Grading criteria of histopathological evaluation in BCOP assay by various staining methods

This study was performed to provide information on classifying eye irritating chemicals using the BCOP assay. After the BCOP assay, bovine corneas were classified by IVIS presented in OECD test guideline 437, and three special staining methods (H&E, MT, and PAS) were performed for histopathological evaluation. Non-irritant chemicals (IVIS ≤ 3), showed intact structures. In the 3 < IVIS ≤ 55 group, epithelial cell edema was observed by H&E staining, and loose collagen bundles were confirmed by MT staining. In PAS staining, bleaching of the epithelium and reduced visibility of the basement membrane were observed. Severe irritant chemicals (IVIS > 55) showed large increases edema and nuclear condensation by H&E staining. Loose collagen bundles and vacuoles around keratocytes were also observed by MT staining. Bleaching of the epithelial layer, reduction in visibility, and thickness of the basement membrane were confirmed by PAS staining. Based on the stepwise histopathological analysis, we set the criteria and grades for histopathological evaluation and found that eye irritation was increased following the irritation degree of test chemicals. Further histopathological study will support and lead to improvements in the BCOP assay.