AOP-Wiki

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<div id="title">
<h2>AOP ID and Title:</h2>
<div class="title">AOP 575: Decreased testosterone synthesis leading to increased nipple retention (NR) in male (rodent) offspring</div>
Short Title: Decreased testosterone synthesis leading to nipple retention
</div>
<h2>Graphical Representation</h2>
<img src="https://aopwiki.org/system/dragonfly/production/2025/09/18/58xmk0fmhl_AOP_575_Graphic.jpg" height="500" width="700" alt=""/>
<div id="authors">
<h2>Authors</h2>
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
</div>
<div id="status">
<h2>Status</h2>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Author status</th>
<th scope="col">OECD status</th>
<th scope="col">OECD project</th>
<th scope="col">SAAOP status</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Under development: Not open for comment. Do not cite</td>
<td></td>
<td></td>
<td></td>
</tr>
</tbody>
</table>
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</div>
<div id="abstract">
<h2>Abstract</h2>
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.
</div>
<div id="background">
<h3>Background</h3>
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.
</div>
<div id="aop_summary">
<h2>Summary of the AOP</h2>
<h3>Events</h3>
<h3>Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)</h3>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sequence</th>
<th scope="col">Type</th>
<th scope="col">Event ID</th>
<th scope="col">Title</th>
<th scope="col">Short name</th>
</tr>
</thead>
<tbody>
<tr><td></td><td></td><td></td><td></td><td></td></tr>
<tr>
<td></td>
<td>KE</td>
<td>2298</td>
<td><a href="/events/2298">Decrease, intratesticular testosterone levels</a></td>
<td>Decrease, intratesticular testosterone</td>
</tr>
<tr>
<td></td>
<td>KE</td>
<td>1690</td>
<td><a href="/events/1690">Decrease, circulating testosterone levels </a></td>
<td>Decrease, circulating testosterone levels</td>
</tr>
<tr>
<td></td>
<td>KE</td>
<td>1614</td>
<td><a href="/events/1614">Decrease, androgen receptor activation</a></td>
<td>Decrease, AR activation</td>
</tr>
<tr>
<td></td>
<td>KE</td>
<td>286</td>
<td><a href="/events/286">Altered, Transcription of genes by the androgen receptor</a></td>
<td>Altered, Transcription of genes by the AR</td>
</tr>
<tr><td></td><td></td><td></td><td></td><td></td></tr>
<tr>
<td></td>
<td>AO</td>
<td>1786</td>
<td><a href="/events/1786">Nipple retention (NR), increased</a></td>
<td>nipple retention, increased</td>
</tr>
</tbody>
</table>
</div>
<h3>Key Event Relationships</h3>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Upstream Event</th>
<th scope="col">Relationship Type</th>
<th scope="col">Downstream Event</th>
<th scope="col">Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/relationships/3448">Decrease, intratesticular testosterone levels</a></td>
<td>adjacent</td>
<td>Decrease, circulating testosterone levels </td>
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/relationships/2131">Decrease, circulating testosterone levels </a></td>
<td>adjacent</td>
<td>Decrease, androgen receptor activation</td>
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/relationships/2124">Decrease, androgen receptor activation</a></td>
<td>adjacent</td>
<td>Altered, Transcription of genes by the androgen receptor</td>
<td>High</td>
<td></td>
</tr>
<tr>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/relationships/3487">Decrease, intratesticular testosterone levels</a></td>
<td>non-adjacent</td>
<td>Nipple retention (NR), increased</td>
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/relationships/3486">Decrease, circulating testosterone levels </a></td>
<td>non-adjacent</td>
<td>Nipple retention (NR), increased</td>
<td>Moderate</td>
<td></td>
</tr>
<tr>
<td><a href="/relationships/3348">Decrease, androgen receptor activation</a></td>
<td>non-adjacent</td>
<td>Nipple retention (NR), increased</td>
<td>High</td>
<td></td>
</tr>
</tbody>
</table>
</div>
<h3>Stressors</h3>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Name</th>
<th scope="col">Evidence</th>
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<tbody class="tbody-striped">
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<td>Dibutyl phthalate</td>
<td></td>
</tr>
<tr>
<td>Di(2-ethylhexyl) phthalate</td>
<td></td>
</tr>
</tbody>
</table>
</div>
</div>
<div id="overall_assessment">
<h2>Overall Assessment of the AOP</h2>
<h3>Domain of Applicability</h3>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
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<tbody class="tbody-striped">
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<td>Foetal</td>
<td>High</td>
</tr>
</tbody>
</table>
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Taxonomic Applicability
<div class="table-responsive">
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<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
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</thead>
<tbody class="tbody-striped">
<tr>
<td>rat</td>
<td>Rattus norvegicus</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10116" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>mouse</td>
<td>Mus musculus</td>
<td>Low</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10090" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
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</thead>
<tbody class="tbody-striped">
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<td>Male</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
The upstream part of the AOP has a broad applicability domain, but the downstream KERs 3487 (‘Decrease, intratesticular testosterone levels leads to increase, nipple retention’), 3486 (‘Decrease, circulating testosterone levels leads to increase, nipple retention’), and 3348 (‘Decrease AR activation leads to increase, nipple retention’) are considered only directly applicable to male rodents (current evidence stems primarily from laboratory rats and mice) during fetal life, restricting the taxonomic applicability of the AOP. Although NR has primarily been investigated in 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 these species, the nipple anlagen are programmed during fetal development by androgens to regress, leading to no visible nipples in males postnatally, while female rats and mice 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 relevant for other taxa, including humans, as NR in male rodents indicates a reduction in fetal masculinization. NR 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.
<h3>Essentiality of the Key Events</h3>
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Event
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<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:200px">
Evidence
</td>
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Uncertainties and inconsistencies
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<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:200px">
KE-2298
Decreased, intratesticular testosterone (ITT) levels
MODERATE: Testis is the primary organ in males for testosterone synthesis and is required for serum testosterone. Studies with exposure to phthalates show reduced ITT levels and increased nipple retention.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:200px">
Biological plausibility provides strong support for the essentiality of this event, as the testes are the primary testosterone producing organs in male mammals and testosterone is a ligand of the AR and a main driver for normal regression of nipple anlagen in male offspring (Goldman et al., 1976).
Indirect evidence of impact of decreased ITT (KE-2298) on decreased circulating T (KE-1690)
• Castrated males have significantly reduced serum T. Although at different life stage, it is highly likely same relationship exists in fetal males, with loss of testosterone from testis resulting in loss of circulating testosterone.
Indirect evidence of impact of decreased ITT (KE-2298) on increased nipple retention (AO-1786):
• Numerous rat studies evidence a relationship between reduced intratesticular testosterone levels caused by exposure to phthalates and increased nipple retention in male offspring (see empirical evidence table in KER-3487).
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:200px">
</td>
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<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:200px">
KE-1690
Decreased, circulating testosterone (CT) levels
MODERATE: CT is substrate for DHT production, also locally, and numerous studies have shown strong relationships between reduced CT and increased nipple retention.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:200px">
Biological plausibility provides strong support for the essentiality of this event. Testosterone is an AR ligand and a main driver for regression of nipple anlagen in male offspring (Goldman et al., 1976), as well as a substrate for local production of DHT (Imperato-McGinley J et al., 1986).
Indirect evidence of the impact of decreased CT (KE-1690) on AR activity in vitro:
• Increasing concentrations of testosterone lead to increasing AR activation in vitro (U. S. EPA, 2018) (see also KER-2131).
Indirect evidence of impact of decreased CT (KE-1690) on increased nipple retention (AO-1786):
• Exposure to the phthalates DEHP and DBP during prenatal development in rats results in reduced fetal testosterone levels and increased nipple retention in male offspring. Literature review on the relationship has judged the link to be strongly evidenced (See empirical evidence in KER-3348).
Indirect evidence of the impact of decreased CT (KE-1690) on increased nipple retention (AO-1786):
• Nipple formation is inhibited in female rat fetuses exposed to testosterone during gestation (Goldman et al., 1976).
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:200px">
Inconsistencies in indirect evidence of impact on the AO:
• Some inconsistencies were observed in the empirical evidence regarding increased nipple retention in male pups after in utero exposure to DEHP. However, all inconsistencies could be explained by differences in exposure doses and statistical power (See empirical evidence in KER-3348)
</td>
</tr>
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<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:200px">
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.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:200px">
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 pattern in a broad range of organs (refer to KER-2124).
Indirect evidence of 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 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)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:200px">
</td>
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<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:200px">
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.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:200px">
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.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:200px">
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).
</td>
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<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:85px">
Event
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:104px">
Direct evidence
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:123px">
Indirect evidence
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:142px">
Contradictory evidence
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:148px">
Overall essentiality assessment
</td>
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<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:85px">
KE-2298
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:104px">
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:123px">
***
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<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:142px">
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:148px">
Moderate
</td>
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<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:85px">
KE-1690
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:104px">
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:123px">
***
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:142px">
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:148px">
Moderate
</td>
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<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:85px">
KE-1614
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:104px">
***
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:123px">
***
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:142px">
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:148px">
High
</td>
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<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:85px">
KE-286
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:104px">
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:123px">
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:142px">
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:148px">
Low (biological plausibility)
</td>
</tr>
</tbody>
</table>
*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).
<h3>Weight of Evidence Summary</h3>
Confidence in KER-3486 is considered 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.
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KER
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Biological Plausibility
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Empirical Evidence
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Rationale
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KER-3448
Decrease, intratesticular testosterone levels leads to a decrease, circulating testosterone levels
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:85px">
High
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:80px">
High (canonical)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:268px">
It is well established that testes are the primary testosterone-producing organs in male mammals.
In vivo studies have shown that exposure to substances that lower intratesticular testosterone also lowers circulating testosterone levels (Svingen et al., 2025).
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:169px">
KER-2131
Decrease, circulating testosterone levels leads to decrease, AR activation
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:85px">
High
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:80px">
High (canonical)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:268px">
It is well established that testosterone activates the AR.
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)
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:169px">
KER-2124
Decrease, AR activation leads to altered, transcription of genes by AR
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:85px">
High
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:80px">
High (canonical)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:268px">
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 (Draskau et al., 2024).
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:169px">
KER-3487
Decrease, intratesticular testosterone leads to an increase, nipple retention
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:85px">
High
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:80px">
High
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:268px">
It is well established that testicular testosterone is one of the primary androgens responsible for the regression of nipple anlagen in male rodent fetuses
In vivo animal studies support that reductions in fetal testicular testosterone can cause NR in male offspring. Temporal concordance is generally supported, while dose concordance is more weakly suggested.
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:169px">
KER-3486
Decrease, circulating testosterone levels leads to increase, nipple retention
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:85px">
High
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:80px">
Moderate
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:268px">
It is well established that testosterone is one of the primary androgens responsible for the regression of nipple anlagen in male rodent fetuses
Two in vivo rat toxicity studies support the relationship and temporal concordance of the KER. Dose concordance is not informed.
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:169px">
KER-3348
Decrease, AR activation leads to increase, nipple retention
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:85px">
High
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:80px">
High
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:268px">
It is well established that activation of AR 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. Empirical evidence combined with theoretical considerations provide some support for dose, temporal, and incidence concordance for the KER, although this evidence is weak and indirect.
</td>
</tr>
</tbody>
</table>
<h3>Quantitative Consideration</h3>
The quantitative understanding of this AOP is judged as low.
A model for phthalate-induced malformations has been developed which aims to predict the degree of NR related to a phthalate’s reduction in ex vivo testosterone production. The model predicted that a 40% reduction in testosterone levels would induce NR in male rats, with increasing number of nipples as testosterone levels decrease (Gray et al., 2024).
</div>
<div id="considerations_for_potential_applicaitons">
<h2>Considerations for Potential Applications of the AOP (optional)</h2>
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).
</div>
<div id="references">
<h2>References</h2>
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
Coviello, A. D., Bremner, W. J., Matsumoto, A. M., Herbst, K. L., Amory, J. K., Anawalt, B. D., Yan, X., Brown, T. R., Wright, W. W., Zirkin, B. R., & Jarow, J. P. (2004). Intratesticular Testosterone Concentrations Comparable With Serum Levels Are Not Sufficient to Maintain Normal Sperm Production in Men Receiving a Hormonal Contraceptive Regimen. Journal of Andrology, 25(6), 931–938. https://doi.org/10.1002/j.1939-4640.2004.tb03164.x
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. J., Lambright, C. S., Evans, N., Ford, J., & Conley, J. M. (2024). Using targeted fetal rat testis genomic and endocrine alterations to predict the effects of a phthalate mixture on the male reproductive tract. Current Research in Toxicology, 7, 100180. https://doi.org/10.1016/j.crtox.2024.100180
Goldman AS, Shapiro B, & Neumann F. (1976). Role of testosterone and its metabolites in the differentiation of the mammary gland in rats. Endocrinology, 99(6), 1490–1495. https://doi.org/10.1210/endo-99-6-1490
Holmer, M. L., Zilliacus, J., Draskau, M. K., Hlisníková, H., Beronius, A., & Svingen, T. (2024). Methodology for developing data-rich Key Event Relationships for Adverse Outcome Pathways exemplified by linking decreased androgen receptor activity with decreased anogenital distance. Reproductive Toxicology, 128, 108662. https://doi.org/10.1016/j.reprotox.2024.108662
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., & 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. <a href="https://doi.org/10.3389/fendo.2017.00002" style="color:#0563c1; text-decoration:underline">https://doi.org/10.3389/fendo.2017.00002</a>
Miller, W. L., & Auchus, R. J. (2011). The Molecular Biology, Biochemistry, and Physiology of Human Steroidogenesis and Its Disorders. Endocrine Reviews, 32(1), 81–151. <a href="https://doi.org/10.1210/er.2010-0013" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1210/er.2010-0013</a>
OECD (2008), Guidance Document on Mammalian Reproductive Toxicity Testing and Assessment, OECD Series on Testing and Assessment, No. 43, OECD Publishing, Paris, https://doi.org/10.1787/d2631d22-en.
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
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
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. <a href="https://doi.org/10.1093/toxsci/kfab113" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1093/toxsci/kfab113</a>
Svingen T, Elmelund E, Holmer ML, Bindel AO, Holbech H, Draskau MK. AOP report: Adverse Outcome Pathway Network for Developmental Androgen Signalling-Inhibition Leading to Short Anogenital Distance in Male Offspring. Environ Toxicol Chem. 2025 Sep 1:vgaf221. doi: 10.1093/etojnl/vgaf221. Epub ahead of print. PMID: 40888748.
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
U. S. EPA. (2018, October). ToxCast & Tox21 AR agonism of testosterone. https://www.epa.gov/comptox-tools/exploring-toxcast-data
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
</div>
<div id="appendicies">
<h2>Appendix 1</h2>
<h3>List of Key Events in the AOP</h3>
<h4><a href="/events/2298">Event: 2298: Decrease, intratesticular testosterone levels</a></h4>
<h5>Short Name: Decrease, intratesticular testosterone</h5>
<h4>Key Event Component</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Process</th>
<th scope="col">Object</th>
<th scope="col">Action</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>testosterone biosynthetic process</td>
<td>testosterone</td>
<td>decreased</td>
</tr>
</tbody>
</table>
</div>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/307">Aop:307 - Decreased testosterone synthesis leading to short anogenital distance (AGD) in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/570">Aop:570 - Decreased testosterone synthesis leading to hypospadias in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/575">Aop:575 - Decreased testosterone synthesis leading to increased nipple retention (NR) in male (rodent) offspring</a></td>
<td>KeyEvent</td>
</tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Organ</td></tr>
</tbody>
</table>
</div>
<h4>Organ term</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Organ term</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>testis</td></tr>
</tbody>
</table>
</div>
<h4>Domain of Applicability</h4>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Vertebrates</td>
<td>Vertebrates</td>
<td>Moderate</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>mammals</td>
<td>mammals</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>During development and at adulthood</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Male</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
This key event (KE) is applicable to all male vertebrates with testis that produce testosterone.
<h4>Key Event Description</h4>
This KE refers to decreased testosterone biosynthesis in the testis (male); i.e. intratesticular testosterone levels. It is therefore considered distinct from KEs describing circulating testosterone levels, or levels in any other tissue or organ of vertebrate animals. It is also distinct from indirect cell-based assays measuring effects on testosterone synthesis, including in vitro Leydig cells.
In males, the testis is the primary site of testosterone biosynthesis via the steroidogenesis pathway – an enzymatic pathway converting cholesterol into all the downstream steroid hormones (Miller and Auchus 2010). In mammals, the Leydig cells are considered the primary site of steroidogenesis in the testis. Although generally correct, there is evidence to suggest the involvement of Sertoli cells during fetal stages in e.g. mouse and human testis, but with Leydig cells being sufficient in adult life (O’Donnell et al 2022).
Testicular testosterone synthesis is primarily regulated by the hypothalamic-pituitary-gonadal (HPG) axis, with Gonadotropin-releasing hormone (GnRH) from the hypothalamus controlling the secretion of Luteinizing hormone (LH) from the pituitary that ultimately binds to the LH receptors on Leydig cells to stimulate steroidogenesis. Notably, the timing of HPG axis activation during development varies between species. In humans, human chorionic gonadotropin (hCG) act similarly to LH and appear to be critical in stimulating testosterone synthesis in the fetal testis (Huhtaniemi 2025), whereas in the mouse testosterone synthesis in the fetal testis appears to be independent of pituitary gonadotropins even though LH is detectable during late gestation O’Shaughnessy et al 1998).  Irrespective of testosterone being stimulated by gonadotropins or occurring de novo, however, it is essential for masculinization of the developing fetus, initiation of puberty, and maintain reproductive, and other, functions in adulthood.
Notably, intratesticular testosterone concentration is significantly higher than serum testosterone levels, typically ranging from 30- to 200-fold greater in mammals, including humans (Turner et al 1984; McLachlan et al 2002; Coviello et al 2004).
<h4>How it is Measured or Detected</h4>
Testosterone levels can be quantified in testis tissue (ex vivo, in vivo). Methods include traditional immunoassays such as ELISA and RIA, advanced techniques like LC-MS/MS, and liquid scintillation spectrometry following radiolabeling (Shiraishi et al., 2008).
