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

• Published in: Toxicology in vitro Vol. 13; no. 1; pp. 125 - 137
• Main Authors: Hatao, M., Murakami, N., Sakamoto, K., Ohnuma, M., Matsushige, C., Kakishima, H., Ogawa, T., Kojima, H., Matsukawa, K., Masuda, K., Chiba, K., Yoshizawa, K., Kaneko, T., Iwabuchi, Y., Matsushima, Y., Momma, J., Ohno, Y.
• Format: Journal Article
• Language: English
• Published: Elsevier Ltd 1999
• Subjects: denaturation test; Draize eye irritation test; haemoglobin; in vitro; protein denaturation; validation study; NIHS=National Institute of Health Sciences; 1% λmax=percent shift of maximum absorption wavelength by 1% of the test substance; DMSO=dimethyl sulfoxide; Draize test=Draize eye irritation test; protein denaturation; RDC 50 (relative denaturation concentration) 50=test substance concentration inducing a 50% relative haemoglobin denaturation of positive control; denaturation test; MAS=maximal average Draize total score; validation study; Draize eye irritation test; haemoglobin; HD=haemoglobin denaturation; 1%RDR=relative haemoglobin denaturation ratio by 1% of the test substance; in vitro; JCIA=Japan Cosmetic Industry Association; λmax=maximum absorption wavelength; MHW=Ministry of Health and Welfare
• ISSN: 0887-2333; 1879-3177
• DOI: 10.1016/S0887-2333(98)00067-8

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% λmax). 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% λmax 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 418 nm; (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.