P28. Inter-laboratories Validation of Alternative Methods to Eye Irritation Test for Safety Evaluation of Cosmetic Ingredients (3) Evaluation of Hemoglobin Denaturation Test

The hemoglobin denaturation (HD) test was evaluated as an alternative method to the eye irritation test by using three indices of protein denaturation such as a test chemical concentration inducing a 50% HD of positive control (RDC50), a relative HD ratio at 1% of the chemical (1%RDR) and a relative change of maximum absorption wavelength (1%λmax). Thirty-nine test chemicals were commonly used in this project. The inter-laboratory variation of HD test among participating laboratories (6 to 8) was within a practically tolerable range. The in vitro test results were compared with the in vivo eye irritation test results and showed a reasonably good correlation. The correlation coefficients (r) between the in vivo maximum average score and log (RDC50), 1%RDR or 1%λmax were -0.91, 0.67 or 0.79, respectively. Also, some limitations have become apparent, i.e. (1) HD test cannot be applied to colored chemicals with a strong absorption around 418nm, (2) water-insoluble chemicals cannot be evaluated by RDC50 and 1%RDR, (3) HD test cannot be applied to strong acids which exceeds the buffering potential of a phosphate buffer solution, 4) HD test cannot detect the eye irritation potential caused by factors other than protein denaturation. As the HD test evaluates only the potential of protein denaturation by chemicals, it is not appropriate to predict eye irritation potential solely by the HD test. Nevertheless, HD test results matched the in vivo irritation scores fairly by itself. In addition, this method is easy to handle, and results can be obtained in a short time. This study was partly supported by a Research Grant for Health Sciences, MHW.