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Relationship: 3486

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, circulating testosterone levels leads to nipple retention, increased

Upstream event
The causing Key Event (KE) in a Key Event Relationship (KER). More help
Decrease, circulating testosterone levels
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 Quantitative Understanding Point of Contact Author Status OECD Status
Decreased testosterone synthesis leading to increased nipple retention (NR) in male (rodent) offspring non-adjacent Moderate 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 Do not cite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 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).

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 approach (Fig. 1, 570lkl0x3z_KER_3486_Figure_1.png (1355×301)) was used to collect evidence based on the methodology described in (Holmer et al., 2024). The evidence collection for this KER was done concurrently with the evidence collection for KER-3487 ‘Decreased, intratesticular testosterone leads to nipple retention, increased’, for which the same search string was used. 

Search strategy

Search strings were synthesized for PubMed and Web of Science based on the review question, ‘Does decreased testosterone during fetal development lead to decreased anogenital distance in male mammals?’  

Search string in PubMed: "testosterone*" AND ("nipple*” OR “areola*”)

Search string in Web of Science: "testosterone*" AND ("nipple*” OR “areola*”)

The searches were performed on 01.10.2024

Title & abstract screening:

Retrieved articles were screened in the online tool RAYYAN https://www.rayyan.ai/. After the removal of duplicates, the titles and abstracts of the remaining 218 articles were screened according to pre-defined inclusion and exclusion criteria:

Inclusion criteria:

  • In vivo studies in male mammals where fetal testosterone is reduced and nipples / areolas are measured *
  • Reviews on nipple/areola retention
  • In vitro, ex vivo, and in vivo mechanistic studies on nipple/areola retention

Exclusion criteria:

  • Papers not in English
  • Abstracts and other non-full-text publications

*In cases where this criterion could not be determined by reading the abstract, the study was included for full text review.  

Full text review, data extraction and reliability evaluation of animal studies:

For the in vivo studies, the full-text papers were reviewed using the same exclusion criteria as in the title & abstract screening, and data were extracted from the included papers into an Excel template. In parallel, methodological reliability was assessed using the online tool Science in Risk Assessment and Policy (SciRAP; http://www.scirap.org, see appendix 1, 8xgu1c69js_KER_3486_Appendix_1.pdf). Based on the SciRAP evaluations, animal studies were assigned a reliability category using the principles outlined in table 1. Studies were divided into different datasets if multiple different chemicals, different exposure windows, or different timepoints of measurement of NR were included.

Moreover, as this KER was made in parallel with several other KERs for other male reproductive endpoints (anogenital distance and hypospadias), five studies retrieved in the searches for these KERs, which also measured NR, but were not detected in the search for this KER were also added, data extracted and evaluated for reliability.

The collected data were then filtered to only include data sets measuring circulating testosterone, either in plasma or serum. Mixture studies were excluded from the final evidence as the chemicals may have many different upstream mechanisms.

The overall strength of the empirical support was assessed according to the principles outlined in the AOP wiki handbook. Only studies in reliability categories 1 (reliable without restriction) and 2 (reliable with restriction) were used for the assessment of overall confidence in the data.

Table 1 Principles for translation of SciRAP evaluations into reliability categories.

Reliability Category

Principles for Categorization

1.Reliable without restriction

SciRAP methodological quality Score > 80 and all key criteriaa are “Fulfilled” and there are no deficiencies in the non-key criteria that might affect study reliability.

2. Reliable with restriction

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 study reliability.

4. Not assignable

Two or more of the key criteria are “Not Determined”

aKey criteria were SciRAP criteria for methodological quality, judged as specifically critical for reliability of the data for this KER and were determined a priori. The key criteria for this data collection are outlined in appendix 1 (8xgu1c69js_KER_3486_Appendix_1.pdf).

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, 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.

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

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.

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
Modulating Factor (MF) MF Specification Effect(s) on the KER Reference(s)
Rat strain   Long-Evans Hooded rats are less sensitive to NR than Sprague Dawley rats (Wolf et al., 1999; You et al., 1998)
Response-response Relationship
Provides sources of data that define the response-response relationships between the KEs.  More help

There is no known information on the response-response relationship for this KER.

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

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 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

There are no known feedback/feedforward loops 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 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).

References

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

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. https://doi.org/10.1016/0031-9384(94)00291-C

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

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. https://doi.org/10.1016/j.bbagrm.2014.05.020

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, 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

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