Statistical Analysis Of In Vivo Alkaline Comet Assay Data - Comparison Of Median And Geometric Mean As Centrality Measures

The comet assay is one of the standard tests for evaluating the genotoxic potential of a test item able to detect DNA strand breaks in cells or isolated nuclei from various tissues. The in vivo alkaline comet assay is part of the standard test battery, given in option 2 of the ICH guidance S2 (R1) and a follow-up test in the EFSA framework on genotoxicity testing. The current OECD guideline for the testing of chemicals No. 489 directly affects the statistical analysis of comet data as it suggests using the median per slide and the mean of all medians per animal. However, alternative approaches can be used if scientifically justified. In this work, we demonstrated that the selection of different centrality measures to describe an average value per slide may lead to fundamentally different statistical test results and contradicting interpretations. Our focus was on geometric means and medians per slide for the primary endpoint "tail intensity". We compared both strategies using original and simulated data in different experimental settings incl. a varying number of animals, slides and cells per slide. In general, it turned out that the chosen centrality measure has an immense impact on the final statistical test result.