<h4>References</h4>
Coviello, A.D., Bremner, W.J., Matsumoto, A.M., Herbst, K.L., Amory, J.K., Anawalt, B.D., Yan, X., Brown, T.R., Wright, W.W., Zirkin, B.R. and Jarow, J.P. (2004). Intratesticular Testosterone Concentrations Comparable With Serum Levels Are Not Sufficient to Maintain Normal Sperm Production in Men Receiving a Hormonal Contraceptive Regimen. J Androl, 25:931-938. <a href="https://doi.org/10.1002/j.1939-4640.2004.tb03164.x" style="color:blue; text-decoration:underline">https://doi.org/10.1002/j.1939-4640.2004.tb03164.x</a>
Huhtaniemi, I.T. (2025). Luteinizing hormone receptor knockout mouse: What has it taught us? Andrology, In Press. <a href="https://doi.org/10.1111/andr.70000" style="color:blue; text-decoration:underline">https://doi.org/10.1111/andr.70000</a>
McLachlan, R.I., O’Donnell, L., Stanton, P.G., Balourdos, G., Frydenberg, M., de Kretser, D.M. and Robertson, D.M. (2002). Effects of testosterone plus medroxyprogesterone acetate on semen quality, reproductive hormones, and germ cell populations in normal young men. J Clin Endocriol Metab, 87:546-556. <a href="https://doi.org/10.1210/jcem.87.2.8231" style="color:blue; text-decoration:underline">https://doi.org/10.1210/jcem.87.2.8231</a>
Miller, W.L. and Auchus, R.J. (2010). The Molecular Biology, Biochemistry, and Physiology of Human Steroidogenesis and Its Disorders. Endocr Rev, 32(1):81-151. <a href="https://doi.org/10.1210/er.2010-0013" style="color:blue; text-decoration:underline">https://doi.org/10.1210/er.2010-0013</a>
O’Donnell, L., Whiley, P.A.F., and Loveland, K.L. (2022). Activin A and Sertoli Cells: Key to Fetal Testis Steroidogenesis. Front Endocrinol, 13:898876. <a href="https://doi.org/10.3389/fendo.2022.898876" style="color:blue; text-decoration:underline">https://doi.org/10.3389/fendo.2022.898876</a>
O’Shaughnessy, P.J., Baker, P., Sohnius, U., Haavisto, A.M., Charlton, H.M. and Huhtaniemi, I. (1998). Fetal development of Leydig cell activity in the mouse is independent of pituitary gonadotroph function. Endocrinology, 139:1141-1146. <a href="https://doi.org/10.1210/endo.139.3.5788" style="color:blue; text-decoration:underline">https://doi.org/10.1210/endo.139.3.5788</a>
Shiraishi, S., Lee, P. W. N., Leung, A., Goh, V. H. H., Swerdloff, R. S., & Wang, C. (2008). Simultaneous Measurement of Serum Testosterone and Dihydrotestosterone by Liquid Chromatography–Tandem Mass Spectrometry. Clinical Chemistry, 54(11), 1855–1863. <a href="https://doi.org/10.1373/clinchem.2008.103846" style="color:blue; text-decoration:underline">https://doi.org/10.1373/clinchem.2008.103846</a>
Turner, T.T., Jones, C.E., Howards, S.S., Ewing, L.L., Zegeye, B. and Gunsalus, G.L. (1984). On the androgen microenvironment of maturing spermatozoa. Endocrinology, 115:1925-1932. <a href="https://doi.org/10.1210/endo-115-5-1925" style="color:blue; text-decoration:underline">https://doi.org/10.1210/endo-115-5-1925</a>
<h4><a href="/events/1690">Event: 1690: Decrease, circulating testosterone levels </a></h4>
<h5>Short Name: Decrease, circulating testosterone levels</h5>
<h4>Key Event Component</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Process</th>
<th scope="col">Object</th>
<th scope="col">Action</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>hormone biosynthetic process</td>
<td>testosterone</td>
<td>decreased</td>
</tr>
<tr>
<td>testosterone biosynthetic process</td>
<td>testosterone</td>
<td>decreased</td>
</tr>
</tbody>
</table>
</div>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/307">Aop:307 - Decreased testosterone synthesis leading to short anogenital distance (AGD) in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/526">Aop:526 - Decreased, Chicken Ovalbumin Upstream Promoter Transcription Factor II (COUP-TFII) leads to Impaired, Spermatogenesis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/124">Aop:124 - HMG-CoA reductase inhibition leading to decreased fertility</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/18">Aop:18 - PPARα activation in utero leading to impaired fertility in males</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/51">Aop:51 - PPARα activation leading to impaired fertility in adult male rodents </a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/496">Aop:496 - Androgen receptor agonism leading to reproduction dysfunction （in zebrafish）</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/64">Aop:64 - Glucocorticoid Receptor (GR) Mediated Adult Leydig Cell Dysfunction Leading to Decreased Male Fertility</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/120">Aop:120 - Inhibition of 5α-reductase leading to Leydig cell tumors (in rat)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/288">Aop:288 - Inhibition of 17α-hydrolase/C 10,20-lyase (Cyp17A1) activity leads to birth reproductive defects (cryptorchidism) in male (mammals)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/570">Aop:570 - Decreased testosterone synthesis leading to hypospadias in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/575">Aop:575 - Decreased testosterone synthesis leading to increased nipple retention (NR) in male (rodent) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
~~<td><a href="/aops/595">Aop:595 - Nanoplastic effect</a></td>~~
<td><a href="/aops/595">Aop:595 - Emerging OPFRS reproductive outcome pathway</a></td>
<td>KeyEvent</td>
</tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Tissue</td></tr>
</tbody>
</table>
</div>
<h4>Organ term</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Organ term</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>blood</td></tr>
</tbody>
</table>
</div>
<h4>Domain of Applicability</h4>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>mammals</td>
<td>mammals</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>During development and at adulthood</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Male</td>
<td>High</td>
</tr>
<tr>
<td>Female</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
This key event (KE) is applicable to all mammals, as the synthesis and role of testosterone are evolutionarily conserved (Vitousek et al., 2018). Both sexes produce and require testosterone, which plays critical roles throughout life, from development to adulthood; albeit there are differences in lief stages when testosterone exert specific effects and function (Luetjens & Weinbauer, 2012; Naamneh Elzenaty et al., 2022). Accordingly, this KE applies to both males and females across all life stages, but life stage should be considered when embedding in AOPs.
Notably, the key enzymes involved in testosterone production first appeared in the common ancestor of amphioxus and vertebrates (Baker, 2011). This suggests that the KE has a broader domain of applicability, encompassing non-mammalian vertebrates. AOP developers are encouraged to integrate additional knowledge to expand its relevance beyond mammals to other vertebrates.
<h4>Key Event Description</h4>
Testosterone is an endogenous steroid hormone that acts by binding the androgen receptor (AR) in androgen-responsive tissues (Murashima et al., 2015). As with all steroid hormones, testosterone is produced through steroidogenesis, an enzymatic pathway converting cholesterol into all the downstream steroid hormones. Briefly, androstenedione or androstenediol is converted to testosterone by the enzymes 17β-hydroxysteroid dehydrogenase (HSD) or 3β-HSD, respectively. Testosterone can then be converted to the more potent androgen, dihydrotestosterone (DHT) by 5α-reductase, or aromatized by CYP19A1 (Aromatase) into estrogens. Testosterone secreted in blood circulation can be found free or bound to SHBG or albumin (Trost & Mulhall, 2016).
Testosterone is produced mainly by the testes (in males), ovaries (in females) and to a lesser degree in the adrenal glands. The output of testosterone from different tissues varies with life stages. During fetal development testosterone is crucial for the differentiation of male reproductive tissues and the overall male phenotype. In adulthood, testosterone synthesis is controlled by the Hypothalamus-Pituitary-Gonadal (HPG) axis. GnRH is released from the hypothalamus inducing LH pulses secreted by the anterior pituitary. This LH surge leads to increased testosterone production, both in testes (males) and ovaries (females). If testosterone reaches low levels, this axis is once again stimulated to increase testosterone synthesis. This feedback loop is essential for maintenance of appropriate testosterone levels (Chandrashekar & Bartke, 1998; Ellis et al., 1983; Rey, 2021).
By disrupting e.g. steroidogenesis or the HPG-axis, testosterone synthesis or homeostasis may be disrupted and can lead to less testosterone being synthesized and released into circulation.
General role in biology
Androgens are essential hormones responsible for the development of the male phenotype during fetal life and for sexual maturation at puberty. In adulthood, androgens remain essential for the maintenance of male reproductive function and behavior but is also essential for female fertility. Apart from their effects on reproduction, androgens affect a wide variety of non-reproductive tissues such as skin, bone, muscle, and brain (Heemers et al 2006). Androgens, principally testosterone and DHT, exert most of their effects by interacting with the AR (Murashima et al 2015).
<h4>How it is Measured or Detected</h4>
Testosterone levels can be quantified in serum (in vivo), cell culture medium (in vitro), or tissue (ex vivo, in vitro). Methods include traditional immunoassays such as ELISA and RIA, advanced techniques like LC-MS/MS, and liquid scintillation spectrometry following radiolabeling (Shiraishi et al., 2008).
The H295R Steroidogenesis Assay (OECD TG 456) is (currently; anno 2025) primarily used to measure estradiol and testosterone production. This validated OECD test guideline uses adrenal H295R cells, with hormone levels measured in the cell culture medium (OECD, 2011). H295R adrenocortical carcinoma cells express the key enzymes and hormones of the steroidogenic pathway, enabling broad analysis of steroidogenesis disruption by quantifying hormones in the medium using LC-MS/MS. Initially designed to assess testosterone and estradiol levels, the assay now extends to additional steroid hormones, such as progesterone and pregnenolone. The U.S. EPA’s ToxCast program further advanced this method, enabling high-throughput measurement of 11 steroidogenesis-related hormones (Haggard et al., 2018). While the H295R assay indirectly reflects disruptions in overall steroidogenesis (e.g., changes in testosterone levels), it does not provide mechanistic insights.
Testosterone can be measured by immunoassays and by isotope-dilution gas chromatography-mass spectrometry in serum (Taieb et al., 2003; Paduch et al., 2014). Testosterone levels may also be measured by: Fish Lifecycle Toxicity Test (FLCTT) (US EPA OPPTS 850.1500), Male pubertal assay (PP Male Assay) (US EPA OPPTS 890.1500), OECD TG 441: Hershberger Bioassay in Rats (H Assay).
<h4>References</h4>
Baker, M.E. (2011). Insights from the structure of estrogen receptor into the evolution of estrogens: implications for endocrine disruption. Biochem Pharmacol, 82(1), 1-8. <a href="https://doi.org/10.1016/j.bcp.2011.03.008" style="color:blue; text-decoration:underline">https://doi.org/10.1016/j.bcp.2011.03.008</a>
Chandrashekar, V., & Bartke, A. (1998). The Role of Growth Hormone in the Control of Gonadotropin Secretion in Adult Male Rats*. Endocrinology, 139(3), 1067–1074. <a href="https://doi.org/10.1210/endo.139.3.5816" style="color:blue; text-decoration:underline">https://doi.org/10.1210/endo.139.3.5816</a>
Ellis, G. B., Desjardins, C., & Fraser, H. M. (1983). Control of Pulsatile LH Release in Male Rats. Neuroendocrinology, 37(3), 177–183. <a href="https://doi.org/10.1159/000123540" style="color:blue; text-decoration:underline">https://doi.org/10.1159/000123540</a>
Haggard, D. E., Karmaus, A. L., Martin, M. T., Judson, R. S., Setzer, R. W., & Paul Friedman, K. (2018). High-Throughput H295R Steroidogenesis Assay: Utility as an Alternative and a Statistical Approach to Characterize Effects on Steroidogenesis. Toxicological Sciences, 162(2), 509–534. <a href="https://doi.org/10.1093/toxsci/kfx274" style="color:blue; text-decoration:underline">https://doi.org/10.1093/toxsci/kfx274</a>
Heemers, H. V, Verhoeven, G., & Swinnen, J. V. (2006). Androgen activation of the sterol regulatory element-binding protein pathway: Current insights. Molecular Endocrinology (Baltimore, Md.), 20(10), 2265–77. doi:10.1210/me.2005-0479
Luetjens, C. M., & Weinbauer, G. F. (2012). Testosterone: biosynthesis, transport, metabolism and (non-genomic) actions. In Testosterone (pp. 15–32). Cambridge University Press. <a href="https://doi.org/10.1017/CBO9781139003353.003" style="color:blue; text-decoration:underline">https://doi.org/10.1017/CBO9781139003353.003</a>
Murashima, A., Kishigami, S., Thomson, A., & Yamada, G. (2015). Androgens and mammalian male reproductive tract development. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 1849(2), 163–170. <a href="https://doi.org/10.1016/j.bbagrm.2014.05.020" style="color:blue; text-decoration:underline">https://doi.org/10.1016/j.bbagrm.2014.05.020</a>
Naamneh Elzenaty, R., du Toit, T., & Flück, C. E. (2022). Basics of androgen synthesis and action. Best Practice & Research Clinical Endocrinology & Metabolism, 36(4), 101665. <a href="https://doi.org/10.1016/j.beem.2022.101665" style="color:blue; text-decoration:underline">https://doi.org/10.1016/j.beem.2022.101665</a>
Paduch, D. A., Brannigan, R. E., Fuchs, E. F., Kim, E. D., Marmar, J. L., & Sandlow, J. I. (2014). The laboratory diagnosis of testosterone deficiency. Urology, 83(5), 980–8. <a href="https://doi.org/10.1016/j.urology.2013.12.024" style="color:blue; text-decoration:underline">https://doi.org/10.1016/j.urology.2013.12.024</a>
Rey, R. A. (2021). The Role of Androgen Signaling in Male Sexual Development at Puberty. Endocrinology, 162(2). <a href="https://doi.org/10.1210/endocr/bqaa215" style="color:blue; text-decoration:underline">https://doi.org/10.1210/endocr/bqaa215</a>
Shiraishi, S., Lee, P. W. N., Leung, A., Goh, V. H. H., Swerdloff, R. S., & Wang, C. (2008). Simultaneous Measurement of Serum Testosterone and Dihydrotestosterone by Liquid Chromatography–Tandem Mass Spectrometry. Clinical Chemistry, 54(11), 1855–1863. <a href="https://doi.org/10.1373/clinchem.2008.103846" style="color:blue; text-decoration:underline">https://doi.org/10.1373/clinchem.2008.103846</a>
Taieb, J., Mathian, B., Millot, F., Patricot, M.-C., Mathieu, E., Queyrel, N., … Boudou, P. (2003). Testosterone measured by 10 immunoassays and by isotope-dilution gas chromatography-mass spectrometry in sera from 116 men, women, and children. Clinical Chemistry, 49(8), 1381–95.
Trost, L. W., & Mulhall, J. P. (2016). Challenges in Testosterone Measurement, Data Interpretation, and Methodological Appraisal of Interventional Trials. The Journal of Sexual Medicine, 13(7), 1029–1046. <a href="https://doi.org/10.1016/j.jsxm.2016.04.068" style="color:blue; text-decoration:underline">https://doi.org/10.1016/j.jsxm.2016.04.068</a>
Vitousek, M. N., Johnson, M. A., Donald, J. W., Francis, C. D., Fuxjager, M. J., Goymann, W., Hau, M., Husak, J. F., Kircher, B. K., Knapp, R., Martin, L. B., Miller, E. T., Schoenle, L. A., Uehling, J. J., & Williams, T. D. (2018). HormoneBase, a population-level database of steroid hormone levels across vertebrates. Scientific Data, 5(1), 180097. <a href="https://doi.org/10.1038/sdata.2018.97" style="color:blue; text-decoration:underline">https://doi.org/10.1038/sdata.2018.97</a>
<h4><a href="/events/1614">Event: 1614: Decrease, androgen receptor activation</a></h4>
<h5>Short Name: Decrease, AR activation</h5>
<h4>Key Event Component</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Process</th>
<th scope="col">Object</th>
<th scope="col">Action</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>androgen receptor activity</td>
<td>androgen receptor</td>
<td>decreased</td>
</tr>
</tbody>
</table>
</div>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/288">Aop:288 - Inhibition of 17α-hydrolase/C 10,20-lyase (Cyp17A1) activity leads to birth reproductive defects (cryptorchidism) in male (mammals)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/305">Aop:305 - 5α-reductase inhibition leading to short anogenital distance (AGD) in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/306">Aop:306 - Androgen receptor (AR) antagonism leading to short anogenital distance (AGD) in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/307">Aop:307 - Decreased testosterone synthesis leading to short anogenital distance (AGD) in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/344">Aop:344 - Androgen receptor (AR) antagonism leading to nipple retention (NR) in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/372">Aop:372 - Androgen receptor antagonism leading to testicular cancer </a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/477">Aop:477 - Androgen receptor (AR) antagonism leading to hypospadias in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/345">Aop:345 - Androgen receptor (AR) antagonism leading to decreased fertility in females</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/111">Aop:111 - Decrease in androgen receptor activity leading to Leydig cell tumors (in rat)</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/570">Aop:570 - Decreased testosterone synthesis leading to hypospadias in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/571">Aop:571 - 5α-reductase inhibition leading to hypospadias in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/575">Aop:575 - Decreased testosterone synthesis leading to increased nipple retention (NR) in male (rodent) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/576">Aop:576 - 5α-reductase inhibition leading to increased nipple retention (NR) in male (rodent) offspring</a></td>
<td>KeyEvent</td>
</tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Tissue</td></tr>
</tbody>
</table>
</div>
<h4>Domain of Applicability</h4>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>mammals</td>
<td>mammals</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>During development and at adulthood</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Mixed</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
This KE is considered broadly applicable across mammalian taxa as all mammals express the AR in numerous cells and tissues where it regulates gene transcription required for developmental processes and functions. It is, however, acknowledged that this KE most likely has a much broader domain of applicability extending to non-mammalian vertebrates. AOP developers are encouraged to add additional relevant knowledge to expand on the applicability to also include other vertebrates.
<h4>Key Event Description</h4>
This KE refers to decreased activation of the androgen receptor (AR) as occurring in complex biological systems such as tissues and organs in vivo. It is thus considered distinct from KEs describing either blocking of AR or decreased androgen synthesis.
The AR is a nuclear transcription factor with canonical AR activation regulated by the binding of the androgens such as testosterone or dihydrotestosterone (DHT). Thus, AR activity can be decreased by reduced levels of steroidal ligands (testosterone, DHT) or the presence of compounds interfering with ligand binding to the receptor (Davey & Grossmann, 2016; Gao et al., 2005).
