Critical Evaluation of an Agarose Diffusion Method for the Assessment of Eye Irritancy

Abstract

The agarose overlay method is often used to study the cytotoxicity of non-liquid products. However, diffusion through agarose is a limiting factor in some cases, and this reduces contact between the cells and the test material. We have developed a new model aimed at reducing this phenomenon. In our technique, rather than being cultured in a monolayer, V79 cells are suspended in 1% agarose medium. We obtained a good linear correlation between this method and the usual L-929 cell monolayer technique (r = 0.95; n = 31). In addition, the new technique is more sensitive. In particular, the cytotoxicity of surfactants is apparent at lower concentrations. Using this technique, we studied 115 miscellaneous raw materials and cosmetic products, for which in vivo historical ocular irritancy data were available. As expected, our method was unsuitable for the study of alkaline and acidic products, as well as water-alcohol-based lotions. We also adapted the method for testing mascaras and obtained a good ranking for eye irritancy. For the other 93 products (surfactants, shampoos, lotions, gels, emulsions and miscellaneous products), the mean diameter of the lysis zone after contact for 18 hours was used as the endpoint for comparison with in vivo maximum average scores. There was a strong linear/rank correlation between the two parameters (r = 0.83/rho = 0.79). Furthermore, the concordance between the in vivo and in vitro data in a three-class ranking system was good (weighed kappa index = 0.81). These results suggest that the agarose diffusion method is a potentially useful screening tool for the ocular safety of the categories of products described above.