Adverse Outcome Pathway Concept Research Articles

In silico toxicology: From structure-activity relationships towards deep learning and adverse outcome pathways.

In silico toxicology is an emerging field. It gains increasing importance as research is aiming to decrease the use of animal experiments as suggested in the 3R principles by Russell and Burch. In silico toxicology is a means to identify hazards of compounds before synthesis, and thus in very early stages of drug development. For chemical industries, as well as regulatory agencies it can aid in gap‐filling and guide risk minimization strategies. Techniques such as structural alerts, read‐across, quantitative structure–activity relationship, machine learning, and deep learning allow to use in silico toxicology in many cases, some even when data is scarce. Especially the concept of adverse outcome pathways puts all techniques into a broader context and can elucidate predictions by mechanistic insights.This article is categorized under:Structure and Mechanism > Computational Biochemistry and BiophysicsData Science > Chemoinformatics

Use of EpiAlveolar Lung Model to Predict Fibrotic Potential of Multiwalled Carbon Nanotubes.

Expansion in production and commercial use of nanomaterials increases the potential human exposure during the lifecycle of these materials (production, use, and disposal). Inhalation is a primary route of exposure to nanomaterials; therefore it is critical to assess their potential respiratory hazard. Herein, we developed a three-dimensional alveolar model (EpiAlveolar) consisting of human primary alveolar epithelial cells, fibroblasts, and endothelial cells, with or without macrophages for predicting long-term responses to aerosols. Following thorough characterization of the model, proinflammatory and profibrotic responses based on the adverse outcome pathway concept for lung fibrosis were assessed upon repeated subchronic exposures (up to 21 days) to two types of multiwalled carbon nanotubes (MWCNTs) and silica quartz particles. We simulate occupational exposure doses for the MWCNTs (1-30 μg/cm2) using an air-liquid interface exposure device (VITROCELL Cloud) with repeated exposures over 3 weeks. Specific key events leading to lung fibrosis, such as barrier integrity and release of proinflammatory and profibrotic markers, show the responsiveness of the model. Nanocyl induced, in general, a less pronounced reaction than Mitsui-7, and the cultures with human monocyte-derived macrophages (MDMs) showed the proinflammatory response at later time points than those without MDMs. In conclusion, we present a robust alveolar model to predict inflammatory and fibrotic responses upon exposure to MWCNTs.

Critical Review of Read-Across Potential in Testing for Endocrine-Related Effects in Vertebrate Ecological Receptors.

Recent regulatory testing programs have been designed to evaluate whether a chemical has the potential to interact with the endocrine system and could cause adverse effects. Some endocrine pathways are highly conserved among vertebrates, providing a potential to extrapolate data generated for one vertebrate taxonomic group to others (i.e., biological read‐across). To assess the potential for biological read‐across, we reviewed tools and approaches that support species extrapolation for fish, amphibians, birds, and reptiles. For each of the estrogen, androgen, thyroid, and steroidogenesis (EATS) pathways, we considered the pathway conservation across species and the responses of endocrine‐sensitive endpoints. The available data show a high degree of confidence in the conservation of the hypothalamus–pituitary–gonadal axis between fish and mammals and the hypothalamus–pituitary–thyroid axis between amphibians and mammals. Comparatively, there is less empirical evidence for the conservation of other EATS pathways between other taxonomic groups, but this may be due to limited data. Although more information on sensitive pathways and endpoints would be useful, current developments in the use of molecular target sequencing similarity tools and thoughtful application of the adverse outcome pathway concept show promise for further advancement of read‐across approaches for testing EATS pathways in vertebrate ecological receptors. Environ Toxicol Chem 2020;39:739–753. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Assessment of developmental neurotoxicity induced by chemical mixtures using an adverse outcome pathway concept

