Interlaboratory validation of in vitro eye irritation tests for cosmetic ingredients. (2) Chorioallantoic membrane...

A chorioallantoic membrane (CAM) assay evaluates the blood vessel reaction and damage to the CAM of a fertilized hen's egg. Two types of CAM assays, the hen's egg test-chorioallantoic membrane (HET-CAM) method and the chorioallantoic membrane-trypan blue staining (CAM-TB) method, were evaluated as alternative methods to the Draize eye irritation test (Draize test). The validation project was composed of three test phases in which 10, 15 and 14 test chemicals, respectively, were evaluated. The test procedure of the five independent laboratories was controlled under the same standard operating procedure (SOP). The interlaboratory variation was relatively high for both methods. However, the rank correlation was relatively high among the values obtained by the five laboratories. The variation associated with the CAM-TB method was smaller than that of the HET-CAM method, which requires macroscopic observation, suggesting that the objectivity and quantitativeness differs between the assay systems. The average values using these two methods were compared with the maximum average Draize total score (MAS). The correlation coefficient (r) between the HET-CAM scores and the MAS was 0.688. This suggests that a simple linear regression may not be appropriate for HET-CAM. However, the Spearman's rank correlation coefficient (rs) was relatively high (rs=0.802). In contrast, the CAM-TB test results showed a good correlation with the MAS when the test chemicals were classified according to their physical properties (r=0.801, liquid and r=0.926, powder). These results suggest that both the HET-CAM and CAM-TB methods may present alternative method of evaluation of eye irritation despite problems of interlaboratory reproducibility.