Aop: 376

Title

Each AOP should be given a descriptive title that takes the form “MIE leading to AO”. For example, “Aromatase inhibition [MIE] leading to reproductive dysfunction [AO]” or “Thyroperoxidase inhibition [MIE] leading to decreased cognitive function [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

Androgen receptor agonism leading to male-biased sex ratio

Short name
A short name should also be provided that succinctly summarises the information from the title. This name should not exceed 90 characters. More help
AR agonism leading to male-biased sex ratio

Graphical Representation

A graphical summary of the AOP listing all the KEs in sequence, including the MIE (if known) and AO, and the pair-wise relationships (links or KERs) between those KEs should be provided. This is easily achieved using the standard box and arrow AOP diagram (see this page for example). The graphical summary is prepared and uploaded by the user (templates are available) and is often included as part of the proposal when AOP development projects are submitted to the OECD AOP Development Workplan. The graphical representation or AOP diagram provides a useful and concise overview of the KEs that are included in the AOP, and the sequence in which they are linked together. This can aid both the process of development, as well as review and use of the AOP (for more information please see page 19 of the Users' Handbook).If you already have a graphical representation of your AOP in electronic format, simple save it in a standard image format (e.g. jpeg, png) then click ‘Choose File’ under the “Graphical Representation” heading, which is part of the Summary of the AOP section, to select the file that you have just edited. Files must be in jpeg, jpg, gif, png, or bmp format. Click ‘Upload’ to upload the file. You should see the AOP page with the image displayed under the “Graphical Representation” heading. To remove a graphical representation file, click 'Remove' and then click 'OK.'  Your graphic should no longer be displayed on the AOP page. If you do not have a graphical representation of your AOP in electronic format, a template is available to assist you.  Under “Summary of the AOP”, under the “Graphical Representation” heading click on the link “Click to download template for graphical representation.” A Powerpoint template file should download via the default download mechanism for your browser. Click to open this file; it contains a Powerpoint template for an AOP diagram and instructions for editing and saving the diagram. Be sure to save the diagram as jpeg, jpg, gif, png, or bmp format. Once the diagram is edited to its final state, upload the image file as described above. More help

Authors

List the name and affiliation information of the individual(s)/organisation(s) that created/developed the AOP. In the context of the OECD AOP Development Workplan, this would typically be the individuals and organisation that submitted an AOP development proposal to the EAGMST. Significant contributors to the AOP should also be listed. A corresponding author with contact information may be provided here. This author does not need an account on the AOP-KB and can be distinct from the point of contact below. The list of authors will be included in any snapshot made from an AOP. More help

Kelvin J. Santana Rodriguez, Oak Ridge Institute for Science and Education, U.S. Environmental Protection Agency, Great Lakes Ecology Divison, Duluth, MN

Point of Contact

Indicate the point of contact for the AOP-KB entry itself. This person is responsible for managing the AOP entry in the AOP-KB and controls write access to the page by defining the contributors as described below. Clicking on the name will allow any wiki user to correspond with the point of contact via the email address associated with their user profile in the AOP-KB. This person can be the same as the corresponding author listed in the authors section but isn’t required to be. In cases where the individuals are different, the corresponding author would be the appropriate person to contact for scientific issues whereas the point of contact would be the appropriate person to contact about technical issues with the AOP-KB entry itself. Corresponding authors and the point of contact are encouraged to monitor comments on their AOPs and develop or coordinate responses as appropriate.  More help
Evgeniia Kazymova   (email point of contact)

Contributors

List user names of all  authors contributing to or revising pages in the AOP-KB that are linked to the AOP description. This information is mainly used to control write access to the AOP page and is controlled by the Point of Contact.  More help
  • Dan Villeneuve
  • Kelvin Santana Rodriguez
  • Gerald Ankley
  • Evgeniia Kazymova

Status

The status section is used to provide AOP-KB users with information concerning how actively the AOP page is being developed, what type of use or input the authors feel comfortable with given the current level of development, and whether it is part of the OECD AOP Development Workplan and has been reviewed and/or endorsed. “Author Status” is an author defined field that is designated by selecting one of several options from a drop-down menu (Table 3). The “Author Status” field should be changed by the point of contact, as appropriate, as AOP development proceeds. See page 22 of the User Handbook for definitions of selection options. More help
Author status OECD status OECD project SAAOP status
Open for citation & comment
This AOP was last modified on May 08, 2022 11:33
The date the AOP was last modified is automatically tracked by the AOP-KB. The date modified field can be used to evaluate how actively the page is under development and how recently the version within the AOP-Wiki has been updated compared to any snapshots that were generated. More help

