Aop: 346

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

Aromatase inhibition leads to male-biased sex ratio via impacts on gonad differentiation

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
Aromatase inhibition leads to male-biased sex ratio via impacts on gonad differentiation

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
Kelvin Santana Rodriguez   (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
  • Kelvin Santana Rodriguez

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
Under Development: Contributions and Comments Welcome
This AOP was last modified on April 22, 2021 10:04
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
Inhibition, Aromatase September 16, 2017 10:14
Reduction, 17beta-estradiol synthesis by the undifferentiated gonad September 15, 2020 13:49
Increased, Differentiation to Testis September 15, 2020 14:01
Increased, Male Biased Sex Ratio August 07, 2020 18:17
Decrease, Population trajectory September 26, 2017 11:33
Inhibition, Aromatase leads to Reduction, E2 Synthesis by the undifferentiated gonad September 30, 2020 09:42
Inhibition, Aromatase leads to Increased, Male Biased Sex Ratio April 13, 2021 11:20
Reduction, E2 Synthesis by the undifferentiated gonad leads to Increased, Differentiation to Testis September 30, 2020 10:15
Inhibition, Aromatase leads to Increased, Differentiation to Testis September 30, 2020 09:34
Increased, Differentiation to Testis leads to Increased, Male Biased Sex Ratio September 17, 2020 09:03
Increased, Male Biased Sex Ratio leads to Decrease, Population trajectory September 17, 2020 06:34
Fadrozole November 29, 2016 18:42
Letrozole November 29, 2016 18:42
Exemestane November 12, 2020 01:53
Stressor:292 Clotrimazole November 12, 2020 01:55
Prochloraz November 29, 2016 18:42

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 inhibition of aromatase activity in teleost fish during gonadogenesis leading to a male-biased sex determination and successively, reduced population sustainability. Most gonochoristic fish species, develop either as males or females, and do not change sex throughout their entire life spans. However, there’s a developmental window in which their sex determination can be sensitive to environmental conditions or chemical pollutants. Treatment with steroid hormones prior to sexual differentiation has shown to induce ovary or testis development according to the type of steroid that is administered. For most vertebrate taxa, aromatase (Cyp19a1) is the rate-limiting enzyme for the biosynthesis of 17β - estradiol from testosterone. Many endocrine disrupting chemicals such as fadrozole, letrozole and exemestane are well known chemicals that inhibit the activity aromatase. Exposure during the critical period of sex differentiation in gonochoristic teleost fish with an aromatase inhibitor that blocks estrogen biosynthesis can induce phenotypic males. Given that females carry the major reproductive production of the population, a male-biased sex ratio can result in a reduced population fitness, particularly for those species present in ecosystems that are heavily impacted by human activities.

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

In fish, sexual differentiation occurs post hatching which makes them susceptible to the action of exogenous factors including hormones, temperature, pH, population density, social cues and more. As a result, the sex phenotype in most fish can be altered depending on the environmental conditions in which they are exposed during development, particularly during the critical period of sexual differentiation. At this stage, the bipotential gonad can be destined to take a testis or an ovary differentiation pathway that is reliant on both the genetic and environmental factors.

Sex steroid hormones are considered the natural inducers of sex differentiation for non-mammalian vertebrates where androgens and estrogens act, respectively, as testis and ovary inducers. In teleost fish, the hormonal balance between estrogens and androgens is essential during the sexual differentiation period and this balance is in turn dependent on the availability and activity of steroid synthesizing enzymes such as aromatase60.

Cytochrome P450 aromatase (CYP19) is the enzyme responsible for the conversion of C19 androgens to C18 estrogens in brain and gonadal tissues of vertebrates52,70. Therefore it a crucial enzyme for the female developmental pathway for many vertebrates. In fish, there are two isoforms of aromatase due to the teleost-specific whole-genome duplication.  Cyp19a1a that is mostly expressed in the gonads and cyp19a1b that is expressed in the brain.

In recent years, there has been growing concern about the potential impacts of endocrine disrupting chemicals in the wildlife. Particularly of important concern, is the effects it can exert in early life stages that can lead to major impacts at the population level.  Many EDC’s are known to alter the sexual phenotype of fish by disrupting sex steroid synthesizing enzymes. Cyp1a1 can be a potential target for endocrine disrupting chemicals as it catalyzes the final step of estrogen biosynthesis which control crucial developmental and physiological processes.

