SNAPSHOT

Evidence for Perturbation by Stressor

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Overview for Molecular Initiating Event

A large number of drugs and chemicals have been shown to antagonise the AR using various AR reporter gene assays. The AR is specifically targeted in AR-sensitive cancers, for example the use of the anti-androgenic drug flutamide in treating prostate cancer (Alapi & Fischer, 2006). Flutamide has also been used in several rodent in vivo studies showing anti-androgenic effects (feminization of male offspring) evident by e.g. short anogenital distance (AGD) in males (Foster & Harris, 2005; Hass et al, 2007; Kita et al, 2016). QSAR models can predict AR antagonism for a wide range of chemicals, many of which have shown in vitro antagonistic potential (Vinggaard et al, 2008).

Triticonazole

Using hAR-EcoScreen Assay, triticonazole showed a LOEC for antagonisms of 0.2 uM and an IC50 of 0.3 (±0.01) uM (Draskau et al, 2019)

 

Flusilazole

Using hAR-EcoScreen Assay, flusilazole showed a LOEC for antagonisms of 0.8 uM and an IC50 of 2.8 (±0.1) uM (Draskau et al, 2019).►

Epoxiconazole

Using transiently AR-transfected CHO cells, epoxiconazole showed a LOEC of 1.6 uM and an IC50 of 10 uM (Kjærstad et al, 2010)

Prochloraz

Using transiently AR-transfected CHO cells, prochloraz showed a LOEC of 6.3 uM and an IC50 of 13 uM (Kjærstad et al, 2010)

Propiconazole

Using transiently AR-transfected CHO cells, propiconazole showed a LOEC of 12.5 uM and an IC50 of 18 uM (Kjærstad et al, 2010)

Tebuconazole

Using transiently AR-transfected CHO cells, tebuconazole showed a LOEC of 3.1 uM and an IC50 of 8.1 uM (Kjærstad et al, 2010)

Flutamide

Using the AR-CALUX reporter assay in antagonism mode, flutamide showed an IC50 of 1.3 uM (Sonneveld et al, 2005).

Cyproterone acetate

Using the AR-CALUX reporter assay in antagonism mode, cyproterone acetate showed an IC50 of 7.1 nM (Sonneveld et al, 2005).

Vinclozolin

Using the AR-CALUX reporter assay in antagonism mode, vinclozolin showed an IC50of 1.0 uM (Sonneveld et al, 2005).

Domain of Applicability

Key Event Description

The androgen receptor (AR) and its function

Development of the male reproductive system and secondary male characteristics is dependent on androgens (foremost testosterone (T) and dihydrotestosterone (DHT). T and the more biologically active DHT act by binding to the AR (MacLean et al, 1993; MacLeod et al, 2010; Schwartz et al, 2019), with human AR mutations and mouse knock-out models having established its pivotal role in masculinization and spermatogenesis (Walters et al, 2010). The AR is a ligand-activated transcription factor belonging to the steroid hormone nuclear receptor family (Davey & Grossmann, 2016). The AR has three domains; the N-terminal domain, the DNA-binding domain and the ligand-binding domain, with the latter being most evolutionary conserved. Apart from the essential role AR plays for male reproductive development and function (Walters et al, 2010), the AR is also expressed in many other tissues and organs such as bone, muscles, ovaries and the immune system (Rana et al, 2014). 

AR antagonism as Key Event

The main function of the AR is to activate gene transcription in cells. Canonical signaling occurs by ligands (androgens) binding to AR in the cytoplasm which results in translocation to the cell nucleus, receptor dimerization and binding to specific regulatory DNA sequences (Heemers & Tindall, 2007). The gene targets regulated by AR activation depends on cell/tissue type and what stage of development activation occur, and is, for instance, dependent on available co-factors. Apart from the canonical signaling pathway, AR can also function through non-genomic modalities, for instance rapid change in cell function by ion transport changes (Heinlein & Chang, 2002). However, with regard to this specific KE the canonical signaling pathway is what is referred to.

How it is Measured or Detected

AR antagonism can be measured in vitro by transient or stable transactivation assays to evaluate nuclear receptor activation. There is already a validated assay for AR (ant)agonism adopted by the OECD, Test No. 458: Stably Transfected Human Androgen Receptor Transcriptional Activation Assay for Detection of Androgenic Agonist and Antagonist Activity of Chemicals (OECD, 2016). The stably transfected AR-EcoScreenTM cell line should be used for the assay and is freely available for the Japanese Collection of Research Bioresources (JCRB) Cell Bank under reference number JCRB1328.

