A validation exercise of the hen's egg test for micronucleus induction was finalised with a very good predictivity based on the analysis of micronuclei in peripheral erythrocytes of fertilised chicken eggs (Reisinger et al. The hen's egg test for micronucleus-induction (HET-MN): validation data set. Mutagenesis, this issue). For transparency reasons this complementary publication provides further details on the assay especially as it 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 in 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.

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).

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 optimizations of the standard in vitro tests, two gaps could merely be addressed with assays based on monolayer cell cultures, that is, the route of exposure and a relevant intrinsic metabolic capacity to transform chemicals into reactive metabolites. Following these considerations, fertilized chicken eggs have been introduced into genotoxicity testing and were combined with a classical readout parameter, i.e., the analysis of micronucleus frequency in erythrocytes, to develop the hen's egg test for micronucleus induction, the HET-MN. As a major advantage the test mirrors the systemic availability of compounds after oral exposure reflecting certain steps of ADME without being considered as an animal experiment. After a successful validation exercise the detailed protocol is given here.

The HET-MN assay (hen's egg test for micronucleus induction) is different from other in vitro genotoxicity assays in that it includes toxicologically important features such as absorption, distribution, metabolic activation, and excretion of the test compound. As a promising follow-up to complement existing in vitro test batteries for genotoxicity, the HET-MN is currently undergoing a formal validation. To optimize the validation, the present study describes a critical analysis of previously obtained HET-MN data to check the experimental design and to identify the most appropriate statistical procedure to evaluate treatment effects. Six statistical challenges (I-VI) of general relevance were identified, and remedies were provided which can be transferred to similarly designed test methods: a Williams-type trend test is proposed for overdispersed counts (II) by means of a square-root transformation which is robust for small sample sizes (I), variance heterogeneity (III), and possible downturn effects at high doses (IV). Due to near-to-zero or even zero-count data occurring in the negative control (V), a conditional comparison of the treatment groups against the mean of the historical controls (VI) instead of the concurrent control was proposed, which is in accordance with US-FDA recommendations. For the modified Williams-type tests, the power can be estimated depending on the magnitude and shape of the trend, the number of dose groups, and the magnitude of the MN counts in the negative control. The experimental design used previously (i.e. six eggs per dose group, scoring of 1000 cells per egg) was confirmed. The proposed approaches are easily available in the statistical computing environment R, and the corresponding R-codes are provided.

The hen's egg test for analysis of micronucleus formation (HET-MN) was developed several years ago to provide an alternative test system to the in vivo micronucleus test. In order to assess its applicability and robustness, a study was carried out at the University of Osnabrueck (lab A) and at the laboratories of Henkel AG & Co. KGaA (lab B). Following transfer of the method to lab B, a range of test substances that had been pre-tested at lab A, were tested at Henkel: the genotoxins cyclophosphamide, dimethylbenz(a)anthracene, methotrexate, acrylamide, azorubin, N-nitroso-dimethylamine and the non-genotoxins, orange G and isopropyl myristate. In a second phase, additional compounds with known in vivo properties were examined in both labs: the non-genotoxin, ampicillin, the "irrelevant" positives, isophorone and 2,4-dichlorophenol ("irrelevant" means positive in standard in vitro tests, but negative in vivo), the clastogen p-chloroaniline, and the aneugens carbendazim and vinorelbine. All substances were correctly predicted in both labs with respect to their in vivo genotoxic properties, indicating that the HET-MN may have an improved predictivity compared with current standard in vitro test systems. The results support the promising role of the HET-MN assay as a supplement to existing test batteries.

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.

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.

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The formation of micronuclei (MN) is a widely used and accepted endpoint of genotoxicity testing. The micronucleus assay provides a simple and rapid indirect measure of the induction of structural or numerical chromosome aberrations. In this work we describe hen's eggs, incubated for 11 days, as ex vivo assay system for the detection of micronucleus formation in young erythrocytes (Hen's Egg Test for Micronucleus Induction, HET-MN). At this stage of development the chick embryo presents a high metabolic competency which allows an adequate activation of several types of promutagens, as previously reported by several authors. As all stages of maturing erythrocytes are present in the bloodstream of the chick embryo, we could conveniently use samples of peripheral blood for scoring micronuclei as well as for determining the ratio between mature and immature erythrocytes as a measure of an undisturbed erythropoiesis. The obtained blood smears were stained by a modified May-Gruenwald-Giemsa procedure and scored microscopically. The examinations were facilitated by using a semiautomatic image analysis system. We could demonstrate a strong increase of the micronucleus frequency after the administration of the promutagens diethylnitrosamine (DENA), 7,12-dimethyl-benz[a]anthracene (DMBA), cyclophosphamide (CP), ifosphamide (IF), mitomycin C (MMC), and the direct-acting mutagen methanesulfonic acid methyl ester (MMS) compared to the concomitant negative controls. CP was used to demonstrate a dose–response relation and the effect of using two different routes of application (air cell and albumen). Nuclear aberrations, other than MN, were demonstrated after application of high doses of CP or IF. Expanded exposure times revealed a similar effect. The HET-MN, as an ex vivo assay, is a simple, inexpensive, and rapid assay system for genotoxicity testing, positioned between pure in vitro and in vivo assays, strictly in line with animal protection regulations and ethical aspects.