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AOP: 32


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

Inhibition of iNOS, hepatotoxicity, and regenerative proliferation leading to liver tumors

Short name
A name that succinctly summarises the information from the title. This name should not exceed 90 characters. More help
Inhibition of iNOS, hepatotoxicity, and regenerative proliferation leading to liver tumors
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 v1.0

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


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

Michelle Embry, HESI

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
Undefined   (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


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

OECD Information Table

Provides users with information concerning how actively the AOP page is being developed and whether it is part of the OECD Workplan and has been reviewed and/or endorsed. OECD Project: Assigned upon acceptance onto OECD workplan. This project ID is managed and updated (if needed) by the OECD. OECD Status: For AOPs included on the OECD workplan, ‘OECD status’ tracks the level of review/endorsement of the AOP . This designation is managed and updated by the OECD. Journal-format Article: The OECD is developing co-operation with Scientific Journals for the review and publication of AOPs, via the signature of a Memorandum of Understanding. When the scientific review of an AOP is conducted by these Journals, the journal review panel will review the content of the Wiki. In addition, the Journal may ask the AOP authors to develop a separate manuscript (i.e. Journal Format Article) using a format determined by the Journal for Journal publication. In that case, the journal review panel will be required to review both the Wiki content and the Journal Format Article. The Journal will publish the AOP reviewed through the Journal Format Article. OECD iLibrary published version: OECD iLibrary is the online library of the OECD. The version of the AOP that is published there has been endorsed by the OECD. The purpose of publication on iLibrary is to provide a stable version over time, i.e. the version which has been reviewed and revised based on the outcome of the review. AOPs are viewed as living documents and may continue to evolve on the AOP-Wiki after their OECD endorsement and publication.   More help
OECD Project # OECD Status Reviewer's Reports Journal-format Article OECD iLibrary Published Version
This AOP was last modified on April 29, 2023 16:02

Revision dates for related pages

Page Revision Date/Time
Production, Critical Metabolites (CGA 330050 and CGA 265307) September 16, 2017 10:14
Induction, Liver “Dysfunctional” Changes by CGA 330050 September 16, 2017 10:14
Inhibition, Inducible Nitric Oxide Synthase by Metabolite CGA 265307 September 16, 2017 10:14
Induction, Sustained Hepatotoxicity December 03, 2016 16:37
Induction, Sustained Cell Proliferation September 16, 2017 10:14
Formation, Liver tumor December 03, 2016 16:33
Production, Critical Metabolites (CGA 330050 and CGA 265307) leads to Induction, Liver “Dysfunctional” Changes by CGA 330050 December 03, 2016 16:37
Induction, Liver “Dysfunctional” Changes by CGA 330050 leads to Inhibition, Inducible Nitric Oxide Synthase by Metabolite CGA 265307 December 03, 2016 16:37
Inhibition, Inducible Nitric Oxide Synthase by Metabolite CGA 265307 leads to Induction, Sustained Hepatotoxicity December 03, 2016 16:37
Induction, Sustained Hepatotoxicity leads to Induction, Sustained Cell Proliferation December 03, 2016 16:37
Induction, Sustained Cell Proliferation leads to Formation, Liver tumor December 03, 2016 16:37


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

Thiamethoxam is a neonicotinoid insecticide that has been extensively tested in animal models for short- and long-term toxicological effects. An increased incidence of liver tumors was seen in male and female Tif:MAGf mice when fed in the diet for 18 months at concentrations up to 2500 ppm. It is a mouse liver specific carcinogen and does not induce tumors at any other site in the mouse. There were no increases in cancer incidences either in the liver, or at any other site, in rats fed on diets containing up to 3000 ppm thiamethoxam for two years. Thiamethoxam was not genotoxic when evaluated in a battery of in vitro and in vivo assays.

Thiamethoxam is metabolize to two key metabolites, CGA 322704 and CGA 330050. These metabolites can be further metabolize to CGA 265307. Basic toxicity studies on these metabolites give clues to the critical events involved in its mode of action resulting in hepatacarcinogenesis. These metabolites were given at doses to mimic systemic exposure that would result following a tumorigenic dose of Thiamethoxam. When administered directly in the rodent bioassay (rats and mice), the CGA 322704 and CGA 265307 metabolites did not result in any tumors or any other effect in the liver including altered serum cholesterol, liver toxicity, apoptosis, or increased cell proliferation. However, Metabolite CGA 265307 is very structurally similar with substrates and inhibitors of the nitric oxide synthases. Direct exposure to metabolite CGA 330050 did not result in tumors but did result in the same liver toxicity effects as for thiamethoxam. It is proposed that the metabolites CGA 330050 and CGA 265307 are involved in thiamethoxam’s hepatocarcinogensis.

