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AOP ID and Title:

AOP 305: 5α-reductase inhibition leading to short anogenital distance (AGD) in male (mammalian) offspring
Short Title: 5α-reductase inhibition leading to short AGD

Graphical Representation

Authors

Terje Svingen; National Food Institute, Technical University of Denmark, Kongens Lyngby, 2800 Denmark


Status

Author status OECD status OECD project SAAOP status
Under development: Not open for comment. Do not cite

Abstract

This AOP links 5α-reductase inhibition during fetal life with short anogenital distance (AGD) in male offspring. A short AGD around birth is a marker for feminization of male fetuses and is associated with male reproductive disorders, including reduced fertility in adulthood. Although a short AGD is not necessarily ‘adverse’ from a human health perspective, it is considered an ‘adverse outcome’ in OECD test guidelines; AGD measurements are mandatory in specific tests for developmental and reproductive toxicity in chemical risk assessment (TG 443, TG 421/422, TG 414).

5α-reductase is an enzyme responsible for the conversion of testosterone to DHT in target tissues. DHT is more potent agonist of the Androgen receptor (AR) than testosterone, so that DHT is necessary for proper masculinization of e.g. male external genitalia. Under normal physiological conditions, testosterone produced mainly by the testicles, is converted in peripheral tissues by 5α-reductase into DHT, which in turn binds AR and activates downstream target genes. AR signaling is necessary for masculinization of the developing fetus, including differentiation of the levator ani/bulbocavernosus (LABC) muscle complex in males. The LABC complex does not develop in the absence, or low levels of, androgen signaling, as in female fetuses.

The key events in this pathway is inhibition of 5α-reductase that converts testosterone into the more potent DHT in androgen sensitive target tissues. This includes developing perineal region, which, when DHT levels are low or absent, leads to inactivation of the AR and failure to properly masculinize the perineum/LABC complex.



Summary of the AOP

Events

Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)

Sequence Type Event ID Title Short name
MIE 1617 5α-reductase, inhibition 5α-reductase, inhibition
KE 1613 Decrease, dihydrotestosterone (DHT) level Decrease, DHT level
KE 1614 Decrease, androgen receptors (AR) activation Decrease, AR activation
KE 1687 decrease, transcription of genes by AR decrease, transcription of genes by AR
AO 1688 decrease, male anogenital distance short male AGD

Key Event Relationships

Upstream Event Relationship Type Downstream Event Evidence Quantitative Understanding
5α-reductase, inhibition adjacent Decrease, dihydrotestosterone (DHT) level High High
Decrease, dihydrotestosterone (DHT) level adjacent Decrease, androgen receptors (AR) activation High Moderate
Decrease, androgen receptors (AR) activation adjacent decrease, transcription of genes by AR Moderate Low
decrease, transcription of genes by AR adjacent decrease, male anogenital distance Moderate Low

Stressors


Name Evidence
Finasteride High

Finasteride

Finasteride is a type II 5alpha-reductase inhibitor that blocks conversion of testosterone to dihydrotestosterone (Clark et al 1990; Imperato-McGinley et al 1992). Intrauterine exposure in rats can result in shorter male AGD in male offspring (Bowman et al 2003; Christiansen et al 2009; Schwartz et al 2019)

References:

Bowman et al (2003), Toxicol Sci 74:393-406; doi: 10.1093/toxsci/kfg128

Christiansen et al (2009), Environ Health Perspect 117:1839-1846; doi: 10.1289/ehp.0900689

Clark et al (1990), Teratology 42:91-100; doi: 10.1002/tera.1420420111

Imperato-McGinley (1992), J Clin Endocrinol Metab 75:1022-1026; doi: 10.1210/jcem.75.4.1400866

Schwartz et al (2019), Toxicol Sci 169:303-311; doi: 10.1093/toxsci/kfz046

Overall Assessment of the AOP

Domain of Applicability

Life Stage Applicability
Life Stage Evidence
Pregnancy High
Taxonomic Applicability
Term Scientific Term Evidence Links
human Homo sapiens Moderate NCBI
rat Rattus norvegicus High NCBI
mouse Mus musculus Moderate NCBI
Sex Applicability
Sex Evidence
Male High

References

  1. Schwartz CL, Christiansen S, Vinggaard AM, Axelstad M, Hass U and Svingen T (2019), Anogenital distance as a toxicological or clinical marker for fetal androgen action and risk for reproductive disorders. Arch Toxicol 93: 253-272.

