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AOP: 307

Title

A descriptive phrase which references both the Molecular Initiating Event and Adverse Outcome.It should take the form “MIE leading to AO”. For example, “Aromatase inhibition leading to reproductive dysfunction” where Aromatase inhibition is the MIE and reproductive dysfunction the AO. In cases where the MIE is unknown or undefined, the earliest known KE in the chain (i.e., furthest upstream) should be used in lieu of the MIE and it should be made clear that the stated event is a KE and not the MIE. More help

Decreased testosterone synthesis leading to short anogenital distance (AGD) in male (mammalian) offspring

Short name

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Decreased testosterone synthesis leading to short AGD

The current version of the Developer's Handbook will be automatically populated into the Handbook Version field when a new AOP page is created.Authors have the option to switch to a newer (but not older) Handbook version any time thereafter. More help

Handbook Version v2.0

Graphical Representation

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Authors

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Monica K. Draskau; National Food Institute, Technical University of Denmark, Lyngby, DK-2800, Denmark

Marie L. Holmer; National Food Institute, Technical University of Denmark, Lyngby, DK-2800, Denmark

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

Point of Contact

The user responsible for managing the AOP entry in the AOP-KB and controlling write access to the page by defining the contributors as described in the next section. More help

Terje Svingen (email point of contact)

Contributors

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Terje Svingen

Coaches

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Judy Choi
Shihori Tanabe

OECD Information Table

Provides users with information concerning how actively the AOP page is being developed and whether it is part of the OECD Workplan and has been reviewed and/or endorsed. OECD Project: Assigned upon acceptance onto OECD workplan. This project ID is managed and updated (if needed) by the OECD. OECD Status: For AOPs included on the OECD workplan, ‘OECD status’ tracks the level of review/endorsement of the AOP . This designation is managed and updated by the OECD. Journal-format Article: The OECD is developing co-operation with Scientific Journals for the review and publication of AOPs, via the signature of a Memorandum of Understanding. When the scientific review of an AOP is conducted by these Journals, the journal review panel will review the content of the Wiki. In addition, the Journal may ask the AOP authors to develop a separate manuscript (i.e. Journal Format Article) using a format determined by the Journal for Journal publication. In that case, the journal review panel will be required to review both the Wiki content and the Journal Format Article. The Journal will publish the AOP reviewed through the Journal Format Article. OECD iLibrary published version: OECD iLibrary is the online library of the OECD. The version of the AOP that is published there has been endorsed by the OECD. The purpose of publication on iLibrary is to provide a stable version over time, i.e. the version which has been reviewed and revised based on the outcome of the review. AOPs are viewed as living documents and may continue to evolve on the AOP-Wiki after their OECD endorsement and publication. More help

OECD Project #	OECD Status	Reviewer's Reports	Journal-format Article	OECD iLibrary Published Version
1.90	Under Development

This AOP was last modified on September 25, 2024 03:33

Revision dates for related pages

Page	Revision Date/Time
Decrease, circulating testosterone levels	December 12, 2024 05:27
Decrease, androgen receptor activation	April 05, 2024 08:19
Altered, Transcription of genes by the androgen receptor	April 05, 2024 09:28
anogenital distance (AGD), decreased	December 22, 2022 05:18
Decrease, circulating testosterone levels leads to Decrease, AR activation	April 05, 2024 09:18
Decrease, AR activation leads to AGD, decreased	August 08, 2024 12:45
Decrease, AR activation leads to Altered, Transcription of genes by the AR	April 05, 2024 08:50
Altered, Transcription of genes by the AR leads to AGD, decreased	May 11, 2020 07:04
Dibutyl phthalate	November 29, 2016 18:42
Bis(2-ethylhexyl) phthalate	November 29, 2016 18:42

Abstract

A concise and informative summation of the AOP under development that can stand-alone from the AOP page. The aim is to capture the highlights of the AOP and its potential scientific and regulatory relevance. More help

