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

Toxicology 332 (2015) 112–123

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Gaining acceptance for the use of in vitro toxicity assays and QIVIVE in regulatory risk assessment M.E. (Bette) Meek a, * , John C. Lipscomb b a Chemical Risk Assessment, McLaughlin Centre for Population Health Risk Assessment, Institute of Population Health, University of Ottawa, Ottawa, ON K1H 8M5, Canada b United States Environmental Protection Agency, Office of Research and Development, National Center for Environmental Assessment, Cincinnati, OH 45268, USA

A R T I C L E I N F O

A B S T R A C T

Article history: Received 7 August 2014 Received in revised form 31 December 2014 Accepted 14 January 2015 Available online 15 January 2015

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. ã 2015 Elsevier Ireland Ltd. All rights reserved.

Keywords: Tiered approach In vitro Risk assessment Extrapolation Dose–response Mode of action Strategy

1. Introduction There is growing recognition of the need for more efficient methods and strategies to assess the hazards, exposures and risks of the wide array of chemicals to which humans are exposed. This has been reflected in, for example, progressive regulatory mandates in Canada, the European Union and, more recently, the Asian Pacific region to systematically consider priorities for risk management from among all existing chemicals (see, for example, Council of Labor Affairs, Taiwan, 2012; Dellarco et al., 2010; European Commission, 2006; Hughes et al., 2009; Lowell Center for Sustainable Production, 2012; Meek and Armstrong, 2007). To meet this objective, there is need to transition toxicity testing and assessment to be more efficient, economical, less animal intensive, and more relevant to human health by utilizing new technologies that have potential to provide better understanding of the underlying biological system (NRC, 2007). Prerequisites for such transition include not only the development of novel methodologies but also early and continuing coordination

* Corresponding author. Tel.: +1 613 276-4134. E-mail addresses: [email protected] (M.E. (. Meek), [email protected] (J.C. Lipscomb). http://dx.doi.org/10.1016/j.tox.2015.01.010 0300-483X/ ã 2015 Elsevier Ireland Ltd. All rights reserved.

among a range of relevant communities (in particular, the regulatory and research communities). This in turn, necessitates effective knowledge dissemination and communication. Envisaged testing strategies are anticipated to increasingly rely on in vitro data as a basis to characterize early key events for toxicity at relevant dose levels in species of interest and quantitative in vitro to in vivo extrapolations. The considerable difficulties faced by the scientific and regulatory communities in developing alternative methodologies in risk assessment in a timely fashion have been previously reported in response to the 7th amendment to the EU Cosmetics Directive which prohibits animal-tested cosmetics in Europe after 2013 (Adler et al., 2011). Identified principles in increasing the potential of timely acceptance of these evolving strategies by the regulatory community are addressed here, based on experience in a number of national and international initiatives. 2. Reliance on in vitro mechanistic data in regulatory risk assessment Risk assessment i.e., the characterization of the potential adverse effects of human exposures, is the requisite basis for the development and implementation of control measures that are

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protective of public health (i.e., risk management). Traditionally, risk assessment has been considered to be composed of four different elements: hazard identification (i.e., the intrinsic capability of a chemical to do harm), dose–response assessment, exposure estimation and risk characterization. Hazard identification has been based principally on the consideration of toxicological studies on individual endpoints in experimental animals conducted at relatively high doses (e.g., cancer, reproductive and developmental effects, etc.) requiring dose and species extrapolations as a basis for comparison with exposure to estimate risk for humans. Earlier focus to include more mechanistically based approaches in which early key events on the path to toxicity are examined in human tissues in vitro at more realistic doses has potential to increase predictivity, while decreasing costs and uncertainty. This necessitates development and acceptance for regulatory application of in vitro testing in human tissues and quantitative in vitro to in vivo extrapolation for dose–response analysis. It also requires evolution of current regulatory risk assessment approaches for characterization of hazard and dose–response. These approaches have been traditionally based on division of a point of departure (e.g., no-or-lowest observed (adverse) effect levels or benchmark doses) for late stage adverse effects in animals exposed to (often) two or three relatively high doses by (often default) uncertainty factors to address aspects such as interspecies differences and human variability. Alternatively, these points of departure are extrapolated through the origin as a basis to estimate probability of harm (i.e., low dose linear extrapolation often adopted for cancer risk). There have been a number of developments which have contributed to the evolution and increasing acceptance of mechanistically informed approaches in regulatory risk assessment (including reliance on in vitro data). These developments include accepted guidance for the assessment of hypothesized modes of action and chemical specific adjustments in place of default (see, for example, Health Canada, 1994; IPCS, 2005; US EPA, 2005, 2014). As reviewed here, this has led to the incorporation in a number of regulatory risk assessments, of more mechanistically based approaches to dose–response analysis and reliance on chemical specific toxicokinetic and toxicodynamic data (often from in vitro studies) as a basis to reduce uncertainties in extrapolations across and within species (e.g., to identify susceptible subgroups) and doses. To some degree, this has led to increasing acceptance in regulatory risk assessment of in vitro mechanistic based testing in recognition of advantages over in vivo tests, which include the extent to which both the concentration and chemical nature of exposure can be well controlled and the capability to isolate specific biological targets within the human system. However, still for the most part, there is continued reliance principally on default models for dose–response extrapolation which is attributable in part to the lack of mechanistic data focused on specific regulatory risk assessment needs. Even in cases where there are considerable robust mechanistic data to inform quantitative risk assessment, the information is often not used in regulatory application. This is likely a function of several contributing factors including familiarity leading to perhaps misplaced or greater than is warranted acceptance of default approaches for dose–response analysis, regulatory pressures to conduct assessments in very limited timeframes or lack of understanding due to a shortage of interdisciplinary consultation of risk assessors (who commonly have backgrounds principally in toxicology), modelers and those that conduct mechanistic investigations. It can also be related to a lack of transparency in the separation of science judgment from science policy choices (i.e., with default often being considered to be more public health