Appendix 1

List of MIEs in this AOP

Event: 1617: 5α-reductase, inhibition

Short Name: 5α-reductase, inhibition

Biological Context

Level of Biological Organization
Molecular

List of Key Events in the AOP


Biological Context

Level of Biological Organization
Cellular

Key Event Description

Reduction in DHT synthesis leads to a reduction in DHT circulating levels. 12


How it is Measured or Detected

DHT levels in a sample can be measured by (High Performance) Liquid Chromatography. After sample fractionation, DHT can be identify by comparison with internal standards spectrum. Quantification of DHT levels can be performed using hormones measurements kits (ELISA), instrumental techniques (LC-MS) or liquid scintillation spectrometry (after radiolabeling).3


References

1 Miller Walter L. (1988) Molecular Biology of Steroid Hormone Synthesis. Endocrine Reviews, 9(3): 295-318.https://doi.org/10.1210/edrv-9-3-295 

 

2 Miller W.L. and Auchus R.J. (2011) The Molecular Biology, Biochemistry, and Physiology of Human Steroidogenesis and Its Disorders. Endocrine Reviews, 32(1): 81-151.https://doi.org/10.1210/er.2010-0013 

 

3 Shiraishi S., Lee P.W., Leung A., Goh V.H., Swerdloff R.S. and Wang C. (2008) Simultaneous measurement of serum testosterone and dihydrotestosterone by liquid chromatography-tandem mass spectrometry. Clinical chemistry, 54(11): 1855-63.https://doi.org/10.1373/clinchem.2008.103846

Event: 1614: Decrease, androgen receptors (AR) activation

Short Name: Decrease, AR activation

Biological Context

Level of Biological Organization
Cellular

Key Event Description

Androgen receptor activation is regulated by the binding of androgens. AR activity can be decreased by either a lack of steroidal ligands (testosterone, DHT) or the presence of antagonist compounds. 12


How it is Measured or Detected

Significance of AR signaling in fetal development can be studied through a conditional deletion of the androgen receptor using a Cre/loxP approach. The recommended animal model for reproductive study is the mouse.3

Also, epidemiological case-studies following mouse or humans expressing a complete androgen insensitivity allow to directly assess the effects of a lack of AR activation on the development.4

Enzyme immunoassay (ELISA) kits for in vitro quantitative measurement of AR activity are available. Androgen receptors activity can be measured using bioassay such as the (Anti-)Androgen Receptor CALUX reporter gene assay.5


References

 

1 Davey R.A and Grossmann M. (2016) Androgen Receptor Structure, Function and Biology: From Bench to Bedside. Clinical Biochemist Reviews, 37(1): 3-15. PCM4810760

2 Gao W., Bohl C.E. and Dalton J.T. (2005) Chemistry and Structural Biology of Androgen Receptor. Chemical Reviews 105(9): 3352-3370https://doi.org/10.1021/cr020456u 

3 Kaftanovskaya E.M., Huang Z., Barbara A.M., De Gendt K., Verhoeven G., Ivan P. Gorlov, and Agoulnik A.I. (2012) Cryptorchidism in Mice with an Androgen Receptor Ablation in Gubernaculum Testis. Molecular Endocrinology, 26(4): 598-607.https://doi.org/10.1210/me.2011-1283 

4 Hutson J.M. (1985) A biphasic model for the hormonal control of testicular descent. Lancet, 24;2(8452): 419-21http://dx.doi.org/10.1016/S0140-6736(85)92739-4 

5 van der Burg B., Winter R., Man HY., Vangenechten C., Berckmans P., Weimer M., Witters M. and van der Linden S. (2010) Optimization and prevalidation of the in vitro AR CALUX method to test androgenic and antiandrogenic activity of compounds. Reproductive Toxicology, 30(1):18-24 https://doi.org/0.1016/j.reprotox.2010.04.012 

