Statistical analysis of the hen's egg test for micronucleus induction (HET-MN assay)

Publikation: Beitrag in FachzeitschriftArtikelForschungPeer-Review

Autoren

  • Ludwig A. Hothorn
  • Kerstin Reisinger
  • Thorsten Wolf
  • Albrecht Poth
  • Dagmar Fieblinger
  • Manfred Liebsch
  • Ralph Pirow

Organisationseinheiten

Externe Organisationen

  • Henkel AG & Co. KGaA
  • Universität Osnabrück
  • Institute for Competence Contract Research – Roßdorf (ICCR-Roßdorf) GmbH
  • Bundesinstitut für Risikobewertung (BfR)
Forschungs-netzwerk anzeigen

Details

OriginalspracheEnglisch
Seiten (von - bis)68-78
Seitenumfang11
FachzeitschriftMutation Research - Genetic Toxicology and Environmental Mutagenesis
Jahrgang757
Ausgabenummer1
Frühes Online-Datum26 Juli 2013
PublikationsstatusElektronisch veröffentlicht (E-Pub) - 26 Juli 2013

Abstract

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.

ASJC Scopus Sachgebiete

Zitieren

Statistical analysis of the hen's egg test for micronucleus induction (HET-MN assay). / Hothorn, Ludwig A.; Reisinger, Kerstin; Wolf, Thorsten et al.
in: Mutation Research - Genetic Toxicology and Environmental Mutagenesis, Jahrgang 757, Nr. 1, 26.07.2013, S. 68-78.

Publikation: Beitrag in FachzeitschriftArtikelForschungPeer-Review

Hothorn, L. A., Reisinger, K., Wolf, T., Poth, A., Fieblinger, D., Liebsch, M., & Pirow, R. (2013). Statistical analysis of the hen's egg test for micronucleus induction (HET-MN assay). Mutation Research - Genetic Toxicology and Environmental Mutagenesis, 757(1), 68-78. Vorabveröffentlichung online. https://doi.org/10.1016/j.mrgentox.2013.04.023
Hothorn LA, Reisinger K, Wolf T, Poth A, Fieblinger D, Liebsch M et al. Statistical analysis of the hen's egg test for micronucleus induction (HET-MN assay). Mutation Research - Genetic Toxicology and Environmental Mutagenesis. 2013 Jul 26;757(1):68-78. Epub 2013 Jul 26. doi: 10.1016/j.mrgentox.2013.04.023
Hothorn, Ludwig A. ; Reisinger, Kerstin ; Wolf, Thorsten et al. / Statistical analysis of the hen's egg test for micronucleus induction (HET-MN assay). in: Mutation Research - Genetic Toxicology and Environmental Mutagenesis. 2013 ; Jahrgang 757, Nr. 1. S. 68-78.
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abstract = "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.",
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AU - Hothorn, Ludwig A.

AU - Reisinger, Kerstin

AU - Wolf, Thorsten

AU - Poth, Albrecht

AU - Fieblinger, Dagmar

AU - Liebsch, Manfred

AU - Pirow, Ralph

N1 - Funding Information: The funding of the present study by the German Federal Institute for Risk Assessment (BfR) is greatfully acknowledged. The experimental data used in this study originate from a validation study, which is funded by the German Federal Ministry for Research and Technology (BMBF Funding Priority “Replacement methods of animal experiments”, funding code: 0315803). The authors are grateful to two anonymous referees for the helpful comments which considerably improved the article. Appendix A

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