Procedural application of mode-of-action and human relevance analysis: styrene-induced lung tumors in mice.

Risk assessment of human health hazards has traditionally relied on experiments that use animal models. Although exposure studies in rats and mice are a major basis for determining risk in many cases, observations made in animals do not always reflect health hazards in humans due to differences in biology. In this critical review, we use the mode-of-action (MOA) human relevance framework to assess the likelihood that bronchiolar lung tumors observed in mice chronically exposed to styrene represent a plausible tumor risk in humans. Using available datasets, we analyze the weight-of-evidence 1) that styrene-induced tumors in mice occur through a MOA based on metabolism of styrene by Cyp2F2; and 2) whether the hypothesized key event relationships are likely to occur in humans. This assessment describes how the five modified Hill causality considerations support that a Cyp2F2-dependent MOA causing lung tumors is active in mice, but only results in tumorigenicity in susceptible strains. Comparison of the key event relationships assessed in the mouse was compared to an analogous MOA hypothesis staged in the human lung. While some biological concordance was recognized between key events in mice and humans, the MOA as hypothesized in the mouse appears unlikely in humans due to quantitative differences in the metabolic capacity of the airways and qualitative uncertainties in the toxicological and prognostic concordance of pre-neoplastic and neoplastic lesions arising in either species. This analysis serves as a rigorous demonstration of the framework's utility in increasing transparency and consistency in evidence-based assessment of MOA hypotheses in toxicological models and determining relevance to human health.

The need for good practice in the application of mechanistic constructs in hazard and risk assessment.

A series of recent and proposed workshops address the interface between key characteristics and mechanistic pathway descriptions (adverse outcome pathways and mode of action) to identify commonalities and potential for complementary application. Informed by different communities, these constructs have collective potential to increase confidence to support the application of mechanistic data in hazard assessment. This forum article summarizes concepts, introduces evolving understanding, and invites future collaboration to contribute to better common understanding and development of good practice in the use of mechanistic data in hazard assessment.

A user-friendly tool to assess combined exposures to indoor air pollutants in public spaces of children.

This manuscript describes the methodology for and early experience in the application of a screening tool to assess health risks from combined exposure to indoor air pollutants in public settings for children such as schools, kindergartens and day-care centres. The user-friendly tool incorporates tiers modified from those of the World Health Organization (WHO) framework for risk assessment of combined exposure to multiple chemicals and includes a spreadsheet for risk calculation as well as a supporting toxicological database of guidance values and points of departure (PODs) for inhalation for selected effects. Supporting resources on exposure assessment include a screening questionnaire to identify optimum sampling strategies and standardized analytical methods. The approach to assessment of combined exposure within the screening tool, including decision rules, assumptions and limitations/uncertainties is addressed, as is the nature of health-effects and reference/toxicity values prioritized for inclusion in the associated toxicological database. Results of early experience in application illustrate how the screening tool contributes as an important component in strategies to assess and manage indoor air pollution in public settings for children.

Towards a qAOP framework for predictive toxicology - Linking data to decisions.

The adverse outcome pathway (AOP) is a conceptual construct that facilitates organisation and interpretation of mechanistic data representing multiple biological levels and deriving from a range of methodological approaches including in silico, in vitro and in vivo assays. AOPs are playing an increasingly important role in the chemical safety assessment paradigm and quantification of AOPs is an important step towards a more reliable prediction of chemically induced adverse effects. Modelling methodologies require the identification, extraction and use of reliable data and information to support the inclusion of quantitative considerations in AOP development. An extensive and growing range of digital resources are available to support the modelling of quantitative AOPs, providing a wide range of information, but also requiring guidance for their practical application. A framework for qAOP development is proposed based on feedback from a group of experts and three qAOP case studies. The proposed framework provides a harmonised approach for both regulators and scientists working in this area.

Evolution of chemical-specific adjustment factors (CSAF) based on recent international experience; increasing utility and facilitating regulatory acceptance.

