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AOP: 263

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

Uncoupling of oxidative phosphorylation leading to growth inhibition via decreased cell proliferation

Short name

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

Uncoupling of OXPHOS leading to growth inhibition 1

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 v2.0

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

You Song^a and Daniel L. Villeneuve^b

^a Norwegian Institute for Water Research (NIVA), Økernveien 94, NO-0579 Oslo, Norway

^b U.S. Environmental Protection Agency, Great Lakes Toxicology and Ecology Division, Duluth, Minnesota 55804, USA

Acknowledgement

This project was funded by the Research Council of Norway (RCN), grant no. 301397 “RiskAOP - Quantitative Adverse Outcome Pathway assisted risk assessment of mitochondrial toxicants” (https://www.niva.no/en/projectweb/riskaop), and supported by the NIVA Computational Toxicology Program, NCTP (www.niva.no/nctp).

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

You Song (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

You Song
Dan Villeneuve

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
1.92	WPHA/WNT Endorsed		Journal link	iLibrary link

This AOP was last modified on April 29, 2023 16:03

Revision dates for related pages

Page	Revision Date/Time
Decrease, Coupling of oxidative phosphorylation	May 28, 2021 07:59
Decrease, Growth	July 06, 2022 07:36
Decrease, Adenosine triphosphate pool	June 14, 2021 13:40
Decrease, Cell proliferation	December 07, 2020 06:55
Decrease, Coupling of OXPHOS leads to Decrease, ATP pool	July 06, 2022 07:39
Decrease, ATP pool leads to Decrease, Cell proliferation	December 07, 2020 07:43
Decrease, Cell proliferation leads to Decrease, Growth	July 06, 2022 07:43
2,4-Dinitrophenol	November 29, 2016 18:42
Pentachlorophenol	November 12, 2020 17:59
Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone	November 12, 2020 17:59
Carbonyl cyanide m-chlorophenyl hydrazone	November 12, 2020 17:59
Triclosan	November 12, 2020 18:00
Dinoseb	November 12, 2020 18:00
3,5-Dichlorophenol	October 10, 2017 07:47
Emodin	November 20, 2020 13:48
Arsenite	March 07, 2019 06:14
Niclosamide ethanolamine	July 06, 2022 07:24
Oxyclozanide	July 06, 2022 07:25

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

Uncoupling of oxidative phosphorylation (OXPHOS) is a well-known mechanism of action of many chemicals. Mitochondrial uncoupler-mediated energetic dysfunction is known to affect growth, a critical process in most organisms and a chronic toxicity endpoint included in many OECD test guidelines. This adverse outcome pathway (AOP) causally links uncoupling of OXPHOS to growth inhibition, through ATP depletion and reduced cell proliferation as the intermediate key events (KEs), with strong weight of evidence support. The AOP is generalized to reflect its expected applicability to a broad range of taxa, ranging from microalga to human. Three out of four KEs included can be quantified using high-throughput methods, making this AOP particularly useful for screening, prioritization and hazard assessment of mitochondrial uncouplers as potential growth inhibiting chemicals. This AOP is of high regulatory relevance, as it is considered applicable to both human health and ecological risk assessments. The AOP also forms the core of a larger AOP network addressing uncoupling of OXPHOS mediated growth inhibition (AOP 263-268).