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protective, though this is not always true based on experience with more mechanistically based approaches). Experience in evolution and adoption of some of these more mechanistically based approaches involving reliance on in vitro data is considered here as a basis to identify “principles” to facilitate their increasing acceptance. This includes the development of frameworks for assessment of mode of action/adverse outcome pathways, chemical specific adjustment factors, physiologically based pharmacokinetic modeling and tiered assessment strategies. 3. The principles Relevant experience is presented here in the context of 5 principles to encourage and facilitate regulatory acceptance of novel methodologies based on in vitro data, identified in summary form as: (1) transitioning in a familiar context, (2) tiering to acquire experience and increase confidence, (3) contextual knowledge transfer to facilitate interpretation and communication in application, (4) coordination and development of expertise and (5) challenge. 3.1. Transitioning in a familiar context The first principle involves stepwise development of a pragmatic path forward that can be iteratively refined as greater understanding is achieved. This builds on past progress in the evolution of regulatory assessment which has resulted from progressive introduction of novel approaches within familiar constructs as a basis to facilitate increased understanding and acceptance. The evolution of chemical specific adjustment factors (referenced as data derived extrapolation factors by US EPA), provides a relevant example (IPCS, 2005; US EPA, 2014). This approach built on the traditional construct for dose– response analysis of dividing a point of departure by uncertainty factors but contributed to advancement of uncertainty factor quantitation based on: (1) the understanding that it is the concentration of the toxicologically active chemical species in the target tissue that produces the response, (2) that toxicokinetic differences between and among species may alter the ratio of applied dose to target tissue concentrations, and (3) that toxicodynamic differences between and among species may alter the level of a given response at a given target tissue concentration of toxicant. Building on previous work conducted principally within the research community (Renwick, 1993), international guidance (IPCS, 2005) was developed concerning the quantity and nature of kinetic and dynamic data considered appropriate for replacement of default uncertainty factors with chemical-specific quantitative values. This guidance on chemical specific adjustment factors (CSAF) was developed and refined through a series of planning and technical meetings and larger workshops. These activities included a broad range of participants representing both technical scientists and risk assessors from academia, government agencies and the private sector and the principles have more recently, been widely incorporated in guidance of regulatory agencies worldwide (Health Canada, 1994; US EPA, 2014; European Commission, 2003). These developments were consistent with but extended concepts introduced by the US EPA in methodology for dosimetric adjustments for development of inhalation reference concentrations based on categorical defaults (i.e., introducing several categories of defaults to address the toxicokinetic subcomponent of interspecies differences and human variability based on chemical characteristics) (US EPA, 1994; Jarabek, 1994). Objectives of the CSAF guidance included encouraging the generation and incorporation of relevant quantitative kinetic and

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dynamic data in a context consistent with traditional approaches to development of measures of dose/concentration – response. As such, the guidance is relevant not only to risk assessors, but also to those who commission, design or conduct relevant studies as a basis for informing risk assessment (IPCS, 2005; Meek, 2001; Meek et al., 2002a,b; Gundert-Remy and Sonich-Mullin, 2002). Consistency with traditional approaches relates to subdividing the existing default values (i.e., ten-fold) for uncertainty factors for interspecies differences and human variability into subcomponents for toxicokinetic or toxicodynamic aspects to permit the application of chemical specific (partial) data. In the absence of adequate, quantitatively valuable data, the approach collapses to the currently well accepted (though not necessarily well justified) defaults. This does not reflect confidence in these pre-existing default (ten-fold) values, per se, but rather, is justified on the basis that it facilitates transition to more data informed approaches by increasing common understanding of the nature of more relevant and predictive data. For example, for kinetic parameters, for simple cases, substitution of default values can involve comparison between mean values for kinetic parameters such as area under the plasma concentration time curve or clearance for the active entity (i.e., parent compound or toxic metabolite) in humans and animals to address interspecies differences. For human variability, data requirements are more demanding, since population distributions for the relevant parameters must be characterized, but are often based on physiologically based pharmacokinetic models incorporating in vitro data on metabolic parameters (IPCS, 2005). While in vitro data cannot fully provide an understanding of in vivo toxicokinetics, in the context of existing frameworks, then, they contribute to characterization of both interspecies differences and human variability (US EPA, 2004; Health Canada, 2001). For example, determination of chemical-specific aspects such as tissue solubility (i.e., partition coefficients), binding, and metabolism in humans and laboratory animals is often based on in vitro studies, the results of which serve as input to physiologically based pharmacokinetic (PBPK) models to address interspecies differences or human variability between applied and internal dose (toxicokinetic aspects). In vitro data are considered to represent a complete enough stand-alone understanding of toxicodynamic differences between test species and humans to serve as the basis for nondefault uncertainty factor values (US EPA, 2010a,b; Health Canada, 2003). Interspecies differences are based on quantitative comparison of the concentrations which cause an effect of defined magnitude in human and animal tissues and for human variability, data could be derived from in vitro studies in tissue of critical effect from average versus sensitive humans. A number of important considerations concerning the adequacy of relevant in vitro data for this purpose have been identified (see, for example, IPCS, 2005; US EPA, 2014). Examples of requlatory assessments in which such data have been applied to develop chemical specific adjustment factors include chloroform (http://www.hc-sc.gc.ca/ewh-semt/pubs/contaminants/psl2-lsp2/chloroform/index-eng.php), boron (http:// www.epa.gov/iris/subst/0410.htm) and 2-butoxyethanol or EGBE. For example, for 2-butoxyethanol, differences in erythrocyte responsiveness have been used to quantify animal to human differences in toxicodynamics (Health Canada, 2003; US EPA, 2010a,b).

3.2. Tiering to acquire experience and increase confidence. Tiered strategies for assessment and associated frameworks provide a unifying construct for transparency for increasingly data informed, value-of-information-driven, “fit for purpose” priority setting and assessment. Normally, this involves greater reliance on

in vitro data at lower tiers progressing to more labor intensive and costly strategies including in vivo data at higher tiers. These strategies provide framing for coordination of tool development and contextual consideration of the likely value of information, based on collective experience. Frameworks for consideration of information on mode of action/adverse outcome pathways and combined exposures provide relevant examples.