Revision dates for related pages

Page Revision Date/Time
Agonism, Androgen receptor March 20, 2017 17:44
Increased, Differentiation to Testis May 05, 2022 16:31
Increased, Male Biased Sex Ratio May 05, 2022 13:40
Decrease, Population growth rate March 29, 2022 11:50
Agonism, Androgen receptor leads to Increased, Differentiation to Testis April 12, 2021 10:24
Agonism, Androgen receptor leads to Increased, Male Biased Sex Ratio April 12, 2021 09:31
Increased, Differentiation to Testis leads to Increased, Male Biased Sex Ratio August 23, 2021 11:55
Increased, Male Biased Sex Ratio leads to Decrease, Population growth rate May 05, 2022 11:17
17beta-Trenbolone November 29, 2016 18:42
Chemical:33664 17-Methyltestosterone March 23, 2021 13:34
5alpha-Dihydrotestosterone March 14, 2017 12:44
Methyldihydrotestosterone May 07, 2021 15:36
11-Keto-testosterone May 07, 2021 15:36

Abstract

In the abstract section, authors should provide a concise and informative summation of the AOP under development that can stand-alone from the AOP page. Abstracts should typically be 200-400 words in length (similar to an abstract for a journal article). Suggested content for the abstract includes the following: The background/purpose for initiation of the AOP’s development (if there was a specific intent) A brief description of the MIE, AO, and/or major KEs that define the pathway A short summation of the overall WoE supporting the AOP and identification of major knowledge gaps (if any) If a brief statement about how the AOP may be applied (optional). The aim is to capture the highlights of the AOP and its potential scientific and regulatory relevance More help

This adverse outcome pathway links  androgen receptor agonism in teleost fish during gonadogenesis to male-biased sexual differentiation and consequently, reduced population growth rate. Sex determination in teleost fishes is highly plastic; it can be genetically or environmentally influenced. Species with environmentally-based sex determination in particular can be very sensitive to environmental pollutants during the period of differentiation. Exogenous hormones are of ecological concern because they have the potential to alter gonad development and sex differentiation. Activation of the androgen receptor (AR) by endogenous androgens plays a crucial role in normal sex differentiation, sexual maturation, and spermatogenesis in vertebrates and inappropirate signaling by exogenous AR agonists can disrupt theses processes. For example, studies have shown that during early development in some teleost species, exposure to androgenic steroids can induce complete gonadal sex inversion, resuting in male-biased sex ratios. This can lead to impacts on population growth rates due to the decreased number of reproductively viable females in the population.

Background (optional)

This optional subsection should be 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. Examples of potential uses of the optional background section are listed on pages 24-25 of the User Handbook. More help

This AOP shares multiple KEs and KERs with AOP 346 which links aromatase inhibition to male-biased sex ratios in vertebrates with environmental sex determination. 

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 stressor and the biological system) of an AOP. More help
Key Events (KE)
This table summarises all of the KEs of the AOP. This table is populated in the AOP-Wiki as KEs are added to the AOP. Each table entry acts as a link to the individual KE description page.  More help
Adverse Outcomes (AO)
An AO is a specialised KE that represents the end (an adverse outcome of regulatory significance) of an AOP.  More help
Sequence Type Event ID Title Short name
1 MIE 25 Agonism, Androgen receptor Agonism, Androgen receptor
2 KE 1790 Increased, Differentiation to Testis Increased, Differentiation to Testis
3 KE 1791 Increased, Male Biased Sex Ratio Increased, Male Biased Sex Ratio
4 AO 360 Decrease, Population growth rate Decrease, Population growth rate

Relationships Between Two Key Events (Including MIEs and AOs)

