Development and intralaboratory evaluation of an in vitro human cell-based test to aid ocular irritancy assessments.
A human cell-based in vitro method was developed to screen for ocular irritancy potential of aqueous compatible and incompatible test agents, such as liquids, insoluble solids, powders, granulars, emulsions, and acids/alkalis. Methods were developed for topical application (an exposure that mimics in vivo testing) and wash-off of test substances on the epithelial surface of human skin derived epithelial-fibroblast cocultures (Skin2 Model ZK1200 from Advanced Tissue Sciences). These cultures contain noncornified stratified squamous epithelium, providing a three-dimensional in vitro model that resembles noncornified mucosal epithelium, such as cornea and conjunctiva. The hypothesis tested and confirmed in this work was that the rate of cytotoxicity induced by topical application of test substances to the stratified epithelial cell cultures would correlate with ocular irritancy. Test substances were applied to the cell system for up to 30 min, and cytotoxicity was measured as decreased 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) vital dye metabolism. The time (in minutes) of exposure to test agent that reduced MTT metabolism to 50% of control levels (the t50 value) was calculated for each test substance, and these values showed a good correlation (r = 0.87) with historic rabbit low-volume eye test maximum average scores (MAS) for a range of liquid, solid, granular, powder, and other test materials, including consumer product formulations and ingredient chemicals. Additionally, t50 values confirmed the mild nature of selected cosmetic formulations. Taken together, these data indicate that this in vitro test, termed the tissue equivalent assay, is a valuable tool to screen for irritancy of test substances to mucosal tissues, such as cornea and conjunctiva.
Osborne
R
R
Human Safety Department, Miami Valley Laboratories, Procter & Gamble Company, Corporate Professional & Regulatory Services, Cincinnati, Ohio 45253-8707, USA.
Perkins
M A
MA
Roberts
D A
DA
D000826:Animal Testing Alternatives; D002470:Cell Survival; D002478:Cells, Cultured; D004847:Epithelial Cells; D004848:Epithelium / Q000187:drug effects; D005128:Eye Diseases / Q000139:chemically induced* / Q000473:pathology; D005347:Fibroblasts / Q000187:drug effects; D006801:Humans; D007509:Irritants / Q000633:toxicity*; D015603:Keratinocytes / Q000187:drug effects; D009499:Neutral Red; D013778:Tetrazolium Salts; D013844:Thiazoles; D018675:Toxicity Tests; D015262:Xenobiotics / Q000633:toxicity
D007509:Irritants; D013778:Tetrazolium Salts; D013844:Thiazoles; D015262:Xenobiotics; D009499:Neutral Red; C022616:thiazolyl blue