The hen's egg test for micronucleus induction (HET-MN): validation data set

Abstract

The classical in vitro genotoxicity test battery is known to be sensitive for indicating genotoxicity. However, a high rate of 'misleading positives' was reported when three assays were combined as required by several legislations. Despite the recent optimisations of the standard in vitro tests, two gaps could hardly be addressed with assays based on 2D monolayer cell cultures: the route of exposure and a relevant intrinsic metabolic capacity to transform pro-mutagens into reactive metabolites. Following these considerations, fertilised chicken eggs have been introduced into genotoxicity testing and were combined with a classical read-out parameter, the micronucleus frequency in circulating erythrocytes, to develop the hen's egg test for micronucleus induction (HET-MN). As a major advantage, the test mirrors the systemic availability of compounds after oral exposure by reflecting certain steps of Absorption, Distribution, Metabolism, Excretion (ADME) without being considered as an animal experiment. The assay is supposed to add to a toolbox of assays to follow up on positive findings from initial testing with classical in vitro assays. We here report on a validation exercise, in which >30 chemicals were tested double-blinded in three laboratories. The specificity and sensitivity of the HET-MN were calculated to be 98 and 84%, respectively, corresponding to an overall accuracy of 91%. A detailed protocol, which includes a picture atlas detailing the cell and micronuclei analysis, is published in parallel (Maul et al. Validation of the hen's egg test for micronucleus induction (HET-MN): detailed protocol including scoring atlas, historical control data and statistical analysis).

Fig. 1.

Representative HET-MN study results—2-aminoanthracene. The lab-specific data of two experiments are shown. The FT-transformed MN rate (circles; left axis) and the egg viability (triangles; right axis) are given in relation to the different treatments. Filled triangles indicate viabilities < 40%. MN data are given as mean ± standard deviation and as raw data (small cross symbols). Dotted horizontal lines refer to the MN rate and indicate the upper acceptance limit for the SC and the lower acceptance limit for the PC. MN data were tested for an increase above the threshold (Th) and for a linear trend using the JT test (PM1). MN data were also analysed using the UW procedure (PM2). Finally, the result of the expert judgement (EJ) is indicated. For each test, a positive outcome is indicated by a crossed check box at the top of graphs. Filled circles above the x-axis (individual or linked) indicate single- or pooled-dose groups for which the UW test indicated a statistically significant increase; circles with a black outline circles indicate single or pooled dose groups with the smallest significant p-value. The used solvent (IPM) is indicated in the low right, and the adjacent label ‘S’ indicates the single-dose regimen.

Fig. 2.

Chemicals re-tested non-coded with the standard protocol after using the ‘repeated-dose regimen’ during validation. One experiment each with 2-AAF and cadmium sulphate is shown. For further graphic details, see legend of Figure 1.

Fig. 3.

Comparison of the standard protocol with the ‘day 9 protocol’ using benzo[a]pyrene. Eggs were either treated on day 8 of egg development (left) or on day 9 (right) with the indicated doses of benzo[a]pyrene, while sampling was done on day 11 in the laboratory, which performed both experiments. For further graphic details, see legend of Figure 1.