3.2.1. Mode of action/adverse outcome pathways Mode of action is conceptually identical to the more recent term of “adverse outcome pathway” (AOP) which was described initially by the computational ecotoxicology community (Ankley et al., 2010). The terms MOA and AOP, represent conceptually the subdivision of the pathway between exposure and effect in either individuals or populations into a series of hypothesized key events at different levels of biological organization (e.g., molecular, subcellular, cellular, tissue) (Meek et al., 2014a). AOPs, on the other hand are considered more in the context of biological pathways initiated by various chemical and/or other stressors (termed as “initiators”); i.e., they are stressor independent and as such, do not take into account toxicokinetics and metabolism specific to an initiator. This facilitates building of networks of biological pathways that are independent of applied stressors. These essentially conceptually identical constructs organize mechanistic knowledge at a range of levels of biological organization to facilitate its evaluation for specified application. Data for organization include those that identify perturbations at more precisely defined molecular (structurally defined biological component such as a DNA base) or biochemical level (e.g., receptor binding characteristics), more traditional measures of intermediate outcomes (e.g., altered function of established biological pathways) and those that characterize adverse health outcomes commonly measured in traditional toxicity studies (e.g., altered renal function). The MOA/AOP construct was proposed principally as a basis to facilitate consideration of mechanistic data in risk assessment. However, it also has potential to contribute to better coordination of the regulatory and research communities. This is essential to transitioning to more efficient mechanistically-based predictive approaches based on better common understanding of critical data gaps in a regulatory risk assessment context. In essence, the MOA/AOP construct combines consideration of traditional biochemical and histological measures of toxicity or adverse outcome (familiar context) with potentially more predictive information from lower levels of biological organization (for example, more recent omics technologies) and non-test methods such as quantitative structure activity relationship analysis. This provides opportunity for tiering, based on specified risk assessment application and has potential to contribute over time, to meet increasing requirement for more efficient testing and assessment of the hazards and risks of much larger numbers of chemicals. The importance of the construct of mode of action in transitioning focus in both testing and hazard/risk assessment strategies for public health protection lies in part, in its distinction with “mechanism” of action. While the former is a description of critical events that lead to induction of the relevant end-point of toxicity for which the weight of evidence supports plausibility, the latter implies a more detailed molecular description of causality. This distinction serves as an important basis to promote tailoring of mechanistic research to address pragmatic risk assessment (i.e., mode of action) needs. Identification of “key” events – i.e., those that are both measurable and necessary to the observed effect is also fundamental to the construct and encourages early interdisciplinary collaboration in consideration and development of data that are much better tailored for risk assessment application.

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3.2.2. Mode of action/species concordance analysis The need to increase understanding of the nature of analysis which supports the use of mechanistic data for risk assessment application was recognized early and has resulted in development of the MOA human relevance (MOA/HR) framework, an analytical tool designed to increase transparency in the systematic consideration of the weight of evidence (WOE) of hypothesized MOA(s) and their relevance to humans. Briefly, the WOE for a hypothesized MOA in animals is assessed based on considerations modified from those proposed by Bradford Hill (Hill, 1965) for assessment of causality in epidemiological studies. HR or species concordance is then considered, based on systematic evaluation of comparability between key events in animals and humans taking into account principally generic information on, for example, biology, physiology and human disease models. If the WOE for the hypothesized MOA is sufficient and relevant to humans (most often the case), implications for dose–response in humans are then considered in the context of kinetic and dynamic data, including the development of chemical specific adjustment factors for interspecies differences and human variability. Delineation of the degree of confidence in the WOE for hypothesized MOAs is critical, as is the delineation of critical research needs. The most significant contribution of mode of action analysis relates, then, principally to increasing common understanding of the importance of considering the likely interspecies differences and human variability and resulting impact on dose–response relationships for a series of toxicokinetic and toxicodynamic key events rather than a single adverse outcome in estimating risk. The framework which was developed in initiatives of the International Life Sciences Institute Risk Sciences Institute (ILSI RSI) and the International Programme on Chemical Safety (IPCS), evolved in a stepwise manner being based on earlier work on weight of evidence for MOA in animals by the US Environmental Protection Agency (US EPA) and IPCS (Sonich-Mullin et al., 2001). Its development and evolution which has involved large numbers of research and risk assessment scientists internationally, is described in several publications and includes approximately 30 associated case studies (see Table 1 in Meek et al., 2014a) (Boobis et al., 2006, 2008; Meek, 2008; Meek et al., 2003; Seed et al., 2005). Examples of documented modes of action for both cancer and non cancer effects include those involving direct interaction with DNA leading to tumors (mutagenic MOAs), mammary tumors associated with suppression of luteinizing hormone, thyroid tumors associated with increased clearance of thyroxine and androgen receptor antagonism leading to developmental effects. The framework has been widely adopted in international and national guidance and assessments (Meek et al., 2008) and communicated through associated training internationally. While it was originally developed as a basis to increase transparency in the rigor and consistency of documentation of modes of action for specific (normally chemical) stressors, the construct is also helpful in transitioning the risk assessment community in the integration of data from evolving technologies. It contributes to increasing the predictive capacity and utility of risk assessment by drawing maximally and early on mechanistic data as a basis to increase predictivity – i.e., data on kinetics/dynamics and the broader biology base. It encourages generation of data more relevant to risk assessment by facilitating iterative dialog between risk assessors and researchers. Based on increasing experience and technological advances, potential application of the framework in a broader range of relevant contexts has been considered (Carmichael et al., 2011; Meek and Klaunig, 2010). It has also been updated to illustrate its utility in emerging areas in toxicity testing and non-testing methods (Meek et al., 2014a). The modified framework can be used

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as originally intended, where toxicological effects are known, or in hypothesizing effects resulting from chemical exposure, using information on putative key events in established modes of action from appropriate in vitro or in silico systems. Most importantly, though, the framework is presented in a roadmap within the context of problem formulation, encouraging continuous refinement of fit-for-purpose testing strategies and risk assessment (also known as integrated approaches to testing and assessment). For example, decisions concerning chemical prioritization for testing and/or assessment will likely allow for higher levels of uncertainty than those related to establishing regulatory standards. In problem formulation, then, the complexity of the envisaged mode of action analysis is tailored to the context of decision-making; approaches are necessarily flexible and iterative, permitting efficient identification and generation of the essential information to serve as a basis to assess and manage risks appropriately. Relevant case examples illustrate existing regulatory applications in different tiers of analysis. These range from the creation of chemical categories through anchoring of the results of in vitro approaches to relevant outcomes based on existing knowledge on mode of action (e.g., cholinesterase inhibition by organophosphate insecticides) to the contribution of in vitro genomic studies designed to address critical issues in species concordance analysis and resulting implications for dose–response analysis. The need for a broader range of increasingly data informed or tiered approaches to address dose–response analysis for a wide variety of different applications based on early consideration of objectives and resources or problem formulation is increasingly recognized (Meek et al., 2014a). 3.2.3. Tiering in mode of action analysis Such staged approaches based on tiered assessment strategies are likely to contribute to increased confidence in the interpretation and application of in vitro data in regulatory risk assessment. Greater reliance on results in vitro to address applications where implications of decision making are less significant and greater uncertainty can be tolerated (e.g., prioritizing or grouping for testing) has potential to increase confidence in their use. Experience in the pragmatic use of such data in order to increase efficiency will undoubtedly also contribute to acceptance and additional refinement of methodology. Early success is most likely for cases where there is supportive in vivo data for related compounds and confidence in the applicability of the mode of action by which critical effects are produced across compounds and species. For example, for a structural class of compounds for which the mode of action for critical effects is similar and well documented, in vitro studies of early key events for a newly synthesized (or previously unstudied) chemical in the structural group could support prediction to estimate relative potency, relative to other compounds for which there are in vivo hazard data (“read-across”). Depending on the significance of the application (e.g., priority setting vs. full risk assessment with implications for risk management), data from in vitro studies of early key events could be supplemented with data on later key events which may become evident in targeted in vivo studies. In combination with basic information on toxicokinetic aspects such as absorption (which could be predicted from lipid solubility, etc.) and metabolic stability (which could be determined in in vitro test systems, such as hepatic microsomal fraction or cultured hepatocytes) the “readacross” prediction could be used, either semiquantitatively or with a physiologically based toxicokinetic model to inform the choice of reference point from among those of the compounds for which information is already available. In this way, the results of in vitro approaches can be anchored in relevant outcomes based on existing in vivo data.