Other assays include the AR-CALUX reporter gene assay that is derived from human U2-OS cells stably transfected with the human AR and an AR responsive reporter gene (van der Burg et al, 2010).

References

Alapi EM, Fischer J (2006) Table of Selected Analogue Classes. In Analogue-based Drug Discovery, Fischer J, Ganellin CR (eds), p 515. Weinheim: Wiley-VCH Verlag GmbH & Co

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

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

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

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

Heemers HV, Tindall DJ (2007) Androgen receptor (AR) coregulators: a diversity of functions converging on and regulating the AR transcriptional complex. Endocr Rev 28: 778-808

Heinlein CA, Chang C (2002) The roles of androgen receptors and androgen-binding proteins in nongenomic androgen actions. Mol Endocrinol 16: 2181-2187

Kita DH, Meyer KB, Venturelli AC, Adams R, Machado DL, Morais RN, Swan SH, Gennings C, Martino-Andrade AJ (2016) Manipulation of pre and postnatal androgen environments and anogenital distance in rats. Toxicology 368-369: 152-161

Kjærstad MB, Taxvig C, Nellemann C, Vinggaard AM, Andersen HR (2010) Endocrine disrupting effects in vitro of conazole antifungals used as pesticides and pharmaceuticals. Reprod Toxicol 30: 573-582

MacLean HE, Chu S, Warne GL, Zajac JD (1993) Related individuals with different androgen receptor gene deletions. J Clin Invest 91: 1123-1128

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

OECD. (2016) Test No. 458: Stably Transfected Human Androgen Receptor Transcriptional Activation Assay for Detection of Androgenic Agonist and Antagonist Activity of Chemicals. OECD Guidelines for the Testing of Chemicals, Section 4, Paris.

Rana K, davey RA, Zajac JD (2014) Human androgen deficiency: insights gained from androgen receptor knockout mouse models. Asian J Androl 16: 169-177

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

Sonneveld E, Jansen HJ, Riteco JA, Brouwer A, van der Burg B (2005) Development of androgen- and estrogen-responsive bioassays, members of a panel of human cell line-based highly selective steroid-responsive bioassays. Toxicol Sci 83: 136-148

van der Burg B, Winter R, Man HY, Vangenechten C, Berckmans P, Weimer M, Witters H, van der Linden S (2010) Optimization and prevalidation of the in vitro AR CALUX method to test androgenic and antiandrogenic activity of compounds. Reprod Toxicol 30: 18-24

Vinggaard AM, Niemelä J, Wedebye EB, Jensen GE (2008) Screening of 397 chemicals and development of a quantitative structure--activity relationship model for androgen receptor antagonism. Chem Res Toxicol 21: 813-823

Walters KA, Simanainen U, Handelsman DJ (2010) Molecular insights into androgen actions in male and female reproductive function from androgen receptor knockout models. Hum Reprod Update 16: 543-558

 

 

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

Key Event Component

Evidence for Perturbation by Stressor

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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

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

Kita DH, Meyer KB, Venturelli AC, Adams R, Machado DL, Morais RN, Swan SH, Gennings C, Martino-Andrade AJ (2016) Manipulation of pre and postnatal androgen environments and anogenital distance in rats. Toxicology 368-369: 152-161

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

Li M, Qiu L, Zhang Y, Hua Y, Tu S, He Y, Wen S, Wang Q, Wei G (2013) Dose-related effect by maternal exposure to di-(2-ethylhexyl) phthalate plasticizer on inducing hypospadiac male rats. Environ Toxicol Pharmacol 35: 55-60

Lin H, Lian QQ, Hu GX, Jin Y, Zhang Y, Hardy DO, Chen GR, Lu ZQ, Sottas CM, Hardy MP, Ge RS (2009) In utero and lactational exposures to diethylhexyl-phthalate affect two populations of Leydig cells in male Long-Evans rats. Biol Reprod 80: 882-888

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

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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

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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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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

Welsh M, Saunders PT, Sharpe RM (2007) The critical time window for androgen-dependent development of the Wolffian duct in the rat. Endocrinology 148: 3185-3195

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

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List of Adjacent Key Event Relationships

List of Non Adjacent Key Event Relationships