Evaluating legacy contaminants and emerging chemicals in marine environments using adverse outcome pathways and biological effects-directed analysis

Outcome of the pesticides peer review meeting on general recurring issues in ecotoxicology

The adverse outcome pathway (AOP) framework contributes to understanding how specific and measurable biological perturbations cause adverse effects on human and environmental health. Recognizing the value of AOPs to support regulatory decisions around the world, the Organisation for Economic Co-operation and Development (OECD)launched the AOP Programme in 2012, which sought to promote and guide the development of AOPs to ensure their suitability for the downstream applications in the context of regulatory safety assessment. The OECD published the initial guidance on AOP development and assessment in 2013, which has been expanded as practices have evolved and matured. Adverse outcome pathway development requires adherence to specific principles and considerations for identifying and describing key events (KEs) and representing and assessing the weight of evidence for the key event relationships. Ultimately, the structured and consistent application of the principles helps build confidence in the applicability of the knowledge represented in the AOP for decision-making in the regulatory context. To assist new AOP developers, in 2019, the OECD introduced a coaching program. This program primarily aims to pair novices with experienced AOP developers (i.e., coaches). International partnerships in the coaching program contribute to harmonizing and promoting AOP development according to OECD guidance. Coaches have also helped to identify and initiate "gardening" efforts that remove redundant/synonymous KEs in the AOP-Wiki, allowing for improved AOP network creation, promoting the reuse of extensively reviewed KEs, and ensuring the development of high-quality AOPs. The AOP Coaching Program represents the latest international activity to ensure that AOPs are developed in a consistent manner that is designed to enhance their use for supporting public health decisions around the world.

The Adverse Outcome Pathway (AOP) concept is expected to guide risk assessors in their work to use all existing information on the effects of chemicals on humans and wildlife, and to target the generation of additional information to the regulatory objective. AOPs will therefore be used in the Organisation for Economic Co-operation and Development (OECD) chemical safety programme, as underlying scientific rationales for the development of alternative methods for hazard assessment, such as read-across, in vitro test methods or the development of integrated testing strategies that have the potential to replace animal tests. As a proof-of-concept, the OECD has developed an AOP for skin sensitisation, and as a follow-up has: a) implemented the AOP into the OECD QSAR Toolbox, so that information related to the Key Events (KEs) in the AOP can be used to group chemicals that are expected to act by the same mechanism and hence have the same skin sensitisation potential; b) developed alternative test methods for the KEs, so that ultimately chemicals can be tested for skin sensitisation without the use of animal tests. The development of integrated testing strategies based on the AOP is ongoing. Building on this proof-of-concept, the OECD has launched an AOP development programme with a first batch of AOPs published in 2016. A number of IT tools, which together form an AOP Knowledge Base, are at various stages of development, and support the construction of AOPs and their use in the development of integrated approaches for testing and assessment. Following the publication of the first batch of AOPs, OECD member countries will decide on priorities for their use in supporting the development of tools for regulatory use.

Increasing Scientific Confidence in Adverse Outcome Pathways: Application of Tailored Bradford-Hill Considerations for Evaluating Weight of Evidence

