Relationship: 3448: Decrease, intratesticular testosterone leads to Decrease, circulating testosterone levels

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

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).

Key Event Relationship Description

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.

Evidence Supporting this KER

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.  

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.

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.

Quantitative Understanding of the Linkage

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).

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).

References

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

Relationship: 2131: Decrease, circulating testosterone levels leads to Decrease, AR activation

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

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.

Key Event Relationship Description

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).

Evidence Supporting this KER

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.

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

• 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).
• 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).

Ligand-independent actions of the AR have been identified. To what extent and of which biological significance is not well defined (Bennesch & Picard, 2015).

Quantitative Understanding of the Linkage

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).

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).

Androgens can upregulate and downregulate AR expression (Lee & Chang, 2003).

References

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. http://www.ncbi.nlm.nih.gov/pubmed/16985920

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.

Relationship: 2124: Decrease, AR activation leads to Altered, Transcription of genes by the AR

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

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.

Key Event Relationship Description

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.

Evidence Supporting this KER

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).

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).

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).

Quantitative Understanding of the Linkage

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).

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.

AR has been hypothesized to auto-regulate its mRNA and protein levels (Mora and Mahesh 1999).

References

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.

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Relationship: 3487: Decrease, intratesticular testosterone leads to nipple retention, increased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

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).

Key Event Relationship Description

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).

Evidence Supporting this KER

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.

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, 2twr7tg67o_KER_3487_Appendix_2_250901_final.pdf). 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.

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.
 

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.

Quantitative Understanding of the Linkage

The quantitative understanding of this KER is classified as low.

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).

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).

There are no known feedback/feedforward loops for this KER.

References

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Relationship: 3486: Decrease, circulating testosterone levels leads to nipple retention, increased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

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).

Key Event Relationship Description

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 Supporting this KER

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.

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, 61b9o238vs_KER_3486_Appendix_2.pdf). Both data sets showed concurrent NR.

Table 2 Empirical evidence for KER 3486 LOAEL: Lowest observed adverse effect level; See appendix 2 for specifications.

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.
 

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.

Quantitative Understanding of the Linkage

The quantitative understanding of this KER is classified as low.

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

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)

There are no known feedback/feedforward loops for this KER.

References

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

Relationship: 3348: Decrease, AR activation leads to nipple retention, increased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

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.

Key Event Relationship Description

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 Supporting this KER

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).

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).

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.
 

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.

Quantitative Understanding of the Linkage

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.

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).

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) .

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)

Not relevant for this KER.

References

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

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

Christiansen S, Scholze M, Dalgaard M, Vinggaard AM, Axelstad M, Kortenkamp A, & Hass U. (2009). Synergistic disruption of external male sex organ development by a mixture of four antiandrogens. Environmental Health Perspectives, 117(12), 1839–1846. https://doi.org/10.1289/ehp.0900689

Christiansen S, Boberg J, Axelstad M, Dalgaard M, Vinggaard AM, Metzdorff SB, & Hass U. (2010). Low-dose perinatal exposure to di(2-ethylhexyl) phthalate induces anti-androgenic effects in male rats. Reproductive Toxicology (Elmsford, N.Y.), 30(2), 313–321. https://doi.org/10.1016/j.reprotox.2010.04.005

Clewell, R. A., Thomas, A., Willson, G., Creasy, D. M., & Andersen, M. E. (2013). A dose response study to assess effects after dietary administration of diisononyl phthalate (DINP) in gestation and lactation on male rat sexual development. REPRODUCTIVE TOXICOLOGY, 35, 70–80. https://doi.org/10.1016/j.reprotox.2012.07.008

Culty, M., Thuillier, R., Li, W., Wang, Y., Martinez-Arguelles, D. B., Benjamin, C. G., Triantafilou, K. M., Zirkin, B. R., & Papadopoulos, V. (2008). In Utero Exposure to Di-(2-ethylhexyl) Phthalate Exerts Both Short-Term and Long-Lasting Suppressive Effects on Testosterone Production in the Rat1. Biology of Reproduction, 78(6), 1018–1028. https://doi.org/10.1095/biolreprod.107.065649

Euling, S. Y. (2002). Response-Surface Modeling of the Effect of 5alpha-Dihydrotestosterone and Androgen Receptor Levels on the Response to the Androgen Antagonist Vinclozolin. Toxicological Sciences, 69(2), 332–343. https://doi.org/10.1093/toxsci/69.2.332

Fussell KC, Schneider S, Buesen R, Groeters S, Strauss V, Melching-Kollmuss S, & van Ravenzwaay B. (2015). Investigations of putative reproductive toxicity of low-dose exposures to flutamide in Wistar rats. Archives of Toxicology, 89(12), 2385–2402. https://doi.org/10.1007/s00204-015-1622-6

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

Goldspiel, B. R., & Kohler, D. R. (1990). Flutamide: An Antiandrogen for Advanced Prostate Cancer. DICP, 24(6), 616–623. https://doi.org/10.1177/106002809002400612

Gray LE Jr, Ostby J, Furr J, Price M, Veeramachaneni DN, & Parks L. (2000). Perinatal exposure to the phthalates DEHP, BBP, and DINP, but not DEP, DMP, or DOTP, alters sexual differentiation of the male rat. Toxicological Sciences : An Official Journal of the Society of Toxicology, 58(2), 350–365. https://doi.org/10.1093/toxsci/58.2.350

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