5
Developing the Science Base and Assays to Implement the Vision

Rapid advances in the understanding of the organization and function of biologic systems provide the opportunity to develop innovative mechanistic approaches to toxicity testing. In comparison with the current system, the new approaches should provide wider coverage of chemicals of concern, reduce the time needed for generating toxicity-test data required for decision-making, and use animals to a far smaller extent. Accordingly, the committee has proposed development of a testing structure that evaluates perturbations in toxicity pathways and relies on a mix of high-and medium-throughput assays and targeted in vivo tests as the foundation of its vision for toxicity testing. This chapter discusses the kinds of applied and basic research needed to support the new toxicity-testing approach, the institutional resources required to support and encourage it, and the valuable products that can be expected during the transition from the current apical end-point testing to a mechanistically based in vivo and in vitro test system.



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Toxicity Testing in the 21st Century: A Vision and a Strategy 5 Developing the Science Base and Assays to Implement the Vision Rapid advances in the understanding of the organization and function of biologic systems provide the opportunity to develop innovative mechanistic approaches to toxicity testing. In comparison with the current system, the new approaches should provide wider coverage of chemicals of concern, reduce the time needed for generating toxicity-test data required for decision-making, and use animals to a far smaller extent. Accordingly, the committee has proposed development of a testing structure that evaluates perturbations in toxicity pathways and relies on a mix of high-and medium-throughput assays and targeted in vivo tests as the foundation of its vision for toxicity testing. This chapter discusses the kinds of applied and basic research needed to support the new toxicity-testing approach, the institutional resources required to support and encourage it, and the valuable products that can be expected during the transition from the current apical end-point testing to a mechanistically based in vivo and in vitro test system.

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Toxicity Testing in the 21st Century: A Vision and a Strategy Most tests in the committee’s vision would be unlike current toxicity tests, which generate data on apical end points. The mix of tests in the vision include in vitro tests that assess critical mechanistic end points involved in the induction of overt toxic effects rather than the effects themselves and targeted in vivo tests that ensure adequate testing of metabolites and coverage of end points. The move toward a mechanism-oriented testing paradigm poses challenges. Implementation will require (1) the availability of suites of in vitro tests—preferably based on human cells, cell lines, or components—that are sufficiently comprehensive to evaluate activity in toxicity pathways associated with the broad array of possible toxic responses; (2) the availability of targeted tests to complement the in vitro tests and ensure overall adequate data for decision-making; (3) models of toxicity pathways to support application of in vitro test results to predict general-population exposures that could potentially cause adverse perturbations; (4) infrastructure changes to support the basic and applied research needed to develop the tests and the pathway models; (5) validation of tests and test strategies for incorporation into chemical-assessment guidelines that will provide direction on interpreting and drawing conclusions from the new assay results; and (6) acceptance of the idea that the results of tests based on perturbations in toxicity pathways are adequately predictive of adverse responses and can be used in decision-making. Development of the new assays and the related basic research—the focus of this chapter—requires a substantial research investment over quite a few years. Institutional acceptance of the new tests and the requisite new risk-assessment approaches—the focus of Chapter 6—also require careful planning. They cannot occur overnight. Ultimately, the time required to conduct the research needed to support large-scale incorporation of the new mechanistic assays into a test strategy that can adequately and rapidly address large numbers of agents depends on the institutional will to commit resources to support the changes. The committee believes that with

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Toxicity Testing in the 21st Century: A Vision and a Strategy a concerted research effort, over the next 10 years high-throughput test batteries could be developed that would substantially improve the ability to identify toxicity hazards caused by a number of mechanisms of action. Those results in themselves would be a considerable advance. The time for full realization of the new test strategy, with its mix of in vitro and in vivo test batteries that can rapidly and inexpensively assess large numbers of substances with adequate coverage of possible end points, could be 20 or more years. This chapter starts by discussing basic research that will provide the foundation for assay development. It then outlines a research strategy and milestones. It concludes by discussing the scientific infrastructure that will support the basic and applied research required to develop the high-throughput and targeted testing strategy envisioned by the committee. SCOPE OF SCIENTIFIC KNOWLEDGE, METHODS, AND ASSAY DEVELOPMENT This section outlines the scientific inquiry required to develop the efficient and effective testing strategy envisioned by the committee. Several basic-research questions need to be addressed to develop the knowledge base from which toxicity-pathway assays and supporting testing technologies can be designed. The discussion here is intended to provide a broad overview, not a detailed research agenda. The committee recognizes the challenges and effort involved in addressing some of these research questions. Knowledge Development Knowledge critical for the development of high-throughput assays is emerging from biologic, medical, and pharmaceutical