This AOP links decreased testosterone levels with short anogenital distance (AGD) in male offspring. It does not yet contain an MIE, as the upstream events leading to ‘reduced testosterone’ synthesis in fetal testis can be many, for example by inhibiting various enzymes of the steroidogenesis pathway. The precursor molecule cholesterol is converted to testosterone via several enzymatic steps and includes, for instance, the CYP enzymes CYP11 and CYP17. Following synthesis, testosterone is released into the circulation and transported to target tissues and organs where it initiates masculinization processes, typically by binding to and activating the androgen receptor (AR) in target cells. Notably, testosterone can be converted to DHT by 5α-reductase, with DHT being a more potent AR agonist than testosterone; this testosterone-to-DHT conversion is critical during development for differentiation of male traits, including masculinization of the developing fetus, including differentiation of the levator ani/bulbocavernosus (LABC) muscle complex (Davey and Grossmann, 2016; Keller et al, 1996; Robitaille and Langlois, 2020). The LABC complex does not develop in the absence, or low levels of, androgen signaling, as in female fetuses.

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 (Schwartz et al, 2019). 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), with measurement guidance provided in OECD guidance documents 43 (OECD, 2008) and 151 (OECD, 2013).

A central event in this pathway is inhibition of testosterone synthesis by fetal testes. In turn, this results in reduced circulating testosterone levels and less DHT (converted by 5α-reductase). Low DHT fails to properly activate AR in target tissues, including the developing perineal region, which leads to failure to properly masculinize the perineum/LABC complex and ultimately a short AGD.

AOP Development Strategy

Context

Used to provide background information for AOP reviewers and users that is considered helpful in understanding the biology underlying the AOP and the motivation for its development.The background should NOT provide an overview of the AOP, its KEs or KERs, which are captured in more detail below. More help

Androgen signaling is critical for male sex differentiation during fetal life and suboptimal action during critical life stages leads to under-masculinized offspring. Androgens, primarily testosterone and dihydro-testosterone (DHT), act by binding to and activating the AR is target cells. Blocking the AR basically blocks androgen signaling and masculinization of tissues and organs that otherwise should masculinize in male fetuses. One morphometric marker for reduced fetal androgen action is a shorter than normal anogenital distance.

Strategy

Provides a description of the approaches to the identification, screening and quality assessment of the data relevant to identification of the key events and key event relationships included in the AOP or AOP network.This information is important as a basis to support the objective/envisaged application of the AOP by the regulatory community and to facilitate the reuse of its components. Suggested content includes a rationale for and description of the scope and focus of the data search and identification strategy/ies including the nature of preliminary scoping and/or expert input, the overall literature screening strategy and more focused literature surveys to identify additional information (including e.g., key search terms, databases and time period searched, any tools used). More help

For the AOP network development, the OECD AOP Developer’s Handbook was followed alongside pragmatic approaches (Svingen et al., 2021). Key events (KEs) and key event relationships (KERs) based on canonical knowledge from the ‘upstream anti-androgenic network’ (Draskau et al., 2024) were treated less stringently, while new units adhered to more rigorous systematic literature retrieval methods.

KER-2820, linking KE-1614 (decreased AR activation) with AO-1688 (decreased AGD), was developed using a systematic weight of evidence (WoE) approach (Holmer et al., 2024). From an initial 826 publications, 557 were retained, with 71 selected for data extraction (82 datasets). Ultimately, 25 reliable datasets from in vivo studies on five model compounds (flutamide, procymidone, vinclozolin, finasteride, di-2-ethylhexyl phthalate) provided strong empirical support. Conversely, KER-2127, linking KE-286 (altered AR transcription) with AO-1688, was developed semi-systematically, yielding only two relevant studies due to limited transcriptional data from perineal tissue exposed to anti-androgenic chemicals.

The overall AOP assessments followed the AOP Developer’s Handbook guidelines.

