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AOP: 344
Title
Androgen receptor (AR) antagonism leading to nipple retention (NR) in male (mammalian) offspring
Short name
Graphical Representation
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Point of Contact
Contributors
- Terje Svingen
- Emilie Elmelund
Coaches
- Judy Choi
OECD Information Table
| OECD Project # | OECD Status | Reviewer's Reports | Journal-format Article | OECD iLibrary Published Version |
|---|---|---|---|---|
| 1.108 | Under Development |
This AOP was last modified on September 18, 2025 03:31
Revision dates for related pages
| Page | Revision Date/Time |
|---|---|
| Decrease, androgen receptor activation | February 04, 2026 16:01 |
| Altered, Transcription of genes by the androgen receptor | April 05, 2024 09:28 |
| Antagonism, Androgen receptor | April 05, 2024 08:04 |
| Nipple retention (NR), increased | January 11, 2023 05:53 |
| Antagonism, Androgen receptor leads to Decrease, AR activation | March 18, 2025 11:58 |
| Antagonism, Androgen receptor leads to nipple retention, increased | September 17, 2025 10:57 |
| Decrease, AR activation leads to Altered, Transcription of genes by the AR | April 05, 2024 08:50 |
| Decrease, AR activation leads to nipple retention, increased | September 18, 2025 03:29 |
| Flutamide | August 14, 2025 05:22 |
| Vinclozolin | May 14, 2020 11:28 |
| Procymidone | May 18, 2020 12:55 |
Abstract
This AOP links androgen receptor (AR) antagonism during fetal life with nipple/areola retention (NR) in male rodent offspring. NR, measured around 2 weeks postpartum I laboratory mice and rats, is a marker for feminization of male offspring.
The AR is a nuclear receptor involved in the transcriptional regulation of various target genes during development and adulthood across species. Its main ligands are testosterone and dihydrotestosterone (DHT). Under normal physiological conditions, testosterone, produced mainly by the testes, is converted by 5α-reductase to DHT locally in tissues; in turn DHT binds AR and activates downstream target genes. 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.
The key events in this pathway are fetal antagonism of the AR in target cells of the nipple anlagen, which leads to inactivation of the AR and failure to suppress development of the nipples, causing retention of nipples, visible postnatally in male offspring. In this instance, the local levels of testosterone or DHT may be normal but prevented from binding to the AR. Downstream of a reduction in AR activation, the molecular mechanisms of nipple retention are unclear, highlighting a knowledge gap in this AOP and potential for further development.
The confidence in each of the 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 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 and vertebrates more broadly due to the conserved nature of the AR and its implication in sexual differentiation across species.
AOP Development Strategy
Context
This AOP is a part of an AOP network for reduced androgen receptor activation leading to retention of nipples/areolas in male offspring. The other AOPs in this network are AOP-575 (‘Decreased intratesticular testosterone leading to increased nipple retention (NR) in male (mouse and rat) offspring’) and AOP-576 (‘5α-reductase inhibition leading to increased nipple retention (NR) in male (mouse and rat) 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 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.
Strategy
The OECD AOP Developer’s Handbook was followed alongside pragmatic approaches (Svingen et al., 2021).
Part of this AOP (MIE-26: Antagonism, androgen receptor; AO 1786: Increase nipple retention, and the non-adjacent KER-2133 linking these) was originally developed based on a literature review conducted in a transparent, semi-systematic manner in peer-reviewed databases using pre-defined inclusion criteria (Pedersen et al., 2022).
MIE 26 was updated in Draskau et al., 2024 as part of an upstream anti-androgenic network developed using mainly key review publications since it was considered canonical knowledge. The anti-androgenic network included upstream KEs (MIE-26: Antagonism, androgen receptor; KE-1614: Decrease, AR activation; KE-286: Altered transcription of genes by the AR) and connecting KERs (KER-2130: Antagonism, AR leads to decrease, AR activation and KER-2124: Decrease, AR activation leads to altered transcription of genes by AR) (Draskau et al., 2024).
The non-adjacent KER-3348, linking reduced AR activation with increased nipple retention, was 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. As there are currently no in vivo methods to measure AR activation in mammals, six chemical substances 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 (‘increased nipple retention’) likely contains a tissue- and life stage-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 NR is captured by KER-3348.
