An in Vitro Method for Measuring Ophthalmic Preservative Cytotoxicity

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Abstract
Using an in vitro model we studied the cytotoxicity of the topical ophthalmic preservatives benzalkonium chloride (BAC), thimerosal (TMS), and chlorobutanol (CHB) on confluent rabbit corneal epithelial cell cultures. Concentrations of the preservatives were BAC 0.0004–0.04%, TMS 0.0001–0.04%, and CHB 0.1–0.5%. Drug exposure times for all agents were 5, 30, and 60 min. Inhibition of 12-hr [3H]thymidine incorporation and phase contrast inverted light microscopy were performed to assess cytotoxic effect. In a concentration- and time-dependent manner all preservatives produced significant (p < 0.05) inhibition of [3H]thymidine incorporation within the range of their clinically used concentrations. This effect was associated with changes in cellular morphology as seen with light microscopy. The correlation of our in vitro data with the findings of previous in vivo studies suggests that our model is a rapid, objective, and quantitative screening method to study the cytotoxic effects of topical agents on corneal epithelium.