EpiOcular’sstrongcorrelationtohistoricalinvivodatasetsandexceptionalreproducibilitymakeitanidealmodelfortheocularsafetytestingofrawingredientsandfinalformulations. TheEpiOcularEyeIrritationTest(EIT)isaninvitroalternativetoanimaltestingandisavalidated,OECDtestguideline(TG492). EpiOcularhasbeenusedformanyyearsbyindustryasanon-animal,invitroalternativetodetermineDraizescores(forwatersolubletestarticles)andtoassessthemildness/ultra-mildness(sub-Draize)ofmaterialscontactingtheeyes.Technology:EpiOculareyeirritationassayshavebeenincorporatedintotoxicologylaboratoriesacrosstheworld.Withstraightforwardprotocolsandsimpleyethighlypredictiveendpoints,EpiOcularassaysareroutinelyutilizedthroughoutthecosmetic,personalcare,chemicalandpharmaceuticalindustries.Developedmorethan20yearsago,EpiOcularhasbeenusedfortestingthousandsofmaterials.Whetherscientistsneedariskassessmentassayfortheirritationpotentialofanoculardeliveredmaterialorthehazardassessmentofchemicalbeingtransportedacrosstheworld,EpiOcularisthetestsystemofchoicetomeetyouroculartestingneeds.Applications:OcularIrritationTestsEpiOcularEyeIrritationTestTheEpiOcularEITisaninvitrohazardassessmentassaythathastheABIlitytodifferentiatematerialsthatareocularnon-irritantsfrommaterialsthatarenotocularirritants.TheEITisapplicabletoabroadrangeoforganicandinorganicchemicals,andisacceptedasOECDTG492.EpiOcularEyeIrritationTestProtocolWatchavideobyJoveonEpiOcularEITEpiOcularSubDraizeUltra-MildnessTestTheEpiOcularUltra-mildnessTestisaninvitroriskassessmentassayroutinelyutilizedformildmaterialsforwhichtheDraizetestisinsensitive.ThishighlyreproducIBLeassayallowsforquantifiablediscriminationamongmild,milderandmildestproductformulations.EpiOcularSubDraizeProtocolEpiOcularMTTET-50(Neat)Thisinvitroriskassessmentassayisbasedontheeffectivetimeatwhichamaterial(appliedneat)causesa50%reductionintissueviability(ET-50).BasedontheET-50,thetestarticleiscategorizedintooneof4classificationsrangingfromnon-irritatingtosevere/extremewhichcorrespondtogroupingsofRabbitDraizeEyeScores(MMAS).EpiOcularMTTET-50NeatProtocolEpiOcularMTTET-50(Dilution)SimilartotheMTTET-50Neatmethod,thisinvitroriskassessmentassayisbasedontheeffectivetimeatwhichamaterial(dilutedto20%inwater)causesa50%reductionintissueviability(ET-50).BasedontheET-50,thetestarticleiscategorizedintooneof4classificationsrangingfromnon-irritatingtosevere/extremewhichcorrespondtogroupingsofRabbitDraizeEyeScores(MMAS).Thismethodisrecommendedforsurfactantbasedsolutionsandisapplicabletowater-solublematerialswithaspecificgravityof>0.95.EpiOcularMTTET-50DilutionProtocolBrowseourreferencelibrarytoseehowourEpiOculartissuehasbeenusedintheseareasofstudy.a[rel~="mtli_filesize259MB"]:after {content:" (2.59 MB)"}a[rel~="mtli_filesize36597kB"]:after {content:" (365.97 kB)"}a[rel~="mtli_filesize24579kB"]:after {content:" (245.79 kB)"}a[rel~="mtli_filesize33345kB"]:after {content:" (333.45 kB)"}TechSpecs:Tissue:Kit:StandardEpiOcular™kit(OCL-200)consistsof24tissues,eachtissue9mmindiameter.(Tissue“kits”containtissues,asmallamountofculturemedium,andplasticware;contactMatTekforspecifickitcontents.)Substrate:Non-coated,9mmIDsinglewelltissuecultureplateinsertsareused(e.g.MillicellPCF,Nuncpolycarbonatesinglewelltissuecultureplateinserts).Culture:Atairliquidinterface.Histology:5-8celllayers(basallayertoapicalsurface).Cornification:None.LotNumbers:Tissuelotsproducedbyeachtechnicianforeachweekareassignedaspecificlotnumber.Typically,therearemultiplelotnumbersforanygivenweek’stissueproduction.Aletterofthealphabetisappendedtotheendofthelotnumbertodifferentiatebetweenindividualkitswithinagivenlotoftissues.Alltissuekitswithinalotareidenticalinregardstocells,medium,handling,cultureconditions,etc.Shipment:At4°Conmedium-supplemented,agarosegelsin24-wellplate.Shipmentday:EveryMonday.ShipmentonThursdayalsopossibleuponspecialrequest.Delivery:TuesdaymorningviaFedExpriorityservice(US).OutsideUS:Tuesday-Thursdaydependingonlocation.Shelflife:Includingtimeintransit,tissuesmaybestoredat4°Cforupto4dayspriortouse.However,extendedstorageperiodsarenotrecommendedunlessabsolutelynecessary.Inaddition,thebestreproducibilitywillbeobtainediftissuesareusedconsistentlyonthesameday,e.g.Tuesdayafternoonorfollowingovernightstorageat4°C(Wednesdaymorning).Alternativetissues:OCL-200-ABF:Antibiotic-freetissue.Forlast3daysofculture,gentamicinisomittedfromculturemedium.Customerreceives200mlofOCL-200-ASY-AFABmedium.OCL-200-AFF:Anti-fungal-freetissue.Forlast3daysofculture,Amphotericinisomittedfromculturemedium.Customerreceives200mlofOCL-200-ASY-AFABmedium.OCL-200-AFAB:Antibiotic,anti-fungal-freetissue.Forlast3daysofculture,gentamicinandAmphotericinareomittedfromculturemedium.Customerreceives200mlofOCL-200-ASY-AFABmedium.OCL-200-EACH:SingleEpiOculartissueinsert.OCL-200-FRZN-EA:SingleOCL-200tissuethathasbeensubjectedto3freeze/thawcycles.DesignedtoserveascontrolforMTTauto-reductiontest.OCL-212:Halfkit(12culturesinsteadofstandard24).OCL-606:SameasOCL-200except6tissuesperkit,eachtissueculturedinalargercellcultureinsert(22mmdiameter).Cells:Type:Normalhumanepidermalkeratinocytes(NHEK).Geneticmake-up:Singledonor.Derivedfrom:Neonatal-foreskintissue.Screenedfor:HIV,Hepatitis-B,Hepatitis-C,mycoplasma.Medium:Basemedium:Dulbecco’sModifiedEagle’sMedium(DMEM).Growthfactors/hormones:Epidermalgrowthfactor,insulin,hydrocortisoneandotherproprietarystimulatorsofnon-keratinizingepithelialdifferentiation.Serum:None.Antibiotics:Gentamicin5µg/ml(10%ofnormalgentamicinlevel).Anti-fungalagent:AmphotericinB0.25µg/ml.pHIndicator:Phenolred.Alternatives:Phenolred-free,antibiotic-free,anti-fungal-free,orhydrocortisone-freemediumandtissueareavailable.Agentsareremovedatleast3dayspriortoshipment.Maintenancemedium:MostassayswithEpiOcular™areperformedwithin24hours.However,forlongerexperiments,thetissuecanbemaintainedinOCL-200-MM(identicaltoEpiOcular™assaymedium).QualityControlandSterility:Visualinspection:Alltissuesarevisuallyinspectedandifphysicalimperfectionsarenoted,tissuesarerejectedforshipment.End-usetesting:Tissuesareexposedto0.3%TritonX-100for5,20,and60minutes.Thetimeofexposurerequiredtoreducethetissueviability(ET-50)usingtheMTTassayisdetermined(SeeMatTekEpiOcularTMET-50protocol)foreachlotoftissue.ET-50’smustfallwithintherangeofthe1996EpiOcular™database:12.2-37.5minutes.ET-50’sincustomers’labsareoftenverysimilartoMatTekresults.Sterility:Allmediausedthroughouttheproductionprocessischeckedforsterility.Maintenancemediumisincubatedwithandwithoutantibioticsfor1weekandcheckedforsterility.Theagarosegelfromthe24-wellplateusedforshippingisalsoincubatedfor1weekandcheckedforanysignofcontamination.Screeningforpathogens:AllcellsarescreenedandarenegativeforHIV,hepatitisBandhepatitisCusingPCR.However,noknowntestmethodcanoffercompleteassurancethatthecellsarepathogenfree.Thus,theseproductsandallhumanderivedproductsshouldbehandledatBSL-2levels(biosafetylevel2)orhigherasrecommendedintheCDC-NIHmanual,“BiosafetyinmicroBIOLOGicalandbiomedicallaboratories,”1998.Forfurtherassistance,pleasecontactyoursiteSafetyOfficerorMatTektechnicalservice.Notificationoflotfailure:IfatissuelotfailsourQCorsterilitytesting,thecustomerwillbenotifiedandthetissueswillbereplacedwithoutcharge.BecauseourQCandsterilitytestingisdonepost-shipment,notificationwillbemadeassoonaspossible(Undernormalcircumstances,ET-50failureswillbenotifiedbyWednesday5pm;sterilityfailureswillbenotifiedwithin8daysofshipment).