The application of chemical-specific toxicokinetic or toxicodynamic data to address interspecies differences and human variability in the quantification of hazard has potential to reduce uncertainty and better characterize variability compared with the use of traditional default or categorically-based uncertainty factors. The present review summarizes the state-of-the-science since the introduction of the World Health Organization/International Programme on Chemical Safety (WHO/IPCS) guidance on chemical-specific adjustment factors (CSAF) in 2005 and the availability of recent applicable guidance including the WHO/IPCS guidance on physiologically-based pharmacokinetic (PBPK) modeling in 2010 as well as the U.S. EPA guidance on data-derived extrapolation factors in 2014. A summary of lessons learned from an analysis of more than 100 case studies from global regulators or published literature illustrates the utility and evolution of CSAF in regulatory decisions. Challenges in CSAF development related to the adequacy of, or confidence in, the supporting data, including verification or validation of PBPK models. The analysis also identified issues related to adequacy of CSAF documentation, such as inconsistent terminology and often limited and/or inconsistent reporting, of both supporting data and/or risk assessment context. Based on this analysis, recommendations for standardized terminology, documentation and relevant interdisciplinary research and engagement are included to facilitate the continuing evolution of CSAF development and guidance.

Adverse outcome pathways: Application to enhance mechanistic understanding of neurotoxicity.

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.

Developing and applying the adverse outcome pathway concept for understanding and predicting neurotoxicity.

The Adverse Outcome Pathway (AOP) concept has recently been proposed to support a paradigm shift in regulatory toxicology testing and risk assessment. This concept is similar to the Mode of Action (MOA), in that it describes a sequence of measurable key events triggered by a molecular initiating event in which a stressor interacts with a biological target. The resulting cascade of key events includes molecular, cellular, structural and functional changes in biological systems, resulting in a measurable adverse outcome. Thereby, an AOP ideally provides information relevant to chemical structure-activity relationships as a basis for predicting effects of structurally similar compounds. AOPs could potentially also form the basis for qualitative and quantitative predictive modeling of the human adverse outcome resulting from molecular initiating or other key events for which higher-throughput testing methods are available or can be developed. A variety of cellular and molecular processes are known to be critical for normal function of the central (CNS) and peripheral nervous systems (PNS). Because of the biological and functional complexity of the CNS and PNS, it has been challenging to establish causative links and quantitative relationships between key events that comprise the pathways leading from chemical exposure to an adverse outcome in the nervous system. Following introduction of the principles of MOA and AOPs, examples of potential or putative adverse outcome pathways specific for developmental or adult neurotoxicity are summarized and aspects of their assessment considered. Their possible application in developing mechanistically informed Integrated Approaches to Testing and Assessment (IATA) is also discussed.

How Adverse Outcome Pathways Can Aid the Development and Use of Computational Prediction Models for Regulatory Toxicology.

Efforts are underway to transform regulatory toxicology and chemical safety assessment from a largely empirical science based on direct observation of apical toxicity outcomes in whole organism toxicity tests to a predictive one in which outcomes and risk are inferred from accumulated mechanistic understanding. The adverse outcome pathway (AOP) framework provides a systematic approach for organizing knowledge that may support such inference. Likewise, computational models of biological systems at various scales provide another means and platform to integrate current biological understanding to facilitate inference and extrapolation. We argue that the systematic organization of knowledge into AOP frameworks can inform and help direct the design and development of computational prediction models that can further enhance the utility of mechanistic and in silico data for chemical safety assessment. This concept was explored as part of a workshop on AOP-Informed Predictive Modeling Approaches for Regulatory Toxicology held September 24-25, 2015. Examples of AOP-informed model development and its application to the assessment of chemicals for skin sensitization and multiple modes of endocrine disruption are provided. The role of problem formulation, not only as a critical phase of risk assessment, but also as guide for both AOP and complementary model development is described. Finally, a proposal for actively engaging the modeling community in AOP-informed computational model development is made. The contents serve as a vision for how AOPs can be leveraged to facilitate development of computational prediction models needed to support the next generation of chemical safety assessment.

Framework for human health risk assessment of non-cancer effects resulting from short-duration and intermittent exposures to chemicals.

Durations of exposure to chemicals, whether for single, repeated or intermittent periods, may vary from those upon which most guidance values are normally based. Because it is presently not feasible to conduct toxicity studies or develop toxicity reference values (TRVs) specific to each scenario of interest, methods are needed to address these various durations, drawing as much as possible on existing TRVs. A working framework was developed to address the potential for non-cancer effects resulting from continuous short-duration and intermittent exposures to chemicals. The framework presents an integrated, tiered approach that assists the user in identifying when existing TRVs can be applied directly, and the adaptations needed to assess the acceptability of short-duration or intermittent exposure scenarios. Descriptions of when and how toxicokinetic and toxicodynamic aspects need to be taken into consideration are also presented. The framework incorporates the use of TRVs based on exposure periods as similar as possible to the "actual" exposure periods and application of dose averaging under limited, specified conditions. This framework has been developed to aid in improving the scientific basis for the evaluation of short-duration and intermittent exposures in a variety of settings. Copyright © 2016 John Wiley & Sons, Ltd.

