Interlaboratory validation of the in vitro eye irritation tests for cosmetic ingredients. (3) evaluation of the haemolysis test

The haemolysis test using sheep red blood cells (RBC) was evaluated as an a lternative method to the Draize rabbit eye irritation test (Draize test) by six to nine laboratories. The participating laboratories performed the tes t according to the standard operating procedure (SOP). Thirty-eight cosmeti c ingredients and isotonic sodium chloride solution were used as test subst ances in this validation study. The concentrations of the test substances t hat induced 50% haemolysis (HC50 value) was obtained to serve as a toxicolo gical index and compared with in vivo Draize scores. HC50 values were not o btained for coloured or water-insoluble (turbid) substances. Three acids ca used denaturation of haemoglobin leaked from RBC and consequently interfere d with the determination of the HC50 value. Interlaboratory reproducibility was relatively good except in the case of water-insoluble substances. The average values of coefficient of variation (CV) was 37%. The correlation co efficient and Spearman's rank correlation between the HC50 value and maximu m average Draize total score (MAS) were -0.631 and 0.641, respectively. The equivalence ratio between the haemolysis test and MAS was 70.0% when MAS 1 5 was set as the in vivo cut-off point. On the other hand, strong irritants (MAS greater than or equal to 50) could be correctly classified by this me thod. These results suggest that the haemolysis test might be applied to co smetic ingredients as a screening method to distinguish strong irritants th at directly affect the cell membrane permeability and do not disturb spectr ophotometrical determination of haemoglobin. In order to evaluate the poten tial for eye irritation of cosmetic ingredients, a combination of haemolysi s with other methods based on different mechanism should be employed to imp rove the predictability. (C) 1999 Elsevier Science Ltd. All rights reserved .