Event: 1687: decrease, transcription of genes by AR

Short Name: decrease, transcription of genes by AR

Biological Context

Level of Biological Organization
Cellular

List of Adverse Outcomes in this AOP

Key Event Component

Process Object Action
androgen receptor signaling pathway Musculature of male perineum disrupted

Stressors

Name
Butylparaben
p,p'-DDE
Bis(2-ethylhexyl) phthalate
Dexamethasone
Fenitrothion
Finasteride
Flutamide
Ketoconazole
Linuron
Prochloraz
Procymidone
Triticonazole
Vinclozolin
di-n-hexyl phthalate
Dicyclohexyl phthalate
butyl benzyl phthalate
monobenzyl phthalate
di-n-heptyl phthalate

Biological Context

Level of Biological Organization
Tissue

Organ term

Organ term
perineum

Evidence for Perturbation by Stressor


Butylparaben

Butylparaben has been shown to cause decreased male AGD in rats following intrauterine exposure to 500 and 1000 mg/kg bw/day (Boberg et al, 2016; Zhang et al, 2014). A separate study using 600 mg/kg bw/day did not see an effect on male AGD (Boberg et al, 2008).


p,p'-DDE

p,p,DDE has been shown to cause decreased male AGD in rats following intrauterine exposure to 100-200 mg/kg bw/day (Loeffler & Peterson, 1999; Wolf et al, 1999).


Bis(2-ethylhexyl) phthalate

DEHP has been shown to cause decreased male AGD in rats following intrauterine exposure to 300-1500 mg/kg bw/day (Christiansen et al, 2010; Gray et al, 2000; Howdeshell et al, 2007; Jarfelt et al, 2005; Kita et al, 2016; Li et al, 2013; Lin et al, 2009; Moore et al, 2001; Nardelli et al, 2017; Saillenfait et al, 2009; Wolf et al, 1999).


Dexamethasone

Dexamethasone has been shown to cause decreased male AGD in rats following intrauterine exposure to 0.1 mg/kg bw/day (Van den Driesche et al, 2012).


Fenitrothion

Fenitrothion has been shown to cause decreased male AGD in rats following intrauterine exposure to 25 mg/kg bw/day (Turner et al, 2002).


Finasteride

Finasteride has been shown to cause decreased male AGD in rats following intrauterine exposure to 100 mg/kg bw/day (Bowman et al, 2003).


Flutamide

Flutamide has been shown to cause decreased male AGD in rats following intrauterine exposure to doses between 16-100 mg/kg bw/day (Foster & Harris, 2005; Hass et al, 2007; Kita et al, 2016; McIntyre et al, 2001; Mylchreest et al, 1999; Scott et al, 2007; Welsh et al, 2007).


Ketoconazole

Ketoconazole has been shown to cause decreased male AGD in rats following intrauterine exposure to 50 mg/kg bw/day in one study (Taxvig et al, 2008), but no effect in another study using same dose (Wolf et al, 1999).


Linuron

Linuron has been shown to cause decreased male AGD in rats following intrauterine exposure to 50-100 mg/kg bw/day (Hotchkiss et al, 2004; McIntyre et al, 2002; Wolf et al, 1999).


Prochloraz

Prochloraz has been shown to cause decreased male AGD in rats following intrauterine exposure to 150-250 mg/kg bw/day (Laier et al, 2006; Noriega et al, 2005).


Procymidone

Procymidone has been shown to cause decreased male AGD in rats following intrauterine exposure to doses between 50-150 mg/kg bw/day (Hass et al, 2012; Hass et al, 2007; Wolf et al, 1999).


Triticonazole

Triticonazole has been shown to cause decreased male AGD in rats following intrauterine exposure to 150 and 450 mg/kg bw/day (Draskau et al, 2019).


