xeredar: An open-source R package for the statistical analysis of endocrine new approach methodologies using fish and amphibian eleutheroembryos

The experimental design of new approach methodologies (NAMs) might deviate from common ecotoxicological studies, often requiring tailored statistical approaches. For instance, in NAMs developed for the detection of endocrine activity using aquatic vertebrate eleutheroembryos (Xenopus eleutheroembryonic thyroid assay [XETA], rapid androgen disruption activity reporter [RADAR] assay and rapid estrogen activity in vivo [REACTIV] assay), all concentration groups are nested within three independent study repeats, named 'runs' in the relevant test guidelines. Here, runs are referred to as replicates to emphasize their role as the repeated, independent entity. By contrast, for most other ecotoxicological studies, the replicates are nested in the concentration groups. This leads to a different dependency structure for XETA, RADAR, and REACTIV assays. Disregarding this violates the basic statistical requirement for independence of observations potentially leading to incorrect conclusions. Unfortunately, in the statistical sections of the test guidelines of the XETA, RADAR and REACTIV assays, it is not clearly recommended to regard this dependency structure, as statistical recommendations using a mixed analysis of variance (ANOVA) are provided only in the annexes. Here, we present xeredar, an open-source R package allowing automated statistical analysis of XETA, RADAR, and REACTIV assays where the dependency structure of the data is correctly regarded through a mixed ANOVA. The xeredar package was validated on 36 XETA ring test studies and further tested on 41 RADAR ring test studies. A power analysis was carried out for the REACTIV assay, demonstrating that ignoring the dependency structure potentially leads to lower power and an increased false-positive rate compared with the mixed ANOVA approach. The open-source R package xeredar also comes with a Shiny application, making it accessible to everyone and thereby enhancing standardization and reproducibility for the statistical analyses of XETA, RADAR, and REACTIV assays.