Aop: 205

Title

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

AOP from chemical insult to cell death

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
AOP on basal cytotoxicity

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

Authors

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

Mathieu Vinken and Bas Blaauboer

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
Cataia Ives   (email point of contact)

Contributors

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
  • Mathieu Vinken
  • Cataia Ives

Status

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 comment. Do not cite Under Development
This AOP was last modified on April 05, 2021 18:16
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
Decompartmentalization September 16, 2017 10:17
Direct mitochondrial inhibition September 16, 2017 10:17
narcosis September 16, 2017 10:17
Mitochondrial impairment September 16, 2017 10:17
Apoptosis February 10, 2020 03:23
Necrosis February 07, 2017 13:22
Decompartmentalization leads to Mitochondrial impairment February 07, 2017 13:23
Direct mitochondrial inhibition leads to Mitochondrial impairment February 07, 2017 13:24
narcosis leads to Mitochondrial impairment February 07, 2017 13:24
Mitochondrial impairment leads to Apoptosis February 07, 2017 13:25
Mitochondrial impairment leads to Necrosis February 07, 2017 13:26
Chemical February 07, 2017 13:22

Abstract

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

Basal cytotoxicity refers to the ability of a chemical substance to damage living cells, in particular by compromising functional and structural features related to general cellular housekeeping. Being a rather comprehensive term, it is not surprising that the pathways leading to basal cytotoxicity are quite generic. Nevertheless, a tentative AOP to basal cytotoxicity could consist of 3 consecutive steps. The first step involves initial cell injury caused by the parent chemical and/or its metabolites. In the second step, a mitochondrial dysfunction takes place as a consequence of the primary insult. This ultimately leads to cell death in the third step.

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

Evaluation of safety is a prerequisite prior introduction of new chemical entities onto the market. Historically, animal testing has formed the basis for such risk assessment exercises. Driven by scientific and ethical constraints, and initiated more than 3 decades ago, however, there is a clear tendency worldwide to increasingly address animal-free methods for this purpose. This has been reinforced by a number of legislative changes over the past few years in the European Union, imposing a ban on animal testing for particular groups of chemicals, in casu in the cosmetics field. This has been followed by other parts of the world, such as in Norway, Israel, India, New Zealand and the state of São Paulo in Brazil. In response to this ubiquitous matter, the scientific community has been urged to develop animal-free methods for evaluating the safety of chemicals, including in vitro and in silico assays, being a research area that is gaining momentum. Interestingly, this has triggered a paradigm shift from classical toxicology, focusing on apical endpoints for toxicity in animal models, to predictive toxicology, relying on information on mechanisms of toxic action.

A major tool adopted in predictive toxicology is the AOP framework, which refers to a conceptual construct that portrays existing knowledge concerning the linkage between a direct molecular initiating event (MIE) and an adverse outcome via a number of key events at a biological level of organization relevant to risk assessment. AOPs can serve several purposes pertinent to non-animal chemical risk assessment, such as read-across methods, integrated approaches to testing and assessment, quantitative structure-activity relationships or the elaboration of prioritization strategies. In fact, AOPs embody a number of proposed frameworks for the implementation of animal-free safety testing of chemicals. Such frameworks typically start with exposure assessment, physico-chemical profiling, read-across and biokinetic evaluation, all which dictate the subsequent selection of in vitro biomarkers and corresponding assays. For many new chemical entities, however, such pre-existing information may be scarce, which thus impedes targeted establishment of an in vitro testing battery. A strategy for setting up basal in vitro cytotoxicity testing of such data-poor chemicals is outlined could be based on the newly proposed generic AOP from chemical insult to cell death.

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 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 1258 Decompartmentalization Decompartmentalization
2 MIE 1260 Direct mitochondrial inhibition Direct mitochondrial inhibition
3 MIE 1259 narcosis narcosis
4 KE 1261 Mitochondrial impairment Mitochondrial impairment
5 AO 1262 Apoptosis Apoptosis
6 AO 1263 Necrosis Necrosis

Relationships Between Two Key Events (Including MIEs and AOs)

TESTINGThis 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
Title Adjacency Evidence Quantitative Understanding
Decompartmentalization leads to Mitochondrial impairment non-adjacent Moderate Not Specified
narcosis leads to Mitochondrial impairment non-adjacent Moderate Not Specified
Mitochondrial impairment leads to Necrosis non-adjacent High Not Specified

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

Stressors

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
Chemical Not Specified

Life Stage Applicability

Identify the life stage for which the KE is known to be applicable. More help
Life stage Evidence
Not Otherwise Specified Not Specified

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
Vertebrates Vertebrates High 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 High

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

Being pragmatic tools, the extent of AOP optimization typically depends on the intended use. In this respect, the postulated AOP from chemical insult to cell death may already be considered fit-for-purpose, namely in vitro basal cytotoxicity testing of new chemical entities with non-substantiated toxicological profiles. Such default in vitro toxicity strategy could consist of at least 2 major testing rounds. In a first run, outlined in the present paper, basal cytotoxicity can be tested using a minimum of 2 assays to assess 2  key events. This may imply a number of repetitive runs in itself in order to optimize exposure conditions, in particular the concentration range of the test compound. This could be followed by global toxicogenomics screening, which will provide more insight into the tentative mechanism of action of the test compound. This outcome, together with the result of the first testing run, largely dictates the selection of methods in the second testing run. In fact, the latter rather addresses suspected tissue-specific toxicity, thereby selecting biomarkers and assays that rely on the respective AOPs, such as available for specific types of hepatotoxicity, renal toxicity and neurotoxicity.

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

1. Initial injury

Chemicals can cause direct cell injury through a variety of mechanisms, which may involve a single specific event, such as altered activation of an ion channel (Gennari et al., 2004; Schoonen et al., 2009) or a receptor (Gennari et al., 2004; Houck and Kavlock, 2008). However, a generic AOP from chemical insult to cell death should preferably encompass more general processes that instantly disrupt cellular homeostasis.

A first mechanism in this respect is disturbance of plasma membrane integrity. A prerequisite for performing cellular functionality includes appropriate physical segregation between the extracellular environment and the cytosol, which contributes to selective passage of substances between both compartments. This is accomplished to a large extent by a solid double phospholipid layer. Damage to this plasma membrane induced by chemicals can occur in a number ways, of which accumulation and binding to the phospholipid bilayer, a process called narcosis, is a prominent one (Escher et al., 2002).

A second mechanism relates to interfering with subcellular architectural organization. In order to maintain homeostasis, cellular functions are restricted to specific organelles within the cell, such as the nucleus, where genetic material is stored and processed, or the rough endoplasmic reticulum, taking care of protein synthesis. This so-called compartmentalization may be compromised by chemicals, thereby jeopardizing overall cellular functionality (Eisenbrand et al., 2002; Schoonen et al., 2009).

