The objective of this grant project is the development and pre-validation of the Replacement Ocular Battery (ROBatt), a tiered testing strategy consisting of a battery of four alternative ocular irritancy assays, which will replace regulatory mandated acute ocular irritation testing using the Draize Rabbit Eye test. ROBatt consists of the Bovine Corneal Opacity and Permeability Assay (BCOP), the Chorioallantoic Membrane Vascular Assay using 10-day fertile chicken eggs (CAMVA), the Porcine Corneal Reversibility Assay (PorCORA) and the Porcine Confocal Assay (PorFocal). This tiered strategy follows a decision tree that allows for a thorough interrogation of possible ocular irritants. Although four assays are recommended, in most cases only two or three will be used depending on the degree of irritation. The Specific Aim of the ROBatt project is to validate the decision tree variables using at least 50 chemicals listed in the European Center for Ecotoxicology and Toxicology of Chemicals (ECETOC) data bank, including Corrosive (EEC R41, GHS/EPA Cat 1), Severe (EEC R36, GHS CaL 2, HMIS 2), Moderate (EPA Cat. 3, HMIS 2), Mild (HMIS 1) and Non-irritating (EPA Cat 4, HMIS 0). The long-term project goal is to submit the ROBatt testing strategy to iCCVAM/ECVAM for consideration as a standalone alternative to the Draize Rabbit Eye test. Validation and acceptance of the ROBatt testing strategy will significantly reduce the number of rabbits used in the toxicological assessment of consumer products, chemicals and raw materials by replacing rabbits with four robust alternative assays. PUBLIC HEALTH RELEVANCE: Ocular irritation testing is extremely relevant to assuring adequate safety levels of public health as new formulations of chemicals and products are introduced. In most cases, these safety assessments are performed using the Draize Rabbit Eye test, resulting in thousands of rabbits used in testing every year. Alternatives have been discussed since the 80s without any appreciable acceptance from regulators.