This table summarises 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.To add a key event relationship click on either Add relationship: events adjacent in sequence or Add relationship: events non-adjacent in sequence.For example, if the intended sequence of KEs for the AOP is [KE1 > KE2 > KE3 > KE4]; relationships between KE1 and KE2; KE2 and KE3; and KE3 and KE4 would be defined using the add relationship: events adjacent in sequence button.  Relationships between KE1 and KE3; KE2 and KE4; or KE1 and KE4, for example, should be created using the add relationship: events non-adjacent button. This helps to both organize the table with regard to which KERs define the main sequence of KEs and those that provide additional supporting evidence and aids computational analysis of AOP networks, where non-adjacent KERs can result in artifacts (see Villeneuve et al. 2018; DOI: 10.1002/etc.4124).After clicking either option, the user will be brought to a new page entitled ‘Add Relationship to AOP.’ To create a new relationship, select an upstream event and a downstream event from the drop down menus. The KER will automatically be designated as either adjacent or non-adjacent depending on the button selected. The fields “Evidence” and “Quantitative understanding” can be selected from the drop-down options at the time of creation of the relationship, or can be added later. See the Users Handbook, page 52 (Assess Evidence Supporting All KERs for guiding questions, etc.).  Click ‘Create [adjacent/non-adjacent] relationship.’  The new relationship should be listed on the AOP page under the heading “Relationships Between Two Key Events (Including MIEs and AOs)”. To edit a key event relationship, click ‘Edit’ next to the name of the relationship you wish to edit. The user will be directed to an Editing Relationship page where they can edit the Evidence, and Quantitative Understanding fields using the drop down menus. Once finished editing, click ‘Update [adjacent/non-adjacent] relationship’ to update these fields and return to the AOP page.To remove a key event relationship to an AOP page, under Summary of the AOP, next to “Relationships Between Two Key Events (Including MIEs and AOs)” click ‘Remove’ The relationship should no longer be listed on the AOP page under the heading “Relationships Between Two Key Events (Including MIEs and AOs)”. More help

Network View

The AOP-Wiki automatically generates a network view of the AOP. This network graphic is based on the information provided in the MIE, KEs, AO, KERs and 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

Stressors

The stressor field is a structured data field that can be used to annotate an AOP with standardised terms identifying stressors known to trigger the MIE/AOP. Most often these are chemical names selected from established chemical ontologies. However, depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). It can also include non-chemical stressors such as genetic or environmental factors. Although AOPs themselves are not chemical or stressor-specific, linking to stressor terms known to be relevant to different AOPs can aid users in searching for AOPs that may be relevant to a given stressor. More help

Life Stage Applicability

Identify the life stage for which the KE is known to be applicable. More help
Life stage Evidence
Development High

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected. In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
Term Scientific Term Evidence Link
zebra fish Danio rerio High NCBI
medaka Oryzias latipes Low NCBI
fathead minnow Pimephales promelas Low NCBI
channel catfish Ictalurus punctatus Low NCBI
Oreochromis niloticus Oreochromis niloticus Low NCBI
Chinook salmon Oncorhynchus tshawytscha Low NCBI

Sex Applicability

The authors must select from one of the following: Male, female, mixed, asexual, third gender, hermaphrodite, or unspecific. More help
Sex Evidence
Unspecific High

Overall Assessment of the AOP

This section addresses the relevant biological domain of applicability (i.e., in terms of taxa, sex, life stage, etc.) and WoE for the overall AOP as a basis to consider appropriate regulatory application (e.g., priority setting, testing strategies or risk assessment). The goal of the overall assessment is to provide a high level synthesis and overview of the relative confidence in the AOP and where the significant gaps or weaknesses are (if they exist). Users or readers can drill down into the finer details captured in the KE and KER descriptions, and/or associated summary tables, as appropriate to their needs.Assessment of the AOP is organised into a number of steps. Guidance on pages 59-62 of the User Handbook is available to facilitate assignment of categories of high, moderate, or low confidence for each consideration. While it is not necessary to repeat lengthy text that appears elsewhere in the AOP description (or related KE and KER descriptions), a brief explanation or rationale for the selection of high, moderate, or low confidence should be made. More help

See details below.

Domain of Applicability

The relevant biological domain(s) of applicability in terms of sex, life-stage, taxa, and other aspects of biological context are defined in this section. Biological domain of applicability is informed by the “Description” and “Biological Domain of Applicability” sections of each KE and KER description (see sections 2G and 3E for details). In essence the taxa/life-stage/sex applicability is defined based on the groups of organisms for which the measurements represented by the KEs can feasibly be measured and the functional and regulatory relationships represented by the KERs are operative.The relevant biological domain of applicability of the AOP as a whole will nearly always be defined based on the most narrowly restricted of its KEs and KERs. For example, if most of the KEs apply to either sex, but one is relevant to females only, the biological domain of applicability of the AOP as a whole would be limited to females. While much of the detail defining the domain of applicability may be found in the individual KE and KER descriptions, the rationale for defining the relevant biological domain of applicability of the overall AOP should be briefly summarised on the AOP page. More help

Life Stage

The life stage applicable to this AOP is developing embryos and juveniles prior to- or during the gonadal developmental stage. This AOP is not applicable to sexually differentiated adults. 

