The authors have designated this AOP as all rights reserved. Re-use in any form requires advanced permission from the authors.

AOP: 499


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 of MEK-ERK1/2 leads to deficits in learning and cognition via disrupted neurotransmitter release

Short name
A name that succinctly summarises the information from the title. This name should not exceed 90 characters. More help
MEK-ERK1/2 activation leading to deficits in learning and cognition
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 v2.6

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


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

Of the originating work:

Katherine von Stackelberg (Harvard Center for Risk Analysis, Boston, MA, USA.) (Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA, USA.)

Elizabeth Guzy (Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA, USA.)

Tian Chu (Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA, USA.)

Birgit Claus Henn (Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA, USA.) (Department of Environmental Health, Boston University School of Public Health, Boston, MA, USA.)

Of the content populated in the AOP-Wiki:

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)


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


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 May 09, 2024 17:09

Revision dates for related pages

Page Revision Date/Time
Activation of mitogen-activated protein kinase kinase, extracellular signal-regulated kinase 1/2 July 28, 2023 09:33
Increase, intracellular calcium July 21, 2023 16:26
Impairment, Learning and memory June 26, 2023 12:44
Disruption, neurotransmitter release July 21, 2023 16:35
Activation of MEK, ERK1/2 leads to Increase, intracellular calcium April 11, 2024 15:20
Increase, intracellular calcium leads to Disruption, neurotransmitter release April 11, 2024 15:20
Disruption, neurotransmitter release leads to Impairment, Learning and memory April 11, 2024 15:20
Lead November 29, 2016 18:42
Arsenic April 27, 2021 00:15
Cadmium October 25, 2017 08:33
Manganese February 04, 2022 14:47
Heavy metals (cadmium, lead, copper, iron, nickel) October 25, 2021 03:21


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

Metal mixture activation of ERK1/2 and JNK1/2 in astrocytes leads to increased Ca2+ release (Asit Rai and others 2010).  Alterations to calcium, an essential nutrient which is required in multiple cellular and physiological functions, such as cell adhesion, signal transduction, and neurotransmission can be expected to have downstream effects in those functions (Antonio et al., 2002). Changes in neurotransmission can then lead to changes in learning and cognitition (Neal and Guilarte 2010).

MEK-ERK1/2 is important in understanding uptake of metals into the brain and its relationship to deficits in learning and cognition from exposure to metals commonly detected at Superfund sites including lead, cadmium, manganese, and arsenic.  Current risk assessment guidance dictates a largely chemical-by-chemical evaluation of exposures and risks, which fails to adequately address potential interactions with other chemicals, nonchemical stressors, and genetic factors. Cumulative risk assessment methods and approaches are evolving to meet regulatory needs, (MacDonell et al., 2013; Backhaus and Faust 2012; IPCS Workshop 2009) but significant challenges remain. As our understanding of complex exposures and interactions continues to grow, synthesis and integration across disciplines and studies focused on different aspects of the environmental fate–exposure–toxicology–health outcome continuum are required to assess the likelihood of adverse effects and to support cumulative risk assessment.  Environmental exposures are virtually always to complex mixtures (Katherine von Stackelberg et al., 2015).

AOP Development Strategy


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 developed as part of an Environmental Protection Agency effort to increase the impact of AOPs published in the peer-reviewed literature, but heretofore unrepresented in the AOP-Wiki, by facilitating their entry and update.  The originating work for this AOP was Katherine von Stackelberg & Elizabeth Guzy & Tian Chu & Birgit Claus Henn, 2015. Exposure to Mixtures of Metals and Neurodevelopmental Outcomes: A Multidisciplinary Review Using an Adverse Outcome Pathway Framework, Risk Analysis, John Wiley & Sons, vol. 35(6), pages 971-1016, June.  This publication, and the work cited within, were used create and support this AOP and its respective KE and KER pages.

An examination of neurodevelopmental disorders and subclinical effects using multi-domain global neurodevelopment assessments is warranted as they can have profound population level implications.  In the context of neurotoxicity, neurodevelopmental pathways in the developing human brain are not fully understood (Schubert et al., 2015; Bal-Price et al., 2015) although there are a number of commonly observed phenomena which may take part in those pathways e.g. changes in intracellular calcium, ROS generation, apoptosis, and neurotransmitter disruption.  This AOP highlights a specific set of response-response relationships using a subset of those commonly observed phenonema related to metals and metal mixture exposures leading to deficits in learning and cognition. 

The focus of the originating work was to conduct a review of the literature on relationships between prenatal and early life exposure to mixtures of lead (Pb), arsenic (As), cadmium (Cd), and manganese (Mn) with neurodevelopmental outcomes and then use an AOP framework to integrate lines of evidence from multiple disciplines based on evolving guidance developed by the Organization for Economic Cooperation and Development (OECD). Importantly, the review considered whether exposures to mixtures of metals was associated with neurodevelopment effects that were greater or less than effects from exposure to each individual metal.


