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Summary Change often involves a pivotal event that builds on previous history and opens the door to a new era. Pivotal events in science include the discovery of penicillin, the elucidation of the DNA double helix, and the development of computers. All were marked by inauspicious beginnings followed by unheralded ad- vances over a period of years but ultimately resulted in a pharma- copoeia of life-saving drugs, a map of the human genome, and a personal computer on almost every desk in todayâs workplace. Toxicity testing is approaching such a scientific pivot point. It is poised to take advantage of the revolutions in biology and bio- technology. Advances in toxicogenomics, bioinformatics, systems biology, epigenetics, and computational toxicology could trans- form toxicity testing from a system based on whole-animal testing to one founded primarily on in vitro methods that evaluate changes in biologic processes using cells, cell lines, or cellular components, preferably of human origin. Anticipating the impact 1
2 Toxicity Testing in the 21st Century of recent scientific advances, the U.S. Environmental Protection Agency (EPA) asked the National Research Council (NRC) to de- velop a long-range vision for toxicity testing and a strategic plan for implementing the vision. This report of the NRC Committee on Toxicity Testing and Assessment of Environmental Agents, prepared in response to EPAâs request, envisions a major campaign in the scientific com- munity to advance the science of toxicity testing and put it on a forward-looking footing. The potential benefits are clear. Fresh thinking and the use of emerging methods for understanding how environmental agents affect human health will promote beneficial changes in testing of these agents and in the use of data for deci- sion-making. The envisioned change is expected to generate more robust data on the potential risks to humans posed by exposure to environmental agents and to expand capabilities to test chemicals more efficiently. A stronger scientific foundation offers the pros- pect of improved risk-based regulatory decisions and possibly greater public confidence in and acceptance of the decisions. With those goals in mind, the committee presents in this re- port a vision for mobilizing the scientific community and marshal- ling scientific resources to initiate and sustain new approaches, some available and others yet to be developed, to toxicity testing. This report speaks to scientists in all sectorsâgovernment, public interest, industry, university, and consulting laboratoriesâwho design and conduct toxicity tests and who use test results to evaluate risks to human health. The report also seeks to inform and engage decision-makers and other leaders who shape the na- ture and scope of government regulations and who establish budgetary priorities that will determine progress in advancing toxicity testing in the future. The full impact of the committeeâs wide-ranging recommendations can be achieved only if both sci- entists and nonscientists work to advance the objectives set forth in the vision.
Summary 3 THE VISION The current approach to toxicity testing relies primarily on a complex array of studies that evaluate observable outcomes in whole animals, such as clinical signs or pathologic changes, that are indicative of a disease state. Partly because that strategy is so time-consuming and resource-intensive, it has had difficulty in meeting many challenges encountered today, such as evaluating various life stages, numerous health outcomes, and large numbers of untested chemicals. The committee debated several options for improving the current system but concluded that a transformative paradigm shift is needed to achieve the design criteria set out in the committeeâs interim report: (1) to provide broad coverage of chemicals, chemical mixtures, outcomes, and life stages, (2) to re- duce the cost and time of testing, (3) to use fewer animals and cause minimal suffering in the animals used, and (4) to develop a more robust scientific basis for assessing health effects of envi- ronmental agents.1 The committee considered recent scientific advances in defin- ing a new approach to toxicity testing. Substantial progress is be- ing made in the elucidation of cellular-response networksâ interconnected pathways composed of complex biochemical inter- actions of genes, proteins, and small molecules that maintain normal cellular function, control communication between cells, and allow cells to adapt to changes in their environment. For ex- ample, one familiar cellular-response network is signaling by es- trogens in which initial exposure results in enhanced cell prolif- eration and tissue growth in specific tissues. Bioscience is enhancing our knowledge of cellular-response networks and al- lowing scientists to begin to uncover how environmental agents perturb pathways in ways that lead to toxicity. Cellular response pathways that, when sufficiently perturbed, are expected to result 1For a further discussion of the options considered by the committee, see Chapter 2, âOptions for a New Toxicity-Testing Paradigm.â