Background The purpose of toxicology is to protect human health and the environment. To support this, the Organisation for Economic Co-operation and Development (OECD), operating via its Extended Advisory Group for Molecular Screening and Toxicogenomics (EAGMST), has been developing the Adverse Outcome Pathway (AOP) approach to consolidate evidence for chemical toxicity spanning multiple levels of biological organization. The knowledge transcribed into AOPs provides a structured framework to transparently organize data, examine the weight of evidence of the AOP, and identify causal relationships between exposure to stressors and adverse effects of regulatory relevance. The AOP framework has undergone substantial maturation in the field of hazard characterization of chemicals over the last decade, and has also recently gained attention from the radiation community as a means to advance the mechanistic understanding of human and ecological health effects from exposure to ionizing radiation at low dose and low dose-rates. To fully exploit the value of such approaches for facilitating risk assessment and management in the field of radiation protection, solicitation of experiences and active cooperation between chemical and radiation communities are needed. As a result, the Radiation and Chemical (Rad/Chem) AOP joint topical group was formed on June 1, 2021 as part of the initiative from the High Level Group on Low Dose Research (HLG-LDR). HLG-LDR is overseen by the OECD Nuclear Energy Agency (NEA) Committee on Radiation Protection and Public Health (CRPPH). The main aims of the joint AOP topical group are to advance the use of AOPs in radiation research and foster broader implementation of AOPs into hazard and risk assessment. With global representation, it serves as a forum to discuss, identify and develop joint initiatives that support research and take on regulatory challenges. Conclusion The Rad/Chem AOP joint topical group will specifically engage, promote, and implement the use of the AOP framework to: (a) organize and evaluate mechanistic knowledge relevant to the protection of human and ecosystem health from radiation; (b) identify data gaps and research needs pertinent to expanding knowledge of low dose and low dose-rate radiation effects; and (c) demonstrate utility to support risk assessment by developing radiation-relevant case studies. It is envisioned that the Rad/Chem AOP joint topical group will actively liaise with the OECD EAGMST AOP developmental program to collectively advance areas of common interest and, specifically, provide recommendations for harmonization of the AOP framework to accommodate non-chemical stressors, such as radiation.

New streamlined approaches that use fewer resources and animals are needed for the safety assessment of chemicals. Data gathering should be streamlined to fit regulatory need and the specific properties of the chemicals being assessed. Toward this goal, the Organization for Economic Cooperation and Development (OECD) has introduced the concept of integrated approaches to testing and assessment (IATA) to inform hazard or risk assessment. An IATA is designed to address a specific question, and may include exposure or regulatory considerations, depending on the context. IATA can be informed by mechanistic information about the chemical and related biology. OECD's of Adverse Outcome Pathways (AOPs) framework is designed to gather biological information related to adverse outcomes of regulatory significance. An AOP is the collected chemical and biological information about a particular biological pathway. The OECD has developed guidance for building and assessing AOPs, and is coordinating development of the AOP Knowledge-base (AOP-KB) for collecting and using this information. The AOP-KB also accepts information about the perturbations of these pathways caused by chemical exposure – information can be used to design prediction models. AOPs can form the logical basis for the integration of information and the design of integrated testing strategies (ITS), within the context of an (IATA), to more effectively and efficiently inform hazard or risk determination.

Background The circulatory system distributes nutrients, signaling molecules, and immune cells to vital organs and soft tissues. Epidemiological, animal, and in vitro cellular mechanistic studies have highlighted that exposure to ionizing radiation (IR) can induce molecular changes in cellular and subcellular milieus leading to long-term health impacts, particularly on the circulatory system. Although the mechanisms for the pathologies are not fully elucidated, endothelial dysfunction is proven to be a critical event via radiation-induced oxidative stress mediators. To delineate connectivities of events specifically to cardiovascular disease (CVD) initiation and progression, the adverse outcome pathway (AOP) approach was used with consultation from field experts. AOPs are a means to organize information around a disease of interest to a regulatory question. An AOP begins with a molecular initiating event and ends in an adverse outcome via sequential linkages of key event relationships that are supported by evidence in the form of the modified Bradford-Hill criteria. Detailed guidelines on building AOPs are provided by the Organisation for Economic Cooperation and Development (OECD) AOP program. Here, we report on the questions and discussions needed to develop an AOP for CVD resulting from IR exposure. A recent workshop jointly organized by the MELODI (Multidisciplinary European Low Dose Initiative) and the ALLIANCE (European Radioecology Alliance) associations brought together experts from the OECD to present the AOP approach and tools with examples from the toxicology field. As part of this workshop, four working groups were formed to discuss the identification of adverse outcomes relevant to radiation exposures and development of potential AOPs, one of which was focused on IR-induced cardiovascular effects. Each working group comprised subject matter experts and radiation researchers interested in the specific disease area and included an AOP coach. Conclusion The CVD working group identified the critical questions of interest for AOP development, including the exposure scenario that would inform the evidence, the mechanisms of toxicity, the initiating event, intermediate key events/relationships, and the type of data currently available. This commentary describes the four-day discussion of the CVD working group, its outcomes, and demonstrates how collaboration and expert consultation is vital to informing AOP construction.

