Test No. 437: Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants [electronic resource]

2009·0.57 MB·English

by  Organisation for Economic Co-operation and Development

OECD/OCDE 437
Adopted:
7September2009
OECDGUIDELINEFORTHETESTINGOFCHEMICALS
BovineCornealOpacityandPermeabilityTestMethodforIdentifyingOcularCorrosivesand
SevereIrritants
INTRODUCTION
c1a.nbeusTedh,euBnodveirneceCrtoamienaclirOcpuamcsittayncaensdaPnedrmewaibtihlistpyec(ifBiCcOlPi)mittaesttiomnse,thtoodclisasasnifiynsvuibtsrotatnecstesmeatsh“oodcutlhaart
corrosivesandsevereirritants”asdefinedbytheU.S.EnvironmentalProtectionAgency(EPA)(Category
o1)f,CtlhaessEiufricoapteiaonnaUnndioLnab(eElUli)ng(CoafteCghoermyicRa4l1s),(aGnHdS)the(CUantietgeodryNa1t)io(1n)s((2U)N()3).GlFoobraltlhyeHpaurrpmoosneizoefdthSisysTteesmt
Guideline,severeirritantsaredefinedasthosethatinduceocularlesionsthatpersistintherabbitforat
lreaabsbtit21eydeaystesatf,tetrhaedmBinCiOstPratiisonr.ecWohimlmeenitdiesdnoftorcounsseidaesrepdavratliodfasaaticeormepd-lteetsetirnegplstarcaetmeegnytffoorrtrheeguilnatvoirvyo
classificationandlabelingwithinaspecificapplicabilitydomain(4)(5).Testsubstances(includingsingle
ucirsorimintpgaontnasesnweitqtsuheonatunitdaflumtruetslhtetirin-gtecssottmriapntogengieynn,rtaasbbfoioturstm.luilAnaetsdiuoibnnsstO)aEn(cC6e)DthcTaaetnsttebsGetusicndleeaglsaistniifevie4ed0w5oaus(7l)od.cnuleaerdtcoorbreostievsetsedoirnrsaebvbeirtes
2. ThepurposeofthisTestGuidelineistodescribetheproceduresusedtoevaluatethepotential
ocularcorrosivityorsevereirritancyofatestsubstanceasmeasuredbyitsabilitytoinduceopacityand
increasedpermeabilityinanisolatedbovinecornea.Toxiceffectstothecorneaaremeasuredby:(i)
decreasedlighttransmission(opacity),and(ii)increasedpassageofsodiumfluoresceindye(permeability).
Theopacityandpermeabilityassessmentsofthecorneafollowingexposuretoatestsubstanceare
combinedtoderiveanInVitroIrritancyScore(IVIS),whichisusedtoclassifytheirritancylevelofthe
testsubstance.
b3f.oerensutbessttaeOndccuuelssairnignirttrhhieetasBnetCscOatthPeagttoerisintedsmu,ecteahsloedds.eifoHinnosewtdehvabteyrr,etshteohlevEeaPciAcnurl(e1as5cs)y,tahEnaUdnr2(e12l6i)ad,baiylasintydanoGdfHtnShoen-(Bi3r)Cr,iOthaPantvtseeshtnaomvteetbaheloesdno
formallyevaluated.
4. DefinitionsareprovidedinAnnexI.
INITIALCONSIDERATIONSANDLIMITATIONS
5A.lternativTehiMsetTehsotdsGu(idIeClCinVeAMis)baBsCedOPontetshtemInettehroadgepnrcoytoCcooolrd(i8n)a,tiwnhgicChomwmaisttdeeeveolnoptehdeVfaollildoawtiinognaonf
AilntteerrnnaattiiovnealMveatlhidoadtsion(sEtCuVdyAM(4))(5a)(n9d),twhiethJacpoanntersibeutCieonntsefrrofmorthteheEuVraolpiedaatnioCnentorfeAflotrertnhaetViavleidMaettihonodosf
(JaCVAM).TheprotocolisbasedoninformationobtainedfromtheInstituteforInVitroSciences(IIVS)
andINVITTOXProtocol124(10),whichrepresentstheprotocolusedfortheEuropeanCommunity-
©OECD,(2009)
Youarefreetousethismaterialforpersonal,non-commercialpurposeswithoutseekingpriorconsentfrom
ptheermOiEssCiDo,npfrroovmitdheedOtEheCDso.urceisdulymentioned.AnycommerciaiuseofthismaterialIssubjecttowritten
437 OECD/OCDE
sponsoredprevalidationstudyoftheBCOPassayconductedin1997-1998.Bothoftheseprotocolsare
basedontheBCOPassaymethodologyfirstreportedbyGautheronetal.(11).