4 Toxicity Testing in the 21st Century in adverse health effects are termed toxicity pathways. The commit- tee envisions a new toxicity-testing system that evaluates biologi- cally significant perturbations in key toxicity pathways by using new methods in computational biology and a comprehensive ar- ray of in vitro tests based on human biology. COMPONENTS OF THE VISION Figure S-1 illustrates the major components of the commit- teeâs vision: chemical characterization, toxicity testing, and dose- response and extrapolation modeling. The components of the vi- sion, which are described in the sections that follow, are distinct but interrelated modules involving specific sets of technologies and scientific capabilities. Some chemical evaluations may pro- ceed in a stepwise mannerâfrom chemical characterization to tox- icity testing to dose-response and extrapolation modelingâbut such a sequential evaluation need not always be followed in prac- tice. A critical feature of the new vision is consideration of the risk context (the decision-making context that creates the need for tox- icity-testing information) at each step and the ability to exit the strategy at any point when sufficient data have been generated for decision-making. The vision emphasizes the generation and use of population-based and human exposure data where possible for interpreting test results and encourages the collection of such data on important chemicals with biomonitoring, surveillance, and epidemiologic studies. Population-based and human exposure data, along with the risk context, will play a role in both guiding and using the toxicity information that is produced. Finally, the vision anticipates the development of a formal process to phase in and phase out test methods as scientific understanding of toxicity- testing methods expands. That process addresses the need for effi- cient testing of all chemicals in a timely, cost-effective fashion.
Summary 5 FIGURE S-1 The committeeâs vision for toxicity testing is a process that includes chemical characterization, toxicity testing, and dose-response and extrapolation modeling. At each step, population-based and human exposure data are consid- ered, as is the question of what data are needed for decision-making. Chemical Characterization Chemical characterization is meant to provide insights to key questions, including a compoundâs stability in the environment, the potential for human exposure, the likely routes of exposure, the potential for bioaccumulation, possible routes of metabolism, and the likely toxicity of the compound and possible metabolites based on chemical structure or physical or chemical characteris- tics. Thus, data would be collected on physical and chemical properties, use, possible environmental concentrations, metabo- lites and breakdown products, initial molecular interactions of compounds and metabolites with cellular components, and possi- ble toxic properties. A variety of computational methods might be used to predict those properties and characteristics. After chemi- cal characterization, decisions might be made about what further testing is required or whether it is needed at all. In most cases,
6 Toxicity Testing in the 21st Century chemical characterization alone is not expected to be sufficient to reach decisions about the toxicity of an environmental agent. Toxicity Testing In the vision proposed (see Figure S-1), toxicity testing has two components: toxicity-pathway assays and targeted testing. The committee expects that when the vision is achieved, predic- tive, pathway-based assays will serve as the central component of a broad toxicity-testing strategy for assessing the biologic activity of new and existing compounds. Targeted testing will serve to complement the assays and support evaluation. Toxicity Pathways Figure S-2 illustrates the activation of a toxicity pathway. The initial perturbations of cell-signaling motifs, genetic circuits, and cellular-response networks are obligatory changes resulting from chemical exposure that might eventually result in disease. The consequences of a biologic perturbation depend on its magnitude, which is related to the dose, the timing and duration of the perturbation, and the susceptibility of the host. Accordingly, at low doses, many biologic systems may function normally within their homeostatic limits. At somewhat higher doses, clear biologic responses occur. They may be successfully handled by adaptation, although some susceptible people may respond. More intense or persistent perturbations may overwhelm the capacity of the system to adapt and lead to tissue injury and possible adverse health effects. The committeeâs vision capitalizes on the identification and use of toxicity pathways as the basis of new approaches to toxicity
Summary 7 Exposure Tissue Dose Biologic Interaction Perturbation Biologic Normal Inputs Biologic Function Early Cellular Changes Adaptive Stress Cell Responses Injury Morbidity and Mortality FIGURE S-2 Biologic responses viewed as results of an intersection of exposure and biologic function. The intersection results in perturbation of biologic path- ways. When perturbations are sufficiently large or when the host is unable to adapt because of underlying nutritional, genetic, disease, or life-stage status, bio- logic function is compromised, and this leads to toxicity and disease. Source: Adapted from Andersen, M.E., J.E. Dennison, R.S. Thomas, and R.B. Conolly. 2005. New directions in incidence-dose modeling. Trends Biotechnol. 23(3):122- 127. Reprinted with permission; copyright 2005, Trends in Biotechnology. testing and dose-response modeling. Accordingly, the vision em- phasizes the development of suites of predictive, high-throughput assays2 that use cells or cell lines, preferably of human origin, to evaluate relevant perturbations in key toxicity pathways. Those assays may measure relatively simple processes, such as binding of environmental agents with cellular proteins and changes in gene expression caused by that binding, or they may measure 2High-throughput assays are efficiently designed experiments that can be automated and rapidly performed to measure the effect of substances on a bio- logic process of interest. These assays can evaluate hundreds to many thousands of chemicals over a wide concentration range to identify chemical actions on gene, pathway, and cell function.