BackgroundIn light of the vulnerability of the developing brain, mixture risk assessment (MRA) for the evaluation of developmental neurotoxicity (DNT) should be implemented, since infants and children are co-exposed to more than one chemical at a time. One possible approach to tackle MRA could be to cluster DNT chemicals in a mixture on the basis of their mode of action (MoA) into ‘similar’ and ‘dissimilar’, but still contributing to the same adverse outcome, and anchor DNT assays to common key events (CKEs) identified in DNT-specific adverse outcome pathways (AOPs). Moreover, the use of human in vitro models, such as induced pluripotent stem cell (hiPSC)-derived neuronal and glial cultures would enable mechanistic understanding of chemically-induced adverse effects, avoiding species extrapolation.MethodsHiPSC-derived neural progenitors differentiated into mixed cultures of neurons and astrocytes were used to assess the effects of acute (3 days) and repeated dose (14 days) treatments with single chemicals and in mixtures belonging to different classes (i.e., lead(II) chloride and methylmercury chloride (heavy metals), chlorpyrifos (pesticide), bisphenol A (organic compound and endocrine disrupter), valproic acid (drug), and PCB138 (persistent organic pollutant and endocrine disrupter), which are associated with cognitive deficits, including learning and memory impairment in children. Selected chemicals were grouped based on their mode of action (MoA) into ‘similar’ and ‘dissimilar’ MoA compounds and their effects on synaptogenesis, neurite outgrowth, and brain derived neurotrophic factor (BDNF) protein levels, identified as CKEs in currently available AOPs relevant to DNT, were evaluated by immunocytochemistry and high content imaging analysis.ResultsChemicals working through similar MoA (i.e., alterations of BDNF levels), at non-cytotoxic (IC20/100), very low toxic (IC5), or moderately toxic (IC20) concentrations, induce DNT effects in mixtures, as shown by increased number of neurons, impairment of neurite outgrowth and synaptogenesis (the most sensitive endpoint as confirmed by mathematical modelling) and increase of BDNF levels, to a certain extent reproducing autism-like cellular changes observed in the brain of autistic children.ConclusionsOur findings suggest that the use of human iPSC-derived mixed neuronal/glial cultures applied to a battery of assays anchored to key events of an AOP network represents a valuable approach to identify mixtures of chemicals with potential to cause learning and memory impairment in children.

A case example of a radiation-relevant adverse outcome pathway to lung cancer

Background Adverse outcome pathways (AOPs) describe how a measurable sequence of key events, beginning from a molecular initiator, can lead to an adverse outcome of relevance to risk assessment. An AOP is modular by design, comprised of four main components: (1) a Molecular Initiating Event (MIE), (2) Key Events (KEs), (3) Key Event Relationships (KERs) and (4) an Adverse Outcome (AO). Purpose Here, we illustrate the utility of the AOP concept through a case example in the field of ionizing radiation, using the Organisation for Economic Cooperation and Development (OECD) Users' Handbook. This AOP defines a classic targeted response to a radiation insult with an AO of lung cancer that is relevant to radon gas exposure. Materials and methods To build this AOP, over 500 papers were reviewed and categorized based on the modified Bradford-Hill Criteria. Data-rich key events from the MIE, to several measurable KEs and KERs related to DNA damage response/repair were identified. Results The components for this AOP begin with direct deposition of energy as the MIE. Energy deposited into a cell can lead to multiple ionization events to targets such as DNA. This energy can damage DNA leading to double-strand breaks (DSBs) (KE1), this will initiate repair activation (KE2) in higher eukaryotes through mechanisms that are quick and efficient, but error-prone. If DSBs occur in regions of the DNA transcribing critical genes, then mutations (KE3) generated through faulty repair may alter the function of these genes or may cause chromosomal aberrations (KE4). This can impact cellular pathways such as cell growth, cell cycling and then cellular proliferation (KE5). This will form hyperplasia in lung cells, leading eventually to lung cancer (AO) induction and metastasis. The weight of evidence for the KERs was built using biological plausibility, incidence concordance, dose-response, time-response and essentiality studies. The uncertainties and inconsistencies surrounding this AOP are centered on dose-response relationships associated with dose, dose-rates and radiation quality. Conclusion Overall, the AOP framework provided an effective means to organize the scientific knowledge surrounding the KERs and identify those with strong dose-response relationships and those with inconsistencies. This case study is an example of how the AOP methodology can be applied to sources of radiation to help support areas of risk assessment.

Development of a Novel AOP for Cyp2F2-Mediated Lung Cancer in Mice.

