This AOP is licensed under the BY-SA license. This license allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use. If you remix, adapt, or build upon the material, you must license the modified material under identical terms.

AOP: 641

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

Activation, estrogen receptor alpha leads to increased, phenotypic female-biased sex ratio via increased, differentiation to ovaries

Short name

A name that succinctly summarises the information from the title. This name should not exceed 90 characters. More help

Activation, ERα leads to female-biased sex ratio via increased, differentiation to ovaries

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 v2.8

Graphical Representation

A graphical representation of the AOP.This graphic should list all KEs in sequence, including the MIE (if known) and AO, and the pair-wise relationships (links or KERs) between those KEs. More help

Click to download graphical representation template Explore AOP in a Third Party Tool

Authors

The names and affiliations of the individual(s)/organisation(s) that created/developed the AOP. More help

Of the content populated in the AOP-Wiki: John R. Frisch and Travis Karschnik, General Dynamics Information Technology; Daniel L. Villeneuve, US Environmental Protection Agency, Risk Assessment Support Division; Scott Lynn, US Environmental Protection Agency, Office of Chemical Safety and Pollution Prevention.

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

John Frisch (email point of contact)

Contributors

Users with write access to the AOP page. Entries in this field are controlled by the Point of Contact. More help

John Frisch

Coaches

This field is used to identify coaches who supported the development of the AOP.Each coach selected must be a registered author. More help

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

This AOP was last modified on April 17, 2026 14:58

Revision dates for related pages

Page	Revision Date/Time
Activation, estrogen receptor alpha	January 28, 2026 14:32
Increased, differentiation to ovaries	April 17, 2026 13:59
Increased, phenotypic female-biased sex ratio	April 17, 2026 14:57
Activation, ERα leads to Increased, differentiation to ovaries	April 17, 2026 14:47
Increased, differentiation to ovaries leads to Increased, female-biased sex ratio	April 17, 2026 14:52

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

Estrogen receptor alpha (ERa) is a nuclear transcription factor involved in regulation of many physiological processes in vertebrates. Binding by estrogen induces the transcription of target genes. Here we focus on the role of ERa in the hypothalamus- pituitary-gonadal (HPG) axis involved in reproductive development. Estrogens have a central role in pathways leading to ovarian differentiation, with androgens causing testicular differentiation (Guiguen et al., 2010).

In species with genetic sex determination (GSD) whether an individual develops into a male or female is based on chromosome composition. In species with environmental sex determination (ESD), environmental factors such as temperature influence whether an individual develops into a male or female (in vertebrates, mainly fish and reptiles per Nagahama et al., 2021). For a review of sex determination systems in invertebrates, see (Picard et al., 2021).

In fish husbandry, long standing practice has demonstrated that adding estrogens during development can increase the number of individuals that develop ovaries. This is of interest when females achieve greater growth than males (in many fish species per review by Piferrer, 2001). Similarly exposure to estrogenic endocrine disrupting compounds during development can result in an increased number of fish that develop ovaries as seen in sex ratios (in many fish species per review by Dang and Kienzler, 2019). An adverse outcome of female-biased sex ratio occurs when a disruptor skews more individuals to developing ovaries and resulting in more females and intersex individuals, which has the potential to reduce effective population size and have negative effects on population dynamics. A 7-year whole lake study showed a near population collapse of fathead minnows (Pimephales promelas) exposed to doses of 5-6 ng/L 17β-ethynylestradiol (EE2) in a treated lake compared to a reference lake due to arrested male testicular development and genotypic males exhibiting ova-testes, followed by loss of young-of-the year age classes (Kidd et al., 2007).

Although the relative importance of genetic and environmental factors for determining the sex of an individual differ among classes of vertebrates, there are some evolutionarily conserved consistencies in development (Ditewig and Yao, 2005; Nichol et al., 2022). The gonadal primordium (genital ridge) develops on the surface of the mesonephros (intermediate mesoderm), which has the capability to develop either into ovaries or testes. Upon receiving cues to undergo female development, cells from the gonadal primordium differentiate into granulosa cells. Subsequent differentiation results in increased specialization of cells in the developing ovary, including germ cells (follicles that develop into oocytes) and somatic cells (granulosa and thecal cells that produce hormones among other duties, stromal cells that provide connective tissue, epithelium surface cells).

This AOP contributes to the scientific understanding of the mechanistic foundations for linking estrogen receptor agonism to the adverse outcome female-biased sex ratio, with female-biased sex ratio representing an apical endpoint in guideline tests associated with the Endocrine Disruptor Screening Program (US EPA, 1998).

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

This AOP was part of an Environmental Protection Agency effort to develop AOPs that establish scientifically supported causal linkages between alternative endpoints measured using new approach methodologies (NAMs) and guideline apical endpoints measured in Tier 1 and Tier 2 test guidelines (U.S. EPA, 2024) employed by the Endocrine Disruptor Screening Program (EDSP). A series of key events that represent significant, measurable, milestones connecting molecular initiation to apical endpoints indicative of adversity were identified based on scientific review articles and empirical studies. Additionally, scientific evidence supporting the causal relationships between each pair of key events was assembled and evaluated. The present effort focused primarily on empirical studies with fish.

