Decreased testosterone synthesis leading to increased nipple retention (NR) in male (rodent) offspring

Emilie Elmelund; National Food Institute, Technical University of Denmark, Lyngby, DK-2800, Denmark

Marie Holmer; National Food Institute, Technical University of Denmark, Lyngby, DK-2800, Denmark

Johanna Zilliacus; Institute of Environmental Medicine, Karolinska Institutet, SE-171 77 Stockholm, Sweden

Anna Beronius; Institute of Environmental Medicine, Karolinska Institutet, SE-171 77 Stockholm, Sweden

Sofie Christiansen; National Food Institute, Technical University of Denmark, Lyngby, DK-2800, Denmark

Eleni Bampari; National Food Institute, Technical University of Denmark, Lyngby, DK-2800, Denmark

Terje Svingen; National Food Institute, Technical University of Denmark, Lyngby, DK-2800, Denmark

This AOP links decreased intratesticular testosterone levels during fetal development with nipple/areola retention (NR) in male rodent offspring. NR, measured around 2 weeks postpartum, is a marker for disrupted masculinization of male offspring, with data primarily from laboratory mice and rats.

Testosterone is one of the two main steroid sex hormones essential for male reproductive development. Testosterone is primarily, but not exclusively, produced in the testes and then secreted into the circulation. In peripheral reproductive tissues, testosterone is either converted to dihydrotestosterone (DHT) or directly activates the androgen receptor (AR). AR is a nuclear receptor involved in the transcriptional regulation of various target genes during development and adulthood across species. AR signaling is necessary for normal masculinization of the developing fetus, and AR action in male rodents signals the nipple anlagen to regress, leaving males with no nipples.

This AOP delineates the evidence that decreasing testicular testosterone production lowers circulating testosterone levels and consequently AR activation, thereby causing retention of nipples in male rodents. In this AOP, the first KE is not considered an MIE, as testicular testosterone production can be obstructed by various mechanisms (Miller & Auchus, 2011). Moreover, the AOP does not discriminate whether the reduction in AR activation is due to a direct lack of testosterone binding AR or due to decreased conversion of testosterone to DHT, as there is not sufficient information on this distinction.  Downstream of a reduction in AR activation, the molecular mechanisms of NR are unclear, highlighting a knowledge gap in this AOP and potential for further development.

The confidence in KER-3486 (‘Decrease, circulating testosterone levels’ leads to ‘Increase, nipple retention’) is moderate due to the limited empirical evidence available. The confidence in each of the remaining KERs comprising the AOP is judged as high, with both high biological plausibility and high confidence in empirical evidence. The mechanistic link between KE-286 (‘altered, transcription of genes by AR’) and AO-1786 (‘increase, nipple retention’) is not established, but given the high confidence in the KERs, the overall confidence in the AOP is judged as high.

The AOP supports the regulatory application of NR as a measure of endocrine disruption relevant for human health and the use of NR as an indicator of anti-androgenicity in environmentally relevant species. Even though NR cannot be directly translated to a human endpoint, the AOP is considered human relevant since NR is a clear readout of reduced androgen action and fetal masculinization during development and is considered an ‘adverse outcome’ in OECD test guidelines (TG 443, TG 421, TG 422). The AOP also holds utility for informing on anti-androgenicity more generally, as this modality is highly relevant across mammalian species.

This AOP is a part of an AOP network for reduced AR activation leading to increased NR in male offspring. The other AOPs in this network are AOP-344 (‘Androgen receptor antagonism leading to increased nipple retention (NR) in male (rodent) offspring’) and AOP 576 (‘5α-reductase inhibition leading to increased nipple retention (NR) in male (rodent) offspring’). The purpose of the AOP network is to organize the well-established evidence for anti-androgenic mechanisms-of-action leading to increased NR. It can be used in the identification and assessment of endocrine disruptors and to inform predictive toxicology, identification of knowledge gaps for investigation and method development.

This work received funding from the European Food and Safety Authority (EFSA) under Grant agreement no. GP/EFSA/PREV/2022/01.

The OECD AOP Developer’s Handbook v2.7was followed alongside pragmatic approaches (Svingen et al., 2021).

The adverse outcome, AO 1786: Increase nipple retention, was originally developed in (Pedersen et al., 2022).

Some upstream KEs and KERs were developed in Draskau et al., 2024 as part of an anti-androgenic network, using mainly key review publications, since it was considered canonical knowledge. This included KE 1690: Decrease, circulating testosterone levels; KE 1614: Decrease, AR activation and KE 286: Altered transcription of genes by the AR as well as the connecting KERs, KER 2131: Decrease, circulating testosterone levels leads to decrease, AR activation and KER 2124: Decrease, AR activation leads to altered transcription of genes by AR (Draskau et al., 2024). 

KE 2298 (Decrease, intratesticular testosterone levels) and KER 3448, connecting this to KE 1690 (Decrease, intratesticular testosterone levels leads to decrease, circulation testosterone levels) were added as part of an AOP network, ultimately leading to the AO decreased anogenital distance (Svingen et al., 2025). This was done to discriminate between the large difference in testosterone levels between the testes and in circulation (Coviello et al., 2004). As for other upstream KEs and KERs, this was considered canonical knowledge, and the KE and KER were developed using mainly key review publications (Draskau et al., 2024).

The non-adjacent KERs 3487, 3486, and 3348 linking reduced decreased intratesticular testosterone, circulating testosterone, and AR activation with increased NR, respectively, were developed using a systematic weight-of-evidence approach, following the methodology outlined in (Holmer et al., 2024). Publications were retrieved by literature searches in PubMed and Web of Science and extensive screening using pre-defined inclusion and exclusion criteria. Evaluation of methodological reliability of in vivo animal studies was performed using the Science in Risk Assessment and Policy (SciRAP) online tool. For KERs 3487 and 3486 regarding testosterone levels, publications were included if there was a decrease in fetal testosterone levels and NR was assessed in male offspring. For KER 3348, there are currently no in vivo methods to measure AR activation in mammals, and instead, six chemicals with known anti-androgenic mechanisms-of-action were chosen for the empirical evidence for this KER.

The rationale for the inclusion of KEs and KERs in the upstream anti-androgenic network is detailed in (Draskau et al., 2024). The link between the upstream network, more specifically KE-286 (‘altered, transcription of genes by AR’), and AO-1786 (‘increase, nipple retention’) likely contains a tissue-specific KE that has not been developed, as sufficient evidence is not yet available. Thus, for now, the most evidence for the link between the upstream anti-androgenic network and increased nipple retention is captured by KERs 3348, 3486 and 3487.

The AOP supports the regulatory application of NR as a measure of endocrine disruption relevant for human health and the use of NR as an indicator of anti-androgenicity in environmentally relevant species. NR is a mandatory endpoint in multiple OECD test guidelines, including TG 443 (extended one-generation reproductive toxicity study) and TGs 421/422 (reproductive toxicity screening studies) (OECD 2025a; OECD 2025b; OECD 2025c). NR can contribute to establishing a No Observed Adverse Effect Level (NOAEL), as outlined in OECD guidance documents No. 43 and 151 (OECD 2008; OECD 2013). The ability to derive a NOAEL for increased NR in male rodent offspring, which can serve as a point of departure for determining human safety thresholds, underscores the regulatory significance of this AOP. The AOP also holds utility for informing on anti-androgenicity more generally, as this modality is highly relevant across mammalian species (Schwartz et al., 2021).

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