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Relationship: 2133
Title
Antagonism, Androgen receptor leads to nipple retention, increased
Upstream event
Downstream event
Key Event Relationship Overview
AOPs Referencing Relationship
| AOP Name | Adjacency | Weight of Evidence | Quantitative Understanding | Point of Contact | Author Status | OECD Status |
|---|---|---|---|---|---|---|
| Androgen receptor (AR) antagonism leading to nipple retention (NR) in male (mammalian) offspring | non-adjacent | High | Terje Svingen (send email) | Under development: Not open for comment. Do not cite | Under Development |
Taxonomic Applicability
Sex Applicability
| Sex | Evidence |
|---|---|
| Male | High |
Life Stage Applicability
| Term | Evidence |
|---|---|
| Foetal | High |
Key Event Relationship Description
This KER links antagonism of the androgen receptor (AR) during fetal development to increased nipple/areola retention (NR) in male rodent offspring.
The KER is not directly applicable to humans, as both males and females have two nipples, and there is no known effect of androgens on their development (Schwartz et al., 2021). However, NR is a clear readout of reduced androgen action, fetal masculinization and sexual differentiation during development, which is relevant to humans, mammals (Schwartz et al., 2021), and vertebrates more broadly (Ogino et al., 2023). It is included as a mandatory endpoint in several rodent OECD Test Guidelines (OECD, 2025a, 2025b, 2025c) and, in OECD GD 151, is considered an adverse outcome applicable to the setting of Points of Departure for use in human health risk assessment (OECD, 2013). NR can also be used as an indicator of anti-androgenicity in mammals and vertebrates in the environment due to the conserved nature of the AR and its implication in sexual differentiation across species (Ogino et al., 2023).
Evidence Collection Strategy
A semi-systematic literature search was conducted during March 2022 in the peer-reviewed databases PubMed and Web of Science, using the search terms “(Nipple) AND (retain* OR retention) AND (androgen)” as well as “(Androgen receptor OR AR) AND (active*) AND (nipple OR areolae) AND (retain* OR retention)”. These searches resulted in 138 papers in total. Upon removal of duplicates, papers were screened according to title, abstract and ultimately full text based on pre-defined inclusion criteria. In vivo studies were included if (i) the study was carried out in mice or rats, (ii) NR in males was investigated as an endpoint, (iii) AR antagonism was the suspected mechanism of action and (iv) anti-androgenic effects of single substance exposures (i.e., not studies on chemical mixtures) were investigated. In vitro studies were included if they contained mechanistic information on AR inhibition by chemical stressors (Pedersen et al., 2022).
Evidence Supporting this KER
Biological Plausibility
The biological plausibility for this KER is judged to be high based on the following:
- Sexual differentiation happens in fetal life. The testes are developed and start to produce testosterone that is converted in other tissues by the enzyme 5-alpha-reductase to the more potent androgen dihydrotestosterone (DHT). Both hormones bind and activate the AR, which in turn drives the masculinization of the male fetus (Schwartz et al., 2021; Welsh et al., 2014).
- Fetal masculinization depends on the activation of androgen signalling during a critical time window, the masculinization programming window (MPW), from gestational day (GD) 16-20 in rats, 14.5-16.5 in mice and presumably gestation weeks (GWs) 8-14 in humans (Amato et al., 2022; Welsh et al., 2008).
- The fetal masculinization process involves a range of tissues and organs, including the nipple anlagen in rodents (primarily investigated in laboratory rats and mice). In humans, both sexes have two nipples. In contrast, rodents such as laboratory rats and mice are sexually dimorphic, with females having 12 (rats) and 10 (mice) nipples, and males generally having none (Mayer et al., 2008; Schwartz et al., 2021). In both male and female mouse embryos, stem cells differentiate into a mammary gland, with nipple anlagen being visible by embryonic day 11.5 (Mayer et al., 2008). In male embryos, the presence of androgen leads the nipple anlagen to regress a few days later (Kratochwil, 1977; Kratochwil & Schwartz, 1976) . The androgen responsiveness in the nipple anlagen is rather short, in mice, starting late embryonic day 13, with loss of responsiveness on embryonic day 15 (Imperato-McGinley et al., 1986; Kratochwil, 1977) and thus roughly following the timing of the MPW.
- Nipple formation is inhibited in female mouse and rat fetuses exposed to androgens during gestation (Goldman et al., 1976; Greene et al., 1941; Imperato-McGinley et al., 1986).
- Male Tfm-mutant mice, which are insensitive to androgens and believed to be so due to a nonfunctional AR, present with retained nipples (Kratochwil & Schwartz, 1976).
- Multiple mechanisms of action may potentially lead to NR in male mouse and rat offspring. DHT is the main androgen responsible for regression of the nipples through interaction with AR in the nipple anlagen (Imperato-McGinley et al., 1986). Inhibition of testosterone synthesis or the conversion of testosterone to DHT, increased metabolism of androgens, or direct interference with AR activation may thus all lead to nipple/areola retention (Imperato-McGinley et al., 1986; Schwartz et al., 2021).
Empirical Evidence
The empirical support from studies in animals for this KER is judged as high overall.
The relationship is supported by numerous studies demonstrating induction of NR in male offspring after in utero exposure to substances known to antagonise AR in vitro (fenitrothion, flutamide, linuron, mancozeb, pp’-DDE, prochloraz, procymidone, pyrifluquinazon, tebuconazole and vinclozolin) (Pedersen et al., 2022; Appendix 1, 12h4h48cc2_KER_2133_Appendix_1.pdf). The empirical evidence includes only studies conducted in rats, although it is believed that the link also exists in mice, and other rodent species (Pedersen et al., 2022). Some inconsistences in the empirical evidence for 3 of the substances were observed. These could, however, be explained by differences in dose levels, and the level of confidence for prenatal exposure to these substances resulting in increased NR in male offspring is judged to be strong (Appendix 1,12h4h48cc2_KER_2133_Appendix_1.pdf).
