TY - JOUR

T1 - Statistical analysis of the hen's egg test for micronucleus induction (HET-MN assay)

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

PY - 2013/9/18

Y1 - 2013/9/18

N2 - 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.

AB - 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.

KW - HET-MN assay

KW - Historical control

KW - Williams-trend test

UR - http://www.scopus.com/inward/record.url?scp=84891552587&partnerID=8YFLogxK

U2 - 10.1016/j.mrgentox.2013.04.023

DO - 10.1016/j.mrgentox.2013.04.023

M3 - Article

C2 - 23892231

AN - SCOPUS:84891552587

VL - 757

SP - 68

EP - 78

JO - Mutation Research - Genetic Toxicology and Environmental Mutagenesis

JF - Mutation Research - Genetic Toxicology and Environmental Mutagenesis

SN - 1383-5718

IS - 1

ER -