6. Theidentifiedlimitationsforthistestmethodarebasedonthehighfalsepositiveratesfor
alcoholsandketonesandthehighfalsenegativerateforsolidsobservedinthevalidationdatabase(see
pdaatraabgarsaep,ht4h4e)a(c5c)u.rWachyenofsBubCsOtaPncaecsrowsisthtihnetEhUes,eEcPhAe,micaanldaGnHdSphcylsaiscsailficcaltaisosnessyarseteemxsclisudseudbsftranotmiatlhley
improved(5).Basedonthepurposeofthisassay{i.e.,toidentifyocularcorrosives/severeirritantsonly),
falsenegativeratesarenotcriticalsincesuchsubstanceswouldbesubsequentlytestedinrabbitsorwith
otheradequatelyvalidatedinvitrotests,dependingonregulatoryrequirements,usingasequentialtesting
strategyinaweightofevidenceapproach.Furthermore,thecurrentvalidationdatabasedidnotallowforan
adequateevaluationofsomechemicalorproductclasses{e.g.,formulations).However,investigatorscould
considerusingthistestmethodforalltypesoftestsubstances(includingformulations),wherebyapositive
resultcouldbeacceptedasindicativeofanocularcorrosiveorsevereirritantresponse.However,positive
resultsobtainedwithalcoholsorketonesshouldbeinterpretedcautiouslyduetoriskofover-prediction.
7. Allprocedureswithbovineeyesandbovinecorneasshouldfollowthetestingfacility’s
applicableregulationsandproceduresforhandlinganimal-derivedmaterials,whichinclude,butarenot
limitedto,tissuesandtissuefluids.Universallaboratoryprecautionsarerecommended(12).
8. Alimitationofthetestmethodisthat,althoughittakesintoaccountsomeoftheoculareffects
evaluatedintherabbitocularirritancytestmethodandtosomedegreetheirseverity,itdoesnotconsider
cseonijnuntchteivBaClOanPdaisrsiadya,liitnjhuarisesb.eeAlnsop,roaplotsheodu,ghbatsheedroevnerrsaibbbiiltiteyyoefsctourdnieesa,ltlheastioannsacsasnensostmbenetevoafltuhaeteidniptiearl
dtheeptBhCoOfPcordnoeeaslniontjuarlylcoawnfboeraunseadstsoesdsimsetinntguoifshthbeetpowteeenntiiarlrefvoerrssiybslteemaincdtroexviecristiyblaessefofceicattsed(1w3i).thFioncaulllya,r
exposure.
9. EffortsareongoingtofurthercharacterizetheusefulnessandlimitationsoftheBCOPassayfor
identifyingnon-severeirritantsandnon-irritants(seealsoparagraph45).ThisTestGuidelinewillbe
updatedperiodicallyasnewinformationanddataareconsidered.Forexample,histopathologymaybe
potentiallyusefulwhenamorecompletecharacterizationofcornealdamageisneeded.Toevaluatethis
possibility,usersareencouragedtopreservecorneasandpreparehistopathologyspecimensthatcanbe
usedtodevelopadatabaseanddecisioncriteriathatmayfurtherimprovetheaccuracyofthistestmethod.
Usersarealsoencouragedtoprovidespecimensand/ordatatovalidationorganizationsforaformal
eirvrailtuanattsioanndofnpoon-sisrirbilteanftust.urTeheuseOsEoCfDtheisBdCevOePlotpeisntgmeatGhuoid,dainncceluDdoincgumfoernttheonidetnhteifuisceatoifoninofvnitorno-soecvuelraer
toxicitytestmethods,whichwillincludedetailedproceduresonthecollectionofhistopathologyspecimens
andinformationonwheretosubmitspecimensand/orhistopathologydata.
pI1Ie0.rsfhoorumlidnFgboetrheausnBeydC.lOaAbPortlaeatsbtoormryaetitnohirtoyidaclplarynieosrutstaeobltsihusebhsmieintgcttihhneigmsiBcasCaslOasyP,totahsdeseapmyroodnfasittcariafetonrecyrtechgehuielrmaittcoearcylhnshiapczaralorvdciodcmelpdaseistniefnAiccnaentieoixnn
purposes.
