To the extent possible under law, AOP-Wiki has waived all copyright and related or neighboring rights to KE:1668

Event: 1668

Key Event Title

The KE title should describe a discrete biological change that can be measured. It should generally define the biological object or process being measured and whether it is increased, decreased, or otherwise definably altered relative to a control state. For example “enzyme activity, decreased”, “hormone concentration, increased”, or “growth rate, decreased”, where the specific enzyme or hormone being measured is defined. More help

Frustrated phagoytosis

Short name
The KE short name should be a reasonable abbreviation of the KE title and is used in labelling this object throughout the AOP-Wiki. The short name should be less than 80 characters in length. More help
Frustrated phagoytosis

Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. Note, KEs should be defined within a particular level of biological organization. Only KERs should be used to transition from one level of organization to another. Selection of the level of biological organization defines which structured terms will be available to select when defining the Event Components (below). More help
Level of Biological Organization

Cell term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help

Organ term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help

Key Event Components

Further information on Event Components and Biological Context may be viewed on the attached pdf.Because one of the aims of the AOP-KB is to facilitate de facto construction of AOP networks through the use of shared KE and KER elements, authors are also asked to define their KEs using a set of structured ontology terms (Event Components). In the absence of structured terms, the same KE can readily be defined using a number of synonymous titles (read by a computer as character strings). In order to make these synonymous KEs more machine-readable, KEs should also be defined by one or more “event components” consisting of a biological process, object, and action with each term originating from one of 22 biological ontologies (Ives, et al., 2017; See List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling). The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signalling by that receptor).Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description. To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons. If a desired term does not exist, a new term request may be made via Term Requests. Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add. More help
Process Object Action
phagocytosis macrophage decreased

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE. Clicking on the name of the AOP will bring you to the individual page for that AOP. More help
AOP Name Role of event in AOP Point of Contact Author Status OECD Status
Frustrated phagocytosis-induced lung cancer MolecularInitiatingEvent Carole Seidel (send email) Under development: Not open for comment. Do not cite Under Development
Frustrated phagocytosis leads to malignant mesothelioma MolecularInitiatingEvent Penny Nymark (send email) Under development: Not open for comment. Do not cite


This is a structured field used to identify specific agents (generally chemicals) that can trigger the KE. Stressors identified in this field will be linked to the KE in a machine-readable manner, such that, for example, a stressor search would identify this as an event the stressor can trigger. NOTE: intermediate or downstream KEs in one AOP may function as MIEs in other AOPs, meaning that stressor information may be added to the KE description, even if it is a downstream KE in the pathway currently under development.Information concerning the stressors that may trigger an MIE can be defined using a combination of structured and unstructured (free-text) fields. For example, structured fields may be used to indicate specific chemicals for which there is evidence of an interaction relevant to this MIE. By linking the KE description to a structured chemical name, it will be increasingly possible to link the MIE to other sources of chemical data and information, enhancing searchability and inter-operability among different data-sources and knowledgebases. The free-text section “Evidence for perturbation of this MIE by stressor” can be used both to identify the supporting evidence for specific stressors triggering the MIE as well as to define broad chemical categories or other properties that classify the stressors able to trigger the MIE for which specific structured terms may not exist. More help

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected from an ontology. 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
mammals mammals NCBI

Life Stages

The structured ontology terms for life-stage are more comprehensive than those for taxa, but may still require further description/development and explanation in the free text section. More help
Life stage Evidence

Sex Applicability

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

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. For example, the biological state being measured could be the activity of an enzyme, the expression of a gene or abundance of an mRNA transcript, the concentration of a hormone or protein, neuronal activity, heart rate, etc. The biological compartment may be a particular cell type, tissue, organ, fluid (e.g., plasma, cerebrospinal fluid), etc. The role in the biology could describe the reaction that an enzyme catalyses and the role of that reaction within a given metabolic pathway; the protein that a gene or mRNA transcript codes for and the function of that protein; the function of a hormone in a given target tissue, physiological function of an organ, etc. Careful attention should be taken to avoid reference to other KEs, KERs or AOPs. Only describe this KE as a single isolated measurable event/state. This will ensure that the KE is modular and can be used by other AOPs, thereby facilitating construction of AOP networks. More help

Phagocytosis is the first line of defence of the organism against foreign matter and therefore is essential for the maintenance of the homeostasis [1]. This process, mainly performed by macrophages, is dividing in two steps, first after recognition and internalization of the foreign matter, the phagosome is formed, and second, this structure is mature in a degradative compartment [1].