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Introduction of novel methodology is facilitated, then, by testing and assessment that is focussed for purpose, as framed in problem formulation. Resulting consideration within a continuum of increasingly complex approaches or tiers to address various applications (e.g., priority setting for testing to considering the adequacy of risk management for public health protection) contributes to increase efficiency through tailored investment of resources. It also contributes to better common understanding of the types of information or “determinants” which contribute most to increasing confidence for applications with greater impact for which more certainty is required (e.g., quantitative assessment of risk). The broader application of an accepted construct emphasizes the importance of interaction among the risk assessment, risk management and research communities, as a basis to transition to consideration of data from different levels of biological organization in mode of action analysis tailored for the purpose at hand (e.g.,“fit for purpose” such as prioritization vs. full assessment), while also highlighting the need to anchor data from evolving technologies and research. 3.2.4. Tiering in combined exposures assessment A recent framework to address combined exposures to multiple chemicals builds on evolving experience in tiered assessment programs, internationally (Meek et al., 2011). Experience in development and application of the framework underscores the importance not only of tiered or “fit for purpose” strategies as a basis to increase efficiency in assessment but illustrates their potential in increasing application of novel data for early tiers where implications of decision making are more limited. This would offer considerable improvement of the status quo, given the limited extent to which combined exposures to multiple chemicals have been formally addressed in regulatory risk assessment. The framework includes problem formulation as a basis to consider appropriate grouping within the context of application followed by stepwise integrated and iterative consideration of both exposure and hazard in several tiers of increasingly data-informed analyses. The objective of tiered framework analyses is to tailor the complexity of assessment to address the issue at hand, to ensure that no more resources are invested than are necessary – either to set an assessment group aside as not a priority for further consideration or to inform risk management. The level of effort is also tailored to the magnitude of potential risks, the objective (e.g., priority setting or screening for additional focus or risk management) and scope (e.g., local, national). Each tier is more refined (i.e. less conservative and uncertain than the previous one), but additionally labor, modeling and data intensive, incorporating less certain predictive approaches in early tiers and increasingly refined, more data-informed and probabilistic analyses in later tiers, for which associated uncertainty is less. For example for exposure, the described tiers range from simple semi-quantitative estimates based often on crude “indicators” or “determinants” (such as use profiles in combination with physical/chemical properties) to conservative quantitative point estimates based on generic exposure scenarios and finally, more refined estimates incorporating much more monitoring data (to provide probabilistic estimates, where possible). Described hazard tiers are based on the assumption of dose addition for components and range from broad groupings including all identified components to those that are more refined based on knowledge of mode of action and/or more certain estimates of potency. In early tiers, in vitro studies have potential to contribute to grouping of compounds that should be considered together based on similar profiles of hazard. In higher tiers, based on documented MOA and measures of early key events for related compounds, in vitro studies can contribute to refined grouping and/or relative potency estimates for

components. At any tier, the outcome can be risk management, no further action, generation of additional data or further assessment (i.e., additional refinement in a higher tier), based on context specific evaluation of the adequacy of margins of exposure. In addition to conserving resources in assessment, the approach is helpful in focusing research in critical areas. The framework was developed in collaborative effort by a broad range of organizations (IPCS, 2009). Case studies test and illustrate the framework (e.g., Boobis et al., 2011; Meek, 2011) and its principles have been adopted in risk assessments by regulatory agencies (see, for example, EFSA, 2008). This illustrates the potential of tiered strategies as a basis to facilitate uptake of mechanistic and quantitative data from in vitro studies, particularly in areas that are not being addressed, currently. Experience in developing and testing the framework identified a number of priorities for consideration in increasing the efficiency of risk assessment (Meek, 2013). For problem formulation, this included a range of aspects to consider in tailoring any assessment to context including objectives, available resources/deadlines and data availability. Development of the framework and associated case studies also illustrated the importance of “framing” of estimates of exposure and hazard in a contextual construct that characterizes their relative degree of conservatism and associated uncertainty. Ultimately, appropriate balance is incurred through the use in early tiers of simplistically informed tools which, while associated with greater uncertainty, meaningfully distinguish priorities for further consideration. This necessitates identification of those parameters which most significantly impact output at each stage (sensitivity analysis), as a basis for efficient and meaningful refinement in higher tiers and contributes to the consideration of the importance or value of generating additional (absent) data. The explicit consideration of the required degree of discrimination to address the identified need in problem formulation and subsequent tailoring of the assessment and testing strategy facilitates integration of in vitro results of in early tiers. Such application is reasonably justified as a basis to set priorities and acquire more experience in their interpretation and refinement, given that their implications for impact are less. Experience in application of the framework is consistent with results of programs to establish priorities from among large numbers of existing substances (see, for example, Meek and Armstrong, 2007; Hughes et al., 2009). Comparison in several increasingly data informed tiers in case studies indicates that exposure is much more discriminating than hazard as a basis to consider priorities for testing (i.e., there are many more orders of magnitude difference between quantitative estimates of exposure in early and late tiers versus those for hazard). Additional development of relatively simple measures which are predictive of potential for exposure (e.g., algorithms which weight potential for direct contact based on use profiles) is likely, then, to effect greater discrimination in early tiers of assessment, compared to quantitation of hazard. Targeted monitoring for verification of predictive exposure measures is critical as a basis to contribute to increased efficiency in targeted testing strategies.