Development of the adverse outcome pathway "alkylation of DNA in male premeiotic germ cells leading to heritable mutations" using the OECD's users' handbook supplement.

The Organisation for Economic Cooperation and Development's (OECD) Adverse Outcome Pathway (AOP) programme aims to develop a knowledgebase of all known pathways of toxicity that lead to adverse effects in humans and ecosystems. A Users' Handbook was recently released to provide supplementary guidance on AOP development. This article describes one AOP-alkylation of DNA in male premeiotic germ cells leading to heritable mutations. This outcome is an important regulatory endpoint. The AOP describes the biological plausibility and empirical evidence supporting that compounds capable of alkylating DNA cause germ cell mutations and subsequent mutations in the offspring of exposed males. Alkyl adducts are subject to DNA repair; however, at high doses the repair machinery becomes saturated. Lack of repair leads to replication of alkylated DNA and ensuing mutations in male premeiotic germ cells. Mutations that do not impair spermatogenesis persist and eventually are present in mature sperm. Thus, the mutations are transmitted to the offspring. Although there are some gaps in empirical support and evidence for essentiality of the key events for certain aspects of this AOP, the overall AOP is generally accepted as dogma and applies broadly to any species that produces sperm. The AOP was developed and used in an iterative process to test and refine the Users' Handbook, and is one of the first publicly available AOPs. It is our hope that this AOP will be leveraged to develop other AOPs in this field to advance method development, computational models to predict germ cell effects, and integrated testing strategies.

Gaining acceptance for the use of in vitro toxicity assays and QIVIVE in regulatory risk assessment.

Testing strategies are anticipated to increasingly rely on in vitro data as a basis to characterize early steps or key events in toxicity at relevant dose levels in human tissues. Such strategies require quantitative in vitro to in vivo extrapolation to characterize dose-response as a basis for comparison with exposure to estimate risk. Current experience in the incorporation of mechanistic and in vitro data in risk assessment is considered here in the context of identified principles to increase the potential for timely acceptance of more progressive and tailored testing strategies by the regulatory community. These principles are outlined as transitioning in a familiar context, tiering to acquire experience and increase confidence, contextual knowledge transfer to facilitate interpretation and communication, coordination and development of expertise and continuing challenge. A proposed pragmatic tiered data driven framework which includes increasing reliance on in vitro data and quantitative in vitro to in vivo extrapolation is considered in the context of these principles. Based on this analysis, possible additional steps that might facilitate timely evolution and potentially, uptake are identified.

Application of benchmark dose modeling to protein expression data in the development and analysis of mode of action/adverse outcome pathways for testicular toxicity.

Reliable quantification of gene and protein expression has potential to contribute significantly to the characterization of hypothesized modes of action (MOA) or adverse outcome pathways for critical effects of toxicants. Quantitative analysis of gene expression by benchmark dose (BMD) modeling has been facilitated by the development of effective software tools. In contrast, protein expression is still generally quantified by a less robust effect level (no or lowest [adverse] effect levels) approach, which minimizes its potential utility in the consideration of dose-response and temporal concordance for key events in hypothesized MOAs. BMD modeling is applied here to toxicological data on testicular toxicity to investigate its potential utility in analyzing protein expression relevant to the proposed MOA to inform human health risk assessment. The results illustrate how the BMD analysis of protein expression in animal tissues in response to toxicant exposure: (1) complements other toxicity data, and (2) contributes to consideration of the empirical concordance of dose-response relationships, as part of the weight of evidence for hypothesized MOAs to facilitate consideration and application in regulatory risk assessment. Lack of BMD analysis in proteomics has likely limited its use for these purposes. This paper illustrates the added value of BMD modeling to support and strengthen hypothetical MOAs as a basis to facilitate the translation and uptake of the results of proteomic research into risk assessment.

