Aop: 360


Each AOP should be given a descriptive title that takes the form “MIE leading to AO”. For example, “Aromatase inhibition [MIE] leading to reproductive dysfunction [AO]” or “Thyroperoxidase inhibition [MIE] leading to decreased cognitive function [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

Chitin synthase 1 inhibition leading to mortality

Short name
A short name should also be provided that succinctly summarises the information from the title. This name should not exceed 90 characters. More help
CHS-1 inhibition leading to mortality

Graphical Representation

A graphical summary of the AOP listing all the KEs in sequence, including the MIE (if known) and AO, and the pair-wise relationships (links or KERs) between those KEs should be provided. This is easily achieved using the standard box and arrow AOP diagram (see this page for example). The graphical summary is prepared and uploaded by the user (templates are available) and is often included as part of the proposal when AOP development projects are submitted to the OECD AOP Development Workplan. The graphical representation or AOP diagram provides a useful and concise overview of the KEs that are included in the AOP, and the sequence in which they are linked together. This can aid both the process of development, as well as review and use of the AOP (for more information please see page 19 of the Users' Handbook).If you already have a graphical representation of your AOP in electronic format, simple save it in a standard image format (e.g. jpeg, png) then click ‘Choose File’ under the “Graphical Representation” heading, which is part of the Summary of the AOP section, to select the file that you have just edited. Files must be in jpeg, jpg, gif, png, or bmp format. Click ‘Upload’ to upload the file. You should see the AOP page with the image displayed under the “Graphical Representation” heading. To remove a graphical representation file, click 'Remove' and then click 'OK.'  Your graphic should no longer be displayed on the AOP page. If you do not have a graphical representation of your AOP in electronic format, a template is available to assist you.  Under “Summary of the AOP”, under the “Graphical Representation” heading click on the link “Click to download template for graphical representation.” A Powerpoint template file should download via the default download mechanism for your browser. Click to open this file; it contains a Powerpoint template for an AOP diagram and instructions for editing and saving the diagram. Be sure to save the diagram as jpeg, jpg, gif, png, or bmp format. Once the diagram is edited to its final state, upload the image file as described above. More help


List the name and affiliation information of the individual(s)/organisation(s) that created/developed the AOP. In the context of the OECD AOP Development Workplan, this would typically be the individuals and organisation that submitted an AOP development proposal to the EAGMST. Significant contributors to the AOP should also be listed. A corresponding author with contact information may be provided here. This author does not need an account on the AOP-KB and can be distinct from the point of contact below. The list of authors will be included in any snapshot made from an AOP. More help

Simon Schmid 1,2, You Song 1, and Knut Erik Tollefsen 1,2,3

1 Norwegian Institute for Water Research (NIVA), Section of Ecotoxicology and Risk Assessment, Økernveien 94, N-0579, Oslo, Norway

2 Faculty of Environmental Science and Resource Management (MINA), Norwegian University of Life Sciences (NMBU), N-1432, Ås, Norway

3 Centre for Environmental Radioactivity (CERAD), Norwegian University of Life Sciences (NMBU), N-1432 Ås, Norway


Acknowledgements: This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 859891 and was supported by NIVA’s Computational Toxicology Program, NCTP (

Point of Contact

Indicate the point of contact for the AOP-KB entry itself. This person is responsible for managing the AOP entry in the AOP-KB and controls write access to the page by defining the contributors as described below. Clicking on the name will allow any wiki user to correspond with the point of contact via the email address associated with their user profile in the AOP-KB. This person can be the same as the corresponding author listed in the authors section but isn’t required to be. In cases where the individuals are different, the corresponding author would be the appropriate person to contact for scientific issues whereas the point of contact would be the appropriate person to contact about technical issues with the AOP-KB entry itself. Corresponding authors and the point of contact are encouraged to monitor comments on their AOPs and develop or coordinate responses as appropriate.  More help
Simon Schmid   (email point of contact)


List user names of all  authors contributing to or revising pages in the AOP-KB that are linked to the AOP description. This information is mainly used to control write access to the AOP page and is controlled by the Point of Contact.  More help
  • Simon Schmid
  • You Song
  • Knut Erik Tollefsen


The status section is used to provide AOP-KB users with information concerning how actively the AOP page is being developed, what type of use or input the authors feel comfortable with given the current level of development, and whether it is part of the OECD AOP Development Workplan and has been reviewed and/or endorsed. “Author Status” is an author defined field that is designated by selecting one of several options from a drop-down menu (Table 3). The “Author Status” field should be changed by the point of contact, as appropriate, as AOP development proceeds. See page 22 of the User Handbook for definitions of selection options. More help
Author status OECD status OECD project SAAOP status
Open for citation & comment WPHA/WNT Endorsed 1.94 Included in OECD Work Plan
This AOP was last modified on June 21, 2022 09:15
The date the AOP was last modified is automatically tracked by the AOP-KB. The date modified field can be used to evaluate how actively the page is under development and how recently the version within the AOP-Wiki has been updated compared to any snapshots that were generated. More help

