n overview of current techniques for ocular toxicity testing

Review

An

overview

of

current

techniques

for

ocular

toxicity

testing

Samantha

L.

Wilson

*

,

Mark

Ahearne

,

Andrew

Hopkinson

a_{AcademicOphthalmology,DivisionofClinicalNeuroscience,UniversityofNottingham,Queen’sMedicalCentreCampusNG72UH,UK} b

TrinityCentreforBioengineering,TrinityBiomedicalSciencesInstitute,TrinityCollegeDublin,Ireland,UK c

DepartmentofMechanicalandManufacturingEngineering,SchoolofEngineering,TrinityCollegeDublin,Ireland,UK

ARTICLE INFO

Articlehistory:

Received25September2014

Receivedinrevisedform5November2014 Accepted6November2014

Availableonline11November2014

Keywords: Cornealequivalents Cytotoxicity Draize

Invitroalternatives Organotypicmodels Regulation Validation

ABSTRACT

Giventhehazardousnatureofmanymaterialsandsubstances,oculartoxicitytestingisrequiredto

evaluatethedangersassociatedwiththesesubstancesaftertheirexposuretotheeye.Historically,animal

testssuchastheDraizetestwereexclusivelyusedtodeterminethelevelofoculartoxicitybyapplyinga

testsubstancetoaliverabbit’seyeandevaluatingthebiologicalresponse.Inrecentyears,legislationin

manydevelopedcountrieshasbeenintroducedtotrytoreduceanimaltestingandpromotealternative

techniques.Thesetechniquesincludeexvivotestsondeceasedanimaltissue,computationalmodelsthat

usealgorithmstoapplyexistingdatatonewchemicalsandinvitroassaysbasedontwodimensional(2D)

andthreedimensional(3D)cellculturemodels.Hereweprovideacomprehensiveoverviewofthelatest

advancesinoculartoxicitytestingtechniques,anddiscusstheregulatoryframeworkusedtoevaluate

theirsuitability.

ã2014TheAuthors.PublishedbyElsevierIrelandLtd.

ThisisanopenaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/3.0/).

Contents

1. Introduction ... 33

2. Draizetesting ... 33

3. Alternativeinvivotests ... 34

3.1. Low-volumeeye-irritationtest(LVET) ... 34

3.2. Humandata ... 34

4. Exvivotests ... 35

4.1. Isolated/enucleatedorgan/organotypicmethods ... 35

4.2. Non-ocularorganotypicmodels ... 37

5. Invitrotests ... 38

5.1. Cytotoxicityassessment ... 38

5.2. Cornealepithelialmodels ... 40

5.3. Cornealequivalents ... 41

Abbreviations:BCOP,BovineCorneaOpacityPermeability(test/assay);CAMVA,Chorioallantoicmembranevascularassay;CEET,Chickenenucleatedeyetest;CK, Cytokeratin;CM,CytosensorMicrophysiometer(test);CPSC,ConsumerProductSafetyCommission;CTPA,Cosmetic,ToiletryandPerfumeryAssociation;DMSO,Dimethyl sulfoxide;DNA,Deoxyribonucleicacid;EC,EuropeanCommission;ECVAM,EuropeanCentrefortheValidationofAlternativeMethods;EET,Enucleatedeyetest;EIT,Eye irritationtest;EURL-ECVAM,EuropeanUnionReferenceLaboratoryforAlternativestoAnimalTests(formallyECVAM);FDA,FoodandDrugAdministration;FL,Fluorescein leakage;GHS,GloballyHarmonizedSystem(ofclassification);HCE,Humancornealepithelium;HET,Hen’seggtest;HET-CAM,Hühner-embryonentestonchorioallantoic membrane;HO,HomeOffice(British);ICCVAM,InteragencyCoordinatingCommitteeontheValidationofAlternativeMethods;ICE,Isolatedchickeneye(test);IRAG, InteragencyRegulatoryAlternativesGroup;IRE,Isolatedrabbiteye(test);IVIS,Invitroirritancyscore;JaCVAM,JapaneseCentrefortheValidationofAlternativeMethods; LDH,Lactatedehydrogenase(leakage);LVET,Lowvolumeeyeirritationtest;MAS,Maximumaveragescore;MDCK,Madin–Dardycaninekidney(cells);MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide;NICEATM, NTPInteragencyCenterfortheEvaluationofAlternativeToxicologicalMethods;NIEHS,National CommitteeofEnvironmentalHealthSciences;NIOSHA,NationalInstituteforOccupationalSafetyandHealthAdministartion;NRC,NationalResearchCouncil;NTP,National ToxicologyProgramme;NZW,NewZealandWhite(rabbits);OECD,OrganizationfortheEconomicCo-operationandCountriesDevelopment;PLLBOA,Prototypelaserlight basedopacitometer;QSAR,Quantitativestructure–activityrelationship;RCE,Rabbitcornealepithelium;REET,Rabbitenucleatedeyetest;SIRC,StatensSeruminstitutrabbit corneal(cells);SMI,Slugmucosalirritation(assay/test);STE,Shorttimeexposure(test);TG,Testguidance;UN,UnitedNations;VMP,ValidationManagementGroup.

*Correspondingauthor.Tel.:+447976063762.

E-mailaddress:[email protected](S.L.Wilson).

http://dx.doi.org/10.1016/j.tox.2014.11.003

0300-483X/ã2014TheAuthors.PublishedbyElsevierIrelandLtd.ThisisanopenaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/3.0/). ContentslistsavailableatScienceDirect

Toxicology

5.4. Futureofinvitroalternatives ... 42

6. Insilicomodels ... 42

7. Regulationandvalidationofoculartoxicitytesting ... 43

7.1. Regulation ... 43

7.2. Validationandregulatoryacceptance ... 43

8. Conclusion ... 44

Conflictofinterest ... 44

Transparencydocument ... 44

Acknowledgment ... 44

References ... 44

1.Introduction

The location, physiological structure and sensitivity of the ocular surface predispose it to exposure from a variety of potentiallyhazardous environmentalconditions and substances onadailybasis.Manydifferentmaterialsandchemicalscanresult in damage to the cornea that may vary from irritation and inflammationcausingmilddiscomforttotissuecorrosionresulting in irreversible blindness. These include household, industrial, agriculturalandmilitaryproducts,cosmetics,toiletriesandmay even include certain ocular drugs and pharmaceuticals if incorrectlyadministered (Wilhelmus,2001).While exposure to such substances may be incidental, accidental or intentional (Vinardell and Mitjans, 2008), most ocular incidents involve accidental exposure either in the workplace or at home via

splashing with concentrated solutions, such as bleach or detergents, followed by rapid washing with water or removal

vialacrimation(Shawetal.,1991).Toreducetheriskofexposureto dangeroussubstancesallmanufacturedconsumerproducts and theiringredientsmustbetestedandtheireyeirritationpotential assessedsothatthepubliccanbeassuredoftheirsafety,orwarned oftheassociateddangers.Eyetoxicitytestsarethereforerequired toensurethat therisksassociatedwithproductsmeetsuitable safetycriteriaandareclearlylabeled.

Historically,astoxicologytestinghasbecomemorecommon,its relianceuponanimalusehasincreased.Thishasprimarilybeendue totheabsenceofmoresophisticatedassessmenttechniquesandthe lowstatusofanimalsinsociety(StephensandMak,2013).Ethical reconsiderationofanimalusefortoxicologystudieswasdrivenby the emergence of the animal rights movement in the 1950s (StephensandMak,2013)anditscriticismofanimal experimenta-tion,inparticulartheuseofDraizetestingforcosmeticstesting.In 1959, Russell and Burch performed a study based upon the philosophicalconcept ofhumanity, inwhichthey observedthat somebiologicalexperimentscouldbeclassedas“inhumane”based uponthelevelsofpain,distressandlastingharmexperiencedbythe test animals (Russell et al.,1959). Their research provided the systematicbasisofthe3R’s:Replace,ReduceandRefinetheuseof sentient beings in experimental biology. This led to a general expansionoffundingsourcesforex vivoandinvitroalternative methods,toreducethedependencyonliveanimaltesting,whilst alsocreatingapolitical climatewhereby alternativeprocedures were incorporated into federal and government legislation (Stephens and Mak, 2013). In this review, we will provide an overviewofestablishedandnewlydevelopedoculartoxicitytests anddiscusstheiradvantagesandpotentiallimitations.

2.Draizetesting

Liveanimalshavebeenusedtoassessandevaluatepotentially harmfulproductstotheeyessincethe18thcentury(Wilhelmus, 2001). The international standard assay foracute ocular toxicity is the rabbit in vivo Draize eye test (Draize et al., 1944) which was developedinthe1940sbytheFoodandDrugsAdministration(FDA)

whichcause reversibleirritation to eyes within 7days.Non irritating chemicals are assigned a GHS No Category classification. The categories are assigned based on calculations of a mean score followingobservational grading at 24, 48 and 72h post application of thetestchemical.TheGHSwasadoptedin2002andpublishedin 2003(Silk,2003).

