In vitro Eye Irritation RhT Test Methods for Dyes. COLIPA Program on Optimization and Validation of HPLC Bio-Analytical Assay to Expand Applicability Domain of in vitro Eye Irritation RhT Test Methods for Dyes.

In vitro eye irritation assays using Human Reconstructed Tissue (RhT) test systems (SkinEthicTM HCE & EpiOcularTM EIT) are currently undergoing ECVAM validation for detection of GHS irritants/non-irritants. In these test methods, irritation potential of a test chemical is determined by measuring cell viability in treated tissues by colorimetric MTT reduction assay. Cell viability is determined by enzymatic reduction of a yellow MTT tetrazolium salt to blue formazan. It is quantified photometrically with results expressed as % viability in the test chemical treated tissues relative to the negative control. A decrease in MTT reduction capacity is used to identify potential irritancy of test chemicals. A known limitation of the MTT test is possible interference of dye with absorbance measurement of formazan . The European Cosmetics Industry Association (COLIPA) is conducting a program to address this limitation. The aims of the program are to: 1) optimise the preliminary analytical approach using High Performance Liquid Chromatography (HPLC) to quantify separately formazan from intrinsically coloured test chemical; 2) demonstrate application of the HPLC analytical method to evaluate dyes in RhT test methods; 3) demonstrate within and between laboratory reproducibility. Results presented describe: validation using the FDA guideline for bio-analytical method and the multi-centric study (6 chemicals tested in 3 labs). This work has supported development of a Standard Operating Procedure that can be applied to extend the applicability domain of RhT test methods.