Revision dates for related pages

Page Revision Date/Time
Inhibition, Chitin synthase 1 February 24, 2021 04:41
Decrease, Cuticular chitin content February 17, 2021 05:37
Increase, Premature molting February 17, 2021 05:30
Increase, Mortality October 26, 2020 05:18
Inhibition, CHS-1 leads to Decrease, Cuticular chitin content February 17, 2021 07:50
Decrease, Cuticular chitin content leads to Increase, Premature molting February 17, 2021 08:20
Increase, Premature molting leads to Increase, Mortality February 17, 2021 08:47
Polyoxin B May 24, 2018 15:54
Polyoxin D October 23, 2020 06:20
Nikkomycins May 24, 2018 15:54
Captan October 23, 2020 06:50
Captafol October 23, 2020 06:52
Folpet October 23, 2020 06:53


In the abstract section, authors should provide a concise and informative summation of the AOP under development that can stand-alone from the AOP page. Abstracts should typically be 200-400 words in length (similar to an abstract for a journal article). Suggested content for the abstract includes the following: The background/purpose for initiation of the AOP’s development (if there was a specific intent) A brief description of the MIE, AO, and/or major KEs that define the pathway A short summation of the overall WoE supporting the AOP and identification of major knowledge gaps (if any) If a brief statement about how the AOP may be applied (optional). The aim is to capture the highlights of the AOP and its potential scientific and regulatory relevance More help

In order to grow and develop, arthropods need to shed their exoskeleton (or cuticle) periodically and replace it with a new one in a process called molting. Successful molting, and therefore a successful development necessitates stability and integrity of the cuticle to support muscular contractions involved in the shedding of the old cuticle. The integrity of the cuticle is largely dependent on the N-acetylglucosamine (GlcNAc) polymer chitin. Therefore, arthropods heavily rely on chitin synthesis as chitin is one of the main constituents of the cuticle. The cuticular chitin synthase (CHS-1) is the key enzyme in the biosynthetic pathway and arthropods are therefore especially dependent on its proper function. The present AOP describes the effects of chemical inhibition of the cuticular chitin synthase (CHS-1) on the molting process leading to increased mortality in arthropods. Inhibition of CHS-1 is the molecular initiating event and leads to a decreased chitin content in the arthropod cuticle which leaves the organism immature at the stage for ecdysis. This phenomenon can be described as premature molting. The organism eventually dies due to being stuck in the old cuticle or due to the consequences of a weak exoskeleton after ecdysis. The AOP is considered to be very consistent. Essentiality of key events was rated as high for every key event and the biological plausibility was rated as high for the whole AOP. However, there does not exist very much empirical evidence that allows to draw a representative conclusion on dose concordance along the AOP whereas time concordance can be supported by knockdown studies of CHS-1. Therefore, empirical evidence was considered to be moderate and the quantitative understanding was considered to be low. The overall confidence in the AOP was valued as moderate. The present AOP will guide assay development for further experimental studies by revealing data and knowledge gaps. One of its primary applications will also be providing guidance in screening strategies in order to identify chemicals directly interacting with CHS-1.

Background (optional)

This optional subsection should be 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. Examples of potential uses of the optional background section are listed on pages 24-25 of the User Handbook. More help

Arthropods (including insects, crustaceans and arachnids) need to shed their exoskeleton in order to grow and reproduce. This process, also called molting or ecdysis, is mediated by behavioural mechanisms which involve the skeletal muscles (Ayali 2009; Song et al. 2017a). In order to properly shed its cuticle, the organism needs to possess a newly synthesized cuticle that possesses a certain integrity to support this process. Since chitin is a major constituent of the cuticle, it contributes substantially to its integrity (Cohen 2001; Vincent and Wegst 2004). Chitin is synthesized from uridine diphosphate-N-Acetylglucosamine (UDP-GlcNAc) in a polymerization reaction by the transmembrane enzyme chitin synthase isoform 1 (CHS-1). CHS-1 is localized on the apical side in the cuticular epithelium. Since chitin and the process of chitin synthesis does not occur in vertebrates, it can and has been exploited for the design of pest controlling agents. Inhibitors of chitin synthesis may not only be of use for the control of unwanted arthropods and fungi, they may also pose a risk for beneficial arthropods such as insects and crustaceans. Disruption of chitin synthesis or the endocrine mechanisms controlling molting generally lead to a disruption of ecdysis (Merzendorfer et al. 2012; Song et al. 2017a; Song et al. 2017b). If the amount of chitin in the cuticle decreases, the affected organism may not be able to molt properly and will most probably die of starvation or suffocation (Camp et al. 2014; Song et al. 2017a). Alternatively, if molting is completed despite an immature cuticle, the organism may be deformed and die as a consequence of a weak cuticle. Therefore, the present AOP should build the basis of a mechanistic approach for the systematic evaluation and the risk assessment of chemicals interfering with chitin synthesis by directly inhibiting CHS-1.

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 stressor and the biological system) of an AOP. More help
Key Events (KE)
This table summarises all of the KEs of the AOP. This table is populated in the AOP-Wiki as KEs are added to the AOP. Each table entry acts as a link to the individual KE description page.  More help
Adverse Outcomes (AO)
An AO is a specialised KE that represents the end (an adverse outcome of regulatory significance) of an AOP.  More help
Sequence Type Event ID Title Short name
1 MIE 1522 Inhibition, Chitin synthase 1 Inhibition, CHS-1
2 KE 1523 Decrease, Cuticular chitin content Decrease, Cuticular chitin content
3 KE 1524 Increase, Premature molting Increase, Premature molting
4 AO 350 Increase, Mortality Increase, Mortality

Relationships Between Two Key Events (Including MIEs and AOs)