In the inactive state, AR is sequestered in the cytoplasm of cells by molecular chaperones. In the classical (genomic) AR signaling pathway, AR activation causes dissociation of the chaperones, AR dimerization and translocation to the nucleus to modulate gene expression. AR binds to the androgen response element (ARE) (Davey & Grossmann, 2016; Gao et al., 2005). Notably, for transcriptional regulation the AR is closely associated with other co-factors that may differ between cells, tissues and life stages. In this way, the functional consequence of AR activation is cell- and tissue-specific. This dependency on co-factors such as the SRC proteins also means that stressors affecting recruitment of co-activators to AR can result in decreased AR activity (Heinlein & Chang, 2002).
In the inactive state, AR is sequestered in the cytoplasm of cells by molecular chaperones. In the classical (genomic) AR signaling pathway, AR activation causes dissociation of the chaperones, AR dimerization and translocation to the nucleus to modulate gene expression. AR binds to the androgen response element (ARE) (Davey & Grossmann, 2016; Gao et al., 2005). Notably, for transcriptional regulation the AR is closely associated with other co-factors that may differ between cells, tissues and life stages. In this way, the functional consequence of AR activation is cell- and tissue-specific. This dependency on co-factors such as the SRC proteins also means that stressors affecting recruitment of co-activators to AR can result in decreased AR activity (Heinlein & Chang, 2002), as shown for the pyrethroid cypermethrin (Wang et al., 2016).
Ligand-bound AR may also associate with cytoplasmic and membrane-bound proteins to initiate cytoplasmic signaling pathways with other functions than the nuclear pathway. Non-genomic AR signaling includes association with Src kinase to activate MAPK/ERK signaling and activation of the PI3K/Akt pathway. Decreased AR activity may therefore be a decrease in the genomic and/or non-genomic AR signaling pathways (Leung & Sadar, 2017).
<h4>How it is Measured or Detected</h4>
This KE specifically focuses on decreased in vivo activation, with most methods that can be used to measure AR activity carried out in vitro. They provide indirect information about the KE and are described in lower tier MIE/KEs (see for example MIE/KE-26 for AR antagonism, KE-1690 for decreased T levels and KE-1613 for decreased dihydrotestosterone levels). Assays may in the future be developed to measure AR activation in mammalian organisms.
<h4>References</h4>
Davey, R. A., & Grossmann, M. (2016). Androgen Receptor Structure, Function and Biology: From Bench to Bedside. The Clinical Biochemist. Reviews, 37(1), 3–15.
Gao, W., Bohl, C. E., & Dalton, J. T. (2005). Chemistry and structural biology of androgen receptor. Chemical Reviews, 105(9), 3352–3370. https://doi.org/10.1021/cr020456u
Heinlein, C. A., & Chang, C. (2002). Androgen Receptor (AR) Coregulators: An Overview. https://academic.oup.com/edrv/article/23/2/175/2424160
Leung, J. K., & Sadar, M. D. (2017). Non-Genomic Actions of the Androgen Receptor in Prostate Cancer. Frontiers in Endocrinology, 8. <a href="https://doi.org/10.3389/fendo.2017.00002" style="color:#0563c1; text-decoration:underline">https://doi.org/10.3389/fendo.2017.00002</a>
OECD (2022). Test No. 251: Rapid Androgen Disruption Activity Reporter (RADAR) assay. Paris: OECD Publishing doi:10.1787/da264d82-en.
Wang Q, Zhou JL, Wang H, Ju Q, Ding Z, Zhou XL, Ge X, Shi QM, Pan C, Zhang JP, Zhang MR, Yu HM, Xu LC. (2016). Inhibition effect of cypermethrin mediated by co-regulators SRC-1 and SMRT in interleukin-6-induced androgen receptor activation. Chemosphere. 158:24-9. doi: 10.1016/j.chemosphere.2016.05.053
<table>
<tbody>
<tr>
<td colspan="1" rowspan="1">
</td>
<td colspan="1" rowspan="1">
</td>
</tr>
</tbody>
</table>
<h4><a href="/events/286">Event: 286: Altered, Transcription of genes by the androgen receptor</a></h4>
<h5>Short Name: Altered, Transcription of genes by the AR</h5>
<h4>Key Event Component</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Process</th>
<th scope="col">Object</th>
<th scope="col">Action</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>regulation of gene expression</td>
<td>androgen receptor</td>
<td>decreased</td>
</tr>
</tbody>
</table>
</div>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/19">Aop:19 - Androgen receptor antagonism leading to adverse effects in the male foetus (mammals)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/307">Aop:307 - Decreased testosterone synthesis leading to short anogenital distance (AGD) in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/344">Aop:344 - Androgen receptor (AR) antagonism leading to nipple retention (NR) in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/345">Aop:345 - Androgen receptor (AR) antagonism leading to decreased fertility in females</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/305">Aop:305 - 5α-reductase inhibition leading to short anogenital distance (AGD) in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/495">Aop:495 - Androgen receptor activation leading to prostate cancer</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/306">Aop:306 - Androgen receptor (AR) antagonism leading to short anogenital distance (AGD) in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/496">Aop:496 - Androgen receptor agonism leading to reproduction dysfunction （in zebrafish）</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/372">Aop:372 - Androgen receptor antagonism leading to testicular cancer </a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/570">Aop:570 - Decreased testosterone synthesis leading to hypospadias in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/571">Aop:571 - 5α-reductase inhibition leading to hypospadias in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/575">Aop:575 - Decreased testosterone synthesis leading to increased nipple retention (NR) in male (rodent) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/576">Aop:576 - 5α-reductase inhibition leading to increased nipple retention (NR) in male (rodent) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/477">Aop:477 - Androgen receptor (AR) antagonism leading to hypospadias in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
</tbody>
</table>
</div>
<h4>Stressors</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Name</th></tr>
</thead>
<tbody>
<tr><td>Bicalutamide</td></tr>
<tr><td>Cyproterone acetate</td></tr>
<tr><td>Epoxiconazole</td></tr>
<tr><td>Flutamide</td></tr>
<tr><td>Flusilazole</td></tr>
<tr><td>Prochloraz</td></tr>
<tr><td>Propiconazole</td></tr>
<tr><td>Stressor:286 Tebuconazole</td></tr>
<tr><td>Triticonazole</td></tr>
<tr><td>Vinclozalin</td></tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Tissue</td></tr>
</tbody>
</table>
</div>
<h4>Domain of Applicability</h4>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>mammals</td>
<td>mammals</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>During development and at adulthood</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Mixed</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Both the DNA-binding and ligand-binding domains of the AR are highly evolutionary conserved, whereas the transactivation domain show more divergence, which may affect AR-mediated gene regulation across species (Davey and Grossmann 2016). Despite certain inter-species differences, AR function mediated through gene expression is highly conserved, with mutation studies from both humans and rodents showing strong correlation for AR-dependent development and function (Walters et al. 2010).
This KE is considered broadly applicable across mammalian taxa, sex and developmental stages, as all mammals express the AR in numerous cells and tissues where it regulates gene transcription required for developmental processes and function. It is, however, acknowledged that this KE most likely has a much broader domain of applicability extending to non-mammalian vertebrates. AOP developers are encouraged to add additional relevant knowledge to expand on the applicability to also include other vertebrates.
<h4>Key Event Description</h4>
This KE refers to transcription of genes by the androgen receptor (AR) as occurring in complex biological systems such as tissues and organs in vivo. Rather than measuring individual genes, this KE aims to capture patterns of effects at transcriptome level in specific target cells/tissues. In other words, it can be replaced by specific KEs for individual adverse outcomes as information becomes available, for example the transcriptional toxicity response in prostate tissue for AO: prostate cancer, perineum tissue for AO: reduced AGD, etc.  AR regulates many genes that differ between tissues and life stages and, importantly, different gene transcripts within individual cells can go in either direction since AR can act as both transcriptional activator and suppressor. Thus, the ‘directionality’ of the KE cannot be either reduced or increased, but instead describe an altered transcriptome.
The Androgen Receptor and its function
The AR belongs to the steroid hormone nuclear receptor family. It is a ligand-activated transcription factor with three domains: the N-terminal domain, the DNA-binding domain, and the ligand-binding domain with the latter being the most evolutionary conserved (Davey and Grossmann 2016). Androgens (such as dihydrotestosterone and testosterone) are AR ligands and act by binding to the AR in androgen-responsive tissues (Davey and Grossmann 2016). Human AR mutations and mouse knockout models have established a fundamental role for AR in masculinization and spermatogenesis (Maclean et al.; Walters et al. 2010; Rana et al. 2014). The AR is also expressed in many other tissues such as bone, muscles, ovaries and within the immune system (Rana et al. 2014).
Altered transcription of genes by the AR as a Key Event
Upon activation by ligand-binding, the AR translocates from the cytoplasm to the cell nucleus, dimerizes, binds to androgen response elements in the DNA to modulate gene transcription (Davey and Grossmann 2016). The transcriptional targets vary between cells and tissues, as well as with developmental stages and is also dependent on available co-regulators (Bevan and Parker 1999; Heemers and Tindall 2007). It should also be mentioned that the AR can work in other ‘non-canonial’ ways such as non-genomic signaling, and ligand-independent activation (Davey & Grossmann, 2016; Estrada et al, 2003; Jin et al, 2013).
A large number of known, and proposed, target genes of AR canonical signaling have been identified by analysis of gene expression following treatments with AR agonists (Bolton et al. 2007; Ngan et al. 2009, Jin et al. 2013).
<h4>How it is Measured or Detected</h4>
Altered transcription of genes by the AR can be measured by measuring the transcription level of known downstream target genes by RT-qPCR or other transcription analyses approaches, e.g. transcriptomics.
Since this KE aims to capture AR-mediated transcriptional patterns of effect, downstream bioinformatics analyses will typically be required to identify and compare effect footprints. Clusters of genes can be statistically associated with, for example, biological process terms or gene ontology terms relevant for AR-mediated signaling. Large transcriptomics data repositories can be used to compare transcriptional patterns between chemicals, tissues, and species (e.g. TOXsIgN (Darde et al, 2018a; Darde et al, 2018b), comparisons can be made to identified sets of AR ‘biomarker’ genes (e.g. as done in (Rooney et al, 2018)), and various methods can be used e.g. connectivity mapping (Keenan et al, 2019).
<h4>References</h4>
Bevan C, Parker M (1999) The role of coactivators in steroid hormone action. Exp. Cell Res. 253:349–356
Bolton EC, So AY, Chaivorapol C, et al (2007) Cell- and gene-specific regulation of primary target genes by the androgen receptor. Genes Dev 21:2005–2017. doi: 10.1101/gad.1564207
Darde, T. A., Gaudriault, P., Beranger, R., Lancien, C., Caillarec-Joly, A., Sallou, O., et al. (2018a). TOXsIgN: a cross-species repository for toxicogenomic signatures. Bioinformatics 34, 2116–2122. doi:10.1093/bioinformatics/bty040.
Darde, T. A., Chalmel, F., and Svingen, T. (2018b). Exploiting advances in transcriptomics to improve on human-relevant toxicology. Curr. Opin. Toxicol. 11–12, 43–50. doi:10.1016/j.cotox.2019.02.001.
Davey RA, Grossmann M (2016) Androgen Receptor Structure, Function and Biology: From Bench to Bedside. Clin Biochem Rev 37:3–15
Estrada M, Espinosa A, Müller M, Jaimovich E (2003) Testosterone Stimulates Intracellular Calcium Release and Mitogen-Activated Protein Kinases Via a G Protein-Coupled Receptor in Skeletal Muscle Cells. Endocrinology 144:3586–3597. doi: 10.1210/en.2002-0164
Heemers H V., Tindall DJ (2007) Androgen receptor (AR) coregulators: A diversity of functions converging on and regulating the AR transcriptional complex. Endocr. Rev. 28:778–808
Jin, Hong Jian, Jung Kim, and Jindan Yu. 2013. “Androgen Receptor Genomic Regulation.” Translational Andrology and Urology 2(3):158–77. doi: 10.3978/j.issn.2223-4683.2013.09.01
Keenan, A. B., Wojciechowicz, M. L., Wang, Z., Jagodnik, K. M., Jenkins, S. L., Lachmann, A., et al. (2019). Connectivity Mapping: Methods and Applications. Annu. Rev. Biomed. Data Sci. 2, 69–92. doi:10.1146/ANNUREV-BIODATASCI-072018-021211.
Maclean HE, Chu S, Warne GL, Zajact JD Related Individuals with Different Androgen Receptor Gene Deletions
MacLeod DJ, Sharpe RM, Welsh M, et al (2010) Androgen action in the masculinization programming window and development of male reproductive organs. In: International Journal of Andrology. Blackwell Publishing Ltd, pp 279–287
Ngan S, Stronach EA, Photiou A, et al (2009) Microarray coupled to quantitative RT&ndash;PCR analysis of androgen-regulated genes in human LNCaP prostate cancer cells. Oncogene 28:2051–2063. doi: 10.1038/onc.2009.68<a name="_Hlk148352925"></a>
Rana K, Davey RA, Zajac JD (2014) Human androgen deficiency: Insights gained from androgen receptor knockout mouse models. Asian J. Androl. 16:169–177
Rooney, J. P., Chorley, B., Kleinstreuer, N., and Corton, J. C. (2018). Identification of Androgen Receptor Modulators in a Prostate Cancer Cell Line Microarray Compendium. Toxicol. Sci. 166, 146–162. doi:10.1093/TOXSCI/KFY187.
Walters KA, Simanainen U, Handelsman DJ (2010) Molecular insights into androgen actions in male and female reproductive function from androgen receptor knockout models. Hum Reprod Update 16:543–558. doi: 10.1093/humupd/dmq003
<h3>List of Adverse Outcomes in this AOP</h3>
<h4><a href="/events/1786">Event: 1786: Nipple retention (NR), increased</a></h4>
<h5>Short Name: nipple retention, increased</h5>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/344">Aop:344 - Androgen receptor (AR) antagonism leading to nipple retention (NR) in male (mammalian) offspring</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/575">Aop:575 - Decreased testosterone synthesis leading to increased nipple retention (NR) in male (rodent) offspring</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/576">Aop:576 - 5α-reductase inhibition leading to increased nipple retention (NR) in male (rodent) offspring</a></td>
<td>AdverseOutcome</td>
</tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Individual</td></tr>
</tbody>
</table>
</div>
<h4>Domain of Applicability</h4>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>rats</td>
<td>Rattus norvegicus</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10116" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>mouse</td>
<td>Mus musculus</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10090" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Birth to < 1 month</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Male</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
The applicability domain of NR is limited to male laboratory strains of rats and mice from birth to juvenile age.
<h4>Key Event Description</h4>
In common laboratory strains of rats and mice, females typically have 6 (rats) or 5 (mice) pairs of nipples along the bilateral milk lines. In contrast, male rats and mice do not have nipples. This is unlike e.g., humans where both sexes have 2 nipples (Schwartz et al., 2021).
In laboratory rats, high levels of dihydrotestosterone (DHT) induce regression of the nipples in males (Imperato-McGinley & Gautier, 1986; Kratochwil, 1977; Kratochwil & Schwartz, 1976). Females, in the absence of this DHT surge, retain their nipples. This relationship has also been shown in numerous rat studies with perinatal exposure to anti-androgenic chemicals (Schwartz et al., 2021). Hence, if juvenile male rats and mice possess nipples, it is considered a sign of perturbed androgen action early in life.
This KE was first published by Pedersen et al (2022).
<h4>How it is Measured or Detected</h4>
Nipple retention (NR) is visually assessed, ideally on postnatal day (PND) 12/13 (OECD, 2018; Schwartz et al., 2021). However, PND 14 is also an accepted stage of examination (OECD, 2013). Depending on animal strain, the time when nipples become visible can vary, but the assessment of NR in males should be conducted when nipples are visible in their female littermates (OECD, 2013).
Nipples are detected as dark spots (or shadows) called areolae, which resemble precursors to a nipple rather than a fully developed nipple. The dark area may or may not display a nipple bud (Hass et al., 2007). Areolae typically emerge along the milk lines of the male pups corresponding to where female pups display nipples. Fur growth may challenge detection of areolae after PND 14/15. Therefore, the NR assessment should be conducted prior to excessive fur growth. Ideally, all pups in a study are assessed on the same postnatal day to minimize variation due to maturation level (OECD, 2013).
NR is occasionally observed in controls. Hence, accurate assessment of NR in controls is needed to detect substance-induced effects on masculine development (Schwartz et al., 2021). It is recommended by the OECD guidance documents 43 and 151 to record NR as a quantitative number rather than a qualitative measure (present/absent or yes/no response). This allows for more nuanced analysis of results, e.g., high control values may be recognized (OECD, 2013, 2018). Studies reporting quantitative measures of NR are therefore considered stronger in terms of weight of evidence.
Reproducibility of NR results is challenged by the measure being a visual assessment prone to a degree of subjectivity. Thus, NR should be assessed and scored blinded to exposure groups and ideally be performed by the same person(s) within the same study.
<h4>Regulatory Significance of the AO</h4>
NR is recognized by the OECD as a relevant measure for anti-androgenic effects and is mandatory in the test guidelines Extended One Generation Reproductive Toxicity Study, TG 443 (OECD, 2018) and the two screening studies for reproductive toxicity, TGs 421/422 (OECD, 2016a, 2016b). The endpoint is also described in the guidance documents 43 (OECD, 2008) and 151 (OECD, 2013). Furthermore, NR data can be used in chemical risk assessment for setting the No Observed Adverse Effect Level (NOAEL) as stated in the OECD guidance document 151 (OECD, 2013): “A statistically significant change in nipple retention should be evaluated similarly to an effect on AGD as both endpoints indicate an adverse effect of exposure and should be considered in setting a NOAEL”.
<h4>References</h4>
Hass, U., Scholze, M., Christiansen, S., Dalgaard, M., Vinggaard, A. M., Axelstad, M., Metzdorff, S. B., & Kortenkamp, A. (2007). Combined exposure to anti-androgens exacerbates disruption of sexual differentiation in the rat. Environmental Health Perspectives, 115(suppl 1), 122–128.
Imperato-McGinley, J., Binienda, Z., Gedney, J., & Vaughan, E. D. (1986). Nipple Differentiation in Fetal Male Rats Treated with an Inhibitor of the Enzyme 5α-Reductase: Definition of a Selective Role for Dihydrotestosterone. Endocrinology, 118(1), 132–137.
