGHS(UNGHSNoCategory)(1)withoutfurthertesting,withinatestingstrategysuchastheBottom-Up/Top-DownapproachsuggestedbyScottetal.e.g.,asaninitialstepinaBottom-UpapproachorasoneofthelaststepsinaTop-Downapproach(15).However,theEpiOcular™EITandSkinEthic™HCEEITarenotintendedtodifferentiatebetweenUNGHSCategory1(seriouseyedamage)andUNGHSCategory2(eyeirritation).Thisdifferentiationwillneedtobeaddressedbyanothertierofateststrategy(15).Atestchemicalthatisidentifiedasrequiringclassificationforeyeirritation/seriouseyedamagewithEpiOcular™EITorSkinEthic™HCEEITwillthusrequireadditionaltesting(invitroand/orinvivo)toreachadefinitiveconclusion(UNGHSNoCategory,Category2orCategory1),usinge.g.,TG437,438,460or491.6.ThepurposeofthisTestGuidelineistodescribetheprocedureusedtoevaluatetheeyehazardpotentialofatestchemicalbasedonitsabilitytoinducecytotoxicityinaRhCEtissueconstruct,asmeasuredbytheMTTassay(16)(seeparagraph21).TheviabilityoftheRhCEtissuefollowingexposuretoatestchemicalisdeterminedincomparisontotissuestreatedwiththenegativecontrolsubstance(%viability),andisthenusedtopredicttheeyehazardpotentialofthetestchemical.7.PerformanceStandards(17)areavailabletofacilitatethevalidationofnewormodifiedinvitroRhCE-basedtestmethodssimilartoEpiOcular™EITandSkinEthic™HCEEIT,inaccordancewiththeprinciplesofGuidanceDocumentNo.34(18),andallowfortimelyamendmentofthisTestGuidelinefortheirinclusion.MutualAcceptanceofData(MAD)willonlybeguaranteedfortestmethodsvalidatedaccordingtothePerformanceStandards,ifthesetestmethodshavebeenreviewedandincludedinthisTestGuidelinebytheOECD.DEFINITIONS8.DefinitionsareprovidedinAnnexI.OECD/OCDE4923©OECD,(2017)INITIALCONSIDERATIONSANDLIMITATIONS9.ThisTestGuidelineisbasedoncommercialthree-dimensionalRhCEtissueconstructsthatareproducedusingeitherprimaryhumanepidermalkeratinocytes(i.e.,EpiOcular™OCL-200)orhumanimmortalizedcornealepithelialcells(i.e.,SkinEthic™HCE/S).TheEpiOcular™OCL-200andSkinEthic™HCE/SRhCEtissueconstructsaresimilartotheinvivocornealepitheliumthree-dimensionalstructureandareproducedusingcellsfromthespeciesofinterest(19)(20).Moreover,thetestmethodsdirectlymeasurecytotoxicityresultingfrompenetrationofthechemicalthroughthecorneaandproductionofcellandtissuedamage;thecytotoxicresponsethendeterminestheoverallinvivoseriouseyedamage/eyeirritationoutcome.Celldamagecanoccurbyseveralmodesofaction(seeparagraph20),butcytotoxicityplaysanimportant,ifnottheprimary,mechanisticroleindeterminingtheoverallseriouseyedamage/eyeirritationresponseofachemical,manifestedinvivomainlybycornealopacity,iritis,conjunctivalrednessand/orconjunctivalchemosis,regardlessofthephysicochemicalprocessesunderlyingtissuedamage.10.Awiderangeofchemicals,coveringalargevarietyofchemicaltypes,chemicalclasses,molecularweights,LogPs,chemicalstructures,etc.,havebeentestedinthevalidationstudyunderlyingthisTestGuideline.TheEpiOcular™EITvalidationdatabasecontained113chemicalsintotal,covering95differentorganicfunctionalgroupsaccordingtoanOECDQSARtoolboxanalysis(8).Themajorityofthesechemicalsrepresentedmono-constituentsubstances,butseveralmulti-constituentsubstances(including3homopolymers,5copolymersand10quasipolymers)werealsoincludedinthestudy.IntermsofphysicalstateandUNGHSCategories,the113testedchemicalsweredistributedasfollows:13Category1liquids,15Category1solids,6Category2Aliquids,10Category2Asolids,7Category2Bliquids,7Category2Bsolids,27NoCategoryliquidsand28NoCategorysolids(8).TheSkinEthic™HCEEITvalidationdatabasecontained200chemicalsintotal,covering165differentorganicfunctionalgroups(8)(10)(11).Themajorityofthesechemicalsrepresentedmono-constituentsubstances,butseveralmulti-constituentsubstances(including10polymers)werealsoincludedinthestudy.IntermsofphysicalstateandUNGHSCategories,the200testedchemicalsweredistributedasfollows:27Category1liquids,24Category1solids,19Category2Aliquids,10Category2Asolids,9Category2Bliquids,8Category2Bsolids,50NoCategoryliquidsand53NoCategorysolids(10)(11).11.ThisTestGuidelineisapplicabletosubstancesandmixtures,andtosolids,liquids,semi-solidsandwaxes.Theliquidsmaybeaqueousornon-aqueous;solidsmaybesolubleorinsolubleinwater.Wheneverpossible,solidsshouldbegroundtoafinepowderbeforeapplication;nootherpre-treatmentofthesampleisrequired.Gasesandaerosolshavenotbeenassessedinavalidationstudy.WhileitisconceivablethatthesecanbetestedusingRhCEtechnology,thecurrentTestGuidelinedoesnotallowtestingofgasesandaerosols.12.TestchemicalsabsorbinglightinthesamerangeasMTTformazan(naturallyoraftertreatment)andtestchemicalsabletodirectlyreducethevitaldyeMTT(toMTTformazan)mayinterferewiththetissueviabilitymeasurementsandneedtheuseofadaptedcontrolsforcorrections.ThetypeofadaptedcontrolsthatmayberequiredwillvarydependingonthetypeofinterferenceproducedbythetestchemicalandtheprocedureusedtoquantifyMTTformazan(seeparagraphs36-42).13.Resultsgeneratedinpre-validation(21)(22)andfullvalidation(8)(10)(11)studieshavedemonstratedthatbothEpiOcular™EITandSkinEthic™HCEEITaretransferabletolaboratoriesconsideredtobenaïveintheconductoftheassaysandalsotobereproduciblewithin-andbetweenlaboratories.Basedonthesestudies,thelevelofreproducibilityintermsofconcordanceofpredictionsthatcanbeexpectedfromEpiOcular™EITfromdataon113chemicalsisintheorderof95%within492OECD/OCDE4©OECD,(2017)laboratoriesand93%betweenlaboratories.ThelevelofreproducibilityintermsofconcordanceofpredictionsthatcanbeexpectedfromSkinEthic™HCEEITfromdataon120chemicalsisintheorderof92%withinlaboratoriesand95%betweenlaboratories.14.TheEpiOcular™EITcanbeusedtoidentifychemicalsthatdonotrequireclassificationforeyeirritationorseriouseyedamageaccordingtotheUNGHSclassificationsystem(1).Consideringthedataobtainedinthevalidationstudy(8),theEpiOcular™EIThasanoverallaccuracyof80%(basedon112chemicals),sensitivityof96%(basedon57chemicals),falsenegativerateof4%(basedon57chemicals),specificityof63%(basedon55chemicals)andfalsepositiverateof37%(basedon55chemicals),whencomparedtoreferenceinvivorabbiteyetestdata(OECDTG405)(2)(14)classifiedaccordingtotheUNGHSclassificationsystem(1).Astudywhere97liquidagrochemicalformulationsweretestedwithEpiOcular™EITdemonstratedasimilarperformanceofthetestmethodforthistypeofmixturesasobtainedinthevalidationstudy(23).The97formulationsweredistributedasfollows:21Category1,19Category2A,14Category2Band43NoCategory,classifiedaccordingtotheUNGHSclassificationsystem(1)basedonreferenceinvivorabbiteyetestdata(OECDTG405)(2)(14).Anoverallaccuracyof82%(basedon97formulations),sensitivityof91%(basedon54formulations),falsenegativerateof9%(basedon54formulations),specificityof72%(basedon43formulations)andfalsepositiverateof28%(basedon43formulations)wereobtained(23).15.TheSkinEthic™HCEEITcanbeusedtoidentifychemicalsthatdonotrequireclassificationforeyeirritationorseriouseyedamageaccordingtotheUNGHSclassificationsystem(1).Consideringthedataobtainedinthevalidationstudy(10)(11),theSkinEthic™HCEEIThasanoverallaccuracyof84%(basedon200chemicals),sensitivityof95%(basedon97chemicals),falsenegativerateof5%(basedon97chemicals),specificityof72%(basedon103chemicals)andfalsepositiverateof28%(basedon103chemicals),whencomparedtoreferenceinvivorabbiteyetestdata(OECDTG405)(2)(14)classifiedaccordingtotheUNGHSclassificationsystem(1).16.ThefalsenegativeratesobtainedwithbothRhCEtestmethods,witheithersubstancesormixtures,fallwithinthe12%overallprobabilitythatchemicalsareidentifiedaseitherUNGHSCategory2orUNGHSNoCategorybytheinvivoDraizeeyetest,inrepeatedtests;thisisduetothemethod'sinherentwithin-testvariability(24).ThefalsepositiveratesobtainedwithbothRhCEtestmethodswitheithersubstancesormixturesarenotcriticalinthecontextofthisTestGuidelinesincealltestchemicalsthatproduceatissueviabilityequalorlowerthantheestablishedcut-offs(seeparagraph44)willrequirefurthertestingwithotherinvitrotestmethods,orasalastoptioninrabbits,dependingonregulatoryrequirements,usingasequentialtestingstrategyinaweight-of-evidenceapproach.Thesetestmethodscanbeusedforalltypesofchemicals,wherebyanegativeresultshouldbeacceptedfornotclassifyingachemicalforeyeirritationandseriouseyedamage(UNGHSNoCategory).TheappropriateregulatoryauthoritiesshouldbeconsultedbeforeusingtheEpiOcular™EITandSkinEthic™HCEEITunderclassificationschemesotherthanUNGHS.17.AlimitationofthisTestGuidelineisthatitdoesnotallowdiscriminationbetweeneyeirritation/reversibleeffectsontheeye(Category2)andseriouseyedamage/irreversibleeffectsontheeye(Category1),norbetweeneyeirritants(optionalCategory2A)andmildeyeirritants(optionalCategory2B),asdefinedbyUNGHS(1).Forthesepurposes,furthertestingwithotherinvitrotestmethodsisrequired.OECD/OCDE4925©OECD,(2017)18.Theterm"testchemical"isusedinthisTestGuidelinetorefertowhatisbeingtested1andisnotrelatedtotheapplicabilityoftheRhCEtestmethodtothetestingofsubstancesand/ormixtures.PRINCIPLEOFTHETEST19.Thetestchemicalisappliedtopicallytoaminimumoftwothree-dimensionalRhCEtissueconstructsandtissueviabilityismeasuredfollowingexposureandapost-treatmentincubationperiod.TheRhCEtissuesarereconstructedfromprimaryhumanepidermalkeratinocytesorhumanimmortalizedcornealepithelialcells,whichhavebeenculturedforseveraldaystoformastratified,highlydifferentiatedsquamousepitheliummorphologicallysimilartothatfoundinthehumancornea.TheEpiOcular™RhCEtissueconstructconsistsofatleast3viablelayersofcellsandanon-keratinizedsurface,showingacornea-likestructureanalogoustothatfoundinvivo.TheSkinEthic™HCERhCEtissueconstructconsistsofatleast4viablelayersofcellsincludingcolumnarbasalcells,transitionalwingcellsandsuperficialsquamouscellssimilartothatofthenormalhumancornealepithelium(20)(26).20.Chemical-inducedseriouseyedamage/eyeirritation,manifestedinvivomainlybycornealopacity,iritis,conjunctivalrednessand/orconjunctivalchemosis,istheresultofacascadeofeventsbeginningwithpenetrationofthechemicalthroughthecorneaand/orconjunctivaandproductionofdamagetothecells.Celldamagecanoccurbyseveralmodesofaction,including:cellmembranelysis(e.g.,bysurfactants,organicsolvents);coagulationofmacromolecules(particularlyproteins)(e.g.,bysurfactants,organicsolvents,alkalisandacids);saponificationoflipids(e.g.,byalkalis);andalkylationorothercovalentinteractionswithmacromolecules(e.g.,bybleaches,peroxidesandalkylators)(15)(27)(28).However,ithasbeenshownthatcytotoxicityplaysanimportant,ifnottheprimary,mechanisticroleindeterminingtheoverallseriouseyedamage/eyeirritationresponseofachemicalregardlessofthephysicochemicalprocessesunderlyingtissuedamage(29)(30).Moreover,theseriouseyedamage/eyeirritationpotentialofachemicalisprincipallydeterminedbytheextentofinitialinjury(31),whichcorrelateswiththeextentofcelldeath(29)andwiththeextentofthesubsequentresponsesandeventualoutcomes(32).Thus,slightirritantsgenerallyonlyaffectthesuperficialcornealepithelium,themildandmoderateirritantsdamageprincipallytheepitheliumandsuperficialstromaandthesevereirritantsdamagetheepithelium,deepstromaandattimesthecornealendothelium(30)(33).ThemeasurementofviabilityoftheRhCEtissueconstructaftertopicalexposuretoatestchemicaltoidentifychemicalsnotrequiringclassificationforseriouseyedamage/eyeirritancy(UNGHSNoCategory)isbasedontheassumptionthatallchemicalsinducingseriouseyedamageoreyeirritationwillinducecytotoxicityinthecornealepitheliumand/orconjunctiva.21.RhCEtissueviabilityisclassicallymeasuredbyenzymaticconversionofthevitaldyeMTT[3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide;Thiazolylbluetetrazoliumbromide;CASnumber298-93-1]bytheviablecellsofthetissueintoablueMTTformazansaltthatisquantitativelymeasuredafterextractionfromtissues(16).ChemicalsnotrequiringclassificationandlabellingaccordingtoUNGHS(NoCategory)areidentifiedasthosethatdonotdecreasetissueviabilitybelowadefinedthreshold(i.e.,tissueviability>60%,inEpiOcular™EITandSkinEthic™HCEEITL2,or>50%,inSkinEthic™HCEEITS3)(seeparagraph44).1InJune2013,theJointMeetingagreedthatwherepossible,amoreconsistentuseoftheterm“testchemical”describingwhatisbeingtestedshouldnowbeappliedinnewandupdatedTestGuidelines.2EITL:EITforliquidsinthecaseofSkinEthic™HCE3EITS:EITforsolidsinthecaseofSkinEthic™HCE492OECD/OCDE6©OECD,(2017)DEMONSTRATIONOFPROFICIENCY22.PriortoroutineuseofRhCEtestmethodsforregulatorypurposes,laboratoriesshoulddemonstratetechnicalproficiencybycorrectlypredictingthefifteenproficiencychemicalslistedinTable1.ThesechemicalswereselectedfromthechemicalsusedinthevalidationstudiesoftheVRMs(8)(10)(11).Theselectionincludes,totheextentpossible,chemicalsthat:(i)coverdifferentphysicalstates;(ii)coverthefullrangeofinvivoseriouseyedamage/eyeirritationresponsesbasedonhighqualityresultsobtainedinthereferenceinvivorabbiteyetest(OECDTG405)(2)(14)andtheUNGHSclassificationsystem(i.e.,Categories1,2A,2B,orNoCategory)(1);(iii)coverthevariousinvivodriversofclassification(24)(25);(iv)arerepresentativeofthechemicalclassesusedinthevalidationstudy(8)(10)(11);(v)coveragoodandwiderepresentationoforganicfunctionalgroups(8)(10)(11);(vi)havechemicalstructuresthatarewell-defined(8)(10)(11);(vii)arecolouredand/ordirectMTTreducers;(viii)producedreproducibleresultsinRhCEtestmethodsduringtheirvalidations;(ix)werecorrectlypredictedbyRhCEtestmethodsduringtheirvalidationstudies;(x)coverthefullrangeofinvitroresponsesbasedonhighqualityRhCEtestmethodsdata(0to100%viability);(xi)arecommerciallyavailable;and(xii)arenotassociatedwithprohibitiveacquisitionand/ordisposalcosts.Insituationswherealistedchemicalisunavailableorcannotbeusedforotherjustifiedreasons,anotherchemicalfulfillingthecriteriadescribedabove,e.g.fromthechemicalsusedinthevalidationoftheVRM,couldbeused.Suchdeviationsshouldhoweverbejustified.Table1:ListofproficiencychemicalsChemicalNameCASRNOrganicFunctionalGroup1PhysicalStateVRM1viability(%)2VRM2viability(%)3VRMPredictionMTTReducerColourinterf.InVivoCategory14Methylthioglycolate2365-48-2Carboxylicacidester;ThioalcoholL10.9±6.45.5±7.4NopredictioncanbemadeY(strong)NHydroxyethylacrylate818-61-1Acrylate;AlcoholL7.5±4.751.6±1.0NopredictioncanbemadeNN2,5-Dimethyl-2,5-hexanediol110-03-2AlcoholS2.3±0.20.2±0.1NopredictioncanbemadeNNSodiumoxalate62-76-0OxocarboxylicacidS29.0±1.25.3±4.1NopredictioncanbemadeNNInVivoCategory2A42,4,11,13-Tetraazatetradecane-diimidamide,N,N''-bis(4-chlorophenyl)-3,12-diimino-,di-D-gluconate(20%,aqueous)618472-51-0Aromaticheterocyclichalide;Arylhalide;Dihydroxylgroup;GuanidineL4.0±1.11.3±0.6NopredictioncanbemadeNY(weak)Sodiumbenzoate532-32-1Aryl;CarboxylicacidS3.5±2.60.6±0.1NopredictioncanbemadeNNInVivoCategory2B4OECD/OCDE4927©OECD,(2017)ChemicalNameCASRNOrganicFunctionalGroup1PhysicalStateVRM1viability(%)2VRM2viability(%)3VRMPredictionMTTReducerColourinterf.Diethyltoluamide134-62-3BenzamideL15.6±6.32.8±0.9NopredictioncanbemadeNN2,2-Dimethyl-3-methylenebicyclo[2.2.1]heptane79-92-5Alkane,branchedwithtertiarycarbon;Alkene;Bicycloheptane;Bridged-ringcarbocycles;CycloalkaneS4.7±1.515.8±1.1NopredictioncanbemadeNNInVivoNoCategory41-Ethyl-3-methylimidazoliumethylsulphate342573-75-5Alkoxy;Ammoniumsalt;Aryl;Imidazole;SulphateL79.9±6.479.4±6.2NoCatNNDicaprylylether629-82-3Alkoxy;EtherL97.8±4.395.2±3.0NoCatNNPiperonylbutoxide51-03-6Alkoxy;Benzodioxole;Benzyl;EtherL104.2±4.296.5±3.5NoCatNNPolyethyleneglycol(PEG-40)hydrogenatedcastoroil61788-85-0Acylal;Alcohol;Allyl;EtherViscous77.6±5.489.1±2.9NoCatNN1-(4-Chlorophenyl)-3-(3,4-dichlorophenyl)urea101-20-2Aromaticheterocyclichalide;Arylhalide;UreaderivativesS106.7±5.3101.9±6.6NoCatNN2,2'-Methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)-phenol)103597-45-1Alkanebranchedwithquaternarycarbon;Fusedcarbocyclicaromatic;Fusedsaturatedheterocycles;Precursorsquinoidcompounds;tert-ButylS102.7±13.497.7±5.6NoCatNNPotassiumtetrafluoroborate14075-53-7InorganicSaltS88.6±3.392.9±5.1NoCatNNAbbreviations:CASRN=ChemicalAbstractsServiceRegistryNumber;UNGHS=UnitedNationsGloballyHarmonizedSystemofClassificationandL