Despite the adoption of the GHS, Draize testing is often criticizedduetoitssubjectiveandtimeconsumingnature,lackof repeatability, variable estimates, insufficient relevance of test chemicalapplication(Davilaetal.,1998),highdosages(Currenand Harbell, 2002) and over-prediction of human responses(Jester etal.,2001),primarilyduetointerspeciesdifferences.Inaddition, formostroutineandacutetoxicitytests,forexampleskintoxicity tests, there are standardized exposure times and/or delivery methodsinplace.Thisisnotthecasewitheyeirritation;liquids, pastesandsolidsallhavedifferentcontacttimeswiththeeye,none ofwhicharewelldefined(Prinsen,2006).Draizetestingalsofails toelucidatetheunderpinningcellularandmolecularmechanisms oftoxicology.SinceDraizeassessmentsarebaseduponpenlightor slit-lamp assessments, they provide very little information regardingtheprimaryorsecondaryresponsesinthecornea,iris orconjunctiva(Maureret al.,2002).Despite its“goldstandard”

status, Draize testing was never formally validated to any significantdegree(Freebergetal.,1986b).Since theanatomyof therabbiteyediffersfromthehumaneyestructurally, physiologi-callyandbiochemically,differencesinsensitivitytoirritantscan occur.Forexample,incomparisontohumans,rabbitcorneasare thinner,havelowertearproduction,blinkingfrequencyandocular surface sensitivity (Huhtala et al., 2008). Rabbits have larger conjunctivalsacsandanictitatingmembrane(thirdeyelid),which mayaidtheremovalofatestsubstancefromtheocularsurface (Calabrese,1987).

There is almost no other field of science in which the fundamental experimental protocols have remained relatively unchanged for more than 40 years (Hartung, 2009), and yet consumerscontinuallyexpect increasedsafety and information regardingtheirproducts.Worldwide,approximately£10billionis spent on animal experimentation per annum, approximately £2billion ofwhich is ontoxicological studies (Hartung, 2009). Thecostassociatedwithusing,housingandmaintainingcolonies oflive animalsfortoxicology testingof asinglecompound can exceed millionsof pounds (Davilaet al.,1998).Ethical (animal welfare),business(timeandcost),scientificadvances (reproduc-ibility, mechanistic understanding) and legal concerns have all driventhedemandforalternative,preferablyanimal-freetesting platformsandprotocolswhicharemorepreciseandrelevantto humans. There has beenmore focus ondeveloping alternative testingtechniques toDraizethanallotherinvivotoxicitytests combined(Huhtalaet al., 2008). However,the development of alternativemodelshasnot advancedin a steady orcontinuous manner(DholakiyaandBarile,2013),althoughthebanonanimal testingforcosmeticsuse(Regulation(EC)No._{1223/2009)hasacted}

asakeydriverforthedevelopmentofalternativemethodssince this sector is constantly havingtoprovide innovative and safe products.In Europe, withdirective 2010/63/EU, thereis a legal requirementtousealternatives wherethey exist.However,the reductionofanimaluseisprimarilyconcentratedontoxicology studiessincenogovernmentagencytodatehaseliminatedanimal useinbasicbiomedicalresearchorpharmaceuticaldevelopment.

3.Alternativeinvivotests

3.1.Low-volumeeye-irritationtest(LVET)

Low-volume eye-irritation tests (LVET) were developed in responsetoarecommendationfromtheNationalResearchCouncil

(NRC,1977).LVETisarefinementofDraizetestingdevelopedby

Griffithetal.(1980).TheprimarydifferencetotheDraizetestisthat lowervolumesoftestsubstances(0.01ml/0.01g)(Lambertetal., 1993)areappliedtotheright-eyeoftheanimal(Maureretal.,2002), with no forced eyelid closure employed (ICCVAM, 2010b). Test substancesarealsoonlyappliedtothecornealsurfaceandnotthe conjunctivalsac.Thetestisbelievedtobelessstressfultothetested animal(Jesteretal.,2001).Pathologicalchangesarecharacterizedin thecornea,conjunctivaandiris/cilliarybody(Maureretal.,2002). Most LVET data is based upon surfactant-based mixtures or responsesthatareassociatedwithmildirritationornon-irritants. This is due to the importance of surfactant use in cosmetic, pharmaceutical and household cleaning products (Davila et al., 1998).However,Gettingsetal.(1996)investigatedLVETinresponse tosevereirritantsandreportedanunder-predictionofresultswhen comparedtoDraizedata.SinceDraizetestingisoftencriticizedforits over-predictionofhumanresponses,itisarguablethatLVETtesting ismoreaccurate(Freebergetal.,1984,1986a;Ghassemietal.,1993; Roggebandetal.,2000).However,LVETisstillcriticizedforitsuseof animals.Inaddition,shouldanegativeirritancyresultoccurusinga lower test volume, the standard procedure is to increase the concentrationofthe drug, effectivelyresortingbackto Draize testing. The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) recently evaluated the validity ofLVETforthereplacementofDraizetesting.Itwasnotconsidered tobeavalid replacementnorrecommendforprospectiveocular safetytesting (ICCVAM, 2010b).As a result, LVET hasyet to be adopted by any regulatory agency as an alternative test. The reluctancetoadoptLVETmaybeduetothefactthatitdoesnot offer the element of “exaggeration” present in Draize testing, thathelpstoassurepublicsafety(Freebergetal.,1986b;Ubelsand Clousing,2005).However,retrospectiveLVETdataisstillusefulto weight-of-evidenceapproaches.

3.2.Humandata

Ithasbeensuggestedthatthe“goldstandard”foreyeirritation shouldbethehumanresponse(Bagleyetal.,2006)andthatideally, atestingstrategytodetermineifasubstanceisharmfultohumans would utilize an extremelyhigh number of humansubjectsin ordertofaithfullyrepresenthumandiversity.Theywouldhaveto beunknowinglyexposedtoasubstanceunderrealisticconditions andtheeffectsassessed(Hartung,2009).However,such experi-mentationisboth unrealisticand unethical. Asa result,human studydataandexperiencesofpotential ocularhazardsareonly available from either accidental exposure or clinical studies. Unfortunately,accidentalexposuredataoftendoesnotrealistically representthemostseverelesionssinceexposureisoftenbriefdue toimmediateflushingoftheeye.Furthermore,detailedstudiesby ophthalmologists and emergency roomclinicians are often not collectedor recorded.Clinical studiesusing undilutedproducts raisebothscientificandethicalconcerns,soexperimentshaveto becarefullycontrolled.Inclinicalstudies,thetestmaterial(often verysmallvolumesand/ordiluted)isusuallyappliedtotheupper orlowerconjunctivalsac,asopposedtotheapexofthecorneaasin

Draize testing was a poor predictor of accidental human eye exposure, whereas LVET correlated well, although still over-predictedresults.

Humanstudiesarelimited,andareusuallycomparinghuman responseswithDraizeorLVET,asproof-of-principlethatLVETis morecrediblethanDraizetesting(Roggebandetal.,2000),andnot as a comparison for the validation of alternative methods. A prevalent problem is that there is no human databasefor the development of the prediction models needed in validation studies, thus in vitro toxicity tests are still being compared to rabbitdata(Bagleyetal.,2006).

4.Exvivotests

4.1.Isolated/enucleatedorgan/organotypicmethods

Ocular organotypic models are isolated systems that aim to maintainshort-termnormalphysiologicalandbiochemicalfunction oftheenucleatedeyeorcornea(Barile,2010).Thetestmaterialis oftenappliedneatsoismorerelevanttoindustrialtesting(Reader etal.,1990)andmorefaithfullyrepresentsaccidentalexposure.The protocolsusuallyutilizeopacitometricandspectroscopicmethods forquantitative assessmentof changestotheisolated corneain responsetoatestmaterialfollowedbyhistologicalanalysis.Corneal opacityisalsoaninvivocornealendpoint,althoughthedatais observational, so often subjective. Corneal opacity acts as an indicator ofprotein denaturation,swelling,vacuolationanddamage totheepitheliumandcornealstroma(Barile,2010).Fluorescein retention/leakage of the cornea is often used as a measure of permeability(PrinsenandKoëter,1993),althoughin vivotheirisand theconjunctivaarealsoinvolvedinocularirritation,socorneal swellingandhistologicalanalysisareoftenincludedasadditional endpointsinorganotypicmodels(OECD,2009a),oftento distin-guish“borderline” cases.Unlikeinvivotesting,thequantitative assessmentofcornealopacityand swellingprovidesmoresolid data,allowing forinter-laboratory variationstobeeasily deter-mined.Thesemeasurementsarethencombinedtoderiveaneye irritationclassificationoraninvitroirritancyscore.Eyeirritationis primarilydeterminedbytheextentofinitialinjurythatcorrelates withtheextentofcelldeathand ultimatelytheoutcomeofan irritantonaneye(Jesteretal., 2001).Generally, slightirritants damagethesuperficialepithelium,mildirritantspenetratefurther todamagethestromaandsevereirritantspenetratethroughthe corneaanddamagetheendothelium(Jesteretal.,2001)(Fig.1).