This table summarises 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.To add a key event relationship click on either Add relationship: events adjacent in sequence or Add relationship: events non-adjacent in sequence.For example, if the intended sequence of KEs for the AOP is [KE1 > KE2 > KE3 > KE4]; relationships between KE1 and KE2; KE2 and KE3; and KE3 and KE4 would be defined using the add relationship: events adjacent in sequence button.  Relationships between KE1 and KE3; KE2 and KE4; or KE1 and KE4, for example, should be created using the add relationship: events non-adjacent button. This helps to both organize the table with regard to which KERs define the main sequence of KEs and those that provide additional supporting evidence and aids computational analysis of AOP networks, where non-adjacent KERs can result in artifacts (see Villeneuve et al. 2018; DOI: 10.1002/etc.4124).After clicking either option, the user will be brought to a new page entitled ‘Add Relationship to AOP.’ To create a new relationship, select an upstream event and a downstream event from the drop down menus. The KER will automatically be designated as either adjacent or non-adjacent depending on the button selected. The fields “Evidence” and “Quantitative understanding” can be selected from the drop-down options at the time of creation of the relationship, or can be added later. See the Users Handbook, page 52 (Assess Evidence Supporting All KERs for guiding questions, etc.).  Click ‘Create [adjacent/non-adjacent] relationship.’  The new relationship should be listed on the AOP page under the heading “Relationships Between Two Key Events (Including MIEs and AOs)”. To edit a key event relationship, click ‘Edit’ next to the name of the relationship you wish to edit. The user will be directed to an Editing Relationship page where they can edit the Evidence, and Quantitative Understanding fields using the drop down menus. Once finished editing, click ‘Update [adjacent/non-adjacent] relationship’ to update these fields and return to the AOP page.To remove a key event relationship to an AOP page, under Summary of the AOP, next to “Relationships Between Two Key Events (Including MIEs and AOs)” click ‘Remove’ The relationship should no longer be listed on the AOP page under the heading “Relationships Between Two Key Events (Including MIEs and AOs)”. More help

Network View

The AOP-Wiki automatically generates a network view of the AOP. This network graphic is based on the information provided in the MIE, KEs, AO, KERs and 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


The stressor field is a structured data field that can be used to annotate an AOP with standardised terms identifying stressors known to trigger the MIE/AOP. Most often these are chemical names selected from established chemical ontologies. However, depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). It can also include non-chemical stressors such as genetic or environmental factors. Although AOPs themselves are not chemical or stressor-specific, linking to stressor terms known to be relevant to different AOPs can aid users in searching for AOPs that may be relevant to a given stressor. More help
Name Evidence Term
Polyoxin B High
Polyoxin D High
Nikkomycins High
Captan Moderate
Captafol Moderate
Folpet Moderate

Life Stage Applicability

Identify the life stage for which the KE is known to be applicable. More help
Life stage Evidence
Larvae High
Juvenile High
Adult Moderate

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 in relation to this KE. More help
Term Scientific Term Evidence Link
Pieris brassicae Pieris brassicae High NCBI
Anopheles gambiae Anopheles gambiae High NCBI
Lucilia cuprina Lucilia cuprina High NCBI
Tribolium castaneum Tribolium castaneum High NCBI
Bombyx mori Bombyx mori High NCBI
Anopheles quadrimaculatus Anopheles quadrimaculatus High NCBI
Trichoplusia ni Trichoplusia ni High NCBI
Artemia salina Artemia salina High NCBI
Daphnia magna Daphnia magna High NCBI
Hyalophora cecropia Hyalophora cecropia High NCBI
Ostrinia nubilalis Ostrinia nubilalis High NCBI
Bradysia hygida Bradysia hygida Moderate NCBI
Mamestra brassicae Mamestra brassicae Moderate NCBI
Chilo suppressalis Chilo suppressalis Moderate NCBI
Locusta migratoria Locusta migratoria Moderate NCBI
Nilaparvata lugens Nilaparvata lugens Moderate NCBI
Aphis glycines Aphis glycines Moderate NCBI
Lepeophtheirus salmonis Lepeophtheirus salmonis Moderate NCBI
Panonychus citri Panonychus citri Moderate NCBI
Grapholita molesta Grapholita molesta Moderate NCBI
Ectropis obliqua Ectropis obliqua Moderate NCBI
Tigriopus japonicus Tigriopus japonicus Moderate NCBI

Sex Applicability

The authors must select from one of the following: Male, female, mixed, asexual, third gender, hermaphrodite, or unspecific. More help
Sex Evidence
Unspecific Moderate

Overall Assessment of the AOP

This section addresses the relevant biological domain of applicability (i.e., in terms of taxa, sex, life stage, etc.) and WoE for the overall AOP as a basis to consider appropriate regulatory application (e.g., priority setting, testing strategies or risk assessment). The goal of the overall assessment is to provide a high level synthesis and overview of the relative confidence in the AOP and where the significant gaps or weaknesses are (if they exist). Users or readers can drill down into the finer details captured in the KE and KER descriptions, and/or associated summary tables, as appropriate to their needs.Assessment of the AOP is organised into a number of steps. Guidance on pages 59-62 of the User Handbook is available to facilitate assignment of categories of high, moderate, or low confidence for each consideration. While it is not necessary to repeat lengthy text that appears elsewhere in the AOP description (or related KE and KER descriptions), a brief explanation or rationale for the selection of high, moderate, or low confidence should be made. More help