A third mechanism involves directly negatively affecting cellular energy supplies, in particular by targeting mitochondria. Thus, chemicals may uncouple the mitochondrial respiratory chain, inhibit adenosine triphosphate (ATP) synthesis, damage mitochondrial deoxyribonucleic acid (DNA), interfere with the replication of mitochondrial DNA or decrease the synthesis and stability of mitochondrial transcripts (Jones et al., 2010; Pessayre et al., 2010).

2. Mytochondrial dysfunction

Mitochondria are considered as the fuel stations of the cell. In this context, pyruvate, produced from glucose through the process of glycolysis, is taken up by mitochondria and is transformed to acetylco-enzyme A. In a parallel pathway, fatty acids bound to acetylco-enzyme A enter the mitochondrion, where they are split by successive beta-oxidation cycles, also yielding acetylco-enzyme A. The latter is then converted into carbon dioxide through the tricarboxylic acid cycle. This is associated with the production of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide. Both are oxidized, whereby electrons are generated that are used to reduce molecular oxygen to water. This reaction, catalyzed by respiratory chain complexes, is coupled with the extrusion of protons from the matrix into the inner membrane space. When energy is needed, the protons re-enter the matrix through ATP synthetase to generate ATP from adenosine diphosphate (Jones et al., 2010; Pessayre et al., 2010).

Being critical and vital, this mitochondrial process inherently is vulnerable, as there are many potential sites for toxic chemicals to act upon. An important one relates to the mitochondrial permeability transition (MPT) pore. This is a complex megachannel that spans across the inner and outer membrane of the mitochondrion and that consists of several proteins, including the voltage-dependent anion channel, adenine nucleotide translocase, cyclophilin D, creatine kinase, hexokinase, the benzodiazepine peripheral receptor and some B-cell lymphoma 2 proteins (Juhaszova et al., 2008; Tsujimoto and Shimizu, 2007). The MPT pore is usually closed, though it can be opened by some xenobiotics. This leads to the release of molecules, such as cytochrome C, into the cytosol, or the uptake of substances, including protons and water, in the mitochondrial matrix. If the MPT pore opens instantly in a large number of mitochondria, severe ATP depletion occurs. This is detrimental for many cellular functions that rely on energy and results in the disequilibrium of critical ion levels, eventually causing necrosis. Alternatively, when the MPT pore only opens in a limited set of mitochondria, unaffected mitochondria continue to produce ATP, while disrupted mitochondria release cytochrome C. This ultimately activates the apoptotic signaling cascade (Jones et al., 2010; Pessayre et al., 2012; Tsujimoto and Shimizu, 2007).

3. Cell death

Apoptosis is a programmed cell death mode that relies on the proteolytic activity of caspases. As such, 2 major apoptotic pathways have been described, namely the extrinsic signaling cascade and the intrinsic pathway. The latter is initiated by stimulating the release of cytochrome C from mitochondria, a process that is controlled by pro-apoptotic and anti-apoptotic B-cell lymphoma 2 proteins. Liberated cytochrome C forms a so-called apoptosome with apoptotic protease activating factor 1, deoxyadenosine triphosphate and procaspase 9. Following activation, the apoptosome triggers caspase 3. In the extrinsic pathway, specific ligands, such as Fas ligand, bind to their corresponding receptors at the cell plasma membrane surface. This induces the recruitment and cleavage of procaspase 8, which in turn activates caspase 3.  Fas-expressing cells are classified as either type I or type II based upon the ability of the intrinsic pathway to contribute to the amplification of the caspase cascade activated through the extrinsic pathway. In type I cells, sufficient amounts of caspase 8 are produced through this route and caspase 3 becomes directly activated. By contrast, in type II cells, only minimal quantities of active caspase 8 can be generated and the execution of the apoptotic response requires mitochondrial amplification. In this scenario, caspase 8 activates the pro-apoptotic Bid protein, which then translocates to mitochondria, where it leads to the release of cytochrome C and apoptosome formation. The overall outcome of the apoptotic pathways is the activation of caspase 3, which is the main executor of apoptosis. In fact, caspase 3 cleaves a broad spectrum of cellular proteins, such as cytoskeletal proteins, which gives rise to the typical apoptotic phenotype, involving blebbing, cell shrinkage, cytoplasmic and nuclear condensation, DNA fragmentation and the formation of apoptotic bodies. These apoptotic bodies are rapidly engulfed by neighboring phagocytes and no inflammatory response is induced (Alkhouri et al., 2011, Au et al., 2011; Guicciardi and Gores, 2005; Mahli et al., 2006; Schulze-Bergkamen et al., 2006; St-Pierre and Dufour, 2012).

Necrosis, as opposed to apoptosis, is a rather passive and unorganized process that is caused by a plethora of stress factors. It usually starts with the loss of ion homeostasis, which activates proteases, endonucleases and phospholipases. This generally results in cell swelling, cell lysis and induction of inflammation (Au et al., 2011; Grattagliano et al., 2009; Mahli et al., 2006; Schulze-Bergkamen et al., 2006).

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

While most of the KERs in the AOP are supported by a plethora of strong evidence, the links between decompartmentalization and mitochondrial impairment as well as between narcosis and mitochondrial impairment are less clear.

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

Concentration-response relationships are likely to underlie most KERs in the AOP.

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

In case of lack of any pre-existing information on potential toxicity of a test compound towards a specific cell type whatsoever, a general cytotoxicity testing scheme could be set up supported by the proposed generic AOP from chemical insult to cell death. A number of factors related to the selection of the cellular system, cytotoxicity assay and exposure conditions must be considered prior organizing such default in vitro cytotoxicity testing trials. These factors will be discussed in the following sections, with many of them being illustrated by knowledge gained from the use of in vitro systems based on liver, which is one of the most prominent target organs for toxicity.

1. Selection of the cellular system

A prerequisite for reliably predicting human real-life cytotoxicity is the use of an in vitro system in which all critical biological targets, as depicted in the generic AOP from chemical insult to cell death, are phenotypically expressed at an in vivo-like level for the entire testing regime. Human-based in vitro models are obviously strongly preferred, although this may be limited because of ethical, financial and other reasons (Fraczek et al., 2013; Vinken et al., 2012). Human-relevant information can also be obtained to a large extent when addressing systems based on other species, such as rodents. Nevertheless, this may introduce interspecies differences, which should be taken into consideration while interpreting testing results. Such inherent discrepancies between humans and rodents not only apply to tissue-specific (patho)physiological functions, but have been equally reported for seemingly more generic processes, such as inflammation (Seok et al., 2013).