Vinclozolin

Vinclozolin has been shown to cause decreased male AGD in rats following intrauterine exposure to doses between 50-200 mg/kg bw/day (Christiansen et al, 2009; Gray et al, 1994; Hass et al, 2007; Matsuura et al, 2005; Ostby et al, 1999; Schneider et al, 2011; Wolf et al, 2004).


di-n-hexyl phthalate

DnHP has been shown to cause decreased male AGD in rats following intrauterine exposure to 500-750 mg/kg bw/day (Saillenfait et al, 2009a; Saillenfait et al, 2009b).


Dicyclohexyl phthalate

DCHP has been shown to cause decreased male AGD in rats following intrauterine exposure to 350-750 mg/kg bw/day (Aydoğan Ahbab & Barlas, 2015; Hoshino et al, 2005; Saillenfait et al, 2009a).


butyl benzyl phthalate

BBP has been shown to cause decreased male AGD in rats following intrauterine exposure to 500-1000 mg/kg bw/day (Ema & Miyawaki, 2002; Gray et al, 2000; Hotchkiss et al, 2004; Nagao et al, 2000; Tyl et al, 2004).


monobenzyl phthalate

MBeP has been shown to cause decreased male AGD in rats following intrauterine exposure to 375 mg/kg bw/day (Ema et al, 2003).


di-n-heptyl phthalate

DHPP has been shown to cause decreased male AGD in rats following intrauterine exposure to 1000 mg/kg bw/day (Saillenfait et al, 2011).


Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
human Homo sapiens Moderate NCBI
rat Rattus norvegicus High NCBI
mouse Mus musculus High NCBI
Life Stage Applicability
Life Stage Evidence
Foetal High
Sex Applicability
Sex Evidence
Male High

A short AGD in male offspring is a marker of insufficient androgen action during critical fetal developmental stages (Schwartz et al, 2019; Welsh et al, 2008). A short AGD is thus a sign of undervirilization, which is also associated with a series of male reproductive disorders, including genital malformations and infertility in humans (Juul et al, 2014; Skakkebaek et al, 2001).

There are numerous human epidemiological studies showing associations with intrauterine exposure to anti-androgenic chemicals and short AGD in newborn boys alongside other reproductive disorders (Schwartz et al, 2019). This underscores the human relevance of this AO. However, in reproductive toxicity studies and chemical risk assessment, rodents (rats and mice) are what is tested on. The list of chemicals inducing short male AGD in male rat offspring is extensive, as evidenced by the ‘stressor’ list and reviewed by (Schwartz et al, 2019).


Key Event Description

The anogenital distance (AGD) refers to the distance between anus and the external genitalia. In rodents and humans, the male AGD is approximately twice the length as the female AGD (Salazar-Martinez et al, 2004; Schwartz et al, 2019). This sexual dimorphisms is a consequence of sex hormone-dependent development of secondary sexual characteristics (Schwartz et al, 2019). In males, it is believed that androgens (primarily DHT) activate AR-positive cells in non-myotic cells in the fetal perineum region to initiate differentiation of the perineal levator ani and bulbocavernosus (LABC) muscle complex (Ipulan et al, 2014). This AR-dependent process occurs within a critical window of development, around gestational days 15-18 in rats (MacLeod et al, 2010). In females, the absence of DHT prevents this masculinization effect from occurring.

The involvement of androgens in masculinization of the male fetus, including the perineum, has been known for a very long time (Jost, 1953), and AGD has historically been used to, for instance, sex newborn kittens. It is now well established that the AGD in newborns is a proxy readout for the intrauterine sex hormone milieu the fetus was developing. Too low androgen levels in XY fetuses makes the male AGD shorter, whereas excess (ectopic) androgen levels in XX fetuses makes the female AGD longer, in humans and rodents (Schwartz et al, 2019).


How it is Measured or Detected

The AGD is a morphometric measurement carried out by trained technicians (rodents) or medical staff (humans).

In rodent studies AGD is assessed as the distance between the genital papilla and the anus, and measured using a stereomicroscope with a micrometer eyepiece. The AGD index (AGDi) is often calculated by dividing AGD by the cube root of the body weight.  It is important in statistical analysis to use litter as the statistical unit. This is done when more than one pup from each litter is examined. Statistical analyses is adjusted using litter as an independent, random and nested factor. AGD are analysed using body weight as covariate as recommended in Guidance Document 151 (OECD, 2013).

