Corneal epithelial and retinal pigment epithelial cell culture assays as potential alternatives to animal experimentation for the evaluation of ocular toxicity

Abstract

The widely criticized Draize rabbit test is the only eye toxicity test officially accepted worldwide for regulatory purposes in the classification of slightly and moderately irritating chemicals. Today, there are no in vitro alternatives that could be used as a complete replacement for the Draize eye test. Moreover, in the current OECD guidelines, there are no tests for retinal toxicity, not even in vivo.

This study was undertaken to develop corneal epithelial and retinal pigment epithelial cell culture assays as pre-screens and potential alternatives in a more comprehensive battery test for in vitro eye toxicity testing. Two basal cytotoxicity tests with effective 96-well techniques and plate readers were established to evaluate the adverse ocular effects of selected test compounds: WST-1 test as an index of mitochondrial function and cell viability/proliferation, and lactate dehydrogenase (LDH) leakage test as an index of cell membrane integrity.

The confluent and post-confluent human corneal epithelial (HCE) cell line in culture medium was found to express cornea-specific cytokeratin 3, but it also expressed simple epithelium-specific cytokeratins 7, 8, 18 and 19. In most cases in the WST-1 and LDH testing, the pre-confluent HCE cell line was as sensitive to the test compounds as the pre-confluent primary cultures of rabbit corneal epithelial epithelium. The WST-1 test appeared to be an earlier indicator of toxicity than the LDH test, which also showed great variations in the cytotoxicity results. In the HCE-WST-1 interlaboratory study, the most reproducible results were obtained when the cells were exposed to the test substances for one hour in the absence of serum. The use of the human retinal pigment epithelial cell line D407 and of pig primary retinal pigment epithelial cell cultures in the WST-1 testing also yielded comparable results.

In all cases, the use of serum in culture medium resulted in lower toxicity, and thus its use is not recommended. The assays studied, especially those based on cell lines, are reliable, transferable, easy-to-perform tests for ocular toxicity screening.