In the lung tissue, macrophages located in the alveolar space are involved in the clearance of foreign matter inhaled. After phagocytosis, cells migrate out of the alveolar space via the mucociliary escalator or the lymphatic system.

High aspect ratio nanoparticles (HARN) are particles with a ratio length – diameter ≥ 3 [2] [3]. Their fibre-shaped, similar to asbestos, is causing concern about their toxicity [4]. HARN include nanotubes, nanorods, nanowires and nanofibers in which carbon nanotubes (CNTs) are the most known and studied. CNT could enter in cells and interact with mitotic spindles as well as nuclei [5]. Macrophages try to phagocytose these particles, however the phagocytosis if incomplete leading to a frustrated phagocytosis. Consequently, the foreign matter is retained in the body because it cannot be cleared by macrophages [6].

How It Is Measured or Detected

One of the primary considerations in evaluating AOPs is the relevance and reliability of the methods with which the KEs can be measured. The aim of this section of the KE description is not to provide detailed protocols, but rather to capture, in a sentence or two, per method, the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements. Methods that can be used to detect or measure the biological state represented in the KE should be briefly described and/or cited. These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA).Key considerations regarding scientific confidence in the measurement approach include whether the assay is fit for purpose, whether it provides a direct or indirect measure of the biological state in question, whether it is repeatable and reproducible, and the extent to which it is accepted in the scientific and/or regulatory community. Information can be obtained from the OECD Test Guidelines website and the EURL ECVAM Database Service on Alternative Methods to Animal Experimentation (DB-ALM). ?

Using in vitro cell models, such as macrophages or epithelial cells, the analysis of interaction between HARN and cells could be performed in time-lapse microscopy [7] or backscatter electron microscopy [8].

The frustrated phagocytosis could be measure by different type of microscopy analysis allowing a direct measurement. For examples, time-lapse video microscopy [7], light microscopy [9], scanning electron microscopy [6, 9], bright-field microscopy [8] and backscatter electron microscopy [8] are used in the literature.

The analysis of phagocytic receptor expression such as MARCO, MSR-1, CD36, TLR4 is an indirect measurement [6].

The study of the capacity of macrophages to complete phagocytosis process could be performed in vitro using different type of macrophage cell lines (THP-1, NR8383, RAW267) and analysis by microscopy or gene expression or in vivo after exposure of rodents to different type of high aspect ratio nanoparticles and analysis of the remaining quantity of material in the body or the ability of macrophages to phagocyte foreign matter.

Domain of Applicability

This free text section should be used to elaborate on the scientific basis for the indicated domains of applicability and the WoE calls (if provided). While structured terms may be selected to define the taxonomic, life stage and sex applicability (see structured applicability terms, above) of the KE, the structured terms may not adequately reflect or capture the overall biological applicability domain (particularly with regard to taxa). Likewise, the structured terms do not provide an explanation or rationale for the selection. The free-text section on evidence for taxonomic, life stage, and sex applicability can be used to elaborate on why the specific structured terms were selected, and provide supporting references and background information.  More help

The frustrated phagocytosis of high aspect ratio particle can occur in mammals, male or female, and is generally measured in adults.

Evidence for Perturbation by Stressor

Overview for Molecular Initiating Event

When a specific MIE can be defined (i.e., the molecular target and nature of interaction is known), in addition to describing the biological state associated with the MIE, how it can be measured, and its taxonomic, life stage, and sex applicability, it is useful to list stressors known to trigger the MIE and provide evidence supporting that initiation. This will often be a list of prototypical compounds demonstrated to interact with the target molecule in the manner detailed in the MIE description to initiate a given pathway (e.g., 2,3,7,8-TCDD as a prototypical AhR agonist; 17α-ethynyl estradiol as a prototypical ER agonist). 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). Known stressors should be included in the MIE description, but it is not expected to include a comprehensive list. Rather initially, stressors identified will be exemplary and the stressor list will be expanded over time. For more information on MIE, please see pages 32-33 in the User Handbook.

High aspect ratio nanoparticles are not completely engulfed by macrophages due to their long shape (> 10µm) [8, 10].

Some studies analysed the effect of the length of nanoparticles (NPs) on the capacity of macrophages to phagocytose them.

