Test no. 438 [electronic resource] : isolated chicken eye test method for identifying

2013·0.61 MB·English

by  Organisation for Economic Co-operation and Development

OECD/OCDE 438
Adopted:
26July2013
OECDGUIDELINESFORTHETESTINGOFCHEMICALS
Isoalnatdedii)CChhiecmkiecnaElsyeNoTetsRteMqeutirhiondgfColrasIsdiefnitciaftyiionngfio)rCEhyeemiIcrarlitsatIinodnuocrinSgerSieoruisouEsyeEyDeaDmaamgaege
INTRODUCTION
1. TheIsolatedChickenEye(ICE)testmethodwasevaluatedbytheInteragencyCoordinating
CommitteeontheValidationofAlternativeMethods(ICCVAM),inconjunctionwiththeEuropean
CentrefortheValidationofAlternativeMethods(ECVAM)andtheJapaneseCentreforthe
ValidationofAlternativeMethods(JaCVAM),in2006and2010(1)(2)(3).Inthefirstevaluation,the
ICEwasendorsedasascientificallyvalidtestmethodforuseasascreeningtesttoidentifychemicals
(substancesandmixtures)inducingseriouseyedamage(Category1)asdefinedbytheUnitedNations
(UN)GloballyHarmonizedSystemofClassificationandLabellingofChemicals(GHS)(1)(2)(4).In
tchheemsieccaolnsdnoetvaclluaastsiiofni,edtfhoerIeyCeEitrersittatmieonthoordsewraiosuseveayleuadtaemdafgoeraussedeafsinaedsbcyreeUnNinGgHteSst(3t)o(i4d)e.ntTihfey
resultsfromthevalidationstudyandthepeerreviewpanelrecommendationsmaintainedtheoriginal
recommendationforusingtheICEforclassificationofchemicalsinducingseriouseyedamage(UN
GtvoaHlaiSldsaotCiiaonntc.elgAuodtreythoatt1h)es,rtacagaset,etgnhooerifeuasrvtawhieelrarberleescuogdgmaemtseatbneaddsa.etAiroernems-aefivonareladunatueinxopcnahonafsnitgoheendionfsivtnihtcereoICtahEnedapiopnrlivigiciavnboaildliattIyaCsdeCotmVuasAieMnd
inthevalidationstudywasmadewiththefocusofevaluatingtheusefulnessoftheICEtoidentify
chemicalsnotrequiringclassificationforeyeirritationorseriouseyedamage(5).Thisre-evaluation
cfoorncelyuedeidrrtihtaattiotnheaIndCEsetersitoumsetehyoeddcaamnagalesoasbedeufsiendedtobiydetnhteifyUcNheGmiHcSals(4)no(t5)r.eqTuhiirsinTgecsltasGsuiifidcealtiinoen
(adoptedin2009andupdatedin2013)includestherecommendedusesandlimitationsoftheICEtest
methodbasedontheseevaluations.Themaindifferencesbetweentheoriginal2009versionandthe
c2h0e1m3icuapldsatneodtrveeqrusiiroinnginccllausdseif,icbauttionaraeccnoortdilnigmittoedthteo,UtNheGuHseSoCflatshseifiIcCaEtiotnestSymsetetmh,odantoupiddaetnteiftoy
thetestreportelements,anupdateofAnnex1ondefinitions,andanupdatetoAnnex2onthe
proficiencychemicals.