Kratochwil, K. (1977). Development and Loss of Androgen Responsiveness in the Embryonic Rudiment of the Mouse Mammary Gland. DEVELOPMENTAL BIOLOGY, 61, 358–365.
OECD. (2008). Guidance document 43 on mammalian reproductive toxicity testing and assessment. Environment, Health and Safety Publications, 16(43).
OECD. (2013). Guidance document supporting OECD test guideline 443 on the extended one-generation reproductive toxicity test. Environment, Health and Safety Publications, 10(151).
OECD. (2016a). Test Guideline 421: Reproduction/Developmental Toxicity Screening Test. OECD Guidelines for the Testing of Chemicals, 421. <a href="http://www.oecd.org/termsandconditions/" style="color:#0563c1; text-decoration:underline">http://www.oecd.org/termsandconditions/</a>
OECD. (2016b). Test Guideline 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test. OECD Guidelines for the Testing of Chemicals, 422. <a href="http://www.oecd.org/termsandconditions/" style="color:#0563c1; text-decoration:underline">http://www.oecd.org/termsandconditions/</a>
OECD. (2018). Test Guideline 443: Extended one-generation reproductive toxicity study. OECD Guidelines for the Testing of Chemicals, 443. <a href="http://www.oecd.org/termsandconditions/" style="color:#0563c1; text-decoration:underline">http://www.oecd.org/termsandconditions/</a>
Pedersen, E. B., Christiansen, S., & Svingen, T. (2022). AOP key event relationship report: Linking androgen receptor antagonism with nipple retention. Current Research in Toxicology, 3, 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. Frontiers in Toxicology, 3, 730752.
<h2>Appendix 2</h2>
<h2>List of Key Event Relationships in the AOP</h2>
<div id="evidence_supporting_links">
<h3>List of Adjacent Key Event Relationships</h3>
<div>
<h4><a href="/relationships/3448">Relationship: 3448: Decrease, intratesticular testosterone leads to Decrease, circulating testosterone levels</a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/307">Decreased testosterone synthesis leading to short anogenital distance (AGD) in male (mammalian) offspring</a></td>
<td>adjacent</td>
<td>High</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/570">Decreased testosterone synthesis leading to hypospadias in male (mammalian) offspring</a></td>
<td>adjacent</td>
~~<td></td>~~
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/aops/575">Decreased testosterone synthesis leading to increased nipple retention (NR) in male (rodent) offspring</a></td>
<td>adjacent</td>
<td>High</td>
<td></td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>mammals</td>
<td>mammals</td>
<td></td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>rat</td>
<td>Rattus norvegicus</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10116" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>mouse</td>
<td>Mus musculus</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10090" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>All life stages</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Male</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
Taxonomic applicability
The KER is assessed applicable to mammals, as testicular testosterone synthesis is common for all mammals. It is, however, acknowledged that this KER most likely has a much broader domain of applicability extending to non-mammalian vertebrates.
Sex applicability
This KER is only applicable to males, as testes are only found in males.
Life stage applicability
This KER is applicable to all life stages.  Once formed, the testes produce and secrete testosterone during fetal development and throughout postnatal life, although testosterone levels do vary between life stages (Vesper et al., 2015).
<h4>Key Event Relationship Description</h4>
This KE describes a decrease in intratesticular testosterone production leading to a decrease in circulating levels of testosterone. Intratesticular testosterone can be measured in whole testicular tissue samples by testing ex vivo testicular testosterone production, and circulating testosterone is measured in plasma or serum. In males, the testes produce and secrete the majority of the circulating testosterone, with only a small contribution from the adrenal gland (Naamneh Elzenaty et al., 2022). In mammals, intratesticular testosterone levels are 30- to 100-fold higher than serum testosterone levels (Coviello et al., 2004; McLachlan et al., 2002; Turner et al., 1984). Reducing testicular testosterone will consequently lead to a reduction in circulating levels as well.
<h4>Evidence Supporting this KER</h4>
Biological Plausibility
The biological plausibility for this KER is considered high. The testes are the primary testosterone-producing organs in male mammals and the main contributors to the circulating testosterone levels in males (Naamneh Elzenaty et al., 2022). A decrease in intratesticular testosterone will therefore lead to a decrease in secretion of testosterone and consequently lower circulating levels of testosterone.
Empirical Evidence
The empirical evidence for this KER is overall judged as high.
In vivo toxicity studies in rats and mice have shown that exposure to substances that lowers intratesticular testosterone also lowers circulating testosterone levels. This includes in utero exposure and measurements in fetal males (Borch J et al., 2004; Vinggaard AM et al., 2005) as well as exposure and measurements postnatally in male rodents (Hou X et al., 2020; Ji et al., 2010; Jiang XP et al., 2017)
Supporting this evidence are castration studies in male rats and monkeys, showing a marked reduction in circulating testosterone levels when removing the testes (Gomes & Jain, 1976; Perachio et al., 1977).
Lastly, in humans, males with hypogonadism or gonadal dysgenesis present with lower circulating testosterone levels (Hirose Y et al., 2007; Jones LW et al., 1970).
Dose concordance
In vivo toxicity studies support dose concordance for this KER, as exemplified below.
In pre-pubertal/pubertal male rats, chlorocholine chloride exposure (postnatal day (PND) 23-60) in three doses reduced both intratesticular and serum testosterone levels at PND60 at all doses tested (Hou X et al., 2020).
Perinatal exposure (gestational day (GD) 10-birth) of male mice to diethylhexyl phthalate (DEHP) in three doses (100, 500, and 1000 mg/kg bw/day) reduced intratesticular testosterone at 500 and 1000 mg/kg bw/day at PND1, while only 1000 mg/kg bw/day reduced serum levels of testosterone, although this was measured later, at PND56 (Xie Q et al., 2024)
In utero exposure (GD7-21) of male rats to DEHP in doses of 300 or 750 mg/kg bw/day reduced intratesticular testosterone levels at GD21, while only the high dose also reduced plasma testosterone levels (Borch J et al., 2004).
Temporal concordance
In vivo toxicity studies moderately support temporal concordance for this KER, as exemplified below.
Several studies show that a decrease in intratesticular and circulating testosterone can be measured at the same time point (Borch J et al., 2004; Hou X et al., 2020; Jiang XP et al., 2017; Vinggaard AM et al., 2005).
In utero exposure of male mice to DEHP from GD10 to birth reduced intratesticular testosterone levels at PND1 with LOAEL 500 mg/kg bw/day, and when measured at PND56, circulating testosterone levels were decreased, but with LOAEL 1000 mg/kg bw/day (Xie Q et al., 2024).
In Fisher JS et al., 2003, exposure of male rats from GD13-21 to 500 mg/kg bw/day dibutyl phthalate reduced intratesticular testosterone by ~90% (measured at GD19). When analyzing circulating testosterone levels at PND4, 10, 15, 25, and 90, only the testosterone levels on PND25 were decreased.
One study report conflicting results on the temporal concordance of this KER (Caceres et al., 2023). Here, male rats were exposed for 20 weeks from PND60 to a mixture of the phytoestrogens genistein and daidzein (combined dose of either 29 or 290 mg/kg bw/day). Intratesticular testosterone was measured every 4 weeks, while serum levels of testosterone were measured every second week. While the mixture caused a reduction of serum testosterone after 2 weeks of exposure, a reduction in intratesticular testosterone was not measured until after 8 weeks. The discrepancy might be explained by the multiple mechanisms of action of the phytoestrogens, as they, besides affecting testicular testosterone synthesis, may also influence peripheral aromatization of testosterone to estrogens (van Duursen et al., 2011).
Incidence concordance
Incidence concordance can not be evaluated for this KER.
Uncertainties and Inconsistencies
There are examples of in vivo studies, in which stressors exposure have caused a reduction in intratesticular testosterone levels without a reduction in circulating testosterone levels.
<h4>Quantitative Understanding of the Linkage</h4>
Time-scale
The time-scale for this KER is likely minutes or hours, as testosterone is secreted into the blood from the testes after synthesis. In vivo, a decrease in intratesticular and circulating testosterone can be measured at the same time, both in fetal and postnatal studies (Borch J et al., 2004; Hou X et al., 2020; Jiang XP et al., 2017; Vinggaard AM et al., 2005). Ex vivo, chemically-induced reduction in testicular production of testosterone can be measured in culture media after 3 hours incubation (earlier time points were not measured) (Wilson et al., 2009).
Known Feedforward/Feedback loops influencing this KER
Testosterone is a part of the hypothalamic-pituitary-gonadal (HPG) axis, which controls testosterone synthesis in puberty and adulthood. In this axis, gonatropin-releasing hormone (GnRH) is released from the hypothalamus and stimulates release of luteinizing hormone (LH) from the pituitary. LH acts on the testes to produce and secrete testosterone. Elevated circulating testosterone levels exerts negative feedback on the HPG axis (decreasing GnRH secretion) to keep testosterone levels in balance (Tilbrook & Clarke, 2001).
Importantly, there are species-specific differences in when the HPG axis is functional during development. In the mouse, fetal testosterone synthesis is independent of pituitary LH (O’Shaughnessy et al., 1998), whereas in humans, human chorionic gonadotropin (hCG) act similarly to LH and appear to be critical in stimulating testosterone synthesis in the fetal testis (Huhtaniemi, 2025).
<h4>References</h4>
Borch J, Ladefoged O, Hass U, & Vinggaard AM. (2004). Steroidogenesis in fetal male rats is reduced by DEHP and DINP, but endocrine effects of DEHP are not modulated by DEHA in fetal, prepubertal and adult male rats. Reproductive Toxicology (Elmsford, N.Y.), 18(1), 53–61. https://doi.org/10.1016/j.reprotox.2003.10.011
Caceres, S., Crespo, B., Alonso-Diez, A., De Andrés, P. J., Millan, P., Silván, G., Illera, M. J., & Illera, J. C. (2023). Long-Term Exposure to Isoflavones Alters the Hormonal Steroid Homeostasis-Impairing Reproductive Function in Adult Male Wistar Rats. Nutrients, 15(5), 1261. https://doi.org/10.3390/nu15051261
Coviello, A. D., Bremner, W. J., Matsumoto, A. M., Herbst, K. L., Amory, J. K., Anawalt, B. D., Yan, X., Brown, T. R., Wright, W. W., Zirkin, B. R., & Jarow, J. P. (2004). Intratesticular Testosterone Concentrations Comparable With Serum Levels Are Not Sufficient to Maintain Normal Sperm Production in Men Receiving a Hormonal Contraceptive Regimen. Journal of Andrology, 25(6), 931–938. https://doi.org/10.1002/j.1939-4640.2004.tb03164.x
Fisher JS, Macpherson S, Marchetti N, & Sharpe RM. (2003). Human “testicular dysgenesis syndrome”: A possible model using in-utero exposure of the rat to dibutyl phthalate. Human Reproduction (Oxford, England), 18(7), 1383–1394. https://doi.org/10.1093/humrep/deg273
Gomes, W. R., & Jain, S. K. (1976). Effect of unilateral and bilateral castration and cryptorchidism on serum gonadotrophins in the rat. The Journal of Endocrinology, 68(02), 191–196. https://doi.org/10.1677/joe.0.0680191
Hirose Y, Sasa M, Bando Y, Hirose T, Morimoto T, Kurokawa Y, Nagao T, & Tangoku A. (2007). Bilateral male breast cancer with male potential hypogonadism. World Journal of Surgical Oncology, 5, 60. https://doi.org/10.1186/1477-7819-5-60
Hou X, Hu H, Xiagedeer B, Wang P, Kang C, Zhang Q, Meng Q, & Hao W. (2020). Effects of chlorocholine chloride on pubertal development and reproductive functions in male rats. Toxicology Letters, 319, 1–10. https://doi.org/10.1016/j.toxlet.2019.10.024
Huhtaniemi, I. T. (2025). Luteinizing hormone receptor knockout mouse: What has it taught us? Andrology, andr.70000. https://doi.org/10.1111/andr.70000
Ji, Y.-L., Wang, H., Liu, P., Wang, Q., Zhao, X.-F., Meng, X.-H., Yu, T., Zhang, H., Zhang, C., Zhang, Y., & Xu, D.-X. (2010). Pubertal cadmium exposure impairs testicular development and spermatogenesis via disrupting testicular testosterone synthesis in adult mice. Reproductive Toxicology, 29(2), 176–183. https://doi.org/10.1016/j.reprotox.2009.10.014
Jiang XP, Tang JY, Xu Z, Han P, Qin ZQ, Yang CD, Wang SQ, Tang M, Wang W, Qin C, Xu Y, Shen BX, Zhou WM, & Zhang W. (2017). Sulforaphane attenuates di-N-butylphthalate-induced reproductive damage in pubertal mice: Involvement of the Nrf2-antioxidant system. Environmental Toxicology, 32(7), 1908–1917. https://doi.org/10.1002/tox.22413
Jones LW, Isaacs H Jr, Edelbrock H, & Donnell GN. (1970). Reifenstein’s syndrome: Hereditary familial hypogonadism with hypospadias and gynecomastia. The Journal of Urology, 104(4), 608–611. https://doi.org/10.1016/s0022-5347(17)61793-2
McLachlan, R. I., O’Donnell, L., Stanton, P. G., Balourdos, G., Frydenberg, M., de Kretser, D. M., & Robertson, D. M. (2002). Effects of Testosterone Plus Medroxyprogesterone Acetate on Semen Quality, Reproductive Hormones, and Germ Cell Populations in Normal Young Men. The Journal of Clinical Endocrinology & Metabolism, 87(2), 546–556. https://doi.org/10.1210/jcem.87.2.8231
Naamneh Elzenaty, R., Du Toit, T., & Flück, C. E. (2022). Basics of androgen synthesis and action. Best Practice & Research Clinical Endocrinology & Metabolism, 36(4), 101665. https://doi.org/10.1016/j.beem.2022.101665
O’Shaughnessy, P. J., Baker, P., Sohnius, U., Haavisto, A.-M., Charlton, H. M., & Huhtaniemi, I. (1998). Fetal Development of Leydig Cell Activity in the Mouse Is Independent of Pituitary Gonadotroph Function*. Endocrinology, 139(3), 1141–1146. https://doi.org/10.1210/endo.139.3.5788
Perachio, A. A., Alexander, M., Marr, L. D., & Collins, D. C. (1977). Diurnal variations of serum testosterone levels in intact and gonadectomized male and female rhesus monkeys. Steroids, 29(1), 21–33. https://doi.org/10.1016/0039-128X(77)90106-4
Tilbrook, A. J., & Clarke, I. J. (2001). Negative Feedback Regulation of the Secretion and Actions of Gonadotropin-Releasing Hormone in Males. Biology of Reproduction, 64(3), 735–742. https://doi.org/10.1095/biolreprod64.3.735
Turner, T. T., Jones, C. E., Howards, S. S., Ewing, L. L., Zegeye, B., & Gunsalus, G. L. (1984). On the androgen microenvironment of maturing spermatozoa. Endocrinology, 115(5), 1925–1932. https://doi.org/10.1210/endo-115-5-1925
van Duursen, M. B. M., Nijmeijer, S. M., de Morree, E. S., de Jong, P. Chr., & van den Berg, M. (2011). Genistein induces breast cancer-associated aromatase and stimulates estrogen-dependent tumor cell growth in in vitro breast cancer model. Toxicology, 289(2), 67–73. https://doi.org/10.1016/j.tox.2011.07.005
Vesper, H. W., Wang, Y., Vidal, M., Botelho, J. C., & Caudill, S. P. (2015). Serum Total Testosterone Concentrations in the US Household Population from the NHANES 2011-2012 Study Population. Clinical Chemisty, 61(12), 1495–1504. https://doi.org/10.1373/clinchem.2015.245969
Vinggaard AM, Christiansen S, Laier P, Poulsen ME, Breinholt V, Jarfelt K, Jacobsen H, Dalgaard M, Nellemann C, & Hass U. (2005). Perinatal exposure to the fungicide prochloraz feminizes the male rat offspring. Toxicological Sciences : An Official Journal of the Society of Toxicology, 85(2), 886–897. https://doi.org/doi.org/10.1093/toxsci/kfi150
Wilson, V. S., Lambright, C. R., Furr, J. R., Howdeshell, K. L., & Gray, L. E., Jr. (2009). The herbicide linuron reduces testosterone production from the fetal rat testis during both in utero and in vitro exposures. TOXICOLOGY LETTERS, 186(2), 73–77. https://doi.org/10.1016/j.toxlet.2008.12.017
Xie Q, Cao H, Liu H, Xia K, Gao Y, & Deng C. (2024). Prenatal DEHP exposure induces lifelong testicular toxicity by continuously interfering with steroidogenic gene expression. Translational Andrology and Urology, 13(3), 369–382. https://doi.org/10.21037/tau-23-503
</div>
<div>
<h4><a href="/relationships/2131">Relationship: 2131: Decrease, circulating testosterone levels leads to Decrease, AR activation</a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/288">Inhibition of 17α-hydrolase/C 10,20-lyase (Cyp17A1) activity leads to birth reproductive defects (cryptorchidism) in male (mammals)</a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
<tr>
<td><a href="/aops/307">Decreased testosterone synthesis leading to short anogenital distance (AGD) in male (mammalian) offspring</a></td>
<td>adjacent</td>
<td>High</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/570">Decreased testosterone synthesis leading to hypospadias in male (mammalian) offspring</a></td>
<td>adjacent</td>
~~<td></td>~~
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/aops/575">Decreased testosterone synthesis leading to increased nipple retention (NR) in male (rodent) offspring</a></td>
<td>adjacent</td>
<td>High</td>
<td></td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>mammals</td>
<td>mammals</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>During development and at adulthood</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Mixed</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
Taxonomic applicability
KER2131 is assessed applicable to mammals, as T and AR activation are known to be related in mammals. It is, however, acknowledged that this KER most likely has a much broader domain of applicability extending to non-mammalian vertebrates. AOP developers are encouraged to add additional relevant knowledge to expand on the applicability to also include other vertebrates.
Sex applicability
KER2131 is assessed applicable to both sexes, as T activates AR in both males and females.
Life-stage applicability
KER2131 is considered applicable to developmental and adult life stages, as T-mediated AR activation is relevant from the AR is expressed.