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


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


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 77 Production, Critical Metabolites (CGA 330050 and CGA 265307) Production, Critical Metabolites (CGA 330050 and CGA 265307)
KE 164 Induction, Liver “Dysfunctional” Changes by CGA 330050 Induction, Liver “Dysfunctional” Changes by CGA 330050
KE 147 Inhibition, Inducible Nitric Oxide Synthase by Metabolite CGA 265307 Inhibition, Inducible Nitric Oxide Synthase by Metabolite CGA 265307
KE 270 Induction, Sustained Hepatotoxicity Induction, Sustained Hepatotoxicity
KE 269 Induction, Sustained Cell Proliferation Induction, Sustained Cell Proliferation
KE 347 Formation, Liver tumor Formation, Liver tumor

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

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
mice Mus sp. High NCBI

Sex Applicability

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

Consider the following criteria (may include references to KE Relationship pages): 1. concordance of dose-response relationships; 2. temporal concordance among the key events and adverse effect; 3. strength, consistency, and specificity of association of adverse effect and initiating event; 4. biological plausibility, coherence, and consistency of the experimental evidence; 5. alternative mechanisms that logically present themselves and the extent to which they may distract from the postulated AOP. It should be noted that alternative mechanisms of action, if supported, require a separate AOP; 6. uncertainties, inconsistencies and data gaps.

The AOP leading to thiamethoxam-induced hepatocarcinogenesis is a species-, time-, dose-, and metabolite-dependent process.

Dose and Temporal Concordance In a 50-week mouse study with thiamethoxam, the earliest change, within one week, was a marked reduction (by up to 40%) in plasma cholesterol. This was followed 10 weeks later by evidence of liver toxicity including increased single-cell necrosis and increased apoptosis. After 20 weeks there was a significant increase in hepatic cell replication rates. All of these changes persisted from the time they were first observed until the end of the study at 50 weeks. Progression of events was consistently seen in several studies of 10, 20, or 50 weeks duration, with the hallmark indicator being a substantial decrease in plasma cholesterol levels [1]. The time-dependent key events occurs in a dose-response relationship that parallels the dose-related, late-life occurrence of tumors in mouse livers [2] and are only found at the carcinogenic dose (i.e., ≥ 500 ppm).

Three metabolites (i.e., CGA 322704 and CGA 330050 which are further metabolize to CGA 265307) were identified and systematically evaluated for toxicological contribution to the sequence of hepatic effects. Mice and rats both produce CGA322704 as a major blood metabolite, which suggests that this particular metabolite is not an indicator of a species difference. However, CGA322704 does not cause liver tumors in mice nor does it cause any of the hepatic changes seen with thiamethoxam, and is thus considered not to be a part of causative chain of hepatic events. In contrast, CGA265307 and CGA330050 are produced in substantially greater quantity by mice than by rats (up to 140-fold and 15-fold greater, respectively), suggesting that the metabolic pathway through CGA330050 is critical to the AOP. In studies where these metabolites were fed to mice for at least ten weeks, CGA330050 was found to induce the same hepatic effects, and to the same degree, as thiamethoxam. CGA265307 alone induced none of the clinical or histopathological changes seen in the thiamethoxam-treated mice.

Strength, Consistency, Specificity of Association All key events are well-defined measured effects with dose response and temporal concordance. The role of specific metabolites and time-dependent progression of hepatic lesions consistently seen including two strains of mice but not in rats. The role of CGA265307 was established by comparing its structural similarity to known inhibitors of inducible nitric oxide synthase (iNOS), by verifying the ability of CGA265307 to inhibit iNOS in vitro, and by assessing the ability of CGA265307 to exacerbate the iNOS-dependent hepatic toxicity of carbon tetrachloride in vivo. Based on structure-activity relationships and in vitro and in vivo experimentation, CGA265307’s role is thought to enhance the relatively mild hepatotoxicity induced by CGA330050, which leads to an increase in cellular death (via necrosis and apoptosis).