Domain of Applicability

The relevant biological domain(s) of applicability in terms of sex, life-stage, taxa, and other aspects of biological context are defined in this section. Biological domain of applicability is informed by the “Description” and “Biological Domain of Applicability” sections of each KE and KER description (see sections 2G and 3E for details). In essence the taxa/life-stage/sex applicability is defined based on the groups of organisms for which the measurements represented by the KEs can feasibly be measured and the functional and regulatory relationships represented by the KERs are operative.The relevant biological domain of applicability of the AOP as a whole will nearly always be defined based on the most narrowly restricted of its KEs and KERs. For example, if most of the KEs apply to either sex, but one is relevant to females only, the biological domain of applicability of the AOP as a whole would be limited to females. While much of the detail defining the domain of applicability may be found in the individual KE and KER descriptions, the rationale for defining the relevant biological domain of applicability of the overall AOP should be briefly summarised on the AOP page. More help

Taxonomic: Since the whole phylum of arthropods is dependent on the synthesis of chitin to molt successfully, it is extremely likely that the AOP is applicable to all arthropods. Effect data along the AOP exist from Dipteran, Lepidopteran and Coleopteran insect species as well as from Branchiopods and Anostracans of the crustacea. Although data is limited, KEs seem to be well conserved across taxa, as shown in available studies with specific stressors known to inhibit CHS and in studies where CHS-1 was knocked down by RNA interference. However, due to limited data availability, it was not possible to cover whole taxa but rather single species in the assessment of KEs. Alignment of amino acid residues in the catalytic center of CHS-1 using the Sequence Alignment to Predict Across Species Susceptibility tool (SeqAPASS,, LaLone et al. 2016), confirmed structural and functional conservation in various insect, arachnid and crustacean species, strenghtening the evidence for the applicability domain to be the whole phylum of arthropods. However, taxonomic applicability may not only be defined by structural conservation of the protein sequence. So the evidence for the taxonomic applicability for species with support only from sequence alignment was judged as moderate, whereas evidence for species with support from sequence alignment and effect data was judged as high.

Life stage: The AOP is applicable for organisms undergoing continuous molt cycles. As insects do not molt in their adulthood, the AOP is only applicable for larval and pupal stages of insects. Crustaceans and arachnids grow and molt throughout their lifetime (Passano 1961; Uhl et al. 2015), which makes the AOP applicable to all life stages, where juvenile life stages might be more susceptible to chemical perturbations due to higher growth rate and therefore more frequent molting.

Sex: The AOP is applicable to all sexes.

Chemical: Substances known to trigger the MIE and leading to the AO are of the family of pyrimidine nucleosides (e.g. polyoxin D, polyoxin B and nikkomycin Z) (Osada 2019). There also exists evidence for phthalimides (captan, captafol and folpet) to inhibit CHS-1 activity and to decrease the cuticular chitin content in vitro (Cohen and Casida 1982; Gelman and Borkovec 1986). However, as these substances are known to covalently bind to thiol groups in proteins (Lukens and Sisler 1958), it is not clear if the inhibition is due to specific CHS-1 inhibition or due to unspecific protein binding.

Essentiality of the Key Events

An important aspect of assessing an AOP is evaluating the essentiality of its KEs. 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.When assembling the support for essentiality of the KEs, authors should organise relevant data in a tabular format. The objective is to summarise briefly the nature and numbers of investigations in which the essentiality of KEs has been experimentally explored either directly or indirectly. See pages 50-51 in the User Handbook for further definitions and clarifications.  More help

The essentiality of all key events was considered as high. Essentiality evaluations were mainly based on specifically designed studies demonstrating the expected effect pattern predicted by the AOP to occur after knockdown of CHS-1.

Inhibition, Chitin synthase 1 (High): Knockdown of the cuticular chitin synthase leads to the expected pattern of effects described in this AOP. It decreases the cuticular chitin content and leads to premature molting associated mortality in insects (Arakane et al. 2005; X. Zhang et al. 2010; Li et al. 2017; Zhai et al. 2017). If the cuticular chitin content was not directly measured as endpoint, knockdown of the CHS-1 led directly to the occurrence of premature molting associated increase of mortality (Chen et al. 2008; X. Zhang et al. 2010; Wang et al. 2012; Yang et al. 2013; Shang et al. 2016; Mohammed et al. 2017; Wang et al. 2019; Ye et al. 2019; Ullah et al. 2020)

Decrease, Cuticular chitin content (High): Abolishment of the cuticular chitin synthesis through knockdown of CHS-1 leads to premature molting associated mortality (Arakane et al. 2005; X. Zhang et al. 2010; Li et al. 2017; Zhai et al. 2017). By knocking down the UDP-GlcNAc pyrophosphorylase (UAP), which catalyzes the last sugar conversion before the polymerization to chitin, it was shown that reduced chitin content leads to the same outcome as the knockdown of CHS-1. Namely premature molting and increased mortality (Arakane et al. 2011; Liu et al. 2013). Knockdown of trehalase genes, which constitutes the start of the chitin synthetic pathway and convert trehalose to glucose, leads to a similar pattern of effects, namely decreased cuticular chitin content and premature molting associated mortality (Chen et al. 2010; Shi et al. 2016).

Increase, Premature molting (High): Several studies show that premature molting is a direct consequence of decreased chitin synthesis and leads to increased mortality. The KE is consistently listed as cause for mortality when CHS-1 is knocked down throughout a number of studies (Arakane et al. 2005; Chen et al. 2008; J. Zhang et al. 2010; X. Zhang et al. 2010; Wang et al. 2012; Yang et al. 2013; Shang et al. 2016; Li et al. 2017; Mohammed et al. 2017; Zhai et al. 2017; Wang et al. 2019; Ye et al. 2019).