International Consortium to Advance Cross-Species Extrapolation of the Effects of Chemicals in Regulatory Toxicology.

Simple SummaryIt is recognized that the current developmental neurotoxicity (DNT) testing paradigm is not fit-for -purpose for the assessment of a large number of chemicals. In the last two decades there have been scientific advances made for evaluating chemical interactions with the developing nervous system that rely on alternative to animal methods. The Organisation for Economic Co-Operation and Development (OECD) provides a forum to develop internationally harmonised guidance to test and assess chemicals for DNT that is primarily based on cellular models. Given the complexity of the developing nervous system and the availability of a number of non-animal methods to address DNT, integration of data from multiple studies is necessary and an OECD framework for organising existing scientific knowledge can be applied as the canvas of this integration. Herein, we provide a brief overview of the OECD DNT project and summarize various achievements of relevance to the project. The review also presents an opportunity to describe considerations for uptake of the DNT non animal methods in a regulatory context.Characterization of potential chemical-induced developmental neurotoxicity (DNT) hazard is considered for risk assessment purposes by many regulatory sectors. However, due to test complexity, difficulty in interpreting results and need of substantial resources, the use of the in vivo DNT test guidelines has been limited and animal data on DNT are scarce. To address challenging endpoints such as DNT, the Organisation for Economic Co-Operation and Development (OECD) chemical safety program has been working lately toward the development of integrated approaches for testing and assessment (IATA) that rely on a combination of multiple layers of data (e.g., in vitro, in silico and non-mammalian in vivo models) that are supported by mechanistic knowledge organized according to the adverse outcome pathway (AOP) framework. In 2017, the OECD convened a dedicated OECD expert group to develop a guidance document on the application and interpretation of data derived from a DNT testing battery that relies on key neurodevelopmental processes and is complemented by zebrafish assays. This review will provide a brief overview of the OECD DNT project and summarize various achievements of relevance to the project. The review also presents an opportunity to describe considerations for uptake of the DNT in an in vitro battery in a regulatory context.

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.

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.