Traditional methods for carcinogenicity testing rely heavily on the rodent bioassay as the standard for identification of tumorigenic risk. As such, identification of species-specific outcomes and/or metabolism are a frequent argument for regulatory exemption. One example is the association of tumor formation in the mouse lung after exposure to Cyp2F2 ligands. The adverse outcome pathway (AOP) framework offers a theoretical platform to address issues of species specificity that is consistent, transparent, and capable of integrating data from new approach methodologies as well as traditional data streams. A central premise of the AOP concept is that pathway progression from the molecular initiating event (MIE) implies a definable "response-response" (R-R) relationship between each key event (KE) that drives the pathway towards a specific adverse outcome (AO). This article describes an AOP for lung cancer in the mouse from an MIE of Cyp2F2-specific reactive metabolite formation, advancing through KE that include protein and/or nucleic acid adducts, diminished Club Cell 10 kDa (CC10) protein expression, hyperplasia of CC10 deficient Club cells, and culminating in the AO of mixed-cell tumor formation in the distal airways. This tumor formation is independent of route of exposure and our AOP construct is based on overlapping mechanistic events for naphthalene, styrene, ethyl benzene, isoniazid, and fluensulfone in the mouse. This AOP is intended to accelerate the explication of an apparent mouse-specific outcome and serve as a starting point for a quantitative analysis of mouse-human differences in susceptibility to the tumorigenic effects of Cyp2F2 ligands.

Effect-based methods in combination with state-of-the-art chemical analysis for assessment of water quality as integrated approach.

Complex mixtures of chemicals in waste and finally in surface water may pose a risk to the environment and also to human health. This contamination of surface water cannot be addressed with chemical analysis alone. Tools are required to detect and assess these micropollutants which might cause adverse effects. Effect-directed analysis (EDA) with effect-based methods in combination with state-of-the-art chemical analysis can meet this challenge. The present paper summarizes and outlines current experiences with analytical tools and bioassays as integrated approach for assessment of water quality. The need for a holistic and solution-oriented procedure of water quality monitoring is described. To integrate and evaluate existing information about toxicity pathways, which are essential for the EDA approach, the adverse outcome pathway (AOP) concept is useful and recommended. An integration of AOP concept in water quality assessment and further requirements are discussed.

Advancing Computational Toxicology in the Big Data Era by Artificial Intelligence: Data-Driven and Mechanism-Driven Modeling for Chemical Toxicity.

In 2016, the Frank R. Lautenberg Chemical Safety for the 21st Century Act became the first US legislation to advance chemical safety evaluations by utilizing novel testing approaches that reduce the testing of vertebrate animals. Central to this mission is the advancement of computational toxicology and artificial intelligence approaches to implementing innovative testing methods. In the current big data era, the terms volume (amount of data), velocity (growth of data), and variety (the diversity of sources) have been used to characterize the currently available chemical, in vitro, and in vivo data for toxicity modeling purposes. Furthermore, as suggested by various scientists, the variability (internal consistency or lack thereof) of publicly available data pools, such as PubChem, also presents significant computational challenges. The development of novel artificial intelligence approaches based on public massive toxicity data is urgently needed to generate new predictive models for chemical toxicity evaluations and make the developed models applicable as alternatives for evaluating untested compounds. In this procedure, traditional approaches (e.g., QSAR) purely based on chemical structures have been replaced by newly designed data-driven and mechanism-driven modeling. The resulting models realize the concept of adverse outcome pathway (AOP), which can not only directly evaluate toxicity potentials of new compounds, but also illustrate relevant toxicity mechanisms. The recent advancement of computational toxicology in the big data era has paved the road to future toxicity testing, which will significantly impact on the public health.

Introducing WikiPathways as a Data-Source to Support Adverse Outcome Pathways for Regulatory Risk Assessment of Chemicals and Nanomaterials.

