Pathway-based Toxicology

Advances in biological understanding as well as experimental technologies (e.g. ‘omics tools, stem cell culturing, reconstructed tissues), have allowed the contemplation of dramatically different approaches to understanding disease and toxicology than those traditionally practiced. One such approach couples existing knowledge of normal biology with new chemical and biological information about the consequences of disturbing that biology, leading to a structured, transparent, and hypothesis-based approach to predicting adverse outcomes resulting from those perturbations. This general approach has been variously termed Mode-of-Action (MoA), Toxicity Pathway, and Adverse Outcome Pathway (AOP) approaches.

The idea of incorporating mechanistic biochemical information into toxicological assessment is not new; it began with dose-response modeling efforts (e.g. Clewell et al., 1995) and mode-of-action frameworks, such as those developed by the International Program on Chemical Safety (IPCS) to determine the human relevance of modes-of-action of pesticides and industrial chemicals leading to carcinogenicity (and later non-carcinogenic toxicity)(Boobis, et al., 2006; Boobis, et al., 2008), and the creation of mode of action pathways commonly used in drug development and applied to human disease. The notion of toxicity pathways as articulated by the National Research Council in 2007 in its report, Toxicity Testing for the 21st Century: a vision and a Strategy, takes this concept a bit further by envisioning a system-wide network of pathways that leads to a predictive, hypothesis-driven assessment paradigm for toxicity in general. The goals of this new approach are to improve efficiency and decrease uncertainty in risk and hazard evaluations. Recently, this concept has been further formalized for toxicological assessment for both human health and ecological endpoints as the Adverse Outcome Pathway (AOP) and has been taken up by the Test Guidelines Program at the Organization for Economic Cooperation and Development (OECD) as an organizing principle for all test guidelines.

Mode-of-Action frameworks

The IPCS cancer and non-cancer MoA frameworks outline a systematic process of describing chemical MoA in animals and comparing those with likely MoA in humans to determine human relevance (Meek, et al., 2003) . Several founding principles of pathway-based approaches are articulated in the original IPCS frameworks: MoA defined as a series of key events along a biological pathway from the initial chemical interaction through to the toxicological outcome; a recognition that a MoA does not need to be complete to be useful and that its use depends on level of completeness (e.g., incomplete MoA can inform testing strategies but is likely not sufficient to support hazard classification); a focus on a single MoA at a time while recognizing that a chemical may have more than one MoA and that several modes may be related; definition of a “key event” as a step in the pathway that is critical to development of the toxicological outcome and is measurable; a requirement to systematically establish causation between key events; the importance of establishing quantitative parameters in order to apply the MoA to risk assessment; and the need to establish relevance to human biology. In this context, mode-of-action is distinguished from mechanism of action: the latter being defined as a more detailed description of the pathway that includes molecular interactions.

Establishing evidence for the MoA hypothesis is based on the Bradford-Hill criteria for establishing causation: strength of association, consistency of the evidence, specificity of the relationship, consistent temporal relationships, dose-response relationships, biological plausibility, coherence of the evidence, and consideration of alternative explanations (Bradford-Hill, 1965). The IPCS frameworks recommend determining human relevance by answering four key questions: 1) is there sufficient weight-of-evidence for the MoA in animals? 2) can human relevance be excluded on the basis of qualitative differences in key events? 3) can human relevance be excluded on the basis of quantitative differences in key events? and 4) do the quantitative differences affect the default uncertainty factors applied in risk assessment?

The MOA framework has been updated to accommodate insights from the expanding application of pathway-based approaches to risk assessment in general (Meek et al., 2014). In this framework, MoA and AOP are conceptually similar, with a distinction in that MoA does not necessarily imply adversity; it can also refer, for example, to efficacy of a drug.

