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Event: 1786

Key Event Title

A descriptive phrase which defines a discrete biological change that can be measured. More help

Nipple retention (NR), increased

Short name
The KE short name should be a reasonable abbreviation of the KE title and is used in labelling this object throughout the AOP-Wiki. More help
nipple retention, increased
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Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. More help
Level of Biological Organization

Key Event Components

The KE, as defined by a set structured ontology terms consisting of a biological process, object, and action with each term originating from one of 14 biological ontologies (Ives, et al., 2017; Biological process describes dynamics of the underlying biological system (e.g., receptor signalling).Biological process describes dynamics of the underlying biological system (e.g., receptor signaling).  The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signaling by that receptor).  Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description.  To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons.  If a desired term does not exist, a new term request may be made via Term Requests.  Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE.Clicking on the name of the AOP will bring you to the individual page for that AOP. More help
AOP Name Role of event in AOP Point of Contact Author Status OECD Status
AR antagonism leading to NR AdverseOutcome Terje Svingen (send email) Under development: Not open for comment. Do not cite Under Development

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 KE.In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
Term Scientific Term Evidence Link
rats Rattus norvegicus High NCBI
mouse Mus musculus High NCBI

Life Stages

An indication of the the relevant life stage(s) for this KE. More help
Life stage Evidence
Birth to < 1 month High

Sex Applicability

An indication of the the relevant sex for this KE. More help
Term Evidence
Male High

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. More help

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).

How It Is Measured or Detected

A description of the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements.These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA). Do not provide detailed protocols. More help

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.

Domain of Applicability

A description of the scientific basis for the indicated domains of applicability and the WoE calls (if provided).  More help

The applicability domain of NR is limited to male laboratory strains of rats and mice from birth to juvenile age.

Regulatory Significance of the Adverse Outcome

An AO is a specialised KE that represents the end (an adverse outcome of regulatory significance) of an AOP. More help

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”.


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

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.

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.

OECD. (2018). Test Guideline 443: Extended one-generation reproductive toxicity study. OECD Guidelines for the Testing of Chemicals, 443.

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.