During a COLIPA multicentre study carried out on ‘Alternatives to Eye Irritation’ two laboratories undertook the evaluation of the samples with the fluorescein leakage test (FLT). The lead laboratory (L'Oréal) proposed a prediction model (PM), which converts the in vitro data from this assay to a prediction of eye irritation. All the surfactant-containing substances were tested using the FLT if they were soluble in Hanks’ balanced salt solution (HBSS). Briefly, confluent MDCK cells were exposed for 15 minutes to five fixed concentrations of the test substance. The amount of damage caused to the cellular monolayer was determined by the amount of fluorescein that leaked through the cell layer after a 4-hour period. The amount of test substance that produced 20% leakage compared with a cell-free control was determined (FL20 H4). The FLT results transformed using the PM, generally predicted the in vivo classification (linear Kappa: 0.87±0.17 and 0.75±0.25). Where there were misclassifications, the category assigned was only one different from the observed in vivo score. There was generally good agreement between the two laboratories in this study. The model seems to be more appropriate for evaluation of non-irritants or moderate irritants than for severe irritants. This is relevant for the selection of cosmetic ingredients. Overall, the results of the FLT assay appeared to be encouraging.

The fluorescein leakage test (FLT) provides information on the effects of xenobiotics on the impermeability (gate function) of epithelial cell monolayers, and their recovery after exposure. The aim of this study was to assess the validity of this test in the ocular safety assessment of surfactant-based products with various irritant potencies. Madin-Darby canine kidney cells were grown to confluency on microporous membranes and exposed for 15 min to increasing concentrations of test substances. Damage was evaluated by measuring the amount of Na-fluorescein that passed through the monolayer in 30 min, starting just after exposure. Recovery was assessed 4, 24, 48 and 72 hr later. For each sample and each time point, the amounts of test substance that produced 10% and 20% leakage (FL10 and FL20) compared with a cell-free control were calculated. For the 43 samples, FL20 values ranged from 0.65 to 1000 mg/ml. These values increased or decreased with time according to the substance. In particular, cell monolayers showed very different recoveries after exposure to anionic and cationic substances with similar initial FL20 values. These in vitro data correlated well with historical Draize in vivo test data (Spearman's ϱ > 0.90). The FLT is therefore useful as a complement to other in vitro methods for the ocular safety evaluation of cosmetics.

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.