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Toxicity Testing in the 21st Century: A Vision and a Strategy research. Further complementary, focused research will be needed to address fully the key questions that when answered will support toxicity-pathway assay development. Those questions are outlined in Box 5-1 and elaborated below. Toxicity-pathway identification. The key pathways that, when sufficiently perturbed, will result in toxicity will be identified primarily from future, current, and completed studies in the basic biology of cell-signaling motifs. Identification will involve the discovery of the protein components of toxicity pathways and how the pathways are altered by environmental agents. Many pathways are under investigation with respect to the basic biology of cellular processes. For example, the National Institutes of Health (NIH) has a major program under way to develop high-throughput screening (HTS) assays based on important biologic responses in in vitro systems. HTS has the potential to identify chemical probes of genes, pathways, and cell functions that may ultimately lead to characterization of the relationship between chemical structure and biologic activity (Inglese et al. 2006). Determining the number and nature of toxicity pathways involved in human disease and impairment is an essential component of the committee’s vision for toxicity testing. Multiple pathways. Adverse biologic change can occur from simultaneous perturbations of multiple toxicity pathways. Environmental agents typically affect more than one toxicity pathway. Although the committee envisions the design of a suite of toxicity tests that will provide broad coverage of biologic perturbations in all key toxicity pathways, biologic perturbations in different pathways may lead to synergistic interactions with important implications for human health. For some adverse health effects, an understanding of the interplay of multiple pathways involved may be important. For others, the research need will be to identify the pathway affected at the lowest dose of the environmental agent.

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Toxicity Testing in the 21st Century: A Vision and a Strategy BOX 5-1 Key Research Questions in Developing Knowledge to Support Pathway Testing Toxicity-Pathway Identification—What are the key pathways whose perturbations result in toxicity? Multiple Pathways—What alteration in response can be expected from simultaneous perturbations of multiple toxicity pathways? Adversity—What adverse effects are linked to specific toxicity-pathway perturbations? What patterns and magnitudes of perturbations are predictive of adverse health outcomes? Life Stages—How can the perturbations of toxicity pathways associated with developmental timing or aging be best captured to enable the advancement of high-throughput assays? Effects of Exposure Duration—How are biologic responses affected by exposures of different duration? Low-Dose Response—What is the effect on a toxicity pathway of adding small amounts of toxicants in light of pre-existing endogenous and exogenous human exposures? Human Variability—How do people differ in their expression of toxicity-pathway constituents and in their predisposition to disease and impairment? Adversity. An understanding of possible diseases or functional losses that may result from specific toxicity-pathway perturbations will support the use of pathway perturbations for decision-making. Current risk assessments rely on toxicity tests that demonstrate apical adverse health effects, such as disease or functional deficits, that are at various distances downstream of the toxicity-pathway perturbations. In the committee’s vision, the assessment of potential human health impact will be based on perturbations in toxicity pathways. For example, activation of estrogenic action to abnormal levels during pregnancy is associated with undescended testes and, in later life, testicular cancer. Research will be needed to understand the patterns and magnitudes of the perturbations that will lead to adverse effects. As part of the

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Toxicity Testing in the 21st Century: A Vision and a Strategy research, biomarkers of effect that can be monitored in humans and studied in whole animals will be useful. Life stages. An understanding of how pathways associated with developmental timing or aging can be adversely perturbed and lead to toxicity will be needed to develop high-throughput assays that can capture and adequately cover developmental and senescing life stages. Many biologic functions require coordination and integration of a wide array of cellular signals that interact through broad networks that contribute to biologic function at different life stages. That complexity of pathway interaction holds for reproductive and developmental functions, which are governed by parallel and sequential signaling networks during critical periods of biologic development. Because of the complexity of such pathways, the challenge will be to identify all important pathways that affect such functions to ensure adequate protection against risks to the fetus and infant. That research will involve elucidating temporal changes in key toxicity pathways that might occur during development and the time-dependent effects of exposure on these pathways. Effects of exposure duration. The dose of and response to exposure to a toxicant in the whole organism depend on the duration of exposure. Thus, conventional toxicity testing places considerable emphasis on characterizing risks associated with exposures of different duration, from a few days to the test animal’s lifetime. The ultimate goal in the new paradigm is to evaluate conditions under which human cells are likely to respond and to ensure that these conditions do not occur in exposures of human populations. Research will be needed to understand how the dose-response relationships for perturbations might change with the duration of exposure and to understand pathway activation under acute, subchronic, and chronic exposure conditions. The research will involve investigating the differential responses of cells of various ages and backgrounds to a toxic compound and

