Evaluation of anogenital distance (AGD) and areola/nipple retention (NR) assessments in extended one generation reproductive toxicity studies - Implications for regulatory toxicology

The Extended One-Generation Reproductive Toxicity Study (EOGRTS) represents the current state-of-the-art in in vivo reproductive toxicity testing. Although its main purpose is to provide information on sexual function and fertility, it can also provide important information for assessing the endocrine disrupting effects of chemical substances. In a collaborative effort between the European Chemicals Agency (ECHA) and nine EU Member States, a comprehensive review of the EOGRTS was conducted to evaluate its role in supporting regulatory hazard assessment in the EU. In a satellite project, measurements of anogenital distance (AGD) and areola/nipple retention (NR), two critical and sensitive endpoints in assessing endocrine disruption, were analysed. From 72 full study reports (FSR), all F1 generation offspring were investigated for AGD and NR. An additional 25 datasets for AGD and 21 datasets for NR were available from the F2 generation offspring, resulting in a total of 97 datasets analysed for AGD and 93 datasets for NR. In the evaluation of the AGD data, the following factors were assessed; coefficient of variation (CoV), measuring precision and overlap between male and female AGDs in the control group. Good or acceptable data quality (i.e. low CoV, sufficient measuring precision and a clear separation between male and female AGDs) was observed in 63% of the datasets. Limited data quality was seen in 12%, while 25% demonstrated insufficient quality. The 20 test facilities performing the studies demonstrated variable proficiency, with approximately one-third of the facilities excelling, one-third showing mixed results, and one-third underperforming. In the evaluation of the areola retention datasets, it was considered a potential limitation if no areolae were seen in any of the investigated males within a study. Likewise, a potential limitation was identified if an unexpectedly high number of areolae was seen in the control males. Surprisingly, these issues were very common and were identified in 83% of the datasets, which emphasises the importance of improving guidance on the assessment of this endpoint. Based on this review, updated guidance for assessing both endpoints is presently being prepared at the Organisation for Economic Co-operation and Development (OECD). Addressing the identified challenges will be crucial for enhancing the utility of the EOGRTS and consequently contributing to more informed decision-making on the potential hazards and risks associated with exposure to endocrine disrupting chemicals.