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 optimizations 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, fertilized 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 ADME (Absorption, Distribution, Metabolism, Excretion) 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 more than 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 (1).

Systems for Genetic Assessment of the Impact of Environmental Factors

“…Scientific papers [222,223] highlight the issues of assessing the genotoxicity of environmental factors using a promising new model that uses the method of micronucleus analysis with fertilized chicken eggs and erythrocytes. The main advantages of the method include the ability to assess genotoxic effects at the in vitro model level, taking into account the parameters of ADME, which are decisive from the point of view of the bioavailability of the chemical compound and associated with its adsorption, distribution, metabolism, release and toxicity.…”

Utility of ToxTracker in animal alternative testing strategy for fragrance materials

“…In order to evaluate the utility of the ToxTracker assay for animal alternative testing strategy, a set of four fragrance materials (supplied at market quality by Research Institute for Fragrance Materials [RIFM] member companies) were selected based on four criteria (1) Material should be a member of an aldehyde or ketone class of fragrance materials, (2) Material should have been tested in a GLP compliant in vitro micronucleus assay in accordance with OECD TG 487 using human peripheral blood lymphocytes (HPBL) in both the presence and absence of an S9 fraction from the livers of male Sprague Dawley rats induced with Aroclor 1254 or phenobarbital and β‐naphthoflavone intraperitoneally, (3) Since the substance is also used as a flavoring agent it was tested in a GLP compliant in vivo micronucleus assay in accordance with OECD TG 474 using groups of male and female Hsd:IRC (CD‐1) mice or Han Wistar rats with the test material administered either two, three, or four times in corn oil or deionized water via oral gavage, and (4) Material has also been tested in a follow‐up animal alternative study such as 3Dskin micronucleus test (MNT; Dahl et al, 2011) or Hen's egg MNT (Maul et al, 2021; Reisinger et al, 2022). The name and structures of all the materials are described below in Figure 2.…”

Cosmetic Formulations from Natural Sources: Safety Considerations and Legislative Frameworks in the European Union

“…The hen's egg model can metabolise test substances, making it comparable to in vivo biological systems [10]. Although the HET-MN has yet to attain regulatory approval, various validation studies have been published [106], and its classification as an animal or non-animal approach depends on the jurisdiction [107]. Ongoing research is exploring the application of the principles of the in vitro micronucleus test and the in vivo comet assay to RHE skin model for genotoxicity testing [102].…”