<h4>Key Event Relationship Description</h4>
This key event relationship links decreased testosterone (T) levels to decreased androgen receptor (AR) activation. T is an endogenous steroid hormone important for, amongst other things, reproductive organ development and growth as well as muscle mass and spermatogenesis (Marks, 2004).T is, together with dihydrotestosterone (DHT), a primary ligand for the AR in mammals (Schuppe et al., 2020). Besides its genomic actions, the AR can also mediate rapid, non-genomic second messenger signaling (Davey & Grossmann, 2016). When T levels are reduced, less substrate is available for the AR, and hence, AR activation is decreased (Gao et al., 2005).
<h4>Evidence Supporting this KER</h4>
Biological Plausibility
The biological plausibility for this KER is considered high
AR activation is dependent on ligand binding (though a few cases of ligand-independent AR activation has been shown, see uncertainties and inconsistencies). T is a primary ligand for the AR, and when T levels are decreased there is less substrate for the AR, and hence, AR activation is decreased. In the male, T is primarily synthesized by the testes, and in some target tissues T is irreversibly metabolized to the more potent metabolite DHT. T and DHT both bind to the AR, but DHT has a higher binding affinity (Gao et al., 2005). The lower binding affinity of T compared to DHT is due to the faster dissociation rate of T from the full-length AR, as T has less effective FXXLF motif binding to AF2 (Askew et al., 2007). Binding of T or DHT has different effects in different tissues. E.g. in the developing male, T is required for development of the internal sex organs (epididymis, vas deferens and the seminal vesicles), whereas DHT is crucial for development of the external sex organs (Keller et al., 1996). In the adult male, androgen action in the reproductive tissues is DHT dependent, whereas action in muscle and bone is DHT independent (Gao et al., 2005). In patients with male androgen deficiency syndrome (AIS), clinically low levels of T leads to reduced AR activation (either due to low T or DHT in target tissue), which manifests as both androgenic related symptoms (such as incomplete or delayed sexual development, loss of body hair, small or shrinking testes, low or zero sperm count) as well as anabolic related symptoms (such as height loss, low trauma fracture, low bone mineral density, reduced muscle bulk and strength, increased body fat). All symptoms can be counteracted by treatment with T, which acts directly on the AR receptor in anabolic tissue (Bhasin et al., 2010). Similarly, removal of the testicles in weanling rats results in a feminized body composition and muscle metabolism, which is reversed by administration of T (Krotkiewski et al., 1980). As this demonstrates, the consequences of low T regarding AR activation will depend on tissue, life stage, species etc.
AR activation is dependent on ligand binding (though a few cases of ligand-independent AR activation has been shown, see uncertainties and inconsistencies). T is a primary ligand for the AR, and when T levels are decreased there is less substrate for the AR, and hence, AR activation is decreased. In the male, T is primarily synthesized by the testes, and in some target tissues T is irreversibly metabolized to the more potent metabolite DHT. T and DHT both bind to the AR, but DHT has a higher binding affinity (Gao et al., 2005). The lower binding affinity of T compared to DHT is due to the faster dissociation rate of T from the full-length AR, as T has less effective FXXLF motif binding to AF2 (Askew et al., 2007). Binding of T or DHT has different effects in different tissues. E.g. in the developing male, T is required for development of the internal sex organs (epididymis, vas deferens and the seminal vesicles), whereas DHT is crucial for development of the external sex organs (Keller et al., 1996). In the adult male, androgen action in the reproductive tissues is DHT dependent, whereas action in muscle and bone is DHT independent (Gao et al., 2005). In patients with male androgen deficiency syndrome, clinically low levels of T leads to reduced AR activation (either due to low T or DHT in target tissue), which manifests as both androgenic related symptoms (such as incomplete or delayed sexual development, loss of body hair, small or shrinking testes, low or zero sperm count) as well as anabolic related symptoms (such as height loss, low trauma fracture, low bone mineral density, reduced muscle bulk and strength, increased body fat). All symptoms can be counteracted by treatment with T, which acts directly on the AR receptor in anabolic tissue (Bhasin et al., 2010). Similarly, removal of the testicles in weanling rats results in a feminized body composition and muscle metabolism, which is reversed by administration of T (Krotkiewski et al., 1980). As this demonstrates, the consequences of low T regarding AR activation will depend on tissue, life stage, species etc.
Empirical Evidence
The empirical evidence for this KER is considered high
Dose concordance
There is a positive dose-response relationship between increasing concentrations of T and AR activation (U.S. EPA., 2023).
Other evidence
<ul>
<li style="text-align:justify">In male patients with androgen deficiency, treatment with T counteracts anabolic (DHT independent) related symptoms such as height loss, low trauma fracture, low bone mineral density, reduced muscle bulk and strength, increased body fat (Bhasin et al., 2010; Katznelson et al., 1996).</li>
<li style="text-align:justify">Removal of the testicles in weanling rats result in a feminized body composition and muscle metabolism, which is reversed by administration of T (Krotkiewski et al., 1980).</li>
</ul>
Uncertainties and Inconsistencies
Ligand-independent actions of the AR have been identified. To what extent and of which biological significance is not well defined (Bennesch & Picard, 2015).
<h4>Quantitative Understanding of the Linkage</h4>
Response-response relationship
There is a positive dose-response relationship between increasing concentrations of T and AR activation (U.S. EPA., 2023). However, there is not enough data, or overview of the data, to define a quantitative linkage in vivo, and such a relationship will differ between biological systems (species, tissue, cell type).
Time-scale
AR 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).
Known modulating factors
<table class="table table-bordered table-fullwidth">
<thead>
<tr>
<th>Modulating Factor (MF)</th>
<th>MF Specification</th>
<th>Effect(s) on the KER</th>
<th>Reference(s)</th>
</tr>
</thead>
<tbody>
<tr>
<td>Age</td>
<td>AR expression changes with aging </td>
<td>Tissue-specific alterations in AR activity with aging</td>
<td>(Supakar et al., 1993; Wu et al., 2009)</td>
</tr>
<tr>
<td>Genotype</td>
<td>Number of CAG repeats in the first exon of AR</td>
<td>Decreased AR activation with increased number of CAGs</td>
<td>(Chamberlain et al., 1994; Tut et al., 1997)</td>
</tr>
<tr>
<td>Male androgen deficiency syndrome</td>
<td>Low circulating testosterone levels due to primary (testicular) or secondary (pituitary-hypothalamic) hypogonadism</td>
<td>Reduced levels of circulating testosterone</td>
<td>(Bhasin et al., 2010)</td>
</tr>
<tr>
<td>Castration</td>
<td>Removal of testicles</td>
<td>Reduced levels of circulating testosterone</td>
<td>(Krotkiewski et al., 1980)</td>
</tr>
</tbody>
</table>
Known Feedforward/Feedback loops influencing this KER
Androgens can upregulate and downregulate AR expression (Lee & Chang, 2003).
<h4>References</h4>
Askew, E. B., Gampe, R. T., Stanley, T. B., Faggart, J. L., & Wilson, E. M. (2007). Modulation of Androgen Receptor Activation Function 2 by Testosterone and Dihydrotestosterone. Journal of Biological Chemistry, 282(35), 25801–25816. https://doi.org/10.1074/jbc.M703268200
Bennesch, M. A., & Picard, D. (2015). Minireview: Tipping the Balance: Ligand-Independent Activation of Steroid Receptors. Molecular Endocrinology, 29(3), 349–363. https://doi.org/10.1210/me.2014-1315
Bhasin, S., Cunningham, G. R., Hayes, F. J., Matsumoto, A. M., Snyder, P. J., Swerdloff, R. S., & Montori, V. M. (2010). Testosterone Therapy in Men with Androgen Deficiency Syndromes: An Endocrine Society Clinical Practice Guideline. The Journal of Clinical Endocrinology & Metabolism, 95(6), 2536–2559. https://doi.org/10.1210/jc.2009-2354
Davey, R. A., & Grossmann, M. (2016). Androgen Receptor Structure, Function and Biology: From Bench to Bedside. The Clinical Biochemist. Reviews, 37(1), 3–15. http://www.ncbi.nlm.nih.gov/pubmed/27057074
Gao, W., Bohl, C. E., & Dalton, J. T. (2005). Chemistry and Structural Biology of Androgen Receptor. Chemical Reviews, 105(9), 3352–3370. https://doi.org/10.1021/cr020456u
Kang, Z., Pirskanen, A., Jänne, O. A., & Palvimo, J. J. (2002). Involvement of Proteasome in the Dynamic Assembly of the Androgen Receptor Transcription Complex. Journal of Biological Chemistry, 277(50), 48366–48371. https://doi.org/10.1074/jbc.M209074200
Katznelson, L., Finkelstein, J. S., Schoenfeld, D. A., Rosenthal, D. I., Anderson, E. J., & Klibanski, A. (1996). Increase in bone density and lean body mass during testosterone administration in men with acquired hypogonadism. The Journal of Clinical Endocrinology & Metabolism, 81(12), 4358–4365. https://doi.org/10.1210/jcem.81.12.8954042
Keller, E. T., Ershler, W. B., & Chang, Chawnshang. (1996). The androgen receptor: A mediator of diverse responses. Frontiers in Bioscience, 1(4), 59–71. https://doi.org/10.2741/A116
Krotkiewski, M., Kral, J. G., & Karlsson, J. (1980). Effects of castration and testosterone substitution on body composition and muscle metabolism in rats. Acta Physiologica Scandinavica, 109(3), 233–237. https://doi.org/10.1111/j.1748-1716.1980.tb06592.x
Lee, D. K., & Chang, C. (2003). Expression and Degradation of Androgen Receptor: Mechanism and Clinical Implication. The Journal of Clinical Endocrinology & Metabolism, 88(9), 4043–4054. https://doi.org/10.1210/jc.2003-030261
Marks, L. S. (2004). 5alpha-reductase: history and clinical importance. Reviews in Urology, 6 Suppl 9(Suppl 9), S11-21. <a href="http://www.ncbi.nlm.nih.gov/pubmed/16985920" style="color:#0563c1; text-decoration:underline">http://www.ncbi.nlm.nih.gov/pubmed/16985920</a>
Schuppe, E. R., Miles, M. C., and Fuxjager, M. J. (2020). Evolution of the androgen receptor: Perspectives from human health to dancing birds. Mol. Cell. Endocrinol. 499, 110577. doi:10.1016/J.MCE.2019.110577.
U.S. EPA. (2023). ToxCast & Tox21 AR agonism of testosterone. Retrieved from Https://Www.Epa.Gov/Chemical-Research/Toxicity-Forecaster-Toxcasttm-Data June 23, 2023. Data Released October 2018.
</div>
<div>
<h4><a href="/relationships/2124">Relationship: 2124: Decrease, AR activation leads to Altered, Transcription of genes by the AR</a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/344">Androgen receptor (AR) antagonism leading to nipple retention (NR) in male (mammalian) offspring</a></td>
<td>adjacent</td>
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/aops/345">Androgen receptor (AR) antagonism leading to decreased fertility in females</a></td>
<td>adjacent</td>
<td>High</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/305">5α-reductase inhibition leading to short anogenital distance (AGD) in male (mammalian) offspring</a></td>
<td>adjacent</td>
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/aops/306">Androgen receptor (AR) antagonism leading to short anogenital distance (AGD) in male (mammalian) offspring</a></td>
<td>adjacent</td>
<td>Moderate</td>
<td></td>
</tr>
<tr>
<td><a href="/aops/307">Decreased testosterone synthesis leading to short anogenital distance (AGD) in male (mammalian) offspring</a></td>
<td>adjacent</td>
<td>Moderate</td>
<td>Low</td>
</tr>
<tr>
<td><a href="/aops/570">Decreased testosterone synthesis leading to hypospadias in male (mammalian) offspring</a></td>
<td>adjacent</td>
~~<td></td>~~
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/aops/571">5α-reductase inhibition leading to hypospadias in male (mammalian) offspring</a></td>
<td>adjacent</td>
~~<td></td>~~
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/aops/576">5α-reductase inhibition leading to increased nipple retention (NR) in male (rodent) offspring</a></td>
<td>adjacent</td>
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/aops/575">Decreased testosterone synthesis leading to increased nipple retention (NR) in male (rodent) offspring</a></td>
<td>adjacent</td>
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/aops/477">Androgen receptor (AR) antagonism leading to hypospadias in male (mammalian) offspring</a></td>
<td>adjacent</td>
<td>High</td>
<td></td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>mammals</td>
<td>mammals</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>During development and at adulthood</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Mixed</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
This KER is applicable for both sexes, across developmental stages into adulthood, in numerous cells and tissues and across mammalian taxa. It is, however, acknowledged that this KER most likely has a much broader domain of applicability extending to non-mammalian vertebrates. AOP developers are encouraged to add additional relevant knowledge to expand on the applicability to also include other vertebrates.
<h4>Key Event Relationship Description</h4>
The androgen receptor (AR) is a ligand-dependent nuclear transcription factor that upon activation translocates to the nucleus, dimerizes, and binds androgen response elements (AREs) to modulate transcription of target genes (Lamont and Tindall, 2010, Roy et al. 2001). Decreased activation of the AR affects its transcription factor activity, therefore leading to altered AR-target gene expression. This KER refers to decreased AR activation and altered gene expression occurring in complex systems, such as in vivo and the specific effect on transcription of AR target genes will depend on species, life stage, tissue, cell type etc.
<h4>Evidence Supporting this KER</h4>
Biological Plausibility
The biological plausibility for this KER is considered high
The AR is a ligand-activated transcription factor part of the steroid hormone nuclear receptor family. Non-activated AR is found in the cytoplasm as a multiprotein complex with heat-shock proteins, immunophilins and, other chaperones (Roy et al. 2001). Upon activation through ligand binding, the AR dissociates from the protein complex, translocates to the nucleus and homodimerizes. Facilitated by co-regulators, AR can bind to DNA regions containing AREs and initiate transcription of target genes, that thus will be different in e.g. different tissues, life-stages, species etc.
Through mapping of AREs and ChIP sequencing studies, several AR target genes have been identified, mainly studied in prostate cells (Jin, Kim, and Yu 2013). Different co-regulators and ligands lead to altered expression of different sets of genes (Jin et al. 2013; Kanno et al. 2022). Alternative splicing of the AR can lead to different AR variants that also affects which genes are transcribed (Jin et al. 2013).
Apart from this canonical signaling pathway, the AR can suppress gene expression, indirectly regulate miRNA transcription, and have non-genomic effects by rapid activation of second messenger pathways in either presence or absence of a ligand (Jin et al. 2013).
Empirical Evidence
The empirical evidence for this KER is considered high
In humans, altered gene expression profiling in individuals with androgen insensitivity syndrome (AIS) can provide supporting empirical evidence (Holterhus et al. 2003; Peng et al. 2021). In rodent AR knockout (KO) models, gene expression profiling studies and gene-targeted approaches have provided information on differentially expressed genes in several organ systems including male and female reproductive, endocrine, muscular, cardiovascular and nervous systems (Denolet et al. 2006; Fan et al. 2005; Holterhus et al. 2003; Ikeda et al. 2005; Karlsson et al. 2016; MacLean et al. 2008; Rana et al. 2011; Russell et al. 2012; Shiina et al. 2006; Wang et al. 2006; Welsh et al. 2012; Willems et al. 2010; Yu et al. 2008, 2012; Zhang et al. 2006; Zhou et al. 2011).
Exposure to known antiandrogens has been shown to alter transcriptional profiles, for example of neonatal pig ovaries (Knapczyk-Stwora et al. 2019).
Dose concordance has also been observed for instance in zebrafish embryos; a dose of 50 µg/L of the AR antagonist flutamide resulted in 674 differentially expressed genes at 96 h post fertilization whereas 500 µg/L flutamide resulted in 2871 differentially expressed genes (Ayobahan et al., 2023).
Uncertainties and Inconsistencies
AR action has been reported to occur also without ligand binding. However, not much is known about the extent and biological implications of such non-canonical, ligand-independent AR activation (Bennesch and Picard 2015).
<h4>Quantitative Understanding of the Linkage</h4>
Response-response relationship
There is not enough data to define a quantitative relationship between AR activation and alteration of AR target gene transcription, and such a relationship will differ between biological systems (species, tissue, cell type, life stage etc).
Time-scale
AR and promoter interactions occur within 15 minutes of ligand binding, RNA polymerase II and coactivator recruitment are proposed to occur transiently with cycles of approximately 90 minutes in LNCaP cells (Kang et al. 2002). RNA polymerase II elongation rates in mammalian cells have been shown to range between 1.3 and 4.3 kb/min (Maiuri et al. 2011). Therefore, depending on the cell type and the half-life of the AR target gene transcripts, changes are to be expected within hours.
Known modulating factors
<div>
<table class="table table-bordered table-fullwidth">
<thead>
<tr>
<th>Modulating Factor (MF)</th>
<th>MF Specification</th>
<th>Effect(s) on the KER</th>
<th>Reference(s)</th>
</tr>
</thead>
<tbody>
<tr>
<td>Age</td>
<td>AR expression in aging male rats</td>
<td>Tissue-specific alterations in AR activity with aging</td>
<td>(Supakar et al. 1993; Wu, Lin, and Gore 2009)</td>
</tr>
<tr>
<td>Genotype</td>
<td>Number of CAG repeats in the first exon of AR</td>
<td>Decreased AR activation with increased number of CAGs</td>
<td>
(Tut et al. 1997; Chamberlain et al. 1994)
</td>
</tr>
</tbody>
</table>
</div>
Known Feedforward/Feedback loops influencing this KER
AR has been hypothesized to auto-regulate its mRNA and protein levels (Mora and Mahesh 1999).
<h4>References</h4>
Ayobahan, S. U., Alvincz, J., Reinwald, H., Strompen, J., Salinas, G., Schäfers, C., et al. (2023). Comprehensive identification of gene expression fingerprints and biomarkers of sexual endocrine disruption in zebrafish embryo. Ecotoxicol. Environ. Saf. 250, 114514. doi:10.1016/J.ECOENV.2023.114514.
Bennesch, Marcela A., and Didier Picard. 2015. “Minireview: Tipping the Balance: Ligand-Independent Activation of Steroid Receptors.” Molecular Endocrinology 29(3):349–63.
Chamberlain, Nancy L., Erika D. Driverand, and Roger L. Miesfeldi. 1994. The Length and Location of CAG Trinucleotide Repeats in the Androgen Receptor N-Terminal Domain Affect Transactivation Function. Vol. 22.
