The ECVAM retrospective validation study on cytotoxicity- and cell function-based in vitro assays for eye irritation.

Current European legislations strongly call for alternatives to replace animal testing. In major evaluation studies conducted in the 1990¿s, no single method was found able to fully replace the Draize eye irritation test. However, some alternatives showed good reproducibility and reliability. In 2006, ECVAM initiated the retrospective validation of four assays based on cytotoxicity and cell function: 1) Neutral Red Release (NRR), 2) Red Blood Cell test, 3) Fluorescein Leakage (FL) and 4) Cytosensor Microphysiometer (CM). The study was coordinated by an international Validation Management Group (VMG) including observers from ICCVAM-NICEATM and JaCVAM. The existing data were collected and compiled according to ECVAM Principles of Weight-of-Evidence Validation. The resulting Background Review Document (BRD) for each assay was structured according to the ECVAM Modular Approach to Validation and underwent independent quality control audits. The VMG evaluated the four BRDs and made recommendations concerning the suitability of the assays to be used in the Bottom-Up and Top-Down Approaches for Eye irritation Testing Strategy proposed in a 2005 ECVAM expert meeting, to reduce and replace in vivo studies. In particular, the CM protocol INVITTOX 102-modified and the FL protocol INVITTOX 71 were recommended as suitable to discriminate severe irritants from other classes. Furthermore, the FL protocol INVITTOX 120 and the NRR protocol INVITTOX 54 and PREDISAFETM were recommended as suitable to discriminate non-irritants from other classes. These assays are currently under Peer Review by the ECVAM Scientific Advisory Committee. The ultimate goal is to combine validated in vitro assays for eye irritation based on their performances and applicability domains, to identify the most suitable testing strategies to classify substances according to their irritation potential and ultimately replace the Draize rabbit eye test.JRC.DDG.I.3-In-vitro method