An in vitro tier evaluation for the identification of cosmetic ingredients which are not ocular irritants.

A tier evaluation system was assessed as an alternative method for the identification of cosmetic ingredients which are not ocular irritants. The system employed monolayer cultures of SIRC cells, an established cell line originally derived from the rabbit cornea, and a three-dimensional living dermal model (LDM), MATREXtrade mark, which consists of human dermal fibroblasts in a contracted collagen lattice. Effects on the cell monolayer cultures were determined by using SIRC cell-Crystal Violet staining (SIRC-CVS), and effects on the LDM were assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A non-irritating ingredient was defined as a compound having a maximal average total score (MAS) of 5 or less in the Draize eye test, as this is the criterion used in the Japanese draft guidance for evaluating cosmetic ingredients. Among 34 test substances with known characteristics, 30 were classified accurately. Based on these encouraging results, the possibility of simplifying the MTT assay on the LDM for more-practical use, by selecting only three concentration levels to discriminate non-irritants from irritants, was assessed. The simplified method, involving a three-dose set (the three-dose method), was confirmed as being suitable for the identification of non-irritating ingredients, with triethanolamine used as a negative reference standard. Finally, the LDM was used to evaluate compounds at similar concentrations to those tested in vivo, aiming to predict the concentration at which an ingredient can be formulated into products without causing eye irritation. On the basis of previous validation data and our additional results, it was found that test samples that resulted in a cell viability of 50% or more in this model, could be classified as non-irritating ingredients. In all, these results indicate that the tier evaluation system may be suitable for the evaluation of ingredients intended to be used in cosmetics and medicated cosmetics in Japan.