The HET-CAM test combined with histological studies for better evaluation of active ingredient innocuity.

In the search for alternative methods to animal testing, the Hen's egg test on chorioallantoic membrane (HET-CAM) plays a central role in evaluating the innocuity of active ingredients. Therefore, in the following studies we combined the HET-CAM test with histological evaluation in order to increase the sensitivity of evaluation. Twenty active ingredients from four different categories of origin (vegetal, marine, biotechnological and chemical synthetic) were subjected to innocuity evaluation at two different concentrations (pure and 10%). We performed the HET-CAM test and histological evaluation after trypan blue and hematoxylin-eosin staining of the chorioallantoic membrane to microscopically evaluate its state of damage after application of each active ingredient. These studies showed that when the active ingredient was diluted (10%), no discrepancy was seen between the classical HET-CAM evaluation and the histological reading of the chorioallantoic membrane. The histological findings corresponded with the visual observation of the CAM. When the active ingredients were tested at pure concentration, 7 out of 20 tested products demonstrated discrepancy between the two tests. In six cases, the histological examination revealed signs of irritation, such as hyperemia, while visual HET-CAM evaluation was negative. In another case, the histological examination revealed a slight hemorrhage whereas the HET-CAM reading showed only hyperemia. Moreover, the results of trypan blue staining corroborated the histological evaluation of the CAM. These results strongly suggest that the combination of histological and visual HET-CAM tests is of interest for a more sensitive evaluation of the innocuity of cosmetic active ingredients. This additional sensitivity may help to prevent some cases of in vivo intolerance reactions.