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AOP: 167

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

Early-life estrogen receptor agonism leading to endometrial adenosquamous carcinoma via promotion of sine oculis homeobox 1 progenitor cells

Short name

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

Early-life ER agonism and endometrial adenosquamous carcinoma via SIX1 expression

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 v2.7

Graphical Representation

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

Authors

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Cancer AOP Workgroup. National Health and Environmental Effects Research Laboratory, Office of Research and Development, Integrated Systems Toxicology Division, US Environmental Protection Agency, Research Triangle Park, NC. Corresponding author for wiki entry (wood.charles@epa.gov)

Travis Karschnik (General Dynamics Information Technology, Duluth, MN, USA.)

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

Travis Karschnik (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

Charles Wood
Travis Karschnik

Coaches

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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 January 11, 2026 16:56

Revision dates for related pages

Page	Revision Date/Time
Promotion, Sine oculis homeobox 1 positive progenitor cells in endometrium	September 12, 2025 12:32
Endometrial squamous metaplasia, Increase	September 18, 2025 15:31
Increased, adenosquamous carcinomas of endometrium	September 18, 2025 15:32
Sine oculis homeobox 1 gene expression, increased	September 11, 2025 16:35
Sine oculis homeobox 1 protein expression, increased	December 17, 2025 14:59
Agonism, Estrogen receptor	September 11, 2025 15:13
Increase, Hyperplasia (glandular epithelial cells of endometrium)	September 18, 2025 15:31
Agonism, Estrogen receptor leads to SIX1 gene expression, increased	September 12, 2025 17:24
SIX1 gene expression, increased leads to SIX1 protein expression, increased	September 18, 2025 11:04
SIX1 protein expression, increased leads to Promotion, SIX1 positive progenitor cells in endometrium	September 18, 2025 11:05
Promotion, SIX1 positive progenitor cells in endometrium leads to Endometrial squamous metaplasia, Increase	September 18, 2025 11:08
Promotion, SIX1 positive progenitor cells in endometrium leads to Increase, Hyperplasia (glandular epithelial cells of endometrium)	September 18, 2025 11:05
Endometrial squamous metaplasia, Increase leads to Increased, adenosquamous carcinomas of endometrium	September 18, 2025 11:06
Increase, Hyperplasia (glandular epithelial cells of endometrium) leads to Increased, adenosquamous carcinomas of endometrium	September 18, 2025 11:06
Diethylstilbestrol	November 29, 2016 18:42
Genistein	November 29, 2016 18:42

Abstract

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

Developmental estrogen receptor agonism induces localized aberrant sine oculis homeobox 1 (six1) progenitor cell populations in the uterine epithelium. In adulthood, these six1+ progenitor cells drive abnormal epithelial differentiation. This differentiation manifests, in part, as squamous metaplasia and glandular hyperplasia which can both undergo malignant transformation resulting in adenosquamous carcinoma.

This AOP illustrates how early lifestage estrogen receptor agonism, and by extension estrogen receptor agonists, can drive molecular changes resulting in long-term adverse outcomes i.e., uterine cancer. The endometrial histological measurements: metaplasia, hyperplasia, and eventual adenosquamous carcinoma, are important markers/endpoints in the human health field. This is particularly relevant to the US EPA Endocrine Disruptor Screening Program (EDSP), which uses Tier 1 assays to identify potential estrogenic compounds and Tier 2 assays to evaluate adverse outcomes across lifestages and generations.

It also applies to a number of international regulatory frameworks concerned with endocrine disruption including but not limited to:

OECD for international harmonization.
The European Union (EU) by way of the European Chemicals Agency (ECHA), and the Registration, Evaluation, Authorization, and Restricting of Chemicals (REACH) regulation.
Canada, who evaluates endocrine disruption under the Canadian Environmental Protection Act (CEPA).

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 as 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 laboratory rodents and other mammals.

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 observed 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

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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

111

Agonism, Estrogen receptor

KE	2329	Sine oculis homeobox 1 gene expression, increased	SIX1 gene expression, increased
KE	2330	Sine oculis homeobox 1 protein expression, increased	SIX1 protein expression, increased
KE	1066	Promotion, Sine oculis homeobox 1 positive progenitor cells in endometrium	Promotion, SIX1 positive progenitor cells in endometrium
KE	1068	Endometrial squamous metaplasia, Increase	Endometrial squamous metaplasia, Increase
KE	772	Increase, Hyperplasia (glandular epithelial cells of endometrium)	Increase, Hyperplasia (glandular epithelial cells of endometrium)

1070

Increased, adenosquamous carcinomas of endometrium

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

Agonism, Estrogen receptor leads to SIX1 gene expression, increased	adjacent	High	Not Specified
SIX1 gene expression, increased leads to SIX1 protein expression, increased	adjacent	High	Not Specified
SIX1 protein expression, increased leads to Promotion, SIX1 positive progenitor cells in endometrium	adjacent	High	Not Specified
Promotion, SIX1 positive progenitor cells in endometrium leads to Endometrial squamous metaplasia, Increase	adjacent	High	Moderate
Promotion, SIX1 positive progenitor cells in endometrium leads to Increase, Hyperplasia (glandular epithelial cells of endometrium)	adjacent	High	Moderate
Endometrial squamous metaplasia, Increase leads to Increased, adenosquamous carcinomas of endometrium	adjacent	Moderate	Not Specified
Increase, Hyperplasia (glandular epithelial cells of endometrium) leads to Increased, adenosquamous carcinomas of endometrium	adjacent	Moderate	Not Specified