Cytotoxicity sensu stricto is not a cell type-specific process, yet some tissues may be more susceptible to this process than others. As the human body consists of a large repertoire of cell types, the selection of the cellular origin of the in vitro model therefore is another critical parameter. When suspecting major mitochondrial damage induced by the chemical under investigation, one might consider a cell type that is naturally endowed with a high number of mitochondria, such as hepatocytes, myocytes or adipocytes. In most cases, however, a more common type of cell is used, such as fibroblasts.

Because of practical reasons, cell lines are more frequently used compared to primary cells for cytotoxicity testing purposes. Indeed, cell lines foresee a virtually unlimited cell supply, perform better in terms or reproducibility, are less labor-intensive and thus more users-friendly in comparison with most primary cell systems, many of which are prone to rapidly progressing dedifferentiation (Vinken et al., 2012). A number of human and rodent cell lines have been shown sensitive to chemical-induced cytotoxicity, including human embryonic kidney HEK293cells, human T-cell leukemia Jurkat cells, human neuroblastoma SH-SY5Y cells, mouse neuroblastoma N2a cells, mouse embryonic NIH3T3 fibroblasts and rat hepatoma H-4-II-E cells (Shukla et al., 2010). Nevertheless, it should be kept in mind that a majority of cell lines originate from cancers, which implies that they may perform aberrant functionality. This specifically holds true for the biotransformation machinery, which may be defective or even lacking in cell lines, but that may be indispensable for bio-activation or de-activation of chemicals (Fraczek et al., 2013; Vinken et al., 2012). Furthermore, cancer cells display altered cell death potential (Vinken et al., 2014). Regarding the latter, the background level of cell death in the selected in vitro model should be reduced as much as possible, as this may interfere with the testing outcome. This has been exemplified for conventional monolayer cultures primary of rat hepatocytes, which cope with substantial spontaneous apoptosis and necrosis (Vinken et al., 2011).

Whenever possible, it may be advisable to seed cells on small format culture plates (i.e. 96-well plates). This carries a number of advantages, including reduction of cellular and testing material, and thus of overall costs, as well as high-throughput potential. A pivotal factor in this respect is the density at which the cells are seeded, as both too low and too high densities may induce cell death (Qiao and Farrell, 1999). Similarly, the substrata used for cell seeding strongly affect viability. Thus, cultivating cells on an extracellular matrix scaffold promotes attachment and thereby cell survival (Vanhaecke et al., 2004). In the same light, the composition of the cell culture medium is of key importance while setting up cytotoxicity tests. A variety of culture media is commercially available, including Dulbecco’s modified Eagle’s medium, William’s medium E  and Leibovitz’s L15 medium, all which are typically supplemented with a number of additives (Elaut et al., 2005). Among those, several ones counteract spontaneous cell death, such as serum, typically added to increase cell attachment, (Tuschl et al., 2009) and glucocorticosteroids, which positively affect the differentiated cellular phenotype (Bailly-Maitre et al., 2002). As many cytotoxicity tests are based on measuring release of components from cells into the cell culture medium as a function of time following injury, the frequency of cell culture media renewal and, linked to this, the time of sampling should be carefully selected. In this regard, while some cultivation protocols only foresee renewal of cell culture media every 3 days, others, especially those using primary cells, require daily refreshment of the cell culture medium.

2. Selection of cytotoxicity assays

In vitro cytotoxicity testing of chemicals for which no pre-existing toxicologically relevant information is available could rely on the proposed generic AOP from chemical insult to cell death and should include at least 2 assays to test 2 KEs. Given their indispensable role in the initiation and perpetuation of cytotoxicity, mitochondria seem ideal biomarkers of chemical-induced cellular damage. Their activity can be monitored by a number of tests, of which the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay undoubtedly is the most commonly used one  (Mosmann, 1983). The MTT cytotoxicity assay is a colorimetric method based on the ability of viable cells to reduce a yellow tetrazolium salt into a blue insoluble formazan that is retained inside cells. The addition of organic solvents, such as dimethylsulfoxide (DMSO), leads to solubilization of the formed formazan and its release into the culture medium allowing its colorimetric measurement. The mitochondrial enzyme succinate dehydrogenase is responsible for the reduction of the tetrazolium salt to formazan. The ability of cells to reduce MTT provides an indication of mitochondrial integrity and activity, which in turn may be interpreted as a measure of viability and/or cell number. The number of surviving cells is directly proportional to the level of the formazan product created. Typically, IC50 and IC10 values are established in MTT testing, thus concentrations of the test compound that trigger cell death in 50% and 10% of the cultured cells, respectively (Tolosa et al., 2015). A number of other tetrazolium salts, such as 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, and water-soluble tetrazolium salts can be used for the same purpose (Gomez-Lechon et al., 2010). While setting up cell cultures for MTT testing, colored culture medium additives, such as phenol red, must be avoided, as they can interfere with formazan color development. Furthermore, for some test compounds, including chemicals that may directly reduce tetrazolium salts, the MTT assay is not applicable (Tolosa et al., 2015). A variety of alternative assays can be addressed instead, such as bioluminescent measurement of ATP content and accumulation of the supravital dye neutral red in lysosomes (Babich and Borenfreund, 1987; https://ecvam-dbalm.jrc.ec.europa.eu/methods-and-protocols/protocol/balb-c-3t3-neutral-red-uptake-cytotoxicity-assay-(3t3-nru)-protocol-no.-139/key/p_1527). In fact, the latter, like the MTT test, has been reported to be most sensitive in detecting cytotoxic events (Fotakis and Timbrell, 2006).

A second KE to be tested could be plasma membrane damage. As a consequence of the compromised cell plasma integrity, cytosolic compounds can freely move outside the cell into the cell culture medium. Among those is lactate dehydrogenase (LDH), a stable enzyme that leaks from the cell in relatively high amounts upon cell plasma membrane damage. LDH catalyzes the interconversion of pyruvate and lactate with concomitant interconversion of reduced and oxidized NADH. The consumption of NADH can be spectrophotometrically assessed and serves as a measure that is proportional to LDH activity (Bergmeyer, 1974). A parameter that is routinely used in this context is the LDH index, which is the ratio of LDH activity in the cell culture medium over the total LDH activity (i.e. in cells and in the cell culture medium). Typically, the cut-off is set at 20%, with an LDH index above this value indicating cytotoxicity (Maes et al., 2015). Other cytotoxicity techniques that are based on chemical insults to the plasma membrane surface use reporter dyes that, upon addition to the cell culture medium, move inside of damaged cells, such as propidium iodide and Trypan blue (Gomez-Lechon et al., 2010).