The study of Sweeney et al 2015 demonstrated that treatment of primary human alveolar macrophages by multi-walled carbon nanotubes (MWCNT) similar in term of diameter, specific surface area and purity but differ by their length differentially alter phagocytosis [6]. Indeed, treatment by the longer CNT (median length 19.3 µm) induced frustrated phagocytosis and receptor expression (MARCO) as well as decreased phagocytic ability and migratory capacity in a more extend manner than the shorter CNT (median length 1.1 µm) [6]. Another study analysed the effect of particle morphology on the ability of human monocytic cell line THP-1 to engulf carbon nanotubes [9]. Cells were treated for 24h by two longs CNTs (men length 13 and 36 µm, dimeter 84.89 and 165.02 nm), two tangled CNTs (length 1-5 and 5-20 µm, dimeter 14.84 and 10.40 nm) and one short CNT (length 1-2 µm, diameter 25.7 nm). The authors observed by light microscopy that only the two long CNTs are protruding from the cells. A study on THP-1 cells treated for 4h was conducted with silver nanowires that possess similar diameter but different length (average length: 3, 5, 10, 14 and 28 µm) [8]. The authors observed by bright-field microscopy that the shorter NPs (3 and 5) were fully enclosed by macrophages, while the longer NPs (14 and 28) caused frustrated phagocytosis. In addition, injection of NPs in mouse pleural cavity followed by pleural lavage demonstrated that the shorter (3 and 5) were fully phagocytosed whereas the longer (10) caused frustrated phagocytosis. The authors observed differences between in vitro and in vivo studies in term of sensitivity for the determination of the length threshold that caused frustrated phagocytosis [8]. Finally, Padmore et al showed by time-lapse video microscopy that immortalized MH-S murine alveolar macrophages were able to internalized short glass fibres (mean length 7 µm) whereas the longer fibres were not (mean length 39.3 µm) [7].

All together, these studies could suggest that the threshold for frustrated phagocytosis should be around 10 µm, close to the suggestion formulated by Donaldson et al that fibres longer than 15 µm cause this process [10].


List of the literature that was cited for this KE description. Ideally, the list of references, should conform, to the extent possible, with the OECD Style Guide ( (OECD, 2015). More help

1.         Montano F, Grinstein S, Levin R. Quantitative Phagocytosis Assays in Primary and Cultured Macrophages. 2018;1784:151-63; doi: 10.1007/978-1-4939-7837-3_15.


3.         Oberdorster G, Oberdorster E, Oberdorster J. Concepts of nanoparticle dose metric and response metric. 2007;115 6:A290; doi: 10.1289/ehp.115-1892118.

4.         Donaldson K, Poland CA. Inhaled nanoparticles and lung cancer - what we can learn from conventional particle toxicology. 2012;142:w13547; doi: 10.4414/smw.2012.13547.

5.         Sargent LM, Shvedova AA, Hubbs AF, Salisbury JL, Benkovic SA, Kashon ML, et al. Induction of aneuploidy by single-walled carbon nanotubes. 2009;50 8:708-17; doi: 10.1002/em.20529.

6.         Sweeney S, Grandolfo D, Ruenraroengsak P, Tetley TD. Functional consequences for primary human alveolar macrophages following treatment with long, but not short, multiwalled carbon nanotubes. International journal of nanomedicine. 2015;10:3115-29; doi: 10.2147/IJN.S77867.

7.         Padmore T, Stark C, Turkevich LA, Champion JA. Quantitative analysis of the role of fiber length on phagocytosis and inflammatory response by alveolar macrophages. Biochimica et biophysica acta. 2017;1861 2:58-67; doi: 10.1016/j.bbagen.2016.09.031.

8.         Schinwald A, Donaldson K. Use of back-scatter electron signals to visualise cell/nanowires interactions in vitro and in vivo; frustrated phagocytosis of long fibres in macrophages and compartmentalisation in mesothelial cells in vivo. Particle and fibre toxicology. 2012;9:34; doi: 10.1186/1743-8977-9-34.

9.         Murphy FA, Schinwald A, Poland CA, Donaldson K. The mechanism of pleural inflammation by long carbon nanotubes: interaction of long fibres with macrophages stimulates them to amplify pro-inflammatory responses in mesothelial cells. Particle and fibre toxicology. 2012;9:8; doi: 10.1186/1743-8977-9-8.

           10.       Donaldson K, Murphy FA, Duffin R, Poland CA. Asbestos, carbon nanotubes and the pleural mesothelium:            a review of the hypothesis regarding the role of long fibre retention in the parietal pleura, inflammation and                        mesothelioma. 2010;7:5; doi: 10.1186/1743-8977-7-5.