Ocularorganotypicmodelsorenucleatedeyetests(EET)were

firstintroduced byBurton et al. (1981)using isolated rabbit eyes (IRE) fromanimalsusedforotherresearchpurposes,orthosethathad beensacrificedcommerciallyasafoodsource(ICCVAM,2010c).The IRE test, or rabbit enucleated eye test (REET) was originally

developedtodetectsevereirritantsthatcauseseriousirreversible eyedamage(Guoetal.,2012).Currently,themostcommonlyused test substances for IRE are active pharmaceutical ingredients, chemical/synthetic intermediates, cleaners,raw materials,soaps and detergents, solvents and surfactants (ICCVAM, 2010c). Lab-specificIREprotocolshavedevelopedovertime,withvariables including the evaluation of one to four different endpoints, differences in prediction models or classification systems, differences in the number of controlsused and methodological variations(ICCVAM,2010c).IREhasbeenextensivelyevaluatedby internationalregulatorybodies includingtheEuropean Commis-sion/British Home Of_fice (EC/HO), the Cosmetic, Toiletry and Perfumery Association (CTPA) and the Interagency Regulatory AlternativesGroup(IRAG)(Guoetal.,2012).However,todate,the IRE protocol is not considered to be adequately validated for classificationofocularirritancy.Instead,itisadvisedthatIREisused fornon-regulatoryoptimizationstudiestofacilitatethecollectionof datatoexpandtoxicologydatabases(ICCVAM,2010c).

Slaughterhousewastehasbeenextensivelyinvestigatedasan alternativetissuesource(Prinsen, 1996)forEETs.Bovineorporcine corneas are often used (Reichl and Muller-Goymann, 2001), althoughchickenenucleatedeyetests(CEET),alsoknownasthe isolatedchickeneye(ICE)testarewidelyacceptedtobeareliable andaccurateslaughterhousetissueforassessingtheeyeirritation potentialoftestmaterials(Prinsen,1996).TheICEtestingprotocol (TG438,(OECD,2013b)isbasedupontheIREmodelandwasfirst describedbyPrinsenandKoëter(1993).Theeyesareisolatedfrom an intact chicken head and processed 2h postmortem. The enucleated eye is then positioned in a clamp, with thecornea positionedverticallyandtransferredtoasuperfusionapparatusfor examination of damage (Maurer et al., 2002) (Fig. 2i). Once approved,theeyesareequilibratedforupto1h(Fig.2ii).Baseline thicknessandopacitymeasurementsarethenrecorded,beforethe eye is positioned horizontally and the test substance applied (0.03mlliquid,0.03gsolid)for10s(Fig.2iii).Thecorneaisthen rinsedwithhypertonicsaline(Fig.2iv)beforebeingreturnedtothe superfusion chamber for analysis (Fig. 2v). Toxic effects are recordedbymeasuringchangesinopacity,fluoresceinretention, tissue thickness (swelling) and a macroscopic evaluation of changestothesurfaceofthetissue(OECD,2013b).

Arecentre-evaluationofICEtestingresultedinanendorsement forthetestasbeingscientificallysoundandthatthetestcanbe successfully used to identify substances that do not require classification (non-irritants, GHSNo Category) as wellas those deemedtocauseseriousirreversibleeyedamage(GHSCategory1). Thisguidancewasadoptedin2009(OECD,2009a)andupdatedin 2013 (OECD, 2013b). Solids (soluble and insoluble), liquids, emulsionsandgelscanallbetested,althoughgasesandaerosols haveyettobeassessedandvalidatedusingthis method.When used to identifyGHS Category 1 chemicals, ICE has an overall accuracyof86%,whenusedtoidentifyGHSNoCategorychemicals ICEhasanoverallaccuracyof82%(OECD,2013b).ICEisoftenused as a pre-screen for Draize testing;although despite promising outcomestheinvivoDraizetestingresults stilloverruleexvivo

results should discrepancies occur. Discrepancies are often associatedwithhighfalsepositiveresultsforalcohols,andhigh false negative results for solids, surfactants and anti-fouling organicsolventcontainingpaints(OECD, 2009a).ICE cannotbe usedtoclassifyGHSCategory2,2Aor2Bchemicals,althoughto date,noexvivoorinvitrotestiscapableofclassifyingchemicalsin thiscategory.

TheBovineCorneaOpacityPermeability(BCOP)assaywasfirst developedbyGautheronetal.(1992)basedonmethodsoriginally describedbyMuir(1984,1985,1987)andTchao(1988).Theintact corneasofhealthyanimalsareheldbetweenO-ringsmountedover a(posterior)chamber;ananteriorchamberispositionedabovethe

cornea,bothofwhichareclampedtogether(Fig.3).Eachchamber hasits owndosing holewhichallows boththe epitheliumand endotheliumto be treated independently. Currently, opacity is measuredusinganOP-KITopacitometer,whichprovidesa center-weightedreadingoflighttransmissionbymeasuringthechanges involtagewhenthetransmissionofwhitelightaltersasitpasses throughthecornea(Verstraelenet al., 2013).However,opacity readingscanbeunderestimatedasopaqueareastendtodevelopin

spotsinanon-homogeneousmanneraroundthecornealperiphery (Verstraelenetal.,2013).InresponsetothisVanGoethemetal.

(2010) developed a prototype laser light based opacitometer

(PLLBOA),whichhasbeenfurtherimprovedviatheintroductionof a camerawith a specklenoise reducer, and anoptimizationof treatmentconditionstomorecloselymimictheinvivoscenario (JamurandOliver,2010;Verstraelenetal.,2013).Aminimumof threeeyesareusedpertest.Twodifferenttreatmentprotocolsare

Fig.2.Schematicrepresentationofthechickenenucleatedeyetest(CEET),alsoknownastheisolatedchickeneye(ICE)protocol,whichisbasedupontheisolatedrabbiteye (IRE)protocol.

useddependentuponwhetherthetestmaterialisasurfactantor not.Anadvantageofthisassayisitsspeed,withresultsusually obtainedwithin24h.

BCOPtestinghasbeenevaluatednumeroustimesbyICCVAM,in conjunction with the European Union reference laboratory for alternativestoanimaltesting(EURL-ECVAM),formallyknownas theEuropean Centrefor the Validation of AlternativeMethods (ECVAM)andtheJapaneseCentrefortheValuationofAlternative Methods (JaCVAM) regarding its suitability in identifying both substances that induce serious damage and those that are classified asnon-irritants.It hasbeen determinedthat BCOPis suitableandscienti_ficallyvalidforbothpurposes(OECD,2013a) and is routinely used by cosmetics and drug development companiesfor in-housetesting ofprocess intermediates(Eskes etal.,2005).Althoughitcannotbeconsideredasastand-alonetest, BCOPreceivedinternational acceptancein2009 (OECDTG437) whichwasthenreviewedandupdatedin2013(OECD,2013a).Itis recommended for identifying severe irritants without further testing(OECD,2009b)andhasreceivedendorsementforbeinga scientificallyvalidalternativetest(OECD,2013a).BCOPandhasan overall accuracy of 79% when used to classify GHS Category 1irritants,whencomparedtoDraizetesting(OECD,2009b,2013a). Lossofaccuracyhasbeenlinkedtohighfalsepositiveratesfor alcohols, ketones and solid test materials. When these are excluded, BCOP accuracy increases to 85%. However, since all alcohols and ketones are not over-predicted, they are not consideredtobeoutoftheapplicabilitydomainofthetest.Solid materialsoftenresultinvariabledataandirrelevantresultswhen usingDraizetesting(Prinsen,2006)sincesolidmaterialscanalso causemechanicaldamage.Withregardstotheclassificationoftest materials that do not promote serious eye damage (GHS No Category),BCOPhasanoverallaccuracyof69%.BCOPdoeshavea highfalsepositiverateof69%whencomparedtoDraizedata,but this value,although seemingly high, is not critical, since non-irritatingchemicalswhichhavealowinvitroirritancyscore(IVIS) willbetestedusinganotheradequatelyvalidatedinvitrotestdata, orasalastoptioninvivorabbittesting(OECD,2013a).

The porcine cornea opacity permeability (PCOP) assay uses porcine corneas, which can be considered as advantageous in comparison to bovine corneas since there are fewer concerns regardingencephalopathydiseases(VandenBergheetal.,2005). Anatomically,itmoreaccuratelyresemblesthehumancorneawith regardstostructureandthickness,andporcinecorneashavebeen regularlyusedinophthalmicresearch(LynchandAhearne,2013). Sincecornealsizeisdifferentbetweenpigsandcows,theholder utilizedinPCOPstudieshasbeenslightlymodified,withsmaller diameters, and reduced volumes of test substances (Van den Bergheetal.,2005).PreliminarystudiesconcerningPCOPrevealed thatitcanbeusedtoaccuratelypredicteyeirritationforliquidand

watersolublesubstances(VandenBergheetal.,2005).However,it hasyettobeadoptedbyregulatorybodiesthatseemtofavorBCOP. Organotypic/enucleatedmodelsareborderlinebetweeninvivo

andinvitrosystemsandareadvantageousinthattheyhavefewer ethicalconnotations(Luepke,1985)withreducedcosts.Although promisingresultshavebeenobtainedfromEETstheyallsharethe commonproblemthatinterspeciesdifferencesregardinganatomy and physiology are still present. Such differences produce discrepancies in permeation studies and toxicity tests (Reichl etal.,2004;ReichlandMuller-Goymann,2003).EETmodelsalso lack, or do not consider conjunctival and irradial issues, in_flammatoryresponseelementsandcornealrecoveryor revers-ibility of lesions(Guo et al., 2012). Theyalso only account for cornealeffectsandcannotpredictsystemiceffectsofsubstances, such as the lethality of certain pesticides (OECD, 2009a). Furthermore they can only be used for relatively short-term assessment periods (4h), and so are not suitable for testing substances that produce effects over extended time frames. However, suchproblemsare associated withall exvivo testing methodsandprotocols.