Summary of the AOP
Events:
Molecular Initiating Events (MIE)
Key Events (KE)
Adverse Outcomes (AO)
| Type | Event ID | Title | Short name |
|---|
| MIE | 26 | Antagonism, Androgen receptor | Antagonism, Androgen receptor |
| KE | 1614 | Decrease, androgen receptor activation | Decrease, AR activation |
| KE | 286 | Altered, Transcription of genes by the androgen receptor | Altered, Transcription of genes by the AR |
| AO | 1786 | Nipple retention (NR), increased | nipple retention, increased |
Relationships Between Two Key Events (Including MIEs and AOs)
| Title | Adjacency | Evidence | Quantitative Understanding |
|---|
| Antagonism, Androgen receptor leads to Decrease, AR activation | adjacent | High | |
| Decrease, AR activation leads to Altered, Transcription of genes by the AR | adjacent | High |
| Antagonism, Androgen receptor leads to nipple retention, increased | non-adjacent | High | |
| Decrease, AR activation leads to nipple retention, increased | non-adjacent | High |
Network View
Prototypical Stressors
Life Stage Applicability
| Life stage | Evidence |
|---|---|
| Foetal | High |
Taxonomic Applicability
Sex Applicability
| Sex | Evidence |
|---|---|
| Male | High |
Overall Assessment of the AOP
Domain of Applicability
The upstream part of the AOP has a broad applicability domain, but the downstream KERs-2133 (Antagonism, AR, leads to increased nipple retention) and KER-3348 (Decrease, AR activation, leads to increased nipple retention) are considered only directly applicable to male rodents (evidence primarily from laboratory rats and mice) during fetal life, restricting the taxonomic applicability of the AOP. Although NR is a feature having been investigated in laboratory rats and mice, it is biologically plausible that the AOP is applicable to other rodent species. The process of retention of nipples by disruption of androgen programming happens in the fetal life stage, but the AO is detected postnatally. In the males of mice and rats, the nipple anlagen are programmed during fetal development by androgens to regress, leading to no visible nipples in males postnatally, while females exhibit nipples. This AOP only contains empirical evidence for the applicability to male rats, but the AOP is considered equally applicable to male mice, as these also normally exhibit nipple regression stimulated by androgens. Moreover, the AOP is indirectly relevant for other taxa, including humans, as nipple retention in male rodents indicates a reduction in fetal masculinization. Nipple retention is therefore included as a mandatory endpoint in multiple OECD Test Guideline studies for developmental and reproductive toxicity and is considered applicable as an adverse outcome to set NOAELs and LOAELs of substances in human health risk assessments.
Essentiality of the Key Events
|
Evidence |
Uncertainties, inconsistencies and contradictory evidence |
|
|
MIE-26 Antagonism, AR receptor HIGH: This MIE is usually measured in vitro, whereas the downstream events in the AOP are, in most cases measured in vivo. Canonical knowledge of normal male reproductive development provides strong support for essentiality, along with AR knockout models.