Sex

The molecular initiating event for this AOP occurs prior to gonad differentiation. Therefore, this AOP is only applicable to sexually undifferentiated individuals

Taxonomic

Most evidence for this AOP is derived from fish in the class Osteichthyes. Both phylogenetic analysis and evaluation of protein sequence conservation via SeqAPASS (https://seqapass.epa.gov/seqapass/) has shown that the structure of the AR is  well conserved among most jawed vertebrates (Gnathostomata). This AOP is not expected to apply to mammals, birds, or other jawed vertebrates with genetic sex determination. However, it may be applicable to fishes, amphibians, and reptiles with environmentally-dependent sex determination, although outcomes may differ across physiologically different taxa. The present AOP is not considered relevant to Agnathans since the AR appears not to be present in jawless fishes (Thornton 2001; Hossain et al 2008). 

Essentiality of the Key Events

An important aspect of assessing an AOP is evaluating the essentiality of its KEs. 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.When assembling the support for essentiality of the KEs, authors should organise relevant data in a tabular format. The objective is to summarise briefly the nature and numbers of investigations in which the essentiality of KEs has been experimentally explored either directly or indirectly. See pages 50-51 in the User Handbook for further definitions and clarifications.  More help

Support for the essentiality of several of the key events in the AOP is provided by both in vivo and in vitro studies with chemicals acting as AR agonists and antagonists. Important in vivo studies typically have been conducted during the critical period of sexual differentiation.

Golan & Levavi-Sivian (2014) exposed genetic females of Nile tilapia (Oreochromis niloticus) to 17α-methyltestosterone (MT) and dihydrotestosterone (DHT), and showed that the two  AR agonists  induced a  male biased sex ratio in a dose-dependent manner.. However, in combined exposures with the AR antagonist flutamide (a pharmaceutical), the sex inversion potency of MT and DHT was reduced in a dose-dependent manner.  This provides direct evidence that activation of the AR is required for the subsequent key events to occur.

Crowder et al. (2018) generated zebrafish with a mutation in the AR gene (aruab105/105). Most mutants developed ovaries and displayed female secondary sexual characteristics. The small percentage of mutants that developed as males displayed female secondary sexual characteristics with structurally disorganized testes, and were unable to produce normal levels of sperm. This demonstrates that the AR is required for proper testis development and fertility and supports the essentiality of AR activation in testis differentiation.

In a similar study with zebrafish, Yu et al. (2018) generated an AR gene mutant line using CRISPR/Cas9 technology. The number of female offspring was increased and the resulting AR-null males had female secondary sex characteristics and were infertile due to defective spermatogenesis. This study again supports the essentiality of AR agonism for the development of testis and how inappropriate (increased) signaling in the pathway could result ina male biased sex ratio.

 

Key Event

Evidence

Essentiality/Assessment

Agonism, Androgen

moderate

There is good evidence from a sex inversion treatment via the direct blocking of AR using androgen antagonist that support the specificity of androgen receptor agonism for the subsequent key events to occur.  

Differentiation to Testis

moderate

Biological plausibility provides strong support for the essentiality of this event for the subsequent key events to occur. By definition, males have testis.

Male Biased Sex Ratio

moderate

Breeding females (and both sexes) are necessary for population sustainability. A male biased sex population suggests a reduced offspring production, due to reduced numbers of females in the population, and consequentially reduced population growth rates.

Population Sustainability

weak

Full life-cycle and even multi-generation tests would be the ideal method for the detection of population-relevant endpoints. Modeling, however, supports this outcome from a conceptual perspective.

 

 

Evidence Assessment

The biological plausibility, empirical support, and quantitative understanding from each KER in an AOP are assessed together.  Biological plausibility of each of the KERs in the AOP is the most influential consideration in assessing WoE or degree of confidence in an overall hypothesised AOP for potential regulatory application (Meek et al., 2014; 2014a). Empirical support entails consideration of experimental data in terms of the associations between KEs – namely dose-response concordance and temporal relationships between and across multiple KEs. It is examined most often in studies of dose-response/incidence and temporal relationships for stressors that impact the pathway. While less influential than biological plausibility of the KERs and essentiality of the KEs, empirical support can increase confidence in the relationships included in an AOP. For clarification on how to rate the given empirical support for a KER, as well as examples, see pages 53- 55 of the User Handbook.  More help

Biological Plausibility

The biological plausibility linking androgen receptor (AR) agonism to increased differentiation to testis is very solid. Actions of androgens are mediated by the AR which is part of the nuclear receptor superfamily. ARs are ligand-dependent transcription factors (Hossain et al., 2008). Steroidal androgens act by entering the cell and forming a complex with the AR, resulting in conformational change (Bohen et al., 1995; Pratt and Toft, 1997). The ligand-AR complex is translocated to the nucleus where it binds to specific short DNA sequences (Androgen Responsive Elements), thereby activating transcripton of androgen regulated genes (Harbott et al., 2009). During sexual development, endogenous androgen can therefore induce the upregulation of many genes involved in the male developmental pathway.

