Anti-androgenicity can only be evaluated using a weight of evidence approach

- Treated or untreated male pups with lower body weights at day 21 p.p. show delayed PPS.
- Delayed PPS are caused by anti-androgenic compounds or occur secondary to impaired growth.
- An individual body weight analysis helps distinguish between direct and secondary PPS delay.
- The validity of the endpoint PPS as a sole measure to assess anti-androgenicity is limited.
- The assessment of anti-androgenicity needs a thorough weight-of-evidence evaluation.

Preputial separation (PPS) is a commonly used external marker for the onset of male puberty in experimental animal studies. While treatment-related delays in PPS may be indicative of specific anti-androgenic activity, impaired general growth also alters the onset of puberty. To differentiate between specific and non-specific effects on the age at PPS - and thereby evaluate the validity of the endpoint PPS-two-generation toxicity studies of 23 substances were evaluated. The 23 substances were assessed regarding anti-androgenicity using all available data and external assessments in a weight-of-evidence evaluation (WoE).Correlation of individual pup body weight with age at PPS revealed that delays in pubertal development coincided with reduced pup body weight. After comparison with the WoE assessment, we concluded that inclusion of body weight analysis into the PPS evaluation of each study was able to correctly identify three compounds which specifically induced delayed PPS and 16 which only showed unspecific changes. A further two compounds which might be categorized as anti-androgens based on delayed PPS, were correctly regrouped using our refined methodology. Based on this analysis and in comparison to the WoE evaluation, it was found, that caution should be exercised when using the endpoint PPS in hazard assessment.