3.3. Contextual knowledge transfer to facilitate communication and evaluation Standardized formats for reporting of relevant information to support regulatory risk assessment are critical as a basis to facilitate knowledge transfer between communities (in this case, the scientific and regulatory risk assessment communities). These formats or “templates” can relate to reporting of individual study results in a risk assessment context or to the assimilation of

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information to consider weight of evidence; they promote the necessary transparency to enable evaluation for risk assessment application. 3.3.1. Mode of action/species concordance analysis An example of the latter is provided in the series of templates that have been developed in as support for the MOA/HR or species concordance framework. These templates have been helpful to those tasked with developing or considering information on mode of action to focus their attention on critical aspects of the basis for weight of evidence and/or associated decision points. They also contribute to increasing focus, transparency and consistency in analysis and have evolved over time based on increasing experience in application and training internationally. The templates include those for the following: a. Preliminary comparative

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3.3.2. Guidance on characterization and application of physiologically based pharmacokinetic models in risk assessment Another example is provided by guidance and case study examples developed in an international initiative to inform the characterization, documentation, evaluation and communication of PBPK models for health risk assessment (IPCS, 2010). Drawing on the output of initiatives within the USA, Canada and Europe (Gentry et al., 2004; US EPA, 2006; Barton et al., 2007; Loizou et al., 2008), the WHO Guidance documents key principles both for risk assessors who need to evaluate the models as well as for model developers to facilitate tailoring for application in risk assessment. It contributes a template developed from dialogue between technical scientists and risk assessors which ensures sufficiency of documentation of model descriptions and supporting data (in a standardized format) to permit independent evaluation. This template is seen as an important tool in facilitating communication between the modeling and risk assessment communities to increase understanding and regulatory acceptance of purpose oriented models. The template addresses four primary areas:

analysis of the extent of data supporting various hypothesized MOAs (Meek and Klaunig, 2010). Assessing dose–response and temporal concordance as part of weight of evidence. Consideration of relevant kinetic and/or dynamic data for each of the key events to assess qualitative and quantitative concordance for humans (and associated critical data gaps) (Meek et al., 2005; Seed et al., 2005; Boobis et al., 2006, 2008) with subsequent extension to dose response (Meek et al., 2014a). Consideration of defining questions for each of the Bradford Hill considerations rank ordered to reflect their importance in assessing weight of evidence (WOE) (Meek et al., 2014b). Consideration of WOE in a comparative context as a basis to determine its adequacy in relation to supporting data for various hypothesized modes of action and/or other chemicals (Meek et al., 2014a,b).

Supporting data required to document the models for regulatory application include the original model code and reproduction of any simulations that form the basis of dose metrics proposed for use in the risk assessment. Supporting files and data sets sufficient to reproduce comparison of the model simulations with the experimental data) and reported numerical results (exposure/dose calculations) should also be provided (see IPCS, 2010; Meek et al., 2013a).

The additional articulation of supporting considerations and comparative analysis of WOE as required in such templates seems essential as a basis to simplify or “codify” experience and knowledge in MOA analyses to facilitate its inclusion in evolving knowledge bases to disseminate information on MOAs/AOPs. Such knowledge bases are currently under development through collaboration of the European Commission Joint Research Centre and the US EPA Office of Research and Development, under the auspices of the Organization of Economic Cooperation and Development (OECD) (see, for example http://ihcp.jrc.ec.europa. eu/our_activities/alt-animal-testing-safety-assessment-chemicals/improved_safety_assessment_chemicals/adverse-outcomepathways-aop). A wiki-based tool provides a user-friendly and open-source interface for rapid, widely accessible, building of new AOPs and collaborative sharing of established AOPs (https://aopkb. org/aopwiki/index.php/Main_Page). The tool is envisaged to provide an interface for early scientific discussions as a basis to build consensus on the weight of evidence of supporting information as well as relevance to regulatory decision making. The AOP Wiki represents the initial phase in a larger effort to build a complete AOP knowledge base which will also contain a graphic module to highlight the interconnections among AOPs via network viewing tools. It will also incorporate quantitative information to facilitate computational modeling of AOPs. An intermediate effects module will provide AOP information in a format acceptable for current regulatory purposes (OECD harmonized templates). As such, then, this evolving knowledge base provides the necessary link between the risk assessment and regulatory communities and is anticipated to contribute significantly in facilitating progression to more efficient integrated assessment and testing strategies.

3.3.3. OECD guidance for describing non guideline in vitro test methods There is also notable progress in the development of templates for in vitro data evaluation as a basis to contribute to integrated assessment and testing strategies. Draft guidance on best practice and documentation of non guideline in vitro tests is available at: http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=ENV/JM/MONO(2014)35&doclanguage=en. The guidance outlines the elements considered relevant for providing a comprehensive description of an in vitro method including those based on manual protocols or those adapted for use on automated platforms or high-throughput screening systems (HTS). Required documentation includes proprietary aspects, test method definition and performance, data interpretation and prediction model and potential regulatory applications. It is anticipated to contribute to the consideration of the practical utility of new in vitro methods at early stages of development based on their evaluation and interpretation in a manner that assures scientific confidence and the interpretation of results to support scientifically defensible tailored applications, for use in decision making by regulators and the scientific community. This requires building on appropriate biological context [e.g. the adverse outcome pathway (AOP)] and should in turn help to inform the use of non guideline in vitro methods in integrated approaches to testing and assessment (IATA) as well as chemical grouping and prioritization. Templates for test methods descriptions for in vitro data are being implemented in databases in countries and regions (e.g., DBALM in Europe (http://www.oecd.org/env/ehs/testing/draftguidanceandreviewdocumentsmonographs.htm).

b. c.

d.

e.

 Background on the chemical, its pharmacokinetics (PK) and

MOA.  Characterization and evaluation of the PBPK model.  Modeling and evaluation of the model-derived dose metrics.  PBPK modeling and comparison with default approaches.