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Toxicity Testing in the 21st Century: A Vision and a Strategy possible differences in responses of cells between people of different ages. Low-dose response. The assessment of the potential for an adverse health effect from a small environmental exposure involves an understanding of how the small exposure adds to preexisting exposures that affect the same toxicity pathways and disease processes. For the more common human diseases and impairments, a myriad of exposures from food, pharmaceuticals, the environment, and endogenous processes have the potential to perturb underlying toxicity pathways. Understanding how a specific environmental exposure contributes, with the other exposures, to modulate a toxicity pathway is critical for the understanding of low-dose response. Because the toxicity tests used in the committee’s long-range vision are based largely on cellular assays involving sensitive biomarkers of alterations in biologic function, it will be possible to study the potential for adverse human health effects at doses lower than is possible with conventional whole-animal tests. Given the cost-effectiveness of the computational methods and in vitro tests that form the core of the toxicity testing, it will be efficient to evaluate effects at multiple doses and so build a basis of detailed dose-response research. Human variability. People differ in their expression of toxicity-pathway constituents and consequently in their predisposition to disease and impairment. An understanding of differences among people in the level of responsiveness of particular toxicity pathways is needed to interpret the importance of small environmental exposures. The comprehensive mapping of toxicity pathways provides an unprecedented opportunity to identify gene loci and other determinants of human sensitivity to environmental exposures. That research will support the development of biomarkers of exposure, effect, and susceptibility for surveillance in the human population, and these discoveries in turn will support an assessment of host susceptibility for use in extrapolating results from the in vitro assays to the general population and susceptible

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Toxicity Testing in the 21st Century: A Vision and a Strategy groups. The enhanced ability to characterize interindividual differences in sensitivity to environmental exposures will provide a firmer scientific basis of the establishment of human exposure guidelines that can protect susceptible subpopulations. Research on most, or all, of the above subjects is going on in the United States and internationally. It is taking place in academe, industry, and government institutions and is funded by foundations and the federal government mainly to understand the basis of human disease and treatment. Private firms, such as pharmaceutical and biotechnology companies, conduct the research for product development. However, efforts directed specifically toward developing toxicity-testing systems are small. Test and Analytic Methods Development The research described above will provide the foundation for the development of toxicity tests and comprehensive testing approaches. The categories of toxicity tests and methods to be developed are outlined below, and the primary questions to be answered in their development are presented in Box 5-2. Methods to predict metabolism. A key issue to address at an early phase will be development of methods to ensure adequate testing for metabolites in high-throughput assays. Understanding the range of metabolic products and the variation in metabolism among humans and being able to simulate human metabolism as needed in test systems are critical for developing valid toxicity-pathway assays. Without such methods, targeted in vivo assays will be needed to evaluate metabolism. Chemical-characterization tools. In addition to metabolism, further development of tools to support chemical characterization

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Toxicity Testing in the 21st Century: A Vision and a Strategy BOX 5-2 Main Questions in Developing Tests and Methods Methods to Predict Metabolism—How can adequate testing for metabolites in the high-throughput assays be ensured? Chemical-Characterization Tools—What computational tools can best predict chemical properties, metabolites, xenobiotic-cellular and molecular interactions, and biologic activity? Assays to Uncover Cell Circuitry—What methods will best facilitate the discovery of the circuitry associated with toxicity pathways? Assays for Large-Scale Application—Which assays best capture the elucidated pathways and best reflect in vivo conditions? What designs will ensure adequate testing of volatile compounds? Suite of Assays—What mix of pathway-based high- and medium-throughput assays and targeted tests will provide adequate coverage? What targeted tests should be developed to complement the toxicity-pathway assays? What are the appropriate positive and negative controls that should be used to validate the assay suite? Human-Surveillance Strategy—What surveillance is needed to interpret the results of pathway tests in light of variable human susceptibility and background exposures? Mathematical Models for Data Interpretation and Extrapolation—What procedures should be used to evaluate whether humans are at risk from environmental exposures? Test-Strategy Uncertainty—How can the overall uncertainty in the testing strategy be best evaluated? will be important. The tools will include computational and structure-activity relationship (SAR) methods to predict chemical properties, potential initial interactions of a chemical and its metabolites with cellular molecules, and biologic activity. A National Research Council report (NRC 2000) indicated that early cellular interactions are important in understanding potential toxicity and include receptor-ligand interactions, covalent binding with DNA and other endogenous molecules, peroxidation of lipids and proteins, interference with sulfhydryl groups, DNA methylation, and

