The hen's egg test for micronucleus induction (HET-MN): novel analyses with a series of well-characterized substances support the further evaluation of the test system.

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 those that are mutagens or promutagens. This assay procedure is in line with demands for animal protection. In three previous publications we presented the scientific rationale and methodological aspects for this assay as well as results for some well-characterized mutagens and promutagens. Here we present the results of new experiments involving further genotoxic and non-genotoxic model substances. Making a comparison with published data we have to date not found any false negatives or false positives in the experiments presented here and in trials published before, thus demonstrating a promising predictivity of genotoxic effects with this assay. We could confirm relevant genotoxicity for the following substances in the HET-MN: acetylamino-fluorene (2-AAF), acrylamide (ACM), cytarabine (AraC), methotrexate (MTX), cadmium chloride (CD), dipotassium monochromate (DPC), and epirubicine (EPI). Negative results were obtained for azorubin (E122), orange G (OG) and starch (STRC). The micronucleus frequencies (MNE II) of the concurrent negative controls were in agreement with the values of the historical negative control (0.87 per thousand+/-0.87; average+/-s.d.). This value is based upon the scoring of 556,500 erythrocytes from 445 eggs. In historical positive controls the administration of 0.05mg cyclophosphamide/egg at d8 resulted in an MNE II-frequency of 12.4 per thousand+/-6.8 (average+/-s.d.) at d 10.5. This value is based upon the scoring of 249,250 erythrocytes from 223 eggs.

Some new methodological aspects of the hen's egg test for micronucleus induction (HET-MN).

In a previous publication we introduced the hen's egg test for micronucleus induction (HET-MN) as an extremely simple, inexpensive and rapid animal free genotoxicity assay which is positioned between pure in vitro and in vivo assays, strictly in line with animal protection regulations and ethical aspects. The HET-MN combines the use of the commonly accepted genetic endpoint "formation of micronuclei" with the well characterized and complex model of the chick embryo. The high metabolic competency provided by this model enables metabolic activation, elimination and excretion of xenobiotics including mutagens and promutagens. In this paper we present some new methodological aspects, which are important for improving the experimental protocol. We used cyclophosphamide (CP) and 7,12-dimethyl-benz[a]anthracene (DMBA) as model substances. Dose-response-relationship for both chemicals and cytotoxic effects for CP are described. In addition to the standard proliferation marker PCE/NCE-ratio we found an increased frequency of primitive erythrocytes (E I) and the appearance of proerythroblasts and erythroblasts as further alerting signals for cytotoxic or erythrosuppressive effects. From the total cell population we could further qualify the group of target cells. We found that all definite erythrocytes (E II), observed at day 11 (d11), are relevant target cells, independent from their stage of maturity (polychromatic as well as normochromatic definite erythrocytes). E I cells do not belong to the group of target cells, however. An additional important methodological aspect is the optimal time frame. We found the time period from d8 of incubation (administration of the test substance) up to d11 (time point of blood sampling) as most favorable. In this way an exposure period of up to 72h is covered. Further results indicate that the air cell route provides a higher response to the test substances than the albumen route. The consideration of the described methodological aspects will contribute to the improvement of the experimental protocol of the HET-MN.