Enumeration of Microscopic Ovarian Structures in Rodents: Best Practices and Toxicological Significance.

BACKGROUND: Reproductive toxicity studies for industrial and agricultural chemicals require enumeration of ovarian follicles (OF). In recent years, the enumeration of corpora lutea (CL) in histological sections has also been requested. Both endpoints are now considered routine quantitative measures to assess female reproductive toxicity of (agro)chemicals and have also been used to support pharmaceutical drug development, when warranted. Procedural guidance for both endpoints from FDA, EPA, OECD, and other regional health authorities is generally limited. While most laboratories follow the generically stated procedures for including a statistically appropriate number of animals and ovarian section selection, methodologies vary significantly across the industry making cross-study data comparisons extremely challenging. In March 2023, the European Chemicals Agency (ECHA) published its report on its EOGRTS Review Project and noted the lack of information on laboratory proficiency and high variability as the main deficiencies in OF and CL count data which resulted in industry-wide discussions regarding the lack of published guidance and the need for updates to regulatory guidelines. In June 2025, OECD TG 443 was updated to include additional information regarding qualitative ovarian evaluation; however, no new information regarding laboratory proficiency or reduction of variability was included. METHODS: We discuss the biological relevance of these endpoints, the need for consistency across the industry, and technical and scientific considerations for quantitative ovarian assessments and present historical control data for these endpoints. RECOMMENDATIONS AND CONCLUSIONS: Based on the authors' collective experience and successful regulatory history, we put forth best practice recommendations for data collection and analysis with the goal to provide a frame of reference for industry laboratories.