Increase, Mortality (High): Increased mortality was observed in all of the abovementioned studies.

Evidence Assessment

The biological plausibility, empirical support, and quantitative understanding from each KER in an AOP are assessed together.  Biological plausibility of each of the KERs in the AOP is the most influential consideration in assessing WoE or degree of confidence in an overall hypothesised AOP for potential regulatory application (Meek et al., 2014; 2014a). Empirical support entails consideration of experimental data in terms of the associations between KEs – namely dose-response concordance and temporal relationships between and across multiple KEs. It is examined most often in studies of dose-response/incidence and temporal relationships for stressors that impact the pathway. While less influential than biological plausibility of the KERs and essentiality of the KEs, empirical support can increase confidence in the relationships included in an AOP. For clarification on how to rate the given empirical support for a KER, as well as examples, see pages 53- 55 of the User Handbook.  More help

Biological Plausibility: The biosynthesis of chitin is well characterized and is conserved among arthropods. Although the exact mode of action of chitin synthases remains elusive, it is widely accepted and well established that the chitin synthase is the key enzyme in the pathway, polymerizing chitin using UDP-N-Acetylglucosamine as substrate (Merzendorfer and Zimoch 2003). Arthropod cuticles mostly consist of chitin embedded into a matrix of cuticular proteins. It is therefore widely accepted that chitin contributes crucially to the quality and function of the cuticle (Reynolds 1987; Muthukrishnan et al. 2012). The molting process requires the new cuticle to be strong enough to withstand the stresses of ecdysis. During ecdysis, arthropods pause food intake and growth. If ecdysis is initiated before the new cuticle is strong enough, the organism likely dies of starvation or growth arrest (Song, Villeneuve, et al. 2017). It was also reported that certain arthropods pause respiration during ecdysis, which may lead to suffocation (Camp et al. 2014). Based on the well-established biological knowledge on the processes this AOP bases on, the biological plausibility for all KER was rated as high.

Empirical Evidence: Empirical evidence assessment was conducted on the basis of in vitro and in vivo experiments performed with stressors affecting key events throughout the AOP. Studies showed that the key events are affected by model stressors such as Polyoxin D and Nikkomycin Z, which are able to competitively inhibit CHS1 (Endo et al. 1970). Several studies provide evidence that polyoxin B, polyoxin D and nikkomycin Z trigger the MIE in cell free systems of coleopteran, lepidopteran and dipteran insect species (Cohen 1982; Turnbull and Howells 1982; Kuwano and Cohen 1984; Cohen and Casida 1990; Zhang and Yan Zhu 2013). Also the cuticular chitin content was shown to be decreased by polyoxin D and nikkomycin Z in lepidopteran and dipteran species as well as in the crustacean Artemia salina (Gijswijt et al. 1979; Calcott and Fatig 1984; Gelman and Borkovec 1986; Zhuo et al. 2014). The AO is supported by in vivo studies with polyoxin D and nikkomycin Z in dipteran insects and Daphnia magna (Tellam et al. 2000; Tellam and Eisemann 2000; Zhu et al. 2007; Zhang and Yan Zhu 2013; New Zealand Environmental Protection Authority 2015). A major data gap constitutes the absence of data covering the KE “Increase, premature molting”. This KE is mentioned in some studies but never assessed as an individual endpoint (Gijswijt et al. 1979; Tellam et al. 2000). Another major data gap is the lacking quantitative data for KERs. As endpoints were only measured as individual endpoints and not in sequence, it makes it nearly impossible to evaluate the dose for the KEs and KERs. However, data from studies where CHS-1 was knocked down are able to support temporal concordance for all KERs. Knockdown of CHS-1 led to decreased chitin content and subsequently to premature molting associated mortality (Arakane et al., 2005; Li et al., 2017). Based on the major data gaps and therefore the lacking information on dose concordance as well as the given time concordance, empirical evidence was evaluated to be moderate for the whole AOP.

Overall confidence in the AOP: Both, essentiality of KEs and the biological plausibility of the whole AOP were considered to be high. However, due to missing quantitative data and the lack of evidence for dose concordance, empirical evidence was judged to be moderate. Therefore the overall confidence in the AOP was evaluated as moderate.

Quantitative Understanding

Some proof of concept examples to address the WoE considerations for AOPs quantitatively have recently been developed, based on the rank ordering of the relevant Bradford Hill considerations (i.e., biological plausibility, essentiality and empirical support) (Becker et al., 2017; Becker et al, 2015; Collier et al., 2016). Suggested quantitation of the various elements is expert derived, without collective consideration currently of appropriate reporting templates or formal expert engagement. Though not essential, developers may wish to assign comparative quantitative values to the extent of the supporting data based on the three critical Bradford Hill considerations for AOPs, as a basis to contribute to collective experience.Specific attention is also given to how precisely and accurately one can potentially predict an impact on KEdownstream based on some measurement of KEupstream. This is captured in the form of quantitative understanding calls for each KER. See pages 55-56 of the User Handbook for a review of quantitative understanding for KER's. More help

Quantitative data are limited for all KER and therefore the whole AOP. Therefore, predictions on the occurrence of downstream KE and the AO on the basis of the occurrence of upstream KEs is not readily feasible. Quantitative understanding of the AOP was therefore considered to be low.