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

The mitochondrial OXPHOS machinery is a key physiological process responsible for producing the primary cellular energy, adenosine triphosphate (ATP). During OXPHOS, a series of redox reactions (oxidation) are mediated by protein complexes in an electron transport chain to create a protonmotive force (PMF) across the inner mitochondrial membrane (Liberman 1969). The PMF acts as a driving force of ATP synthesis through phosphorylation of adenosine diphosphate (ADP). Mitochondrial oxidation and phosphorylation are coupled to ensure continuous ATP supply for various physiological processes. A number of chemicals can bind to the inner mitochondrial membrane and dissipate the PMF, thus leading to uncoupling of OXPHOS and reduction in ATP synthetic efficiency. Classical “uncouplers” are normally protonophores with major characteristics of bulky hydrophobic moiety, an acid dissociable group and a strong electron-withdrawing group (Terada 1990). With the rapid development of in silico (Russom 1997; Schultz 1997; Naven 2012; Dreier 2019; Troger 2020) and in vitro (Escher 2002; Attene-Ramos 2013; Attene-Ramos 2015; Xia 2018) approaches, more and more uncouplers have been identified. However, their hazards to biota remain to be assessed. Uncoupling of OXPHOS can affect many ATP-dependent biological functions. In particular, cell proliferation as a major process to achieve organismal growth is positively correlated with the cellular ATP level and highly susceptible to energy depletion (Ramaiah 1964; Bonora 2012). Therefore, a link between uncoupling of OXPHOS and growth inhibition can be established with ATP depletion and reduced cell proliferation as the intermediate steps.

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

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

1446

Decrease, Coupling of oxidative phosphorylation

Decrease, Coupling of OXPHOS

KE	1771	Decrease, Adenosine triphosphate pool	Decrease, ATP pool
KE	1821	Decrease, Cell proliferation	Decrease, Cell proliferation

1521

Decrease, Growth

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

Decrease, Coupling of OXPHOS leads to Decrease, ATP pool	adjacent	High	High
Decrease, ATP pool leads to Decrease, Cell proliferation	adjacent	Moderate	Moderate
Decrease, Cell proliferation leads to Decrease, Growth	adjacent	Moderate	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

Name
2,4-Dinitrophenol
Pentachlorophenol
Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone
Carbonyl cyanide m-chlorophenyl hydrazone
Triclosan
Dinoseb
3,5-Dichlorophenol
Emodin
Arsenite
Niclosamide ethanolamine
Oxyclozanide

Life Stage Applicability

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

Life stage	Evidence
Embryo	High
Juvenile	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
zebrafish	Danio rerio	High	NCBI
Lemna minor	Lemna minor	Moderate	NCBI
human	Homo sapiens	Moderate	NCBI
mouse	Mus musculus	High	NCBI
rat	Rattus norvegicus	High	NCBI
Caenorhabditis elegans	Caenorhabditis elegans	Moderate	NCBI

Sex Applicability

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

Sex	Evidence
Unspecific	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

The weight of evidence (WoE) assessment of the AOP was conducted based on the evolved Bradford-Hill considerations (Becker 2015) and according to the criteria in OECD’s Guidance Document for Developing and Assessing AOPs (OECD 2018). In terms of evidence for the essentiality of the key events, the MIE (Event 1446) and KE1 (Event 1771) were scored as high, whereas KE2 (Event 1821) was scored as moderate due to a lack of solid evidence to support its essentiality. The overall WoE of KER1 (Relationship 2203) is considered high, as strong biological plausibility, empirical evidence and fairly good quantitative understanding were evidenced from multiple studies. The overall WoE of KER2 (Relationship 2204) is considered moderate, due to high biological plausibility, acceptable empirical concordance and some biological understanding. The overall WoE of KER3 (Relationship 2205) is scored as moderate, mainly due to biological plausibility, but there is presently a lack of empirical evidence and quantitative understanding to further support causality. The AOP is considered applicable to a wide range of species as well as a broad domain of chemicals. The rationales for making these judgements will be discussed in detail in the following sections.

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

The taxonomic application domain of the AOP potential covers all animals, plants and some microorganisms such as fungus and protists, as mitochondrial OXPHOS is highly conserved in eukaryotes (Roger 2017).

The life stage applicability domain of the AOP mainly contains embryos and juveniles, as growth is more relevant to developing organisms. It should be noted that fully grown adults are also susceptible to uncouplers, as tissue/organ (e.g., adipose tissue) growth and regeneration still occur in adults (Yun 2015; Demine 2019). Classical uncouplers such as 2,4-DNP have been reported to cause weight loss in adult humans (Grundlingh 2011). In fact, 2,4-DNP was sold for weight loss until its legal sale was banned over toxicity and abuse concerns (Baker 2020). These suggest that adults are in the applicability domain of this AOP.

