Investigating genotoxic and hematotoxic effects of N-nitrosodimethylamine, N-nitrosodiethylamine and N-nitrosodiethanolamine in the hen's egg–micronucleus test (HET–MN)

Abstract

The hen's egg test for micronucleus induction (HET–MN) combines the use of the commonly accepted genetic endpoint “formation of micronuclei” with the well-characterized and complex model of the incubated hen's egg, which enables metabolic activation, elimination and excretion of xenobiotics including mutagens and promutagens and does not conflict with animal protection regulations and ethical aspects. N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) produced clearly positive, dose-dependent and reproducible results in this assay. NDMA revealed, in accordance with literature data, a much higher mutagenicity than NDEA. For both compounds the sensitivity of HETMN was to a large extent higher than published for the rodent micronucleus test, which is not capable of unequivocally identifying NDEA as positive. Additionally, NDEA induced severe anemia without obscuring the formation of micronucleated cells. N-nitrosodiethanolamine (NDELA), which in the literature is described as a non-mutagenic animal carcinogen, could clearly be confirmed as non-mutagenic in the HETMN without showing any disturbing effects on the formation of erythrocytes. The micronucleus frequencies of the concurrent negative controls of all experiments was in agreement with the historic negative control from 302 eggs and 412,532 cells. The same is true for the historic control of proliferation marker from 61 eggs and 13,020 cells. We interpret these results, which correspond well to published data from animal tests, as being further support for using the HET–MN as a reliable alternative genotoxicity assay system, which is physiologically closer to in vivo conditions than in vitro genotoxicity tests, and allows the observation of further local and systemic effects.