Summary of the AOP

This section is for information that describes the overall AOP.The information described in section 1 is entered on the upper portion of an AOP page within the AOP-Wiki. This is where some background information may be provided, the structure of the AOP is described, and the KEs and KERs are listed. More help

Events:

Molecular Initiating Events (MIE)

An MIE is a specialised KE that represents the beginning (point of interaction between a prototypical stressor and the biological system) of an AOP. More help

Key Events (KE)

A measurable event within a specific biological level of organisation. More help

Adverse Outcomes (AO)

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

Type	Event ID	Title	Short name

KE	1690	Decrease, circulating testosterone levels	Decrease, circulating testosterone levels
KE	1614	Decrease, androgen receptor activation	Decrease, AR activation
KE	286	Altered, Transcription of genes by the androgen receptor	Altered, Transcription of genes by the AR

1688

anogenital distance (AGD), decreased

AGD, decreased

Relationships Between Two Key Events (Including MIEs and AOs)

This table summarizes all of the KERs of the AOP and is populated in the AOP-Wiki as KERs are added to the AOP.Each table entry acts as a link to the individual KER description page. More help

Title	Adjacency	Evidence	Quantitative Understanding

Decrease, circulating testosterone levels leads to Decrease, AR activation	adjacent	High	Moderate
Decrease, AR activation leads to Altered, Transcription of genes by the AR	adjacent	Moderate	Low

Decrease, AR activation leads to AGD, decreased	non-adjacent	High	Moderate
Altered, Transcription of genes by the AR leads to AGD, decreased	non-adjacent	Moderate	Low

Network View

This network graphic is automatically generated based on the information provided in the MIE(s), KEs, AO(s), KERs and Weight of Evidence (WoE) summary tables. The width of the edges representing the KERs is determined by its WoE confidence level, with thicker lines representing higher degrees of confidence. This network view also shows which KEs are shared with other AOPs. More help

Prototypical Stressors

A structured data field that can be used to identify one or more “prototypical” stressors that act through this AOP. Prototypical stressors are stressors for which responses at multiple key events have been well documented. More help

Name
Dibutyl phthalate
Bis(2-ethylhexyl) phthalate

Life Stage Applicability

The life stage for which the AOP is known to be applicable. More help

Life stage	Evidence
Foetal	High
Pregnancy	High

Taxonomic Applicability

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Term	Scientific Term	Evidence	Link
human	Homo sapiens	Moderate	NCBI
rat	Rattus norvegicus	High	NCBI
mouse	Mus musculus	Moderate	NCBI

Sex Applicability

The sex for which the AOP is known to be applicable. More help

Sex	Evidence
Male	High

Overall Assessment of the AOP

Addressess the relevant biological domain of applicability (i.e., in terms of taxa, sex, life stage, etc.) and Weight of Evidence (WoE) for the overall AOP as a basis to consider appropriate regulatory application (e.g., priority setting, testing strategies or risk assessment). More help

Domain of Applicability

Addressess the relevant biological domain(s) of applicability in terms of sex, life-stage, taxa, and other aspects of biological context. More help

The upstream part of the AOP, culminating at KE-286 (altered transcription of genes by the AR), has a broad applicability domain. It is built primarily on mammalian data and includes all life stages and both sexes. It could be extended to cover non-mammalian vertebrates by adding additional relevant knowledge, as previously discussed (Draskau et al, 2024). The overall applicability domain is limited by AO-1688 (decreased AGD). The AGD is strongly influenced by androgen action during critical fetal stages in mammals, with evidence from humans (Murashima et al, 2015; Thankamony et al, 2016), and from numerous gestational exposure studies in rats and mice to anti-androgenic chemicals (Gray et al, 2001; Schwartz et al, 2019). The male masculinisation programming window occurs at a developmental stage included in the applicability domain of these AOPs and corresponds to around gestational day 16-20 in rats and gestation weeks 8-14 in humans (Welsh et al, 2008). Only males are included in the applicability domain since the male AGD, but not the female AGD, is shortened by decreased androgen action (Schwartz et al, 2019).