Considerations for Potential Applications of the AOP (optional)

At their discretion, the developer may include in this section discussion of the 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. While it is challenging to foresee all potential regulatory application of AOPs and any application will ultimately lie within the purview of regulatory agencies, potential applications may be apparent as the AOP is being developed, particularly if it was initiated with a particular application in mind. This optional section is intended to provide the developer with an opportunity to suggest potential regulatory applications and describe his or her rationale.To edit the “Considerations for Potential Applications of the AOP” section, on an AOP page, in the upper right hand menu, click ‘Edit.’ This brings you to a page entitled, “Editing AOP.” Scroll down to the “Considerations for Potential Applications of the AOP” section, where a text entry box allows you to submit text. In the upper right hand menu, click ‘Update AOP’ to save your changes and return to the AOP page or 'Update and continue' to continue editing AOP text sections.  The new text should appear under the “Considerations for Potential Applications of the AOP” section on the AOP page. More help

Arthropods are responsible for many functions in terrestrial as well as aquatic ecosystems and are therefore jointly responsible for ecosystem health (Seastedt and Crossley 1984; Losey and Vaughan 2006; LeBlanc 2007). Therefore, it is important to develop AOPs which enhance the mechanistic knowledge on chemicals, such as chitin synthesis inhibitors, which may pose a risk to non-target arthropods. Those AOPs will contribute to the systematic use of mechanistic data to preserve beneficial arthropod populations and ecosystem health. The present AOP will help to guide future experimental studies by identifying data gaps. This will lead to the identification and development suitable bioassays in order to populate the AOP with (quantitative) experimental data which may allow for predictions of regulatory relevant endpoints on the basis of the occurrence of the MIE. The present AOP may also guide screening strategies in order to identify chemicals inhibiting CHS-1. The identified substances may then be prioritized and undergo a thorough hazard assessment. As there already exist approaches to assess mixture toxicity using the AOP framework (Altenburger et al. 2012; Beyer et al. 2014), the present AOP could be employed for the effect assessment  of mixtures of chemicals that share the same KEs (e.g. AOP #361,, AOP #358,, and AOP #359,


List the bibliographic references to original papers, books or other documents used to support the AOP. More help

Altenburger R, Scholz S, Schmitt-Jansen M, Busch W, Escher BI. 2012. Mixture toxicity revisited from a toxicogenomic perspective. Environ Sci Technol. 46(5):2508–2522. doi:10.1021/es2038036.

Arakane Y, Baguinon MC, Jasrapuria S, Chaudhari S, Doyungan A, Kramer KJ, Muthukrishnan S, Beeman RW. 2011. Both UDP N-acetylglucosamine pyrophosphorylases of Tribolium castaneum are critical for molting, survival and fecundity. Insect Biochem Mol Biol. 41(1):42–50. doi:10.1016/j.ibmb.2010.09.011.

Arakane Y, Muthukrishnan S, Kramer KJ, Specht CA, Tomoyasu Y, Lorenzen MD, Kanost M, Beeman RW. 2005. The Tribolium  chitin synthase genes TcCHS1 and TcCHS2 are specialized for synthesis of epidermal cuticle and midgut peritrophic matrix. Insect Mol Biol. 14(5):453–463. doi:10.1111/j.1365-2583.2005.00576.x.

Ayali A. 2009. The role of the arthropod stomatogastric nervous system in moulting behaviour and ecdysis. J Exp Biol. 212(4):453–459. doi:10.1242/jeb.023879.

Beyer J, Petersen K, Song Y, Ruus A, Grung M, Bakke T, Tollefsen KE. 2014. Environmental risk assessment of combined effects in aquatic ecotoxicology: A discussion paper. Mar Environ Res. 96:81–91. doi:10.1016/j.marenvres.2013.10.008.

Calcott PH, Fatig RO. 1984. Inhibition of Chitin metabolism by Avermectin in susceptible Organisms. J Antibiot (Tokyo). 37(3):253–259. doi:10.7164/antibiotics.37.253.

Camp AA, Funk DH, Buchwalter DB. 2014. A stressful shortness of breath: Molting disrupts breathing in the mayfly Cloeon dipterum. Freshw Sci. 33(3):695–699. doi:10.1086/677899.

Chen Jie, Tang B, Chen H, Yao Q, Huang X, Chen Jing, Zhang D, Zhang W. 2010. Different functions of the insect soluble and membrane-bound trehalase genes in chitin biosynthesis revealed by RNA interference. PLoS One. 5(4). doi:10.1371/journal.pone.0010133.

Chen X, Tian H, Zou L, Tang B, Hu J, Zhang W. 2008. Disruption of Spodoptera exigua larval development by silencing chitin synthase gene A with RNA interference. Bull Entomol Res. 98(6):613–619. doi:10.1017/S0007485308005932.

Cohen E. 1982. In vitro chitin synthesis in an insect: formation and structure of microfibrils. Eur J Cell Biol. 26(2):289–294.

Cohen E. 2001. Chitin synthesis and inhibition: A revisit. Pest Manag Sci. 57(10):946–950. doi:10.1002/ps.363.

Cohen E, Casida JE. 1982. Properties and inhibition of insect integumental chitin synthetase. Pestic Biochem Physiol. 17(3):301–306. doi:10.1016/0048-3575(82)90141-9.

