Inter-laboratory Study of the Reproducibility of the Bovine Corneal Opacity and Permeability Assay: Investigations of Solid Test Substances

The Bovine Corneal Opacity and Permeability Assay (BCOP) is an ex vivo assay which may be used to assess the eye irritation potential of new chemicals and finished products. The BCOP assay has been accepted by several regulatory agencies for the identification severe and corrosive ocular irritants, replacing the rabbit eye test. According to OECD Test Guideline 437 two treatment protocols may be used; one for liquids and one for solids. Solids are tested as 20% (w/v) solutions or suspensions in deionized water. Freshly excised bovine corneas are mounted in special corneal holders and are treated with the 20% (w/v) test material dilutions for four hours at approximately 32°C. Changes in corneal opacity are measured using an opacitometer, and impairment of the corneal barrier function is determined by measuring fluorescein passage through the corneas. Histological evaluation of the treated corneas may be used to determine the degree and depth of injury at the tissue level. In this study, the reference standard solids recommended in the OECD TG 437 were tested in an inter-laboratory study. Overall, the results from the evaluation of solids were highly congruent between the two laboratories and to the historical data and for several substances histological evaluation improved the understanding of eye irritation effect. However, for chlorhexidine and dibenzoyl-L-tartaric acid there were inter-laboratory differences which were further evaluated. For chorhexidine, differences in results were attributed to different sources of the chemical. This study demonstrates the reproducibility of the BCOP assay when evaluating solid test substances. In parallel, the study compared the opacity scores from a newly developed opacitometer (BASF-OP2.0) to those of the standard device (OP-KIT). The comparison between the BASF-OP2.0 and OP-KIT demonstrated that the BASF-OP2.0 showed very little variability and overall corresponded very well with the OP-KIT values.