4.2.Non-ocularorganotypicmodels

The chorioallantoicmembrane vascularassay (CAMVA),also knownastheHen’seggtest(HET),orHühner-embryonenteston CAM(HET-CAM),orsimplyCAMassaywasfirstproposedbyLuepe andKemper(Luepke,1985;LuepkeandKemper,1986).CAMisthe vascularizedrespiratorymembranefoundwithinthemembraneof a fertilized chicken egg, with a vasculature and inflammatory processsimilartotheconjunctivaltissueofrabbit’seyes.Thetestis used toprovide qualitativeinformation onthe potentialeffects occurringintheconjunctiva followingexposure toa substance, whilstevaluationofcoagulationcanbeusedtoreflectpotential corneal damage (NICEATM, 2006). Although CAM models are usuallyclassifiedalongsideICE,BCOPandIREmodels,theydifferin evaluationcriteriaused(Barile,2010)sincetheyhavetheaddition of vasculature(Curren andHarbell,2002).The generalprotocol involves exposing theCAM (Fig.4i), theapplication ofthe test materialtothesurface(0.2–0.3mlliquid,0.1–0.3gsolid)(Fig.4ii), followedbyrinsing(Fig.4iii)and observationofchangestothe membranemorphologywhichareassessedandscored(Fig.4iv). Incubationtimes,relativehumidity,numberofreplicates,breedof hen(althoughwhiteleghornbreedismostoftenused),criteriafor eggselection(age/weight),eggrotation,methodofopeningthe eggshell, volume/weight/concentration of test substance used (althoughusuallyappliedneat),useofpositive/negativecontrols andexposuretimesmayvarydependentupontheprotocolused. This inadvertentlyleadsto problemsregarding intra-laboratory reproducibility. Most protocols observe the time in seconds

wherebyasubstancecauseshemorrhage,vasoconstrictionand/or coagulation that is measured, scored and then categorized (VinardellandMitjans,2008).Otherendpointsincludeinjection (mildhemorrhage), vasoconstriction,dilationand lysis (disinte-grationofvessels)(Gettingsetal.,1996;Luepke,1985;Luepkeand Kemper,1986;Macianetal.,1996;Spielmann,1995;Sterzeletal., 1990).Theirritationscoringvariesdependentupontheclassifi ca-tionsystembeingused.Theuseofcolored,turbidorsubstances thatadheretotheCAMhavebeenlinkedtocompromisedresults sincetheyimpairvisualization(NICEATM,2006).TheCAMassay has yet to receive international regulatory acceptance. Instead

ICCVAM (2010a) recommends that the test is used for

non-regulatoryvalidationoroptimizationstudies.

Theslugmucosalirritation(SMI)assaywasdevelopedatthe laboratory of pharmaceutical toxicology, Ghent University, Belgiumtopredictthemucosalirritancypotencyof pharmaceuti-cal formulations and ingredients (Adriaens et al., 2001, 2008; Adriaens and Remon, 1999). It uses the terrestrial slug Arion lusitanicus,whichisconsideredtohavelimitedsentienceandsois not protected by legislation covering animal experiments

(Adriaens and Remon, 1999). Slugs produce mucous and lose

bodyweightwhenplacedupon irritatingsurfaces.Whentissue damageoccurstheslugreleasesadditionalproteinsandenzymes from its mucosal surface. Both of these factors allow for quantifiable endpoints, and for substances to be classified as non-irritating, irritating or severely irritating. In general, mild irritantscauseanincreaseinmucousproduction,whereassevere irritantsresult intissue damage and protein/enzymerelease in additiontoincreasedmucousproduction(Adriaensetal.,2008). Ina previousstudyusing20 knownreferencechemicalsit was shownthattheSMIassaywasareliableandreproducibletesting system(Adriaensetal.,2008).HowevertheSMIassayfailedtopass aformalvalidationstudy,soiscurrentlyonlyusedasapre-screen forsimpletoxicologicalendpoints.

5.Invitrotests

Invitrotoxicitytestingmodelsandassaysusingculturedcells areadvantageouscomparedtoinvivoandexvivotestinginthat theyarerelativelyinexpensive,simple,andquicktomanufacture. Thisallowsforreplicationandquantifiabledatatobegathered, whilstalsolendingitselftoautomation.Invitrosystemsmayalso allowforamechanisticunderstandingoftoxicityatthecellularor molecular level (Davila et al., 1998). They are also capable of creatingabroaderrangeoftoxicologytestingcapabilitiessuchas multipleendpoints,concentrations,exposuremethodsandtimes tobebettercontrolledandtailoredaccordingly.

Oneoftheoriginalalternativeocularirritationmodelswasthe EYTEXTM_{systemwhichwasdeveloped,testedandevaluatedinthe}

1990s(Courtellemontetal.,1999;Gordonetal.,1990;Matsukawa etal.,1999;Royetal.,1994).AlthoughEYTEXTM_{wasunreliableat}

predicting ocular irritancy, primarily due to the lack of an appropriate prediction model; it did set the stage for the developmentof oculartoxicity models. The OcularIrritection1 assayis an updated protocolbased upon the former EYTEXTM

system (Eskes et al., 2005, 2014). The test is based upon the principle that eye irritation and corneal opacity caused by exposuretoirritating chemicalsalter thefundamental function oftheproteinsthatmakeupthehighlyorganizedcornealtissue (Eskesetal.,2005).Theassayisavailableasanoff-the-shelfkit comprisedofamacromolecularreagentofproteins,lipids,andlow molecular weight proteins which when rehydrated form an orderedmatrixsimilartothatof thenativetissue, amembrane discwhichallowsfordeliveryofthetestchemical, instrumenta-tionandcomputersoftware.Testchemicalsaregraduallyadded using thedefined membrane disc, resulting in turbidityof the

matrix,duetothechangeinconformationandhydration(Eskes et al., 2005). Spectroscopic methods are used to measure the turbidityofthereagentat405nm.Prospectiveandretrospective validationstudieshavebeenperformedtoevaluatethesuitability of the Ocular Irritection1 assay for discriminating between chemicalsthatdo notrequireclassification fromchemicalsthat do(Eskesetal.,2014).Limitationsincludelimitedusefulnesswith respecttointenselycoloredchemicals,underestimationofsome cationic surfactants and overestimation of surfactant based formulations containing magnesium and multi-carboxylated carbohydratechemicals(Eskesetal.,2005).Currently,theresults of prospective and retrospective validation studies have been submittedforformalvalidation(Eskesetal.,2014).

5.1.Cytotoxicityassessment

Mostinvitrooculartoxicityassaysconsistofamonolayerof culturedcellsandacytotoxicityassessmentinresponsetoatest material.Ingeneral,cytotoxicitymeasurementsarequick,simple andinexpensive(Takahashietal.,2008).Amongthemethodsof assessing cytotoxicity are thymidine incorporation, Coomassie brilliant blue protein measurements, crystal violet and Lowry reagent, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays (MTT assays), lactate dehydrogenase leakage (LDH), fluorescein leakage(FL)trypanblueexclusion,florescent staining withpropidium iodide and neutral red uptake/release tests (Huhtala et al., 2008). Each of these methods has their advantagesandlimitations. Ingeneral,a combinationoftwo or moreofthesemethodsisnormallyusedtoassesscytotoxicity.

Several assays combine cell staining with fluorescence or absorbancemeasurementtomonitorchangesincellnumberand determinewhetherasubstanceiscytotoxic.Assaysthatusedyes suchastrypanblueorpropidiumiodidearebasedontheconcept thatthesedyeswillbepreventedfromenteringthecellunlessthere isdisruptiontothecellsmembrane(Strober,2001).Hencehealthy cellswillremainunstained,whiledeadcellswillstainpositive.The amountofdyewithinacellpopulationcanbemeasuredandusedto determinethepercentageofcytotoxiccells.Onelimitationwiththis approachisthatitonlystainsdeadcellswhilstdyingorunhealthy cellsmayremainunstained.Alternativelyadyesuchascrystalviolet canstaindeoxyribonucleicacid(DNA)withinacellasshown(Fig.5). In this assay the color absorbance of the stained cells can be measuredatawavelengthofapproximately570nm,whichcanthen beusedtoassessthenumberofcellspresent(Gilliesetal.,1986;

Rothman, 1986). A reduction in cell number would indicate a

cytotoxiceffect.Intheneutralredassay,lysosomesratherthanDNA inhealthycellsarestainedpositive.Thedyecanthenbeextracted andusedtoquantifythenumberofviablecells(Repettoetal.,2008).