Dose concordance
Dose concordance is challenging to assess for this KER since the upstream event is measured in vitro and the downstream event is measured in vivo.
Temporal concordance
Temporal concordance can only be considered from a theoretical perspective since the downstream event, ‘increased NR’, is a result of the disruption of the normal regression of nipple anlagen in male rodents induced during a short window of gestational development (in mice of approximately 2 days), but usually measured at PND12-14 in rats. Earlier than this, the areolae are not yet visible through the skin and later than this, the animals grow fur and need to be shaved for proper examination. This is supported by several of the studies in the empirical evidence, where the test substance was administered during a short period during gestation and nipple retention was observed postnatally.
Based on current knowledge, it is understood that the upstream event – antagonism of the AR – takes place minutes to hours after exposure to an anti-androgenic substance.
Uncertainties and Inconsistencies
A major challenge with using NR as a biomarker is the subjectivity of the measurement. In juvenile rat pups, nipples are only present as areolae, i.e., dark shadows with or without a nipple bud. This means that the experience of the personnel assessing the presence and number of areolae/nipples can influence the results. Furthermore, the results are likely prone to larger variation if several assessors are used to record NR within the same study. To minimise these sources of uncertainty, assessors must be trained to recognise areolae and not look for fully developed nipples. Moreover, the number of assessors should be limited to one or two, and they should always be blinded to exposure groups.
Another factor that may affect NR results is the age of the rat pups at the time of assessment. OECD guidelines have standardised the time for measuring the occurrence of NR to be optimal at PD 12 or 13, when they are visible in female littermates (OECD, 2013).
Known modulating factors
A well-established modulating factor for androgen action is genetic variations in the AR, which decrease the function of the receptor. For example, longer CAG repeat lengths have been associated with decreased AR activation (Chamberlain et al., 1994; Tut et al., 1997).
Rat strain is another important modulating factor, with studies showing that the Long-Evans Hooded rat is less sensitive to nipple/areola retention than the Sprague-Dawley rat (Wolf et al., 1999; You et al., 1998).
Quantitative Understanding of the Linkage
The quantitative understanding of the relationship between decreased AR activity and NR is challenged by the fact that the potency of AR antagonism in vitro is not proportional to the magnitude of NR observed in vivo (Gray et al., 2019).
Response-response Relationship
The difficulties in extrapolating potency from in vitro to in vivo 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).
Time-scale
AR activation operates on a time-scale of minutes. The AR is a ligand-activated nuclear receptor and transcription factor. Upon ligand binding a conformational change and subsequent dimerisation of the AR take place within 3-6 minutes (Schaufele et al., 2005). Nuclear translocation (Nightingale et al., 2003) and promoter interactions occur within 15 minutes of ligand binding, and RNA polymerase II and coactivator recruitment are then proposed to occur transiently with cycles of approximately 90 minutes (Kang et al., 2002).
For the downstream event, the time-scale for observing a measurable effect on nipple/areola retention is closer to days and weeks, depending on species. For instance, in mice, the nipple anlage are responsive to androgen action at embryonic day 13-15, while a sexual dimorphism of the nipples/areolas can first be observed after birth (Imperato-McGinley et al., 1986) .
Known Feedforward/Feedback loops influencing this KER
Not relevant for this KER.
Domain of Applicability
Taxonomic
The KER is considered directly applicable to rats and mice, in which males normally have no nipples due to high levels of androgens during development, leading to regression of nipple anlagen. The empirical evidence supports the relevance to rats, whereas the relevance in mice is assumed based on knowledge about developmental biology in this species. Applicability may extend to most rodents.
While NR is not directly translatable to humans, it serves as a clear indicator of diminished androgen activity causing disrupted fetal masculinisation and sexual differentiation during development - an effect considered relevant to mammals, humans (Schwartz et al., 2021) and vertebrates more broadly (Ogino et al., 2023). NR is included as a mandatory endpoint in several rodent OECD Test Guidelines (OECD 2025a; OECD 2025b, OECD 2025c) and in OECD GD 151 considered an adverse outcome applicable to the setting of Points of Departure for use in human health risk assessment (OECD, 2013). NR can also be used as an indicator of anti-androgenicity in mammals and vertebrates in the environment due to the conserved nature of the AR and its implication in sexual differentiation across species (Ogino et al., 2023).
Life stage
Programming of nipple/areola regression in males occurs during a short window of sensitivity to androgens in the nipple anlagen during fetal life. This takes place in rats around GD16-20 (Imperato-McGinley et al., 1986), which is, therefore, the relevant window of exposure. The relevant timing for the investigation of NR is PND12-14 in male rat offspring when the nipples are visible in the female littermates. At this time in development, the nipples/areolas are visible through the skin without excessive fur that may interfere with the investigation (Schwartz et al., 2021). It should be mentioned that though the occurrence of nipples/areolas in male offspring is believed to be relatively stable throughout life, it may be responsive to postnatal changes. Permanent nipple/areola retention is observed in some but not all in utero exposure studies with antiandrogens inducing nipple/areola retention at PND 12-14. Most of the differences between studies seem explainable by the window of exposure, dose levels and methods for investigation used, but the responsiveness of nipple/areola retention to postnatal changes remains to be fully explored (Schwartz et al., 2021).
Sex
Data presented in this KER support that disruption of androgen action during fetal life can lead to increased nipple/areola retention in male rat offspring. Since female mice and rat offspring, in general, have 10 (mice) or 12 (rats) nipples at the relevant time of investigation, increased nipple/areola retention at that time point is not a relevant endpoint for females.
References
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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
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