2. Itiscurrentlygenerallyacceptedthat,intheforeseeablefuture,nosingleinvitroeye
irritationtestwillbeabletoreplacetheinvivoDraizeeyetesttopredictacrossthefullrangeof
irritationfordifferentchemicalclasses.However,strategiccombinationsofseveralalternativetest
Dmeotwhnodasppwriotahcihn(a7)(tiisedreeds)igtneestdintgobsetruatseegdywmhaeny,bbeasaebdleontoexriesptliancgeitnhfeorDmrataiiozne,eaycehetemsitca(6l).isTehxepeTcotpe-d
tohavehighirritancypotential,whiletheBottom-Upapproach(7)isdesignedtobeusedwhen,based
onexistinginformation,achemicalisexpectednottocausesufficienteyeirritationtorequirea
classification.TheICEtestmethodisaninvitrotestmethodthatcanbeused,undercertain
circumstancesandwithspecificlimitationsasdescribedinparagraphs8to10foreyehazard
classificationandlabellingofchemicals.Whileitisnotconsideredvalidasastand-alonereplacement
©OECD,(2013) 1
YftoroouwmriattrhteeenfOrpEeeeCrmtDoi,suspsireoonvthifidrseodmmattthehereisaOolEufCroDcre.peisrsdounlayl,mennotni-ocnoemdm.eArnciyaclopmumreprocsieaslwuistehoouftthsiesekmiantegripariloriscsounbsjeenctt
438 OECD/OCDE
fortheinvivorabbiteyetest,theICEtestmethodisrecommendedasaninitialstepwithinatesting
ssterraitoeugsyesyuecdhaamsagtehe,Ti.oe.p,-cDheomwincaalpsptrooabcehclsausgsgiefsitededasbyUNScoGtHtSetCaal.te(g7o)rtyo1idweintthiofyutchfuermtihcearltsesitnidnugc(i4n)g.
TeyheeiIrCriEtatteisotnmoertsheordioiussaelsyoerdeacmoamgmeenadseddeftionieddenbtyifythceheUmNicaGlHsStha(tNodoCnaottegroerqyu,irNeCc)las(s4i)f,icaantdionmafoyr
thereforebeusedasaninitialstepwithinaBottom-Uptestingstrategyapproach(7).However,a
chemicalthatisnotpredictedascausingseriouseyedamageorasnotclassifiedforeye
irritation/seriouseyedamagewiththeICEtestmethodwouldrequireadditionaltesting(invitroand/or
invivo)toestablishadefinitiveclassification.Furthermore,theappropriateregulatoryauthorities
sthhaonultdhebeUNconGsHuSlt.edbeforeusingtheICEinabottomupapproachunderotherclassificationschemes
3. ThepurposeofthisTestGuidelineistodescribetheproceduresusedtoevaluatetheeye
hazardpotentialofatestchemicalasmeasuredbyitsabilitytoinduceornottoxicityinanenucleated
chickeneye.Toxiceffectstothecorneaaremeasuredby(i)aqualitativeassessmentofopacity,(ii)a
qualitativeassessmentofdamagetoepitheliumbasedonapplicationoffluoresceintotheeye
(fluoresceinretention),(iii)aquantitativemeasurementofincreasedthickness(swelling),and(iv)a
qualitativeevaluationofmacroscopicmorphologicaldamagetothesurface.Thecornealopacity,
swelling,anddamageassessmentsfollowingexposuretoatestchemicalareassessedindividuallyand
thencombinedtoderiveanEyeIrritancyClassification.
4. DefinitionsareprovidedinAnnex1.
INITIALCONSIDERATIONSANDLIMITATIONS
51.60(8),wThhiicshTweastsGdueivdeelloipneedifsoblalosweidngonthteheICpCroVtoAcMolisnutgegrensattieodnailnvtahleidOatEiConDstGuudiyda(1n)ce(3)Do(9c)u,mweintth
contributionsfromtheEuropeanCentrefortheValidationofAlternativeMethods,theJapanese
CenterfortheValidationofAlternativeMethods,andTNOQualityofLifeDepartmentofToxicology
paunbdliAsphpeldiperdotoPchoalrs,maacsowleolglyas(tNheethceurrlraenndts)p.rotTohceolpursoetdocboylTiNsOba(s1e0d)(o1n1)i(n1f2o)r(m1a3t)io(n14)o.btainedfrom
6. AwiderangeofchemicalshasbeentestedinthevalidationunderlyingthisTestGuideline
andtheempiricaldatabaseofthevalidationstudyamountedto152chemicalsincluding72substances
and80mixtures(5).TheTestGuidelineisapplicabletosolids,liquids,emulsionsandgels.The
liquidsmaybeaqueousornon-aqueous;solidsmaybesolubleorinsolubleinwater.Gasesand
aerosolshavenotbeenassessedyetinavalidationstudy.