FotakisandTimbrell(2006)foundthattheneutralredassaywas moresensitivetocytotoxiceffectsoncellsthanseveralotherassays tested.

In addition to staining, DNA can be quantified using other techniques. For example in a thymidine incorporation assay, 3H-thymidine (a radioactive nucleoside) is incorporated into newlysynthesizedDNAduringmitosis.Inhabitationofthymidine incorporationwouldindicatecytotoxicity.

Enzymaticassaysareamongthemostcommonlyusedtoassess cytotoxicity. LDH assays quantify the release of LDH following ruptureofthecellmembranebyusingittocatalyzetheconversion oflactatetopyruvatewhichcanbemeasuredcolormetricallyand usedtoquantifycelldeath.MTTassaysmeasuresthereductionof yellow MTT to purple formazan by mitochondrial succinate dehydrogenase.Thischangeincolor ismeasurablevia spectro-photometry.AsMTTreductiononlyoccursinmetabolicallyactive cells,thespectrophotometerreadingcangiveanestimateofthe numberofviablecellspresent.

Theshorttimeexposuretest(STE)isarelativelysimpleassay methodthatestimatescell cytotoxicityand viabilityusingMTT (Kojima et al., 2013; Takahashiet al.,2008, 2011).It utilizes a confluentmonolayerofrabbitcornealcelllines(Statens Serumin-stitutrabbitcorneal,SIRC)ona96wellpolycarbonatemicroplate (Takahashietal.,2008).Testchemicalsaredissolvedoruniformly distributedin either physiologicalsaline, 5%dimethyl sulfoxide (DMSO)inphysiologicalsaline,ormineraloilsastestsolvents,as opposedtoculturemediumwhichisoftenusedincytotoxictests. Thisallowsforwaterinsolublematerials,acidsandamidestobe evaluated(Takahashietal.,2008), whichwouldotherwisehave weakenedeffects when mediais used asa solvent, due tothe bufferingeffectthatthemediamayhave.Asthenamesuggests,the exposuretimetoagivenchemicalisveryshort,itisonly5min, comparedtolongerexposuretimesusedintheFLassay(15min) andtheneutralredassay(1,5or30min)(Takahashietal.,2008)for example.Itisbelievedthatthattheshortexposureismoresimilar toactualexposureconditionstoaconsumerproduct,whilstalso providingfastresults(Kojimaetal.,2013;Takahashietal.,2011). ThisalsoallowstheSTEtobeusedforhigh-throughputscreening toevaluatemanychemicals.Twodifferentconcentrationsofthe testmaterialareevaluated,5and0.5%,respectively.Postexposure cellviabilityiscompared toasolventcontrol(relativeviability) (OECD,2014a;Takahashietal.,2011).Ifthecellviabilityis70%at both0.5and5%concentration,thenthechemicalisclassifiedas GHSCategory1.Ifcellviabilityif70%atbothconcentrationsthen thechemicalisclassifiedasGHSNoCategory(OECD,2014a).The STE was submittedto theOECD in 2011 as a method of high-throughputscreening(Kojimaet al.,2013)toevaluateminimal, moderateand severeeye irritation. The STE is currentlyunder investigationviatheOECDforregulatoryacceptanceaspartofa tiered-testing strategy for either top–down or bottom–up approaches. It is recommended that STE is used for the identification of GHS Category 1, severe irritants and GHS No Category,non-irritants,althoughinbothinstancesfurthertesting isrequiredtoestablishadefinitiveclassification(OECD,2014a).It isnotrecommendedfortheidentificationofGHSCategory2(Aor B)chemicals.

Penetrationof a dye or reagent througha barrier of cells is anotherapproachtoassesscytotoxicity(Fig.6).TheFLassay(TG 460, (OECD, 2012c) can reveal the toxic effects of chemicals followingashortexposure.AmonolayerofMadin–Darbycanine kidney(MDCK)cellsaregrownonpermeablecellinserts.Thetest worksbymeasuringtheamountoffluoresceinleakagethroughthe cellmonolayerwhichcanbeusedtodeterminetheintegrityofthe barrierformedbythecells.Cytotoxicitywouldresultinanincrease inthepenetrationof_fluoresceinthroughthemonolayer.Increased

invivopermeabilityofthecornealepitheliumcorrelateswiththe degree of inflammation and surface damage as eye irritation occurs. The volumeof fluorescin leakage is measured

spectro-fluorometricallyandcomparedtocontrols.TheFL20,whichrefers

totheconcentrationthatcauses20%FLrelativetoanuntreated controliscalculatedandincorporatedintoapredictionmodelto identifyirritation.FLisrecommendedforuseinidentifyingsevere, GHSCategory1watersolublechemicalsaspartofatiered-testing strategy (OECD, 2012c). Any chemical that is not predicted as severelyirritatingusingFLwouldrequirefurtherinvitroorinvivo

methods,sinceitisnotcapableofdistinguishingsuchchemicals. AnotherlimitationisthatFLcannotbeusedtoclassifystrongacids andbases,cell_fixativesandhighlyvolatilechemicalssincetheir modes of actioncannot bemeasured using this mechanism.In addition,viscousandcoloredmaterialsarenotsuitedtothistest (OECD,2012c).

The Cytosensor Microphysiometer (CM) test method is a cytometricandcell basedassay whichutilizesa sub-confluent monolayer of mouse L3929 fibroblasts culturedon a transwell insertinasensorchamber.Changesinacidityinresponsetoan irritant are measured using a pH meter, ocular toxicity is evaluatedbycalculatingthereductioninmetabolicratecaused by the addition of a test chemical to the culture media compared to the basal metabolic state. CM has been recommended for the identificationof GHS Category 1, severe irritantsandGHSnonirritants(Alépéeetal.,2013;OECD,2012a). The test is limited for use with test substances which do not settle or separate during analysis, primarily water solublesurfactantsandsurfactant-containingmixtures,butalso somenon-water-solublesolids,viscouschemicalsorsuspensions thatmaintainuniformityduringanalysis(OECD,2012a).Adraft OECD test guidance for CM is currently under review (OECD, 2012a).

Cellculturesusingbothtargetcellsandnon-targetcells,usually exposecells totest materialsthat havebeendilutedin culture media (Reader et al., 1990), although both water soluble and insolublematerialscanbeassessed(VanGoethemetal.,2006).In general, cell culture methods are based upon long term cell survival, proliferation and function, including the release of

specificcytokines.Usingpermanentorimmortalizedcellslinesis advantageouswithregardstoavailability,reproducibility,easeof maintenanceandeaseofdamagedetection(Readeretal.,1990).

5.2.Cornealepithelialmodels

Several in vitro toxicity models have been developed using cornealepithelialcells.Invivotheepitheliumistheoutermostlayer of thecorneathat protectstheunderlying tissue by restricting foreignmaterialfromenteringwhilestillallowinggasandnutrient exchangetotheunderlyinglayersofthecornea.Thusitisthefirst point ofcontactforpotentiallyhazardous materials.In vitrocultured epitheliumiscapableofretainingtheinvivorepairmechanisms foundinthe nativecornea(Davilaet al.,1998), althoughthese mechanismsarenotalwaysgiventhelevelofattentiontheydeserve (DholakiyaandBarile,2013).Epithelialmodelscanbeconstructed fromanimalcells(commonlySIRCcells(UbelsandClousing,2005)) suchasintheSTEtest,humanepidermalcells,orhumancorneal cells,whichareusuallyculturedinde_finedmediumoncellculture membranesusingair-liftingtechniques(Alépéeetal.,2013;Cotovio

etal.,2007;Kaluzhny etal.,2011;Matsudaetal.,2009)tocreatea3D stratified epithelium.Cytotoxicity following topical exposure is generallyused as anendpoint (Curren and Harbell, 2002), and epithelialmodelshavethepotentialtoidentifynon-classifi ed/non-irritatingsubstancesfrommildirritants(Scottetal.,2010). Time-to-toxicitymeasurements(ET50),whichaccountforthetimerequired

fora50%reductionincellortissueviabilityfollowingexposure when compared to a negative control (Kaluzhny et al., 2011; Osborneetal.,1995),areoften usedasanendpoint.Althoughhuman primary epithelial cells have been investigated (Tripathi and Tripathi,1988,1989;Tripathietal.,1989),theiruseislimitedin toxicologymodelsduetothelackofavailabilityofhumancorneas anddifficultiesassociatedwithexpandingandpassagingprimary epithelialcells.Thus,rabbitcornealcellsormousefibroblastsare oftenutilizedasanalternativesource.

Matsudaetal.(2009)culturedrabbitcornealepithelium(RCE) cellsontocollagenhydrogels,whichactsasaperabasalmembrane. Tovalidate themodel 30chemicalswithknowndegreesofeye irritation (from Draize testing), ranging from non-irritating to severely irritating were tested. Inconsistencies occurred when

testingacidsandalcohols,whichwasthoughttobeduetoapH dilution,thevolatilityofthealcohol,orareactionwiththebuffer solutionpriortotesting(Matsudaetal.,2009).