Denolet, Evi, Karel De Gendt, Joke Allemeersch, Kristof Engelen, Kathleen Marchal, Paul Van Hummelen, Karen A. L. Tan, Richard M. Sharpe, Philippa T. K. Saunders, Johannes V. Swinnen, and Guido Verhoeven. 2006. “The Effect of a Sertoli Cell-Selective Knockout of the Androgen Receptor on Testicular Gene Expression in Prepubertal Mice.” Molecular Endocrinology 20(2):321–34. doi: 10.1210/me.2005-0113.
Fan, Wuqiang, Toshihiko Yanase, Masatoshi Nomura, Taijiro Okabe, Kiminobu Goto, Takashi Sato, Hirotaka Kawano, Shigeaki Kato, and Hajime Nawata. 2005. Androgen Receptor Null Male Mice Develop Late-Onset Obesity Caused by Decreased Energy Expenditure and Lipolytic Activity but Show Normal Insulin Sensitivity With High Adiponectin Secretion. Vol. 54.
Holterhus, Paul-Martin, Olaf Hiort, Janos Demeter, Patrick O. Brown, and James D. Brooks. 2003. Differential Gene-Expression Patterns in Genital Fibroblasts of Normal Males and 46,XY Females with Androgen Insensitivity Syndrome: Evidence for Early Programming Involving the Androgen Receptor. Vol. 4.
Ikeda, Yasumasa, Ken Ichi Aihara, Takashi Sato, Masashi Akaike, Masanori Yoshizumi, Yuki Suzaki, Yuki Izawa, Mitsunori Fujimura, Shunji Hashizume, Midori Kato, Shusuke Yagi, Toshiaki Tamaki, Hirotaka Kawano, Takahiro Matsumoto, Hiroyuki Azuma, Shigeaki Kato, and Toshio Matsumoto. 2005. “Androgen Receptor Gene Knockout Male Mice Exhibit Impaired Cardiac Growth and Exacerbation of Angiotensin II-Induced Cardiac Fibrosis.” Journal of Biological Chemistry 280(33):29661–66. doi: 10.1074/jbc.M411694200.
Jin, Hong Jian, Jung Kim, and Jindan Yu. 2013. “Androgen Receptor Genomic Regulation.” Translational Andrology and Urology 2(3):158–77.
Kang, Zhigang, Asta Pirskanen, Olli A. Jänne, and Jorma J. Palvimo. 2002. “Involvement of Proteasome in the Dynamic Assembly of the Androgen Receptor Transcription Complex.” Journal of Biological Chemistry 277(50):48366–71. doi: 10.1074/jbc.M209074200.
Kanno, Yuichiro, Nao Saito, Ryota Saito, Tomohiro Kosuge, Ryota Shizu, Tomofumi Yatsu, Takuomi Hosaka, Kiyomitsu Nemoto, Keisuke Kato, and Kouichi Yoshinari. 2022. “Differential DNA-Binding and Cofactor Recruitment Are Possible Determinants of the Synthetic Steroid YK11-Dependent Gene Expression by Androgen Receptor in Breast Cancer MDA-MB 453 Cells.” Experimental Cell Research 419(2). doi: 10.1016/j.yexcr.2022.113333.
Karlsson, Sara A., Erik Studer, Petronella Kettunen, and Lars Westberg. 2016. “Neural Androgen Receptors Modulate Gene Expression and Social Recognition but Not Social Investigation.” Frontiers in Behavioral Neuroscience 10(MAR). doi: 10.3389/fnbeh.2016.00041.
Knapczyk-Stwora, Katarzyna, Anna Nynca, Renata E. Ciereszko, Lukasz Paukszto, Jan P. Jastrzebski, Elzbieta Czaja, Patrycja Witek, Marek Koziorowski, and Maria Slomczynska. 2019. “Flutamide-Induced Alterations in Transcriptional Profiling of Neonatal Porcine Ovaries.” Journal of Animal Science and Biotechnology 10(1):1–15. doi: 10.1186/s40104-019-0340-y.
Lamont, K. R., and Tindall, D. J. (2010). Androgen Regulation of Gene Expression. Adv. Cancer Res. 107, 137–162. doi:10.1016/S0065-230X(10)07005-3.
MacLean, Helen E., W. S. Maria Chiu, Amanda J. Notini, Anna-Maree Axell, Rachel A. Davey, Julie F. McManus, Cathy Ma, David R. Plant, Gordon S. Lynch, and Jeffrey D. Zajac. 2008. “ Impaired Skeletal Muscle Development and Function in Male, but Not Female, Genomic Androgen Receptor Knockout Mice .” The FASEB Journal 22(8):2676–89. doi: 10.1096/fj.08-105726.
Maiuri, Paolo, Anna Knezevich, Alex De Marco, Davide Mazza, Anna Kula, Jim G. McNally, and Alessandro Marcello. 2011. “Fast Transcription Rates of RNA Polymerase II in Human Cells.” EMBO Reports 12(12):1280–85. doi: 10.1038/embor.2011.196.
Mora, Gloria R., and Virendra B. Mahesh. 1999. Autoregulation of the Androgen Receptor at the Translational Level: Testosterone Induces Accumulation of Androgen Receptor MRNA in the Rat Ventral Prostate Polyribosomes.
Peng, Yajie, Hui Zhu, Bing Han, Yue Xu, Xuemeng Liu, Huaidong Song, and Jie Qiao. 2021. “Identification of Potential Genes in Pathogenesis and Diagnostic Value Analysis of Partial Androgen Insensitivity Syndrome Using Bioinformatics Analysis.” Frontiers in Endocrinology 12. doi: 10.3389/fendo.2021.731107.
Rana, Kesha, Barbara C. Fam, Michele V Clarke, Tammy P. S. Pang, Jeffrey D. Zajac, and Helen E. Maclean. 2011. “Increased Adiposity in DNA Binding-Dependent Androgen Receptor Knockout Male Mice Associated with Decreased Voluntary Activity and Not Insulin Resistance.” Am J Physiol Endocrinol Me-Tab 301:767–78. doi: 10.1152/ajpendo.00584.2010.-In.
Roy, Arun K., Rakesh K. Tyagi, Chung S. Song, Yan Lavrovsky, Soon C. Ahn, Tae Sung Oh, and Bandana Chatterjee. 2001. “Androgen Receptor: Structural Domains and Functional Dynamics after Ligand-Receptor Interaction.” Pp. 44–57 in Annals of the New York Academy of Sciences. Vol. 949. New York Academy of Sciences.
Russell, Patricia K., Michele V. Clarke, Jarrod P. Skinner, Tammy P. S. Pang, Jeffrey D. Zajac, and Rachel A. Davey. 2012. “Identification of Gene Pathways Altered by Deletion of the Androgen Receptor Specifically in Mineralizing Osteoblasts and Osteocytes in Mice.” Journal of Molecular Endocrinology 49(1):1–10. doi: 10.1530/JME-12-0014.
Shiina, Hiroko, Takahiro Matsumoto, Takashi Sato, Katsuhide Igarashi, Junko Miyamoto, Sayuri Takemasa, Matomo Sakari, Ichiro Takada, Takashi Nakamura, Daniel Metzger, Pierre Chambon, Jun Kanno, Hiroyuki Yoshikawa, and Shigeaki Kato. 2006. Premature Ovarian Failure in Androgen Receptor-Deficient Mice. Vol. 103.
Supakar, P. C., C. S. Song, M. H. Jung, M. A. Slomczynska, J. M. Kim, R. L. Vellanoweth, B. Chatterjee, and A. K. Roy. 1993. “A Novel Regulatory Element Associated with Age-Dependent Expression of the Rat Androgen Receptor Gene.” Journal of Biological Chemistry 268(35):26400–408. doi: 10.1016/s0021-9258(19)74328-2.
Tut, Thein G., Farid J. Ghadessy, M. A. Trifiro, L. Pinsky, and E. L. Yong. 1997. Long Polyglutamine Tracts in the Androgen Receptor Are Associated with Reduced Trans-Activation, Impaired Sperm Production, and Male Infertility*. Vol. 82.
Wang, Ruey Sheng, Shuyuan Yeh, Lu Min Chen, Hung Yun Lin, Caixia Zhang, Jing Ni, Cheng Chia Wu, P. Anthony Di Sant’Agnese, Karen L. DeMesy-Bentley, Chii Ruey Tzeng, and Chawnshang Chang. 2006. “Androgen Receptor in Sertoli Cell Is Essential for Germ Cell Nursery and Junctional Complex Formation in Mouse Testes.” Endocrinology 147(12):5624–33. doi: 10.1210/en.2006-0138.
Welsh, M., L. Moffat, K. Belling, L. R. de França, T. M. Segatelli, P. T. K. Saunders, R. M. Sharpe, and L. B. Smith. 2012. “Androgen Receptor Signalling in Peritubular Myoid Cells Is Essential for Normal Differentiation and Function of Adult Leydig Cells.” International Journal of Andrology 35(1):25–40. doi: 10.1111/j.1365-2605.2011.01150.x.
Willems, Ariane, Sergio R. Batlouni, Arantza Esnal, Johannes V. Swinnen, Philippa T. K. Saunders, Richard M. Sharpe, Luiz R. França, Karel de Gendt, and Guido Verhoeven. 2010. “Selective Ablation of the Androgen Receptor in Mouse Sertoli Cells Affects Sertoli Cell Maturation, Barrier Formation and Cytoskeletal Development.” PLoS ONE 5(11). doi: 10.1371/journal.pone.0014168.
Wu, D. I., Grace Lin, and Andrea C. Gore. 2009. “Age-Related Changes in Hypothalamic Androgen Receptor and Estrogen Receptor in Male Rats.” The Journal of Comparative Neurology 512:688–701. doi: 10.1002/cne.21925.
Yu, I. Chen, Hung Yun Lin, Ning Chun Liu, Ruey Shen Wang, Janet D. Sparks, Shuyuan Yeh, and Chawnshang Chang. 2008. “Hyperleptinemia without Obesity in Male Mice Lacking Androgen Receptor in Adipose Tissue.” Endocrinology 149(5):2361–68. doi: 10.1210/en.2007-0516.
Yu, Shengqiang, Chiuan Ren Yeh, Yuanjie Niu, Hong Chiang Chang, Yu Chieh Tsai, Harold L. Moses, Chih Rong Shyr, Chawnshang Chang, and Shuyuan Yeh. 2012. “Altered Prostate Epithelial Development in Mice Lacking the Androgen Receptor in Stromal Fibroblasts.” Prostate 72(4):437–49. doi: 10.1002/pros.21445.
Zhang, Caixia, Shuyuan Yeh, Yen-Ta Chen, Cheng-Chia Wu, Kuang-Hsiang Chuang, Hung-Yun Lin, Ruey-Sheng Wang, Yu-Jia Chang, Chamindrani Mendis-Handagama, Liquan Hu, Henry Lardy, Chawnshang Chang, and &dagger; &dagger; George. 2006. Oligozoospermia with Normal Fertility in Male Mice Lacking the Androgen Receptor in Testis Peritubular Myoid Cells.
Zhou, Wei, Gensheng Wang, Christopher L. Small, Zhilin Liu, Connie C. Weng, Lizhong Yang, Michael D. Griswold, and Marvin L. Meistrich. 2011. “Erratum: Gene Expression Alterations by Conditional Knockout of Androgen Receptor in Adult Sertoli Cells of Utp14bjsd/Jsd (Jsd) Mice (Biology of Reproduction (2010) 83, (759-766) DOI: 10.1095/Biolreprod.110.085472).” Biology of Reproduction 84(2):400–408.
</div>
<h3>List of Non Adjacent Key Event Relationships</h3>
<div>
<h4><a href="/relationships/3487">Relationship: 3487: Decrease, intratesticular testosterone leads to nipple retention, increased</a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/575">Decreased testosterone synthesis leading to increased nipple retention (NR) in male (rodent) offspring</a></td>
<td>non-adjacent</td>
<td>High</td>
<td></td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>rat</td>
<td>Rattus norvegicus</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10116" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>mouse</td>
<td>Mus musculus</td>
<td>Low</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10090" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Foetal</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Male</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
Taxonomic applicability
NR is observed in male mice and rats. Male rodents (mostly investigated in laboratory rats and mice) do not have nipples, a feature that is androgen-dependent in these species with fetal androgen action impeding development of nipple anlagen. The empirical evidence supports the applicability to rats, and the KER is considered equally applicable to mice based on the biological knowledge of nipple development in this species. 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 and fetal masculinization during development, which is relevant to humans and mammals in general (Schwartz et al., 2021). It is included as a mandatory endpoint in several rodent OECD Test Guidelines (OECD, 2025a, 2025b, 2025c) and considered an adverse outcome applicable to the setting of Points of Departure for use in human health risk assessment (OECD, 2013).
Sex applicability
This KER is only applicable to males, as the testes are male sex organs. Moreover, females usually have the maximum number of nipples (12 in rats, 10 in mice) (Schwartz et al., 2021)
Life stage applicability
The testes start producing testosterone in fetal life, around gestational day (GD) 15 in rats. Programming by androgens of the peripheral reproductive tissues, including the nipple anlagen, mainly occurs within the masculinization programming window, GD16-20 in rats. Morphological development of the mammary glands also starts in fetal life in both sexes, and upon programming by androgens, the mammary glands of males regress, causing a blockade of nipple formation (Kratochwil, 1986; Watson & Khaled, 2008). Nipples in females and retained nipples in males can first be observed postnatally, ideally at postnatal day (PND) 12-14 in rats (Schwartz et al., 2021).
<h4>Key Event Relationship Description</h4>
This non-adjacent KER describes a fetal decrease in intratesticular testosterone leading to NR in male offspring. In this KER, intratesticular testosterone includes measurements of testosterone in homogenates of testes after in vivo exposure to chemicals, as well as measurements of testosterone production in testes ex vivo from exposed animals.
In male mammals, the testes are the first sex organs to develop. Once formed, they produce testosterone by steroidogenesis. The adrenal glands have been shown to synthesize testosterone, but on a much smaller scale, and the testes are the main site of testosterone production (Naamneh Elzenaty et al., 2022). Testicular testosterone is secreted into the blood to initiate masculinization of the peripheral reproductive tissues. In rats and mice, this includes effects on the developing mammary glands, which develop sexually dimorphic. Testosterone either directly activates the AR in the mammary glands or is converted to the more potent androgen dihydrotestosterone (DHT) (Murashima et al., 2015). Activation of AR by androgens in the mammary glands causes apoptosis of epithelial cells and thus separation of the glands from the overlying epidermis. Consequently, no nipples are formed (Kratochwil, 1986). During low androgen levels, such as in female rodents, nipple development progresses to form up to 10 (mice) and 12 (rats) nipples.
As suppression of nipple development in male rats and mice is dependent on androgens, marked reductions in testicular testosterone production can thus cause nipple retention.
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 and fetal masculinization during development, which is relevant to humans (Schwartz et al., 2021). It is included as a mandatory endpoint in several rodent OECD Test Guidelines (OECD, 2025a, 2025b, 2025c) and considered an adverse outcome applicable to the setting of Points of Departure for use in human health risk assessment (OECD, 2013).
<h4>Evidence Supporting this KER</h4>
Biological Plausibility
The biological plausibility for this KER is judged to be high given the canonical biological knowledge on normal reproductive development in rodents.
In common strains of rats and mice, females have 12 and 10 pairs of nipples, respectively, while males usually do not have nipples, although in rare instances control male rats may display a retained nipple (Schwartz et al., 2021). The sexual dimorphism of the mammary tissue is regulated by the androgens testosterone and DHT, during fetal life. Testosterone is produced by the fetal testes by the steroidogenesis pathway, starting from ~GD15. The testes are the primary male sex organs and produce most of the circulating testosterone, although a minor part may be contributed by other organs such as the adrenals (Naamneh Elzenaty et al., 2022). DHT is produced from testosterone in peripheral tissues by the enzyme 5α-reductase. Both testosterone and DHT activate AR in reproductive tissues to initiate masculinization. The programming of the tissues mainly happens within the masculinization programming window (GD16-20 in rats) (Murashima et al., 2015; Welsh et al., 2014).
The mammary glands start out the same in both sexes, developing along two milk lines in early fetal life. In males, androgens activate AR in the mammary gland mesenchymal cells, and in turn, the cells activate apoptosis of epithelial cells that otherwise would contribute to the development of nipples (Kratochwil, 1986; Schwartz et al., 2021).
Given the dependency of testosterone for regression of the nipples, either through direct AR activation or conversion to DHT, it is highly plausible that a decrease in intratesticular testosterone levels will lead to nipple retention in males.
Empirical Evidence
The empirical evidence from studies in animals for this KER is overall judged as strong.
From the data collection, 8 data sets were extracted. The data sets included different stressors causing reduced fetal intratesticular testosterone, all in rats (Table 2 and Appendix 2, <a href="https://aopwiki.org/system/dragonfly/production/2025/09/17/2twr7tg67o_KER_3487_Appendix_2_250901_final.pdf">2twr7tg67o_KER_3487_Appendix_2_250901_final.pdf</a>). Of these 8 data sets, seven showed concurrent nipple retention.
Table 2 Empirical evidence for KER 3487 LOAEL: Lowest observed adverse effect level; NOAEL: No observed adverse effect level. See Appendix 2, for specifications.