7cl.ihmeimtiactailonsTshtofeobrIeCthEcelatIsessCitEfmieteedtsthasomdeUtcNhaonGdbHeaSruesCbeaadtsetegodorioydnen1ttih(fe4y)h.cihWgehhmeifcanlasluesspeiodnsdifutoicrvientghriassteeprsuirofuposorseeay,lecotdhhoealmsiadgeaennt,difiti.ehe.ed,
hcteoisngtthecxfhatelms(iecUanNlesgGatHthiSavteCcraoattmeegesofrooyurt1snoieldigedansttiiavfneidedwsouaursflnadcottbaenbtessiunb(gs1)eUq(uN3e)nGt(l9H)y.StHeCosatwteeedvgewoirrt,yhf1oa)tlhsaeerrenaendgoeatqtuciraviteteirlcaaytlevssailinincdeatthaeilsdl
invitrotest(s),orasalastoptioninrabbits,dependingonregulatoryrequirements,usingasequential
testingstrategyinaweight-of-evidenceapproach.Itshouldbenotedthatsolidsmayleadtovariable
andextremeexposureconditionsintheinvivoDraizeeyeirritationtest,whichmayresultinirrelevant
predictionsoftheirtrueirritationpotential(15).Investigatorscouldconsiderusingthistestmethodfor
alltypesofchemicals,wherebyapositiveresultshouldbeacceptedasindicativeofseriouseye
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OECD/OCDE 438
damage,i.e.,UNGHSCategory1classificationwithoutfurthertesting.However,positiveresults
obtainedwithalcoholsshouldbeinterpretedcautiouslyduetoriskofover-prediction.
I8.CEtestmWehtehonduhsaesdatnooivdeernatlilfyaccchuermaiccyalosfi8n6du%ci(n1g20s/e1r4i0o)u,saeyfealsdeampoasgietiv(eUrNatGeHofS6C%at(e7g/o1r1y3)1)a,ndthae
faaclcsoerdnienggattiovetheraUteNofGH48S%cl(a1s3si/f2i7c)atwiohnesnysctoemmp(a4r)e(d5)t.oinvivorabbiteyetestmethoddataclassified
9f.oreyeirTrihteatiIoCnEortessetrmioeutsheoydecdanamaalgsoebuenduesredthteoiUdNentGifHySchcelmasisciafliscatthiaotndsoysntoetmr(e4q)u.iTreheclaaspspirfoipcraitaitoen
regulatoryauthoritiesshouldbeconsultedbeforeusingtheICEinabottomupapproachunderother
classificationschemes.Thistestmethodcanbeusedforalltypesofchemicals,wherebyanegative
resultcouldbeacceptedfornotclassifyingachemicalforeyeirritationandseriouseyedamage.
However,onthebasisofoneresultfromthevalidationdatabase,anti-foulingorganicsolvent-
containingpaintsmaybeunder-predicted(5).
10. Whenusedtoidentifychemicalsthatdonotrequireclassificationforeyeirritationand
seriouseyedamage,theICEtestmethodhasanoverallaccuracyof82%(125/152),afalsepositive
rmaettehoofd3d3a%ta(c2l6a/s7s9i)f,ieadnadccaofradlisnegnteogatthieveUrNateGHofS1(%4)(1(5/)7.3)W,hwehnentecstomcphaemriecdaltsoiwnitvhiivnocrearbtbaiitnecyleastseesst
t(riah.teee.,oIfaCn0tEi%-tfe(os0ut/l7i1nm)geftoorhrgotahdneiciUssN8ol3vG%eHnt(S1c2co3ln/at1sas4ii9nf)ii,cnagtthipeoanifnastlyssse)tpaeormsei(t4ei)xvc(el5)ur.adteed3f3r%om(2t6h/e78d)a,taabnasde,thethfealascecunregaactyivoef
bsnU1ehu1Nom.cubGlleadHsrsSbiofeTfiNehcodUelaNaCsIsasCitGiEferHigretiSodetsratyatCismanctgehmeteitghmolooidrecdlyyayielsss1irnrc(ooiivht.teeea.rrmt,ciielncUcgaaosNltsmsoimGfeueeHnndydeSdeasersdCc(laUafUtNsoeNrsgiGtofGhriHeHyeSdiS2daCesnoaCtrtaiUetfCgeNiaogctaroeGtyrigyHoo2nS,r2yoB2fC)2AatAtedo)seurtgeooc2rrhBtye.tome2isFt,tcoharec2lhAstcehomitonsihrsacpitaud2lrseBsphrooatasubhnellad,edt
furthertestingwithanothersuitablemethodmayberequired.