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3.4. Coordination and development of expertise Efficient evolution of test methods requires interdisciplinary consultation/communication and training of risk assessors (who commonly have backgrounds principally in toxicology), modelers and those that conduct mechanistic investigations. Communication between the research and regulatory risk assessment communities to address a mutually agreed objective to increase the quantitative reliance of risk assessment practices on in vitro data is a priority. Such consultation/coordination is essential to identifying barriers that impede progress, to increase understanding of priorities and constraints in application and to agree on standards of technical practice. Collective input in the development, for example, of “best practices” documents for the conduct of in vitro studies is essential to maintain flexibility, which is critical to increased uptake of evolving methods in application. This obviates the need for time consuming formal validation of “one size fits all” toxicity tests but necessarily requires continuing commitment to share expertise. Gaps in understanding between those trained/experienced in generating in vitro data and those charged with protecting public health often relates to risk assessors having traditionally received graduate training in environmental health sciences, general pharmacology, toxicology and related biomedical sciences, which may best be supplemented by additional expertise in applied mathematics, statistics and computational methods required to appropriately evaluate and integrate more predictive computational models. Development of training materials and hiring of personnel with appropriate expertise may appreciably stimulate the implementation of more predictive MOA- and PBPK-based risk assessment by regulatory agencies as will additional investment of the research communities in both the receipt of and contribution to training in risk assessment. While the necessary coordination between the research and regulatory communities is effected in part by the standardized templates addressed in principle 3 above, continuing access to relevant expertise is also critical. Peer engagement to assess the suitability of assessment/testing for specific applications in risk assessment is essential as is prioritization of access to relevant expertise through recruitment, training, and/or retraining (Meek et al., 2007; IPCS, 2010). Continuing evolution of the MOA/AOP construct has highlighted the need also for multidisciplinary interaction within the scientific community to address significant challenges in transitioning to more progressive and efficient approaches. Concepts and experience concerning MOA and AOP analysis are similar, for example, between the human health and environmental communities, though in somewhat different spheres of application. Continued collaboration of these two communities is likely to increase efficiency in transition to more predictive approaches. The important need for early access to adequate expertise in facilitating regulatory acceptance of evolving technology has been noted previously in several of the initiatives considered here, being identified as, perhaps, the most critical aspect limiting regulatory uptake of PBPK models (IPCS, 2010). The staging of the transition of knowledge is key to success in uptake by the risk assessment/ regulatory community and necessarily dependent on committed and continuing contribution from the research community. In early stages of transition, identifying and gaining access to (often) limited expertise is critical. In intermediate stages, effective communication and dissemination of training is important and for the longer term, redesign of undergraduate and postgraduate training programs is necessary. In relation to early stages, it has been recommended, for example, that an international standing committee be convened to provide continuing feedback on key aspects regarding the

credibility and reliability of PBPK models in risk assessment (IPCS, 2010; Bessems et al., 2014). This would undoubtedly facilitate their acceptance and uptake, consistent with previous experience in the contribution of the FOrum for Co-ordination of pesticide fate models and their USe (FOCUS) in regulatory acceptance of environmental fate and transport modeling in Europe (Loizou et al., 2008). For the intermediate term, assembly of generic models may prove beneficial, as a basis to communicate fundamental principles as would dissemination of step-by-step instructions to develop focused, web-based training (and retraining) that is easily accessible by risk assessors and other interested audiences. While such web-based training materials can contribute to collective regulatory experience, longer-term goals need to include more quantitative, computationally based study of toxicology in university curricula. 3.5. The importance of challenge Exercise of the challenge function (i.e., continuing pressure to summon to action) is an essential element in prompting regulatory uptake of novel technology as a basis to increase efficiency in testing and assessment. While reports such as those of the US National Research Council on “Toxicity Testing in the 21st Century: A Vision and a Strategy” (NRC, 2007) have provided major stimulus to the scientific community in initiating movement to reassess how toxicity testing and risk assessment are performed, the most significant gains have resulted from legislative imperative. The latter often provides the essential incentive and associated resources to change the status quo. Though it is commonly surmised that legislative mandates do not evolve in short enough timeframes to accommodate efficient application of new technology, in fact, they are the principal drivers for such change. This has been demonstrated repeatedly in jurisdictions where progressive regulatory mandates require priority setting and assessment for many more chemicals much more efficiently. Once established, progressive national legislated trends are often reflected subsequently internationally with relevant mandates being expanded and tailored to the priorities of other countries. For example, a regulatory mandate in Canada introduced during the revision of the Canadian Environmental Protection Act (1999) necessitated considering priorities from among all 23,000 existing substances on the Domestic Substances List by September, 2006. This was followed by the introduction of the Registration, Evaluation and Authorization of Chemicals (REACH) in Europe in 2007 and the Japan Stepwise Assessment under the Chemical Substances Control Law (CSCL) in 2009. This harmonized identification of priorities through legislated imperative is seemingly essential to making meaningful progress in transitioning to more efficient and effective testing and assessment strategies incorporating novel technology. A related example is provided by the continuing long standing challenge to the regulatory risk assessment community to take into account combined exposures to multiple chemicals in addressing risk. In fact, there are very limited numbers of accessible examples of such assessments internationally with much of the recent experience being driven by legislative requirements such as the US Food Quality Protection Act or national challenges to consider not only combined exposures to multiple chemicals but their combination with other non-chemical stressors (NRC, 2008). Paradoxically, immediate pressures to respond to short term deadlines to meet legislated risk assessment requirements (without adequate resources to address longer term strategies) often impedes planning and implementation of necessary change to incorporate potentially valuable evolving technologies. Limited resources are often focused on processing assessments based on

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the status quo – i.e., the application of established and seldomquestioned test strategies for identification of hazard based on standardized or mandated study designs. Another significant barrier to regulatory uptake of novel, potentially more efficient methodology is the associated increasing complexity of communication concerning their basis to others, such as risk managers (addressed in principle 4). This includes the perceived increased complexity of communicating approaches that require tailored testing and assessment based on defined objectives in problem formulation versus the status quo where standardized testing protocols minimize the contribution of scientific judgment. Consideration of several possible approaches may have potential to facilitate common understanding and communication among a range of audiences (e.g., risk assessors in the regulatory community, their managers and policy makers and stakeholders). Undoubtedly, in early stages as a basis to increase confidence, there will be continuing need to “anchor” results at lower levels of biological organization with those from traditional testing approaches. Continuing challenge is critical, then, in advancing testing and assessment strategies. For example, where there are relevant toxicokinetic and toxicodynamic data for data rich substances, it is important that confidence/uncertainties associated with their incorporation be explicitly compared with those of default approaches and critical data gaps identified. In essence, this is the critical objective of more transparent framework analyses as a basis to better coordinate and integrate input of the research and regulatory communities in the translation of relevant mechanistic data into quantitative characterization of risk. Similarly, in tiered analysis, the uncertainties of early tiers of priority setting and assessment (incorporating novel technology) needs to be contrasted with the benefits/risks of lack of reliance on preliminary information to contribute to assessment. Requirement for justification of the use of default methodology or the implications of not conducting even a preliminary assessment based on evolving methods should be an integral component of guidance in risk assessment [as is the case for the IPCS guidance on CSAF and PBPK modeling (IPCS, 2005, 2010)]. This argues for formal requirement in all risk assessments to contrast the confidence and/or uncertainty of various options including lack of assessment or reliance on default methodology. 4. Discussion: a pragmatic path forward? Recently, a series of studies have been proposed in a data driven framework that provides a risk-based and animal-sparing approach to evaluate chemical safety, drawing broadly from previous experience in hazard testing and risk assessment but incorporating technological advances to increase efficiency (Thomas et al., 2013). The approach incorporates a number of the principles outlined above and is addressed here as a basis for consideration of potential additional steps that might facilitate its evolution and uptake. Based on data from high-throughput in vitro (ToxCast) assays, Thomas et al. (2013) have designed the first tier of the framework to separate chemicals based on their relative selectivity in interacting with biological targets (mode of action) and to identify the concentration at which these interactions occur (dose– response). The concept of selectivity relates to the subset of chemicals for which there is evidence that they preferentially target specific biological systems at comparatively low doses. This initial tier also includes in vitro-to-in vivo extrapolation (IVIVE) for calculation of the point of departure and comparisons to human exposure estimates to yield a MOE, in addition to pharmacokinetic modeling and exposure modeling. The second tier involves shortterm in vivo studies, expanded pharmacokinetic evaluations, and refined human exposure estimates. The third tier contains the