The adverse outcome pathway (AOP) framework, at its core, is a way of organizing biological and toxicological knowledge in a manner that facilitates inference and extrapolation. Much of the interest in AOPs is driven by the need to enhance the use of mechanistic or pathway-based data and predictive approaches as a basis for regulatory decision making 1-4. Although AOP description was initially rather ad hoc, the international community, through coordination with the Organisation for Economic Co-operation and Development's (OECD's) Extended Advisory Group on Molecular Screening and Toxicogenomics, has transformed adverse outcome pathway description into a formalized yet flexible process, employing standardized templates, terminology, and conventions (3-5. Both the efficiency of AOP development and transparency in AOP application have been advanced through development of a common internationally harmonized and publicly accessible knowledge base as a central repository for AOP descriptions 6. Together, these developments have fostered more consistent, systematic, and transparent AOP description, which facilitates regulatory application. Concepts of weight-of-evidence assembly and evaluation employing Bradford Hill considerations 7 have been incorporated as a fundamental aspect of AOP description. This is critical in that the amount of support for the relationships depicted in an AOP and the precision and confidence with which one can extrapolate from one element in the AOP to another ultimately dictate the type(s) of regulatory decision making it can reasonably be applied to. The development of AOPs and their representation in the AOP knowledge base employ a modular structure in which the basic elements of an AOP description, key events and key event relationships, are assembled into AOPs that describe a single trajectory of biological failure connecting a molecular initiating event to an adverse outcome (AO) considered relevant to regulatory decision making 3. Because key events and key event relationships can be shared by multiple AOPs, construction of simple AOP descriptions in the AOP knowledge base leads to de facto creation of networks of AOPs that capture the intrinsic complexity of potential biological and toxicological interactions that can modulate toxicological outcomes. Thus, the framework employs an elegant structure in which more sophisticated and complex understanding can be assembled from simpler units. Although the AOP framework has evolved considerably, further development is needed for it to play a central role in the 21st-century practice of regulatory toxicology. Advances are needed in 4 critical areas. First, the AOP knowledge base needs to be populated with AOP descriptions. As of January 2015, the AOP knowledge base contained 8 AOP descriptions that had been formally developed according to OECD guidance. Other AOP descriptions (n ≈ 40) had been initiated but were in early stages of development that did not include a transparent presentation of supporting evidence. The initial contents represent a mere fraction of the universe of toxicologically relevant pathways. There are many plausible AOPs which could be added to the AOP knowledge base and supported with existing knowledge. In an effort to address the need for improved coverage in the AOP knowledge base and to enhance the utility of data being generated through pioneering high-throughput screening programs (e.g., ToxCast, Tox21 8), the US Environmental Protection Agency's Chemical Safety for Sustainability program has initiated an effort to develop putative AOPs related to more than 300 unique molecular targets. Additional AOP development efforts are under way under the auspices of the OECD AOP development program 9. Because these AOPs are being developed in the publicly available AOP knowledge base 6, the ongoing AOP development efforts will be transparent. This serves both to minimize redundant efforts and to provide an open and collaborative environment in which a wide range of scientists can contribute their expertise. Consequently, there are many opportunities for the scientific community to engage and address the need for more AOP descriptions. Population of the AOP knowledge base with a robust set of AOPs and properly linking their descriptions through the use of shared key events and key event relationships is a critical prerequisite for a second important advance in the application of the AOP framework—development of approaches for extracting and analyzing AOP networks. Relevant human and ecological exposures almost universally involve exposure to multiple chemicals. Furthermore, many, if not the majority of, chemicals in the environment have the potential to interact with and perturb multiple biological targets. Consequently, to reasonably predict consequences of real-world exposures, it will be critical to contextualize how interactions among multiple AOPs within complex spatial and temporal domains of an organism will influence their cumulative outcome(s). The challenge of mixture toxicology is long-standing and well recognized. Although AOP networks alone do not provide all the tools needed to address that challenge, they do provide a mode of action–based road map that can aid evaluation of whether cumulative effects of chemicals may be independent, additive, reinforcing, or counteractive. Even if only through qualitative evaluation, consideration of points of interaction/intersection among AOPs, represented as shared key events in an AOP network 3, 4, facilitate hypothesis-driven assessments of potential mixture effects. Such approaches are needed in an environment where comprehensive testing of all individual chemicals, let alone all relevant mixtures, is unrealistic. Third, there is a need to develop computational frameworks that can take chemical-specific property information and/or biological effects measurements and translate them into a predicted probability or severity of an AO (generally for a specific exposure scenario). This has been termed the development of quantitative AOPs. A critical part of this process is developing a quantitative understanding of “points of departure” that define the magnitude and/or duration of change in an upstream key event needed to elicit a state change in a downstream key event. It involves understanding the adaptive mechanisms through which organisms respond to and compensate for different types of stressors and what their limitations are. This requires different kinds of toxicology research from those commonly employed in the past. In many cases, more intensive characterization of dose–response time-course surfaces across a range of biological scales and endpoints will be required to develop this quantitative understanding (e.g., Ankley and Villeneuve 10). Although such experiments may be technically challenging and resource-intensive, the ultimate goal is to unveil generalizable quantitative relationships that can be applied across a broad diversity of chemical, toxicological, and taxonomic space. Effective analysis of AOP networks may help reveal critical convergent nodes for which quantitative characterization of state-change conditions would provide the most broadly applicable improvements in AOP–based quantitative predictions. A final challenge to the AOP framework is that of regulatory acceptance. For the AOP framework to be broadly applied in regulatory (eco)toxicology, it is critical to establish predictive utility by developing AOP–derived hypotheses and testing them. An AOP is in essence a conceptual model based on biological understanding and experience. From it, a specific set of expectations regarding the response to a defined biological perturbation can be derived. Because the key events used to define an AOP are, by definition, measurable and causally related to one another, those expectations can be explicitly tested. In addition, by elucidating the cause(s) of significant deviations from expected results, AOPs can be refined toward greater predictive sophistication. Although we may never achieve perfect prediction of toxicological outcomes for all chemicals in all scenarios, the AOP framework is positioned to help us more effectively leverage new data, in the context of past experience and existing knowledge, to support regulatory decision making. The contents of this article neither constitute nor necessarily reflect official US Environmental Protection Agency policy. Daniel L. Villeneuve US Environmental Protection Agency Mid-Continent Ecology Division, Duluth, MN