 


Regulatory Significance of the AO

In regulatory toxicology, the AGD is mandatory inclusions in OECD test guidelines used to test for developmental and reproductive toxicity of chemicals. Guidelines include ‘TG 443 extended one-generation study’, ‘TG 421/422 reproductive toxicity screening studies’ and ‘TG 414 developmental toxicity study’.


References

Aydoğan Ahbab M, Barlas N (2015) Influence of in utero di-n-hexyl phthalate and dicyclohexyl phthalate on fetal testicular development in rats. Toxicol Lett 233: 125-137

Boberg J, Axelstad M, Svingen T, Mandrup K, Christiansen S, Vinggaard AM, Hass U (2016) Multiple endocrine disrupting effects in rats perinatally exposed to butylparaben. Toxicol Sci 152: 244-256

Boberg J, Metzdorff S, Wortziger R, Axelstad M, Brokken L, Vinggaard AM, Dalgaard M, Nellemann C (2008) Impact of diisobutyl phthalate and other PPAR agonists on steroidogenesis and plasma insulin and leptin levels in fetal rats. Toxicology 250: 75-81

Bowman CJ, Barlow NJ, Turner KJ, Wallace DG, Foster PM (2003) Effects of in utero exposure to finasteride on androgen-dependent reproductive development in the male rat. Toxicol Sci 74: 393-406

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. Reprod Toxicol 30: 313-321

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. Environ Health Perspect 117: 1839-1846

Draskau MK, Boberg J, Taxvig C, Pedersen M, Frandsen HL, Christiansen S, Svingen T (2019) In vitro and in vivo endocrine disrupting effects of the azole fungicides triticonazole and flusilazole. Environ Pollut 255: 113309

Ema M, Miyawaki E (2002) Effects on development of the reproductive system in male offspring of rats given butyl benzyl phthalate during late pregnancy. Reprod Toxicol 16: 71-76

Ema M, Miyawaki E, Hirose A, Kamata E (2003) Decreased anogenital distance and increased incidence of undescended testes in fetuses of rats given monobenzyl phthalate, a major metabolite of butyl benzyl phthalate. Reprod Toxicol 17: 407-412

Foster PM, Harris MW (2005) Changes in androgen-mediated reproductive development in male rat offspring following exposure to a single oral dose of flutamide at different gestational ages. Toxicol Sci 85: 1024-1032

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. Toxicol Sci 58: 350-365

Gray LEJ, Ostby JS, Kelce WR (1994) Developmental effects of an environmental antiandrogen: the fungicide vinclozolin alters sex differentiation of the male rat. Toxicol Appl Pharmacol 129: 46-52

Hass U, Boberg J, Christiansen S, Jacobsen PR, Vinggaard AM, Taxvig C, Poulsen ME, Herrmann SS, Jensen BH, Petersen A, Clemmensen LH, Axelstad M (2012) Adverse effects on sexual development in rat offspring after low dose exposure to a mixture of endocrine disrupting pesticides. Reprod Toxicol 34: 261-274

Hass U, Scholze M, Christiansen S, Dalgaard M, Vinggaard AM, Axelstad M, Metzdorff SB, Kortenkamp A (2007) Combined exposure to anti-androgens exacerbates disruption of sexual differentiation in the rat. Environ Health Perspect 115 Suppl. 1: 122-128

Hoshino N, Iwai M, Okazaki Y (2005) A two-generation reproductive toxicity study of dicyclohexyl phthalate in rats. J Toxicol Sci 30 Spec No: 79-96

Hotchkiss AK, Parks-Saldutti LG, Ostby JS, Lambright C, Furr J, Vandenbergh JG, Gray LEJ (2004) A mixture of the "antiandrogens" linuron and butyl benzyl phthalate alters sexual differentiation of the male rat in a cumulative fashion. Biol Reprod 71: 1852-1861

