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Relationship: 3774

Title

A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

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

Upstream event

The causing Key Event (KE) in a Key Event Relationship (KER). More help

Increased, differentiation to ovaries

Downstream event

The responding Key Event (KE) in a Key Event Relationship (KER). More help

Increased, female-biased sex ratio

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes.Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding	Point of Contact	Author Status	OECD Status
Activation, estrogen receptor alpha leads to increased, phenotypic female-biased sex ratio via increased, differentiation to ovaries	adjacent	High		John Frisch (send email)	Under development: Not open for comment. Do not cite

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER. More help

Term	Scientific Term	Evidence	Link
Animals	Metazoa	Moderate	NCBI

Sex Applicability

An indication of the the relevant sex for this KER. More help

Sex	Evidence
Female	High
Mixed	Moderate

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER. More help

Term	Evidence
Development	Moderate

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. More help

Ovaries are female organs responsible for producing eggs and secreting hormones that regulate development and reproduction. When proportionally more individuals in a population are phenotypic females per development of ovaries, an increased female-biased sex ratio occurs.

Evidence Collection Strategy

Include a description of the approach for identification and assembly of the evidence base for the KER. For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings. Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

This Key Event Relationship 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, 2025) 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.

Empirical studies are focused on increased development of ovaries and resulting increased female-biased sex ratio, in support of development of AOP 641. Authors determined that as ovaries are female organs, relative increase in numbers of phenotypic females in a population will result in an increased female-biased sex ratio.

Following initial human effort AOP development, artificial Intelligence (AI)-assisted literature search and synthesis, using EPA-AI GPT5, was used to identify additional literature, and draft Uncertainties and Inconsistencies content of this KER page. Additionally, EPA-AI GPT5 was also used to check for additional text improvement in other sections. All content generated through this process were reviewed and verified by the KER author against literature sources, prior to inclusion.

Evidence Supporting this KER

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help

Biological Plausibility

Addresses the biological rationale for a connection between KEupstream and KEdownstream. This field can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured. More help

Sex ratio is determined by the proportion of females to males in a population. Development of more organisms with ovaries will lead to a phenotypic female-biased sex ratio.

Empirical Evidence

Provides specific (citable) evidence that supports the idea of a change in the upstream KE (KEupstream) leading to, or being associated with, a subsequent change in the downstream KE (KEdownstream), assuming the perturbation of KEupstream is sufficient. More help

In fish husbandry, long-standing practice has demonstrated that adding estrogens during development can increase the number of individuals that develop ovaries, of interest when females achieve greater growth than males (in many fish species per review by Piferrer, 2001). Similarly exposure to 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).

Uncertainties and Inconsistencies

Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

Methodology for determining phenotypic females.
- Ideally, standard histology techniques should be used to determine whether organisms are phenotypic females based on observation of ovaries and oocytes rather than relying on secondary sex characteristics which can more frequently lead to incorrect classification (OECD, 2011; Dang and Kenzler, 2019).
Sex reversal in fish.
- Sex ratio should be determined immediately at the end of stressor exposure, as sex ratios determined after depuration in clean water may allow time for sex reversal due to gonad plasticity in teleost fish (Dang and Kenzler, 2019; Nagahama et al., 2021).

Known modulating factors

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references. More help

Quantitative Understanding of the Linkage

Captures information that helps to define how much change in the upstream KE, and/or for how long, is needed to elicit a detectable and defined change in the downstream KE. More help

Response-response Relationship

Provides sources of data that define the response-response relationships between the KEs. More help

Time-scale

Information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). More help

Known Feedforward/Feedback loops influencing this KER

Define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits. More help

Domain of Applicability

A free-text section of the KER description that the developers can use to explain their rationale for the taxonomic, life stage, or sex applicability structured terms. More help

Life Stage: Development.

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

Taxonomic: Animals.

References

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

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

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.

Organisation for Economic Co-operation and Development. 2011. Test no. 234: fish sexual development test. https://doi.org/10.1787/9789264122369-en (retrieved 24 June 2026).

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

U.S. Environmental Protection Agency. 2025. 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).

Italics indicate edits from John Frisch April 2026. A full list of updates can be found in the Change Log on the View History page.