An In Vitro Tier Evaluation for the Identification of Cosmetic Ingredients Which are Not Ocular Irritants

Abstract

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), MATREX™, 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a maximal average total score (MAS) of 5 or less in the Draize eye test, as this is the criterion used in the Japanese draft guidance for evaluating cosmetic ingredients.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.

Definition of the Applicability Domain of the Short Time Exposure (STE) Test for Predicting the Eye Irritation of Chemicals

“…The complete replacement of the Draize eye irritation test by a single in vitro test is unlikely to be possible, because the Draize test evaluates a range of criteria for detecting injury to, and inflammation of, the eye. Therefore, a tiered approach combining several in vitro assays was proposed, in order to estimate the eye irritation potential of a wide range of chemical classes (22)(23)(24)(25). In our previous study, a bottom-up tiered approach that combined the STE test and the EpiOcular and the BCOP assays, allowed us to estimate the eye irritation potential of not only insoluble substances, but also volatile substances (13,14).…”

Further Verification of an In Vitro Tier System for the Identification of Cosmetic Ingredients that are Not Ocular Irritants

“…The tier evaluation system, comprising the SIRC-CVS assay and the LDM-MTT assay, for the identification of ingredients which are not ocular irritants, was designed on the basis of the results of = UV absorbers (4); = surfactants (19); = macromolecules (10); = humectants (7); = oils (20); = medicants (13). Japanese validation studies (7). Factors taken into account in developing the tier evaluation system were as follows: the resources required to perform the tests (i.e.…”

“…An 80μl (or 80mg, in the case of a solid) aliquot of test substance was then applied to the surface of the LDM, within the polyethylene ring. For discrimination between non-irritants and irritants at the concentration of 10%, the concentrations of the test samples used were usually 1, 5 and 10%, as according to the three-dose method of Hagino et al (7). For prediction of the concentration at which an ingredient can be formulated into products without causing eye irritation, the concentration of the test samples was set at a similar concentration to that at which ingredi-ents had been previously tested in in vivo tests.…”

“…The solvent used for diluting the test substances was either distilled water, 50% (v/v) DMSO or ethylene glycol, selected on the basis of the solubility characteristics of the substance, as reported by Ohuchi et al (63). When none of these solvents were suitable, liquid paraffin was employed (7). The stepwise determination of whether a test substance dissolved, or whether it was suspended uniformly, was based on visual observation.…”

“…We re-analysed the results of the Japanese validation study, and designed a novel tier evaluation system, by combining SIRC monolayer cell cultures and a three-dimensional LDM (7). The cytotoxic endpoints were measured by means of Crystal Violet staining (in the SIRC-CVS assay) and MTT reduction (in the LDM-MTT assay), respectively.…”