TheMatTekCorporationdevelopedacommerciallyavailable3D corneal epithelialmodel (OCL-200)based upon humanderived epidermalkeratinocytesfromneonatalhumanforeskin( McLaugh-lin et al.,2009; Sheasgreenet al., 2009) grownon cell-culture inserts in serum-free media, to form a stratified, squamous epithelium,marketedasEpiOcularTM_{.Testsubstancesaredirectly}

appliedtothemodels,andcytotoxicity ismeasuredusing MTT. Substancesthatcausethemostrapidinjurytocellsgenerallyhave higher irritationpotentials (Matsudaet al., 2009).The original protocolhassincebeendevelopedintoasingletime-pointprotocol knownastheEpiOcularTM_{eyeirritationtest(EIT)(Pfannenbecker}

etal.,2012).Ifthetreatedcellshaveviabilitygreaterthan60%post treatmentthen thetest substanceisclassifiedasnon-irritating. EpiOcularTM _{is currently used by numerous contract research}

laboratories, industrial cosmetic, personal care, and household chemical companies in place of Draize testing for product development.

SkinEthicLaboratories(Nice,France)developedastandardized 3D reconstitutedhuman corneal epithelium (HCE) model from immortallized human corneal epithelial mucosa cells (Cotovio etal.,2007;Doucetetal.,2006;VanGoethemetal.,2006),thatis structurallyverysimilartothecornealmucosaofthehumaneye. Substancesaretestedusingtwodifferentexposuretimes:(i)short term,wherebytheHCEisexposedtoachemicalorsubstancefor 10min, (ii) longterm, whereby theHCE is exposed for 60min followedby16hincubation.IfthetreatedHCEhasviabilitygreater than50%posttreatmentthenitisclassifiedasnon-irritating.

Both EpiOcularTM EIT and SkinEthicTM HCE models have undergoneprospectivevalidationbyEURL-ECVAMandCosmetics Europeto distinguishirritants(GHS Classification1/Category 2) from non-irritants(GHSNoCategory)(Pfannenbeckeretal.,2012;Zuang etal.,2013).Over100chemicalsweretestedand bothmethods showed high reproducibility (>90%) (Zuang et al., 2013). The EpiOcularTM_{EITmetallthepredictivecapacityacceptancecriteria}

for the testing of liquids protocol, butnot all ofthese criteria were met bythesolidsprotocolnorbyanyoftheSkinEthicTM_HCEprotocols

(Zuangetal.,2013).TheEpiOcularTM_{EITsolidsprotocolwasfurther}

optimized for solids and further validationwas conducted.Atpresent theSkinEthicTM_{HCEmodelisstillundergoingfurtheroptimisation}

foritssolidsprotocol(Zuangetal.,2013).Thefinal sensitivityof EpiOcularTMEITwasdeterminedtobe96%,withspecificityof63% andaccuracyof80%,thuswasconsideredvalidfordistinguishing non-irritantsfromirritants(OECD,2014a).However,EpiOcularTM

EITisnotintendedtodifferentiatebetweenGHSCategory1(serious eye damage) and GHS Category 2 (eye irritation). A draft test guidanceforEpiOcularTM_{EITandperformancestandardshasbeen}

deliveredto theOECD(2014a),and thefinaltestguidelines are expectedtobeadoptedin2015.

Recently Katoh et al. (2012, 2013) and Jung et al. (2011)

developed the LabCyte CORNEA-MODEL (Japanese Tissue Engi-neeringCo.,Ltd.,Japan)andMCTT-HCEmodel(MCTT,Seol,Korea), respectively. Unlikethe commerciallyavailable EpiOcularTM _EIT

andSkinEthicTM_{HECmodels,bothutilizenormalhumancorneal}

epithelial cells isolated from the human limbus of remaining cornealrimfollowingtransplantationthatareculturedaboveand supportedbycellfeederlayers.Theyhavebeenshowntoexpress similarmorphologyandbiomarkerexpressiontotheintacthuman cornealepithelium.Pre-validationstudies havebeenperformed for theLabCyte CORNEA-MODEL, to determineoptimum treat-ment time, volume,post-incubation time and rinsingprotocols (Jungetal.,2011).AlthoughbothMCTT-HCEandLabCyte CORNEA-MODELhave reportedpromising results witha high degreeof accuracy,neitherhasyettoenteraformalvalidationassessment.

Oneofthelimitationswithepithelialmodelsisthattheinitial enzymaticormechanicaldissociationofcornealtissueprecluding cellularisolationitselfmayevoketraumaticstimulithatresultina diverserangeofresponsesfromthecell(Davilaetal.,1998).This may include the disruption of cellular structural integrity, the release of inflammatorymediators or cell differentiation,all of which can lead to differences between in vivo and in vitro

substance biokinetics (Davila et al., 1998). This needs to be consideredwhenusinganycellsforinvitrotoxicologytesting.The useofimmortalizedepithelialcelllinesdoesnotalwaysfaithfully representcornealcellbehaviorinvivo,sincetheimmortalization process or subsequent culturing conditions alter expression patterns.Forinstance,celllinesdonotexpresscytokeratins(CK) suchasCK3,7,8,18and19(Huhtalaetal.,2008).Thismaymake theidentificationofspecificbiomarkersoftoxicologysomewhat morechallenging.

Epithelialmodelsareoftenfragileandhavetobehandledvery carefullytoavoiddryinganddamagingthetissues.Cell detach-mentinculturecanleadtoamisinterpretationofdatadependent upontheexperimentalendpoint(Davilaetal.,1998).Theyarealso somewhatlimitedinthattheyonlymodeltheepitheliallayerand socannotbeusedtodeterminethepossibleeffectsofsubstances that in vivo penetrate the stroma and endothelium, or the reversibility of the irritation.They also do not account for the factthatsomematerialsorchemicalsmayaffectthevariousparts of the eye differently (Reader et al., 1990) and that cell–cell interactions,namelythosebetweentheepitheliumandadjoining stromaarepivotaltocornealresponses(McLaughlinetal.,2009; Wilsonetal.,1999,2014).Invitrocellbasedassaysarealsodevoid ofhormonal,immuneandneuralinfluences.Althoughthismakes them simplerand easier tointerpret, it canalso be seen as a limitationsinceitdoesnotaccountfortheinteractionsthatoccur throughout the whole tissue, especially when considering the complexityinanorganasspecializedastheeye(Barile,2010).

5.3.Cornealequivalents

In response to the limitations incurred from using in vitro

corneal epithelialmodels,morecomplexmulticellularassaysor cornealequivalents,termedassuchduetotheirsimilaritytoreal corneas,havebeenunderdevelopmentinordertomoreaccurately replicatethecomplexityandinherentcharacteristicsofthenative cornea.Bothanimalandhumancellshavebeenincorporatedinto corneal equivalents. Many studies have attempted to culture human primary cells under the premise that they will have a greatercapacity for determininghumanirritancy (Zieske et al., 2004).However,problemsassociatedwiththeisolation,growth, maintenance anddifferentiationofcornealcellshasmeantthat many researchers choose to use transformed or immortalized humancelllinesoranimalcells,sincetheyareeasiertoculture.

Grif_fithetal.(1999)producedthe_firstworkingequivalentofa humancorneausingimmortalizedhumancornealcells.Themodel wasoriginallydevelopedtohelptounderstandwhycorneasfailto heal properly after laser eye surgery. A collagen_–chondroitin sulfatesubstratecross-linkedwithglutaraldehydewasusedasa tissuematrix.Initiallyathinlayerofendothelialcellswasgrownin aculturedish.Keratocytesandsupportproteinswereaddedbefore

2004). Dorsal root ganglia isolated from chick embryos were utilizedasaneuralsource,sinceoptimalfunction,maintenance andwoundhealingofmanytissuesisdependenttosomeextenton peripheral sensory innervations (Suuronen et al., 2004). The innervatedcorneal constructs werereportedtohavelowercell deathrateswhen exposed totest chemicals compared to non-innervatedequivalents.Thissuggeststhatthepresenceofnerves protects the epithelium from chemical irritation and possibly explainswhypreviousnon-innervatedcornealmodelshavebeen deemedover-sensitivewhenusedintoxicitystudies.Thismodel stillrequires furtherdevelopmentsince manyof thefunctional propertiesofthenervesremainunclear.These typesofmodels may demonstrate more promise for clinical development as cadavericalternativesforcornealtransplantation ratherthanas modelsfortoxicologicaltesting.

Reichletal.(2005)manufacturedahumancornealequivalent for invitro drugpermeationstudies byculturing immortalized epithelial, endothelialand stromal cells in a collagen hydrogel matrix.Three reagentscommonly usedin ophthalmic drugs to treat glaucoma and inflammatory diseases were tested and permeationdataobtainedwascomparedwiththosefromexcised porcinecorneaandaporcinecorneaconstruct(Reichletal.,2004; ReichlandMuller-Goymann,2003).Porcinecorneaswere investi-gatedduetotheirrelativelysimilaranatomyandphysiologytothe humancornea.Thehumancorneaconstructhadsimilarepithelial barrierpropertiestoanativecorneawithonlysmallultrastructural differences,possiblyduetolackoftearsandblinking.Therewas increasedpermeabilityinthecornealequivalentscomparedtothe exercised porcine cornea for all reagents tested, although the differenceswererelativelyminor.Unfortunatelytherewasnodata availabletocompare thesecorneal equivalentswithanexcised humancornea(asinthestudiesbyGriffithetal.(1999).