The direct link between increased differentiation to testis leading to a male biased sex ratio is highly plausible. If the conditions that favored a male producing phenotype (in this case, exposure to AR agonsts) overlap with the critical period of sex differentiation in a given population, it is reasonable that more phenotypic males will be produced (Orn et al., 2003; Seki et al., 2004; Bogers et al., 2006; Morthorst et al., 2010; Baumann et al., 2014; Golan & Levavi-Sivian 2014). Therefore, androgen exposure for repeated or prolonged periods of time conceptually will result in a male-biased population. Empirical evidence supporting the direct link between male-biased sex ratio and reduced population growth rate in fish species is lacking. However, altered sex ratios have the potential to affect fish populations (Marty et al. 2017). For example, a male-biased sex ratio suggests that the number of breeding females would be reduced. If the male-biased sex ratio persists and/or increases over time, the offspring produced per reproductive cycle would decrease, eventually leading to a diminished population size, assuming other demongraphic parameters remained largely constant (Brown et al. 2015; Grayson et al. 2014; Miller et al. 2021).

Concordance of Dose Response

The concentration-dependence of the key event responses with regard to the concentration of exogenous AR agonists has been established in vivo for some key events in the AOP. In general, effects on downstream key events occurred at concentrations equal to or greater than those at which upstream events occurred. However, binding to the androgen receptor (the MIE) was not directly measured in any of the in vivo studies. Nonetheless, AR binding of several of the agonists tested in vivo has been documented with in vitro studies with fish ARs (e.g., Wilson et al. 2004). In fish, phenotypic masculinization of female secondary sex characteristics has been used as an indirect measurement of in vivo AR agonism. However, in the of case this AOP specifically, AR agonism is occurring prior to sexual differentiation and the resultant “phenotypic measurement” for the in vivo study (gonad differentiation) is a discrete downstream key event. Consequently, in vitro evidence can reliably be used to identify specific chemicals as AR agonists (i.e., able to activate the MIE). That is, dependence of the severity of the downstream in vivo responses on concentration and potency of chemicals activating the AR in vitro can be used as indirect evidence of dose-response concordance between the MIE and downstream key events.

  1. Concentration dependent androgen receptor agonism (in vitro)
  • COS Whole Cell Binding Assay with fathead minnow AR (fhAR) were used in competitive binding experiments testing several natural and synthetic steroids, some of which are environmental contaminants, such as R1881, 17beta-trenbolone , and 17alpha-trenbolone. All showed a concentration dependent displacement of [3H]R1881 binding proving to be high affinity ligands for the fmAR. (Wilson et al., 2004).
    •  The synthetic steroids, R1881 and methytestosterone, had the highest affinities of all the chemicals tested with IC50 values of about 1.6 nM, followed by the synthetic steroids 17α- and 17β-trenbolone with IC50 values of about 8 and 16 nM, respectively.
    • Of the natural steroids, dihydrotestosterone was the strongest competitor with an IC50 of about 20 nM. The IC50 for the fish specific androgen, 11- ketotestosterone, was approximately 40 nM, followed by both testosterone and androstenedione at about 100 nM
  • Important to note that all of the above steroids tested were used in the in vivo studies that were selected to support this AOP demonstrating that all bound to the fhAR with a higher affinity than 11- ketotestosterone.
  1. Concentration dependent increased differentiation to testes:
  • Studies with zebrafish (Danio rerio) and Japanese medaka (Oryzias latipes) exposed to 17β-trenbolone during development resulted in masculinization in a concentration-dependent manner as evidenced from a significantly increased maturity of testes (Orn et al., 2006; Morthorst et al., 2010; Baumann et al., 2014) for some studies, this was determined ether by the abundance of spermatozoa and/or by the area of the testis.
  1. Concentration dependent increased male biased sex ratio:
  • Exposure of developing zebrafish (Danio rerio) to different concentrations of 17β-trenbolone and dihydrotestosterone led to an increased number of males in a dose-dependent fashion (Orn et al., 2003; Morthorst et al., 2010; Baumann et al., 2013; Baumann et al., 2014)
  1. Concentration dependent decline in population trajectory:
    • Modeled population trajectories show a concentration-dependent reduction in projected population size (Brown et al 2015, Miller et al. 2021) based on the ratio of male to female. Population-level effects exposure of fish to AR agonists have not been measured directly. 