|
Biological plausibility provides strong support for the essentiality of this event, as androgens, acting through AR, are the primary drivers of regression of nipple anlagen in male rat and mice embryos (Imperato-McGinley et al., 1986; Kratochwil, 1977; Kratochwil & Schwartz, 1976). Indirect evidence of the impact of AR antagonism (MIE-26) in vitro on AR activity in vitro: • Several chemical substances, including flutamide and vinclozolin, are known AR antagonists and have been shown to decrease AR activity in vitro (Pedersen et al., 2022; Sonneveld et al., 2004). Indirect evidence of the impact of AR antagonism (MIE-26) in vivo on increased nipple retention (AO-1786): • Rat in vivo exposure to vinclozolin, procymidone and flutamide, which are known AR antagonists, leads to increased nipple retention in offspring (see KER-3348). Direct evidence of the impact of AR antagonism (MIE-26) in vivo on increased nipple retention (AO-1786): • Male Tfm mutant mice, which are insensitive to androgens and believed to be so due to a nonfunctional androgen receptor, present with retained nipples (Kratochwil & Schwartz, 1976) |
|
|
KE-1614 Decreased, AR activation HIGH: There is experimental evidence from mutant mice insensitive to androgens showing that the AR is essential for nipple retention in male offspring. There is also evidence from exposure studies in animals that substances antagonizing AR induce nipple retention in male pups. |
Biological plausibility provides strong support for the essentiality of this event, as AR activation is critical for normal regression of nipple anlagen in male embryos. Indirect evidence of the impact of decreased AR activation (KE-1614) on altered gene transcription by AR (KE-286): • Exposure to known anti-androgenic chemicals induces a changed gene expression pattern, e.g. in neonatal pig ovaries (Knapczyk-Stwora et al., 2019). Direct evidence of the impact of decreased AR activation (KE-1614) on altered gene transcription by AR (KE-286): • Male AR KO mice have altered gene expression patterns in a broad range of organs (refer to KER-2124). Indirect evidence of the impact of decreased AR activation (KE-1614) on increased nipple retention (AO-1786): • Rat in vivo exposure to vinclozolin, procymidone and flutamide, which are known AR antagonists, leads to increased nipple retention in offspring (see KER-3348). Direct evidence of the impact of decreased AR activation (KE-1614) on increased nipple retention (AO-1786): • Male Tfm mutant mice, which are insensitive to androgens and believed to be so due to a nonfunctional androgen receptor, present with retained nipples (Kratochwil & Schwartz, 1976) |
|
|
KE-286 Altered, trans. of genes by AR LOW: Strongest support for essentiality comes from biological plausibility. However, exact transcriptional effects and causality remain to be fully characterized. |
Biological plausibility provides support for the essentiality of this event. AR is a nuclear receptor and transcription factor regulating transcription of genes, and androgens, acting through AR, are essential for normal regression of nipple anlagen in male fetuses. |
There are currently no AR-responsive genes proven to be causally involved in nipple retention, and it is known that AR can also signal through non-genomic actions (Leung & Sadar, 2017). |
|
Direct evidence |
Indirect evidence |
Contradictory evidence |
Overall essentiality assessment |
|
|
MIE-26 |
*** |
** |
|
High |
|
KE-1614 |
*** |
*** |
|
High |
|
KE-286 |
|
|
|
Low (biological plausibility) |
*Low level of evidence (some support for essentiality), ** Intermediate level of evidence (evidence for impact on one or more downstream KEs), ***High level of evidence (evidence for impact on AO).
Evidence Assessment
The confidence in each of the 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.
|
KER |
Biological Plausibility |
Empirical Evidence |
Rationale |
|
KER-2130 Antagonism, AR leads to decrease, AR activation |
High |
High (canonical) |
It is well established that antagonism of the AR leads to decreased AR activity. Direct evidence for this KER is not possible since KE-1614 can currently not be measured and is considered an in vivo effect. Indirect evidence using proxy read-outs of AR activation, either in vitro or in vivo, strongly supports the relationship (Draskau et al., 2024) |
|
KER-2124 Decrease, AR activation leads to altered, transcription of genes by AR |
High |
High (canonical) |
It is well established that the AR regulates gene transcription. In vivo animal studies and human genomic profiling show tissue-specific changes to gene expression upon disruption of AR. |
|
KER-2133 Antagonism, AR leads to increased nipple retention |
High |
High |
It is well established that androgens drive the regression of nipple anlagen in male rat and mouse fetuses through interaction with the AR receptor. The biological plausibility is high, and so is the empirical evidence, which includes numerous rat studies showing increased nipple retention in male offspring after exposure to well-known anti-androgens. |
|
KER-3348 Decrease, AR activation leads to increased nipple retention. |
High |
High |
It is well established that activation of AR drives the regression of nipple anlagen in males. The empirical evidence includes numerous in vivo toxicity studies showing that decreased AR activation leads to increased NR in male offspring, with few inconsistencies. The empirical evidence combined with theoretical considerations provides some support for dose, temporal, and incidence concordance for the KER, although this evidence is weak and indirect. |
Known Modulating Factors
| Modulating Factor (MF) | Influence or Outcome | KER(s) involved |
|---|---|---|
| Genotype | Extended CAG repeat length in AR is associated with reduced AR activity (Chamberlain et al., 1994; Tut et al., 1997). This MF could initiate the AOP at lower stressor doses. | KER-2124, KER-3348 |
| Rat strain | Long-Evans Hooded rat is less sensitive to NR than the Sprague-Dawley rat (Wolf et al., 1999; You et al., 1998). Thus, the effects on AO at certain stressor doses may vary between strains. | KER-3348 |
Quantitative Understanding
The quantitative understanding of the AOP is limited. A key difficulty lies in the challenge of extrapolating from in vitro to in vivo events since these cannot be captured within the same experimental framework. Specifically, MIE-26 is evaluated in vitro, while both the AO (NR) and KE-1614 are in vivo endpoints. KE-1614 pertains to AR activation in vivo - currently lacking viable methods for direct measurement.