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Toxicity Testing in the 21st Century: A Vision and a Strategy inhibition of protein function. Good predictive methods for chemical characterization will reduce the need for targeted testing and enhance the efficiency of the testing. Assays to uncover cell circuitry. Development of methods to facilitate the discovery of the circuitry associated with toxicity pathways will involve functional genomic techniques for integrating and interpreting various data types and for translating dose-response relationships from simple to complex biologic systems, for example, from the pathway to the tissue level. It will most likely require improved methods in bioinformatics, systems biology, and computational toxicology. Some advances in overexpression with complementary DNA (cDNA) and gene knockdown with small inhibitory RNAs are likely to allow improved pathway mapping and will also lead to studies with cells or cell lines that are more readily transfectable. Assays for large-scale application. Several substantive issues will need to be considered in developing assays for routine application in a testing strategy. First, as pathways are identified, medium- and high-throughput assays that adequately evaluate pathways and human biology will be developed, including new, preferably human, cell-based cultures for assessment of perturbations. Second, the assay designs that best capture the elucidated pathways and can be applied for rapid large-scale testing of chemicals will need to be identified. Third, an important design criterion for assays will be that they are adequately reflective of the in vivo cellular environment. For any given assay, that will involve an understanding of the elements of the human cellular environment that must be simulated and of culture conditions that affect response. Fourth, the molecular evolution of cell lines during passage in culture and related interlaboratory differences that can result will have to be controlled for. Fifth, approaches for the testing of volatile compounds will require early attention in the development of high-throughput assays; this has been a challenge for in vitro test systems in general. Sixth, assay sensitivity

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Toxicity Testing in the 21st Century: A Vision and a Strategy (the probability that the assay identifies the phenomenon that it is designed to identify) and assay specificity (the probability that the assay does not identify a phenomenon as occurring when it does not) will be important considerations in assay design. Individual assays and test batteries should have the capability to predict accurately the effects that they are designed to measure without undue numbers of false positives and false negatives. And seventh, it will be important to achieve flexibility to expand or contract the suites of assays as more detailed biologic understanding of health and disease states emerges from basic research studies. Suite of assays. An important criterion for the development of a suite of assays for assessing the potential for a substance to cause a particular type of disease or group of toxicities will be adequate coverage of causative mechanisms, affected cell types, and susceptible individuals. Ensuring the right mix of pathway-based high-throughput assays and targeted tests will involve research. For diseases for which toxicity pathways are not fully understood, targeted in vivo or other tests may be included to ensure adequate coverage. Human-surveillance strategy. Human data on the fundamental biologic events involved in the activation of toxicity pathways will aid the interpretation of the results of high-throughput assays. They will provide the basis of understanding of determinants of human susceptibilities related to a toxicity pathway and of background exposures to compounds affecting the pathway. Research will be needed to assess how population-based studies can best be designed and conducted to complement high-throughput testing and provide the information necessary for data interpretation. Mathematical models for data interpretation and extrapolation. Procedures for evaluating the impact of human exposure concentrations will involve pharmacokinetic and other modeling methods to relate cell media concentrations to human tissue doses and biomonitoring data and to account for exposure patterns and interindividual variabilities. To facilitate interpretation of high-