8 Toxicity Testing in the 21st Century more integrated responses, such as cell division and cell differen- tiation. Although the majority of toxicity tests in the vision are ex- pected to use high-throughput methods, other tests could include medium-throughput assays of more integrated cellular responses, such as cytotoxicity, cell proliferation, and apoptosis. Over time, the need for traditional animal testing should be greatly reduced and possibly even eliminated. Targeted Testing Targeted testing would be used to complement toxicity- pathway tests and to ensure adequate evaluation. It would be used (1) to clarify substantial uncertainties in the interpretation of toxicity-pathway data; (2) to understand effects of representative prototype compounds from classes of materials, such as nanopar- ticles, that may activate toxicity pathways not included in a stan- dard suite of assays; (3) to refine a risk estimate when the targeted testing can reduce uncertainty, and a more refined estimate is needed for decision-making; (4) to investigate the production of possibly toxic metabolites; and (5) to fill gaps in the toxicity- pathway testing strategy to ensure that critical toxicity pathways and end points are adequately covered. One of the challenges of developing an in vitro test system to evaluate toxicity is the cur- rent inability of cell assays to mirror metabolism in the integrated whole animal. For the foreseeable future, any in vitro strategy will need to include a provision to assess likely metabolites through whole-animal testing. Targeted testing might be conducted in vivo or in vitro, de- pending on the toxicity tests available. Although targeted tests could be based on existing toxicity-test systems, they will proba- bly differ from traditional tests in the future. They could use transgenic species, isogenic strains, new animal models, or other novel test systems and could include a toxicogenomic evaluation
Summary 9 of tissue responses over wide dose ranges. Whatever system is used, testing protocols would maximize the amount of informa- tion gained from whole-animal toxicity testing. Dose-Response and Extrapolation Modeling In the vision proposed (see Figure S-1), dose-response mod- els would be developed for environmental agents primarily on the basis of data from mechanistic, in vitro assays as described in the toxicity-testing component. The dose-response models would de- scribe the relationship between concentration in the test medium and degree of in vitro response. In some risk contexts, a dose- response model based on in vitro results might provide adequate data to support a risk-management decision. An example could involve compounds for which host-susceptibility factors in hu- mans are well understood and human biomonitoring provides good information about tissue or blood concentrations of the com- pound and other related exposures that affect the toxicity path- way in a human population. Extrapolation modeling estimates the environmental expo- sures or human intakes that would lead to human tissue concen- trations similar to those associated with perturbations of toxicity pathways in vitro and would account for host susceptibility fac- tors. In the vision proposed, extrapolation modeling has three primary components. First, a toxicity-pathway model would pro- vide a quantitative, mechanistic understanding of the dose- response relationship for the perturbations of the pathways by environmental agents. Second, physiologically based pharma- cokinetic modeling would then be used to predict human expo- sures that lead to tissue concentrations that could be compared with the concentrations that caused perturbations in vitro. Third, human data would provide information on background chemical exposures and disease processes that would affect the same toxic-
10 Toxicity Testing in the 21st Century ity pathway and provide a basis for addressing host susceptibility quantitatively. Population-Based and Human Exposure Data Population-based and human exposure data are important components of the committeeâs toxicity-testing strategy (see Fig- ure S-1). Those data can help to inform each component of the vi- sion and ensure the integrity of the overall testing strategy. The shift toward the collection of more mechanistic data on fundamen- tal biologic perturbations in human cells will require greater use of biomonitoring and human-surveillance studies for data inter- pretation. Moreover, the interaction between population-based studies and toxicity tests will improve the design of each study type for answering questions about the importance of molecular, cellular, and genetic factors that influence individual and popula- tion-level health risks. Because the vision emphasizes studies con- ducted in human cells that indicate how environmental agents can affect human