3. Selection of exposure conditions

In quantifying the cytotoxic potential of a chemical, it is important to take into account the relevance of the tested concentrations. Two issues must be considered in this respect, namely the relevant concentration in the in vitro system and its relationship to the in vivo situation. The first issue relates to the biokinetic behaviour of the compound in the in vitro system. The actual concentration causing the toxic effect is not only determined by the amount of the compound added to the cell culture medium divided by the volume thereof (i.e. the nominal concentration). Processes like evaporation, binding to culture devices or cell culture medium constituents, such as proteins, might lead to deviations in the actual cellular exposure concentrations, sometimes even higher than 2 orders of magnitude. This may result in a much lower free concentration in the cell culture medium and consequently a considerable underestimation of the compound’s cytotoxic potential (Kramer et al., 2007). On the other hand, compounds might specifically accumulate in cells or in cellular compartments, including at the outer membrane, thereby leading to a higher narcotic potential. Therefore, these in vitro biokinetic processes are essential in determining the relevance of the in vitro testing conditions (Groothuis et al, 2015; Kramer et al., 2015).

The second issue is the proper translation of the in vitro cytotoxicity results in a risk assessment context (Blaauboer et al., 2012). This is the process of so-called quantitative in vitro-in vivo extrapolation and needs to take into account any differences in the exposure of cells in the in vitro setting as compared to the in vivo situation as well as the in vivo biokinetics (Yoon et al., 2015). For the latter issue, the use of physiologically-based biokinetic modelling is an important tool (Blaauboer, 2010).

In case no information at all is available about the anticipated cytotoxic concentrations, such as to be derived from structural or physico-chemical properties, it is recommend to perform at least 2 testing rounds, each including 3 biological (i.e. different cell batches) and 3 technical (i.e. different wells on a multiwell plate) repeats. In the first run, a minimum of 10 concentrations spread over a broad concentration range (i.e. 1 nM to 10 mM) should be tested. In the second run, the concentration frame should be narrowed down, usually in the µM range, and, if necessary, further fine-tuned in additional testing rounds. Perhaps an even more important aspect of the testing regime includes the time of exposure. For some toxicological responses, critical changes in gene expression patterns are already induced and detectable within 1 hour of exposure of cultured cells to test chemicals and only vary marginally with increasing concentration (Shinde et al., 2015). In most general cytotoxicity testing procedures, however, exposure times between 1 hour and 72 hours are applied, but as holds for concentration, this parameter may also warrant optimization. Furthermore, it is strongly recommended to determine the biokinetics in vitro, such as by measuring the free concentration, for which tools like solid-phase micro-extraction are available (Kramer et al., 2007; Vaes et al., 1997).

Implementation of an appropriate set of controls is of utmost importance for sound interpretation of in vitro cytotoxicity testing results. In a majority of cases, test compounds are not fully soluble in cell culture media and thus require a co-solvent, such as DMSO, ethanol or methanol. While the latter 2 are well known to act as cytotoxicants, DMSO is sometimes added to cell culture media, such as of cultured hepatocytes, because of its beneficial effects on cell functionality. However, DMSO may also cause cell damage. In a recent study, it was found that concentrations of both DMSO and ethanol exceeding 0.5% v/v induce cyototoxicity in cultures of human breast cancer MCF-7 cells, human umbilical vein endothelial cells and mouse RAW264.7 macrophages after 24 hours of exposure as judged on MTT testing (Jamalzadeh et al., 2016). Therefore, it is strictly necessary to include a solvent control in cytotoxicity testing when using such organic liquids to improve the solubility of test compounds. Simultaneously, a positive control must be tested, being a compound known to potentially trigger the biomarker of interest. Tamoxifen has been proven an appropriate positive control in MTT testing (Tolosa et al., 2015) and ATP-based cytotoxicity assays (Shukla et al., 2010), while compounds that harm the cell plasma membrane surface, such as sodium lauryl sulphate, may be applied as positive controls for the LDH leakage assay and reporter dye uptake tests (https://ecvam-dbalm.jrc.ec.europa.eu/methods-and-protocols/protocol/balb-c-3t3-neutral-red-uptake-cytotoxicity-assay-(3t3-nru)-protocol-no.-139/key/p_1527; Maes et al., 2015). Negative controls, although sometimes ignored, are equally important as their positive counterparts. The most obvious negative control is cell culture medium, yet for a number of reasons, it may be advisable to address specific chemicals as true negative controls. Typical negative controls in in vitro cytotoxicity tests are seemingly innocuous molecules, such as mannitol. Complying with Paracelsus’ basic principle stating that the dose makes the poison, however, it should be kept in mind that such apparent harmless chemicals may become toxic in sufficiently high concentrations, being osmotic stress in case of mannitol. Furthermore, care must be taken while adding controls and test compounds to the cell culture medium as well as while handling cell culture plates, since mechanical stress can also cause cytotoxicity. In case of suspected phototoxicity or temperature instability of the test compound, specific incubation measures could be necessary (Coecke et al., 2005; Cooper-Hannan et al., 1999).

References

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

Alkhouri, N., Carter-Kent, C., Feldstein, A.E., 2011. Apoptosis in nonalcoholic fatty liver disease: diagnostic and therapeutic implications. Expert. Rev. Gastroenterol. Hepatol. 5, 201-212.

Au, J.S., Navarro, V.J., Rossi, S., 2011. Drug-induced liver injury: its pathophysiology and evolving diagnostic tools. Aliment. Pharmacol. Ther. 34, 11-20.

Babich, H., Borenfreund, E., 1987. Structure-activity relationship (SAR) models established in vitro with the neutral red cytotoxicity assay. Toxicol. In Vitro 1, 3-9.

Bailly-Maitre, B., de Sousa, G., Zucchini, N., Gugenheim, J., Boulukos, K.E., Rahmani, R., 2002. Spontaneous apoptosis in primary cultures of human and rat hepatocytes: molecular mechanisms and regulation by dexamethasone. Cell Death Differ. 9, 945-955.

Bergmeyer, H.U., 1974. Lactate dehydrogenase. In: Methods of enzymatic analysis, (ed. H.U., Bergmeyer), pp. 574-579. New York, USA: Academic Press.

Blaauboer, B.J., 2010. Biokinetic modeling and in vitro-in vivo extrapolations. J. Toxicol. Environ. Health B Crit. Rev. 13, 242-252.

Blaauboer, B.J., Boekelheide, K., Clewell, H.J., Daneshian, M., Dingemans, M.M., Goldberg, A.M., Heneweer, M., Jaworska, J., Kramer, N.I., Leist, M., Seibert, H., Testai, E., Vandebriel, R.J., Yager, J.D., Zurlo, J., 2012. The use of biomarkers of toxicity for integrating in vitro hazard estimates into risk assessment for humans. Altern. Lab. Anim. 29, 411-425.

Coecke, S., Balls, M., Bowe, G., Davis, J., Gstraunthaler, G., Hartung, T., Hay, R., Merten, O.W., Price, A., Schechtman, L., Stacey, G., Stokes, W., 2005. Guidance on good cell culture practice. a report of the second ECVAM task force on good cell culture practice. Altern. Lab. Anim. 33: 261-287.