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traditional animal studies currently used to assess chemical safety. In each tier, the point of departure (POD) for biologically selective chemicals is based primarily on endpoints associated with a proposed mode of action, whereas the POD for biologically nonselective chemicals is based on potential biological perturbation. In the first tier, as a basis to consider internal dose, data from in vitro hepatic metabolic clearance and plasma protein binding assays are included. Steady-state blood concentrations are estimated based on a parameterized inherently conservative IVIVE model. Incorporation of Monte Carlo sampling enables estimation of the daily human oral dose and associated interindividual variability, which produces steady-state in vivo blood concentrations equivalent to the in vitro AC50 value (concentration at 50% of maximum activity) for each of the high-throughput in vitro assays (Rotroff et al., 2010b; Wetmore et al., 2012b). When appropriate, other biokinetic factors within the in vitro assays as reported in Blaauboer (2010) are proposed to be taken into account. Identified limitations of the proposed approach are a function largely of the early stage of development of in vitro hazard screening and exposure profiling. The inclusiveness of the current battery of in vitro ToxCast assays to represent critical MOAs for selectivity has been noted as have other limitations of the current range of ToxCast assays (Judson et al., 2011; Thomas et al., 2012a). These include the lack of metabolism, use of single cell types that fail to replicate tissue-level cell–cell interactions, lack of biological context and their short-term nature. Approaches to estimation of exposure in early tiers are also not well developed currently, a function in large part of historical focus of regulatory risk assessment principally on hazard identification with much more limited development and/or consideration of exposure (or the dose dependent nature of developed events/effects). Early consideration of both MOA and dose–response relationships in the approach proposed by Thomas et al. (2013) pragmatically embraces the realistic view that early development of adverse outcome pathways for all relevant effects as a basis to be predictive for much broader ranges of chemicals is precluded by the constraints of both current knowledge and resources. That said, there is an important need to incorporate much more of what we have learned on key events/effects at different levels of biological organization to address impending requisite timeframes to deliver progressive regulatory mandates. The proposed approach is also consistent with current practice in risk assessment, where detailed consideration of mode of action is often restricted to cases where margins of exposure are small, based on dose–response analysis in traditional in vivo toxicity studies. The approach also meets objectives for public health protection with prevention appropriately focussing on avoiding disruption of important biological processes, rather than late stage adverse responses and making conservative choices in the absence of data. In essence, then, the approach of Thomas et al. (2013) addresses many of the principles delineated here. In relation to the first principle (i.e., transitioning in familiar context), the approach is a reasonable and pragmatic path forward that builds on current constructs in regulatory assessment including margins of exposure (MOEs) and linear low dose extrapolation, but incorporating concomitant consideration of exposure and more quantitative dose response modeling early in the process, as a basis to be more discriminating in setting priorities for testing and assessment. This enables tailoring of hazard testing to application, while taking into account early, important components of risk assessment, that are often conducted either subsequently and/or separately. Early incorporation of QVIVE builds on previous experience in incorporating PBPK modeling as a basis to more accurately estimate internal dose, thereby reducing one of the sources of variability in dose–

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response assessment. Early identification of streams for “selective” and “non selective” acting chemicals is consistent with the need to more robustly address hazard and dose–response for those substances which selectively interact at relatively low doses with specific targets. The corollary in current practice is development of mechanistic data to address mode of action, though this has traditionally been conducted at later stage following identification of hazard in studies of standardized test design. Consistent with the important second principle of tiering to acquire experience and increase confidence, Thomas et al. (2013) suggest that their tiered testing strategy be introduced initially into programs tasked with evaluating chemicals with limited toxicity data, such as the US Environmental Protection Agency’s (US EPA’s) Superfund Health Risk Technical Support Center (STSC; http://www.epa.gov/superfund/health/research.htm). This program evaluates chemicals of interest to Superfund for which toxicity data are normally far more limited in comparison with those, for example, which are assessed within the US EPA Integrated Risk Information System (IRIS). Potential for success in increasing acceptance of in vitro data and QVIVE is likely to be increased, however, by proposal for their use in tiered strategies in areas such as combined exposures for which there is continuing public pressure but limited progress. In this manner, the strategy has potential to present a solution (e.g., at least as an interim strategy) rather than competing with existing approaches, where it may not be perceived that there is an issue, currently or for which barriers are likely to be significant. Alternatively, potential for uptake might be maximized through proposal for use in programs where there is an essential need to increase efficiency through legislated mandates such as those to consider much larger numbers of existing chemicals within mandated timeframes. Advantage over the status quo (i.e., making little or no progress in addressing the issue) is self-evident. As a basis to increase efficiency, the complexity of, computational modeling and the nature of in vitro data in the tiered strategy are appropriately tailored to be no more than is required for the risk assessment application envisaged in each tier. For example, the complexity of the toxicokinetic data and PBPK models is counterbalanced by the need for increased accuracy, biological basis and scientific justifiability of risk assessment applications in various tiers. Consistent with previous guidance (IPCS, 2010), while complex PBPK models may be relevant to chemicals for which estimated exposure approaches reference doses, preliminary assessments or screening exercises may be suitably based on simpler models. The results of these simpler models may indicate the need for more complex and resource intensive approaches, like PBPK modeling. Missing from the proposed path forward, however, is any consideration of principles (3) contextual knowledge transfer to facilitate interpretation and communication in application and (4) coordination and development of expertise. While a number of collaborators from both the regulatory and risk assessment communities contributed to the consideration of the testing and assessment strategy proposed in Thomas et al. (2013), discussion of practical implementation is restricted to that mentioned above – i.e., that the tiered testing strategy be introduced initially into programs tasked with evaluating chemicals with limited toxicity data. In addition to proposal for consideration in areas where limited progress has been made despite continuing public pressures (as recommended above), preliminary consideration of envisaged next steps for knowledge sharing between the research and regulatory risk assessment communities and provision of access to relevant expertise is also advised. Initial steps could include convening of a workshop to increase common understanding and solicit feedback with subsequent development

