IN-HOUSE VALIDATION OF THE EPIOCULAR™ EYE IRRITATION TEST AND ITS COMBINATION WITH THE BOVINE CORNEAL OPACITY AND PERMEABILITY TEST FOR THE ASSESSMENT OF OCULAR IRRITATION.

• TR Number: 673
• Authors: Kolle1, S.N., Kandárová2, H., Wareing1, B., van Ravenzwaay1, B., and Landsiedel1, R. 1BASF SE, Experimental Toxicology and Ecology, Ludwigshafen, Germany; 2Mattek In Vitro Life Science Laboratories, Bratislava, Slovakia. ATLA, 39, 365–387, (2011).
• Materials Tested: 1-naphthalene acetic acid, 2,4-Dochloro-5-sulfamoylbenzoic acid, 2,6-Dichlorobenzoyl chloride, 4-carboxybenzaldehyde, Acetone, Agrochemical formulation, Aliphatic carbamate, Aliphatic phosphor-rothioic triamide, Alkoxylated aliphatic alcohol, Ammonium nitrate, Aromatic carboxylic ester, N-containing, Benzalkonium CI (5%), Butyrolactone, Cationic polymer, Chlorhexidine, Cyclohexanone, Dibenzoyl-L-tartaric acid, Dimethyl sulfoxide, Ethanol, Ether, Ethyl-2-methylacetoacetate, Glycerol , HaCaT keratinocytes, Heterocyclic, lmidazole, Metal oxide, n-Hexane, OECD TG 437, Organic compound, Organophosphate, Peptide, Pigment/dye, Polyethylene glycol 400, Pyridine, Salt of aromatic acid and amine, SDS (15%), SDS (3%), Sodium oxalate, Toluene, Triton X-100(5%), Tween 20

In 2009, the Bovine Corneal Opacity and Permeability (BCOP) test was accepted by the regulatory bodies for the identification of corrosive and severe ocular irritants (Global Harmonised System [GHS] Category 1). However, no in vitro test is currently accepted for the differentiation of ocular irritants (GHS Category 2) and non-irritants (GHS No Category). Human reconstructed tissue models have been suggested for incorporation into a tiered testing strategy to ultimately replace the Draize rabbit eye irritation test (OECD TG 405). The purpose of this study was to evaluate whether the EpiOcular™ reconstructed cornealike tissue model and the COLIPA pre-validated EpiOcular Eye Irritation Test (EpiOcular-EIT) could be used as suitable components of this testing strategy. The in-house validation of the EpiOcular-EIT was performed by using 60 test substances, including a broad variety of chemicals and formulations for which in vivo data (from the Draize rabbit eye irritation test) were available. The test substances fell into the following categories: 18 severe irritants/corrosives (Category 1), 21 irritants (Category 2), and 21 non-irritants (No Category). Test substances that decreased tissue viability to ≤ 60% (compared to the negative control tissue) were considered to be eye irritants (Category 1/2). Test substances resulting in tissue viability of > 60% were considered to be non-irritants (No Category). For the assessed dataset and the classification cut-off of 60% viability, the EpiOcular-EIT provided 98% and 84% sensitivity, 64% and 90% specificity, and 85% and 86% overall accuracy for the literature reference and BASF proprietary substances, respectively. Applying a 50% tissue viability cut-off to distinguish between irritants and non-irritants resulted in 93% and 82% sensitivity, 68% and 100% specificity, and 84% and 88% accuracy for the literature reference and BASF proprietary substances, respectively. Further, in the EpiOcular-EIT (60% cut-off), 100% of severely irritating substances under-predicted by the BCOP assay were classified as Category 1/2. The results obtained in this study, based on 60 test substances, indicate that the EpiOcular-EIT and the BCOP assay can be combined in a testing strategy to identify strong/severe eye irritants (Category 1), moderate and mild eye irritants (Category 2), and non-irritants (No Category) in routine testing. In particular, when the bottom-up strategy with the 60% viability cut-off was employed, none of the severely irritating substances (Category 1) were under-predicted to be non-irritant. Sensitivity for Category 1/2 substances was 100% for literature reference substances and 89% for BASF SE proprietary substances.