Dose Concordance Table

Temporal concordance

Because this AOP involves actions during a specific development transition from an undifferentiated to differentiated gonad, the temporal concordance of the events is implicit. A male biased sex ratio cannot be observed untl the population has undergone sexual differentiation. Likewise, reproduction and associated population growth rate cannot be assessed until the animals achieve sexual maturity.

Consistency

We are aware of no cases where substantial exposure of susceptible teleost fish species  during sexual differentiation to known AR agonists  did not impact male sex ratios (for reviews see Pandian and Sheela 1995 and Leet et al. 2011). There are cases however, in which exposure to aromatizable androgens such as methyltestosterone can lead both to masculinization and feminization of fish (e.g., Piferrer et al. 1993); this most likely is due to conversion of the androgen to its corresponding estrogen analogue (i.e., methylestradiol; e.g., Hornung et al. 2004 ). In other instances, non-aromatizable androgens (e.g., dihydrotestosterone) have been reported to feminize fish exposed during early development (e.g., Davis et al. 1992; Bogers et al. 2006). The mechanism underlying this is uncertain, but plausibly could involve binding to the estrogen receptor which is known to interact with a variety of steroids, including androgens at comparatively test concentrations.

The adverse outcome is not wholly specific to this AOP.  Key events included overlap with AOPs linking other MIEs (e.g., aromatase inhibition, AOP 346) that can lead  to male biased sex ratios.

Uncertainties, inconsistencies, and data gaps

Overall, there is strong empirical support for the proposed AOP. We did not find notable inconsistencies in the literature reviewed as part of this AOP development. However, there were several notable data gaps which could potentially be addressed through further research:

(1) The detailed gene expression and signaling mechanisms via with AR activation induces differentiation to testes is not well understood or defined. If key steps in this process could be defined, one or more additional key events could potentially be inserted between Event 25 (agonism, androgen receptor) and Event 1790 (differentiation to testes, increased).

(2) Population-level consequences of a male biased sex ratio are based on modeling. At present, we found no empirical studies that establish the effect of a male-biased sex ratio on population growth rates. Current models assume that other demographic variables such as predation rates, prey availability, habitat availability, etc. are unaffected by sex ratio. This may or may not be the base in real-world populations.

Quantitative Understanding

Some proof of concept examples to address the WoE considerations for AOPs quantitatively have recently been developed, based on the rank ordering of the relevant Bradford Hill considerations (i.e., biological plausibility, essentiality and empirical support) (Becker et al., 2017; Becker et al, 2015; Collier et al., 2016). Suggested quantitation of the various elements is expert derived, without collective consideration currently of appropriate reporting templates or formal expert engagement. Though not essential, developers may wish to assign comparative quantitative values to the extent of the supporting data based on the three critical Bradford Hill considerations for AOPs, as a basis to contribute to collective experience.Specific attention is also given to how precisely and accurately one can potentially predict an impact on KEdownstream based on some measurement of KEupstream. This is captured in the form of quantitative understanding calls for each KER. See pages 55-56 of the User Handbook for a review of quantitative understanding for KER's. More help

At this time available data are insufficient to develop a quantitative AOP linking AR activation to male biased fish populations.

Considerations for Potential Applications of the AOP (optional)

At their discretion, the developer may include in this section discussion of the 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. While it is challenging to foresee all potential regulatory application of AOPs and any application will ultimately lie within the purview of regulatory agencies, potential applications may be apparent as the AOP is being developed, particularly if it was initiated with a particular application in mind. This optional section is intended to provide the developer with an opportunity to suggest potential regulatory applications and describe his or her rationale.To edit the “Considerations for Potential Applications of the AOP” section, on an AOP page, in the upper right hand menu, click ‘Edit.’ This brings you to a page entitled, “Editing AOP.” Scroll down to the “Considerations for Potential Applications of the AOP” section, where a text entry box allows you to submit text. In the upper right hand menu, click ‘Update AOP’ to save your changes and return to the AOP page or 'Update and continue' to continue editing AOP text sections.  The new text should appear under the “Considerations for Potential Applications of the AOP” section on the AOP page. More help

Sex ratios can be a useful endpoint in risk and hazard assessment of chemicals. In July 2011, the Fish Sexual Development Test (FSDT) has officially been adopted as OECD test guideline no. 234 for the detection of EDCs within the OECD conceptual framework at level 4 (OECD, 2011b). The Fish Sexual Development Test(FSDT; OECD TG 234, OECD, 2011) covers endocrine disruption during the developmental period of sexual differentiation of particularly zebrafish and uses gonadal differentiation and sex ratio as endocrine disruption-associated endpoints for indicating EAS (estrogen, androgen and steroidogenesis) activity (Dang & Kienzler 2019). Therefore, this AOP can provide additional support to the use of alternative measurements in this type of tests by screening for aromatase inhibitors.