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Toxicity Testing in the 21st Century: A Vision and a Strategy Improvements in targeted human disease surveillance and exposure biomonitoring. BUILDING A TRANSFORMATIVE RESEARCH PROGRAM Instituting Focused Research A long-term, large-scale concerted effort is needed to bring the new toxicity-testing paradigm to fruition. A critical element is the conduct of transformative research to provide the scientific basis of creating the new testing tools and to understand the implications of test results and how they may be applied in risk assessments used in environmental decision-making. What type of institutional structure would be most appropriate for conducting and managing the research effort? It is beyond the committee's charge and expertise to make specific recommendations either to change or to create government institutions or to alter their funding decisions. The committee will simply sketch its thoughts on an appropriate institutional structure for implementing the vision. Other approaches may also be appropriate. The committee notes that an institutional structure should be selected with the following considerations in mind: The realization of the vision will entail considerable research over many years and require substantial funding—hundreds of millions of dollars. Much of the research will be interdisciplinary and consequently, to be most effective, should not be dispersed among discipline-specific laboratories. The research will need high-level coordination to tackle the challenges presented in the vision efficiently. The research should be informed by the needs of the regulatory agencies that would adapt and use the emerging testing

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Toxicity Testing in the 21st Century: A Vision and a Strategy procedures, but the research program should be insulated from the short-term orientation and varied mandates of the agencies. Interdisciplinarity, Adaptability, and Timeline The need for an institutional structure that encourages and coordinates the necessarily multidisciplinary research cannot be overstated, and a spirit of interdisciplinarity should infuse the research program. Accordingly, the effort would need to draw on a variety of technologies and a number of disciplines, including basic biology, bioinformatics, biostatistics, chemistry, computational biology, developmental biology, engineering, epidemiology, genetics, pathology, structural biology, and toxicology. Good communication and problem-solving across disciplines are a must, as well as leadership adept at fostering interdisciplinary efforts. The effort will have to be monitored continually, with the necessary cross-interactions engineered, managed, and maintained. The testing paradigm would be progressively elaborated over many years or decades as experience and successes accumulate. It should continue to evolve with scientific advances. Its evolution is likely to entail midcourse changes in the direction of research as breakthroughs in technology and science open more promising leads. Neither this committee nor any other constituted committee will be able to foresee the full suite of possibilities or potential limitations of new approaches that might arise with increasing biologic knowledge. The research strategy outlined above provides a preview to the future and suggests general steps needed to arrive at a new toxicity-testing paradigm. Some of the suggested steps would need to be reconsidered as time passes and experience is developed with new cell-based assays and interpretive tools, but no global change in the vision, which the committee regards as robust, is expected.

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Toxicity Testing in the 21st Century: A Vision and a Strategy The transition from existing tests to the new tests would require active management, involvement of the regulatory agencies, and coherent long-range planning that invests in the creation of new knowledge while refining current testing and, correspondingly, stimulating changes in risk-assessment procedures and guidelines. Over time, the research expertise and infrastructure involved in testing regimes could be transformed in important ways as the need for animal testing decreases and pathway-related testing increases. The committee envisions that the new knowledge and technology generated from the proposed research program will be translated to noticeable changes in toxicity-testing practices within 10 years. Within 20 years, testing approaches will more closely reflect the proposed vision than current approaches. That projection assumes adequate and sustained funding. As in the Human Genome Project, progress is expected to be nonlinear, with the pace increasing as technologic and scientific breakthroughs are applied to the effort. Cross-Institution and Sector Linkages The research to describe cellular-response networks and toxicity pathways and to develop the complementary human biomonitoring and surveillance strategy would be part of larger current efforts in medicine and biotechnology. Funding of that research is substantial in medical schools and other academic institutions, some U.S. federal and European agencies, and pharmaceutical, medical, and biotechnology industries. Links among different elements in the research community involved in relevant research will be needed to capitalize on the new knowledge, technologies, and analytic tools as they develop. Mechanisms for ensuring sustained communication and collaboration, such as data-sharing, will also be needed.