The sex applicability domain of the AOP is unspecific, as the AOP is mainly targeting growth effects in sexually immature organisms and the KEs are therefore harmonized between male and females. However, male and females may have different sensitivities to OXPHOS uncoupling, as strategies for allocating energy for developmental processes may be gender specific (Demarest 2015).

The chemical applicability domain of the AOP mainly includes weak acids, such as phenols, benzimidazoles, N-phenylanthranilates, salicylanilides, phenylhydrazones, salicylic acids, acyldithiocarbazates, cumarines, and aromatic amines, which are well-known protonophoric uncouplers. Uncouplers typically have properties as both weak acids and hydrophobic substances. As weak acids, they are capable of gaining and losing an electron. As hydrophobic substances, they are capable of distributing a negative charge over a number of atoms (often by π-orbitals which delocalize a proton's charge when it attaches to the molecule), so that they can diffuse back and forth across the inner mitochondrial membrane in either the charged or uncharged state, thus moving protons back across the concentration gradient generated by the electron transport chain. Classical uncouplers, such as 2,4-dinitrophenol (2,4-DNP), carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone (FCCP), carbonyl cyanide m-chlorophenyl hydrazone (CCCP), pentachlorophenol (PCP), 3,5-dichlorophenol (3,5-DCP), 6-sec-butyl-2,4-dinitrophenol (dinoseb), SF 6847 (3,5-di-t-butyl-4-hydroxybenzylidinemalononitrile) have been widely used as positive controls in (eco)toxicological tests, whereas the hazards of “new” uncouplers, such as triclosan, emodin and metabolites of polybrominated diphenyl ethers (PBDEs) are also under extensive assessments. Other types of uncouplers that are SH-reactive chemicals or hydrophobic ions may also be in the applicability domain of this AOP. A number of potential uncouplers have been identified by in silico (Russom 1997; Schultz 1997; Naven 2012; Dreier 2019; Troger 2020) and in vitro (Escher 2002; Attene-Ramos 2013; Attene-Ramos 2015; Xia 2018) approaches, and are considered in the chemical applicability domain of the AOP.

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

Support for Essentiality of KEs	Defining Question	What is the impact on downstream KEs and/or the AO if an upstream KE is modified or prevented?
	High	Direct evidence from specifically designed experimental studies illustrating prevention or impact on downstream KEs and/or the AO if upstream KEs are blocked or modified.
	Moderate	Indirect evidence that modification of one or more upstream KEs is associated with a corresponding (increase or decrease) in the magnitude or frequency of downstream KEs.
	Low	No or contradictory experimental evidence of the essentiality of any of the KEs.
Event 1446: (Decrease, Coupling of OXPHOS)	Essentiality of Event 1446 is high. Rationale: There is direct evidence from several specifically designed studies showing that removal of an uncoupler from exposure, or addition of a “recoupler” can lead to recovery of the mitochondrial membrane potential (MMP) and total ATP caused by the uncoupler. Evidence: Removal of the classical uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) led to recovery of both MMP and ATP in rat cerebellar granule cells (Weisová 2012). In the red abalone (Haliotis rufescens) larvae, removal of the uncoupler pentachlorophenol also led to recovery of the ATP level (Shofer 2002). Addition of the recoupler GDP led to a rapid increase in ATP/ADP ratio in isolated guinea pig brown-adipose-tissue mitochondria where high activities of natural coupling by the UCPs were expected (Rafael 1976). Addition of octanoate to 2,4-DNP exposed rat hepatocytes mitigated the uncoupling effect and partial restored the ATP/ADP ratio (Sibille 1995). Removal of FCCP led to recovery from FCCP-mediated MMP and ATP reduction in Swiss mouse embryos (Zander-Fox 2015).
Event 1771: (Decrease, ATP pool)	Essentiality of Event 1771 is moderate. Rationale: There is limited direct evidence from specifically designed studies. However, multiple lines of indirect evidence show that modulation of ATP levels by uncouplers can also lead to corresponding changes in cell proliferation. Evidence: Addition of emodin blunted ATP-induced cell proliferation in a concentration-dependent manner in human lung adenocarconoma (A549) cells (Wang 2017), hence providing direct evidence to support the essentiality of this KE. Positive relationships between uncoupler-mediated ATP depletion and reduced cell proliferation have been documented by multiple studies(Sweet 1999; Fine 2009; Guimarães 2012; Sugiyama 2019).
Event 1821: (Decrease, Cell proliferation)	Essentiality of Event 1821 is moderate. Rationale: There is no direct evidence from specifically designed studies to support this KE. However, there are multiple lines of indirect evidence showing positive relationships between cell proliferation and growth. Evidence: Indirect evidence can be obtained from a limited number of relevant studies showing a positive role of cell proliferation in mammalian tumor (Figarola 2018) zebrafish embryo growth(Bestman 2015). Indirect evidence showing that the mitochondrial uncouplers niclosamide ethanolamine and oxyclozanide either completely prevented or drastically reduced hepatic metastasis of colon cancer cells from spleen (Alasadi et al., 2018).
Inconsistencies & uncertainties	There is an uncertainty related to KE1446 that mild uncoupling of OXPHOS may also increase the ATP pool in some cases (Desquiret 2006), possibly as a compensatory response. The underlying mechanism remains to be further elucidated.