PRINCIPLEOFTHETEST
p1h1y.siologiTchaelBanCdObPiotcehstemmiectahlofdunisctainonorogfanthoetybpoivcinmeodceolrntehaatipnrovivtirdo.esInshtohrits-tteesrtmmmeatihnotde,nadnacmeaogfenboyrmtahle
testsubstanceisassessedbyquantitativemeasurementsofchangesincomealopacityandpermeability
withanopacitometerandavisiblelightspectrophotometer,respectively.Bothmeasurementsareusedto
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calculateanIVIS,whichisusedtoassignaninvitroirritancyhazardclassificationcategoryforprediction
oftheinvivoocularirritationpotentialofatestsubstance(seeDecisioncriteria).
12. TheBCOPtestmethodusesisolatedcorneasfromtheeyesoffreshlyslaughteredcattle.Corneal
opacityismeasuredquantitativelyastheamountoflighttransmissionthroughthecornea.Permeabilityis
measuredquantitativelyastheamountofsodiumfluoresceindyethatpassesacrossthefullthicknessofthe
cornea,asdetectedinthemediumintheposteriorchamber.Testsubstancesareappliedtotheepithelial
surfaceofthecorneabyadditiontotheanteriorchamberofthecornealholder.AnnexIIIprovidesa
descriptionandadiagramofacornealholderusedintheBCOP.Cornealholderscanbeobtained
commerciallyfromdifferentsourcesorcanbeconstructed.
SourceandAgeofBovineEyesandSelectionofAnimalSpecies
13. Cattlesenttoslaughterhousesaretypicallykilledeitherforhumanconsumptionorforother
commercialuses.Onlyhealthyanimalsconsideredsuitableforentryintothehumanfoodchainareusedas
asourceofcorneasforuseintheBCOP.Becausecattlehaveawiderangeofweights,dependingonbreed,
age,andsex,thereisnorecommendedweightfortheanimalatthetimeofslaughter.
CCg1eo4Cn.reTnreaa<ls9l0yw0Voiabtprthiamaiatnaiheroodenrsfigzreiononnemtrcacaloaltmldtyeliaeaolmboetldtdaeieirmrneet>ndh3sa0fin.ro5oenimsmghccmtaatyntaelaenrrdeslse,ucsleswtnhttiwrhlaahelnetcnfhoiovmuseeseiaywnlegiattrhehsyiaoeclkshdnoerf(sir1sz4oo)m(n.tCaaFClnoTird)miatavhlmaisselutroeeefsrasd<>oi21nf8,1f.e05er0eymneptsmmafgraeaonsrm.de
cattlegreaterthan60monthsoldarenottypicallyused.Eyesfromcattlelessthan12monthsofagehave
nottraditionallybeenusedsincetheeyesarestilldevelopingandthecornealthicknessandcorneal
diameterareconsiderablysmallerthanthatreportedforeyesfromadultcattle.However,theuseofcorneas
fromyounganimals{i.e.,6to12monthsold)ispermissiblesincetherearesomeadvantages,suchas
increasedavailability,anarrowagerange,anddecreasedhazardsrelatedtopotentialworkerexposureto
BovineSpongiformEncephalopathy(15).Asfurtherevaluationoftheeffectofcornealsizeorthicknesson
responsivenesstocorrosiveandirritantsubstanceswouldbeuseful,usersareencouragedtoreportthe
estimatedageand/orweightoftheanimalsprovidingthecorneasusedinastudy.
CollectionandTransportofEyestotheLaboratory
15. Eyesarecollectedbyslaughterhouseemployees.Tominimizemechanicalandothertypesof
damagetotheeyes,theeyesshouldbeenucleatedassoonaspossibleafterdeath.Topreventexposureof
theeyestopotentiallyirritantsubstances,theslaughterhouseemployeesshouldnotusedetergentwhen
rinsingtheheadoftheanimal.
16. EyesshouldbeimmersedcompletelyinHanks’BalancedSaltSolution(HBSS)inasuitably
sizedcontainer,andtransportedtothelaboratoryinsuchamannerastominimizedeteriorationand/or
bacterialcontamination.Becausetheeyesarecollectedduringtheslaughterprocess,theymightbe
exposedtobloodandotherbiologicalsubstances,includingbacteriaandothermicroorganisms.Therefore,
ictonitsaiinmipnogrttahneteytoeseonsnurweetthicaet,tbhyeardidskinogfanctoinbtiaotmiicnsattoiotnheisHBmiSnSimuiszeeddto{e.sgt.o,rebtyhekeeeypeisndgurtihnegctornatnasipnoertr
[e.g.,penicillinat100lU/mLandstreptomycinat100pg/mL]).