<table cellspacing="0" class="MsoTableGrid" style="border-collapse:collapse; border:none; width:629px">
<tbody>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; height:53px; vertical-align:top; width:98px">
Species
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:53px; vertical-align:top; width:157px">
Stressors(s)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:53px; vertical-align:top; width:180px">
Effect on upstream event
(circulating testosterone)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:53px; vertical-align:top; width:104px">
Effect on downstream event (NR)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:53px; vertical-align:top; width:91px">
Reference
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:98px">
Rat
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:157px">
Butyl benzyl phthalate
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:180px">
LOAEL 500 mg/kg bw/day
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:104px">
No effect
NOAEL 500 mg/kg bw/day
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:91px">
(Hotchkiss et al., 2004)
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:98px">
Rat
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:157px">
Dibutyl phthalate
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:180px">
LOAEL 500 mg/kg bw/day
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:104px">
LOAEL 500 mg/kg bw/day
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:91px">
(Martino-Andrade et al., 2009)
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:98px">
Rat
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:157px">
Diethylhexyl phthalate
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:180px">
LOAEL 750 mg/kg bw/day
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:104px">
LOAEL 750 mg/kg bw/day
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:91px">
(Borch et al., 2004)
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:98px">
Rat
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:157px">
Diisonyl phthalate
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:180px">
LOAEL 600 mg/kg bw/day
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:104px">
LOAEL 750 mg/kg bw/day
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:91px">
(Boberg et al., 2011)
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:98px">
Rat
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:157px">
Linuron
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:180px">
LOAEL 75 mg/kg bw/day
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:104px">
LOAEL 75 mg/kg bw/day
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:91px">
(Hotchkiss et al., 2004)
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:18px; vertical-align:top; width:98px">
Rat
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:18px; vertical-align:top; width:157px">
Prochloraz
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:18px; vertical-align:top; width:180px">
LOAEL 50 mg/kg bw/day
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:18px; vertical-align:top; width:104px">
LOAEL 50 mg/kg bw/day
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:18px; vertical-align:top; width:91px">
(Laier et al., 2006)
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:18px; vertical-align:top; width:98px">
Rat
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:18px; vertical-align:top; width:157px">
Prochloraz
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:18px; vertical-align:top; width:180px">
LOAEL 30 mg/kg bw/day
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:18px; vertical-align:top; width:104px">
LOAEL 30 mg/kg bw/day
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:18px; vertical-align:top; width:91px">
(Vinggaard et al., 2005)
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:18px; vertical-align:top; width:98px">
Rat
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:18px; vertical-align:top; width:157px">
Tebuconazole
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:18px; vertical-align:top; width:180px">
LOAEL 100 mg/kg bw/day
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:18px; vertical-align:top; width:104px">
LOAEL 50 mg/kg bw/day
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:18px; vertical-align:top; width:91px">
(Taxvig et al., 2007)
</td>
</tr>
</tbody>
</table>
Dose concordance
The one study that did not find NR after fetal reduction in intratesticular testosterone levels only tested one dose of stressor, which therefore could be an indication of dose concordance (Hotchkiss et al., 2004).
Four datasets tested multiple doses of stressors. In two datasets, the LOAEL was the same for intratesticular testosterone and NR (Laier et al., 2006; Martino-Andrade et al., 2009). In the study by Martino-Andrade et al. (2009), rats were exposed in utero to DPB during gestation days (GD) 13 to 21. Fetal testicular testosterone levels were assessed at GD21, while NR was evaluated at postnatal day (PND) 13. At a dose of 500 mg/kg/day, both a reduction in fetal testicular testosterone and NR were observed. In contrast, at the lower dose of 100 mg/kg/day, only a slight, non-significant decrease in testosterone levels was reported, with no effect on NR. Additionally, the same publication noted a slight but non-significant reduction in fetal testicular testosterone levels following exposure to 150 mg/kg/day of DEHP, without any observed effects on NR (Martino-Andrade et al., 2009).
Further, one study found a lower LOAEL for NR than intratesticular testosterone (Taxvig et al., 2007) and another reported only dose (600 mg/kg bw/day), but not higher doses) significant for reduced intratesticular testosterone levels (Boberg et al., 2011). In both cases, there was a tendency for lower testosterone levels at other doses as well, and the inconsistency may therefore be due to low sample size and/or high variance in the testosterone data.
Overall, the data could suggest dose concordance for this KER, although the evidence for this is not strong.
Temporal concordance
The empirical evidence supports temporal concordance between the events.
NR is generally first observable in postnatal animals, while the reductions in intratesticular testosterone are measured early in fetal life during exposure. This was demonstrated with prochloraz-induced NR, which was observed at PND13, when intratesticular testosterone levels were reduced (Vinggaard AM et al., 2005). NR was also observed postnatally in studies, where exposure to the stressor was only during fetal life (Boberg et al., 2011; Hotchkiss AK et al., 2004; Martino-Andrade AJ et al., 2009; Taxvig C et al., 2007)
Incidence concordance
The data does not inform incidence concordance.
Uncertainties and Inconsistencies
In several of the studies supporting this KER, intratesticular testosterone was measured in ex vivo testis cultures. This means that fetal testis from animals exposed in utero were cultured for ~3 hours, and the culture media were then collected for testosterone measurement. This creates uncertainty in the exact intratesticular testosterone values. However, in these studies, intratesticular testosterone levels were also measured with largely similar outcomes from the methods. The large translatability is clear from the measurements in (Borch et al., 2004).
As discussed above, the study with negative results for NR might be an indication of dose concordance, as only one stressor dose was tested (Hotchkiss AK et al., 2004). The uncertainty in the study on diisonyl phthalate (Boberg et al., 2011) has also briefly been discussed. Exposure to the phthalate only reduced intratesticular testosterone in the dose of 600 mg/kg bw/day, but not 750 or 900 mg/kg bw/day. For these two higher doses, testosterone also tended to be lower, and lack of statistical significance may be explained by a low sample size.
The empirical evidence for this KER includes stressors with more than one known mechanism of action. In particular, the pesticides prochloraz and linuron are known to also be AR antagonists (Andersen et al., 2002; Lambright et al., 2000), and for these studies, it can therefore not be excluded whether the observed effect on NR is due to the chemicals lowering intratesticular testosterone levels or due to direct antagonism of the AR or a mixture of effects.
<h4>Quantitative Understanding of the Linkage</h4>
The quantitative understanding of this KER is classified as low.
Response-response relationship
There are no direct models for reductions in intratesticular testosterone levels and NR. A model for the phthalates has been developed, showing an induction of NR when ex vivo testosterone production is reduced to  ~40% of control males. After this point, the number of nipples per male increases significantly as testosterone levels decrease. Other chemicals than phthalates have not been tested on this model, and it therefore does not inform of a direct relationship between intratesticular testosterone and NR (Gray et al., 2024).
Time-scale
The time scale of this KER is weeks. In rodents, the mammary glands start developing in both sexes during fetal life. However, once the testes start producing testosterone (~GD15 in rats), the androgen hormones block the further development of the nipple anlagen in males. While the programming of the tissue happens during fetal development, the development of the nipples is not finished until after birth. In females and males with retained nipples, the nipples do not appear until after birth and are optimally assessed at PND12-14, when they have emerged, but the pups have not yet developed thick fur (Schwartz et al., 2021; Welsh et al., 2014).
Known modulating factors
<div>
<table class="table table-bordered table-fullwidth">
<thead>
<tr>
<th>Modulating Factor (MF)</th>
<th>MF Specification</th>
<th>Effect(s) on the KER</th>
<th>Reference(s)</th>
</tr>
</thead>
<tbody>
<tr>
<td>Rat strain</td>
<td> </td>
<td>Long-Evans Hooded rats are less sensitive to NR than Sprague-Dawley rats</td>
<td>
(Wolf et al., 1999; You et al., 1998)
</td>
</tr>
</tbody>
</table>
</div>
Known Feedforward/Feedback loops influencing this KER
There are no known feedback/feedforward loops for this KER.
<h4>References</h4>
Andersen, H. R., Vinggaard, A. M., Rasmussen, T. H., Gjermandsen, I. M., & Bonefeld-Jørgensen, E. C. (2002). Effects of currently used pesticides in assays for estrogenicity, androgenicity, and aromatase activity in vitro. Toxicology and Applied Pharmacology, 179(1), 1–12. https://doi.org/10.1006/taap.2001.9347
Boberg, J., Christiansen, S., Axelstad, M., Kledal, T. S., Vinggaard, A. M., Dalgaard, M., Nellemann, C., & Hass, U. (2011). Reproductive and behavioral effects of diisononyl phthalate (DINP) in perinatally exposed rats. REPRODUCTIVE TOXICOLOGY, 31(2), 200–209. https://doi.org/10.1016/j.reprotox.2010.11.001
Borch J, Ladefoged O, Hass U, & Vinggaard AM. (2004). Steroidogenesis in fetal male rats is reduced by DEHP and DINP, but endocrine effects of DEHP are not modulated by DEHA in fetal, prepubertal and adult male rats. Reproductive Toxicology (Elmsford, N.Y.), 18(1), 53–61. https://doi.org/10.1016/j.reprotox.2003.10.011
Gray L E Jr, Lambright CS, Evans N, Ford J, & Conley JM. (2024). Using targeted fetal rat testis genomic and endocrine alterations to predict the effects of a phthalate mixture on the male reproductive tract. Current Research in Toxicology, 7, 100180. https://doi.org/10.1016/j.crtox.2024.100180
Holmer, M. L., Zilliacus, J., Draskau, M. K., Hlisníková, H., Beronius, A., & Svingen, T. (2024). Methodology for developing data-rich Key Event Relationships for Adverse Outcome Pathways exemplified by linking decreased androgen receptor activity with decreased anogenital distance. Reproductive Toxicology, 128, 108662. https://doi.org/10.1016/j.reprotox.2024.108662
Hotchkiss AK, Parks-Saldutti LG, Ostby JS, Lambright C, Furr J, Vandenbergh JG, & Gray LE Jr. (2004). A mixture of the “antiandrogens” linuron and butyl benzyl phthalate alters sexual differentiation of the male rat in a cumulative fashion. Biology of Reproduction, 71(6), 1852–1861. https://doi.org/10.1095/biolreprod.104.031674
Kratochwil, K. (1986). Tissue Combination and Organ Culture Studies in the Development of the Embryonic Mammary Gland. In R. B. L. Gwatkin (Ed.), Manipulation of Mammalian Development (pp. 315–333). Springer US. https://doi.org/10.1007/978-1-4613-2143-9_11
Laier P, Metzdorff SB, Borch J, Hagen ML, Hass U, Christiansen S, Axelstad M, Kledal T, Dalgaard M, McKinnell C, Brokken LJ, & Vinggaard AM. (2006). Mechanisms of action underlying the antiandrogenic effects of the fungicide prochloraz. Toxicology and Applied Pharmacology, 213(2), 160–171. https://doi.org/10.1016/j.taap.2005.10.013
Lambright, C., Ostby, J., Bobseine, K., Wilson, V., Hotchkiss, A. K., Mann, P. C., & Gray, L. E. J. (2000). Cellular and molecular mechanisms of action of linuron: An antiandrogenic herbicide that produces reproductive malformations in male rats. Toxicological Sciences : An Official Journal of the Society of Toxicology, 56(2), 389–399. https://doi.org/10.1093/toxsci/56.2.389
Martino-Andrade AJ, Morais RN, Botelho GG, Muller G, Grande SW, Carpentieri GB, Leão GM, & Dalsenter PR. (2009). Coadministration of active phthalates results in disruption of foetal testicular function in rats. International Journal of Andrology, 32(6), 704–712. https://doi.org/10.1111/j.1365-2605.2008.00939.x
Murashima, A., Kishigami, S., Thomson, A., & Yamada, G. (2015). Androgens and mammalian male reproductive tract development. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 1849(2), 163–170. https://doi.org/10.1016/j.bbagrm.2014.05.020
Naamneh Elzenaty, R., Du Toit, T., & Flück, C. E. (2022). Basics of androgen synthesis and action. Best Practice & Research Clinical Endocrinology & Metabolism, 36(4), 101665. https://doi.org/10.1016/j.beem.2022.101665
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. 443: Extended One-Generation Reproductive Toxicity Study, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/9789264185371-en.
OECD (2025a), Test No. 421: Reproduction/Developmental Toxicity Screening Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/9789264264380-en.
OECD (2025c), Test No. 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/9789264264403-en.
Schwartz CL, Christiansen S, Hass U, Ramhøj L, Axelstad M, Löbl NM, & Svingen T. (2021). On the Use and Interpretation of Areola/Nipple Retention as a Biomarker for Anti-androgenic Effects in Rat Toxicity Studies. Frontiers in Toxicology, 3, 730752. https://doi.org/10.3389/ftox.2021.730752
Taxvig C, Hass U, Axelstad M, Dalgaard M, Boberg J, Andeasen HR, & Vinggaard AM. (2007). Endocrine-disrupting activities in vivo of the fungicides tebuconazole and epoxiconazole. Toxicological Sciences : An Official Journal of the Society of Toxicology, 100(2), 464–473. https://doi.org/10.1093/toxsci/kfm227
Vinggaard AM, Christiansen S, Laier P, Poulsen ME, Breinholt V, Jarfelt K, Jacobsen H, Dalgaard M, Nellemann C, & Hass U. (2005). Perinatal exposure to the fungicide prochloraz feminizes the male rat offspring. Toxicological Sciences : An Official Journal of the Society of Toxicology, 85(2), 886–897. https://doi.org/doi.org/10.1093/toxsci/kfi150
Watson, C. J., & Khaled, W. T. (2008). Mammary development in the embryo and adult: A journey of morphogenesis and commitment. Development, 135(6), 995–1003. https://doi.org/10.1242/dev.005439
Welsh M, Suzuki H, & Yamada G. (2014). The masculinization programming window. Endocrine Development, 27, 17–27. https://doi.org/10.1159/000363609
Wolf C Jr, Lambright C, Mann P, Price M, Cooper RL, Ostby J, & Gray LE Jr. (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(1), 94–118. https://doi.org/10.1177/074823379901500109
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
</div>
<div>
<h4><a href="/relationships/3486">Relationship: 3486: Decrease, circulating testosterone levels leads to nipple retention, increased</a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/575">Decreased testosterone synthesis leading to increased nipple retention (NR) in male (rodent) offspring</a></td>
<td>non-adjacent</td>
<td>Moderate</td>
<td></td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>rat</td>
<td>Rattus norvegicus</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10116" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>mouse</td>
<td>Mus musculus</td>
<td>Low</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10090" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Foetal</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Male</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
Taxonomic applicability
This KER is considered applicable to rodents (evidence primarily from laboratory rats and mice), where males normally lack nipples due to suppressed differentiation by high levels of androgens. The empirical evidence in this KER supports that reduction in testosterone causes NR in rats, whereas relevance in mice is assumed based on knowledge about developmental biology in this species. In humans, both sexes have two nipples, and there is no known androgen-driven sexual dimorphism (Schwartz et al., 2021). The KER is thus not considered directly applicable to humans. However, NR is a clear readout of reduced androgen action and fetal masculinization during development in rodents, which is relevant to humans (Schwartz et al., 2021). It is included as a mandatory endpoint in several rodent OECD Test Guidelines (OECD, 2025a, 2025b, 2025c) and considered an adverse outcome applicable to the setting of Points of Departure for use in human health risk assessment (OECD, 2013).
Sex applicability
This KER is only applicable to males, as female rats and mice develop 12 and 10 nipples, respectively (Schwartz et al., 2021). Females do have circulating testosterone in fetal life, but the levels are much lower than in males (Houtsmuller et al., 1995), and do therefore not suppress nipple formation.
Life stage applicability
The programming for androgen-driven suppression of nipple development in rodents occurs during fetal life, around gestational days (GD) 16-20 in rats (Imperato-McGinley et al., 1986). In both male and female rodents, the development of the mammary glands starts in fetal life, including initial growth and subsequent sexual differentiation (Kratochwil, 1986; Watson & Khaled, 2008). 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).
<h4>Key Event Relationship Description</h4>
This KER describes a fetal decrease in circulating testosterone (often measured in serum or plasma) leading to NR in male rodent offspring.  In rats and mice, females develop 10 and 12 nipples, respectively, with males typically displaying zero. In male rodents, testosterone is primarily produced by the fetal testes, secreted into the bloodstream and transported to the peripheral reproductive tissues, including the preliminary mammary tissue. Testosterone can bind directly to the AR in the tissue or first being converted to DHT by 5α-reductase (Murashima et al., 2015). AR activation by androgens in mesenchymal cells of the developing mammary glands causes cell death and subsequent separation of the tissue from the epidermis, resulting in no formation of nipples (Kratochwil, 1986). In females, where androgen levels are low, nipple formation is not blocked. The dependency of androgens for suppression of nipple development in males means that reductions in circulating testosterone levels can lead to retention of nipples.
In humans, both sexes have two nipples, and there is no known androgen-driven sexual dimorphism (Schwartz et al., 2021). The KER is thus not considered directly applicable to humans, but is a clear readout of reduced androgen action and fetal masculinization during development, which is relevant to humans (Schwartz et al., 2021). It is included as a mandatory endpoint in several rodent OECD Test Guidelines (OECD, 2025a, 2025b, 2025c) and considered an adverse outcome applicable to the setting of Points of Departure for use in human health risk assessment (OECD, 2013).
<h4>Evidence Supporting this KER</h4>
Biological Plausibility
The biological plausibility for this KER is judged to be high, given the canonical biological knowledge on normal reproductive development in rodents.
Sexual differentiation in males, including blocking of nipple development in rodents, is programmed in fetal life. Once formed, the fetal testes synthesize testosterone through the steroidogenesis pathway. Testosterone is secreted and transported in the bloodstream either as free testosterone or bound to plasma proteins (albumin or sex-hormone binding globulin). Testosterone binds AR in peripheral tissues and can also be converted to DHT by the enzyme 5α-reductase. Binding of testosterone and DHT to AR program the fetal reproductive tissue to male differentiation (Murashima et al., 2015).
In most rodents, development of the nipples is a sexually dimorphic process. In female rats, where androgen levels are low, the nipples develop along the milk lines forming 12 nipples, which are visible around postnatal day 12-14 (Schwartz et al., 2021). In male rats, AR activation in fetal life suppresses the formation of the nipples through apoptosis of epithelial cells in the developing mammary glands. Normally, male rats do therefore not have nipples, although in rare occasions male control rats may display one or more retained nipples (Kratochwil, 1986; Schwartz et al., 2021).
Testosterone is produced from around GD15 in fetal rats and is present in circulation around the same time. Programming of the nipple tissue to regress mainly occurs within the masculinization programming window (GD16-20 in rats) (Welsh et al., 2014).
Given the dependency of testosterone for the regression of the nipples, either through direct AR activation or conversion to DHT, it is highly plausible that a decrease in circulating levels of testosterone will lead to nipple retention in males.
Empirical Evidence
The empirical evidence from studies in animals for this KER is overall judged as moderate
From the data collection, two data sets were extracted. The data set included two different stressors causing reduced fetal levels of circulating testosterone in rats (Table 2 and appendix 2, <a href="https://aopwiki.org/system/dragonfly/production/2025/09/17/61b9o238vs_KER_3486_Appendix_2.pdf">61b9o238vs_KER_3486_Appendix_2.pdf</a>). Both data sets showed concurrent NR.