Essentiality of the Key Events

The essentiality of KEs can only be assessed relative to the impact of manipulation of a given KE (e.g., experimentally blocking or exacerbating the event) on the downstream sequence of KEs defined for the AOP. Consequently, evidence supporting essentiality is assembled on the AOP page, rather than on the independent KE pages that are meant to stand-alone as modular units without reference to other KEs in the sequence. The nature of experimental evidence that is relevant to assessing essentiality relates to the impact on downstream KEs and the AO if upstream KEs are prevented or modified. This includes: Direct evidence: directly measured experimental support that blocking or preventing a KE prevents or impacts downstream KEs in the pathway in the expected fashion. Indirect evidence: evidence that modulation or attenuation in the magnitude of impact on a specific KE (increased effect or decreased effect) is associated with corresponding changes (increases or decreases) in the magnitude or frequency of one or more downstream KEs. More help

The essentiality of each key event (KE) was evaluated, meaning that if an upstream KE is blocked or does not occur, subsequent downstream KEs or the adverse outcome (AO) are prevented or altered. Both direct and indirect evidence of essentiality were assessed according to the OECD developer’s handbook, with a summary provided in Table 1.

Table 1: Essentiality assessment of KEs of AOP 307.

Event	Direct evidence	Indirect evidence	Contradictory evidence	Overall essentiality assessment
KE-1690		***		High
KE-1614	***	***		High
KE-286		***		High

*Low level of evidence (some support for essentiality), ** Intermediate level of evidence (evidence for impact on one or more downstream KEs), ***High level of evidence (evidence for impact on AO).

Evidence Assessment

Addressess the biological plausibility, empirical support, and quantitative understanding from each KER in an AOP. More help

Evidence for anti-androgenicity, by antagonizing the AR, is strong. In this AOP, most KERs are considered highly biologically plausible with strong empirical evidence in support of this assessment, both from human data and animal studies. The overall evidence assessment scores for each KER are summarized in the below Table:

ID	Assessment score	Rationale
KER-2131	High	It is well established that testosterone activates the AR and that decreased testosterone levels leads to decreased AR activation.
KER-2124	High	It is well established that the AR regulates gene transcription, and that decreased AR activity leads to altered gene transcription.
KER-2820	High	It is well established that decreased AR activity leads to decreased AGD in male offspring.
KER-2127	Moderate	It is highly plausible that altered gene transcription in the perineum leads to decreased AGD in male offspring.

Known Modulating Factors

Modulating factors (MFs) may alter the shape of the response-response function that describes the quantitative relationship between two KES, thus having an impact on the progression of the pathway or the severity of the AO.The evidence supporting the influence of various modulating factors is assembled within the individual KERs. More help

Modulating Factor (MF)	Influence or Outcome	KER(s) involved
Age	Tissue-specific changes in AR expression with aging (Supakar et al, 1993; Wu et al, 2009).	Identified in KER-2131 and KER-2124, but also relevant for KER-2127.
Genotype	Decreased AR activation with increased number of CAG repeats in the first axon of the AR (Chamberlain et al, 1994; Tut et al, 1997).	Identified in KER-2131, KER-2124 and KER-2820.
Androgen deficiency syndrome	Low level of circulating testosterone in affected individuals (Bhasin et al, 2010).	Identified in KER-2131.
Castration	Reduced level of circulating testosterone in affected individuals.	Identified in KER-2131.

Quantitative Understanding

Optional field to provide quantitative weight of evidence descriptors. More help

The quantitative understanding between in vitro test data and in vivo is low. There is good quantitative understanding about the magnitude of reduction in explanted fetal testis testosterone production and effect on AGD (and other masculinization parameters) in rats, related to phthalate exposures. The dose-response relationship appears non-linear, with a low incidence rate of male under-virilization effects when testosterone production is reduced to around 46%, but with a steep increase in rate of malformations when testosterone is reduced by more than 75% (Earl Gray 2023; Earl Gray et al, 2024). This relationship has not been systematically evaluated for other chemicals.

Considerations for Potential Applications of the AOP (optional)

Addressess potential applications of an AOP to support regulatory decision-making.This may include, for example, possible utility for test guideline development or refinement, development of integrated testing and assessment approaches, development of (Q)SARs / or chemical profilers to facilitate the grouping of chemicals for subsequent read-across, screening level hazard assessments or even risk assessment. More help

References

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

Bhasin S, Cunningham GR, Hayes FJ, Matsumoto AM, Snyder PJ, Swerdloff RS, Montori VM; Task Force, Endocrine Society (2010). Testosterone therapy in men with androgen deficiency syndromes: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 95(6):2536-59.