Cooper-Hannan, R., Harbell, J.W., Coecke, S., Balls, M., Bowe, G., Cervinka, M., Clothier, R., Hermann, F., Klahm, L.K., de Lange, J., Liebsch, M., Vanparys, P., 1999. The principles of good laboratory practice: application to in vitro toxicology studies. Altern. Lab. Anim. 27, 539-577.

Ekwall, B., 1995. The basal cytotoxicity concept. In: Alternative methods in toxicology and the life sciences: the World Congress on Alternatives and Animal Use in the Life Sciences: Education, Researchers, Testing, (eds. A.M., Goldberg, L.F.M., van Zupthen), pp. 721-725. New York, USA: Mary Ann Liebert.

Elaut, G., Vanhaecke, T., Heyden, Y.V., Rogiers, V., 2005. Spontaneous apoptosis, necrosis, energy status, glutathione levels and biotransformation capacities of isolated rat hepatocytes in suspension: effect of the incubation medium. Biochem. Pharmacol. 69, 1829-1838.

Eisenbrand, G., Pool-Zobel, B., Baker, V., Balls, M., Blaauboer, B.J., Boobis, A., Carere, A., Kevekordes, S., Lhuguenot, J.C., Pieters, R., Kleiner, J., 2002. Methods of in vitro toxicology. Food Chem. Toxicol. 40, 193-236.

Escher, B.I., Eggen, R.I., Schreiber, U., Schreiber, Z., Vye, E., Wisner, B., Schwarzenbach, R.P., 2002. Baseline toxicity (narcosis) of organic chemicals determined by in vitro membrane potential measurements in energy-transducing membranes. Environ. Sci. Technol. 36, 1971-1979.

EU, 2003. Directive 2003/15/EC of the European Parliament and of the Council of 27 February 2003 amending Council Directive 76/768/EEC on the approximation of the laws of the Member States relating to cosmetic products, Official Journal of the European Union L066, 26-35.

EU, 2009. Regulation (EC) No. 1223/2009 of the European Parliament and of the Council of 30 November 2009 on Cosmetic Products (recast), Official Journal of the European Union L342, 59-209.

Fotakis, G., Timbrell, J.A., 2006. In vitro cytotoxicity assays: comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride. Toxicol. Lett. 160, 171-177.

Fraczek, J., Bolleyn, J., Vanhaecke, T., Rogiers, V., Vinken, M., 2013. Primary hepatocyte cultures for pharmaco-toxicological studies: at the busy crossroad of various anti-dedifferentiation strategies. Arch. Toxicol. 87, 577-610.

Gennari, A., van den Berghe, C., Casati, S., Castell, J., Clemedson, C., Coecke, S., Colombo, A., Curren, R., Dal Negro, G., Goldberg, A., Gosmore, C., Hartung, T., Langezaal, I., Lessigiarska, I., Maas, W., Mangelsdorf, I., Parchment, R., Prieto, P., Sintes, J.R., Ryan, M., Schmuck, G., Stitzel, K., Stokes, W., Vericat, J.A., Gribaldo, L., 2004. Strategies to replace in vivo acute systemic toxicity testing: the report and recommendations of ECVAM Workshop 50. Altern. Lab. Anim. 32, 437-459.

Gomez-Lechon, M.J., Lahoz, A., Gombau, L., Castell, J.V., Donato, M.T., 2010. In vitro evaluation of potential hepatotoxicity induced by drugs. Curr. Pharm. Des. 16, 1963-1977.

Grattagliano, I., Bonfrate, L., Diogo, C.V., Wang, H.H., Wang, D.Q., Portincasa, P., 2009. Biochemical mechanisms in drug-induced liver injury: certainties and doubts. World J. Gastroenterol. 15, 4865-4876.

Groothuis, F.A., Heringa, M.B., Nicol, B., Hermens, J.L., Blaauboer, B.J., Kramer, N.I., 2015. Dose metric considerations in in vitro assays to improve quantitative in vitro-in vivo dose extrapolations. Toxicology 332, 30-40.

Guicciardi, M.E., Gores, G.J., 2005. Apoptosis: a mechanism of acute and chronic liver injury. Gut 54, 1024-1033.

Houck, K.A., Kavlock, R.J., 2008. Understanding mechanisms of toxicity: insights from drug discovery research. Toxicol. Appl. Pharmacol. 227, 163-178.

https://aopwiki.org/wiki/index.php/Main_Page (consulted November 2016).

https://ecvam-dbalm.jrc.ec.europa.eu/methods-and-protocols/protocol/balb-c-3t3-neutral-red-uptake-cytotoxicity-assay-(3t3-nru)-protocol-no.-139/key/p_1527 (consulted November 2016).

https://www.epa.gov/chemical-research/toxicity-forecasting (consulted November 2016).

Jamalzadeh, L., Ghafoori, H., Sariri, R., Rabuti, H., Nasirzade, J., Hasani, H., Aghamaali, M.R., 2016. Cytotoxic effects of some common organic solvents on MCF-7, RAW-264.7 and human umbilical vein endothelial cells. Avicenna J. Med. Biochem., e33453.

Jones, D.P., Lemasters, J.J., Han, D., Boelsterli, U.A., Kaplowitz, N., 2010. Mechanisms of pathogenesis in drug hepatotoxicity putting the stress on mitochondria. Mol. Interv. 10, 98-111.

Juhaszova, M., Wang, S., Zorov, D.B., Nuss, H.B., Gleichmann, M., Mattson, M.P., Sollott, S.J., 2008. The identity and regulation of the mitochondrial permeability transition pore: where the known meets the unknown. Ann. N.Y. Acad. Sci. 1123, 197-212.

Kramer, N.I., Van Eijkeren, J.C.H., Hermens, J.L.M., 2007. Influence of albumin on sorption kinetics in solid-phase microextraction: consequences for chemical analyses and uptake processes. Anal. Chem. 79, 6941-6948.

Kramer, N.I., Di Consiglio, E., Blaauboer, B.J., Testai, E., 2015. Biokinetics in repeated-dosing in vitro drug toxicity studies. Toxicol. In Vitro 30, 217-224.

Laquieze, L., Lorencini, M., Granjeiro, J.M., 2015. Alternative methods to animal testing and cosmetic safety: an update on regulations and ethical considerations in Brazil. Appl. In Vitro Toxicol. 1, 243-253.

Maes, M., Vanhaecke, T., Cogliati, B., Yanguas, S.C., Willebrords, J., Rogiers, V., Vinken, M., 2015. Measurement of apoptotic and necrotic cell death in primary hepatocyte cultures. Methods Mol. Biol. 1250, 349-361.