References

List the bibliographic references to original papers, books or other documents used to support the AOP. More help

Baumann, L., Holbech, H., Keiter, S., Kinnberg, K. L., Knörr, S., Nagel, T., & Braunbeck, T. (2013). The maturity index as a tool to facilitate the interpretation of changes in vitellogenin production and sex ratio in the Fish Sexual Development Test. Aquatic toxicology (Amsterdam, Netherlands), 128-129, 34–42.

Baumann, L., Knörr, S., Keiter, S., Nagel, T., Rehberger, K., Volz, S., Oberrauch, S., Schiller, V., Fenske, M., Holbech, H., Segner, H., & Braunbeck, T. (2014). Persistence of endocrine disruption in zebrafish (Danio rerio) after discontinued exposure to the androgen 17β-trenbolone. Environmental toxicology and chemistry, 33(11), 2488–2496. https://doi.org/10.1002/etc.2698

Bogers, R., De Vries-Buitenweg, S., Van Gils, M., Baltussen, E., Hargreaves, A., van de Waart, B., De Roode, D., Legler, J., & Murk, A. (2006). Development of chronic tests for endocrine active chemicals. Part 2: an extended fish early-life stage test with an androgenic chemical in the fathead minnow (Pimephales promelas). Aquatic toxicology (Amsterdam, Netherlands), 80(2), 119–130. https://doi.org/10.1016/j.aquatox.2006.07.020

Crowder, C. M., Lassiter, C. S., & Gorelick, D. A. (2018). Nuclear Androgen Receptor Regulates Testes Organization and Oocyte Maturation in Zebrafish. Endocrinology, 159(2), 980–993. https://doi.org/10.1210/en.2017-00617

Davis, K. B., Goudie, C. A., Simco, B. A., Tiersch, T. R., & Carmichael, G. J. (1992). Influence of dihydrotestosterone on sex determination in channel catfish and blue catfish: period of developmental sensitivity. General and comparative endocrinology, 86(1), 147–151. https://doi.org/10.1016/0016-6480(92)90136-8

Davis, K. B., Simco, B. A., Goudie, C. A., Parker, N. C., Cauldwell, W., & Snellgrove, R. (1990). Hormonal sex manipulation and evidence for female homogamety in channel catfish. General and comparative endocrinology, 78(2), 218–223. https://doi.org/10.1016/0016-6480(90)90008-a

Dušková, M., & Pospíšilová, H. (2011). The role of non-aromatizable testosterone metabolite in metabolic pathways. Physiological research, 60(2), 253–261. https://doi.org/10.33549/physiolres.932080

Galvez, J., Mazik, P., Phelps, R., Mulvaney, D. (1995) Masculinization of Channel Catfish Ictalurus punctatus by Oral Administration of Trenbolone Acetate. World Aquaculture Society, 26(4), 378-383. https://doi.org/10.1111/j.1749-7345.1995.tb00832.x

Golan, M., & Levavi-Sivan, B. (2014). Artificial masculinization in tilapia involves androgen receptor activation. General and comparative endocrinology, 207, 50–55. https://doi.org/10.1016/j.ygcen.2014.04.026

Harbott, L. K., Burmeister, S. S., White, R. B., Vagell, M., & Fernald, R. D. (2007). Androgen receptors in a cichlid fish, Astatotilapia burtoni: structure, localization, and expression levels. The Journal of comparative neurology, 504(1), 57–73. https://doi.org/10.1002/cne.21435

Hossain, M. S., Larsson, A., Scherbak, N., Olsson, P. E., & Orban, L. (2008). Zebrafish androgen receptor: isolation, molecular, and biochemical characterization. Biology of reproduction, 78(2), 361–369. https://doi.org/10.1095/biolreprod.107.062018

Kemppainen, J. A., Langley, E., Wong, C. I., Bobseine, K., Kelce, W. R., & Wilson, E. M. (1999). Distinguishing androgen receptor agonists and antagonists: distinct mechanisms of activation by medroxyprogesterone acetate and dihydrotestosterone. Molecular endocrinology (Baltimore, Md.), 13(3), 440–454. https://doi.org/10.1210/mend.13.3.0255