Toxicity pathways and the NRC “Vision”

In its, by now, seminal 2007 report Toxicity Testing for the 21st Century: a Vision and a Strategy, the National Research Council panel describes a “transformative paradigm shift” that “envisions a new toxicity-testing system that evaluates biologically significant perturbations in key toxicity pathways by using new methods in computational biology and a comprehensive array of in vitro tests based on human biology” (NRC, 2007). In this context, a “toxicity pathway” is a normal biological pathway that becomes perturbed beyond the point of homeostatic correction leading to toxicity. A description of toxicity begins with chemical characterization, progresses through elucidation of the chemical interaction with the biological system (the pathway), involves targeted testing to query effects at critical steps of the pathway, and dose-response extrapolation to estimate human exposures required to elicit the effect. Additional population-based modeling is required to predict ecological effects. The level of assessment required depends on the risk context (e.g., will the information be used for prioritization or for risk assessment?).

The Adverse Outcome Pathway (AOP) approach

A similar approach, the AOP approach, arose from the field of ecotoxicology (primarily at the International QSAR Foundation and the Mid-Continent Ecology Division of the EPA in Duluth, MN) as a way of addressing uncertainty in risk assessment for an increasing number of chemicals and endpoints – and as required by new legislation .–. As described by Ankley et al., an AOP is a flexible framework that can include linking relationships that are “causal, mechanistic, inferential, or correlation based, and the information on which they are based may derive from in vitro, in vivo, or computational systems” and can encompass both mechanism and mode-of-action (Ankley, et al., 2010). An AOP describes the events that occur following chemical exposure, beginning with the molecular interaction of the chemical with a biomolecule (e.g., a protein, receptor, etc.) – the molecular initiating event (MIE) – followed by a description of the sequential cellular and tissue perturbations that lead to eventual toxicological effect – or adverse outcome – which is at the individual level for most human health endpoints or at the population level for environmental endpoints (Figure 1). The AOP framework allows for the integration of all types of information at different levels of biological organization, from molecular to population level, to provide a rational, biologically based argument (or series of hypotheses) to predict the outcome of an initiating event. In this description, the AOP builds on the MoA concepts and includes the “toxicity pathways” as described in the 2007 NRC report.

The AOP concept is useful in developing a predictive toxicological framework in several ways. In the near-term, even incomplete AOPs can inform chemical grouping or categories and structure activity relationships, they can aid in increasing certainty of interpretation of both existing and new information, and they can be used to inform integrated testing strategies that maximize useful information gained from minimal testing (OECD, 2013). In the longer-term, as they become more completely described and quantitative information is added to the links between steps, AOPs can be used to identify intermediate or key events for which non-animal tests can be developed, thereby facilitating transparent, mechanism-based, predictive toxicological assessments with low uncertainty and high human relevance, and ultimately without the use of animals. As AOPs are developed, it is important to clearly identify its completeness, as that will determine its potential use applicability.

Figure 1

The Organization for Economic Cooperation and Development (OECD) AOP Project

The OECD held a Workshop on Using Mechanistic Information in Forming Chemical Categories in 2010, one of the first workshops to gather scientific expertise to guide further AOP development (OECD 2011). The 2010 workshop focused on the use of AOPs in building chemical categories for read-across, how AOPs might be used in Approaches to Integrated Testing and Assessment (IATA) and in identifying Key Events. For the purposes of the Workshop, an AOP was defined as “a narrative which delineates the documented, plausible, and testable processes by which a chemical induces molecular perturbations and the associated biological responses which describe how the molecular perturbations cause effects at the subcellular, cellular, tissue, organ, whole animal and (if required) population levels of observation” (Table 1; OECD, 2011). Based on discussion of several case studies, AOPs were further defined as being based on a single, defined MIE that is linked to a specific in vivo hazard outcome. The workshop also discussed development of a template that would summarized all of the information supporting the AOP, including “1) the level of qualitative understanding of the AOP; 2) consistency of the experimental data; 3) confidence in the AOP, and 4) level of quantitative understanding of the AOP (OECD, 2011).

Since the 2010 workshop, OECD has published guidance and a template for development that is currently undergoing further revision (OECD, 2013). The published Guidance includes a description of the elements and uses of AOPs, a glossary of terms, and a template for developing an AOP. The goals of this guidance are to provide consistency in structure and facilitate harmonized use of AOPs.