Ingeneral,invitromodelsthatincludeastromallayerhavethe greatestpotentialtodistinguishsevere/corrosiveeyeirritantsfrom otherclasses(Scottetal.,2010).Howeverrecentvalidationstudies havedemonstratedthatthereisnosingleinvitroocularirritation test, combination of tests, or testing strategies capable of completely replacing Draize testing (Huhtala et al., 2008) for predictingtheresponseofthefullrangeofirritationclasses.Thisis partlyduetoalackofunderstandingoftheunderlyingcellularand molecularmechanisms of eye irritation (Matsuda et al., 2009; Maureretal.,2002),apossiblelackofinnervation(Suuronenetal., 2004), difficultiesassociatedwhencomparinginvitrodatawith historicalanimaldataduetothesubjectivescoringsystemsused andthefactthatinvitrosystemsonlypartiallymodelinvivotests, insufficient prediction models,inappropriate statistical analysis (Eskesetal.,2005)andanapparentreluctanceofregulatorybodies toacceptnewinvitrocornealconstructs.

The principle disadvantages of using multicellular in vitro

modelsfortoxicityassays,isthatlikeepithelialbasedassays,they stilllackthecomplexityofacompleteorgan(Beckeretal.,2006). Forexample,thecompositionoftheaqueoushumorandtearfluid, orthemechanicalstressoftheeyelidsandtearflow(Tegtmeyer etal.,2001),intrinsicclearingmechanisms(tearingandblinking) (Davilaetal.,1998)arenottakenintoaccount.Inanaturalcornea all of these factors are important to protect the eye and are increasedwhenexposedtoirritation.Invitrofalsepositiveresults canbeattributedtothecontinuouscontactwithatestcompound

(Davila et al., 1998), thus the mechanisms that mimic tear

productionandblinkingmayneedtobeincorporatedintoinvitro

toxicitymodels.Alternatively,invitroassessmentofthe concen-trationinwhichatestsubstanceispharmacologicallyortoxicology activeandrelevantinvivoshouldbeassessed(Davilaetal.,1998) sincetheextent of theinitialresponse is apivotal mechanistic factorthatdeterminestheoutcomeofocularirritation(Jesteretal., 2001;Maureretal.,2002).

5.4.Futureofinvitroalternatives

It is unlikely that any single test, cell monolayer, three-dimensional epithelium,or multicellularcorneal equivalent will becapableofmimickingthecomplexitiesandnumerous physiolog-icalparametersofaninvivosystemfollowingexposuretoagiven substance(BorenfreundandPuerner,1985;Pfannenbeckeretal., 2012).Infact,havinga“one-sizefitsall”approachhaslargelybeen abandoned,withtheintentionofmanyinvitrosystemsistobe utilized as part of an integrated testing strategy using either top–downorbottom–uptiered-testingapproaches(Engelkeetal.,

2013; Scott et al., 2010). Top_–down approaches are for the

identification of severeirritants, bottom–up approaches arefor theidentificationofnon-irritatingsubstances(Barile,2010;Engelke etal.,2013).Thisresultsintwotestingstrategiestoreplaceonein vivotestandasofyet,neitherstrategycanbeusedtodistinguish between non-irritants and mild irritants and this remains an on-goingchallenge(Barile,2010;Nóbregaetal.,2012;Scottetal., 2010).Thecommercialaimsofinvitrotestingaretobefasterand cheaper,althoughcurrently,thecostsare roughly on parwithDraize testing.Itispreferablethatthetestingprocedurescanbeperformed withouttheneedforspecialisttrainingorexpensiveequipment (DholakiyaandBarile,2013).Fromacorporatestandpointinvitro

testsrequirethesamelevelof investmentastheyarecurrently makingusinginvivotests,sotheyeitherdon'tcare,orfailtoseethe benefitsinswitching.Alargefactorthataffectsthedecisionmaking ofcorporatecompaniesisthattheyaresellingtoalocalmarket,not justcountrieswithintheEU.Fordevelopingornewlyindustrialized countries, Brazil, Russia, India, China and South Africa, the underlyingchallengeisgetting themtounderstandtherolesof

in vitrotests,whichisacontinuingeducationalchallenge.Inorderto overcometheseissues,are-evaluationofcurrentlyusedinvitro

testsmayberequired(Nóbregaetal.,2012).

Invitroassaysandmodelsprovideusefuldatathatcomplement

invivostudiesallowingforsignificantreductionsinthenumbersof animalsused.Inorderrealizethis,itmustbeensuredthatclear endpointscorrelatebetweeninvivoandinvitrotests(Maureretal., 2002).Ingeneral,invitrotestsarevalidatedagainsttheDraizetest (Lenoir et al., 2011), with few actually investigating their predictability compared to humans. Despite the lackof formal validation,invitrotestsstillarecommonlyusedbyindustry.For example,industrial toxicologistsoftenuseinvitroprotocolsfor prioritizing products and ingredients for further development (CurrenandHarbell,2002).However,useoftheDraizetestisstill permittedworldwide,withtheexceptionofthecosmeticssection withinEurope.Althoughinvitroalternativestestsareavailable, whethertheyareactuallybeingusedinpracticeisquestionable. Everycountryhasitsownregulationsanddatarequirements.The EUmaybeconsolidated,buteverywhereelseisnotandregulations havetobenegotiatedonebyone–thisisaveryslowprocess,with noonecountryworsethantheother.Regulationsare aimedat protectinghumans, and regulatorsfocus onthis, thecultureof animalwelfareisdifferentineverycountry.

6.Insilicomodels

Insilicomodelsarecomputergeneratedmodelsthatcanplaya usefulroleinpredictingtheoculartoxicityofasubstance.Insilico

modelsutilizerepositoriesofexistinginvitroandinvivotoxicology data topredict thetoxicity of samples.Quantitative structure–

isincorporatedintocomputeralgorithmsthat arethen usedto generate predictive models. Once enough data for a specific toxicologicalendpointiscollected,evaluatedandweighted,then a generalized relationship between the test substances and its biological activity can be defined (Simon-Hettich et al., 2006). Several different commercially available and freely available modelingsoftwarepackageshavebeendeveloped,theapplicability ofwhichhavebeenpreviouslyevaluatedindetail(LoPiparoand

Worth,2010). This type of modeling is also dependenton the

availabilityofsuitablehighqualitydatabases,severalofwhichhave beenpreviouslydiscussed(Valerio,2009).

The primaryadvantages in using in silico modelsto predict toxicity;otherthanthefactthattheydo notrequiretheuseof animalsoranimaltissues;istheirspeedandrelativelowcost.In vitroand invivotoxicity modelsmaytakeweeksor monthsto generateresultsatconsiderableexpensewhileinsilicomodelscan generateresultsinminutesusingjustacomputerandsoftware. The continuing increase in computer processing speeds over recent years has enabled more sophisticated software to be developed.Amongthelimitationsassociatedwithinsilicomodels are its reliance on high quality data. This can be a particular problemwhencompilingdatafromdifferentlaboratoriesthatmay haveproduceddifferingresults.Sincethemodelsarerelianton datagenerated using animal modelsand cellbased assays, the limitationsassociatedwiththese,suchasinterspeciesvariationsin toxicologicalresponse,stillexist.Otherlimitationswithinsilico

modelshavebeendescribedpreviously(Valerio,2009).Ingeneral,

insilicomodels tendtobe moreuseful inpredictinga specific endpointratherthan a broadrangeof toxicologicaleffects that maybeproducedfromatestsubstance(Nigschetal.,2009)and they generally used with other test methods rather than exclusivelybythemselves.

7.Regulationandvalidationofoculartoxicitytesting

Finding suitable, regulatory approvedand validated alterna-tivestoanimaltesting isa crucialaimoftoxicological research (Alépéeetal.,2013)withregulatorybodieskeentoadopttheuseof protocolsthatmodifyandreducethenumberofanimalsusedin oculartestingprocedures.Foralternativemethodstobe success-fullyincorporatedintosafetyassessmentprocedures,theyneedto demonstrate that they can provide at least an equivalent or preferably superior level of protection to that obtained with currentmethods(VinardellandMitjans,2008).Invitroandother alternative testing methods have a long history in corporate decision-makingregardingchemicalsafetyandproduct formula-tion.However,formanyyears,alternativetestingstrategieswere neglectedasadefinitivetestingstrategyintheregulatorycontext

(Stephens and Mak, 2013). Thus, corporations would often

accompany alternative testing methods with more historical animal-based methods (Stephens and Mak, 2013). In order to moveawayfromthisstatusquooftoxicitytesting,itisimportant tohaveanunderstandingofregulatorytestingrequirementsand assessmentandwhytheyweredeveloped(Fowleetal.,2013).