17. ThetimeintervalbetweencollectionoftheeyesanduseofcorneasintheBCOPshouldbe
minimized(typicallycollectedandusedonthesameday)andshouldbedemonstratedtonotcompromise
theassayresults.Theseresultsarebasedontheselectioncriteriafortheeyes,aswellasthepositiveand
negativecontrolresponses.Alleyesusedintheassayshouldbefromthesamegroupofeyescollectedona
specificday.
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SelectionCriteriaforEyesUsedintheBCOP
18. Theeyes,oncetheyarriveatthelaboratory,arecarefullyexaminedfordefectsincluding
increasedopacity,scratches,andneovascularization.Onlycorneasfromeyesfreeofsuchdefectsaretobe
used.
19. Thequalityofeachcorneaisalsoevaluatedatlaterstepsintheassay.Corneasthathavean
opacitygreaterthansevenopacityunits(NOTE:theopacitometershouldbecalibratedwithopacity
standardsthatareusedtoestablishtheopacityunits,seeAnnexIII)afteraninitialonehourequilibration
periodaretobediscarded.
m20i.nimuEmaocfhthtrreeeatemyeenst.Tghrroeuepc(otrensteassubsshtoaunlcde,becounsceudrrfoerntthneengeagtaitvieveancdontprooslitciovrenecaosntirnoltsh)ecBoCnsOisPtsasosfay.a
Sinceallcorneasareexcisedfromthewholeglobe,andmountedinthecomealchambers,thereisthe
potentialforartifactsfromhandlinguponindividualcornealopacityandpermeabilityvalues(including
negativecontrol).Furthermore,theopacityandpermeabilityvaluesfromthenegativecontrolcorneasare
usedtocorrectthetestarticleandpositivecontrol-treatedcornealopacityandpermeabilityvaluesinthe
IVIScalculations.
PROCEDURE
PreparationoftheEyes
21. Corneasfreeofdefectsaredissectedwitha2to3mmrimofscleraremainingtoassistin
subsequenthandling,withcaretakentoavoiddamagetothecornealepitheliumandendothelium.Isolated
corneasaremountedinspeciallydesignedcomealholdersthatconsistofanteriorandposterior
compartments,whichinterfacewiththeepithelialandendothelialsidesofthecornea,respectively.Both
chambersarefilledtoexcesswithpre-warmedEagle'sMinimumEssentialMedium(EMEM)(posterior
chamberfirst),ensuringthatnobubblesareformed.Thedeviceisthenequilibratedat32±1°Cforatleast
onehourtoallowthecorneastoequilibratewiththemediumandtoachievenormalmetabolicactivity,to
theextentpossible(theapproximatetemperatureofthecornealsurfaceinvivois32°C).
22. Followingtheequilibrationperiod,freshpre-warmedEMEMisaddedtobothchambersand
baselineopacityreadingsaretakenforeachcornea.Anycorneasthatshowmacroscopictissuedamage
o{ep.agc.,itsycroaftcahlelse,qupiilgimbernattaetdiocno,rnneeaosvaissccuallacruilzaatteid.onA)omrianniompuamciotfyt>h7reoepcaocritnyeausniwtistharoepdaicsictayrdveadl.ueTshcelomseeatno
themedianvalueforallcorneasareselectedasnegative(orsolvent)controlcorneas.Theremaining
corneasarethendistributedintotreatmentandpositivecontrolgroups.
23. Becausetheheatcapacityofwaterishigherthanthatofair,waterprovidesmorestable
temperatureconditionsforincubation.Therefore,theuseawaterbathformaintainingthecomealholder
anditscontentsat32±ITlisrecommended.However,airincubatorsmightalsobeused,assuming
precautiontomaintaintemperaturestability{e.g.,bypre-warmingofholdersandmedia).
ApplicationoftheTestSubstance
24. Twodifferenttreatmentprotocolsareused,oneforliquidsandsurfactants(solidsorliquids),and
onefornon-surfactantsolids.
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25. Liquidsaretestedundiluted,whilesurfactantsaretestedataconcentrationof10%w/vina0.9%
sodiumchloridesolution,distilledwater,orothersolventthathasbeendemonstratedtohavenoadverse
effectsonthetestsystem.Semi-solids,creams,andwaxesaretypicallytestedasliquids.Appropriate
justificationshouldbeprovidedforalternativedilutionconcentrations.Corneasareexposedtoliquidsand
surfactantsfor10minutes.Useofotherexposuretimesshouldbeaccompaniedbyadequatescientific
rationale.