Howdeshell KL, Furr J, Lambright CR, Rider CV, Wilson VS, Gray LE, Jr. (2007) Cumulative effects of dibutyl phthalate and diethylhexyl phthalate on male rat reproductive tract development: altered fetal steroid hormones and genes. Toxicol Sci 99: 190-202

Ipulan LA, Suzuki K, Sakamoto Y, Murashima A, Imai Y, Omori A, Nakagata N, Nishinakamura R, Valasek P, Yamada G (2014) Nonmyocytic androgen receptor regulates the sexually dimorphic development of the embryonic bulbocavernosus muscle. Endocrinology 155: 2467-2479

Jarfelt K, Dalgaard M, Hass U, Borch J, Jacobsen H, Ladefoged O (2005) Antiandrogenic effects in male rats perinatally exposed to a mixture of di(2-ethylhexyl) phthalate and di(2-ethylhexyl) adipate. Reprod Toxicol 19: 505-515

Jost A (1953) Problems of fetal endocrinology: The gonadal and hypophyseal hormones. Recent Prog Horm Res 8: 379-418

Juul A, Almstrup K, Andersson AM, Jensen TK, Jorgensen N, Main KM, Rajpert-De Meyts E, Toppari J, Skakkebaek NE (2014) Possible fetal determinants of male infertility. Nat Rev Endocrinol 10: 553-562

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Laier P, Metzdorff SB, Borch J, Hagen ML, Hass U, Christiansen S, Axelstad M, Kledal T, Dalgaard M, McKinnell C, Brokken LJ, Vinggaard AM (2006) Mechanisms of action underlying the antiandrogenic effects of the fungicide prochloraz. Toxicol Appl Pharmacol 213: 2

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Loeffler IK, Peterson RE (1999) Interactive effects of TCDD and p,p'-DDE on male reproductive tract development in in utero and lactationally exposed rats. Toxicol Appl Pharmacol 154: 28-39

MacLeod DJ, Sharpe RM, Welsh M, Fisken M, Scott HM, Hutchison GR, Drake AJ, van den Driesche S (2010) Androgen action in the masculinization programming window and development of male reproductive organs. Int J Androl 33: 279-287

Matsuura I, Saitoh T, Ashina M, Wako Y, Iwata H, Toyota N, Ishizuka Y, Namiki M, Hoshino N, Tsuchitani M (2005) Evaluation of a two-generation reproduction toxicity study adding endpoints to detect endocrine disrupting activity using vinclozolin. J Toxicol Sci 30 Spec No: 163-168

McIntyre BS, Barlow NJ, Foster PM (2001) Androgen-mediated development in male rat offspring exposed to flutamide in utero: permanence and correlation of early postnatal changes in anogenital distance and nipple retention with malformations in androgen-dependent tissues. Toxicol Sci 62: 236-249

McIntyre BS, Barlow NJ, Sar M, Wallace DG, Foster PM (2002) Effects of in utero linuron exposure on rat Wolffian duct development. Reprod Toxicol 16: 131-139

Melching-Kollmuss S, Fussell KC, Schneider S, Buesen R, Groeters S, Strauss V, van Ravenzwaay B (2017) Comparing effect levels of regulatory studies with endpoints derived in targeted anti-androgenic studies: example prochloraz. Arch Toxicol 91: 143-162

Moore RW, Rudy TA, Lin TM, Ko K, Peterson RE (2001) Abnormalities of sexual development in male rats with in utero and lactational exposure to the antiandrogenic plasticizer Di(2-ethylhexyl) phthalate. Environ Health Perspect 109: 229-237

Mylchreest E, Sar M, Cattley RC, Foster PM (1999) Disruption of androgen-regulated male reproductive development by di(n-butyl) phthalate during late gestation in rats is different from flutamide. Toxicol Appl Pharmacol 156: 81-95

Nagao T, Ohta R, Marumo H, Shindo T, Yoshimura S, Ono H (2000) Effect of butyl benzyl phthalate in Sprague-Dawley rats after gavage administration: a two-generation reproductive study. Reprod Toxicol 14: 513-532

