Validation of the hen’s egg test for micronucleus induction (HET-MN): detailed protocol including scoring atlas, historical control data and statistical analysis

A validation exercise of the hen's egg test for micronucleus induction (HET-MN) was finalised with a very good predictivity based on the analysis of micronuclei in peripheral erythrocytes of fertilised chicken eggs [1]. For transparency reasons this complementary publication provides further details on the assay especially as this was the first validation study in the field of genotoxicity testing involving the use of chicken eggs. Thus, the experimental protocol is described in detail and is complemented by a scoring atlas for microscopic analysis of blood cells. In addition, general characteristics of the test system, which is able to mirror the systemic availability of test compounds, are delineated: the test compound passes the egg membrane and is taken up by the blood vessels of the underlying chorioallantoic membrane. Subsequently, it is distributed by the circulating blood, metabolised by the developing liver and the yolk sac membrane, and finally excreted into the allantois, a bladder equivalent. In specific, the suitability of the test system for genotoxicity testing is shown by, inter alia, a low background DNA damage in a comprehensive historical control database. In addition, the state-of-the-art statistical method used to evaluate obtained data is delineated. It combines laboratory-specific effect threshold with the Umbrella-Williams test a statistical model also of interest for other genotoxicity test methods.