by a drafting group or groups of guidance addressing not only content but process to ensure consideration of pragmatic implications of introduction of such strategies for training within both the research and regulatory communities. Additional strategies to address short term, intermediate and long term needs for coordination and development of expertise are included under principle 4 above. With respect to the challenge function (principle 5), Thomas et al. (2013) note the recognition in government agencies of the need for greater efficiency in the testing and assessment of chemicals. Release of the National Research Council Report “Toxicity Testing in the 21st Century: A Vision and a Strategy” (NRC, 2007) was highlighted which, among other initiatives, was credited as being a major stimulus in initiating “a broad-based movement in the toxicology community to reassess how toxicity testing and risk assessment are performed”. However, the authors acknowledge the visioning nature of such efforts and essentially collectively challenge the research and regulatory risk assessment communities to consider developing a pragmatic path forward that can be iteratively refined as greater understanding is achieved. What has been proposed to address this need for consideration by Thomas et al. (2013) maximally integrates existing knowledge and experience. And while it has potential to meaningfully advance efficiency in toxicological testing and risk assessment, advancement requires concerted additional collective effort to coordinate expertise within the research and regulatory communities. 5. Conclusions Much has been learned in previous initiatives concerning the importance of transparency and consistency in communication among the regulatory, research and stakeholder communities as a basis to increase confidence essential to effecting progress in regulatory risk assessment. Contributing factors include appropriate communication of the nature of scientific support and robust process for purpose specific review, ensuring continuing access to relevant expertise. The review and analysis here identifies a number of principles critical to effecting collective progress in advancing testing and assessment strategies incorporating best practice and technological advances. This includes the importance of continuing challenge including legislative imperative as a basis to improve the status quo and the early prioritization of efforts to meaningfully coordinate (particularly) between the research and regulatory communities. These aspects coupled with the development of effective strategies for efficient knowledge dissemination and transparent communication, is essential to progress. Indeed, without concerted, harmonized efforts to respond to collective international challenge, it seems unlikely that there will be significant strides in necessary timeframes to embrace evolving technology in the assessment and management of much larger numbers of chemicals. Essential to meaningful progress, then, is a multifaceted communication and uptake strategy building on previous work in this area and incorporating technological advances to increase efficiency in not only testing but also assessment and in the dissemination and communication of knowledge to facilitate common understanding. This requires tiered strategies with introduction of evolving testing and assessment methodology principally in early stages as a basis to acquire pragmatic experience to contribute to their refinement. Legislative mandates requiring much more efficient prioritization and assessment of larger numbers of substances such as that introduced early within Canada and the European Union and more recently in the Asian Pacific region, are important “drivers” in this evolution. They lend themselves well to introduction in staged fashion of increasingly

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data informed testing and assessment strategies, for which early stages are well suited to incorporation of evolving test methods as do areas such as combined exposures where progress to date to address continuing challenge for more encompassing assessment from stakeholder communities has been limited. In addition, in all stages of testing and assessment, the incorporation of novel tools and methodology needs to be contrasted with the broader scale consequences of maintaining the status quo. For example, in early tiers, the result of lack of progress in increasing efficiency through reliance on evolving in vitro methods is that most chemicals will remain unassessed. At all tiers, consistency in documentation of scientific support as a basis to distinguish most proactive and least uncertain options is required. This necessarily requires continuing efficient and coordinated access to sometimes limited relevant expertise within the scientific community. Transition from resource intensive testing approaches to identify late stage adverse outcome to more predictive application based on reliance on earlier key events identified by novel technology also requires focus and assimilation at a much earlier stage of information gathering on mechanistic underpinnings of potential effects. It necessitates earlier consideration of preliminary information on exposure, toxicokinetics and dose–response as a basis to be more discriminating in tiered testing and assessment strategies. This would meaningfully reflect the breadth of experience in tiered assessment conducted to date that indicates that exposure and toxicokinetics are far more discriminating determinants of risk than is hazard; as a result, earlier focus on these aspects is seemingly essential as a basis to increasing efficiency. The importance of tiering to gain both efficiencies and acceptance cannot be overemphasized with potential to provide substantial benefits in the development, interpretation and quantitative reliance on in vitro data for health risk assessment. However, such a framework cannot be developed without identifying a familiar context or without open dialogue between research scientists and risk assessors. It is also anticipated that additional dialogue between research scientists and regulatory risk assessors, perhaps centered on problem formulation, will reveal common ground upon which familiar contexts can be identified. This may require a detailed examination of the historical basis of the status quo (including established policies and their basis on scientific findings as well as on health conservative assumptions), efforts to identify common goals with measurable milestones and the establishment of decision points. A careful review of the development of regulatory guidance and science policy may reveal significant target areas for the application of well designed, conducted and reported in vitro experimentation. Considerations of “fit for purpose” should be explored, and it may be determined that data initially envisioned as having quantitative value may be limited by constraints not immediately evident to research scientists. This of course includes separate discussions of “fit” and “purpose”. In tiered testing and assessment strategies, focus in early tiers should also be on purpose-specific “evaluation” rather than generic “validation” as a basis to gain additional experience in application. This can include “ground truthing” or “anchoring” of model predictions with existing data, supplemented with appropriate analyses of variability, uncertainty, and sensitivity. Explicit (though necessarily simplistic) consideration of uncertainty and sensitivity is important as a basis to enable development of additional data or prompt more robust assessment on those aspects which are most influential and uncertain in determining outcome of early tiers. At all tiers, documentation should be sufficient to enable an experienced assessor, expert reviewer, or interested end user to evaluate the methodology.

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