7.1.Regulation

Numerousregulatoryauthoritiesandsystemsexistworldwide for the assessment and classification of potentially hazardous substances. Theirprincipal objectiveis toassess thehazardous potentialofsubstancesthatmaycomeintocontactwiththeeyein ordertosupplyregulations,guidelinesandrecommendationsfor theirsafeuse.Thisoffersconsumersortheenduserprotectionvia

the communication of hazardous information and protective measures(ICCVAM,2010b;Wilhelmus,2001)toprevent misap-plication and to minimize accidental exposure. Regulatory

assessment is based upon “informed decisions” that are not purely scientific in nature. They have to take into account congressional directives, legal precedent, benefit/cost considerations and public values (Fowle et al., 2013). This sometimes frustrates scientists, alternative-testing supporters andstakeholdersalike,since“goodscience”doesnotalwaysdrive decisionmaking(Fowleetal.,2013).

EURL-EVCAM aims to promote scientific and regulatory acceptance of non-animal tests. Similarly, ICCVAM is an interagency committee made up of 15 US Federal agencies including the Consumer Product Safety Commission (CPSC), NationalInstituteforOccupationalSafetyandHealth Administra-tion (NIOSHA) and the FDA. ICCVAM aims to facilitate the development, validation and regulatoryacceptance of new and revisedregulatorytestmethodsthatreduce,refineandreplacethe useofanimals.Itwasoriginallydevelopedasacommitteeofthe NationalCommitteeofEnvironmentalHealthSciences(NIEHS)in 1997,and wasmadepermanentin 2000under NICEATM.Since thenICCVAMhascontributedto63alternativetestingmethods, 38ofwhichdonotrequireliveanimals,althoughnotallofthem areconcernedwithoculartests.

Severaldirectivesrestrictandevenprohibittheuseofanimal testing, forexampletheAmendment of theCosmeticEuropean Directive(2003/15/EC)imposedabanontheuseofanimalsforthe testingofcosmeticsandtheiringredients.However,untilrecently companiescouldstillmarketproductsthathadbeenanimaltested outside of the EU. A new cosmetic regulation replaced the Cosmetics Directive in 2009 (Regulation (EC) No. 1223/2009) andsinceJuly2013,cosmeticsandcosmeticingredientstestedon animals can no longer be sold in Europe, even if they have been tested elsewhere. This has promoted considerable progress in replacing animal models for chemical toxicology (Alépéeetal.,2013).

7.2.Validationandregulatoryacceptance

EURL-ECVAM(formallyECVAM)andICCVAMwereformedin Europe and theUS, respectively, todeal withthe validation of alternative testing methods (Vinardell and Mitjans, 2008). Validation refers to the formal assessment, or rigorous set of policiesthatchallengethespecificobjectivesofatestmethodor model with regardto itsrelevance and reliability. Thisin turn provides the foundation to facilitate regulatory adoption and acceptance(Corvietal.,2006;StephensandMak,2013).Relevance referstotheextenttowhich atestormodelcorrectlypredicts/ measuresthebiologicaleffectofinterest;reliabilityisthedegreeto which the data in the protocol is reproducible within the guidelinesorprotocolofthemethod(Barile,2010).

Most protocols undergo a pre-validation stage, designed to prepareatestmodelorassayforfurtherprogressionintoaformal validation study. These mayinvolve intra-laboratorystudies to addressprotocoloptimization(PhaseI),transferability(PhaseII) andperformance (PhaseIII)(VanGoethemetal.,2006),sothat prioragreementscanbemadeondetailedprotocolsthatprepares andaidsthetestmodelortestintheformalvalidationprocess.

modelortest,amechanisticbasis,aspecificprotocolincludingall standard operating procedures with clearly defined endpoints, methodsofresultsinterpretationviapredictionmodelsandspecific controls used must be clearly defined; (ii) intra-laboratory variabilityassessment, todeterminepotentialvariationsindata incurredduetodifferentoperatorscarryingouttheprotocolwithin thesamelaboratoryset-up.Thisassessmentstageisusuallynotso problematic,sincelaboratoriesdevelopingamodelortestwould usually abandonor modify an irreproducible protocol prior to assessmentsubmission(UbelsandClousing,2005);(iii) transfer-ability,todemonstratethatthetestcanberepeatedindifferent laboratoryset-ups.Inthecaseofinsilicomodels,thisistheabilityof different operators to reproduce the model definition and predictions,which isoftendependentupon thestrengthofthe explanatorydocumentationprovided; (iv) inter-laboratory vari-ability,wherebythreetofourlaboratoriesaretypicallyaskedtotest adefinednumber ofsubstancesusingtheassessedmethodormodel tohighlightdiscrepancies.Thisalsoaddsfurtherinformationtoa test'spredictivecapacity;(v)predictivecapacity,whichutilizesa predictionmodel,todemonstratehowaccuratelyatestcanpredict toxicitycomparedtoareferencetest(i.e.,Draizetesting).Usuallya definednumberofsubstancesinatleastthreedifferentlaboratories areassessed.Ironically,thisstageofassessmentcanbehinderedby thelowreliabilityofDraizetesting(UbelsandClousing,2005);(vi) applicabilitydomain,whichinvolvesdefiningthepurposetowhich atestcanbeappliedincludingendpoints, chemicalclasses,test materialandphysiochemicalproperties;(vii)performance stand-ards,theseneedtobeestablishedforeachtest.However,ifasimilar, previouslyvalidatedmethodormodelexists,thenthevalidation processismuchfaster(Hartungetal.,2004).Theassessmentofeach moduleisledbyavalidationmanagementgroup(VMP),whowill thenmakerecommendationstoeitherensuetopeerreviewwitha completeddossieroftheinformation,ortocollectadditionaldata (IHCP,2013).AtestcannotproceedtopeerreviewwithoutaVMG recommendation.A formalregulatory validation cantakemore thanfiveyearstoachieve(Sheasgreenetal.,2009)andmayonly thenbeconsideredforregulatoryacceptanceonceachieved.

Regulatoryacceptanceisaformalrecognitionthatindicatesa test method or model may be used for a specific purpose. AcceptanceisusuallyfollowedbyaformaladoptionbytheEUand theOECD,andinclusionintotheEUtestmethodregulationsanda publicallyavailable OECDtest guideline(IHCP,2013).TheOECD continuously updates existing test guidelines and restructures draftproposals forfuture adoption (Barile, 2010), toencourage industriestouseupdatedvalidatedtests,whilstsubmittingdata baseduponthem(StephensandMak,2013).

Mostassessmentsofvalidationandregulatoryacceptancehave occurredsince2000,followingtheestablishmentofvitalalternative testingcentersandthedriveinitiatedEuropeanCosmeticDirective (StephensandMak,2013).However,thelackofhumandatahas arguablyledtodelaysinestablishingthevalidityofalternativetests (Freebergetal.,1986b).Currentlyonlyalimitednumberofocular toxicityassayshaveundergone validation andregulatory accep-tance.BCOP, ICE and FLhavebeenaccepted by ICCVAM, EURL-ECVAMandOECDfortestingocularcorrosionandsevereirritation. CMhasalsobeenacceptedbutisstillawaitingfinalpublicationof OECDtestguidelines.DholakiyaandBarile(2013)summarizedthe validationstatusofseveralinvitrooculartoxicityassays.Sincethat timeanumberofchangeshavebeenmadetothevalidationstatusof thesetests. For example,updated guidelineshave beenissuedby the OECDfortheBCOP(OECD,2013b)andICEtests(OECD,2013a).For bothtestschangeshavebeenmadeconcerningtheidentificationof chemicalthatdonotrequireclassificationtoUNGHS.Inaddition, clarificationshavebeenprovidedontheapplicabilityofBCOPfor testingalcohols,ketonesandsolidsandanupdatedlistofchemicals tobeusedtodemonstratetechnicalproficiencywhenapplyingthe

ICEtest.ArecentreportbyEURL-ECVAMonalternativemethods (Zuanget al., 2013)also providedan update ontheregulatory validationofseveralinvitroassays.TodateEpiOcularEIThaspassed initialvalidationstudies(Zuanget al.,2013)andguidelines are currentlybeingdraftedbytheOECD(2014b).TheHETandIREhave beenrejectedbyICCVAMwhiletheEURL-ECVAMhasrequested furtheroptimizationofthetestprotocol.

8.Conclusion

Wehavepresentedanoverviewofcurrentpracticesinocular toxicitytesting. While progresshasbeenmadein developinga rangeofalternativetechniquestoinvivotesting,furtherprogressis required to reduce the dependency of toxicity testing on live animals.Amongtheissuesthatneedtobeaddressedbyregulatory bodiesiswhetherDraizetestingshouldstillbeconsideredasthe

“goldstandard”and whetherresultsobtainedfromsuchtesting usedtovalidateorevaluatealternativetests.Inordertoadvance alternative testing methodologies, there needs to be active engagement and dialog between scientific and regulatory communities.

Conflictofinterest

Theauthorsdeclarethattherearenoconflictsofinterest.

Transparencydocument

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Acknowledgment

Funding from EPSRC Engineering, Tissue Engineering and Regenerative Medicine Fellowship (E-TERM, Grant number:EP/ 1017801/1)andtheUniversityofNottinghamHERMESFellowship (Grantnumber:13b/I9)isgratefullyacknowledged.

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