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Relationship: 3348

Title

A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

Decrease, AR activation leads to nipple retention, increased

Upstream event

The causing Key Event (KE) in a Key Event Relationship (KER). More help

Decrease, AR activation

Downstream event

The responding Key Event (KE) in a Key Event Relationship (KER). More help

nipple retention, increased

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes.Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Point of Contact	Author Status	OECD Status
5α-reductase inhibition leading to increased nipple retention (NR) in male (rodent) offspring	non-adjacent	High	Terje Svingen (send email)	Under development: Not open for comment. Do not cite
Androgen receptor (AR) antagonism leading to nipple retention (NR) in male (mammalian) offspring	non-adjacent	High	Terje Svingen (send email)	Under development: Not open for comment. Do not cite	Under Development
Decreased testosterone synthesis leading to increased nipple retention (NR) in male (rodent) offspring	non-adjacent	High	Terje Svingen (send email)	Under development: Not open for comment. Do not cite

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER. More help

Term	Scientific Term	Evidence	Link
rat	Rattus norvegicus	High	NCBI
mouse	Mus musculus	Low	NCBI

Sex Applicability

An indication of the the relevant sex for this KER. More help

Sex	Evidence
Male	High

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER. More help

Term	Evidence
Foetal	High

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. More help

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).

Evidence Collection Strategy

Include a description of the approach for identification and assembly of the evidence base for the KER. For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings. Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

A systematic weight-of-evidence approach was applied to collect, evaluate, extract, and integrate evidence from in vivo studies (Figure 1, 71z732d7be_KER_3348_figure_1.png (752×296)).

Search terms and search strings were developed for PubMed and Web of Science.

PubMed: ("androgen receptor*" OR "testosterone receptor*" OR "receptors, androgen"[MeSH Terms] OR "androgen*" OR "testosterone*" OR "dihydrotestosterone*" OR "androgens"[MeSH Terms] OR "androgen antagonists"[MeSH Terms]) AND ("nipple*" OR "areola*")

Web of Science: ("androgen receptor*" OR "testosterone receptor*" OR "androgen*" OR "testosterone*" OR "dihydrotestosterone*") AND ("nipple*" OR "areola*")

Literature searches in PubMed and Web of Science were performed on January 16, 2024. The obtained publications were imported into RAYYAN software (https://www.rayyan.ai) and duplicates were removed, resulting in 376 publications (Figure 1).

The following inclusion and exclusion criteria were used for screening the titles and abstracts in RAYYAN.

Inclusion criteria:

Primary literature on
- exposure to androgenic or antiandrogenic compounds and nipple/areola retention as an outcome in in vivo studies in mammals
- measurement of androgen levels, AR activity or other androgen biomarkers, and nipple/areola retention in in vivo studies in mammals
- In vitro and in vivo mechanistic studies on nipple/areola retention
Reviews on nipple/areola retention

Exclusion criteria:

Not in English
Abstracts and other non-full-text publications

139 publications were included based on the screening of the titles and abstracts by two reviewers. In addition, 21 publications from the reference lists of two previous reviews on NR were included (Pedersen et al., 2022; Schwartz et al., 2021).

Study quality assessment and data extraction for in vivo studies on nipple/areola retention

To cover decreased AR activation resulting from AR antagonism, decreased testosterone production/levels, and decreased dihydrotestosterone (DHT) production/levels, information on model substances acting through these different pathways was included. Thus, full-text publications on in vivo studies of effects on NR in mammals after prenatal exposure to DEHP, DBP, finasteride, flutamide, procymidone, or vinclozolin were analysed by one reviewer per model substance. In short, data from the publications was extracted into an Excel template. Studies were divided into different datasets if different exposure windows or time points for observation of NR were used. The reliability of the datasets was assessed using the SciRAP tool (http://www.scirap.org). In addition to the guidance for evaluation of each methodological quality criterion available in the SciRAP tool, specific considerations were developed and applied in this case and are listed in Table 1 in Appendix 1 (hcnfc4qts_KER_3348_Appendix_Decreased_AR_activation_to_NR.pdf).

For each dataset, the result of the SciRAP evaluation was translated into reliability categories using the principles laid out in Table 1.

Table 1. Principles for translation of SciRAP evaluation results to reliability categories.