12. Allprocedureswithchickeneyesshouldfollowthetestfacility’sapplicableregulationsand
proceduresforhandlingofhumanoranimal-derivedmaterials,whichinclude,butarenotlimitedto,
tissuesandtissuefluids.Universallaboratoryprecautionsarerecommended(16).
13. WhilsttheICEtestmethoddoesnotconsiderconjunctivalandiridalinjuriesasevaluatedin
tclhaessriafbibciattioocnuilnarviivroriwthanecnyctoenstsimdeetrihnogd,thietUadNdrGeHssSesClcaosrsniefaiclateifofne.ctAslswoh,icalhthaorueghthtehemarejvoerrsidbriilvietryooff
craobrbnietaleyleessitoundsiecsa,nnthoattbaeneavsasleusastmeedntpeorfsteheinintihtiealIdCeEptthestofmectohrnoeda,liitnhjaursybmeaenypbreopuosseedd,tobaisdeendtiofny
sometypesofirreversibleeffects(17).Inparticular,furtherscientificknowledgeisrequiredto
understandhowirreversibleeffectsnotlinkedwithinitialhighlevelinjuryoccur.Finally,theICEtest
methoddoesnotallowforanassessmentofthepotentialforsystemictoxicityassociatedwithocular
exposure.
14. ThisTestGuidelinewillbeupdatedperiodicallyasnewinformationanddataareconsidered.
Forexample,histopathologymaybepotentiallyusefulwhenamorecompletecharacterizationof
cornealdamageisneeded.Toevaluatethispossibility,usersareencouragedtopreserveeyesand
pfurretphaerreihmipsrtoopvaeththoeloagcycsupreaccyimoefnsthitshattesctanmebtehouds.edThtoedOevEeCloDphaadsatdaebvaesleopaenddadeGcuiisdiaonncceritDeoricautmheatntmaoyn
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438 OECD/OCDE
theuseofinvitrooculartoxicitytestmethods,whichincludesdetailedproceduresonthecollectionof
histopathologyspecimensandinformationonwheretosubmitspecimensand/orhistopathologydata
(8).
A15n.nex2Fsohroualndybleabuosreadt.oryAinlitaibaolrlayteosrtyabclainshiunsgethtihsesaessacyh,emtihcealpsroftiocideenmcoynscthreamtiecalthseiprrotveicdhenidcailn
competenceinperformingtheICEtestmethodpriortosubmittingICEdataforregulatoryhazard
classificationpurposes.
PRINCIPLEOFTHETEST
16. TheICEtestmethodisanorganotypicmodelthatprovidesshort-termmaintenanceofthe
chickeneyeinvitro.Inthistestmethod,damagebythetestchemicalisassessedbydeterminationof
cornealswelling,opacity,andfluoresceinretention.Whilethelattertwoparametersinvolvea
qualitativeassessment,analysisofcornealswellingprovidesforaquantitativeassessment.Each
measurementiseitherconvertedintoaquantitativescoreusedtocalculateanoverallIrritationIndex,
oeirthaesrsiagsnUedNaGqHuaSliCtaattievgeorcayte1goorriazsatUiNonGthHaStinsoun-sceldastsoifaisesdi.gnEiathnerinofvitthreoseocouultacrohmaezsarcdancltahsesnifbiceatuisoend,
topredictthepotentialinvivoseriouseyedamageornorequirementforeyehazardclassificationofa
testchemical(seeDecisionCriteria).However,noclassificationcanbegivenforchemicalsnot
predictedascausingseriouseyedamageorasnotclassifiedwiththeICEtestmethod(see
paragraph11).
SourceandAgeofChickenEyes
17. Historically,eyescollectedfromchickensobtainedfromaslaughterhousewheretheyare
killedforhumanconsumptionhavebeenusedforthisassay,eliminatingtheneedforlaboratory
animals.Onlytheeyesofhealthyanimalsconsideredsuitableforentryintothehumanfoodchainare
used.
