Abstract:

To address the shortcomings of the isolated rabbit eyes （IRE） assay which is only suitable for screening for severe eye irritation （UN GHS Category 1）, an IRE test combined with a fluorescent staining method based on the biomarkers of cell viability was developed for evaluating the eye irritation potential of cosmetic products. Cosmetic products all contain surfactants and other ingredients, and the surfactants may cause damage in a different way compared to other chemicals, in which the exposure procedures for eye irritation evaluation may be different. The IRE test conditions are explored, including the test concentration and post-exposure time. Eye irritation of the commercially available cosmetics were tested by the optimized IRE assay, processed for cryosectioning and staining using phalloidin to detect F-actin and DAPI staining to detect nuclei, and the depth of injury （DOI） were measured. To evaluate the applicability of eye irritation classification of cosmetics based on the measurement of DOI by live cell fluorescent staining, the correlation between in vitro and in vivo DOI measurements was evaluated, and pearson correlation analysis shows a good correlation between the above two methods （r=0.851 1, p<0.001）. Finally, we compared the in vitro DOI, in vivo DOI and Draize test results to establish a prediction model. The results show that the classification of eye irritation of cosmetic products from non-irritating to moderate level can be distinguished by DOI measuring, suggesting that in isolated rabbit eyes, DOI measurements based on cell viability can be used as a biomarker of corneal damage, and suitable for the evaluation of cosmetic eye irritation.

Key words: isolated rabbit eyes, cosmetics, eye irritation, phalloidin, depth of injury （DOI）