Development of Sensitive In Vitro Assays to Assess the Ocular Toxicity Potential of Chemicals and Ophthalmic Products
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Date
2009-04-27T14:06:30Z
Authors
McCanna, David J.
Journal Title
Journal ISSN
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Publisher
University of Waterloo
Abstract
The 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.
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Keywords
in vitro, Alternative Methods, alamarBlue, rhodamine, tight junctions, bovine lens, viability, toxicity, mitochondria, mitochondria integrity, benzalkonium chloride, sodium dodecyl sulphate, sodium lauryl sulfate, scanning electron microscopy, Draize, Draize maximal average scores, zonula occludens, cosmetic directive, PETA, humane society, animal league, people for the ethical treatment of animals, interagency coordinating committee on the validation of alternative methods, ICCVAM, ECVAM, JaCVAM, BCOP, bovine cornea, ocular irritants, ocular irritation, ocular toxicity, human corneal epithelial cells, vitro, confocal, confocal microscopy, metabolic activity, optical quality, reactive oxygen species, contact lens, contact lens care solutions, barrier function, microbial keratitis, mulitipurpose solutions, tight junction, cell damage, claudin, ZO-1, occludin, MDCK, Madin, Madin-Darby, canine kidney cells, cornea, human cornea, human corneal epithelium, epithelial cell line, human corneal epithelial cell line, sodium fluorescein, sodium fluorescein permeability, fluorescein permeability, ocular surface, cell monolayer, Araki-Sasaki, cell physiology, risk assessment, safety assessment, ScanTox, scanning laser, lens epithelium, corneal cells, in vitro model, ophthalmic formulations, multiple instillation, back vertex, animal testing, rabbit testing, alternatives to animal testing, cultured bovine lens, toxicity of chemicals, irritation, irritants, laser scanner, organ culture, delayed toxicity, toxins, hydrogen peroxide, cornea toxicity, cornea toxicity models, prediction of human toxicity, no observable adverse effect level, lowest observable adverse effect level, NOAEL, LOAEL, toxicity thresholds, safety factors, cornea, uncertainty factors, preservatives, disinfectants, ophthalmic products, preclinical, preclinical testing, epithelial barrier, drug penetration, clinical confocal microscopy, animal rights, rabbit cornea, human cornea, human clinical effects, toxic, animal rights activist, sensitive measures, toxic effect, toxicity threshold, agar overlay, agar diffusion, agar overlay method, agar diffusion method, cytochrome, cytochrome c, apoptosis, necrotic, apoptotic, necrosis, caspase, rhodamine 123, resazuran, resorufin, cell death, cell viability, metabolic dye, microsomal, microsomal enzymes, cytotoxicity, cytotoxic, cytotoxic effect, MTT, XTT, WST-1, plasma membrane, mitochondrial, mitochondrial morphology, ocular toxicity potential, ocular toxicity, human corneal epithelial, cell line, confocal analysis, corneal epithelium, cell fluorescence, alamarBlue assay, rhodamine dye, animal welfare, toxic injury, degraded mitochondria, epithelial monolayer, disinfectants, membrane integrity, eye toxicity, eye, viability dye, toxicity in humans, human toxicity, effects on the mitochondria, mitochondrial toxicity, in vitro battery, in vitro test battery, ophthalmic eye drop, direct contact, product safety, cytotoxicity potential, molecular, molecular biology, refine reduce replace, sensitivity and relevance, sensitivity, relevance, rabbit ocular irritation test, product development, cytotoxicity models, cytotoxicity alternative methods, replacements for animal testing, three r's, beagle, tiered testing, tiered testing strategy, replacements animal testing, mechanistic toxicity, cornea mitochondria, dose response, threshold