26. Non-surfactantsolidsaretypicallytestedassolutionsorsuspensionsat20%concentrationina
0.9%sodiumchloridesolution,distilledwater,orothersolventthathasbeendemonstratedtohaveno
adverseeffectsonthetestsystem.Incertaincircumstancesandwithproperscientificjustification,solids
mayalsobetestedneatbydirectapplicationontothecornealsurfaceusingtheopenchambermethod(see
paragraph29).Corneasareexposedtosolidsforfourhours,butaswithliquidsandsurfactants,alternative
exposuretimesmaybeusedwithappropriatescientificrationale.
27. Differenttreatmentmethodscanbeused,dependingonthephysicalnatureandchemical
characteristics{e.g.,solids,liquids,viscousvs.non-viscousliquids)ofthetestsubstance.Thecritical
fraecmtoorveidsednusruirnigngthtehartintshiengtessttespsu.bsAtanccleosaedde-qcuhaatmebleyrcmoveetrhsodtheisetpyiptihceallilaylsuusrefdacfeoranndont-hvaitscitouissatdoeqsuliagthetllyy
viscousliquidtestsubstances,whileanopen-chambermethodistypicallyusedforsemi-viscousand
viscousliquidtestsubstancesandforneatsolids.
28. Intheclosed-chambermethod,sufficienttestsubstance(750pL)tocovertheepithelialsideof
thecorneaisintroducedintotheanteriorchamberthroughthedosingholesonthetopsurfaceofthe
chamber,andtheholesaresubsequentlysealedwiththechamberplugsduringtheexposure.Itisimportant
toensurethateachcorneaisexposedtoatestsubstancefortheappropriatetimeinterval.
29. Intheopen-chambermethod,thewindow-lockingringandglasswindowfromtheanterior
chamberareremovedpriortotreatment.Thecontrolortestsubstance(750pL,orenoughtestsubstanceto
completelycoverthecornea)isapplieddirectlytotheepithelialsurfaceofthecorneausingamicropipet.If
atestsubstanceisdifficulttopipet,thetestsubstancecanbepressure-loadedintoapositivedisplacement
pipettoaidindosing.Thepipettipofthepositivedisplacementpipetisinsertedintothedispensingtipof
thesyringesothatthematerialcanbeloadedintothedisplacementtipunderpressure.Simultaneously,the
syringeplungerisdepressedasthepipetpistonisdrawnupwards.Ifairbubblesappearinthepipettip,the
testarticleisremoved(expelled)andtheprocessrepeateduntilthetipisfilledwithoutairbubbles.If
necessary,anormalsyringe(withoutaneedle)canbeusedsinceitpermitsmeasuringanaccuratevolume
oftestsubstanceandaneasierapplicationtotheepithelialsurfaceofthecornea.Afterdosing,theglass
windowisreplacedontheanteriorchambertorecreateaclosedsystem.
Post-ExposureIncubation
30. Aftertheexposureperiod,thetestsubstance,thenegativecontrol,orthepositivecontrol
sviusbusatlanecveidisenrceemoovfedtesftrosmubtshteanancteerciaonrcbheamobbeserravnedd)thweitehpitEhMelEiuMmw(acsohnteadinaitnlgeapshtetnhorleerteidm).esP(hoernuonltilrendo-
containingmediumisusedforrinsingsinceacolorchangeinthephenolredmaybemonitoredto
determinetheeffectivenessofrinsingacidicoralkalinematerials.Thecorneasarewashedmorethanthree
tcmEiheMmademEisbuMeimfr(tipwhsrieiftorphrehoeeuttnoooftplhtheeersnetoodpslauicsbrisetstdtyai)lnmliceseda,iusssutcehrodeelomcareosenrdatn.e(fayiTsnehalalelroreawingntosieervreiptnoourraepcnlfhsei)aun,ramleboerrrirentmshieosevtwtaheilesttnhosrfEeuftbMihsletElaepMndhcewe(niwtoiishlthsfrtorieueldltshvfpirEhsoieMmbnloEetl.hMerOenawdn)ict.teehrTotihuhoeter
phenolred.
5 ©OCDE,(2009)
437 OECD/OCDE
31. Forliquidsorsurfactants,afterrinsing,thecorneasareincubatedforanadditionaltwohoursat
32±rc.Longerpost-exposuretimemaybeusefulincertaincircumstancesandcouldbeconsideredona
case-by-casebasis.Corneastreatedwithsolidsarerinsedthoroughlyattheendofthefour-hourexposure
period,butdonotrequirefurtherincubation.