Malhi, H., Gores, G.J., Lemasters, J.J., 2006. Apoptosis and necrosis in the liver: a tale of two deaths? Hepatology 43, S31-S44.

Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55-63.

NRC, 2007. Toxicity testing in the 21st century: a vision and a strategy. The National Academies Press, Washington, DC.

Pessayre, D., Mansouri, A., Berson, A., Fromenty, B., 2010. Mitochondrial involvement in drug-induced liver injury. Handb. Exp. Pharmacol. 196, 311-365.

Pessayre, D., Fromenty, B., Berson, A., Robin, M.A., Letteron, P., Moreau, R., Mansouri, A., 2012. Central role of mitochondria in druginduced liver injury. Drug Metab. Rev. 44, 34-87.

Qiao, L., Farrell, G.C., 1999. The effects of cell density, attachment substratum and dexamethasone on spontaneous apoptosis of rat hepatocytes in primary culture. In Vitro Cell. Dev. Biol. Anim. 35, 417-424.

Schoonen, W.G., Westerink, W.M., Horbach, G.J., 2009. High-throughput screening for analysis of in vitro toxicity. EXS 99, 401-452.

Schulze-Bergkamen, H., Schuchmann, M., Fleischer, B., Galle, P.R., 2006. The role of apoptosis versus oncotic necrosis in liver injury: facts or faith? J. Hepatol. 44, 984-993.

Seok, J., Warren, H.S., Cuenca, A.G., Mindrinos, M.N., Baker, H.V., Xu, W., Richards, D.R., McDonald-Smith, G.P., Gao, H., Hennessy, L., Finnerty, C.C., Lopez, C.M., Honari, S., Moore, E.E., Minei, J.P., Cuschieri, J., Bankey, P.E., Johnson, J.L., Sperry, J., Nathens, A.B., Billiar, T.R., West, M.A., Jeschke, M.G., Klein, M.B., Gamelli, R.L., Gibran, N.S., Brownstein, B.H., Miller-Graziano, C., Calvano, S.E., Mason, P.H., Cobb, J.P., Rahme, L.G., Lowry, S.F., Maier, R.V., Moldawer, L.L., Herndon, D.N., Davis, R.W., Xiao, W., Tompkins, R.G., 2013. Genomic responses in mouse models poorly mimic human inflammatory diseases. Proc. Natl. Acad. Sci. U.S.A. 110, 3507-3512.

Shinde, V., Stöber, R., Nemade, H., Sotiriadou, I., Hescheler, J., Hengstler, J., Sachinidis, A., 2015. Transcriptomics of hepatocytes treated with toxicants for investigating molecular Mechanisms underlying hepatotoxicity. Methods Mol. Biol. 1250, 225-240.

Shukla, S.J., Huang, R., Austin, C.P., Xia, M., 2010. The future of toxicity testing: a focus on in vitro methods using a quantitative high-throughput screening platform. Drug Discov. Today 15, 997-1007.

St-Pierre, M.V., Dufour, J.F., 2012. Biomarkers for hepatocellular apoptosis in the management of liver diseases. Curr. Pharm. Biotechnol. 13, 2221-2227.

Tolosa, L., Donato, M.T., Gomez-Lechon, M.J., 2015. General cytotoxicity assessment by means of the MTT assay. Methods Mol. Biol. 1250, 333-348.

Tsujimoto, Y., Shimizu, S., 2007. Role of the mitochondrial membrane permeability transition in cell death. Apoptosis 12, 835-840.

Tuschl, G., Hrach, J., Walter, Y., Hewitt, P.G., Mueller, S.O., 2009. Serum-free collagen sandwich cultures of adult rat hepatocytes maintain liver-like properties long term: a valuable model for in vitro toxicity and drug-drug interaction studies. Chem. Biol. Interact. 181, 124-137.

Vaes, W.H.J., Ramos, E.U., Hamwijk, C., Van Holsteijn, I., Blaauboer, B.J., Seinen, W., Verhaar, H.J.M., Hermens, J.L.M., 1997. Solid phase microextraction as a tool to determine membrane/water partition coefficients and bioavailable concentrations in in vitro systems. Chem. Res. Toxicol. 10, 1067-1072.

Vanhaecke, T., Henkens, T., Kass, G.E., Rogiers, V., 2004. Effect of the histone deacetylase inhibitor trichostatin A on spontaneous apoptosis in various types of adult rat hepatocyte cultures. Biochem. Pharmacol. 68, 753-760.

Vinken, M., Decrock, E., Doktorova, T., Ramboer, E., De Vuyst, E., Vanhaecke, T., Leybaert, L., Rogiers, V., 2011. Characterization of spontaneous cell death in monolayer cultures of primary hepatocytes. Arch. Toxicol. 85, 1589-1596.

Vinken, M., Vanhaecke, T., Rogiers, V., 2012. Primary hepatocyte cultures as in vitro tools for toxicity testing: quo vadis? Toxicol. In Vitro 26, 541-544.

Vinken, M., 2013. The adverse outcome pathway concept: a pragmatic tool in toxicology. Toxicology 312, 158-165.

Vinken, M., Maes, M., Oliveira, A.G., Cogliati, B., Marques, P.E., Menezes, G.B., Dagli, M.L., Vanhaecke, T., Rogiers, V., 2014. Primary hepatocytes and their cultures in liver apoptosis research. Arch. Toxicol. 88, 199-212.

Vinken, M., 2015. Adverse outcome pathways and drug-induced liver injury testing. Chem. Res. Toxicol. 28, 1391-1397.

Yoon, M., Blaauboer, B.J., Clewell, H.J., 2015. Quantitative in vitro to in vivo extrapolation (QIVIVE): an essential element for in vitro-based risk assessment. Toxicology 332, 1-3.

Alkhouri, N., Carter-Kent, C., Feldstein, A.E., 2011. Apoptosis in nonalcoholic fatty liver disease: diagnostic and therapeutic implications. Expert. Rev. Gastroenterol. Hepatol. 5, 201-212.

Au, J.S., Navarro, V.J., Rossi, S., 2011. Drug-induced liver injury: its pathophysiology and evolving diagnostic tools. Aliment. Pharmacol. Ther. 34, 11-20.

Babich, H., Borenfreund, E., 1987. Structure-activity relationship (SAR) models established in vitro with the neutral red cytotoxicity assay. Toxicol. In Vitro 1, 3-9.

Bailly-Maitre, B., de Sousa, G., Zucchini, N., Gugenheim, J., Boulukos, K.E., Rahmani, R., 2002. Spontaneous apoptosis in primary cultures of human and rat hepatocytes: molecular mechanisms and regulation by dexamethasone. Cell Death Differ. 9, 945-955.

