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Key Event Title
decrease, male anogenital distance
Key Event Components
|androgen receptor signaling pathway||Musculature of male perineum||disrupted|
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP||Point of Contact||Author Status||OECD Status|
|5α-reductase inhibition leading to short AGD||AdverseOutcome||Terje Svingen (send email)||Under development: Not open for comment. Do not cite||Under Development|
|AR antagonism leading to short AGD||AdverseOutcome||Terje Svingen (send email)||Under development: Not open for comment. Do not cite||Under Development|
|Decreased testosterone synthesis leading to short AGD||AdverseOutcome||Terje Svingen (send email)||Under development: Not open for comment. Do not cite||Under Development|
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).
Domain of Applicability
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).
Evidence for Perturbation by Stressor
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).
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 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 has been shown to cause decreased male AGD in rats following intrauterine exposure to 25 mg/kg bw/day (Turner et al, 2002).
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 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 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).
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 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).
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).
MBeP has been shown to cause decreased male AGD in rats following intrauterine exposure to 375 mg/kg bw/day (Ema et al, 2003).
DHPP has been shown to cause decreased male AGD in rats following intrauterine exposure to 1000 mg/kg bw/day (Saillenfait et al, 2011).
Regulatory Significance of the Adverse Outcome
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’.
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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, 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
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
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
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
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
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
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
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
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
Schneider S, Kaufmann W, Strauss V, van Ravenzwaay B (2011) Vinclozolin: a feasibility and sensitivity study of the ILSI-HESI F1-extended one-generation rat reproduction protocol. Regulatory Toxicology and Pharmacology 59: 91-100
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
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
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
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