32. Attheendofthepost-exposureincubationperiodforliquidsandsurfactantsandattheendofthe
four-hourexposureperiodfornon-surfactantsolids,theopacityandpermeabilityofeachcorneaare
recorded.Also,eachcorneaisobservedvisuallyandpertinentobservationsrecorded(e.g.,tissuepeeling,
residualtestsubstance,non-uniformopacitypatterns).Theseobservationscouldbeimportantastheymay
bereflectedbyvariationsintheopacitometerreadings.
ControlSubstances
33. Concurrentnegativeorsolvent/vehiclecontrolsandpositivecontrolsareincludedineach
experiment.
c3h4.loridesWolhuetinontoerstdiinsgtialleldiqwuaitders)ubisstiannccleudaetdi1n0t0h%e,BaCOcoPnctuesrtremnetthnoedgastoivtehactonnotnrsoplec{ie.fgi.c,c0h.a9n%gessoidnituhme
testsystemcanbedetectedandtoprovideabaselinefortheassayendpoints.Italsoensuresthattheassay
conditionsdonotinappropriatelyresultinanirritantresponse.
i35n.cludediWnhtehneBteCstOiPngtaesdtilmuettehdoldiqsuiod,thsautrfnaocntsapnetc,ifoircsoclhiad,ngaescoinncutrhreetnetstsoslyvsetnetm/vechainclbeecdoenttercotlegdraonudptios
provideabaselinefortheassayendpoints.Onlyasolvent/vehiclethathasbeendemonstratedtohaveno
adverseeffectsonthetestsystemcanbeused.
t3h6a.tanappArokpnrioawtneroecsuploanrsierriistainntdiusceidn.clAusdetdheasBaCcOoPncausrsraeyntispobseiitnigveusceodntirnolthiinseTaescthGeuxipdeerliimneenttotoidveenrtiiffyy
corrosiveorsevereirritants,ideallythepositivecontrolshouldbeareferencesubstancethatinducesa
severeresponseinthistestmethod.However,toensurethatvariabilityinthepositivecontrolresponse
acrosstimecanbeassessed,themagnitudeofirritantresponseshouldnotbeexcessive.
37. Examplesofpositivecontrolsforliquidtestsubstancesare 1% sodiumhydroxideor
dimethylformamide.Anexampleofapositivecontrolforsolidtestsubstancesis20%(weighttovolume)
imidazolein0.9%sodiumchloridesolution.
38. Benchmarksubstancesareusefulforevaluatingtheocularirritancypotentialofunknown
chemicalsofaspecificchemicalorproductclass,orforevaluatingtherelativeirritancypotentialofan
ocularirritantwithinaspecificrangeofirritantresponses.
EndpointsMeasured
39. Opacityisdeterminedbytheamountoflighttransmissionthroughthecornea.Cornealopacityis
measuredquantitativelywiththeaidofanopacitometer,resultinginopacityvaluesmeasuredona
continuousscale.
40. Permeabilityisdeterminedbytheamountofsodiumfluoresceindyethatpenetratesallcorneal
csseuulrrlffaalccaety)ae.nrts1so{mil.ieLd.,s,storhedesipeuepmcittifhvleeulloiyru)emsicsoeianndtdsheoedluottuoitotenhre(ca4onrtonereria5orsmucgrhf/aamcmeLbetwrhhroeofuntghhteetscthoiernngeenaldlioqhtuohildedsleira,unmwdhosinucrhtfhaiecnttiaennrtnfsearcoecrsonrwoinnte-ha
theepithelialsideofthecornea,whiletheposteriorchamber,whichinterfaceswiththeendothelialsideof
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thecornea,isfilledwithfreshEMEM.Theholderisthenincubatedinahorizontalpositionfor90+5min
namtema3s2aurr±eerd1e®wcCio.trhdTethdheeasaamiodoputoinfctaUlVofAde^snIosSditiysupm(eOcfDtl4ru9oo0pr)ehsocoteroimnaebttrshyoa.trbcSarpnoecscestervsoaplihuneotsot,otmhweehtirpicocshtmeareriaeosrmuecraehsmauemrnbetedsreoivsnaqlauuaacntoteindttaiatntiuv4oe9ul0sy
scale.ThefluoresceinpermeabilityvaluesaredeterminedusingOD490valuesbaseduponavisiblelight
spectrophotometerusingastandard1cmpathlength.
41. Alternatively,a96-wellmicrotiterplatereadermaybeusedprovidedthat;(i)thelinearrangeof
tfhleuoprleastceeirneasdaemrpfloersdaerteerumsiendinignfthleuo9r6e-scweeilnlOpDla4t9e0tvoalreuseusltcainnObDe4e9s0tavballiusehesd;eqaunidva(liei)n,ttthoetchoerrsetcatnvdoarldum1ecomf
pathlength(thiscouldrequireacompletelyfullwell[usually360pLJ).