biologic responses, the studies will suggest bio- markers (indicators of human exposure, effect, or susceptibility) that can be monitored and studied in human populations. As toxicity testing shifts to cell-based studies, human expo- sure data from biomonitoring studies (such as those recom- mended in the NRC report Human Biomonitoring for Environmental Chemicals3) may prove pivotal. Such data can be used to select doses for toxicity testing that can provide information on biologic effects at environmentally relevant exposures. More important, comparison of concentrations that activate toxicity pathways with concentrations of agents in blood, urine, or other tissues from hu- man populations will help to identify potentially important expo- NRC (National Research Council). 2006. Human Biomonitoring for Environ- 3 mental Chemicals. Washington, DC: The National Academies Press.
Summary 11 sures to ensure an adequate margin of safety in setting human ex- posure guidelines. Risk Context Toxicity testing is useful ultimately only if it can be used to facilitate more informed and efficient responses to the public- health concerns of regulators, industry, and the public. Common scenarios, defined by the committee as ârisk contexts,â for which toxicity testing is used to make decisions include evaluation of potential environmental agents, existing environmental agents, sites of environmental contamination, environmental contribu- tors to a human disease, and the relative risk of different environmental agents. Some risk contexts require rapid screening of tens of thousands of environmental agents; some require highly refined dose-response data, extending down to environmentally relevant exposure concentrations; and some require the ability to test chemical mixtures or to use assays focused on specific mechanisms. Some risk contexts might require the use of population-based approaches, including population health surveillance and biomonitoring. With its emphasis on high- throughput assays that use human cells, cell lines, and components to evaluate biologically significant perturbations in key toxicity pathways, the vision presented here will assist the decision-making process in each risk context. IMPLEMENTATION OF THE VISION Implementation of the vision will require (1) the availability of suites of in vitro testsâpreferably based on human cells, cell lines, or componentsâthat are sufficiently comprehensive to evaluate activity in toxicity pathways associated with the broad
12 Toxicity Testing in the 21st Century array of possible toxic responses; (2) the availability of targeted tests to complement the in vitro tests and ensure an adequate tox- icity database for risk-management decision-making; (3) computa- tional models of toxicity pathways to support application of in vitro test results to predict exposures in the general population that could potentially lead to adverse changes; (4) infrastructure changes to support the basic and applied research needed to de- velop the tests and the pathway models; (5) validation of tests and test strategies for incorporation into chemical-assessment guide- lines that will provide direction in interpreting and drawing con- clusions from the new assay results; and (6) evidence justifying that the results of tests based on perturbations in toxicity path- ways are adequately predictive of adverse health outcomes to be used in decision-making. A substantial and focused research effort will be needed to meet those requirements. The research will need to develop both new scientific knowledge and new toxicity-testing methods. Key questions that need to be addressed regarding knowledge and method development are highlighted in Box S-1. The research and development needed to implement the vi- sion would progress in phases whose timelines would overlap. Phase I would focus on elucidating toxicity pathways; developing a data-storage, -access, and -management system; developing standard protocols for research methods and reporting; and plan- ning a strategy for human surveillance and biomonitoring to sup- port the toxicity-pathway testing approach. Phase II would in- volve development and validation of toxicity-pathway assays and identification of markers of exposure, effect, and susceptibility for use in surveillance and biomonitoring of human populations. Phase III would evaluate assays by running them in parallel with traditional toxicity tests, on chemicals with large datasets, and on chemicals that would not otherwise be tested as a screening proc- ess. Parallel testing will allow identification of toxicities that might