Network View

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

Prototypical Stressors

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

Name
Diethylstilbestrol
Genistein

Life Stage Applicability

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

Life stage	Evidence
Juvenile	Moderate
Embryo	High
Fetal	High
Adult	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. More help

Term	Scientific Term	Evidence	Link
mouse	Mus musculus	High	NCBI
human	Homo sapiens	High	NCBI
Vertebrates	Vertebrates	Not Specified	NCBI
mammals	mammals	Low	NCBI

Sex Applicability

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

Sex	Evidence
Female	High

Overall Assessment of the AOP

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

Defining Question	High (Strong)	Moderate	Low (Weak)
Is there a mechanistic relationship between KEup and KEdown consistent with established biological knowledge?	Extensive understanding of the KER based on extensive previous documentation and broad acceptance.	KER is plausible based on analogy to accepted biological relationships, but scientific understanding is incomplete	Empirical support for association between KEs , but the structural or functional relationship between them is not understood.

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
Relationship 3561: Agonism, Estrogen receptor leads to SIX1 gene expression, increased	Strong support. A number of plausible mechanisms have been proposed for this relationship.
Relationship 3562: SIX1 gene expression, increased leads to SIX1 protein expression, increased	Strong support. This is an example of the broadly accepted and well-known transcription -> translation process.
Relationship 3563: SIX1 protein expression, increased leads to Promotion, SIX1 positive progenitor cells in endometrium	Strong support. Protein expression levels and their relationship to the cells where that protein is measured are well documented. Six1’s developmental role is also plausibly linked to the increased incidence of progenitor cells specifically.
Relationship 3626: Promotion, SIX1 positive progenitor cells in endometrium leads to Endometrial squamous metaplasia, Increase	Moderate/Strong support. Six1’s role in tissue developmental and the mechanisms by which that occurs are plausibly linked in subsequent metaplasia.
Relationship 3627: Promotion, SIX1 positive progenitor cells in endometrium leads to Increase, Hyperplasia (glandular epithelial cells of endometrium)	Moderate/Strong support. Six1’s role in tissue developmental and the mechanisms by which that occurs are plausibly linked in subsequent hyperplasia.
Relationship 3610: Endometrial squamous metaplasia, Increase leads to Increased, adenosquamous carcinomas of endometrium	Strong support. Well supported from a biological standpoint given the nature cell progression to malignancy.
Relationship 3608: Increase, Hyperplasia (glandular epithelial cells of endometrium) leads to Increased, adenosquamous carcinomas of endometrium	Strong support. Well supported from a biological standpoint given the nature cell progression to malignancy.
Overall:	Strong support. Well established plausibility arguments at each step in the pathway.

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

Taxonomic Applicability

The empirical evidence presented throughout is derived from mice and human models. The mechanisms by which estrogen receptor agonism interacts with six1 expression leading to altered cell differentiation and eventually adenosquamous carcinoma could plausibly be extended to all uterine bearing mammals.

Lifestage Applicability

The empirical evidence presented throughout extends from developmental periods (embryogenesis and neonatal periods) through adulthood. Estrogen receptor agonism and the subsequent changes to six1 gene, protein, and progenitor populations occur early in development. The conditions that they create are then re-encountered at adulthood to kick-off the changes to cellular differentiation which can result in uterine cancer.

Sex Applicability

Limited to females as the outcome is limited by a sex-specific organ (uterus).

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

Defining question	High (Strong)	Moderate	Low (Weak)
Are downstream KEs and/or the AO prevented if an upstream KE is blocked?	Direct evidence from specifically designed experimental studies illustrating essentiality for at least one of the important KEs	Indirect evidence that sufficient modification of an expected modulating factor attenuates or augments a KE	No or contradictory experimental evidence of the essentiality of any of the KEs.