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 originating authors conducted a literature search to develop a database of publications categorized by discipline or field of study: toxicology, epidemiology, exposure, and gene-environment interaction. The literature search relied on standard search engines such as PubMed, Web of Science, Google Scholar, Environmental Index, Scopus, Toxline, and Toxnet and the search strategy included terms related to metal mixtures, individual metals (e.g., arsenic, lead, manganese, and cadmium), neurodevelopmental health outcomes, and associated Medical Subject Headings (MeSH) terms. The originating authors reviewed references from individual citations to identify additional studies not captured through the literature search itself. They then included all relevant publications through September 2013. Only studies focused primarily on developmental or neurotoxic endpoints were included; those focused on carcinogenesis or other systemic effects were not included unless there was a particular relevance to a neurotoxic or developmental outcome.

The scope of the aforementioned EPA project was limited to re-representing the AOP(s) as presented in the originating publication. The literature used to support this AOP and its constituent pages began with the originating publication and followed to the primary, secondary, and tertiary works cited therein. 

KE and KER page creation and re-use was determined using Handbook principles where page re-use was preferred.  Once a baseline level of information was populated for the AOP the authors of the originating publication were contacted for collaboration.

Efforts were made not to editorialize or otherwise add any content to the AOP or its constituent pages that weren’t provided in the primary, secondary, or tertiary literature.  In some cases, however, descriptive content was added to pages e.g., assays on a KE page, even if they weren’t specifically provided in the literature stemming from the originating publication.

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


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
KE 1339 Increase, intracellular calcium Increase, intracellular calcium
KE 2151 Disruption, neurotransmitter release Disruption, neurotransmitter release
AO 341 Impairment, Learning and memory Impairment, Learning and memory

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

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
All life stages 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
Rattus norvegicus Rattus norvegicus Moderate NCBI
Mus musculus Mus musculus Moderate NCBI
Homo sapiens Homo sapiens Moderate NCBI

Sex Applicability

The sex for which the AOP is known to be applicable. More help
Sex Evidence
Unspecific 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 KERs

Defining Question

High (Strong)


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.

Relationship 2942: Activation of MEK, ERK1/2 (2146) leads to Increase, intracellular calcium (1339)


Empirical evidence indicates a complex relationship between MEK, ERK1/2 activation and inhibition and Ca2+ response including Ca2+ feeding back into a ERK1/2 activation.  This relationship appears to vary across species and cell type.

Relationship 2954: Increase, intracellular calcium (1339) leads to Disruption, neurotransmitter release (2151)


Intracellular calcium regulation is broadly known as being an important aspect of a number of processes in a variety of cells and is particularly critical in nerve cell terminals where it mediates transmitter release.

Relationship 2955: Disruption, neurotransmitter release (2151) leads to Impairment, Learning and memory (341)


The role of various neurotransmitters and receptors in cognitive function and memory formation are well studied.


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

Life stages applicable to this AOP encompass the full life cycle.  Many of the key events are measured in pregnant females with the adverse outcome (impairment, learning and memory) measured at all life stages.

Taxonomic Applicability

Most evidence for this AOP is derived from rodents and humans where rodents were selected with their ability to model human responses.   

Sex Applicability

This AOP is applicable to all sexes.

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 KEs

Defining question

High (Strong)


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.

MIE 2146:Activation of MEK, ERK1/2


MEK, ERK1/2 activation is fundamental in delivering signals which regulate the cell cycle, proliferation, differentiation, adhesion, and more.  Disruptions in this activation have wide reaching effects however, there is evidence that downstream KEs can also activate this KE.

KE 1339: Increase, intracellular calcium


Calcium, as a primary intracellular messenger in neurons and regulator of cell responses to stress has been shown to directly affect neurotransmitter release with manipulation.

KE 2151: Disruption, neurotransmitter release


Neurotransmitter receptor blocking experiments have shown to directly impair learning and memory tasks in rodents.

AO 341: Impairment, Learning and memory


AOP 499


There is direct evidence contained KER 2955.

Evidence Assessment

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

3. Empirical Support for KERs

Defining Questions

High (Strong)


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

Relationship 2942: Activation of MEK, ERK1/2 (2146) leads to Increase, intracellular calcium (1339)


The evidence collection strategy for this AOP focused mainly on metal and metal mixture exposures, of which, there were many that showed dependent change in both these events following exposure.

Relationship 2954: Increase, intracellular calcium (1339) leads to Disruption, neurotransmitter release (2151)


The evidence collection strategy for this AOP focused mainly on metal and metal mixture exposures, of which, there were many that showed dependent change in both these events following exposure.

Relationship 2955: Disruption, neurotransmitter release (2151) leads to Impairment, Learning and memory (341)


The evidence collection strategy for this AOP focused mainly on metal and metal mixture exposures, of which, there were many that showed dependent change in both these events following exposure. 

