The BCOP assay was optimized to its present form by Drs. Pierre Gautheron and Joe Sina to address ocular irritation potential of pharmaceutical intermediates and is now widely applied across industries and chemical/formulation classes. Membrane lysis, protein coagulation, and saponification are common modes of action that lead to ocular irritation. In our experience, the opacity and permeability endpoints (generally combined into an “in vitro score”) have been able to identify the epithelial and stromal changes associated with these types of damage. However, chemicals that react with nucleic acids, mitochondrial proteins, or other cellular targets, that do not lead to immediate loss of cellular integrity or protein precipitation, have proven more difficult to identify without the addition of histological evaluation of the treated corneas. In this study, a series of reference chemicals (i.e., surfactants, solvents, acids, alkalis and oxidizers) were exposed to the corneas under standard assay conditions. The opacity and permeability to fluorescein were measured and the corneas fixed for histological evaluation. The test substances were chosen to induce corneal lesions consistent with the various modes of action. Histological evaluation was performed on the three layers of the cornea to provide a direct measure of the depth of injury. This evaluation was used to compare depth of injury with the opacity and permeability scores and to identify lesions that were not fully expressed in opacity or permeability changes. For materials inducing membrane lysis, coagulation, and saponification, the opacity and permeability scores paralleled the depth of injury and histology provided data on specific changes in the tissue. Furthermore, the action of reactive materials (i.e., peroxide) was detected with histology and its selective action on the stromal keratocytes demonstrated. Histological evaluation of the treated corneas can be useful in directly evaluating lesions and enhancing the interpretation of the opacity and permeability endpoints.