According to the OECD Guidance, an AOP consists of three main elements: one molecular initiating event (Anchor 1), one Adverse Outcome (Anchor 2), and any number of intermediate events. While in reality an MIE can be associated with a number of adverse effects, and similarly an adverse effect can result from a number of different MIE, to streamline the development and use of AOPs, OECD has defined an AOP as being a linear pathway from one MIE to one adverse outcome. A full description of an MIE should include cellular/tissue location – as immediately elicited intermediate events may be similar in two different AOPs, but differ in cell-type or tissue location (for example, metabolic transformation of a chemical to an electrophilic species may occur in both skin sensitization and liver fibrosis – only in keratinocytes for the former and hepatocytes for the latter). In order for an intermediate event to be identified as a “key event,” it must be able to be evaluated experimentally and causally linked to the adverse outcome. A key event may be shared between two or more AOPs. The Guidance describes the process for evaluating weight-of-evidence for each step in development of an AOP.

Similar to the IPCS MoA frameworks described above, the OECD guidance suggests evaluation following the Bradford-Hill principles. In addition, the guidance recommends evaluating the confidence in each element of the AOP, specifically to address the following: how well characterized are the MIE, adverse outcome, and each intermediate or key event? What are the limitations of the evidence used to support the AOP? What are the specific parameters of the AOP (e.g., life stage, tissue, etc.)? To what degree are the AOP elements conserved across species?

AOPs have a number of potential uses, including 1) supporting chemical category formation and “read-across” (predicting the toxicity of one chemical based on results from a related chemical), 2) priority setting for further testing, 3) hazard identification 4) classification and labeling, and 5) risk assessment. As use progresses from 1 – 5, a corresponding increase in the level of evidence and certainty is necessary for adequate confidence. An AOP can also form the basis for an integrated approach to testing and assessment (IATA) or an integrated testing strategy (ITS) that would be designed to increase the certainty of any decision made regarding a particular substance (OECD, 2013).

There are currently more than 20 AOP projects in the OECD work plan, including pathways and case studies (an AOP that is related to one specific chemical).   In addition, OECD is collaborating with the European Commission Joint Research Centre and the US EPA to develop the AOP Knowledge base (AOP KB; described in more detail in the next section).

The AOP Knowledge-Base and related projects

To develop a system of AOPs that covers the broad spectrum of biological pathways that are likely to be involved in human health and ecological risk assessment, it is necessary to create a unified knowledge base for AOPs and their supporting evidence that integrates information from all scientific sectors, including toxicology, drug development, disease, medicine and research. An Adverse Outcome Pathway Knowledge Base (AOP KB) is currently being developed by the OECD Extended Advisory Group on Molecular Screening and Toxicogenomics (EAG MST) and is being implemented by the European Commission’s Joint Research Centre (JRC) and the US Environmental Protection Agency (US-EPA).2, , , ,   The AOP KB is an IT system to capture, manage and share AOP information and will consist of three modules: 1) AOP-WIKI, a text-based tool allowing the management of AOP-related knowledge (AOPs, Key Events, relationships between them) in a Wikipedia-like environment, 2) a graphical tool implementing quantitative models depicting the relationship between two events in an AOP (Effectopedia) and 3) an Intermediate Effects Database (AOP Network tool) to manage information about intermediate effects triggered by chemicals.

The first available module, the AOP Wiki, leads AOP developers through the steps necessary to capture the scientific information needed to document an AOP – following and implementing OECD guidance on how to describe AOPs. The Wiki also provides a collaborative space for groups to develop AOPs independent of geography or organizational boundaries. Currently a beta version of the AOP Wiki is available only for OECD AOP development teams but the goal is to eventually make this publicly accessible with registration.

(Note: this section is based on excerpts from the following: Willett, C.2014. Adverse Outcome Pathways: Development and Use in Toxicology. In: Wexler, P. (Ed.), Encyclopedia of Toxicology, 3rd edition vol 1. Elsevier Inc., Academic Press, pp. 95–99, and Willett, et al. 2014. Pathway-Based Toxicity: History, Current Approaches and Liver Fibrosis and Steatosis as Prototypes. ALTEX Online first, published June 23, 2014, http://www.altex.ch/resources/epub_Willett_1400623f1.pdf.)

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