Evidence Assessment

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

Biological plausibility

Support for Biological Plausibility of KERs	Defining Question	Is there a mechanistic (i.e., structural or functional) relationship between KE_up and KE_down consistent with established biological knowledge?
	High	Extensive understanding based on extensive previous documentation and broad acceptance -Established mechanistic basis.
	Moderate	The KER is plausible based on analogy to accepted biological relationships but scientific understanding is not completely established.
	Low	There is empirical support for a statistical association between KEs, but the structural or functional relationship between them is not understood.
Relationship 2203: (Decrease, Coupling of OXPHOS leads to Decrease, ATP pool)	Biological Plausibility of Relationship 2203 is high. Rationale: In eukaryotic cells, the major metabolic pathways responsible for ATP production are OXPHOS, citric acid (TCA) cycle, glycolysis and photosynthesis. Oxidative phosphorylation is much (theoretically 15-18 times) more efficient than the rest due to high energy derived from oxygen during aerobic respiration (Schmidt-Rohr 2020). As the ATP level is relatively balanced between production and consumption (Bonora 2012), ATP depletion is a plausible consequence of reduced ATP synthetic efficiency following uncoupling of OXPHOS.
Relationship 2204: (Decrease, ATP pool leads to Decrease, Cell proliferation)	Biological Plausibility of Relationship 2204 is high. Rationale: Cell proliferation is a well-known ATP-dependent process. Cell division processes, such as the mitotic cell cycle uses ATP for chromosome movements and DNA replication (Kingston 1999). The synthetic processes of major cellular components that are necessary for cell structure and growth, such as proteins and lipids, also require sufficient ATP supply (Bonora 2012). Depletion of ATP therefore has a negative impact on these processes.
Relationship 2205: (Decrease, Cell proliferation leads to Decrease, Growth)	Biological Plausibility of Relationship 2205 is high. Rationale: The biological causality between cell proliferation and growth has also been well established. It is commonly accepted that the size of an organism, organ or tissue is dependent on the total number and volume of the cells it contains, and the amount of extracellular matrix and fluids (Conlon 1999). Impairment to cell proliferation can logically affect tissue and organismal growth.
Inconsistencies & uncertainties	There are currently no inconsistencies and uncertainties identified by the authors.