The difficulties with in vitro-to-in vivo potency extrapolation from studies were exemplified by a comparison of the effects of pyrifluquinazon and bisphenol C in vitro and in utero. In vitro, bisphenol C antagonized the androgen receptor with a much higher potency than pyrifluquinazon, but in vivo the potencies were reversed with pyrifluquinazon exposure leading to NR at lower exposure levels than bisphenol C (Gray et al., 2019).
Considerations for Potential Applications of the AOP (optional)
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 mammals and other vertebrates in the environment. 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) and vertebrates more broadly due to the conserved nature of the AR and its implication in sexual differentiation across species (Ogino et al., 2023).
References
Chamberlain, N. L., Driver, E. D., & Miesfeld, R. L. (1994). The length and location of CAG trinucleotide repeats in the androgen receptor N-terminal domain affect transactivation function. Nucleic Acids Research, 22(15), 3181–3186. https://doi.org/10.1093/nar/22.15.3181
Draskau, M. K., Rosenmai, A. K., Bouftas, N., Johansson, H. K. L., Panagiotou, E. M., Holmer, M. L., Elmelund, E., Zilliacus, J., Beronius, A., Damdimopoulou, P., van Duursen, M., & Svingen, T. (2024). AOP Report: An Upstream Network for Reduced Androgen Signaling Leading to Altered Gene Expression of Androgen Receptor–Responsive Genes in Target Tissues. Environmental Toxicology and Chemistry, 43(11), 2329–2337. https://doi.org/10.1002/etc.5972
Gray, L. E., Furr, J. R., Conley, J. M., Lambright, C. S., Evans, N., Cardon, M. C., Wilson, V. S., Foster, P. M., & Hartig, P. C. (2019). A Conflicted Tale of Two Novel AR Antagonists in Vitro and in Vivo: Pyrifluquinazon Versus Bisphenol C. Toxicological Sciences, 168(2), 632–643. https://doi.org/10.1093/toxsci/kfz010
Holmer ML, Zilliacus J, Draskau MK, Hlisníková H, Beronius A, Svingen T. Methodology for developing data-rich Key Event Relationships for Adverse Outcome Pathways exemplified by linking decreased androgen receptor activity with decreased anogenital distance. Reprod Toxicol. 2024 Sep;128:108662. doi: 10.1016/j.reprotox.2024.108662 . Epub 2024 Jul 8. PMID: 38986849.