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

The scope of the aforementioned EPA project was to develop AOP(s) relevant to apical endpoints employed in the test guidelines, based on mechanisms consistent with empirical studies. The literature used to support this AOP and its constituent pages began with the test guidelines and followed to primary, secondary, and/or tertiary works concerning the relevant underlying biology. KE and KER page creation and re-use was determined using Handbook principles where page re-use was preferred.

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

MIE

1065

Activation, estrogen receptor alpha

Activation, ERα

2417

Increased, differentiation to ovaries

2418

Increased, phenotypic female-biased sex ratio

Increased, female-biased sex ratio

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

Increased, differentiation to ovaries leads to Increased, female-biased sex ratio

adjacent

High

Activation, ERα leads to Increased, differentiation to ovaries

non-adjacent

Moderate

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

Life Stage Applicability

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

Life stage	Evidence
Development	Moderate

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. More help

Term	Scientific Term	Evidence	Link
fish	fish	High	NCBI
Amphibia	Amphibia	High	NCBI

Sex Applicability

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

Sex	Evidence
Female	High
Mixed	Moderate

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

1. Support for Biological Plausibility of Key Event Relationships: Is there a mechanistic relationship between KEup and KEdown consistent with established biological knowledge?
Key Event Relationship (KER)	Level of Support Strong = Extensive understanding of the KER based on extensive previous documentation and broad acceptance.
Relationship 3773: ERa activation AVPV leads to increased, differentiation to ovaries	Strong support. The relationship between activation of estrogen receptor alpha and increased differentiation to ovaries is broadly accepted and supported among animals, particularly fish data. Activation of estrogen receptor alpha is often studied in vitro, with activation of estrogen receptor alpha inferred in organism studies when downstream effects are consistent with in vitro observations.
Relationship 3774: Increased, differentiation to ovaries leads to Increased, female-biased sex ratio	Strong support. The relationship between increased differentiation to ovaries and increased female biased sex ratio is broadly accepted and supported among animal data. When an increased number of individuals develop ovaries there is an increase in females in a population, directly resulting in increased female biased sex ratio.
Overall	Strong support. Extensive understanding of the relationships between events from empirical studies from animals, particularly fish, with some inference of estrogen receptor alpha activation from in vitro studies when performing in vivo studies.

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

Life Stage: Development.

Sex: Applies to females, with some mixed genders observed.

Taxonomic: Possible in any animal species that has females and males. Largely studied in vertebrates, with some research in sexually reproducing invertebrate. Vertebrates differ in prevalence of environmental sex determination (Nagahama et al., 2021), with amphibians, reptiles and fish most likely to have increased differentiation to ovaries from environmental factors.

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

2. Essentiality of Key Events: Are downstream KEs and/or the AO prevented if an upstream KE is blocked?
Key Event (KE)	Level of Support Moderate = Direct evidence from specifically designed experimental studies illustrating essentiality between key events, best characterized as non-adjacent due to known hormone effects. Strong = Direct evidence from specifically designed experimental studies illustrating essentiality and direct relationship between key events.
MIE 1065 Activation estrogen receptor alpha	Moderate support. Activation of estrogen receptor alpha leads to increased differentiation to ovaries. Evidence is available from in vitro studies to establish estrogenicity by estrogen receptor competitive binding assays and from in vivo exposure to estrogens, endocrine-disrupting compounds, and toxicants. Best evidence for essentiality for activation of estrogen receptor alpha is from fish husbandry studies that have shown that in a wide variety of species adding estrogen compounds during development results in an increase in the number of individuals that develop ovaries, of economic interest when females achieve greater growth than males (Reviewed in Piferrer, 2001). Gene knockout studies suggest that the relationship between activation of estrogen receptor alpha and development of ovaries is best characterized as non-adjacent, with Estrogen receptor alpha having a key signalling role for enzyme production responsible for hormone levels leading to differentiation of ovaries with normal function (e.g. aromatase (cyp19a1a); Lau et al, 2016; Zhang et al., 2017; Chen et al. 2018; Yan et al. 2019).
KE 2417 Increased, differentiation to ovaries	Strong support. Increased differentiation to ovaries leads to increased female biased sex ratio. Sex ratio is determined by the proportion of females to males in a population. Development of more organisms with ovaries will lead directly to a female-biased sex ratio.
AO 2418 Increased, female-biased sex ratio	This is the final event of the AOP.
Overall	Moderate to strong support. Direct evidence from empirical studies from animals, particularly fish, and cell lines for all key events.