Mode of action human relevance (species concordance) framework: Evolution of the Bradford Hill considerations and comparative analysis of weight of evidence.

The mode of action human relevance (MOA/HR) framework increases transparency in systematically considering data on MOA for end (adverse) effects and their relevance to humans. This framework continues to evolve as experience increases in its application. Though the MOA/HR framework is not designed to address the question of "how much information is enough" to support a hypothesized MOA in animals or its relevance to humans, its organizing construct has potential value in considering relative weight of evidence (WOE) among different cases and hypothesized MOA(s). This context is explored based on MOA analyses in published assessments to illustrate the relative extent of supporting data and their implications for dose-response analysis and involved comparisons for chemical assessments on trichloropropane, and carbon tetrachloride with several hypothesized MOA(s) for cancer. The WOE for each hypothesized MOA was summarized in narrative tables based on comparison and contrast of the extent and nature of the supporting database versus potentially inconsistent or missing information. The comparison was based on evolved Bradford Hill considerations rank ordered to reflect their relative contribution to WOE determinations of MOA taking into account increasing experience in their application internationally. This clarification of considerations for WOE determinations as a basis for comparative analysis is anticipated to contribute to increasing consistency in the application of MOA/HR analysis and potentially, transparency in separating science judgment from public policy considerations in regulatory risk assessment.

Incorporating new technologies into toxicity testing and risk assessment: moving from 21st century vision to a data-driven framework.

Based on existing data and previous work, a series of studies is proposed as a basis toward a pragmatic early step in transforming toxicity testing. These studies were assembled into a data-driven framework that invokes successive tiers of testing with margin of exposure (MOE) as the primary metric. The first tier of the framework integrates data from high-throughput in vitro assays, in vitro-to-in vivo extrapolation (IVIVE) pharmacokinetic modeling, and exposure modeling. The in vitro assays are used to separate chemicals based on their relative selectivity in interacting with biological targets and identify the concentration at which these interactions occur. The IVIVE modeling converts in vitro concentrations into external dose for calculation of the point of departure (POD) and comparisons to human exposure estimates to yield a MOE. The second tier involves short-term in vivo studies, expanded pharmacokinetic evaluations, and refined human exposure estimates. The results from the second tier studies provide more accurate estimates of the POD and the MOE. The third tier contains the traditional animal studies currently used to assess chemical safety. In each tier, the POD for selective chemicals is based primarily on endpoints associated with a proposed mode of action, whereas the POD for nonselective chemicals is based on potential biological perturbation. Based on the MOE, a significant percentage of chemicals evaluated in the first 2 tiers could be eliminated from further testing. The framework provides a risk-based and animal-sparing approach to evaluate chemical safety, drawing broadly from previous experience but incorporating technological advances to increase efficiency.

Case study illustrating the WHO IPCS guidance on characterization and application of physiologically based pharmacokinetic models in risk assessment.

The World Health Organization (WHO) International Programme on Chemical Safety (IPCS) Guidance on Characterization and Application of Physiologically Based Pharmacokinetic Models in Risk Assessment (IPCS, 2010) describes key principles for risk assessors and model developers. In the WHO Guidance, a template for model documentation was developed and a case study included. Here the WHO Guidance, including the template, is summarized and an additional case study is presented to illustrate its application, based upon an existing risk assessment for 2-butoxyethanol (CAS NO. 111-76-2). The goal of the WHO Guidance and the current paper is to increase regulatory acceptance of complex biologically descriptive pharmacokinetic (or toxicokinetic) models, such as PBPK models, by facilitating communication and successful interaction between modelers and risk assessors.

International experience in addressing combined exposures: increasing the efficiency of assessment.

More efficient methodology for assessing the impact of combined exposures to multiple chemicals has been considered in a project of the World Health Organization (WHO) International Programme on Chemical Safety (IPCS). Recommendations regarding terminology and the status of development of the framework, its content, review and application are described. Evolving experience in its application is illustrated by example (polybrominated diphenyls) with special emphasis on the critical content of problem formulation, the role of predictive tools in grouping of chemicals for consideration and the importance of explicit delineation of relative uncertainty and sensitivity for tiered assessment. Priorities in increasing the efficiency of risk assessment not only for combined exposures, but more generally based on experience acquired in developing the framework and its application in case studies are identified and recommendations included.