A paradigm shift is taking place in risk assessment to replace animal models, reduce the number of economic resources, and refine the methodologies to test the growing number of chemicals and nanomaterials. Therefore, approaches such as transcriptomics, proteomics, and metabolomics have become valuable tools in toxicological research, and are finding their way into regulatory toxicity. One promising framework to bridge the gap between the molecular-level measurements and risk assessment is the concept of adverse outcome pathways (AOPs). These pathways comprise mechanistic knowledge and connect biological events from a molecular level toward an adverse effect outcome after exposure to a chemical. However, the implementation of omics-based approaches in the AOPs and their acceptance by the risk assessment community is still a challenge. Because the existing modules in the main repository for AOPs, the AOP Knowledge Base (AOP-KB), do not currently allow the integration of omics technologies, additional tools are required for omics-based data analysis and visualization. Here we show how WikiPathways can serve as a supportive tool to make omics data interoperable with the AOP-Wiki, part of the AOP-KB. Manual matching of key events (KEs) indicated that 67% could be linked with molecular pathways. Automatic connection through linkage of identifiers between the databases showed that only 30% of AOP-Wiki chemicals were found on WikiPathways. More loose linkage through gene names in KE and Key Event Relationships descriptions gave an overlap of 70 and 71%, respectively. This shows many opportunities to create more direct connections, for example with extended ontology annotations, improving its interoperability. This interoperability allows the needed integration of omics data linked to the molecular pathways with AOPs. A new AOP Portal on WikiPathways is presented to allow the community of AOP developers to collaborate and populate the molecular pathways that underlie the KEs of AOP-Wiki. We conclude that the integration of WikiPathways and AOP-Wiki will improve risk assessment because omics data will be linked directly to KEs and therefore allow the comprehensive understanding and description of AOPs. To make this assessment reproducible and valid, major changes are needed in both WikiPathways and AOP-Wiki.

The Adverse Outcome Pathway (AOP) concept as a basis for the development of mechanism-based animal sparing approaches: Kidney toxicity due to lysosomal overload and inhibition of mtDNA polymerase-γ as case studies

Humans are exposed to disinfection by-products (DBPs) mainly through drinking water ingestion and dermal contact. As an emerging class of nitrogenous DBPs (N-DBPs), haloacetamides (HAcAms) have been found to have significantly higher cytotoxicity than regulated DBPs. In this study, we investigated the cytotoxicity of HAcAms on two exposure pathway-related cell lines: human gastric epithelial GES-1 cells and immortalized keratinocytes HaCaT. Our results showed that the ranking order of cytotoxicity of 13 HAcAms was different between HaCaT and GES-1 cells. In addition, the 50% inhibitive concentration in HaCaT was 1.01–3.29 times that in GES-1. Further comparison among GES-1, HaCaT and CHO cell lines confirmed that different cell lines exhibited different sensitivity to the same compound. Importantly, HAcAms showed 5.83–7.13 × 104 times higher toxicity than the well-clarified DBP chloroform, clearly demonstrating the increased toxicity of HAcAms. Finally, using a novel high-content screening (HCS) analysis, we found that 39.29% of chlorinated HAcAms, 42.86% of brominated HAcAms and 16.07% of iodinated HAcAms significantly affected at least one of the cell-health parameters, such as nuclear size, membrane permeability, mitochondrial membrane potential, or cytochrome c release, in GES-1 or HaCaT cells. Thus, brominated HAcAms appear to have stronger effects under the sublethal exposure dose, possibly causing cytotoxicity via apoptosis. Together, our study provides new insights to the toxicity of HAcAms and a comprehensive toxicology dataset for health risk assessment.

Cytochrome P450 Induction and Xeno-Sensing Receptors Pregnane X Receptor, Constitutive Androstane Receptor, Aryl Hydrocarbon Receptor and Peroxisome Proliferator-Activated Receptor α at the Crossroads of Toxicokinetics and Toxicodynamics.

