Employing histopathology to enhance the BCOP test: The emerging role of stromal thickness as a quantitative endpoint

The Bovine Corneal Opacity and Permeability (BCOP) assay is a validated ex vivo method widely used to assess the ocular irritation potential of chemical substances. It is useful for identifying both chemicals that cause serious eye damage and those that are not classified for eye irritation. This study incorporated histopathological parameters into the BCOP assay to enhance the characterization of corneal damage. Fifteen reference chemicals were tested, and IVIS scores were obtained from corneal opacity and permeability measurements. Fixed corneal tissues were then analyzed histologically. Three complementary models were applied: Depth of Injury (DOI), Stromal Thickness (ST), and Total Histopathological Damage (THD). The DOI model helped categorize damage by affected corneal layers and provided relevant information about the depth of the lesions. ST showed a strong correlation with IVIS scores and emerged as a robust indicator of edema. In contrast, the more complex THD model did not improve the classification. Overall, integrating ST and DOI into the BCOP framework provides complementary and informative endpoints that enhance the interpretation of ocular irritation potential. In particular, we highlight ST as a quantitative, and easy-to-measure endpoint that does not rely on expert interpretation.