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Toxicity Testing in the 21st Century: A Vision and a Strategy Some form of participation by industry and public-interest groups should be ensured. Firms have a long-term interest in the new paradigm, and most stand to gain from more efficient testing requirements. Public-health and environmental interest groups, as well as those promoting alternatives to animal testing, should also be engaged. Funding A large-scale, long-term research program is needed to elucidate the cellular-response networks and individual toxicity pathways within them. Given the scientific challenges and knowledge development required, moderately large funding will be required. The committee envisions a research and test-development program similar in scale to the NTP or the Institute for Systems Biology in Seattle, Washington. The success of the project will depend on attracting the best thinkers to the task, and the endeavor would compete with related research programs in medicine, industry, and government for these researchers. Attracting the best researchers in turn would depend on an adequately funded and managed venture that appears well placed to succeed. Institutional Framework The committee concludes that an appropriate institutional structure for the proposed vision is a research institute that fosters multidisciplinary research intramurally and extramurally. A strong intramural research program is essential. The effort cannot succeed merely by creating a virtual institution to link and integrate organizations that are performing relevant research and by dispersing funding on relevant research projects. A mission-

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Toxicity Testing in the 21st Century: A Vision and a Strategy oriented, intramural program with core multidisciplinary programs to answer the critical research questions can foster the kind of cross-discipline activity essential for the success of the initiative. There would be far less chance of success within a reasonable period if the research were dispersed among different locations and organizations without a core integrating and organizing institute. A collocated, strong intramural research initiative will enable the communication and problem-solving across disciplines required for the research and assay development. Similarly, a strong, well-coordinated, targeted extramural program will leverage the expertise that already exists within academe, pharmaceutical companies, the biotechnology sector, and elsewhere and foster research that complements the intramural program. Through its intramural and highly targeted extramural activities, the envisioned research institute would provide the nexus through which the new testing tools would be conceived, developed, validated, and incorporated into coherent testing schemes. The committee sees the research institute funded and coordinated primarily by the federal government, given the scale of the necessary funding, the multiyear nature of the project, and links to government regulatory agencies. That does not mean that there will be no role for other stakeholders. Biotechnology companies, for example, could cofund specific projects. Academic researchers could conduct research with the program’s extramural funds. Moreover, researchers in industry and academe will continue making important progress in fields related to the proposed vision independently of the proposed projects. The key institutional question is where to house the government research institute that carries out the intramural program of core multidisciplinary research and manages the extramural program of research. Should it be an existing entity, such as the National Institute of Environmental Health Sciences (NIEHS), or a new entity devoted exclusively to the proposed vision? The com-

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Toxicity Testing in the 21st Century: A Vision and a Strategy mittee notes that the recognized need for research and institutional structures that transcend disciplinary boundaries to address critical biomedical research questions has spawned systems-biology institutes and centers at biomedical firms and several leading universities in the country. However, the committee found few examples in the government sector. The Department of Energy (DOE) Genomics GTL Program seeks to engineer systems for energy production, site remediation, and carbon sequestration based on systems-biology research on microorganisms. In its review of this DOE program, NRC (2006) found collocated, integrated vertical research to be essential to its success. If one were to place the proposed research program into an existing government entity, a possible choice would be the NTP, a multiagency entity administered and housed in NIEHS. The NTP has several features that suggest it as a possible institutional home for the research program envisioned here, including its mandate to develop innovative testing approaches, its multiagency character, the similarities between its Vision and Roadmap for the Future and what is envisioned here, and its expertise in validating new tests through the NTP Interagency Center for the Evaluation of Alterative Toxicological Methods and its sister entity, the Interagency Coordinating Committee on the Validation of Alternative Methods, and in -omics testing at its Center for Toxicogenomics. It is conceivable that the NTP could absorb the research mandate outlined here if its efforts dramatically scaled up to accommodate the focused program envisioned. If it were placed in the NTP, structures would have to be in place to ensure that the day-to-day technical focus on short-term problems of high-volume chemical testing would not impede progress in evolving testing strategies. As the new test batteries and strategies are developed and validated, they would be moved out of the research arm and be made available for routine application. The committee considered housing the proposed research institute in a regulatory agency and notes that this could be prob-