18. Althoughacontrolledstudytoevaluatetheoptimumchickenagehasnotbeenconducted,
theageandweightofthechickensusedhistoricallyinthistestmethodarethatofspringchickens
traditionallyprocessedbyapoultryslaughterhouse{i.e.,approximately7weeksold,1.5-2.5kg).
CollectionandTransportofEyestotheLaboratory
s1h9.ock,andHeiandcissisohnooufldthbeenercekmofvoredbleiemdmiendgi.aAtellyocaalftseorursceedaotfiocnhiocfketnhsecclhoisceketnost,heusluaablolryatboyryeslhecoturlidc
belocatedsothattheirheadscanbetransferredfromtheslaughterhousetothelaboratoryquickly
enoughtominimizedeteriorationand/orbacterialcontamination.Thetimeintervalbetweencollection
ofthechickenheadsandplacingtheeyesinthesuperfusionchamberfollowingenucleationshouldbe
minimized(typicallywithintwohours)toassuremeetingassayacceptancecriteria.Alleyesusedin
theassayshouldbefromthesamegroupofeyescollectedonaspecificday.
20. Becauseeyesaredissectedinthelaboratory,theintactheadsaretransportedfromthe
slaughterhouseatambienttemperature(typicallybetween18°Cand25°C)inplasticboxeshumidified
withtissuesmoistenedwithisotonicsaline.
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SelectionCriteriaandNumberofEyesUsedintheICE
2(i1..e.,>0.5E)yaefsterthtahteyhaarveeenhuicglheabtaesdelairneerefjleucotrede.sceinstaining(i.e.,>0.5)orcornealopacityscore
22. Eachtreatmentgroupandconcurrentpositivecontrolconsistsofatleastthreeeyes.The
negativecontrolgrouporthesolventcontrol(ifusingasolventotherthansaline)consistsofatleast
oneeye.
23. InthecaseofsolidmaterialsleadingtoaGHSNCoutcome,asecondrunofthreeeyesis
recommendedtoconfirmordiscardthenegativeoutcome.
PROCEDURE
PreparationoftheEyes
q24u.icklyasTsheesseeydelwiidtshaaredrcoarpefoufll2y%ex(cwi/sve)d,sotdakiiunmgfclauroerensocteitnodapapmlaigedettohethceorcnoeran.eaClorsnurefaalceinftoergraitfyeiws
seconds,andthenrinsedwithisotonicsaline.Fluorescein-treatedeyesarethenexaminedwithaslit-
lampmicroscopetoensurethatthecorneaisundamaged(i.e.,fluoresceinretentionandcorneal
opacityscores<0.5).
25. Ifundamaged,theeyeisfurtherdissectedfromtheskull,takingcarenottodamagethe
cornea.Theeyeballispulledfromtheorbitbyholdingthenictitatingmembranefirmlywithsurgical
forceps,andtheeyemusclesarecutwithabent,blunt-tippedscissor.Itisimportanttoavoidcausing
cornealdamageduetoexcessivepressure(i.e.,compressionartifacts).
26. Whentheeyeisremovedfromtheorbit,avisibleportionoftheopticnerveshouldbeleft
attached.Onceremovedfromtheorbit,theeyeisplacedonanabsorbentpadandthenictitating
membraneandotherconnectivetissuearecutaway.
27. Theenucleatedeyeismountedinastainlesssteelclampwiththecorneapositioned
vertically.Theclampisthentransferredtoachamberofthesuperfusionapparatus(18).Theclamps
shouldbepositionedinthesuperfusionapparatussuchthattheentirecorneaissuppliedwiththe
isotonicsalinedrip(3-4dropsperminuteor0.1to0.15mL/min).Thechambersofthesuperfusion
apparatusshouldbetemperaturecontrolledat32±1.5°C.Annex3providesadiagramofatypical
superfusionapparatusandtheeyeclamps,whichcanbeobtainedcommerciallyorconstructed.The
apparatuscanbemodifiedtomeettheneedsofanindividuallaboratory(e.g.,toaccommodatea
differentnumberofeyes).
