Toxicity Testing in the 21st Century A Vision and a Strategy (2007) / Chapter Skim
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5 Developing the Science Base and Assays to Implement the Vision
5 Developing the Science Base and Assays to Implement the Vision
Pages 120-165
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From page 120... ...
Accordingly, the committee has proposed development of a testing structure that evaluates perturbations in toxicity pathways and relies on a mix of highand 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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Developing the Science Base and Assays 121 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.
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122 Toxicity Testing in the 21st Century a concerted research effort, over the next 10 years highthroughput 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.
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Many pathways are under investigation with respect to the basic biology of cellular processes. For example, the National Institutes of Health (NIH)
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124 Toxicity Testing in the 21st Century 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?
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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.
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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.
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Developing the Science Base and Assays 127 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.
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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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Developing the Science Base and Assays 129 inhibition of protein function. Good predictive methods for chemical characterization will reduce the need for targeted testing and enhance the efficiency of the testing.
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130 Toxicity Testing in the 21st Century (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)
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Developing the Science Base and Assays 131 throughput assay results, models of toxicity pathways (see Chapter 3) and other techniques will be needed to address differences among people in their levels of activation of particular response pathways.
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132 Toxicity Testing in the 21st Century Aspects of those endeavors are discussed in the following sections. STRATEGY FOR KNOWLEDGE AND ASSAY DEVELOPMENT AND VALIDATION The research strategy to develop the computational tools, suites of in vitro assays, and complementary targeted tests envisioned by the committee will likely involve contributions on multiple fronts, including the following: • Basic biologic research to obtain the requisite knowledge of toxicity pathways and the potential human health impacts when the pathways are perturbed.
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Developing the Science Base and Assays 133 In each broad field of toxicity testing, such as neurotoxicology and reproductive and developmental toxicity, systematic approaches to assay development, assay validation, and generalized acceptance of the assays will be organized and pursued. As the research questions presented in the previous section are answered, milestones would be achieved in an orderly manner.
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134 Toxicity Testing in the 21st Century BOX 5-3 Some Science and Technology Milestones in Developing Toxicity-Pathway Tests As the Cornerstone of Future Toxicity-Testing Strategies Develop rapid methods and systems to enable in vitro dosing with chemical stressors (including important metabolites and volatile compounds)
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Developing the Science Base and Assays 135 tested and would begin the biomonitoring and surveillance of human populations. Phase IV: Assembly and validation of test batteries.
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136 Toxicity Testing in the 21st Century Elucidate toxicity pathways. Establish data-storing and –management systems.
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Developing the Science Base and Assays 137 ceptor translocation, transcriptional activation, and integrated cellular responses. Some of the processes are outlined here.
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138 Toxicity Testing in the 21st Century • Processes that lead to estrogenic transgenerational epigenetic effects. Assay development to address estrogen-induced transgenerational epigenetic effects would involve understanding how early-life exposures to estrogenic compounds permanently alter transcriptional control of genes, understanding how such earlylife exposures might be priming events for later-life alterations in reproductive competence or the development of cancer, and understanding how such exposures may produce transgenerational effects.
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Developing the Science Base and Assays 139 pathway responses, a dose-response model would be based on key, nodal points in the circuits that control perturbations rather than on the overall detail of all steps in the signaling process. Assessing Validity of Pathway Knowledge and Linkage to Adversity at the Organism Level The next step in pathway elucidation would be the assessment of the validity of the pathway knowledge, which would proceed in two steps and involve the broader scientific community.
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140 Toxicity Testing in the 21st Century altered-estrogen-signaling case study indicates, the acquisition of the knowledge to develop high-throughput testing assays would involve the discovery of toxicity pathways and networks from vast amounts of data from studies of biologic circuitry and interactions of environmental agents with the circuitry. Organization of that knowledge would require data analysis and exploration by interdisciplinary teams of scientists.
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Developing the Science Base and Assays 141 such efforts are moving more quickly with the Minimum Information About a Microarray Experiment formats now in use (Brazma et al.
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142 Toxicity Testing in the 21st Century phisticated computational structural models of ligand interactions with receptor and receptor-complex conformational changes. Further sets of assays would be needed to address the wide variety of toxicity pathways by which estrogenic compounds can operate.
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Developing the Science Base and Assays 143 • Consideration of the relationship of the test-method end points to the biologic effect of interest. • Characterization of reproducibility in and among laboratories, transferability among laboratories, sources of variability, test limits, and other factors related to the reliability of test measurements (sometimes referred to as internal validity)
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144 Toxicity Testing in the 21st Century beyond the technical performance of an assay may be viewed differently by different regulatory authorities. Challenges in Validating Mechanistically Based Assays Validation of the mechanistically based tests envisioned by the committee may be especially challenging for several reasons.
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Developing the Science Base and Assays 145 Fourth, the committee envisions that a suite of new tests typically will be needed to replace an individual in vivo test, given that apical findings can be triggered by multiple mechanisms. Consequently, although it is current practice to validate a single test against the corresponding conventional test and then to look for one-to-one correspondence, the new paradigm would routinely entail validation of test batteries and would use multivariate comparisons.
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146 Toxicity Testing in the 21st Century tool to use as the definitive test. Similarly, a new test may be intended to model one or a few toxicity mechanisms for a given apical end point but not the full array of mechanisms.
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. The Environmental Protection Agency (EPA)
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the associated risks with missing important toxicities with the new assays or incorporating a new assay that detects meaningless physiologic alterations that are not useful for predicting human risk. Third, as the new assays are developed further and validated, they should be deployed as screens for evaluation of chemicals that would not currently undergo toxicity testing, such as existing high-production-volume chemicals that have not been tested or have been evaluated only with the screening information dataset, or new chemicals that are not currently subject to test requirements.
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Developing the Science Base and Assays 149 Chemical Characterization and No regulatory or other Screening Assay Suite action. No further Risk-Management Decision testing.
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In the long run, using upstream indicators of toxicity from high-throughput assays based on toxicity pathways can be more sensitive and hence more protective of public health then using apical-end-point observations from assays in small numbers of
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Phase IV: Assembly and Validation of Test Batteries Once toxicity pathways are elucidated and translated into high-throughput assays for a broad field of toxicity testing, such as neurotoxicology, a progressively more comprehensive suite of validated medium- to high-throughput tests would become available to cover the field. Single assays would not be comprehensive
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152 Toxicity Testing in the 21st Century or predictive in isolation but would be assembled into suites with targeted tests that would cover the field. The suite or "panel" of assays and the scoring of the assays would need to be assessed.
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Developing the Science Base and Assays 153 Caenorhabditis elegans, are in development. Combined mammalian assays that incorporate a broader array of sensitive end points in a more efficient manner have been developed.
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154 Toxicity Testing in the 21st Century • Early cell-based replacements for some in vivo tests, such as those for acute toxicity. • Work to develop consensus approaches for DNAreactivity and mutagenicity assays and strategies for using mechanistic studies in cancer risk assessment.
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Developing the Science Base and Assays 155 • 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.
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156 Toxicity Testing in the 21st Century 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.
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Developing the Science Base and Assays 157 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 pathwayrelated testing increases.
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158 Toxicity Testing in the 21st Century 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.
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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)
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160 Toxicity Testing in the 21st Century mittee notes that the recognized need for research and institutional structures that transcend disciplinary boundaries to address critical biomedical research questions has spawned systemsbiology institutes and centers at biomedical firms and several leading universities in the country. However, the committee found few examples in the government sector.
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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 highand 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.
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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.
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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.
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03-4508. National Institute of Environmental Health Sciences, National Institutes of Health.
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National Toxicology Program, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC[online]
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