Disruption of aromatase expression will alter the production rate of estrogens in the developing gonads, increasing an imbalance in the androgen-t­o-estrogen ratio leading to the disruption of estrogen related biological processes that lead to the determination and differentiation of the ovary. Therefore, as aromatase inhibitors block the synthesis of estrogens (by inhibiting the conversion of androgens to estrogens), the level of androgens in the developing organism increases, inducing testis differentiation and male maturation instead of a female developing pathway7 .  When the conditions that favor a male differentiation pathway persists, male biased sex ratios can occur. As a result, the number of breeding females can decrease over time and the population productivity can be affected. Therefore, altered sex ratios can have negative impacts on population growth and sustainability.

The present AOP provides the evidence framework of the negative impacts of aromatase inhibition at early developmental stage of teleost fish particularly during the critical period of sexual differentiation and how can this ongoing exposure on population can lead to a population dysfunction.

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
MIE 36 Inhibition, Aromatase Inhibition, Aromatase
KE 1789 Reduction, 17beta-estradiol synthesis by the undifferentiated gonad Reduction, E2 Synthesis by the undifferentiated gonad
KE 1790 Increased, Differentiation to Testis Increased, Differentiation to Testis
KE 1791 Increased, Male Biased Sex Ratio Increased, Male Biased Sex Ratio
AO 360 Decrease, Population trajectory Decrease, Population trajectory

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
Name Evidence Term
Fadrozole High
Letrozole High
Exemestane Moderate
Stressor:292 Clotrimazole Low
Prochloraz High

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
zebrafish Danio rerio High NCBI
Oreochromis niloticus Oreochromis niloticus High NCBI
Chinook salmon Oncorhynchus tshawytscha Low NCBI
fathead minnow Pimephales promelas Low NCBI
European sea bass Dicentrarchus labrax 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

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. Since the sexually dimorphic expression of aromatase plays a crucial role in the differentiation to either testis or ovaries in the undifferentiated bipotential gonad, this key event relationship can be applicable to the exact stage of development at which the aromatase enzyme works to influence gonadal differentiation. This AOP is not applicable to sexually differentiated adults. 

Studies with zebrafish have shown that both brain and gonadal aromatase expression can be observed at 20 days post-fertilization with and increase in expression at 25 days post-fertilization in zebrafish destined to become females which also coincided with onset of gonadal differentiation period (Lau et al. 2016). In tilapia, aromatase expression can be observed as early as 3-4 days post fertilization with and increase in expression starting at 11 days post-fertilization in genetic females (Kwon, J. et al. 2001). Additionally, it has been shown that the period of 7-14 days post-fertilization is the most sensitive towards an aromatase inhibitor and that a consecutive exposure of 2-3 weeks is sufficient for the masculinization of the majority of genetic female tilapia fish (Kwon, J. et al. 2000). This suggest that to redirect the sexual differentiation pathway from ovary to testis, an alteration of aromatase expression will be most effective during the early developmental stage prior and during the critical sex differentiation period. 

Sex

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

Taxonomic

The taxonomic applicability of this AOP is the class Osteichthyes. However, phylogenetic analysis among mammalian, amphibian, reptile, bird, and fish has shown that aromatase is well conserved among all vertebrates (Wilson JY et al., 2005). Additionally, CYP19 was detected in the amphioxus suggesting that it has possible origin in primitive chordates. Therefore, because all key events in the present AOP can be applicable to most non-mammalian vertebrates, it is probable that this AOP could be relevant to amphibians, reptiles and birds as well.  Though, the outcomes mind differ due to species-specific differences. 

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 was provided mainly by gene knockout of the cyp1a1 gene in zebrafish and tilapia. Teleost fish have two genes encoding for aromatase; cyp1a1a that is mainly expressed in the gonads and cyp1a1b expressed in the brain. Studies have demonstrated that mutant lines of cyp1a1b develop as females while cypa1a mutants develop as males suggesting that gonadal aromatase inhibition is crucial step for the subsequent key events to occur. 