28. Afterbeingplacedinthesuperfusionapparatus,theeyesareagainexaminedwithaslit-lamp
microscopetoensurethattheyhavenotbeendamagedduringthedissectionprocedure.Corneal
thicknessshouldalsobemeasuredatthistimeatthecornealapexusingthedepthmeasuringdeviceon
theslit-lampmicroscope.Eyeswith;(i),afluoresceinretentionscoreof>0.5;(ii)corneal
opacity>0.5;or,(iii),anyadditionalsignsofdamageshouldbereplaced.Foreyesthatarenot
rejectedbasedonanyofthesecriteria,individualeyeswithacornealthicknessdeviatingmorethan
10%fromthemeanvalueforalleyesaretoberejected.Usersshouldbeawarethatslit-lamp
microscopescouldyielddifferentcornealthicknessmeasurementsiftheslit-widthsettingisdifferent.
Theslit-widthshouldbesetat0.095mm.
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29. Oncealleyeshavebeenexaminedandapproved,theeyesareincubatedforapproximately
45to60minutestoequilibratethemtothetestsystempriortodosing.Followingtheequilibration
period,azeroreferencemeasurementisrecordedforcornealthicknessandopacitytoserveasa
baseline(i.e.,time=0).Thefluoresceinscoredeterminedatdissectionisusedasthebaseline
measurementforthatendpoint.
ApplicationoftheTestChemical
30. Immediatelyfollowingthezeroreferencemeasurements,theeye(initsholder)isremoved
fromthesuperfusionapparatus,placedinahorizontalposition,andthetestchemicalisappliedtothe
cornea.
3(e1.g..,aspLairqtuoifdttehsetscthuedmyicdaelssiganr)e.tTyhpiecaplrleyfteersrteeddsuonldvielnuttefdo,rbduitlumtaeydtbeestdiclhuetmeidciafldseiesmpehdysnieocleosgsiacrayl
saline.However,alternativesolventsmayalsobeusedundercontrolledconditions,butthe
appropriatenessofsolventsotherthanphysiologicalsalineshouldbedemonstrated.
32. Liquidtestchemicalsareappliedtothecorneasuchthattheentiresurfaceofthecorneais
evenlycoveredwiththetestchemical;thestandardvolumeis0.03mL.
33. Ifpossible,solidtestchemicalsshouldbegroundasfinelyaspossibleinamortarandpestle,
orcomparablegrindingtool.Thepowderisappliedtothecorneasuchthatthesurfaceisuniformly
coveredwiththetestchemical;thestandardamountis0.03g.
34. Thetestchemical(liquidorsolid)isappliedfor10secondsandthenrinsedfromtheeye
withisotonicsaline(approximately20mL)atambienttemperature.Theeye(initsholder)is
subsequentlyreturnedtothesuperfusionapparatusintheoriginaluprightposition.Incaseofneed,
additionalrinsingmaybeusedafterthe10-secapplicationandatsubsequenttimepoints(e.g.,upon
discoveryofresiduesoftestchemicalonthecornea).Ingeneraltheamountofsalineadditionallyused
forrinsingisnotcritical,buttheobservationofadherenceofchemicaltothecorneaisimportant.
ControlSubstances
35. Concurrentnegativeorsolvent/vehiclecontrolsandpositivecontrolsshouldbeincludedin
eachexperiment.
36. Whentestingliquidsat100%orsolids,physiologicalsalineisusedastheconcurrent
negativecontrolintheICEtestmethodtodetectnon-specificchangesinthetestsystem,andtoensure
thattheassayconditionsdonotinappropriatelyresultinanirritantresponse.
37. Whentestingdilutedliquids,aconcurrentsolvent/vehiclecontrolgroupisincludedinthe
testmethodtodetectnon-specificchangesinthetestsystem,andtoensurethattheassayconditions
donotinappropriatelyresultinanirritantresponse.Asstatedinparagraph31,onlyasolvent/vehicle
thathasbeendemonstratedtohavenoadverseeffectsonthetestsystemcanbeused.
38. Aknownocularirritantisincludedasaconcurrentpositivecontrolineachexperimentto
verifythatanappropriateresponseisinduced.AstheICEassayisbeingusedinthisTestGuidelineto
identifycorrosiveorsevereirritants,thepositivecontrolshouldbeareferencesubstancethatinducesa
severeresponseinthistestmethod.However,toensurethatvariabilityinthepositivecontrolresponse
acrosstimecanbeassessed,themagnitudeofthesevereresponseshouldnotbeexcessive.Sufficient
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invitrodataforthepositivecontrolshouldbegeneratedsuchthatastatisticallydefinedacceptable
rangeforthepositivecontrolcanbecalculated.IfadequatehistoricalICEtestmethoddataarenot
availableforaparticularpositivecontrol,studiesmayneedtobeconductedtoprovidethis
information.