Chamberlain NL, Driver ED, Miesfeld RL (1994). The length and location of CAG trinucleotide repeats in the androgen receptor N-terminal domain affect transactivation function. Nucleic Acids Res 22(15):3181-6.

Davey RA, Grossmann M (2016). Androgen Receptor Structure, Function and Biology: From Bench to Bedside. Clin Biochem Rev 37(1):3-15.

Draskau MK, Rosenmai AK, Bouftas N, Johansson HKL, Panagiotou EM, Holmer ML, Elmelund E, Zilliacus J, Beronius A, Damdimopolou P, van Duursen M, Svingen T (2024). AOP Report: An Upstream Network for Reduced Androgen Signaling Leading to Altered Gene Expression of Androgen Receptor-Responsive Genes in Target Tissues. Environ Toxicol Chem In Press (doi: 10.1002/etc.5972).

Earl Gray L Jr (2023). Biologically relevant reductions in fetal testosterone and Insl3 induced by in utero exposure to high levels of di-isononyl phthalate (DINP) in male rats. Toxicol Appl Pharmacol 465:116454.

Earl Gray L Jr, Lambright CS, Evans N, Ford J, Conley JM (2024). Using targeted fetal rat testis genomic and endocrine alterations to predict the effects of a phthalate mixture on the male reproductive tract. Curr Res Toxicol. 7:100180. doi: 10.1016/j.crtox.2024.100180

Gray LE, Ostby J, Furr J, Wolf CJ, Lambright C, Parks L, Veeramachaneni DN, Wilson V, Price M, Hotchkiss A, Orlando E, Guillette L (2001). Effects of environmental antiandrogens on reproductive development in experimental animals. Hum Reprod Update 7(3):248-64.

Holmer ML, Zilliacus J, Draskau MK, 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. Reprod Toxicol 128:108662.

Keller ET, Ershler WB, Chang C (1996). The androgen receptor: a mediator of diverse responses. Front Biosci 1:d59-71.

Murashima A, Kishigami S, Thomson A, Yamada G (2015). Androgens and mammalian male reproductive tract development. Biochim Biophys Acta 1849(2):163-70.

OECD (2008), Guidance Document on Mammalian Reproductive Toxicity Testing and Assessment, OECD Series on Testing and Assessment, No. 43, OECD Publishing, Paris.

OECD (2013) Guidance document in support of the test guideline on the extended one generation reproductive toxicity study no. 151.

Robitaille J, Langlois VS (2020). Consequences of steroid-5α-reductase deficiency and inhibition in vertebrates. Gen Comp Endocrinol 290:113400.

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(2):253-272.

Supakar PC, Song CS, Jung MH, Slomczynska MA, Kim JM, Vellanoweth RL, Chatterjee B, Roy AK (1993). A novel regulatory element associated with age-dependent expression of the rat androgen receptor gene. J Biol Chem 268(35):26400-8.

Svingen T, Villeneuve DL, Knapen D, Panagiotou EM, Draskau MK, Damdimopoulou P, O'Brien JM (2021). A Pragmatic Approach to Adverse Outcome Pathway Development and Evaluation. Toxicol Sci 184(2):183-190.

Thankamony A, Pasterski V, Ong KK, Acerini CL, Hughes IA (2016). Anogenital distance as a marker of androgen exposure in humans. Andrology 4(4):616-25.

Tut TG, Ghadessy FJ, Trifiro MA, Pinsky L, Yong EL (1997). Long polyglutamine tracts in the androgen receptor are associated with reduced trans-activation, impaired sperm production, and male infertility. J Clin Endocrinol Metab 82(11):3777-82.

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(4):1479-90.

Wu D, Lin G, Gore AC (2009). Age-related changes in hypothalamic androgen receptor and estrogen receptor alpha in male rats. J Comp Neurol 512(5):688-701.