  1. Lau et al. 201613 generated indel mutations in zebrafish cyp19a1a gene using TALEN and CRISPR/Cas9 approaches. All mutant cyp19a1a-/- developed as males. Histological examination (at 120 days post-fertilization) of the cyp1a1a-/- mutant showed that all exhibited normal spermatogenesis in the testis with no observable difference to the wild type (+/+) and heterozygous (+/-) males. However, to prove the role of E2 synthesis for ovarian differentiation, they performed an experiment to rescue the phenotype of cyp19a1a mutant by E2 treatment (0.05, 0.50 and 5.00 nM) over the time of gonadal differentiation (15–30 days port-fertilization). The result showed that exposure to E2 caused normal ovarian formation with fully developed perinucleolar oocytes and little amount of stromal tissues, and the effect could be observed in some individuals even at the lowest concentration (0.05 nM). This supports the essentiality of aromatase inhibition relative to E2 synthesis reduction as a critical step for testis differentiation.
  2. On a similar study with zebrafish, Muth-Köhne et al. 2016generated cyp19a1a and cyp19a1b gene mutant lines and a cyp19a1a;cyp19a1b double-knockout line in zebrafish using transcription activator-like effector nucleases (TALENs). All cyp19a1a mutants and cyp19a1a;cyp19a1b double mutants developed as males whereas cyp1a1b double mutant (-/-) had a 1:1 sex ratio similar to the wild type controls. This supports the essentiality of gonadal aromatase inhibition for testis differentiation leading to a male biased sex ratio population. Additionally, a rescue experiment was performed using 17 β-estradiol on all male mutant cyp1a1a-/-  and the results suggested that treatment could rescue the sex ratio defect  (9 females among 14 fish).
  3. Similar support using Nile tilapia (Oreochromis niloticus) was provided in a study by Zhang et al. 201712.   Using genetic female mutant for cypa1a and cyp1a1b. Results showed that all cyp19a1a+/- XX and cyp19a1a+/+ XX fish developed as females, whereas all cyp19a1a-/- XX and cyp19a1a-/- XY fish developed as males. The cyp19a1a-/- XX tilapia shifted to the male pathway at as early as 5 days after hatch (dah), as reflected by the gonadal expression and were fertile. This supports the essentiality of gonadal aromatase inhibition during early development for a testis differentiation pathway to be induced. 

Key Event

Evidence

Essentiality/Assessment

Inhibition, Aromatase 

strong

There is good evidence from gene knockout experiments of the two different isoforms of aromatase that support the specificity of gonadal aromatase inhibition for the subsequent key events to occur. 

E2 Synthesis by the undifferentiated gonad 

moderate

There is evidence from a stop (by cyp19a1knockout) and recovery (through compensation) experiment where Ecan rescue the sex ratio altered due to the gonadal aromatase gene knockout suggesting that E2 depletion is necessary for the subsequent key events to occur.

Differentiation to Testis 

strong

Biological plausibility provides strong support for the essentiality of this event for the subsequent key events to occur. 

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 and consequentially reduced population sustainability. 

.

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

Aromatase is the key enzyme in the conversion of C19 androgens to C18 estrogens and the biological plausibility linking aromatase inhibition to E2 reduction is very solid. Additionally, the role of E2 as a major regulator for downstream estrogen-responsive genes necessary for proper female gonad development is well documented in literature (Gorelick et al. 2011; Guiguen et al. 2010). The link between E2 reduction for the undifferentiated gonad leading to an increased differentiation to testis is highly plausible. As the levels of estradiol are reduced, ER responsive genes required for proper ovarian differentiation will be downregulated in the bipotential gonad and instead allowing gene expression that leads to the morphological development of the testes due to an imbalance in the androgen to estrogen ratio (Shi et al., 2018; Yin et al. 2017; Zhang et al. 2017). Therefore, it is plausible that estradiol reduction in the undifferentiated gonad at the onset of sexual differentiation promotes testis differentiating in a concentration dependent manner (Baumann et al., 2015; Morthorst et al., 2010). The direct link between increased differentiation to testis leading to a male biased sex ratio is also well supported by biological plausibility. If the conditions that favored a male producing phenotype (in this case, the aromatase inhibitor) overlap with the critical period of sex differentiation in a given population, it is reasonable that more male offspring will be produced (D'Cotta et al., 2001, Kwon et al., 2000; Luzio et al. 2016). Therefore, persistence of such conditions for repeated or prolong periods of times within the habitat of given species, can result in a male-biased population. Empirical evidence supporting the direct link between male biased population and a reduced population sustainability in fish species is lacking. However, increasing or permanent biased sex ratios can definitely have significant effects in sustainable fish populations (Marty et al. 2017). A male-biased sex ratio already suggests that the number of breeding females is reduced. If the male-biased sex ratio persists and/or increases over time, the offspring production for such population could eventually decrease and consequently, population productivity would be reduced (Brown et al. 2015; Grayson et al. 2014).