Reliability Category	Principles
1. Reliable without restriction	SciRAP methodological quality Score > 80 and all key criteria* are “Fulfilled” and there are no deficiencies in the non-key criteria that might affect study reliability.
2. Reliable with restrictions	SciRAP methodological quality Score > 65 and one or several of the key criteria are “Partially Fulfilled”, or there are minor deficiencies in the non-key criteria that might affect study reliability.
3. Not reliable	SciRAP methodological quality Score < 65 or one or several of the key criteria are “Not Fulfilled”, or there are major deficiencies in the non-key criteria that affect reliability.
4. Not assignable	Two or more of the key criteria are “Not Determined”

*Key criteria are criteria judged as specifically critical for the reliability of the data in a certain case and are determined a priori. The following five SciRAP criteria for methodological quality were considered as key criteria: A concurrent negative control group was included; The timing and duration of administration were appropriate for investigating the included endpoints; Measurements were collected at suitable time points in order to generate sensitive, valid, and reliable data; A sufficient number of animals per dose group were subjected to separate tests/data collection/measurements to generate reliable and valid results; and The statistical methods have been clearly described and do not seem inappropriate, unusual or unfamiliar.

The level of confidence in the overall data for each substance was categorised using the principles laid out in Table 2.

Table 2. Principles for categorisation of the level of confidence.

Level of confidence	Principles for Categorisation**
Strong	Effects were observed in one or more datasets judged as reliable without restriction or reliable with restriction; there are no conflicting results from datasets judged as reliable with or without restriction. OR Effects were observed in one or more datasets judged as reliable without restriction or reliable with restriction, but conflicting results, i.e. no or opposite effects were observed in other datasets judged as reliable with or without restriction. However, conflicts in results could be explained by differences in study design, for example, different exposure periods, doses or animal species or cell models.
Moderate	Effects were observed in one or more datasets judged as reliable without restriction or reliable with restriction, but conflicting results, i.e., no or opposite effects were observed in other datasets judged as reliable with or without restriction. Conflicts of results could not be explained by differences in study design, for example, different exposure periods, doses, or animal species or cell models. Effects were observed in at least half of the datasets.
Weak	Effects were observed in one or more datasets judged as reliable without restriction or reliable with restriction but conflicting results, i.e., no or opposite effects were observed in other datasets judged as reliable with or without restriction. Conflicts of results could not be explained by differences in study design, for example, different exposure periods, doses or animal species or cell models. Effects were observed in fewer than half of the datasets. OR Effects were only observed in one or more datasets judged as not reliable or not assignable.
No effect	No effects were observed in any of the datasets reviewed.

**Conflicting results from datasets judged as not reliable did not impact the categorisation.

Identification of studies on the mechanistic understanding of substances causing increased nipple/areola retention

Relevant studies describing mechanisms for the substances flutamide, procymidone, vinclozolin, finasteride and DEHP were derived from the empirical evidence for the shared upstream KE decreased AR activation described recently for KER 2820 (Holmer et al., 2024). For DBP, which was not used as a model substance in KER 2820, studies describing mechanisms were identified from 1) the publications included on the in vivo studies of DBP in this KER and 2) publications on mechanisms of DEHP and DBP included in (Holmer et al., 2024).

Evidence Supporting this KER

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help

Biological Plausibility

Addresses the biological rationale for a connection between KEupstream and KEdownstream. This field can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured. More help

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

Provides specific (citable) evidence that supports the idea of a change in the upstream KE (KEupstream) leading to, or being associated with, a subsequent change in the downstream KE (KEdownstream), assuming the perturbation of KEupstream is sufficient. More help

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 (6djoma9gmj_KER_3348_Appendix_2_.pdf).

Stressor(s)	Upstream effect (decreased AR activation)	Downstream effect (Increased nipple/areola retention)
Flutamide	AR antagonism in in vitro assay, receptor binding and transactivation assays	Increased NR in males after prenatal exposure in studies in rat
Procymidone	AR antagonism in in vitro assay receptor binding and transactivation assays	Increased NR in males after prenatal exposure in studies in rat
Vinclozolin	AR antagonism in in vitro assay receptor binding and transactivation assays	Increased NR in males after prenatal exposure in studies in rat
Finasteride	Inhibition of 5-alpha-reductase enzyme in in vitro assays	Increased NR in males after prenatal exposure in studies in rat
DEHP	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	Increased NR in males after prenatal exposure in studies in rat
DBP	Reduced production of testosterone in fetal testis measured in ex vivo testis assays and reduced testosterone levels in fetal testis	Increased NR in males after prenatal exposure in studies in rat

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, 6djoma9gmj_KER_3348_Appendix_2_.pdf

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

Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

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.

