Interlaboratory Validation of the in vitro Eye Irritation Tests for Cosmetic Ingredients. (4) Haemoglobin Denaturation Test.

Interlaboratory validation of the haemoglobin denaturation (HD) test on 38 cosmetic ingredients was conducted by five to eight participating laboratories. The HD test was evaluated as an alternative method to the Draize eye irritation test (Draize test) based on three indices of protein denaturation: the test substance concentration that induces 50% HD of the positive control (RDC(50)), a relative HD rate at 1% of the test substance (1%RDR) and a relative change in maximum absorption wavelength (1% lambdamax). The coefficients of variation associated with a positive HD test among the participating laboratories were within an acceptable range for practical application. The in vitro test results were in relatively good agreement with the Draize test. The correlation coefficient (r) between the in vivo maximal average Draize total score (MAS) and log (RDC(50)), 1%RDR and 1% lambdamax were -0.91, 0.67 and 0.79, respectively. The results revealed several limitations associated with the HD test: (1) the HD test cannot be applied to coloured test substances with a strong absorption, around 418nm; (2) water-insoluble test substances cannot be evaluated by RDC(50) or 1%RDR; (3) the HD test cannot be applied to strong acids that exceed the buffering capacity of a phosphate buffer solution; (4) the HD test cannot be used to determine the potential for eye irritation caused by factors other than protein denaturation, for example, polyoxyethylene octylphenylether (10 E.O.). Thus, the HD test alone is not appropriate for predicting eye irritation potential. Nevertheless, the good agreement between the HD test results and in vivo irritation scores as well as the ease of application suggest that this test may play an important role in a test system to determine eye irritation potential.