Evidence Assessment

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

3. Empirical Support for Key Event Relationship: Does empirical evidence support that a change in KEup leads to an appropriate change in KEdown?
Key Event Relationship (KER)	Level of Support Strong = Experimental evidence from exposure to toxicant shows consistent change in both events across taxa and study conditions.
Relationship 3773: ERa activation AVPV leads to increased, differentiation to ovaries	Strong support. Activation of estrogen receptor alpha leads to increased differentiation to ovaries. Evidence is available from in vivo estrogen compound studies, endocrine disruptor studies, toxicant studies, temperature alteration studies, and in vitro studies to establish estrogenicity by estrogen receptor competitive binding assays. Activation of estrogen receptor alpha occurred earlier in the time-course of exposure than increased differentiation to ovaries, and the concentrations that activated estrogen receptor alpha were equal to or lower than the concentrations that increased differentiation to ovaries. Therefore, the data support a causal relationship. In some in vivo laboratory studies, activation of estrogen receptor alpha is inferred by response from a stressor known to be an ERa agonist from in vitro studies.
Relationship 3774: Increased, differentiation to ovaries leads to Increased, female-biased sex ratio	Strong support. Increased differentiation to ovaries leads to increased female biased sex ratio. Evidence is available from vivo estrogen compound studies, endocrine disruptor studies, toxicant studies, and temperature alteration studies. Increased differentiation to ovaries occurred earlier in the time-course of exposure than increased female biased sex ratio, and the concentrations of stressors that increased differentiation to ovaries were equal to or lower than the concentrations that increased female biased sex ratio. When an increased number of individuals develop ovaries there is an increase in females in a population, directly resulting in increased female biased sex ratio. Therefore, the data support a causal relationship.
Overall	Strong support. Evidence from empirical studies shows consistent relationships in upstream and downstream events, with upstream events occurring earlier in the time-course of exposure and at equal or lower concentrations than downstream events, supporting causal relationships.

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

Quantitative Understanding

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

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

References

Chen Y, Tang H, Wang L, He J, Guo Y, Liu Y, Liu X, Lin H. 2018. Fertility Enhancement but Premature Ovarian Failure in esr1-Deficient Female Zebrafish. Frontiers in Endocrinolology 9: 567.

Dang Z, Kienzler A. 2019. Changes in fish sex ratio as a basis for regulating endocrine disruptors. Environment International 130: 104928.

Ditewig AC, Yao HH. 2005. Organogenesis of the ovary: a comparative review on vertebrate ovary formation. Organogenesis 2(2): 36-41.

Guiguen Y, Fostier A, Piferrer F, Chang CF. 2010. Ovarian aromatase and estrogens: a pivotal role for gonadal sex differentiation and sex change in fish. General and Comparative Endocrinology 165(3): 352-366.

Kidd KA, Blanchfield PJ, Mills KH, Palace VP, Evans RE, Lazorchak JM, Flick RW. 2007. Collapse of a fish population after exposure to a synthetic estrogen. Proceedings of the National Academy of Sciences of the United States of America 104(21): 8897-8901.

Lau ES, Zhang Z, Qin M, Ge W. Knockout of Zebrafish Ovarian Aromatase Gene (cyp19a1a) by TALEN and CRISPR/Cas9 Leads to All-male Offspring Due to Failed Ovarian Differentiation. 2016. Scientific Reports 6:37357.

Nagahama Y, Chakraborty T, Paul-Prasanth B, Ohta K, Nakamura M. 2021. Sex determination, gonadal sex differentiation, and plasticity in vertebrate species. Physiological Reviews 101(3): 1237-1308.

Nicol B, Estermann MA, Yao HH, Mellouk N. 2022. Becoming female: Ovarian differentiation from an evolutionary perspective. Frontiers in Cell and Development Biology 10: 944776.

Piferrer F. 2001. Endocrine sex control strategies for the feminization of teleost fish. Aquaculture 197: 229–281.

Picard MAL, Vicoso B, Bertrand S, Escriva H. 2021. Diversity of Modes of Reproduction and Sex Determination Systems in Invertebrates, and the Putative Contribution of Genetic Conflict. Genes 12(8): 1136.

U.S. Environmental Protection Agency. 1998. Health Effects Test Guidelines OPPTS 870.3800 Reproduction and Fertility Effects. https://ntp.niehs.nih.gov/sites/default/files/iccvam/suppdocs/feddocs/epa/epa_870_3800.pdf (retrieved 24 December 2025)

U.S. Environmental Protection Agency. 2004. EDSP Test Guidelines and Guidance Document. https://www.epa.gov/test-guidelines-pesticides-and-toxic-substances/edsp-test-guidelines-and-guidance-document (retrieved 25 July 2025).

Yan L, Feng H, Wang F, Lu B, Liu X, Sun L, Wang D. 2019. Establishment of three estrogen receptors (esr1, esr2a, esr2b) knockout lines for functional study in Nile tilapia. The Journal of Steroid Biochemistry and Molecular Biology 191:105379.

Zhang X, Li M, Ma H, Liu X, Shi H, Li M, Wang D. Mutation of foxl2 or cyp19a1a Results in Female to Male Sex Reversal in XX Nile Tilapia. 2017. Endocrinology. 158(8): 2634-2647.