Table 2 Empirical evidence for KER 3486 LOAEL: Lowest observed adverse effect level; See appendix 2 for specifications.
<table cellspacing="0" class="MsoTableGrid" style="border-collapse:collapse; border:none; width:629px">
<tbody>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; height:53px; vertical-align:top; width:140px">
Species
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:53px; vertical-align:top; width:145px">
Stressors(s)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:53px; vertical-align:top; width:130px">
Effect on upstream event
(circulating testosterone)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:53px; vertical-align:top; width:121px">
Effect on downstream event (NR)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:53px; vertical-align:top; width:93px">
Reference
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:140px">
Rat
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:145px">
Diethylhexyl phthalate
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:130px">
LOAEL 750 mg/kg bw/day
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:121px">
LOAEL 750 mg/kg bw/day
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:93px">
(Borch et al., 2004)
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:140px">
Rat
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:145px">
Prochloraz
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:130px">
LOAEL 30 mg/kg bw/day
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:121px">
LOAEL 30 mg/kg bw/day
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:53px; vertical-align:top; width:93px">
(Vinggaard et al., 2005)
</td>
</tr>
</tbody>
</table>
Dose concordance
The empirical evidence for this KER does not inform dose concordance, as no study uses more than one dose of stressor.
Temporal concordance
NR is generally first observable in postnatal animals, while the reductions in intratesticular testosterone are measured early in fetal life during exposure. This was demonstrated with prochloraz-induced NR, which was observed at PND13, but not when examining the males at GD21, when circulating testosterone levels were reduced (Vinggaard AM et al., 2005).
Incidence concordance
The data does not inform incidence concordance.
Uncertainties and Inconsistencies
The low number of studies retrieved in the empirical evidence collection, this KER is in itself an uncertainty, and both studies only investigated one stressor dose.
An uncertainty in the empirical evidence is that prochloraz is also known to be an AR antagonist (Andersen et al., 2002), and it can therefore not be excluded that the effects of prochloraz on NR is, at least partly, due to direct antagonism of AR and not due to the low testosterone levels.
<h4>Quantitative Understanding of the Linkage</h4>
The quantitative understanding of this KER is classified as low.
Response-response relationship
There is no known information on the response-response relationship for this KER.
Time-scale
The time scale of this KER is weeks. Testosterone is secreted from ~GD15 in rats, which is at the beginning of the masculinization programming window. The mammary glands start developing during fetal life as well, but cannot be observed in female rodents until weeks after birth, which is also the time at which NR can be observed in males (Schwartz et al., 2021)
Known modulating factors
<table class="table table-bordered table-fullwidth">
<thead>
<tr>
<th>Modulating Factor (MF)</th>
<th>MF Specification</th>
<th>Effect(s) on the KER</th>
<th>Reference(s)</th>
</tr>
</thead>
<tbody>
<tr>
<td>Rat strain</td>
<td> </td>
<td>Long-Evans Hooded rats are less sensitive to NR than Sprague Dawley rats</td>
<td>(Wolf et al., 1999; You et al., 1998)</td>
</tr>
</tbody>
</table>
Known Feedforward/Feedback loops influencing this KER
There are no known feedback/feedforward loops for this KER.
<h4>References</h4>
Andersen, H. R., Vinggaard, A. M., Rasmussen, T. H., Gjermandsen, I. M., & Bonefeld-Jørgensen, E. C. (2002). Effects of currently used pesticides in assays for estrogenicity, androgenicity, and aromatase activity in vitro. Toxicology and Applied Pharmacology, 179(1), 1–12. https://doi.org/10.1006/taap.2001.9347
Borch J, Ladefoged O, Hass U, & Vinggaard AM. (2004). Steroidogenesis in fetal male rats is reduced by DEHP and DINP, but endocrine effects of DEHP are not modulated by DEHA in fetal, prepubertal and adult male rats. Reproductive Toxicology (Elmsford, N.Y.), 18(1), 53–61. https://doi.org/10.1016/j.reprotox.2003.10.011
Holmer, M. L., Zilliacus, J., Draskau, M. K., Hlisníková, H., Beronius, A., & Svingen, T. (2024). Methodology for developing data-rich Key Event Relationships for Adverse Outcome Pathways exemplified by linking decreased androgen receptor activity with decreased anogenital distance. Reproductive Toxicology, 128, 108662. https://doi.org/10.1016/j.reprotox.2024.108662
Houtsmuller, E. J., de Jong, F. H., Rowland, D. L., & Slob, A. K. (1995). Plasma testosterone in fetal rats and their mothers on day 19 of gestation. Physiology & Behavior, 57(3), 495–499. <a href="https://doi.org/10.1016/0031-9384(94)00291-C" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1016/0031-9384(94)00291-C</a>
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. <a href="https://doi.org/10.1210/endo-118-1-132" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1210/endo-118-1-132</a>
Kratochwil, K. (1986). Tissue Combination and Organ Culture Studies in the Development of the Embryonic Mammary Gland. In R. B. L. Gwatkin (Ed.), Manipulation of Mammalian Development (pp. 315–333). Springer US. https://doi.org/10.1007/978-1-4613-2143-9_11
Murashima, A., Kishigami, S., Thomson, A., & Yamada, G. (2015). Androgens and mammalian male reproductive tract development. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 1849(2), 163–170. <a href="https://doi.org/10.1016/j.bbagrm.2014.05.020" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1016/j.bbagrm.2014.05.020</a>
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. 443: Extended One-Generation Reproductive Toxicity Study, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/9789264185371-en.
OECD (2025b), Test No. 421: Reproduction/Developmental Toxicity Screening Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/9789264264380-en.
OECD (2025c), Test No. 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, <a href="https://doi.org/10.1787/9789264264403-en" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1787/9789264264403-en</a>.
Schwartz CL, Christiansen S, Hass U, Ramhøj L, Axelstad M, Löbl NM, & Svingen T. (2021). On the Use and Interpretation of Areola/Nipple Retention as a Biomarker for Anti-androgenic Effects in Rat Toxicity Studies. Frontiers in Toxicology, 3, 730752. https://doi.org/10.3389/ftox.2021.730752
Vinggaard AM, Christiansen S, Laier P, Poulsen ME, Breinholt V, Jarfelt K, Jacobsen H, Dalgaard M, Nellemann C, & Hass U. (2005). Perinatal exposure to the fungicide prochloraz feminizes the male rat offspring. Toxicological Sciences : An Official Journal of the Society of Toxicology, 85(2), 886–897. https://doi.org/doi.org/10.1093/toxsci/kfi150
Watson, C. J., & Khaled, W. T. (2008). Mammary development in the embryo and adult: A journey of morphogenesis and commitment. Development, 135(6), 995–1003. https://doi.org/10.1242/dev.005439
Welsh M, Suzuki H, & Yamada G. (2014). The masculinization programming window. Endocrine Development, 27, 17–27. https://doi.org/10.1159/000363609
Wolf C Jr, Lambright C, Mann P, Price M, Cooper RL, Ostby J, & Gray LE Jr. (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(1), 94–118. https://doi.org/10.1177/074823379901500109
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
</div>
<div>
<h4><a href="/relationships/3348">Relationship: 3348: Decrease, AR activation leads to nipple retention, increased</a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/576">5α-reductase inhibition leading to increased nipple retention (NR) in male (rodent) offspring</a></td>
<td>non-adjacent</td>
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/aops/344">Androgen receptor (AR) antagonism leading to nipple retention (NR) in male (mammalian) offspring</a></td>
<td>non-adjacent</td>
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/aops/575">Decreased testosterone synthesis leading to increased nipple retention (NR) in male (rodent) offspring</a></td>
<td>non-adjacent</td>
<td>High</td>
<td></td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>rat</td>
<td>Rattus norvegicus</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10116" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>mouse</td>
<td>Mus musculus</td>
<td>Low</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10090" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Foetal</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Male</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
<a name="_Hlk152319700">Taxonomic</a>
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 embryonic days 13-15 (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.
<h4>Key Event Relationship Description</h4>
This KER  links a decrease in androgen receptor (AR) activation during fetal development to increased nipple/areola retention (NR) in male rodent offspring. It should be noted that the upstream Key Event (KE) ‘decrease, androgen receptor activation’ (KE-1614 in AOP Wiki) specifically focuses on decreased activation of the AR in vivo, while most methods that can be used to measure AR activity are carried out in vitro. Indirect information about this KE may, for example, be provided from assays showing in vitro AR antagonism, decreased in vitro or in vivo testosterone production/levels, or decreased in vitro or in vivo dihydrotestosterone (DHT) production/levels.
The KER is not directly applicable to humans as both sexes 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).
<h4>Evidence Supporting this KER</h4>
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 nuclear receptor and transcription factor 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 rats and mice. In humans, both sexes have two nipples. In contrast, common laboratory mice and rats 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 mice 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 androgen receptor, present with retained nipples (Kratochwil & Schwartz, 1976).
- Multiple mechanisms of action may potentially lead to nipple retention in male mouse and rat offspring. DHT is the main androgen responsible for nipple/areola regression 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.
It should be noted that the KE decreased AR activation (KE 1614 in AOP Wiki) specifically focuses on decreased activation of the AR in vivo, with no methods currently available to measure this. Examples of assays that provide indirect information about KE 1614 are described in upstream MIE/KEs.
The empirical evidence for this KER from animal studies in vivo is based on studies using six different substances that result in decreased AR activation by different mechanisms. Flutamide, procymidone and vinclozolin bind to the AR and inhibit the receptor activity and thereby act as AR antagonists, see MIE 26. Finasteride inhibits the 5-alpha-reductase enzyme that converts testosterone to DHT, see MIE 1617. DEHP and DBP exposure during prenatal development in rats results in reduced fetal testosterone levels, see KE-2298 and KE1690. (MIE 26, MIE 1617 and KE 1690 can be found in AOP-Wiki).
The evidence for the upstream KE is mainly based on data from in vitro assays (AR antagonism or 5-alpha-reductase inhibition in vitro), whereas the evidence for the downstream KE is based on in vivo studies, and there is generally no evidence for both KEs from the same study. However, decreased testosterone levels can be measured in vivo, and (Howdeshell et al., 2007; Martino-Andrade et al., 2009) measured the effect of developmental phthalate exposure on both testosterone levels and nipple/areola retention (see the section about “Dose concordance”).
The empirical evidence for the six substances is summarised in Table 3.
Table 3. Summary of empirical evidence for decreased androgen receptor activation, leading to decreased nipple/areola retention. References for the studies supporting the empirical evidence are found in the section “Evidence for decreased AR activation (KE 1614) by flutamide, procymidone, and vinclozolin, finasteride, DEHP and DBP” and in Table 4 in Appendix 2 (<a href="https://aopwiki.org/system/dragonfly/production/2025/09/18/6djoma9gmj_KER_3348_Appendix_2_.pdf">6djoma9gmj_KER_3348_Appendix_2_.pdf</a>).
<table cellspacing="0" class="Table" style="border-collapse:collapse; border:none; width:590px">
<tbody>
<tr>
<td style="background-color:#e7e6e6; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:94px">
Stressor(s)
</td>
<td style="background-color:#e7e6e6; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:245px">
Upstream effect
(decreased AR activation)
</td>
<td style="background-color:#e7e6e6; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:251px">
Downstream effect
(Increased nipple/areola retention)
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:94px">
Flutamide
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:245px">
AR antagonism in in vitro assay, receptor binding and transactivation assays
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:251px">
Increased NR in males after prenatal exposure in studies in rat
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:94px">
Procymidone
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:245px">
AR antagonism in in vitro assay receptor binding and transactivation assays
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:251px">
Increased NR in males after prenatal exposure in studies in rat
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:94px">
Vinclozolin
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:245px">
AR antagonism in in vitro assay receptor binding and transactivation assays
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:251px">
Increased NR in males after prenatal exposure in studies in rat
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:94px">
Finasteride
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:245px">
Inhibition of 5-alpha-reductase enzyme in in vitro assays
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:251px">
Increased NR in males after prenatal exposure in studies in rat
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:7px; vertical-align:top; width:94px">
DEHP
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:7px; vertical-align:top; width:245px">
Reduced production of testosterone in fetal testis measured in ex vivo testis assays, reduced testosterone levels in testis, and reduced fetal plasma or serum testosterone levels
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:7px; vertical-align:top; width:251px">
Increased NR in males after prenatal exposure in studies in rat
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:7px; vertical-align:top; width:94px">
DBP
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:7px; vertical-align:top; width:245px">
Reduced production of testosterone in fetal testis measured in ex vivo testis assays and reduced testosterone levels in fetal testis
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:7px; vertical-align:top; width:251px">
Increased NR in males after prenatal exposure in studies in rat
</td>
</tr>
</tbody>
</table>
From Table 3, it can be deduced that fetal exposure to substances known to decrease androgen receptor activation through antagonism of the AR (vinclozolin, procymidone, flutamide), inhibition of testosterone synthesis (DEHP, DBP) or inhibition of the conversion of testosterone to DHT (finasteride), results in increased nipple/areola retention in rat male offspring.
Evidence for decreased AR activation (KE 1614) by flutamide, procymidone, vinclozolin, finasteride, DEHP and DBP.
Flutamide, a pharmaceutical, binds the AR and inhibits its activity, thereby acting as an AR antagonist. It has been used as an antiandrogen for the treatment of prostate cancer and is used as a reference chemical for antiandrogenic activity in the AR transactivation assays in the OECD test guideline No 458 (Goldspiel & Kohler, 1990; Labrie, 1993; OECD, 2023; Simard et al., 1986)
Procymidone and vinclozolin are fungicides that have been shown to be AR antagonists. Procymidone binds to the AR and inhibits the agonist binding, as shown in AR binding assays using rat prostate cytosol (Hosokawa et al., 1993) or AR transfected cells (Ostby et al., 1999). Procymidone also inhibits agonist activated transcription in AR reporter assays (Hass et al., 2012; Kojima et al., 2004; Orton et al., 2011; Ostby et al., 1999; Scholze et al., 2020). Vinclozolin binds to the AR and inhibits the agonist binding, as shown in AR binding assays using rat epididymis cytosol (Kelce & Wilson, 1997) or AR transfected cells (Wong et al., 1995). Vinclozolin also inhibits agonist activated transcription in AR reporter assays (Euling, 2002; Kojima et al., 2004; Molina-Molina et al., 2006; Orton et al., 2011; Scholze et al., 2020; Shimamura et al., 2002; Wong et al., 1995).
Finasteride is a pharmaceutical that inhibits the 5-alpha-reductase enzyme that converts testosterone to DHT. Finasteride is used to treat benign prostatic hypertrophy (Andersson & Russell, 1990; Stoner, 1990; Wood & Rittmaster, 1994).
Prenatal exposure to DEHP in rats has been shown to reduce the production of testosterone in fetal testis measured in ex vivo testis assays, and to reduce testosterone levels in testis and in fetal plasma and serum (Borch et al., 2006; Borch J et al., 2004; Culty et al., 2008; Hannas et al., 2011, 2012; Howdeshell et al., 2007; Klinefelter et al., 2012; Parks, 2000; VO et al., 2009; Wilson et al., 2004, 2007). Conversely, prenatal DEHP exposure did not result in any effects on testosterone levels in the testis at PND1 in one study by Andrade et al. (2006) (Andrade et al., 2006). Similar to DEHP, prenatal exposure to DBP has been shown to reduce the production of testosterone in fetal rat testis measured in ex vivo testis studies (Howdeshell et al., 2007; Wilson et al., 2004) and reduce testosterone levels in the fetal rat testis (Martino-Andrade et al., 2009). The precise underlying mechanism for these effects of DEHP and DPB is presently unknown.
Evidence for increased nipple/areola retention in males (AO-1786) by prenatal exposure to flutamide, procymidone, vinclozolin, finasteride, DEHP and DBP.
All datasets that were used for the weight of evidence assessment were judged as reliable without or with restriction. The majority of datasets assessed showed an increased nipple/areola retention in male offspring after gestational exposure. The conclusion was that the level of confidence was strong for all six substances. The studies are summarised in Table 4 in Appendix 2, <a href="https://aopwiki.org/system/dragonfly/production/2025/09/18/6djoma9gmj_KER_3348_Appendix_2_.pdf">6djoma9gmj_KER_3348_Appendix_2_.pdf</a>
Dose concordance
Dose concordance is challenging to assess for this KER since in vivo AR activity is currently not possible to measure, but can only be inferred indirectly by measures of upstream events. In some studies, fetal (testicular) testosterone levels during, or close to, the fetal masculinization programming window are measured in a subset of animals exposed similarly to those investigated for NR post-natally. Such information may inform on dose concordance if more doses are included.
In a rat in utero exposure study (GD13-21) with DPB and DEHP, testosterone levels in the fetal testes were investigated at GD21, and NR was investigated at PND13 (Martino-Andrade et al., 2009). For DBP, both reduced testosterone levels in fetal testes and NR were observed at 500 mg/kg/d, whereas no effect on NR and only a slight non-significant reduction of testosterone was observed at the lower dose (100 mg/kg/d). For DEHP, a slight but non-significant decrease in testosterone levels in fetal rat testis was observed after exposure to 150 mg/kg/d DEHP, with no effects on nipple/areola retention.
Such data could suggest dose concordance for this part of the KER, although the evidence for this is not strong.
Temporal concordance
Temporal concordance can only be considered from a theoretical perspective since the downstream event, increased NR, is a result of disruption to 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 – decreased AR activation in vivo – takes place minutes to hours after exposure to an anti-androgenic substance. If a substance decreases AR activation through inhibition of the AR, the upstream event is expected to happen immediately after exposure. If a substance decreases androgen receptor activation through inhibition of testosterone synthesis, the upstream event is expected to happen minutes to hours after the exposure.
Uncertainties and Inconsistencies
For DEHP and DBP, there were some inconsistencies in the empirical evidence, but they could be explained by differences in study designs and uncertainties in measurements (see Appendix 1). Some uncertainty is imposed by the poorly supported dose-concordance. However, the dose-concordance is well supported by the current understanding of biological processes.
<h4>Quantitative Understanding of the Linkage</h4>
The quantitative understanding of the linkage is low. This is a consequence of it not being possible to measure the upstream and the downstream events in the same study.
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 dimerization of the AR takes 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 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)
Known Feedforward/Feedback loops influencing this KER
Not relevant for this KER.
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