39. Examplesofpositivecontrolsforliquidtestchemicalsare10%aceticacidor5%
benzalkoniumchloride,whileexamplesofpositivecontrolsforsolidtestchemicalsaresodium
hydroxideorimidazole.
40. Benchmarksubstancesareusefulforevaluatingtheocularirritancypotentialofunknown
chemicalsofaspecificchemicalorproductclass,orforevaluatingtherelativeirritancypotentialofan
ocularirritantwithinaspecificrangeofirritantresponses.
EndpointsMeasured
41. Treatedcorneasareevaluatedpriortotreatmentandat30,75,120,180,and240minutes(±
5minutes)afterthepost-treatmentrinse.Thesetimepointsprovideanadequatenumberof
measurements over the four-hour treatment period, while leaving sufficient time between
measurementsfortherequisiteobservationstobemadeforalleyes.
42. The endpoints evaluated are corneal opacity, swelling, fluorescein retention, and
e4m5xo.crepphtoiloongiocfalflueoffreecstcsei(ne.rge.t,epnittitoinng(wohriclhooissedneintgeromfintehdeoenpliythperliiourm)t.oAtlrleatomfenttheanenddp3o0inmtisn,utweisthafttheer
testchemicalexposure)aredeterminedateachoftheabovetimepoints.
43. Photographsareadvisabletodocumentcornealopacity,fluoresceinretention,morphological
effectsand,ifconducted,histopathology.
4464.. Afterthefinalexaminationatfourhours,usersareencouragedtopreserveeyesinan
appropriatefixative{e.g.,neutralbufferedformalin)forpossiblehistopathologicalexamination(see
4p7a.ragraph14andreference(8)fordetails).
Cornealswellingisdeterminedfromcornealthicknessmeasurementsmadewithanoptical
pachymeteronaslit-lampmicroscope.Itisexpressedasapercentageandiscalculatedfromcorneal
thicknessmeasurementsaccordingtothefollowingformula:
cornealthicknescsoartnetailmethtick-nescsoartnetailmeth=i0cknessattime=0 100
Themeanpercentageofcornealswellingforalltesteyesiscalculatedforallobservation
timepoints.Basedonthehighestmeanscoreforcornealswelling,asobservedatanytimepoint,an
overallcategoryscoreisthengivenforeachtestchemical(seeparagraph51).
Cornealopacityisevaluatedbyusingtheareaofthecorneathatismostdenselyopacified
forscoringasshowninTable1.Themeancornealopacityvalueforalltesteyesiscalculatedforall
observationtimepoints.Basedonthehighestmeanscoreforcornealopacity,asobservedatanytime
point,anoverallcategoryscoreisthengivenforeachtestchemical(seeparagraph51).
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Table1.Comealopacityscores.
Score Observation
0 Noopacity
0.5 Veryfaintopacity
1 Scatteredordiffuseareas;detailsoftheirisareclearlyvisible
2 Easilydiscernibletranslucentarea;detailsoftheirisareslightly
obscured
48. 3 Severecomealopacity;nospecificdetailsoftheirisarevisible;size
ofthepupilisbarelydiscernible
4 Completecornealopacity;irisinvisible
Fluoresceinretentionisevaluatedatthe30minuteobservationtimepointonlyasshownin
Table2.Themeanfluoresceinretentionvalueofalltesteyesisthencalculatedforthe30-minute
observationtimepoint,andusedfortheoverallcategoryscoregivenforeachtestchemical(see
paragraph51).
Table2.Fluoresceinretentionscores.
Score Observation
0 Nofluoresceinretention
0.5 Veryminorsinglecellstaining
1 Singlecellstainingscatteredthroughoutthetreatedareaofthe
cornea
2 Focalorconfluentdensesinglecellstaining
3 Confluentlargeareasofthecornearetainingfluorescein
49. Morphologicaleffectsinclude'‘pitting’'ofcomealepithelialcells,“loosening”ofepithelium,
“roughening”ofthecomealsurfaceand“sticking”ofthetestchemicaltothecornea.Thesefindings
canvaryinseverityandmayoccursimultaneously.Theclassificationofthesefindingsissubjective
accordingtotheinterpretationoftheinvestigator.