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Toxicity Testing in the 21st Century: A Vision and a Strategy lematic. The science and technology budgets of regulatory agencies have been under considerable stress and appear unlikely to sustain such an effort. Although EPA’s NCCT has initiated important work in this field, the scale of the endeavor envisioned by the committee is substantially larger and could not be sufficiently supported if recent trends in congressional budgeting for EPA continue. For example, EPA’s science and technology research budget has been suboptimal and decreasing in real dollars for a number of years (EPA 2006, 2007). The research portfolio entailed by the committee’s vision will also require active management to maintain relevance and the scientific focus needed for knowledge development. Although sufficient input from regulatory agencies is needed, insulation of the institute from the short-term orientation of regulatory-agency programs that depend on the results of toxicologic testing is important. In the end, the committee noted that wherever the institute is housed, it should be structured along the lines of the NTP, with intramural and focused extramural components and interagency input but with its own focused mission and funding stream. Scientific Surprises and the Need for Midcourse Corrections Research often brings surprises, and today’s predictions concerning the promise of particular lines of research are probably either pessimistic or optimistic in some details. For example, the committee’s vision of toxicity testing stands on the presumption that a relatively small number of pathways can provide sufficiently broad coverage to allow a moderately sized set of high-and medium-throughput assays to be developed for the scientific community to use with confidence and that any important gaps in coverage can be addressed with a relatively small set of targeted assays. That presumption may be found to be incorrect. Further-

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Toxicity Testing in the 21st Century: A Vision and a Strategy more, the establishment of links between perturbations and apical end points may prove especially challenging for some end points. Thus, as the research proceeds and learning takes place, adjustments in the vision and the research focus can be anticipated. In addition to program oversight noted above, the research program should be assessed every 3-5 years by well-recognized scientific experts independently of vested interests in the public and private sectors. The assessment would weigh practical progress, the promise of methods on the research horizon, and the place of the research in the context of other research, and it would recommend midcourse corrections. CONCLUDING REMARKS In the traditional approach to toxicity testing, the whole animal provides for the integration and evaluation of many toxicity pathways. Yet each animal study is time-consuming and expensive and results in the use of many animals. In addition, many animal studies need to be done to evaluate different end points, life stages, and exposure durations. The new approach may require individual assays for hundreds of relevant toxicity pathways. Despite that apparent complexity, emerging methods allow testing of many pathways extremely rapidly and efficiently (for example, in microarrays or wells). If positive signals from the assays can be used with confidence to guide risk management, the new approach will ultimately prove more efficient than the traditional one. It is clear, however, that much development and refinement will be needed before a new and efficient system could be in place. For some kinds of toxicity, such as developmental toxicity and neurotoxicity, the identification of replacement toxicity-pathway assays might be particularly challenging, and some degree of targeted testing might continue to be necessary. In addition, the

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Toxicity Testing in the 21st Century: A Vision and a Strategy validation process may uncover unexpected and challenging technical problems that will require targeted testing. Finally, the parallel interim process may discover that some categories of chemicals or of toxicity cannot yet be evaluated with toxicity-pathway testing. Nonetheless, the committee envisions the steady evolution of toxicity testing from apical end-point testing to a system based largely on toxicity-pathway batteries in a manner mindful of information needs and of the capacity of the test system to provide information. In the long term, the committee expects toxicity pathways to become sufficiently well understood and calibrated for batteries of high-throughput assays to provide a substantial fraction of the toxicity-testing data needed for environmental decision-making. Exposure monitoring, human surveillance for early perturbations of toxicity-response pathways, and epidemiologic studies should provide an additional layer of assurance that early indications of adverse effects would be detected if they occurred. The research conducted to realize the committee’s vision would support a series of substantial improvements in toxicity testing in the relatively near term. REFERENCES Balls, M., P. Amcoff, S. Bremer, S. Casati, S. Coecke, R. Clothier, R. Combes, R. Corvi, R. Curren, C. Eskes, J. Fentem, L. Gribaldo, M. Halder, T. Hartung, S. Hoffmann, L. Schectman, L. Scott, H. Spielmann, W. Stokes, R. Tice, D. Wagner, and V. Zuang. 2006. The principles of weight of evidence validation of test methods and testing strategies. The report and recommendations of ECVAM workshop 58. Altern. Lab. Anim. 34(6):603-620. Blount, B.C., J.L. Pirkle, J.D. Osterloh, L. Valentin-Blasini, and K.L. Caldwell. 2006. Urinary perchlorate and thyroid hormone levels in adolescent and adult men and women living in the United States. Environ. Health Perspect. 114(12):1865–1871. Brazma, A., P. Hingamp, J. Quackenbush, G. Sherlock, P. Spellman, C. Stoeckert, J. Aach, W. Ansorge, C.A. Ball, H.C. Causton, T. Gaasterland, P. Glenisson, F.C. Holstege, I.F. Kim, V. Markowitz, C. Matese, H. Parkinson, A.

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