Development of an in vitro model to investigate repeat ocular exposure
The Draize eye irritation test has been widely adopted as the "gold standard" to evaluate the potential eye irritation of a wide range of chemicals and formulations, including; pharmaceuticals, cosmetics, and their raw ingredients. The rationale for pursuing the development of human ocular based in vitro alternatives is to provide greater confidence in the prediction of human reactions to mild and moderate chemicals. This is particularly important with the implementation of the EC White Paper, "Strategy for a future chemicals policy" (2001) that is estimated to require the testing of approximately 30,000 'existing' chemicals by 2012. The development of in vitro alternatives for toxicity testing has mainly focused upon tests for quantitative measurement of acute toxicity following a single high-dose exposure. However, the degree of toxicity of any exposure is a function of; the dose which target cells receive, the duration of the exposure and the ability of the exposed cells to recover from the exposure. However, little account is taken of the potential role of long-term effects in modulating the toxic response. This study aims to generate an in vitro model utilizing a human corneal cell line monolayer to investigate the effects chronic exposure to exogenous chemicals has upon toxicity of a subsequent acute challenge. Surfactants are ubiquitous within our daily environment, being significant active components in both household and personal care products, cosmetics and pharmaceuticals. Initially the effects of four representative surfactants (sodium dodecyl sulphate; anionic, tween 20; non-ionic, cocamidopropylbetaine; amphoteric and benzalkonium chloride; cationic) were examined following chronic exposure. Although the measured endpoints (neutral red uptake, resazurin reduction, fluorescein leakage and total protein content) revealed no alterations in J-HCET morphology, barrier function or biochemistry as a consequence of chronic exposure, it was determined that pre-exposure modulated the toxicity of subsequent acute exposures. The observed modulation in toxicity could have significant health implications for personal care products, cosmetics and pharmaceuticals intended for use near or within the eye. However, the mechanism(s) by which the toxicity of subsequent surfactant exposures was modulated remains to be elucidated. Whilst standard surfactants are good indicators of the effects following chronic exposure, there are pharmaceuticals designed for repeat use in the eye that have been associated with long term ocular irritancy, and a discontinuation of use i.e. timolol maleate. J-HCET cultures exposed to BSO in vitro confirm that the toxicity of timolol to the human corneal cells line was enhanced by suppressing the activity of γ-glutamylcysteine synthetase through irreversible inhibition, resulting in a decrease in intracellular glutathione (GSH) levels. In addition, chronic exposures to timolol maleate were also associated with an increase in toxicity of subsequent acute challenges. The long term use of Timolol maleate in the treatment of glaucoma in vivo may result in similar alterations in the intracellular GSH concentrations, resulting in discontinuation of treatment as consequence of ocular irritation through the generation of reactive oxidation species beyond the threshold that depleted intracellular GSH can respond. Since in vitro methods have been, and are being developed as alternatives to animal experiments, the use of bovine serum as a source of growth factors can seem to be contradictory to the purposes of the Three R's concept of Russell and Burch (1959). This study was conducted using culture media that contain animal derived growth supplements and a media where these had been substituted for plant derived materials. Comparisons were made between the effects these two supplements had upon a number of biological factors including morphology, biochemistry, barrier function and the response to exogenous chemicals. No alterations were observed in these parameters as a consequence of using culture medium containing plant derived materials compared to those containing animal derived growth supplements. This study has demonstrated that the development of a reliable and reproducible in vitro assay in keeping with the principles of the Three R's for modeling chronic – repeat ocular irritation is possible. However, the mechanistic relevance of the endpoints chosen and cell layer ultrastructure is considered to be an essential component. The further development of cell based in vitro systems to predict human responses to chronic/repeat ocular irritation is required.