Imperato-McGinley J, Binienda Z, Gedney J, & Vaughan ED Jr. (1986). Nipple differentiation in fetal male rats treated with an inhibitor of the enzyme 5 alpha-reductase: definition of a selective role for dihydrotestosterone. Endocrinology, 118(1), 132–137. https://doi.org/10.1210/endo-118-1-132
Knapczyk-Stwora, K., Nynca, A., Ciereszko, R. E., Paukszto, L., Jastrzebski, J. P., Czaja, E., Witek, P., Koziorowski, M., & Slomczynska, M. (2019). Flutamide-induced alterations in transcriptional profiling of neonatal porcine ovaries. Journal of Animal Science and Biotechnology, 10(1), 35. https://doi.org/10.1186/s40104-019-0340-y
Kratochwil, K. (1977). Development and loss of androgen responsiveness in the embryonic rudiment of the mouse mammary gland. Developmental Biology, 61(2), 358–365. https://doi.org/10.1016/0012-1606(77)90305-0
Kratochwil, K., & Schwartz, P. (1976). Tissue interaction in androgen response of embryonic mammary rudiment of mouse: identification of target tissue for testosterone. Proceedings of the National Academy of Sciences, 73(11), 4041–4044. https://doi.org/10.1073/pnas.73.11.4041
Leung, J. K., & Sadar, M. D. (2017). Non-Genomic Actions of the Androgen Receptor in Prostate Cancer. Frontiers in Endocrinology, 8. https://doi.org/10.3389/fendo.2017.00002
OECD (2013), Guidance Document Supporting OECD Test Guideline 443 on the Extended One-Generational Reproductive Toxicity Test, OECD Series on Testing and Assessment, No. 151, OECD Publishing, Paris, ENV/JM/MONO(2013)10
OECD (2025a). Test No. 421: Reproduction/Developmental Toxicity Screening Test. https://doi.org/10.1787/9789264264380-en
OECD (2025b). Test No. 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test. https://doi.org/10.1787/9789264264403-en
OECD (2025c). Test No. 443: Extended One-Generation Reproductive Toxicity Study. https://doi.org/10.1787/9789264185371-en
Ogino, Y., Ansai, S., Watanabe, E. et al. Evolutionary differentiation of androgen receptor is responsible for sexual characteristic development in a teleost fish. Nat Commun 14, 1428 (2023). https://doi.org/10.1038/s41467-023-37026-6
Pedersen, E. B., Christiansen, S., & Svingen, T. (2022). AOP key event relationship report: Linking androgen receptor antagonism with nipple retention. In Current Research in Toxicology (Vol. 3). Elsevier B.V. https://doi.org/10.1016/j.crtox.2022.100085
Schwartz, C. L., Christiansen, S., Hass, U., Ramhøj, L., Axelstad, M., Löbl, N. M., & Svingen, T. (2021). On the Use and Interpretation of Areola/Nipple Retention as a Biomarker for Anti-androgenic Effects in Rat Toxicity Studies. In Frontiers in Toxicology (Vol. 3). Frontiers Media S.A. https://doi.org/10.3389/ftox.2021.730752
Sonneveld, E., Jansen, H. J., Riteco, J. A., Brouwer, A., & van der Burg, B. (2004). Development of Androgen- and Estrogen-Responsive Bioassays, Members of a Panel of Human Cell Line-Based Highly Selective Steroid-Responsive Bioassays. Toxicological Sciences, 83(1), 136–148. https://doi.org/10.1093/toxsci/kfi005
Svingen, T., Villeneuve, D. L., Knapen, D., Panagiotou, E. M., Draskau, M. K., Damdimopoulou, P., & O’Brien, J. M. (2021). A Pragmatic Approach to Adverse Outcome Pathway Development and Evaluation. Toxicological Sciences, 184(2), 183–190. https://doi.org/10.1093/toxsci/kfab113
Tut, T. G., Ghadessy, F. J., Trifiro, M. A., Pinsky, L., & Yong, E. L. (1997). Long Polyglutamine Tracts in the Androgen Receptor Are Associated with Reduced Trans -Activation, Impaired Sperm Production, and Male Infertility 1. The Journal of Clinical Endocrinology & Metabolism, 82(11), 3777–3782. https://doi.org/10.1210/jcem.82.11.4385
Wolf, C., Lambright, C., Mann, P., Price, M., Cooper, R. L., Ostby, J., & Earl Gray, L. J. (1999). Administration of potentially antiandrogenic pesticides (procymidone, linuron, iprodione, chlozolinate, p,p-DDE, and ketoconazole) and toxic substances (dibutyl-and diethylhexyl phthalate, PCB 169, and ethane dimethane sulphonate) during sexual differentiation produces diverse profiles of reproductive malformations in the male rat. Toxicology and Industrial Health, 15, 94–118. www.stockton-press.co.uk
You L, Casanova M, Archibeque-Engle S, Sar M, Fan LQ, & Heck HA. (1998). Impaired male sexual development in perinatal Sprague-Dawley and Long-Evans hooded rats exposed in utero and lactationally to p,p’-DDE. Toxicological Sciences : An Official Journal of the Society of Toxicology, 45(2), 162–173. https://doi.org/10.1093/toxsci/45.2.162