Bergmeyer, H.U., 1974. Lactate dehydrogenase. In: Methods of enzymatic analysis, (ed. H.U., Bergmeyer), pp. 574-579. New York, USA: Academic Press.

Blaauboer, B.J., 2010. Biokinetic modeling and in vitro-in vivo extrapolations. J. Toxicol. Environ. Health B Crit. Rev. 13, 242-252.

Blaauboer, B.J., Boekelheide, K., Clewell, H.J., Daneshian, M., Dingemans, M.M., Goldberg, A.M., Heneweer, M., Jaworska, J., Kramer, N.I., Leist, M., Seibert, H., Testai, E., Vandebriel, R.J., Yager, J.D., Zurlo, J., 2012. The use of biomarkers of toxicity for integrating in vitro hazard estimates into risk assessment for humans. Altern. Lab. Anim. 29, 411-425.

Coecke, S., Balls, M., Bowe, G., Davis, J., Gstraunthaler, G., Hartung, T., Hay, R., Merten, O.W., Price, A., Schechtman, L., Stacey, G., Stokes, W., 2005. Guidance on good cell culture practice. a report of the second ECVAM task force on good cell culture practice. Altern. Lab. Anim. 33: 261-287.

Cooper-Hannan, R., Harbell, J.W., Coecke, S., Balls, M., Bowe, G., Cervinka, M., Clothier, R., Hermann, F., Klahm, L.K., de Lange, J., Liebsch, M., Vanparys, P., 1999. The principles of good laboratory practice: application to in vitro toxicology studies. Altern. Lab. Anim. 27, 539-577.

Ekwall, B., 1995. The basal cytotoxicity concept. In: Alternative methods in toxicology and the life sciences: the World Congress on Alternatives and Animal Use in the Life Sciences: Education, Researchers, Testing, (eds. A.M., Goldberg, L.F.M., van Zupthen), pp. 721-725. New York, USA: Mary Ann Liebert.

Elaut, G., Vanhaecke, T., Heyden, Y.V., Rogiers, V., 2005. Spontaneous apoptosis, necrosis, energy status, glutathione levels and biotransformation capacities of isolated rat hepatocytes in suspension: effect of the incubation medium. Biochem. Pharmacol. 69, 1829-1838.

Eisenbrand, G., Pool-Zobel, B., Baker, V., Balls, M., Blaauboer, B.J., Boobis, A., Carere, A., Kevekordes, S., Lhuguenot, J.C., Pieters, R., Kleiner, J., 2002. Methods of in vitro toxicology. Food Chem. Toxicol. 40, 193-236.

Escher, B.I., Eggen, R.I., Schreiber, U., Schreiber, Z., Vye, E., Wisner, B., Schwarzenbach, R.P., 2002. Baseline toxicity (narcosis) of organic chemicals determined by in vitro membrane potential measurements in energy-transducing membranes. Environ. Sci. Technol. 36, 1971-1979.

EU, 2003. Directive 2003/15/EC of the European Parliament and of the Council of 27 February 2003 amending Council Directive 76/768/EEC on the approximation of the laws of the Member States relating to cosmetic products, Official Journal of the European Union L066, 26-35.

EU, 2009. Regulation (EC) No. 1223/2009 of the European Parliament and of the Council of 30 November 2009 on Cosmetic Products (recast), Official Journal of the European Union L342, 59-209.

Fotakis, G., Timbrell, J.A., 2006. In vitro cytotoxicity assays: comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride. Toxicol. Lett. 160, 171-177.

Fraczek, J., Bolleyn, J., Vanhaecke, T., Rogiers, V., Vinken, M., 2013. Primary hepatocyte cultures for pharmaco-toxicological studies: at the busy crossroad of various anti-dedifferentiation strategies. Arch. Toxicol. 87, 577-610.

Gennari, A., van den Berghe, C., Casati, S., Castell, J., Clemedson, C., Coecke, S., Colombo, A., Curren, R., Dal Negro, G., Goldberg, A., Gosmore, C., Hartung, T., Langezaal, I., Lessigiarska, I., Maas, W., Mangelsdorf, I., Parchment, R., Prieto, P., Sintes, J.R., Ryan, M., Schmuck, G., Stitzel, K., Stokes, W., Vericat, J.A., Gribaldo, L., 2004. Strategies to replace in vivo acute systemic toxicity testing: the report and recommendations of ECVAM Workshop 50. Altern. Lab. Anim. 32, 437-459.

Gomez-Lechon, M.J., Lahoz, A., Gombau, L., Castell, J.V., Donato, M.T., 2010. In vitro evaluation of potential hepatotoxicity induced by drugs. Curr. Pharm. Des. 16, 1963-1977.

Grattagliano, I., Bonfrate, L., Diogo, C.V., Wang, H.H., Wang, D.Q., Portincasa, P., 2009. Biochemical mechanisms in drug-induced liver injury: certainties and doubts. World J. Gastroenterol. 15, 4865-4876.

Groothuis, F.A., Heringa, M.B., Nicol, B., Hermens, J.L., Blaauboer, B.J., Kramer, N.I., 2015. Dose metric considerations in in vitro assays to improve quantitative in vitro-in vivo dose extrapolations. Toxicology 332, 30-40.

Guicciardi, M.E., Gores, G.J., 2005. Apoptosis: a mechanism of acute and chronic liver injury. Gut 54, 1024-1033.

Houck, K.A., Kavlock, R.J., 2008. Understanding mechanisms of toxicity: insights from drug discovery research. Toxicol. Appl. Pharmacol. 227, 163-178.

https://aopwiki.org/wiki/index.php/Main_Page (consulted November 2016).

https://ecvam-dbalm.jrc.ec.europa.eu/methods-and-protocols/protocol/balb-c-3t3-neutral-red-uptake-cytotoxicity-assay-(3t3-nru)-protocol-no.-139/key/p_1527 (consulted November 2016).

https://www.epa.gov/chemical-research/toxicity-forecasting (consulted November 2016).

Jamalzadeh, L., Ghafoori, H., Sariri, R., Rabuti, H., Nasirzade, J., Hasani, H., Aghamaali, M.R., 2016. Cytotoxic effects of some common organic solvents on MCF-7, RAW-264.7 and human umbilical vein endothelial cells. Avicenna J. Med. Biochem., e33453.

Jones, D.P., Lemasters, J.J., Han, D., Boelsterli, U.A., Kaplowitz, N., 2010. Mechanisms of pathogenesis in drug hepatotoxicity putting the stress on mitochondria. Mol. Interv. 10, 98-111.

Juhaszova, M., Wang, S., Zorov, D.B., Nuss, H.B., Gleichmann, M., Mattson, M.P., Sollott, S.J., 2008. The identity and regulation of the mitochondrial permeability transition pore: where the known meets the unknown. Ann. N.Y. Acad. Sci. 1123, 197-212.