Risk assessment of combined exposure to multiple chemicals: A WHO/IPCS framework.

This paper describes a framework for the risk assessment of combined exposure to multiple chemicals based on and developed subsequent to the World Health Organization/International Programme on Chemical Safety Workshop on Aggregate/Cumulative Risk Assessment (Combined Exposures to Multiple Chemicals) held in 2007. The framework is designed to aid risk assessors in identifying priorities for risk management for a wide range of applications where co-exposures to multiple chemicals are expected. It is based on a hierarchical (phased) approach that involves integrated and iterative consideration of exposure and hazard at all phases, with each tier being more refined (i.e., less cautious and more certain) than the previous one, but more labor and data intensive. It includes reference to predictive and probabilistic methodology in various tiers in addition to tiered consideration of uncertainty. The paper also annexes two case studies that have been developed to test and refine the framework.

Proposed mode of action of benzene-induced leukemia: Interpreting available data and identifying critical data gaps for risk assessment.

Mode of action is defined as a series of key biological events leading to an observed toxicological effect (for example, metabolism to a toxic entity, cell death, regenerative repair and tumors). It contrasts with mechanism of action, which generally involves a detailed understanding of the molecular basis for an effect. A framework to consider the weight of evidence for hypothesized modes of action in animals and their relevance to humans, has been widely adopted and used by government agencies and international organizations. The framework, developed and refined through its application in case studies for principally non-DNA-reactive carcinogens, has more recently been extended to DNA-reactive carcinogens, non-cancer endpoints and different life stages. In addition to increasing transparency, use of the framework promotes consistency in decision-making concerning adequacy of weight of evidence, facilitates peer input and review and identifies critical research needs. The framework provides an effective tool to facilitate discussion between the research and risk assessment communities on critical data gaps, which if filled, would permit more refined estimates of risk. As a basis for additionally coordinating and focusing research on critical data gaps in a risk assessment context, five key events in the mode of action for benzene-induced leukemia are proposed: (1) benzene metabolism via Cytochrome P450, (2) the interaction of benzene metabolites with target cells in the bone marrow, (3) formation of initiated, mutated target cells, (4) selective proliferation of the mutated cells and (5) production of leukemia. These key events are considered in a framework analysis of human relevance as a basis to consider appropriate next steps in developing research strategies.

IPCS framework for analyzing the relevance of a noncancer mode of action for humans.

Structured frameworks are extremely useful in promoting transparent, harmonized approaches to the risk assessment of chemicals. One area where this has been particularly successful is in the analysis of modes of action (MOAs) for chemical carcinogens in experimental animals and their relevance to humans. The International Programme on Chemical Safety (IPCS) recently published an updated version of its MOA framework in animals to address human relevance (cancer human relevance framework, or HRF). This work has now been extended to noncancer effects, with the eventual objective of harmonizing framework approaches to both cancer and noncancer endpoints. As in the cancer HRF, the first step is to determine whether the weight of evidence based on experimental observations is sufficient to establish a hypothesized MOA. This comprises a series of key events causally related to the toxic effect, identified using an approach based on the Bradford Hill criteria. These events are then compared qualitatively and, next, quantitatively between experimental animals and humans. The output of the analysis is a clear statement of conclusions, together with the confidence, analysis, and implications of the findings. This framework provides a means of ensuring a transparent evaluation of the data, identification of key data gaps and of information that would be of value in the further risk assessment of the compound, such as on dose-response relationships, and recognition of potentially susceptible subgroups, for example, based on life-stage considerations.

Recent developments in frameworks to consider human relevance of hypothesized modes of action for tumours in animals.

This paper summarizes recent developments in the continuing evolution of Human Relevance Frameworks to systematically consider the weight of evidence of hypothesized modes of action in animals and their potential human relevance for both cancer and non-cancer effects. These frameworks have been developed in initiatives of the International Life Sciences Institute Risk Sciences Institute and the International Programme on Chemical Safety engaging large numbers of scientists internationally. They are analytical tools designed to organize information in hazard characterization as a basis to clarify the extent of the weight of evidence for mode of action in animals and human relevance and subsequent implications for dose-response. They are also extremely helpful in identifying critical data gaps. These frameworks which are illustrated by an increasing number of case studies, have been widely adopted into international and national guidance and assessments and continue to evolve, as experience increases in their application.