Empirical support

Empirical Support for KERs	Defining Question	Does KE_up occur at lower doses and earlier time points than KE down and at the same dose of stressor, is the incidence of KE_up >than that for KE_down? Are there inconsistencies in empirical support across taxa, species and stressors that don’t align with expected pattern for hypothesized AOP?
	High	Multiple studies showing 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 critical data gaps or conflicting data.
	Moderate	Demonstrated dependent change in both events following exposure to a small number of specific stressors and some evidence inconsistent with expected pattern that can be explained by factors such as experimental design, technical considerations, differences among laboratories, etc.
	Low	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 significant inconsistencies in empirical support across taxa and species that don’t align with expected pattern for hypothesised AOP.
Relationship 2203: (Decrease, Coupling of OXPHOS leads to Decrease, ATP pool)	Empirical support of Relationship 2203 is high. Rationale: The majority of relevant studies show good incidence, temporal and/or dose concordance in different organisms and cell types after exposure to known uncouplers, with relatively few exceptions (see the Relationship 2203 page and concordance table for detailed evidence).
Relationship 2204: (Decrease, ATP pool leads to Decrease, Cell proliferation)	Empirical support of Relationship 2204 is moderate. Rationale: Although only a few studies were found to be relevant, incidence concordance was found for mammalian cells (see the Relationship 2204 page and concordance table for detailed evidence).
Relationship 2205: (Decrease, Cell proliferation leads to Decrease, Growth)	Empirical support of Relationship 2205 is low. Rationale: This KER was included in a very limited number of studies, as it addresses effects occurring at the apical level that in vitro studies cannot cover. There is one zebrafish study reporting concordant relationship between reduced cell proliferation and embryo growth with some inconsistencies (see the Relationship 2205 page and concordance table for detailed evidence).
Inconsistencies & uncertainties	There are some inconsistencies regarding temporal and dose concordance: A significant decrease followed by a significant increase of total ATP (KE1) was observed in human RD cells during a 48h exposure to the uncoupler FCCP(Kuruvilla 2003), possibly due to the enhancement of other ATP synthetic pathways (e.g., glycolysis) as a compensatory action to impaired OXPHOS (Jose 2011). In zebrafish embryos exposed to 2,4-DNP, significant growth inhibition (AO) was identified after 21h, whereas non-significant reductions in ATP (KE1) and cell proliferation (KE2) were reported(Bestman 2015).