Cohen E, Casida JE. 1990. Insect and Fungal Chitin Synthetase Activity: Specificity of Lectins as Enhancers and Nucleoside Peptides as Inhibitors. Pestic Biochem Physiol. 37(3):249–253. doi:10.1016/0048-3575(90)90131-K.

Endo A, Kakiki K, Misato T. 1970. Mechanism of action of the antifugal agent polyoxin D. J Bacteriol. 104(1):189–196. doi:10.1128/jb.104.1.189-196.1970.

Gelman DB, Borkovec AB. 1986. The pharate adult clasper as a tool for measuring chitin synthesis and for identifying new chitin synthesis inhibitors. Comp Biochem Physiol Part C, Comp. 85(1):193–197. doi:10.1016/0742-8413(86)90073-3.

Gijswijt MJ, Deul DH, de Jong BJ. 1979. Inhibition of chitin synthesis by benzoyl-phenylurea insecticides, III. Similarity in action in Pieris brassicae (L.) with Polyoxin D. Pestic Biochem Physiol. 12(1):87–94. doi:10.1016/0048-3575(79)90098-1.

Kuwano E, Cohen E. 1984. The use of a Tribolium chitin synthetase assay in studying the effects of benzimidazoles with a terpene moiety and related compounds. Agric Biol Chem. 48(6):1617–1620. doi:10.1080/00021369.1984.10866362.

LaLone, C.A., Villeneuve, D.L., Lyons, D., Helgen, H.W., Robinson, S.L., Swintek, J.A., Saari, T.W., Ankley, G.T., 2016. Sequence alignment to predict across species susceptibility (seqapass): A web-based tool for addressing the challenges of cross-species extrapolation of chemical toxicity. Toxicol. Sci. 153, 228–245.

LeBlanc GA. 2007. Crustacean endocrine toxicology: A review. Ecotoxicology. 16(1):61–81. doi:10.1007/s10646-006-0115-z.

Li T, Chen J, Fan X, Chen W, Zhang W. 2017. MicroRNA and dsRNA targeting chitin synthase A reveal a great potential for pest management of the hemipteran insect Nilaparvata lugens. Pest Manag Sci. 73(7):1529–1537. doi:10.1002/ps.4492.

Liu X, Li F, Li D, Ma E, Zhang W, Zhu KY, Zhang J. 2013. Molecular and functional analysis of UDP-N-acetylglucosamine Pyrophosphorylases from the Migratory Locust, Locusta migratoria. PLoS One. 8(8). doi:10.1371/journal.pone.0071970.

Losey JE, Vaughan M. 2006. The economic value of ecological services provided by insects. Bioscience. 56(4):311–323. doi:10.1641/0006-3568(2006)56[311:TEVOES]2.0.CO;2.

Lukens RJ, Sisler HD. 1958. 2-Thiazolidinethione-4-carboxylic acid from the reaction of captan with cysteine. Science (80- ). 127(3299):650. doi:10.1126/science.127.3299.650.

Merzendorfer H, Kim HS, Chaudhari SS, Kumari M, Specht CA, Butcher S, Brown SJ, Robert Manak J, Beeman RW, Kramer KJ, et al. 2012. Genomic and proteomic studies on the effects of the insect growth regulator diflubenzuron in the model beetle species Tribolium castaneum. Insect Biochem Mol Biol. 42(4):264–276. doi:10.1016/j.ibmb.2011.12.008.

Merzendorfer H, Zimoch L. 2003. Chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases. J Exp Biol. 206(24):4393 LP – 4412. doi:10.1242/jeb.00709.

Mohammed AMA, DIab MR, Abdelsattar M, Khalil SMS. 2017. Characterization and RNAi-mediated knockdown of Chitin Synthase A in the potato tuber moth, Phthorimaea operculella. Sci Rep. 7(1):1–12. doi:10.1038/s41598-017-09858-y.

Muthukrishnan S, Merzendorfer H, Arakane Y, Kramer KJ. 2012. Chitin Metabolism in Insects. Elsevier B.V.

New Zealand Environmental Protection Authority. 2015. Application for approval to import ESTEEM for release.

Osada H. 2019. Discovery and applications of nucleoside antibiotics beyond polyoxin. J Antibiot (Tokyo). 72(12):855–864. doi:10.1038/s41429-019-0237-1.

Passano LM. 1961. The regulation of crustacean metamorphosis. Integr Comp Biol. 1(1):89–95. doi:10.1093/icb/1.1.89.

Reynolds SE. 1987. The cuticle, growth and moulting in insects: The essential background to the action of acylurea insecticides. Pestic Sci. 20(2):131–146. doi:10.1002/ps.2780200207.

Seastedt TR, Crossley DA. 1984. Influence of on arthropods ecosystems. Bioscience. 34(3):157–161.

Shang F, Xiong Y, Xia WK, Wei DD, Wei D, Wang JJ. 2016. Identification, characterization and functional analysis of a chitin synthase gene in the brown citrus aphid, Toxoptera citricida (Hemiptera, Aphididae). Insect Mol Biol. 25(4):422–430. doi:10.1111/imb.12228.

Shi JF, Xu QY, Sun QK, Meng QW, Mu LL, Guo WC, Li GQ. 2016. Physiological roles of trehalose in Leptinotarsa larvae revealed by RNA interference of trehalose-6-phosphate synthase and trehalase genes. Insect Biochem Mol Biol. 77:52–68. doi:10.1016/j.ibmb.2016.07.012.