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

Developmental neurotoxicity (DNT) is an adverse outcome of concern to multiple regulatory agencies. In vitro screening assays for MEK-ERK1/2 activation would not be recommended as a direct alternative or replacement to established DNT assays like OECD Test No. 426 (OECD 2007). However, detection of MEK-ERK1/2 activation in neuronal cell types may be used to prioritize chemicals with potential to elicit neurotoxicity and flag them for testing in ortogonal assays for evaluating DNT, including proposed alternative test methods (Bal-Price et al. 2018; Crofton et al 2022).  


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

Antonio, M. Teresa, Noelia López, and M. Luisa Leret. "Pb and Cd poisoning during development alters cerebellar and striatal function in rats." Toxicology 176.1-2 (2002): 59-66.

Asit Rai and others, Characterization of Developmental Neurotoxicity of As, Cd, and Pb Mixture: Synergistic Action of Metal Mixture in Glial and Neuronal Functions, Toxicological Sciences, Volume 118, Issue 2, December 2010, Pages 586–601,

Backhaus T, Faust M. Predictive environmental risk assessment of chemical mixtures: A conceptual framework. Environmental Science & Technology, 2012; 46(5):2564–2573.

Bal-Price A, Crofton KM, Sachana M, Shafer TJ, Behl M, Forsby A, Hargreaves A, Landesmann B, Lein PJ, Louisse J, Monnet-Tschudi F, Paini A, Rolaki A, Schrattenholz A, Sunol C, van Thriel C, Whelan M, Fritsche E. Putative adverse outcome pathways relevant to neurotoxicity. Critical Reviews in Toxicology, 2015; 45(1):83–91.

Bal-Price A, Hogberg HT, Crofton KM, Daneshian M, FitzGerald RE, Fritsche E, Heinonen T, Hougaard Bennekou S, Klima S, Piersma AH, Sachana M, Shafer TJ, Terron A, Monnet-Tschudi F, Viviani B, Waldmann T, Westerink RHS, Wilks MF, Witters H, Zurich MG, Leist M. Recommendation on test readiness criteria for new approach methods in toxicology: Exemplified for developmental neurotoxicity. ALTEX. 2018;35(3):306-352. doi: 10.14573/altex.1712081.  Erratum in: ALTEX. 2019;36(3):506. 

Crofton KM, Bassan A, Behl M, Chushak YG, Fritsche E, Gearhart JM, Marty MS, Mumtaz M, Pavan M, Ruiz P, Sachana M, Selvam R, Shafer TJ, Stavitskaya L, Szabo DT, Szabo ST, Tice RR, Wilson D, Woolley D, Myatt GJ. Current status and future directions for a neurotoxicity hazard assessment framework that integrates in silico approaches. Comput Toxicol. 2022 May;22:100223. doi: 10.1016/j.comtox.2022.100223. 

International Programme on Chemical Safety (IPCS),World Health Organization (WHO). Assessment of combined exposures to multiple chemicals. Report of a WHO/IPCS  International Workshop, 2009.

Izquierdo, Ivan. Role of NMDA receptors in memory. Trends in Pharmacological Sciences 12.4 (1991): 128-129

Katherine von Stackelberg & Elizabeth Guzy & Tian Chu & Birgit Claus Henn, 2015. "Exposure to Mixtures of Metals and Neurodevelopmental Outcomes: A Multidisciplinary Review Using an Adverse Outcome Pathway Framework," Risk Analysis, John Wiley & Sons, vol. 35(6), pages 971-1016, June.

Lupușoru CE, Popa EG, Sandu RB, Buca BR, Mititelu-Tarțău L, Lupușoru RV, The influence of Bidens tripartita extracts on psychomotor abilities and cognitive functions in rats.  Farmacia, 2017; 65(2): 284-288.

MacDonell MM, Haroun LA, Teuschler LK, Rice GE, Hertzberg RC, Butler JP, Chang Y-S, Clark SL, John AP, Perry CS, Garcia SS, Jacob JH, Scofield MA. 2013. Cumulative risk assessment toolbox:Methods and approaches for the practitioner. Journal of Toxicology, 2013; Article ID 310904, doi:10.1155/2013/310904.

Navarette M, Perea G, Maglio L, Pastor J, de Sola RG, Araque A. Astrocyte calcium signal and gliotransmission in human brain tissue. Cerebral Cortex, 2013; 23:1240–1246.

Neal, A.P., Guilarte, T.R. Molecular Neurobiology of Lead (Pb2+): Effects on Synaptic Function. Mol Neurobiol 42, 151–160 (2010).

OECD (2007), Test No. 426: Developmental Neurotoxicity Study, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris,

Schubert D, Martens GJM, Kolk SM. Molecular underpinnings of prefrontal cortex development in rodents provide insights into the etiology of neurodevelopmental disorders. Molecular Psychiatry, 2013; 2014:1–15.