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

Quantitative Understanding

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

Quantitative understanding of the KERs	High	Change in KE_downstream can be precisely predicted based on a relevant measure of KE_upstream. Uncertainty in the quantitative prediction can be precisely estimated from the variability in the relevant measure of KE_upstream. Known modulating factors and feedback/feedforward mechanisms are accounted for in the quantitative description. There is evidence that the quantitative relationship between the KEs generalises across the relevant applicability domain of the KER.
	Moderate	Change in KE_downstream can be precisely predicted based on a relevant measure of KE_upstream. Uncertainty in the quantitative prediction is influenced by factors other than the variability in the relevant measure of KE_upstream. Quantitative description does not account for all known modulating factors and/or known feedback/feedforward mechanisms. The quantitative relationship has only been demonstrated for a subset of the overall applicability domain of the KER (e.g., based on a single species).
	Low	Only a qualitative or semi-quantitative prediction of the change in KE_downstream can be determined from a measure of KE_upstream. Known modulating factors and/or known feedback/feedforward mechanisms are not accounted for. The quantitative relationship has only been demonstrated for a narrow subset of the overall applicability domain of the KER (e.g., based on a single species).
Relationship 2203: (Decrease, Coupling of OXPHOS leads to Decrease, ATP pool)	Quantitative understanding of Relationship 2203 is high. Rationale: The theoretical quantitative relationship between OXPHOS and ATP yield has been well established. There are also published computational/mathematical models in which modulating factors known to affect OXPHOS and ATP synthesis are considered. Evidence: A biophysical computational model developed for mitochondrial respiration and OXPHOS (Beard 2005). Continuous development of the mitochondrial energy transduction models since 1967 (Schmitz 2011). A comprehensive mathematical model developed for OXPHOS and ATP production under different physiological and pathological conditions (Heiske 2017). A comprehensive analysis of the quantitative relationships between protonmotive force, ATP synthase rotation, ATP synthesis and hydrolysis (Kubo 2020). A regression based response-response relationship for uncoupling of OXPHOS and ATP depletion (Song 2020).
Relationship 2204: (Decrease, ATP pool leads to Decrease, Cell proliferation)	Quantitative understanding of Relationship 2204 is moderate. Rationale: The total ATP level has been used as an indicator of cell proliferation. Several studies have reported the quantitative relationships between the two events, as well as a threshold value for KE1 to trigger KE2. However, not all modulating factors have been accounted and no well-established computational/mathematical models are found. Evidence: Quantitative understanding of ATP level, cell viability and colony growth (Ahmann 1987). Quantitative relationship between ATP level and cell proliferation (Crouch 1993). Thresholds for ATP depletion (85-90% reduction) to determine cell cycle arrest (<85-90%) or cell death (>85-90%) (Nieminen 1994).
Relationship 2205: (Decrease, Cell proliferation leads to Decrease, Growth)	Quantitative understanding of Relationship 2205 is moderate. Rationale: Multiple mathematical models describing the quantitative relationships between cell proliferation and tissue growth exist for both animals (Binder 2008) and plants (Mosca 2018). There are also numerous models that are specifically developed for predicting tumor growth based on the proliferation rate (Jarrett 2018). However, there is currently a lack of quantitative model to link cell proliferation and individual growth in the presence of uncouplers. Evidence: A mathematical model developed for describing the quantitative relationship between cell proliferation and tissue growth (Binder 2008). A mathematical model developed for cell division and plant tissue growth (Mosca 2018). Multiple mathematical models developed for cell proliferation and tumor growth (Jarrett 2018).

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

The present AOP has several potential applications. First, the AOP anchors a recognized endpoint of regulatory concern (i.e., growth), at least in OECD member countries, and is directly relevant for a number of OECD test guidelines (e.g., TG206, 208, 201, 210, 211, 212, 215, 221, 228, 241, 407, 408, 416, 422, 443 and 453). These guidelines cover a diversity of taxonomic groups including mammals, birds, fish, amphibians, terrestrial plants, aquatic plants and algae, and various invertebrates. Second, the AOP anchors an important molecular initiating event (e.g., uncoupling of oxidative phosphorylation) and can be used to support several initiatives (e.g., Tox21 and ToxCast) for identification of mitochondrial toxicants. The present AOP helps establish the utility of such assays for identifying chemicals with potential to cause growth impacts. Third, three out of four key events in this AOP can be measured using high-throughput in vitro assays, hence offering a tiered testing strategy (i.e., in silico→in vitro→in vivo) or integrated approaches to testing and assessment (IATA) for efficient screening, classification and assessment of potential mitochondrial uncouplers and growth-regulating chemicals. The key events can be considered as useful biomarkers in (eco)toxicological studies. However, it is not recommended to use a single key event (e.g., ATP level alone) as a biomarker for classification and hazard assessment of chemicals, as key events such as decreased ATP pool and cell proliferation can also be the consequences of other biological processes. A combined measurement of 2-3 key events can normally yield more reliable results. Finally, the quantitative relationships of the key events in this AOP have been relatively well defined, allowing it to be further developed into quantitative prediction models for higher tier assessments. This is a range of potential applications that were conceived during the development of the present AOP. However, it is neither an exhaustive list of potential applications, nor can explicit examples of these applications in practice be cited at this time.

We invite users of this AOP to share their applications of this AOP via the Discussion so that practical examples of use can be added.

References

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

Ahmann FR, Garewal HS, Schifman R, Celniker A, Rodney S. 1987. Intracellular adenosine triphosphate as a measure of human tumor cell viability and drug modulated growth. In Vitro Cellular & Developmental Biology 23:474-480. DOI: 10.1007/BF02628417.

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