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 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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Kirkland D, Aardema M, Henderson L et al (2005) Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens I. Sensitivity, specificity and relative predictivity. Mutat Res 584(1–2):1–256
OECD (2016) Guidelines for the testing of chemicals, test no. 487: in vitro mammalian cell micronucleus test. In vitro mammalian cell micronucleus test. https://www.oecd-ilibrary.org/environment/test-no-487-in-vitro-mammalian-cell-micronucleus-test_9789264264861-en
OECD (2016) Guideline for the testing of chemicals, test no. 474: mammalian erythrocyte micronucleus test. http://www.oecd-ilibrary.org/environment/test-no-474-mammalian-erythrocyte-micronucleus-test_9789264071285-en;jsessionid=adeces45nfr16.x-oecd-live-02
OECD (2016) Guidelines for the testing of chemicals, test no. 489: in vivo mammalian alkaline comet assay. http://www.oecd-ilibrary.org/environment/test-no-489-in-vivo-mammalian-alkaline-comet-assay_9789264224179-en;jsessionid=npkmv8v01k1s.x-oecd-live-02
Pfuhler S, Kirst A, Aardema M et al (2010) A tiered approach to the use of alternatives to animal testing for the safety assessment of cosmetics: genotoxicity. A COLIPA analysis. Regul Toxicol Pharmacol 57(2–3):315–324
Greywe D, Kreutz J, Banduhn N et al (2012) Applicability and robustness of the hen’s egg test for analysis of micronucleus induction (HET-MN): results from an inter-laboratory trial. Mutat Res 747(1):118–134
Wolf T, Luepke NP (1997) Formation of micronuclei in incubated hen’s eggs as a measure of genotoxicity. Mutat Res 394(1–3):163–175
Wolf T, Niehaus-Rolf C, Banduhn N et al (2008) 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. Mutat Res 650(2):150–164
Wolf T, Niehaus-Rolf C, Luepke NP (2002) Some new methodological aspects of the hen’s egg test for micronucleus induction (HET-MN). Mutat Res 514(1–2):59–76
Wolf T, Niehaus-Rolf C, Luepke NP (2003) Investigating genotoxic and hematotoxic effects of N-nitrosodimethylamine, N-nitrosodiethylamine and N-nitrosodiethanolamine in the hen's egg-micronucleus test (HET-MN). Food Chem Toxicol 41(4):561–573
Dieterlein-Lievre F (ed) (1988) Birds. Vertebrate blood cells. Cambridge University Press, Cambridge
Jones JF (1983) Development of the spleen. Lymphology 16(2):83–89
Hamburger V, Hamilton HL (1951) A series of normal stages in the development of the chick embryo. J Morphol 88:49–92
Peters JJ, Vonderrahe AR, Powers TH (1956) The functional chronology in developing chick nervous system. J Exp Zool 133:505–518
Rosenbruch M (1997) The sensitivity of chicken embryos in incubated eggs. ALTEX 14:111–113
CH (2008). https://www.admin.ch/opc/de/classified-compilation/20080796/index.html
EU (2010). http://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32010L0063&from=EN
UK (2006). http://www.legislation.gov.uk/ukpga/2006/45/contents
US (2013). http://www.legislation.govt.nz/bill/government/2013/0107/latest/whole.html#DLM5174807
Maul K, Fieblinger D, Heppenheimer A et al The hen’s egg test for micronucleus-induction (HET-MN): detailed protocol and scoring atlas for microscopic analysis. Mutat Res. Submitted for publication
Reisinger K, Fieblinger D, Heppenheimer A et al The hen’s egg test for micronucleus-induction (HET-MN): validation data set. Mutat Res. Submitted for publication
Fenech M (2000) The in vitro micronucleus technique. Mutat Res 455(1–2):81–95
Freeman MF, Tukey JW (1950) Transformations related to the angular and the square root. Math Stat 21(4):607–611
Jonckheere A (1954) A distribution-free k-sample test against ordered alternatives. Biometrika 41:133–145
Hothorn LA, Reisinger K, Wolf T (2013) Statistical analysis of the hen's egg test for micronucleus induction (HET-MN assay). Mutat Res 757(1):68–78
Acknowledgments
The optimization and validation of the HET-MN was funded by the German Federal Ministry for Research and Education as well as by Cosmetics Europe.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Reisinger, K., Dony, E., Wolf, T., Maul, K. (2019). Hen’s Egg Test for Micronucleus Induction (HET-MN). In: Dhawan, A., Bajpayee, M. (eds) Genotoxicity Assessment. Methods in Molecular Biology, vol 2031. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9646-9_10
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9646-9_10
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9645-2
Online ISBN: 978-1-4939-9646-9
eBook Packages: Springer Protocols