 

Concordance of Dose Response Relationship

Concentration dependence of the key events in response to the concentration of the aromatase inhibitor has been established for major key events in several teleost fish species using in vivo studies. The best supporting evidence would be studies that considered multiple key events in an in vivo study. However, in most of them there were exceptions. The differential sensitivity to inhibition of Cytochrome P450 Aromatase (CYP19) is best measured in vitro (Doering et al. 2019) but most studies that support this AOP are performed in vivo. There are cases in which the significant effect of reduced E2 was either not measured, measured at a time period outside the critical differentiation period, only one concentration of an aromatase inhibitor was used (Ruksana et al. 2010) or were gene knockout studies (Yin et al. 2017; Zhang et al. 2017) therefore these could not be considered for the dose-response relationship. Additionally, increased differentiation to testes is observed via histological examinations in which most studies using aromatase inhibitors only determined the general presence of male or female first and secondary characteristics but a degree of differentiation or differentiation stage of the gonads was not measured nor reported in most studies based on the doses. The most observable dose response relationship for this aop was for the non-adjacent relationship between aromatase inhibition and an increased male biased sex ratio in which several studies using multiple concentrations of an aromatase inhibitor leading to increased number of males in a dose-dependent way.

Concentration-dependent aromatase inhibition:

  • Immunohistochemical analyses revealed that fish at 35 dah treated with higher concentrations of EM (500, 1000 and 2000 μg/g feed) had no reaction against P450arom but cells with strongly immunopositive responses against P450arom were evident in the lowest dose of EM (100 μg/g feed) similar to the differentiating ovaries of the control fish; these cells occurred as clusters in the vicinity of blood vessels (Ruksana et al. 2010)

Concentration dependent increased male biased sex ratio:

  • Nile tilapia (Oreochromis niloticus), Fathead minnow (Pimephales promelas), Zebrafish (Danio rerio) exposed to different concentrations of aromatase inhibitors (Exemestane, Fadrozole, Prochloraz) lead to increased number of males in a dose-dependent way (Kwon et al., 2000; Uchida et al., 2004; Ruksana et al. 2010; Thorpe et al., 2011, Holbech et al., 2012).

Concentration dependent decline in population trajectory:

  • Modeled population trajectories for male skews of zebrafish exposed to clotrimazole show a concentration-dependent reduction in projected population growth and viability (Brown et al. 2015). Population-level effects have not been measured directly.

 

Temporal Concordance

Temporal concordance of the AOP from aromatase inhibition to decreased E2 production, increased differentiation to testes and increased male-biased sex ratio (e.g., (Ruksana et al., 2010; Yin et al. 2017; Zhang et al. 2017) has been established. However, beyond that key event, temporal concordance has not yet been established possibly due limiting capability to test and/or document particular population viability in situ. From the evidence gathered for this specific AOP, the best way to determine population viability is via multifactorial population viability analyses that generate the distribution of likely fates for a population exposed to endocrine disrupting chemicals that affect aromatase activity at the developmental stage.

 

Consistency

We are aware of no cases where the pattern of key events described was observed without also observing a significant impact on male sex ratios. The adverse outcome is not specific to this AOP. Many of the key events included in this AOP overlap with AOPs linking other molecular initiating events during the period of development (ie. androgen receptor agonism, AOP 376) to male biased sex ratios.

 

Uncertainties, inconsistencies, and data gaps

Currently the major uncertainty in this AOP is the biological linkage between E2 synthesis reduction by the undifferentiated gonad leading to an increased, differentiation to testis. Biological plausibility connections have been established, but experimental measurements of E2 during the particular period of differentiation is lacking.

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

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 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. 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
Citation
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