DATAANDREPORTING
DataEvaluation
42. Oncetheopacityandmeanpermeability{OD490)valueshavebeencorrectedforbackground
opacityandthenegativecontrolpermeabilityOD490values,themeanopacityandpermeabilityOD490
valuesforeachtreatmentgroupshouldbecombinedinanempirically-derivedformulatocalculateanin
vitroirritancyscore(IVIS)foreachtreatmentgroupasfollows:
IVIS=meanopacityvalue+(15xmeanpermeabilityOD490value)
Sinaetal.(16)reportedthatthisfonnulawasderivedduringin-houseandinter-laboratorystudies.The
datageneratedforaseriesof36compoundsinamulti-laboratorystudyweresubjectedtoamultivariate
analysistodeterminetheequationofbestfitbetweeninvivoandinvitrodata.Thisanalysiswasperformed
byscientistsattwoseparatecompanies,whoderivednearlyidenticalequations.
43. Theopacityandpermeabilityvaluesshouldalsobeevaluatedindependentlytodetermine
whetheratestsubstanceinducedcorrosivityorsevereirritationthroughonlyoneofthetwoendpoints(see
DecisionCriteria).
DecisionCriteria
44. AsubstancethatinducesanIVIS>55.1isdefinedasacorrosiveorsevereirritant.Asstatedin
psahroaulgdrapbhe1c,oinfdtuhcetteedstfsourbsctlaanscseifiiscantoitonideanntdifileadbealsinagnpoucruploasrecso.rrTohsieveBoCrOsPevetreestirrmietatnht,odaddhiatsioannalotveesrtailnlg
accuracyof79%(113/143)to81%(119/147),afalsepositiverateof19%(20/103)to21%(22/103),anda
fcallassesinfieegdataicvceorrdatiengofto1t6h%eE(7P/A43)(1t)o,2E5U%(2()1,0/o4r0)G,HwShe(n3)ccolmaspsairfeicdattiooninsyvsitveomsr.abbWihteenyesutebssttamnectehsowditdhaitna
tcheertaaicncucrhaecmyicoaflB{Ci.eO.,Palaccorhooslss,thkeetEoUn,esE)PoAr,pahynsdicGaHlS{i.cel.,asssoilfiidcsa)ticolnasssyessteamrseerxacngleusdefdrformo8m7t%he(d7a2t/a8b3a)set,o
92%(78/85),thefalsepositiveratesrangefrom12%(7/58)to16%(9/56),andthefalsenegativerates
rangefrom0%(0/27)to12%(3/26).
4Ba5dC.eqOuPatedlaytEavveacnlainidfabtaeenduosicenufluvalir,trcoionrtrecosots,nijvtueoncoftruirsotenhveewrrieetvhiarlrtuietasattnetdacttlhaaessfuisrfeiofcmautltinhoenessiisnannvoditvlooibmrtiaatbiabntieitodnesfyoerofatettshetestoBrsCufbOsrPtoamntceaesn,t
methodforidentifyingnon-severeirritantsandnon-irritants(aGuidanceDocumentontheuseofinvitro
oculartoxicitytestmethodsisunderdevelopment).
7 ©OCDE,(2009)
437 OECD/OCDE
StudyAcceptanceCriteria
46. AtestisconsideredacceptableifthepositivecontrolgivesanIVISthatfallswithintwostandard
deviationsofthecurrenthistoricalmean,whichistobeupdatedatleasteverythreemonths,oreachtime
anacceptabletestisconductedinlaboratorieswheretestsareconductedinfrequently{i.e.,lessthanoncea
month).Thenegativeorsolvent/vehiclecontrolresponsesshouldresultinopacityandpermeabilityvalues
thatarelessthantheestablishedupperlimitsforbackgroundopacityandpermeabilityvaluesforbovine
corneastreatedwiththerespectivenegativeorsolvent/vehiclecontrol.
TestReport
47. Thetestreportshouldincludethefollowinginformation,ifrelevanttotheconductofthestudy:
TestandControlSubstances
Chemicalname(s)suchasthestructuralnameusedbytheChemicalAbstractsService
(CAS),followedbyothernames,ifknown;
TheCASRegistryNumber(RN),ifknown;
Purityandcompositionofthesubstanceorpreparation(inpercentage(s)byweight),tothe
extentthisinformationisavailable;
Physicochemicalpropertiessuchasphysicalstate,volatility,pH,stability,chemicalclass,
watersolubilityrelevanttotheconductofthestudy;
Treatmentofthetest/controlsubstancespriortotesting,ifapplicable{e.g.,warming,
grinding);
Stability,ifknown.
