McCanna, David J.2009-04-272009-04-272009-04-272009http://hdl.handle.net/10012/4338The utilization of in vitro tests with a tiered testing strategy for detection of mild ocular irritants can reduce the use of animals for testing, provide mechanistic data on toxic effects, and reduce the uncertainty associated with dose selection for clinical trials. The first section of this thesis describes how in vitro methods can be used to improve the prediction of the toxicity of chemicals and ophthalmic products. The proper utilization of in vitro methods can accurately predict toxic threshold levels and reduce animal use in product development. Sections two, three and four describe the development of new sensitive in vitro methods for predicting ocular toxicity. Maintaining the barrier function of the cornea is critical for the prevention of the penetration of infections microorganisms and irritating chemicals into the eye. Chapter 2 describes the development of a method for assessing the effects of chemicals on tight junctions using a human corneal epithelial and canine kidney epithelial cell line. In Chapter 3 a method that uses a primary organ culture for assessing single instillation and multiple instillation toxic effects is described. The ScanTox system was shown to be an ideal system to monitor the toxic effects over time as multiple readings can be taken of treated bovine lenses using the nondestructive method of assessing for the lens optical quality. Confirmations of toxic effects were made with the utilization of the viability dye alamarBlue. Chapter 4 describes the development of sensitive in vitro assays for detecting ocular toxicity by measuring the effects of chemicals on the mitochondrial integrity of bovine cornea, bovine lens epithelium and corneal epithelial cells, using fluorescent dyes. The goal of this research was to develop an in vitro test battery that can be used to accurately predict the ocular toxicity of new chemicals and ophthalmic formulations. By comparing the toxicity seen in vivo animals and humans with the toxicity response in these new in vitro methods, it was demonstrated that these in vitro methods can be utilized in a tiered testing strategy in the development of new chemicals and ophthalmic formulations.enin vitroAlternative MethodsalamarBluerhodaminetight junctionsbovine lensviabilitytoxicitymitochondriamitochondria integritybenzalkonium chloridesodium dodecyl sulphatesodium lauryl sulfatescanning electron microscopyDraizeDraize maximal average scoreszonula occludenscosmetic directivePETAhumane societyanimal leaguepeople for the ethical treatment of animalsinteragency coordinating committee on the validation of alternative methodsICCVAMECVAMJaCVAMBCOPbovine corneaocular irritantsocular irritationocular toxicityhuman corneal epithelial cellsvitroconfocalconfocal microscopymetabolic activityoptical qualityreactive oxygen speciescontact lenscontact lens care solutionsbarrier functionmicrobial keratitismulitipurpose solutionstight junctioncell damageclaudinZO-1occludinMDCKMadinMadin-Darbycanine kidney cellscorneahuman corneahuman corneal epitheliumepithelial cell linehuman corneal epithelial cell linesodium fluoresceinsodium fluorescein permeabilityfluorescein permeabilityocular surfacecell monolayerAraki-Sasakicell physiologyrisk assessmentsafety assessmentScanToxscanning laserlens epitheliumcorneal cellsin vitro modelophthalmic formulationsmultiple instillationback vertexanimal testingrabbit testingalternatives to animal testingcultured bovine lenstoxicity of chemicalsirritationirritantslaser scannerorgan culturedelayed toxicitytoxinshydrogen peroxidecornea toxicitycornea toxicity modelsprediction of human toxicityno observable adverse effect levellowest observable adverse effect levelNOAELLOAELtoxicity thresholdssafety factorscorneauncertainty factorspreservativesdisinfectantsophthalmic productspreclinicalpreclinical testingepithelial barrierdrug penetrationclinical confocal microscopyanimal rightsrabbit corneahuman corneahuman clinical effectstoxicanimal rights activistsensitive measurestoxic effecttoxicity thresholdagar overlayagar diffusionagar overlay methodagar diffusion methodcytochromecytochrome capoptosisnecroticapoptoticnecrosiscaspaserhodamine 123resazuranresorufincell deathcell viabilitymetabolic dyemicrosomalmicrosomal enzymescytotoxicitycytotoxiccytotoxic effectMTTXTTWST-1plasma membranemitochondrialmitochondrial morphologyocular toxicity potentialocular toxicityhuman corneal epithelialcell lineconfocal analysiscorneal epitheliumcell fluorescencealamarBlue assayrhodamine dyeanimal welfaretoxic injurydegraded mitochondriaepithelial monolayerdisinfectantsmembrane integrityeye toxicityeyeviability dyetoxicity in humanshuman toxicityeffects on the mitochondriamitochondrial toxicityin vitro batteryin vitro test batteryophthalmic eye dropdirect contactproduct safetycytotoxicity potentialmolecularmolecular biologyrefine reduce replacesensitivity and relevancesensitivityrelevancerabbit ocular irritation testproduct developmentcytotoxicity modelscytotoxicity alternative methodsreplacements for animal testingthree r'sbeagletiered testingtiered testing strategyreplacements animal testingmechanistic toxicitycornea mitochondriadose responsethresholdDoctoral ThesisVision Science and Biology