Pregnane X receptor (PXR), constitutive androstane receptor (CAR), aryl hydrocarbon receptor (AHR) and peroxisome proliferator-activated receptor α (PPARα) are ligand-activated transcription factors that regulate expression of many xenobiotic-metabolizing enzymes including several cytochrome P450 (CYP) enzymes. Many xenobiotics induce CYP enzymes through these intracellular receptors and consequently affect toxicokinetics and possible metabolic activation of the receptor ligands and other xenobiotics utilizing similar metabolic pathways. However, it is now apparent that the xenobiotic receptors regulate also many endogenous functions and signalling pathways, and xenobiotic exposure thus may dysregulate an array of fundamental cell functions. This MiniReview surveys and discusses the multifaceted roles of xenobiotic receptors, for which CYP induction may serve as the first alert and possibly a biomarker for exposure to xenobiotics. With the current emergence of the adverse outcome pathway (AOP) concept, these receptors are being and will be assigned as molecular initiating events or key events in numerous discrete toxicity pathways.

Toxic and Genomic Influences of Inhaled Nanomaterials as a Basis for Predicting Adverse Outcome.

An immense variety of different types of engineered nanomaterials are currently being developed and increasingly applied to consumer products. Importantly, engineered nanomaterials may pose unexplored adverse health effects because of their small size. Particularly in occupational settings, the dustiness of certain engineered nanomaterials involves risk of inhalation and influences on lung function. These facts call for quick and cost-effective safety testing practices, such as that obtained through multiparametric high-throughput screening using cultured human lung cells. The predictive value of such in vitro-based testing depends partly on the effectiveness of coverage of the mechanisms underlying toxicity effects. The concept of adverse outcome pathways covers the array of causative effects starting from a molecular initiating event via cellular-, organ-, individual-, and population-level effects. Screening for adverse outcome pathway-related effects that drive the eventual toxic outcome provides a good basis for developing predictive testing methods and data-driven integrated testing strategies for hazard and risk assessment. Temporal and inherited genomic changes are likely to drive many adverse responses to engineered nanomaterials, such as multiwalled carbon nanotubes, of which one specific form has recently been evaluated as possibly carcinogenic. Here, we briefly describe current state-of-the-art strategies for analyzing and understanding genomic influences of engineered nanomaterial exposure, including the selected focus on lung disease, and strategies for using mechanistic knowledge to predict and prevent adverse outcome.

Development of the Adverse Outcome Pathway (AOP): Chronic binding of antagonist to N-methyl-d-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities of children

The Adverse Outcome Pathways (AOPs) are designed to provide mechanistic understanding of complex biological systems and pathways of toxicity that result in adverse outcomes (AOs) relevant to regulatory endpoints. AOP concept captures in a structured way the causal relationships resulting from initial chemical interaction with biological target(s) (molecular initiating event) to an AO manifested in individual organisms and/or populations through a sequential series of key events (KEs), which are cellular, anatomical and/or functional changes in biological processes. An AOP provides the mechanistic detail required to support chemical safety assessment, the development of alternative methods and the implementation of an integrated testing strategy.An example of the AOP relevant to developmental neurotoxicity (DNT) is described here following the requirements of information defined by the OECD Users' Handbook Supplement to the Guidance Document for developing and assessing AOPs. In this AOP, the binding of an antagonist to glutamate receptor N-methyl-d-aspartate (NMDAR) receptor is defined as MIE. This MIE triggers a cascade of cellular KEs including reduction of intracellular calcium levels, reduction of brain derived neurotrophic factor release, neuronal cell death, decreased glutamate presynaptic release and aberrant dendritic morphology. At organ level, the above mentioned KEs lead to decreased synaptogenesis and decreased neuronal network formation and function causing learning and memory deficit at organism level, which is defined as the AO. There are in vitro, in vivo and epidemiological data that support the described KEs and their causative relationships rendering this AOP relevant to DNT evaluation in the context of regulatory purposes.

Effects of Harmful Algal Blooms on Fish: Insights from Prymnesium parvum

Blooms of the planktonic alga Prymnesium parvum pose a global threat, causing fish kills worldwide. Early studies on the exposure of fish to P. parvum indicate that toxic effects are related to gill damage. The more strictly defined concept of adverse outcome pathways has been suggested as a replacement for the mode of action in toxicology studies. In this study, rainbow trout (Onchorhyncus mykiss) were exposed to P. parvum. During exposure, oxygen consumption was determined by respirometry, and ventilation and coughing rate were determined via video surveillance. Per breath oxygen consumption was calculated to assess the ventilation effort to obtain a unit of oxygen. A second experiment monitored fish behavior to assess recovery. The results indicated that oxygen consumption initially increased, but on average fell below the standard oxygen consumption at 70% relative exposure. Being a function of ventilation frequency and oxygen consumption, the per breath oxygen consumption decreased throughout exposure. Behavioral results determined that short-term P. parvum exposure subsequently caused the exposed fish to seek flow refuge immediately and to a greater extent than unexposed fish. The adverse outcome pathway of P. parvum on rainbow trout is that P. parvum acts as a gill irritant resulting in non-recoverable respiratory failure.