InformationConcerningtheSponsorandtheTestFacility
Nameandaddressofthesponsor,testfacilityandstudydirector;
Identificationofthesourceoftheeyes(i.e.,thefacilityfromwhichtheywerecollected);
Storageandtransportconditionsofeyes(e.g.,dateandtimeofeyecollection,timeinterval
priortoinitiatingtesting,transportmediaandtemperatureconditions,anyantibiotics
used);
Ifavailable,specificcharacteristicsoftheanimalsfromwhichtheeyeswerecollected(e.g.,
age,sex,weightofthedonoranimal).
JustificationoftheTestMethodandProtocolUsed
TestMethodIntegrity
Theprocedureusedtoensuretheintegrity(i.e.,accuracyandreliability)ofthetestmethod
overtime(e.g.,periodictestingofproficiencysubstances,useofhistoricalnegativeand
positivecontroldata).
CriteriaforanAcceptableTest
Acceptableconcurrentpositiveandnegativecontrolrangesbasedonhistoricaldata;
Ifapplicable,acceptableconcurrentbenchmarkcontrolrangesbasedonhistoricaldata.
©OCDE,(2009) 8
OECD/OCDE 437
TestConditions
Descriptionoftestsystemused;
Typeofcornealholderused;
Calibrationinformationfordevicesusedformeasuringopacityandpermeability{e.g.,
opacitometerandspectrophotometer);
Informationonthebovinecorneasused,includingstatementsregardingtheirquality;
Detailsoftestprocedureused;
Testsubstanceconcentration(s)used;
Descriptionofanymodificationsofthetestprocedure;
Referencetohistoricaldataofthemodel{e.g.,negativeandpositivecontrols,proficiency
substances,benchmarksubstances);
Descriptionofevaluationcriteriaused.
Results
Tabulationofdatafromindividualtestsamples{e.g.,opacityandOD490valuesand
calculatedIVISforthetestsubstanceandthepositive,negative,andbenchmarkcontrols
[ifincluded],reportedintabularform,includingdatafromreplicaterepeatexperimentsas
appropriate,andmeans±thestandarddeviationforeachexperiment);
Descriptionofothereffectsobserved.
DiscussionoftheResults
Conclusion
9 ©OCDE,(2009)
437 OECD/OCDE
LITERATURE
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(6)EC(2006).Regulation(EC)No1907/2006oftheEuropeanParliamentandoftheCouncilof18
December2006concerningtheRegistration,Evaluation,AuthorisationandRestrictionofChemicals
(REACH),establishingaEuropeanChemicalsAgency,amendingDirective1999/45/ECandrepealing
CouncilRegulation(EEC)No793/93andCommissionRegulation(EC)No1488/94aswell as Council
Directive76/769/EECandCommissionDirectives91/155/EEC,93/67/EEC,93/105/ECand2000/21/EC.
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irritation/corrosion.Available:
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(8)ICCVAM(2007).ICCVAMRecommendedBCOPTestMethodProtocol.In:ICCVAMTestMethod
EvaluationReport-InVitroOcularToxicityTestMethodsforIdentifyingOcularSevereIrritantsand
Cori'osives.InteragencyCoordinatingCommitteeontheValidationofAlternativeMethods(ICCVAM)
andtheNationalToxicologyProgram(NTP)InteragencyCenterfortheEvaluationofAlternative
Toxicological Methods (NICEATM). NIH Publication No.: 07-4517. Available:
rhttp://iccvam.niehs.nih.gov/mcthods/ocutox/ivocutox/ocutmer.html
(S9e)veIrCeCIVrrAitMan.ts:(2B0o0v6i).neCuCrormeenatlStOaptaucsitoyfaInndVPiterromeTaebsitliMtyetTheosdtsMeftorhoIdd.enNtiIfHyinPgubOliccualtaironCoNror.o:si0v6e-s45a1n2.d
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[http://iccvam.niehs.nih.gov/methods/ocutox/ivocutox/ocubrdbcop.htm1
(10)INVITTOX(1999).Protocol124:BovineComealOpacityandPermeabilityAssay-SOPof
MicrobiologicalAssociatesLtd.Ispra,Italy:EuropeanCentrefortheValidationofAlternativeMethods
(ECVAM).
©OCDE,(2009) 10