Essentiality of Key Events: Are downstream KEs and/or the AO prevented if an upstream KE is blocked?
Key Event (KE)	Level of Support
MIE 111: Agonism, Estrogen receptor	Strong support. KE2329 is not prevented, generally, in the absence of early lifestage ER agonism. However, KE2329 localized to the uterus and specific tissues in the uterus is. AO1070 does not require early lifestage ER agonism as it can be cause through a number of other mechanisms.
KE 2329: SIX1 gene expression, increased	Strong support. KE2330 is prevented in the absence of six1 gene expression. AO1070 does not require aberrant SIX1 gene expression as it can be caused through a number of other mechanisms.
KE 2330: SIX1 protein expression, increased	Strong support. KE1066 is prevented in the absence of six1 protein expression. AO1070 does not require aberrant SIX1 protein expression as it can be caused through a number of other mechanisms.
KE 1066: Promotion, SIX1 positive progenitor cells in endometrium	Moderate support. For both KE1068 and KE772, a promotion of six1 positive progenitor cells is but one mechanism to create the conditions for squamous metaplasia and glandular hyperplasia. Both can occur independently of six1+ progenitor cells.
KE 1068: Endometrial squamous metaplasia, Increase	Strong support. AO1070 can’t be diagnosed without a malignant squamous metaplasia element.
KE 772: Increase, Hyperplasia (glandular epithelial cells of endometrium)	Strong support. AO1070 can’t be diagnosed without a malignant glandular hyperplasia element.
AO 1070: Increased, adenosquamous carcinomas of endometrium	Strong support. This AO isn’t possible in subjects without malignant squamous metaplasia and glandular hyperplasia of the epithelial endometrium. However, the pathway leading from aberrant six1 expression leading to altered cell differentiation is far from the only one documented to achieve the AO.
Overall:	Moderate support. MIE111 and the downstream KEs 2329, 2330, and 1066 are supported by direct evidence, some including causal elements. AO1070 requires malignant versions of KE1068 and KE772 to be diagnosed so those are inextricably linked. AO1070 can come about by mechanisms outside of six1 expression leading to the required aberrant differentiation.

Evidence Assessment

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

Defining Questions	High (Strong)	Moderate	Low (Weak)
Does empirical evidence support that a change in KEup leads to an appropriate change in KEdown? Does KEup occur at lower doses and earlier time points than KE down and is the incidence of KEup > than that for KEdown? Inconsistencies?	if there is dependent change in both events following exposure to a wide range of specific stressors (extensive evidence for temporal, dose- response and incidence concordance) and no or few data gaps or conflicting data	if there is demonstrated dependent change in both events following exposure to a small number of specific stressors and some evidence inconsistent with the expected pattern that can be explained by factors such as experimental design, technical considerations, differences among laboratories, etc.	if there are limited or no studies reporting dependent change in both events following exposure to a specific stressor (i.e., endpoints never measured in the same study or not at all), and/or lacking evidence of temporal or dose- response concordance, or identification of significant inconsistencies in empirical support across taxa and species that don’t align with the expected pattern for the hypothesized AOP

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
Relationship 3561: Agonism, Estrogen receptor leads to SIX1 gene expression, increased	Strong support. Experimentally confirmed in mice including an ER antagonist exposure producing the opposite result.
Relationship 3562: SIX1 gene expression, increased leads to SIX1 protein expression, increased	Strong support. Experimentally confirmed in human cell lines including knockout experiments producing the opposite result.
Relationship 3563: SIX1 protein expression, increased leads to Promotion, SIX1 positive progenitor cells in endometrium	Strong support. Mice and human data are presented supporting the plausibility arguments that six1 protein expression encourages proliferation, expands progenitor populations, suppresses or otherwise alters differentiation, and stabilizes a transcriptional feedback loop.
Relationship 3626: Promotion, SIX1 positive progenitor cells in endometrium leads to Endometrial squamous metaplasia, Increase	Moderate/Strong support. Evidence presented here focus on concurrent six1+ cells and squamous metaplasia. One study is included demonstrates six1 deletion results in less metaplasia implying that DES induced six1 could be partially causal.
Relationship 3627: Promotion, SIX1 positive progenitor cells in endometrium leads to Increase, Hyperplasia (glandular epithelial cells of endometrium)	Moderate support. Evidence presented here focus on concurrent six1+ cells and glandular hyperplasia. The evidence supports the idea that more six1+ cells are found in areas of more hyperplasia.
Relationship 3610: Endometrial squamous metaplasia, Increase leads to Increased, adenosquamous carcinomas of endometrium	Moderate support. This is empirically supported in humans. Although, the evidence is presented as co-occuring rather than strictly causal in nature.
Relationship 3608: Increase, Hyperplasia (glandular epithelial cells of endometrium) leads to Increased, adenosquamous carcinomas of endometrium	Moderate support. This is empirically supported in humans. Although, the evidence is presented as co-occuring rather than strictly causal in nature.
Overall:	Moderate/Strong support. These KERs are a blend of direct causal evidence and co-occurrence at various stages. Because the AO is a histological diagnosis the two required constituent findings, malignant squamous metaplasia and malignant glandular hyperplasia are directly tied.

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

As it pertains to OECD TG440 this AOP could inform the refinement of assay endpoints to include transcriptional and/or protein biomarkers. The timescale at which the overall AOP plays out, depending on species, could inform changes to, or the inclusion of, additional medium-term measurements related to those transcriptional and/protein changes.

The information could be used as justification for testing Diethylstilbestrol and Genistein at the Tier 2 level in the EDSP.

The AOP establishing links between short-term, early-life changes resulting in increased long-term cancer risk supports greater focus on early key events to establish safe exposure levels/timing. Six1 and any other associated genetic and protein markers could also serve as mechanistic biomarkers for early-life estrogenic perturbations and as predictive indicators for long-term uterine cancer outcomes.

References

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

OECD (2007), Test No. 440: Uterotrophic Bioassay in Rodents: A short-term screening test for oestrogenic properties, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/9789264067417-en