Introduction: Humanitarian concern, scientific progress and legislative action have lead to the development, validation and regulatory acceptance of alternative in vitro ocular models. However, to date not a single in vitro alternative ocular toxicity test has been validated as a full replacement for the in vivo Draize rabbit eye test for all classes of chemicals across whole irritancy ranges. Since the 1990s, ocular alternative methods have been validated but few have been accepted for regulatory purposes. These assays include: organotypic models, such as the bovine corneal opacity and permeability (BCOP) assay, the isolated chicken eye (ICE) test method and cell function-based in vitro assays, such as the cytosensor microphysiometer (CM) and the fluorescein leakage (FL) test methods. Some refinements to in vivo testing methods have been accepted by regulatory agencies, including humane endpoints to avoid or minimize pain and distress.

Areas covered: The authors provide a review of the background, protocol overview, applications and their validation status of the tier-testing approach. Furthermore, the authors provide expert analysis and provide their perspective on this approach and potential future developments.

Expert opinion: In the search for a battery of methods that replaces the in vivo Draize test, it is necessary to prioritize techniques, define related mechanisms and justify statistical approaches. Overall, only when the reliability and relevance of a method is unequivocally supported will any technique be ready for regulatory acceptance.