In vitro quantitative determination of ophthalmic irritancy by the chorioallantoic membrane test with trypan blue staining as alternative to eye irritation test

The damage provoked by some substances on the chicken egg’s chorioallantoic membrane (CAM) is used as an alternative assay to determine ocular irritation. There is good prediction of the eye irritation when compared to the in vivo Draize method. Nevertheless, this assay has some limitations, such as subjectivity. Hagino et al. developed an objective evaluation technique using the amount of trypan blue absorbed at the site of treatment as an indicator of injury to the CAM. The present work was aimed at the determination of ocular irritation of 21 substances (chemicals and cosmetics). We used the spectrophotometric quantification by trypan blue staining of the damage produced on CAM, of fertile chicken eggs. Results were compared with the values obtained by the traditional Draize assay. We observed a good correlation (r = 0.835; p < 0.0001) between the amount of dye absorbed by the CAM and the Draize eye irritation test score. The r values were 0.688; p < 0.05 for cosmetics and 0.925; p < 0.0001 for chemicals. Three chemicals turned as false positive and one cosmetic substance as false negative. The CAM-TBS assay is inexpensive, simple and provides an in vitro alternative method to predict the damage that chemical substances or cosmetics can cause to the ocular structures.