Comparison of Human Corneal Epithelial and Human Conjunctival Epithelial Cell Line Cultures in Cytotoxicity Testing

Abstract

Abstract: : Purpose: Alternatives to the Draize eye test have widely been investigated for several decades now. Despite all, no test, combination of tests, or testing strategy have been found to be capable of replacing the Draize eye test completely, but some of the assays have shown a considerable promise as screens for ocular irritancy. In the present study, human corneal epithelial and human conjunctival epithelial cell lines were compared as potential pre–screen alternatives to the Draize test. Methods: Ocular toxicity was tested by using effective multititer techniques and two basal cytotoxicity tests, WST–1 test as an index of mitochondrial function and cell viability/proliferation and ATP test as an index of ATP measurement. Cells were grown in 96–well plates with different cell densities for 24 hours and consequently exposed to BAC for one hour in similar serum–free conditions. Benzalkonium chloride (BAC), a cationic surfactant and a known severe eye irritant, was used as a model compound. Results: Both cell lines yielded comparable results with both test methods. ATP test showed increasing EC₅₀ values with increasing cell numbers while in the WST–1 test the trend was not as clear. In the ATP test, EC₅₀ values varied from 0.0011% to 0.0036%, and in the WST–1 test from 0.0010% to 0.0021%. In the ATP test, the EC₅₀ values for the NHC cells were twice as high as for the HCE cells while in the WST–1 assay the EC₅₀ values were in the same level and more comparable. Conclusions: The cytotoxicity assays used were simple and reproducible, and yielded a defined endpoint. Both cell lines show considerable promise as potential pre–screen alternative methods. As cell lines are more manageable as primary cultures these cell lines are promising tools for toxicity testing.

Keywords: ocular irritancy/toxicity testing • cornea: epithelium • conjunctiva