Known modulating factors

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references. More help

A well established modulating factor for androgen action is genetic variations in the AR, which decrease the function of the receptor. For example, longer CAG repeat lengths have been associated with decreased AR activation (Chamberlain et al., 1994; Tut et al., 1997). Rat strain is another important modulating factor, with studies showing that the Long-Evans Hooded rat is less sensitive to nipple/areola retention than the Sprague-Dawley rat (Wolf et al., 1999; You et al., 1998)

Quantitative Understanding of the Linkage

Captures information that helps to define how much change in the upstream KE, and/or for how long, is needed to elicit a detectable and defined change in the downstream KE. More help

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

Provides sources of data that define the response-response relationships between the KEs. More help

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

Information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). More help

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 Feedforward/Feedback loops influencing this KER

Define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits. More help

Not relevant for this KER.

Domain of Applicability

A free-text section of the KER description that the developers can use to explain their rationale for the taxonomic, life stage, or sex applicability structured terms. More help

Taxonomic

The KER is considered directly applicable to rats and mice, in which males normally have no nipples due to high levels of androgens during development, leading to regression of nipple anlagen. The empirical evidence supports the relevance to rats, whereas the relevance in mice is assumed based on knowledge about developmental biology in this species. Applicability may extend to most rodents.

While NR is not directly translatable to humans, it serves as a clear indicator of diminished androgen activity causing disrupted fetal masculinisation and sexual differentiation during development - an effect considered relevant to mammals, humans (Schwartz et al., 2021), and vertebrates more broadly (Ogino et al., 2023). NR is included as a mandatory endpoint in several rodent OECD Test Guidelines (OECD 2025a; OECD 2025b, OECD 2025c) and in OECD GD 151 considered an adverse outcome applicable to the setting of Points of Departure for use in human health risk assessment (OECD, 2013). NR can also be used as an indicator of anti-androgenicity in mammals and vertebrates in the environment due to the conserved nature of the AR and its implication in sexual differentiation across species (Ogino et al., 2023).

Life stage

Programming of nipple/areola regression in males occurs during a short window of sensitivity to androgens in the nipple anlagen during fetal life. This takes place in rats around 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.

References

List of the literature that was cited for this KER description. More help

Amato, C. M., Yao, H. H.-C., & Zhao, F. (2022). One Tool for Many Jobs: Divergent and Conserved Actions of Androgen Signaling in Male Internal Reproductive Tract and External Genitalia. Frontiers in Endocrinology, 13. https://doi.org/10.3389/fendo.2022.910964

Andersson, S., & Russell, D. W. (1990). Structural and biochemical properties of cloned and expressed human and rat steroid 5 alpha-reductases. Proceedings of the National Academy of Sciences, 87(10), 3640–3644. https://doi.org/10.1073/pnas.87.10.3640

Andrade, A. M., Grande, S. W., Talsness, C. E., Grote, K., & Chahoud, I. (2006). Developmental exposure to high and low doses of di-(2-ethylhexyl) phthalate (DEHP); Effects on male rat offspring. NAUNYN-SCHMIEDEBERGS ARCHIVES OF PHARMACOLOGY, 372, 100–101.

Barlow, N. J., McIntyre, B. S., & Foster, P. M. D. (2004). Male reproductive tract lesions at 6, 12, and 18 months of age following in utero exposure to di(n-butyl) phthalate. TOXICOLOGIC PATHOLOGY, 32(1), 79–90. https://doi.org/10.1080/01926230490265894

Borch, J., Axelstad, M., Vinggaard, A. M., & Dalgaard, M. (2006). Diisobutyl phthalate has comparable anti-androgenic effects to di-n-butyl phthalate in fetal rat testis. Toxicology Letters, 163(3), 183–190. https://doi.org/10.1016/j.toxlet.2005.10.020

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

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