Integrated Approaches to Testing and Assessment: OECD Activities on the Development and Use of Adverse Outcome Pathways and Case Studies

The Organisation for Economic Co-operation and Development (OECD) works with member countries and other stakeholders to improve and harmonize chemical assessment methods. In 2012, the OECD Adverse Outcome Pathways (AOPs) Development Programme started. The Programme has published six AOPs thus far and more than 60 AOPs are under various stages of development under the Programme. This article reviews recent OECD activities on the use of AOPs in developing Integrated Approaches to Testing and Assessments (IATAs). The guidance document for the use of AOPs in developing IATA, published in 2016, provides a framework for developing and using IATA and describes how IATA can be based on an AOP. The guidance document on the reporting of defined approaches to be used within IATA, also published in 2016, provides a set of principles for reporting defined approaches to testing and assessment to facilitate their evaluation. In the guidance documents, the AOP concept plays an important role for building IATA approaches in a science-based and transparent way. In 2015, the IATA Case Studies Project was launched to increase experience with the use of IATA and novel hazard methodologies by developing case studies, which constitute examples of predictions that are fit-for-regulatory use. This activity highlights the importance of international collaboration for harmonizing and improving chemical safety assessment methods.

Knotting nets: Molecular junctions of interconnecting endocrine axes identified by application of the adverse outcome pathway concept.

To be defined as an endocrine disruptor, a substance has to meet several criteria, including the induction of specific adverse effects, a specific endocrine mode of action, and a plausible link between both. The latter criterion in particular might not always be unequivocally determined, especially because the endocrine system consists of diverse endocrine axes. The axes closely interact with each other, and manipulation of one triggers effects on the other. The present review aimed to identify some of the many interconnections between these axes. The focus was on fish, but data obtained in studies on amphibians and mammals were considered if they assisted in closing data gaps, because most of the endocrine mechanisms are evolutionarily conserved. The review includes data both from ecotoxicological studies and on physiological processes and gives information on hormone/hormone receptor interactions or gene transcription regulation. The key events and key event relationships identified provide explanations for unexpected effects on one axis, exerted by substances suspected to act specifically on another axis. Based on these data, several adverse outcome pathway (AOP) segments are identified, describing connections between the hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary-thyroid (HPT) axes, the HPG and hypothalamic-pituitary-adrenal/interrenal (HPA/I) axes, and the HPT and HPA/I axes. Central key events identified across axes were altered aromatase activity as well as altered expression and function of the proteins 11β-hydroxysteroid dehydrogenase (11β-HSD) and steroidogenic acute regulatory (StAR) protein. Substance classes that act on more than one endocrine axis were, for example, goitrogens or aromatase inhibitors. Despite the wealth of information gathered, the present review only provides a few insights into the molecular nets of endocrine axes, demonstrating the complexity of their interconnections. Environ Toxicol Chem 2018;37:318-328. © 2017 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Applying adverse outcome pathways and species sensitivity-weighted distribution to predicted-no-effect concentration derivation and quantitative ecological risk assessment for bisphenol A and 4-nonylphenol in aquatic environments: A case study on Tianjin City, China.