DATAANDREPORTING
DataEvaluation
50. Resultsfromcornealopacity,swelling,andfluoresceinretentionshouldbeevaluated
separatelytogenerateanICEclassforeachendpoint.TheICEclassesforeachendpointarethen
combinedtogenerateanIrritancyClassificationforeachtestchemical.
DecisionCriteria
51. Onceeachendpointhasbeenevaluated,ICEclassescanbeassignedbasedona
predeterminedrange.Interpretationofcornealswelling(Table3),opacity(Table4),andfluorescein
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retention(Table5)usingfourICEclassesisdoneaccordingtothescalesshownbelow.Itisimportant
tonotethatthecornealswellingscoresshowninTable3areonlyapplicableifthicknessismeasured
dwsieifttftheirnaegntHatacao9grV-n2Se,tarleeqituthailcBlkiPnn9egs0s00.m0es9la5istu-mrlmeam.mpenUmtsisecrrisfostshcheoopsuellidtw-ibwtiehdtahdwesaperttethi-ntmgheaiatssudsirlfiiftne-grleandmtep.vimciecrnoos.co1peasndcosullidt-ywiiedltdh
Table3,ICEclassificationcriteriaforcornealswelling.
MeanCornealSwelling(%) ICEClass
0to5 I
>5to12 II
>12to18(>75minaftertreatment) II
>12to18(<75minaftertreatment) III
>18to26 m
>26to32(>75minaftertreatment) HI
>26to32(<75minaftertreatment) IV
>32 IV
Highestmeanscoreobservedatanytimepoint
Table4,ICEclassificationcriteriaforopacity.
MaximumMeanOpacityScore ICEClass
0.0-0.5 I
0.6-1.5 II
1.6-2.5 III
2.6-4.0 IV
Maximummeanscoreobservedatanytimepoint(basedonopacityscoresas
definedinTable1).
Table5.ICEclassificationcriteriaformeanfluoresceinretention.
Meaatn30FlmuionruetsecseipnoRsett-ternetaitomneSnctore ICEClass
0.0-0.5 I
0.6-1.5 II
1.6-2.5 III
2.6-3.0 IV
BasedonscoresasdefinedinTable2.
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438 OECD/OCDE
52.
TheinvitroclassificationforatestchemicalisassessedbyreadingtheGHSclassification
thatcorrespondstothecombinationofcategoriesobtainedforcornealswelling,cornealopacity,and
fluoresceinretentionasdescribedinTable6.
Table6.Overallinvitroclassifications.
UNGHS Combinationsofthe3Endpoints
Classification
NoCategory 3x1
2x1.1xTT
Noprediction Othercombinations
canbemade
Category1 3xTV
2xIV,1xIII
2xIV.1xn*
2xIV.1xI*
Cornealopacity>3at30min(inatleast2eyes)
Cornealopacity=4atanytimepoint(inatleast2eyes)
Severelooseningoftheepithelium(inatleast1eye)
*Combinationslesslikelytooccur.
StudyAcceptanceCriteria
5c3o.ncurrentAptoessittiisveccoonnstirdoelrsedaraecicdeepnttaibflieedifasthGeHcSoncNuornr-eCnltasnseigfaiteidveanodrGveHhSiclCea/tseoglovernyt1c,ornetsrpoelcstiavnedlyt.he
TestReport
54. Thetestreportshouldincludethefollowinginformation,ifrelevanttotheconductofthe
study:
TestChemicalandControlSubstances
- Chemicalname(s)suchasthestructuralnameusedbytheChemicalAbstractsService
(CAS),followedbyothernames,ifknown;
- TheCASRegistryNumber(RN),ifknown;
- Purityandcompositionofthetestchemical/controlsubstanceorpreparation(in
percentage(s)byweight),totheextentthisinformationisavailable;
- Physicochemicalpropertiessuchasphysicalstate,volatility,pH,stability,chemicalclass
watersolubilityrelevanttotheconductofthestudy;
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©OECD,(2013).