Kramer, N.I., Van Eijkeren, J.C.H., Hermens, J.L.M., 2007. Influence of albumin on sorption kinetics in solid-phase microextraction: consequences for chemical analyses and uptake processes. Anal. Chem. 79, 6941-6948.

Kramer, N.I., Di Consiglio, E., Blaauboer, B.J., Testai, E., 2015. Biokinetics in repeated-dosing in vitro drug toxicity studies. Toxicol. In Vitro 30, 217-224.

Laquieze, L., Lorencini, M., Granjeiro, J.M., 2015. Alternative methods to animal testing and cosmetic safety: an update on regulations and ethical considerations in Brazil. Appl. In Vitro Toxicol. 1, 243-253.

Maes, M., Vanhaecke, T., Cogliati, B., Yanguas, S.C., Willebrords, J., Rogiers, V., Vinken, M., 2015. Measurement of apoptotic and necrotic cell death in primary hepatocyte cultures. Methods Mol. Biol. 1250, 349-361.

Malhi, H., Gores, G.J., Lemasters, J.J., 2006. Apoptosis and necrosis in the liver: a tale of two deaths? Hepatology 43, S31-S44.

Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55-63.

NRC, 2007. Toxicity testing in the 21st century: a vision and a strategy. The National Academies Press, Washington, DC.

Pessayre, D., Mansouri, A., Berson, A., Fromenty, B., 2010. Mitochondrial involvement in drug-induced liver injury. Handb. Exp. Pharmacol. 196, 311-365.

Pessayre, D., Fromenty, B., Berson, A., Robin, M.A., Letteron, P., Moreau, R., Mansouri, A., 2012. Central role of mitochondria in druginduced liver injury. Drug Metab. Rev. 44, 34-87.

Qiao, L., Farrell, G.C., 1999. The effects of cell density, attachment substratum and dexamethasone on spontaneous apoptosis of rat hepatocytes in primary culture. In Vitro Cell. Dev. Biol. Anim. 35, 417-424.

Schoonen, W.G., Westerink, W.M., Horbach, G.J., 2009. High-throughput screening for analysis of in vitro toxicity. EXS 99, 401-452.

Schulze-Bergkamen, H., Schuchmann, M., Fleischer, B., Galle, P.R., 2006. The role of apoptosis versus oncotic necrosis in liver injury: facts or faith? J. Hepatol. 44, 984-993.

Seok, J., Warren, H.S., Cuenca, A.G., Mindrinos, M.N., Baker, H.V., Xu, W., Richards, D.R., McDonald-Smith, G.P., Gao, H., Hennessy, L., Finnerty, C.C., Lopez, C.M., Honari, S., Moore, E.E., Minei, J.P., Cuschieri, J., Bankey, P.E., Johnson, J.L., Sperry, J., Nathens, A.B., Billiar, T.R., West, M.A., Jeschke, M.G., Klein, M.B., Gamelli, R.L., Gibran, N.S., Brownstein, B.H., Miller-Graziano, C., Calvano, S.E., Mason, P.H., Cobb, J.P., Rahme, L.G., Lowry, S.F., Maier, R.V., Moldawer, L.L., Herndon, D.N., Davis, R.W., Xiao, W., Tompkins, R.G., 2013. Genomic responses in mouse models poorly mimic human inflammatory diseases. Proc. Natl. Acad. Sci. U.S.A. 110, 3507-3512.

Shinde, V., Stöber, R., Nemade, H., Sotiriadou, I., Hescheler, J., Hengstler, J., Sachinidis, A., 2015. Transcriptomics of hepatocytes treated with toxicants for investigating molecular Mechanisms underlying hepatotoxicity. Methods Mol. Biol. 1250, 225-240.

Shukla, S.J., Huang, R., Austin, C.P., Xia, M., 2010. The future of toxicity testing: a focus on in vitro methods using a quantitative high-throughput screening platform. Drug Discov. Today 15, 997-1007.

St-Pierre, M.V., Dufour, J.F., 2012. Biomarkers for hepatocellular apoptosis in the management of liver diseases. Curr. Pharm. Biotechnol. 13, 2221-2227.

Tolosa, L., Donato, M.T., Gomez-Lechon, M.J., 2015. General cytotoxicity assessment by means of the MTT assay. Methods Mol. Biol. 1250, 333-348.

Tsujimoto, Y., Shimizu, S., 2007. Role of the mitochondrial membrane permeability transition in cell death. Apoptosis 12, 835-840.

Tuschl, G., Hrach, J., Walter, Y., Hewitt, P.G., Mueller, S.O., 2009. Serum-free collagen sandwich cultures of adult rat hepatocytes maintain liver-like properties long term: a valuable model for in vitro toxicity and drug-drug interaction studies. Chem. Biol. Interact. 181, 124-137.

Vaes, W.H.J., Ramos, E.U., Hamwijk, C., Van Holsteijn, I., Blaauboer, B.J., Seinen, W., Verhaar, H.J.M., Hermens, J.L.M., 1997. Solid phase microextraction as a tool to determine membrane/water partition coefficients and bioavailable concentrations in in vitro systems. Chem. Res. Toxicol. 10, 1067-1072.

Vanhaecke, T., Henkens, T., Kass, G.E., Rogiers, V., 2004. Effect of the histone deacetylase inhibitor trichostatin A on spontaneous apoptosis in various types of adult rat hepatocyte cultures. Biochem. Pharmacol. 68, 753-760.

Vinken, M., Decrock, E., Doktorova, T., Ramboer, E., De Vuyst, E., Vanhaecke, T., Leybaert, L., Rogiers, V., 2011. Characterization of spontaneous cell death in monolayer cultures of primary hepatocytes. Arch. Toxicol. 85, 1589-1596.

Vinken, M., Vanhaecke, T., Rogiers, V., 2012. Primary hepatocyte cultures as in vitro tools for toxicity testing: quo vadis? Toxicol. In Vitro 26, 541-544.

Vinken, M., 2013. The adverse outcome pathway concept: a pragmatic tool in toxicology. Toxicology 312, 158-165.

Vinken, M., Maes, M., Oliveira, A.G., Cogliati, B., Marques, P.E., Menezes, G.B., Dagli, M.L., Vanhaecke, T., Rogiers, V., 2014. Primary hepatocytes and their cultures in liver apoptosis research. Arch. Toxicol. 88, 199-212.

Vinken, M., 2015. Adverse outcome pathways and drug-induced liver injury testing. Chem. Res. Toxicol. 28, 1391-1397.

Yoon, M., Blaauboer, B.J., Clewell, H.J., 2015. Quantitative in vitro to in vivo extrapolation (QIVIVE): an essential element for in vitro-based risk assessment. Toxicology 332, 1-3.