Adverse outcome pathways (AOPs) are a novel concept that effectively considers the toxic modes of action and guides the ecological risk assessment of chemicals. To better use toxicity data including biochemical or molecular responses and mechanistic data, we further developed a species sensitivity-weighted distribution (SSWD) method for bisphenol A and 4-nonylphenol. Their aquatic predicted-no-effect concentrations (PNECs) were derived using the log-normal statistical extrapolation method. We calculated aquatic PNECs of bisphenol A and 4-nonylphenol with values of 4.01 and 0.721 µg/L, respectively. The ecological risk of each chemical in different aquatic environments near Tianjin, China, a coastal municipality along the Bohai Sea, was characterized by hazard quotient and probabilistic risk quotient assessment techniques. Hazard quotients of 7.02 and 5.99 at 2 municipal sewage sites using all of the endpoints were observed for 4-nonylphenol, which indicated high ecological risks posed by 4-nonylphenol to aquatic organisms, especially endocrine-disrupting effects. Moreover, a high ecological risk of 4-nonylphenol was indicated based on the probabilistic risk quotient method. The present results show that combining the SSWD method and the AOP concept could better protect aquatic organisms from adverse effects such as endocrine disruption and could decrease uncertainty in ecological risk assessment. Environ Toxicol Chem 2018;37:551-562. © 2017 SETAC.

Adverse outcome pathways: opportunities, limitations and open questions.

Adverse outcome pathways (AOPs) are a recent toxicological construct that connects, in a formalized, transparent and quality-controlled way, mechanistic information to apical endpoints for regulatory purposes. AOP links a molecular initiating event (MIE) to the adverse outcome (AO) via key events (KE), in a way specified by key event relationships (KER). Although this approach to formalize mechanistic toxicological information only started in 2010, over 200 AOPs have already been established. At this stage, new requirements arise, such as the need for harmonization and re-assessment, for continuous updating, as well as for alerting about pitfalls, misuses and limits of applicability. In this review, the history of the AOP concept and its most prominent strengths are discussed, including the advantages of a formalized approach, the systematic collection of weight of evidence, the linkage of mechanisms to apical end points, the examination of the plausibility of epidemiological data, the identification of critical knowledge gaps and the design of mechanistic test methods. To prepare the ground for a broadened and appropriate use of AOPs, some widespread misconceptions are explained. Moreover, potential weaknesses and shortcomings of the current AOP rule set are addressed (1) to facilitate the discussion on its further evolution and (2) to better define appropriate vs. less suitable application areas. Exemplary toxicological studies are presented to discuss the linearity assumptions of AOP, the management of event modifiers and compensatory mechanisms, and whether a separation of toxicodynamics from toxicokinetics including metabolism is possible in the framework of pathway plasticity. Suggestions on how to compromise between different needs of AOP stakeholders have been added. A clear definition of open questions and limitations is provided to encourage further progress in the field.

The AOP Concept: How Novel Technologies Can Support Development of Adverse Outcome Pathways

Abstract Although the introduction of the adverse outcome pathway (AOP) concept has already triggered a strong increase in the awareness that toxicological activity also needs to be studied at the molecular and cell biological level, the practical implementation of the AOP concept faces a multitude of challenges. The AOP concept first requires the dissection of the observed adverse outcome (AO) into individual events and their subsequent composition into simplified linear AOPs. However, for most adverse effects there is hardly sufficient information about the underlying molecular mechanisms available. To describe relevant events that lead to AOs, the use of high-throughput, high-content, and omics approaches seems to be particularly suitable, if they can be integrated in a standardized and systematic way. Subsequently, the complex molecular and cellular interactions of a linear AOP have to be combined in AOP networks to describe toxicological responses in an intact organism. Finally, essential key events ...

Building Predictive Adverse Outcome Pathway Models: Role of Molecular Initiating Events and Structure–Activity Relationships

Abstract The original contribution of the adverse outcome pathway (AOP) concept to predictive toxicology is that the use of a range of assays covering the main events of the whole toxicity pathway would improve the prediction of the apical endpoints with alternative tests. This would permit more solid, mechanistically based predictions. However, explicit and systematic quantitative analysis of the AOPs and their integration into larger schemes is at times lacking. This leaves room to subjective implementations. We have investigated the quantitative modeling aspects of AOPs related to important apical endpoints, that is, skin sensitization, endocrine disruptors, and carcinogenicity. A common trend is that the major contribution to the final predictivity is given by in vitro or in silico models of molecular initiating events (MIEs), which appear to be the rate-limiting steps of the toxicity pathways, whereas models for intermediate events have poor correlations with apical endpoints. Since MIEs are in gener...