Song Y, Evenseth LM, Iguchi T, Tollefsen KE. 2017b. Release of chitobiase as an indicator of potential molting disruption in juvenile Daphnia magna exposed to the ecdysone receptor agonist 20-hydroxyecdysone. J Toxicol Environ Heal - Part A Curr Issues. 80(16–18):954–962. doi:10.1080/15287394.2017.1352215.

Song Y, Villeneuve DL, Toyota K, Iguchi T, Tollefsen KE. 2017a. Ecdysone Receptor Agonism Leading to Lethal Molting Disruption in Arthropods: Review and Adverse Outcome Pathway Development. Environ Sci Technol. 51(8):4142–4157. doi:10.1021/acs.est.7b00480.

Tellam RL, Eisemann C. 2000. Chitin is only a minor component of the peritrophic matrix from larvae of Lucilia cuprina. Insect Biochem Mol Biol. 30(12):1189–1201. doi:10.1016/S0965-1748(00)00097-7.

Tellam RL, Vuocolo T, Johnson SE, Jarmey J, Pearson RD. 2000. Insect chitin synthase. cDNA sequence, gene organization and expression. Eur J Biochem. 267(19):6025–6043. doi:10.1046/j.1432-1327.2000.01679.x.

Turnbull IF, Howells AJ. 1982. Effects of several larvicidal compounds on chitin biosynthesis by isolated larval integuments of the sheep blowfly Lucilia cuprina. Aust J Biol Sci. 35(5):491–504. doi:10.1071/BI9820491.

Uhl G, Zimmer SM, Renner D, Schneider JM. 2015. Exploiting a moment of weakness: Male spiders escape sexual cannibalism by copulating with moulting females. Sci Rep. 5(July):1–7. doi:10.1038/srep16928.

Vincent JFV, Wegst UGK. 2004. Design and mechanical properties of insect cuticle. Arthropod Struct Dev. 33(3):187–199. doi:10.1016/j.asd.2004.05.006.

Wang Y, Fan HW, Huang HJ, Xue J, Wu WJ, Bao YY, Xu HJ, Zhu ZR, Cheng JA, Zhang CX. 2012. Chitin synthase 1 gene and its two alternative splicing variants from two sap-sucking insects, Nilaparvata lugens and Laodelphax striatellus (Hemiptera: Delphacidae). Insect Biochem Mol Biol. 42(9):637–646. doi:10.1016/j.ibmb.2012.04.009.

Wang Z, Yang H, Zhou C, Yang WJ, Jin DC, Long GY. 2019. Molecular cloning, expression, and functional analysis of the chitin synthase 1 gene and its two alternative splicing variants in the white-backed planthopper, Sogatella furcifera (Hemiptera: Delphacidae). Sci Rep. 9(1):1–14. doi:10.1038/s41598-018-37488-5.

Yang WJ, Xu KK, Cong L, Wang JJ. 2013. Identification, mRNA expression, and functional analysis of chitin synthase 1 gene and its two alternative splicing variants in oriental fruit fly, Bactrocera dorsalis. Int J Biol Sci. 9(4):331–342. doi:10.7150/ijbs.6022.

Ye C, Jiang Y Di, An X, Yang L, Shang F, Niu J, Wang JJ. 2019. Effects of RNAi-based silencing of chitin synthase gene on moulting and fecundity in pea aphids (Acyrthosiphon pisum). Sci Rep. 9(1):1–10. doi:10.1038/s41598-019-39837-4.

Zhai Y, Fan X, Yin Z, Yue X, Men X, Zheng L, Zhang W. 2017. Identification and Functional Analysis of Chitin Synthase A in Oriental Armyworm, Mythimna separata. Proteomics. 17(21):1–11. doi:10.1002/pmic.201700165.

Zhang J, Liu X, Zhang Jianqin, Li D, Sun Y, Guo Y, Ma E, Zhu KY. 2010. Silencing of two alternative splicing-derived mRNA variants of chitin synthase 1 gene by RNAi is lethal to the oriental migratory locust, Locusta migratoria manilensis (Meyen). Insect Biochem Mol Biol. 40(11):824–833. doi:10.1016/j.ibmb.2010.08.001.

Zhang X, Yan Zhu K. 2013. Biochemical characterization of chitin synthase activity and inhibition in the African malaria mosquito, Anopheles gambiae. Insect Sci. 20(2):158–166. doi:10.1111/j.1744-7917.2012.01568.x.

Zhang X, Zhang J, Zhu KY. 2010. Chitosan/double-stranded RNA nanoparticle-mediated RNA interference to silence chitin synthase genes through larval feeding in the African malaria mosquito (Anopheles gambiae). Insect Mol Biol. 19(5):683–693. doi:10.1111/j.1365-2583.2010.01029.x.

Zhu KY, Heise S, Zhang J, Anderson TD, Starkey SR. 2007. Comparative Studies on Effects of Three Chitin Synthesis Inhibitors on Common Malaria Mosquito (Diptera: Culicidae). J Med Entomol. 44(6):1047–1053. doi:10.1093/jmedent/44.6.1047.

Zhuo W, Fang Y, Kong L, Li X, Sima Y, Xu S. 2014. Chitin synthase A: A novel epidermal development regulation gene in the larvae of Bombyx mori. Mol Biol Rep. 41(7):4177–4186. doi:10.1007/s11033-014-3288-1.