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Noriega NC, Ostby J, Lambright C, Wilson VS, Gray LE, Jr. (2005) Late gestational exposure to the fungicide prochloraz delays the onset of parturition and causes reproductive malformations in male but not female rat offspring. Biol Reprod 72: 1324-1335

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Ostby J, Kelce WR, Lambright C, Wolf CJ, Mann P, Gray CLJ (1999) The fungicide procymidone alters sexual differentiation in the male rat by acting as an androgen-receptor antagonist in vivo and in vitro. Toxicol Ind Health 15: 80-93

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Saillenfait AM, Roudot AC, Gallissot F, Sabaté JP (2011) Prenatal developmental toxicity studies on di-n-heptyl and di-n-octyl phthalates in Sprague-Dawley rats. Reprod Toxicol 32: 268-276

Saillenfait AM, Sabaté JP, Gallissot F (2009b) Effects of in utero exposure to di-n-hexyl phthalate on the reproductive development of the male rat. Reprod Toxicol 28: 468-476

Salazar-Martinez E, Romano-Riquer P, Yanez-Marquez E, Longnecker MP, Hernandez-Avila M (2004) Anogenital distance in human male and female newborns: a descriptive, cross-sectional study. Environ Health 3: 8

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Schwartz CL, Christiansen S, Vinggaard AM, Axelstad M, Hass U, Svingen T (2019) Anogenital distance as a toxicological or clinical marker for fetal androgen action and risk for reproductive disorders. Arch Toxicol 93: 253-272

Scott HM, Hutchison GR, Mahood IK, Hallmark N, Welsh M, De Gendt K, Verhoeven H, O'Shaughnessy P, Sharpe RM (2007) Role of androgens in fetal testis development and dysgenesis. Endocrinology 148: 2027-2036

Skakkebaek NE, Rajpert-De Meyts E, Main KM (2001) Testicular dysgenesis syndrome: an increasingly common developmental disorder with environmental aspects. Hum Reprod 16: 972-978

Taxvig C, Vinggaard AM, Hass U, Axelstad M, Metzdorff S, Nellemann C (2008) Endocrine-disrupting properties in vivo of widely used azole fungicides. Int J Androl 31: 170-177

Turner KJ, Barlow NJ, Struve MF, Wallace DG, Gaido KW, Dorman DC, Foster PM (2002) Effects of in utero exposure to the organophosphate insecticide fenitrothion on androgen-dependent reproductive development in the Crl:CD(SD)BR rat. Toxicol Sci 68: 174-183

Tyl RW, Myers CB, Marr MC, Fail PA, Seely JC, Brine DR, Barter RA, Butala JH (2004) Reproductive toxicity evaluation of dietary butyl benzyl phthalate (BBP) in rats. Reprod Toxicol 18: 241-264

Van den Driesche S, Kolovos P, Platts S, Drake AJ, Sharpe RM (2012) Inter-relationship between testicular dysgenesis and Leydig cell function in the masculinization programming window in the rat. PloS one 7: e30111

Welsh M, Saunders PT, Fisken M, Scott HM, Hutchison GR, Smith LB, Sharpe RM (2008) Identification in rats of a programming window for reproductive tract masculinization, disruption of which leads to hypospadias and cryptorchidism. J Clin Invest 118: 1479-1490

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Wolf CJ, LeBlanc GA, Gray LE, Jr. (2004) Interactive effects of vinclozolin and testosterone propionate on pregnancy and sexual differentiation of the male and female SD rat. Toxicol Sci 78: 135-143

Wolf CJJ, Lambright C, Mann P, Price M, Cooper RL, Ostby J, Gray CLJ (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. Toxicol Ind Health 15: 94-118

Zhang L, Dong L, Ding S, Qiao P, Wang C, Zhang M, Zhang L, Du Q, Li Y, Tang N, Chang B (2014) Effects of n-butylparaben on steroidogenesis and spermatogenesis through changed E₂ levels in male rat offspring. Environ Toxicol Pharmacol 37: 705-717


Appendix 2

List of Key Event Relationships in the AOP