Larsen, M. G., & Baatrup, E. (2010). Functional behavior and reproduction in androgenic sex reversed zebrafish (Danio rerio). Environmental toxicology and chemistry, 29(8), 1828–1833. https://doi.org/10.1002/etc.214

Leet, J. K., Gall, H. E., & Sepúlveda, M. S. (2011). A review of studies on androgen and estrogen exposure in fish early life stages: effects on gene and hormonal control of sexual differentiation. Journal of applied toxicology : JAT, 31(5), 379–398. https://doi.org/10.1002/jat.1682

Martyniuk, C. J., & Denslow, N. D. (2012). Exploring androgen-regulated pathways in teleost fish using transcriptomics and proteomics. Integrative and comparative biology, 52(5), 695–704. https://doi.org/10.1093/icb/ics072

Morthorst, J. E., Holbech, H., & Bjerregaard, P. (2010). Trenbolone causes irreversible masculinization of zebrafish at environmentally relevant concentrations. Aquatic toxicology (Amsterdam, Netherlands), 98(4), 336–343. https://doi.org/10.1016/j.aquatox.2010.03.008

Örn, S., Holbech, H., & Norrgren, L. (2016). Sexual disruption in zebrafish (Danio rerio) exposed to mixtures of 17α-ethinylestradiol and 17β-trenbolone. Environmental toxicology and pharmacology, 41, 225–231. https://doi.org/10.1016/j.etap.2015.12.010

Orn, S., Holbech, H., Madsen, T. H., Norrgren, L., & Petersen, G. I. (2003). Gonad development and vitellogenin production in zebrafish (Danio rerio) exposed to ethinylestradiol and methyltestosterone. Aquatic toxicology (Amsterdam, Netherlands), 65(4), 397–411. https://doi.org/10.1016/s0166-445x(03)00177-2

Orn, S., Yamani, S., & Norrgren, L. (2006). Comparison of vitellogenin induction, sex ratio, and gonad morphology between zebrafish and Japanese medaka after exposure to 17alpha-ethinylestradiol and 17beta-trenbolone. Archives of environmental contamination and toxicology, 51(2), 237–243. https://doi.org/10.1007/s00244-005-0103-y

Piferrer, F., Baker, I. J., & Donaldson, E. M. (1993). Effects of natural, synthetic, aromatizable, and nonaromatizable androgens in inducing male sex differentiation in genotypic female chinook salmon (Oncorhynchus tshawytscha). General and comparative endocrinology, 91(1), 59–65. https://doi.org/10.1006/gcen.1993.1104

Seki, M., Yokota, H., Matsubara, H., Maeda, M., Tadokoro, H., & Kobayashi, K. (2004). Fish full life-cycle testing for androgen methyltestosterone on medaka (Oryzias latipes). Environmental toxicology and chemistry, 23(3), 774–781. https://doi.org/10.1897/03-26

Shi, W. J., Jiang, Y. X., Huang, G. Y., Zhao, J. L., Zhang, J. N., Liu, Y. S., Xie, L. T., & Ying, G. G. (2018). Dydrogesterone Causes Male Bias and Accelerates Sperm Maturation in Zebrafish ( Danio rerio). Environmental science & technology, 52(15), 8903–8911. https://doi.org/10.1021/acs.est.8b02556

Swerdloff, R. S., & Wang, C. (1998). Dihydrotestosterone: a rationale for its use as a non-aromatizable androgen replacement therapeutic agent. Bailliere's clinical endocrinology and metabolism, 12(3), 501–506. https://doi.org/10.1016/s0950-351x(98)80267-x

Thornton J. W. (2001). Evolution of vertebrate steroid receptors from an ancestral estrogen receptor by ligand exploitation and serial genome expansions. Proceedings of the National Academy of Sciences of the United States of America, 98(10), 5671–5676. https://doi.org/10.1073/pnas.091553298

Wilson, V. S., Cardon, M. C., Thornton, J., Korte, J. J., Ankley, G. T., Welch, J., Gray, L. E., Jr, & Hartig, P. C. (2004). Cloning and in vitro expression and characterization of the androgen receptor and isolation of estrogen receptor alpha from the fathead Minnow (Pimephales promelas). Environmental science & technology, 38(23), 6314–6321. https://doi.org/10.1021/es049771j

Yu, G., Zhang, D., Liu, W., Wang, J., Liu, X., Zhou, C., Gui, J., & Xiao, W. (2018). Zebrafish androgen receptor is required for spermatogenesis and maintenance of ovarian function. Oncotarget, 9(36), 24320–24334. https://doi.org/10.18632/oncotarget.24407