The latest chemical management policies require toxicological evaluation of marketed but untested chemicals. Furthermore, in Europe, for animal welfare reasons sales of cosmetics and raw materials for which animal experiments were conducted were totally banned, in 2013. Responding to these regulatory trends, a strong demand exists to develop new in vitro test methods and to improve in silico prediction models for safety assessments. In recent years, the development of adverse outcome pathways (AOPs) has been actively promoted in the Organisation for Economic Co-operation and Development (OECD). Since it is difficult to replace a particular in vivo animal test with a single in vitro test method or in silico prediction model, integrated approaches to testing and assessment (IATA) have been studied based on AOP information. With regard to skin sensitization, several in vitro test methods that measure key events of AOP have been established, and integrated strategies using in vitro tests have been examined using AOP. Currently, numerous AOPs are under development for a wide range of complex toxicity endpoints in the OECD AOP program. The AOPs are expected to contribute to the development of many accurate in vitro test methods and to establish IATA as well as to evaluate safety in humans of many substances, including household chemicals, food-related chemicals, cosmetics, and pharmaceuticals.

Recent developments have prompted the transition of empirically based testing of late stage toxicity in animals for a range of different endpoints including neurotoxicity to more efficient and predictive mechanistically based approaches with greater emphasis on measurable key events early in the progression of disease. The adverse outcome pathway (AOP) has been proposed as a simplified organizational construct to contribute to this transition by linking molecular initiating events and earlier (more predictive) key events at lower levels of biological organization to disease outcomes. As such, AOPs are anticipated to facilitate the compilation of information to increase mechanistic understanding of pathophysiological pathways that are responsible for human disease.In this review, the sequence of key events resulting in adverse outcome (AO) defined as parkinsonian motor impairment and learning and memory deficit in children, triggered by exposure to environmental chemicals has been briefly described using the AOP framework. These AOPs follow convention adopted in an Organization for Economic Cooperation and Development (OECD) AOP development program, publically available, to permit tailored application of AOPs for a range of different purposes.Due to the complexity of disease pathways, including neurodegenerative disorders, a specific symptom of the disease (e.g. parkinsonian motor deficit) is considered as the AO in a developed AOP. Though the description is necessarily limited by the extent of current knowledge, additional characterization of involved pathways through description of related AOPs interlinked into networks for the same disease has potential to contribute to more holistic and mechanistic understanding of the pathophysiological pathways involved, possibly leading to the mechanism-based reclassification of diseases, thus facilitating more personalized treatment.

The Organisation for Economic Co-operation and Development (OECD) has initiated the adverse outcome pathway (AOP) Development Program in which the concept of AOP is applied to evaluate the safety of molecules such as chemicals. This program aims to assist regulatory needs and construct a knowledge base by accumulating AOP case studies. AOP consists of a molecular initiating event (MIE) as the initiating event of the pathway; key events (KEs) as the events themselves, such as cellular-molecular interactions; and adverse outcome (AO), such as signaling transduction-induced toxicity, as adverse events. KEs are extracted as important events at various levels, such as the molecular, cellular, tissue, organ, individual, and species levels; measurement of KEs and key event relationships (KERs), including mechanisms, plausibility, species differences, and empirical support information, are gathered. The development status of the AOP relating to histone deacetylase inhibition-induced testicular toxicity, currently being reviewed by the OECD, has been introduced. The AOP describing malignancies by Wnt ligand stimulation and Wnt signaling activation using gene expression network analysis-based mechanisms in molecular pathway elucidation has been suggested.