Differences in metabolism between mice and rats, the contributory role of specific metabolites, and the time- dependent progression of hepatic lesions were consistently seen in a series of separate studies, including two strains of mice. [3] [4]

Plausibility and Coherence The phenomenon of a non-genotoxic mouse liver specific carcinogen is not uncommon in rodent bioassay studies. [5] Cytotoxicity and consequent regeneration is a well-known and well-documented mode of carcinogenic action for a variety of chemicals and for a variety of tissues including liver in laboratory animals. [6] [7] [8]

Alternative modes of action Genotoxicity, cytohrome P-450 induction, peroxisomal beta oxidation, and oxidative stress were considered experimentally and shown not to be viable.[9]

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

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

Evidence Assessment

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

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

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


List of the literature that was cited for this AOP. More help
  1. Green, T., Toghill, A., Lee, R., Waechter, F., Weber, E., and Noakes, J. (2005a). Thiamethoxam induced mouse liver tumors and their relevance to humans. Part 1: mode of action studies in the mouse. Toxicol. Sci. 86, 36–47.
  2. Green, T., Toghill, A., Lee, R., Waechter, F., Weber, E., and Noakes, J. (2005a). Thiamethoxam induced mouse liver tumors and their relevance to humans. Part 1: mode of action studies in the mouse. Toxicol. Sci. 86, 36–47.
  3. Green, T., Toghill, A., Lee, R., Waechter, F., Weber, E., and Noakes, J. (2005a). Thiamethoxam induced mouse liver tumors and their relevance to humans. Part 1: mode of action studies in the mouse. Toxicol. Sci. 86, 36–47.
  4. Pastoor, T., Rose, P., Lloyd, S., Peffer, R., and Green T. (2005). Thiamethoxam induced mouse liver tumors and their relevance to humans, Part 3: Weight of evidence evaluation of the human health relevance of thiamethoxam-related mouse liver tumors. Toxicological Sciences 86(1), 56–60.
  5. Carmichael, N. G., Enzmann, H., Pate, I., and Waechter, F. (1997). The significance of mouse liver tumor formation for carcinogenic risk assess- ment: Results and conclusions from a survey of ten years of testing. Environ. Health Perspect. 105, 1196–1203.
  6. Holsapple, M.P., Pitot, H.C., Cohen, S.H., Boobis, A.R., Klaunig, J.E., Pastoor, T., Dellarco, V.L., and Dragan, Y.P. (2006). Mode of Action in Relevance of Rodent Liver Tumors to Human Cancer Risk. Toxicological Sciences 89(1), 51–56.
  7. Bogdanffy, M.S. (2002). Vinyl acetate-induced intracellular acidification: implications for risk assessment. Toxicol Sci. 2002 Apr;66(2):320-6.
  8. Meek, M., Bucher, J., Cohen, S., Dellarco, V., Hill, R., Lehman-McKeeman, L., Longfellow, D., Pastoor, T., Seed, J., and Patton, D. (2003). A Framework for human relevance analysis of information on carcinogenic modes of action. Crit. Rev. Toxicol. 33, 591–653.
  9. Green, T., Toghill, A., Lee, R., Waechter, F., Weber, E., and Noakes, J. (2005a). Thiamethoxam induced mouse liver tumors and their relevance to humans. Part 1: mode of action studies in the mouse. Toxicol. Sci. 86, 36–47.

Confidence in the AOP

Information from this section should be moved to the Key Event Relationship pages!  

The coherence and extent of the database on thiamethoxam and its metabolites clearly demonstrates the mode of action for mouse liver tumorigenesis involving hepatocytotoxicity and regenerative cell proliferation [1] [2] The responses seen with thiamethoxam have been reproduced in studies of 50 and 20 weeks duration, the latter in two strains of mouse. The metabolite studies were internally consistent in that CGA330050 is only formed from thiamethoxam and not from the non-carcinogenic metabolite CGA322704. In all of the studies the key events had logical dose and temporal relationships. The metabolite studies were clear and consistent with the known carcinogenicity profiles of thiamethoxam and CGA322704. Studies on the metabolite CGA265307 and the inhibition of inducible nitric oxide synthase were limited, but showed that CGA265307 inhibits iNOS in vitro and enhances the toxicity of carbon tetrachloride in vivo. It is reasonable to conclude that CGA265307, although not toxic alone, could enhance the hepatotoxicity of metabolite CGA330050. Based on comparative metabolic studies neither rats nor humans would produce sufficient level of the two critical metabolites (CGA 330050 and CGA 265307) to initiate the progression of hepatic key events. As a consequence, it is unlikely that humans would be at risk of developing liver tumors as a result of exposure to thiamethoxam. Cite error: <ref> tags exist, but no <references/> tag was found