Summary 13 BOX S-1 Key Questions to Address in Implementation Knowledge Development Toxicity-Pathway IdentificationâWhat are the key pathways whose perturbations result in toxicity? Multiple PathwaysâWhat alteration in response can be expected from simultaneous perturbations of multiple toxicity pathways? AdversityâWhat adverse effects are linked to specific toxicity-pathway perturbations? What patterns and magnitudes of perturbations are predictive of adverse health outcomes? Life StagesâHow 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 DurationâHow are biologic responses affected by exposures of different duration? Low-Dose ResponseâWhat is the effect on a toxicity pathway of adding small amounts of toxicants in light of pre-existing endogenous and exogenous human exposures? Human VariabilityâHow do people differ in their expression of toxicity-pathway constituents and in their predisposition to disease and impairment? Method Development Methods to Predict MetabolismâHow can adequate testing for metabolites in the high-throughput assays be ensured? Chemical-Characterization ToolsâWhat computational tools can best predict chemical properties, metabolites, xenobiotic-cellular and molecular interactions, and biologic activity? Assays to Uncover Cell CircuitryâWhat methods will best facilitate the discovery of the circuitry associated with toxicity pathways? Assays for Large-Scale ApplicationâWhich assays best capture the elucidated pathways and best reflect in vivo conditions? What designs will ensure adequate testing of volatile compounds? (Continued on next page)
14 Toxicity Testing in the 21st Century BOX S-1 Continued Suite of AssaysâWhat 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 StrategyâWhat 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 ExtrapolationâWhat procedures should be used to evaluate whether humans are at risk from environmental exposures? Test-Strategy UncertaintyâHow can the overall uncertainty in the testing strategy be best evaluated? be missed if the new assays were used alone and will compel the development of assays to address these gaps. Surveillance and biomonitoring of human populations would also begin during Phase III. Finally, the validated assays would be assembled into panels in Phase IV for use in place of identified traditional toxici- ty tests. Validation will be a critical component of the research and development phases. Establishing the validity of any new toxicity assay can be a formidable processâexpensive, time-consuming, and logistically and technically demanding. For several reasons, validation will be especially challenging for the mechanistically based tests envisioned by the committee. First, the test results to be generated in the new paradigm depart from the traditional data used by regulatory agencies to set health advisories and guidelines. Second, the many new technologies developed will need to be standardized and refined before specific applications are validated for regulatory purposes. Third, because new tech- nologies are evolving rapidly, the decision to halt optimization of
Summary 15 a particular application and begin a formal validation study will be somewhat subjective. Fourth, the committee envisions that a suite of new tests will typically be needed to replace a specific tra- ditional test. Fifth, existing guidelines focus on concordance be- tween the results of new and existing assays; the difficulty will be to find standards for comparison that can assess the relevance and predictivity of the new assays. Sixth, because virtually all envi- ronmental agents will perturb signaling pathways to some degree, a key challenge will be to determine when such perturbations are likely to lead to toxic effects and when they are not. A long-term, large-scale concerted effort is needed to bring the committeeâs vision for toxicity-testing to fruition. A critical factor for success is the conduct of the transformative research to establish the scientific basis of new toxicity-testing tools and to understand the implications of test results and their application in risk assessments used in decision-making. The committee con- cludes that an appropriate institutional structure that fosters mul- tidisciplinary intramural and extramural research is needed to achieve the vision. The effort will not succeed merely by creating a virtual institution to link and integrate organizations that perform relevant research and by dispersing funding on relevant research projects. Mission-oriented intramural and extramural programs with core multidisciplinary activities within the institute to an- swer the critical research questions listed above can foster the kind of interdisciplinary activity essential for the success of the initiative. There would be far less chance of success within a rea- sonable time if the research were dispersed among different loca- tions and organizations without a core integrating and organizing institute to enable the communication and problem-solving re- quired across disciplines. Research frequently brings surprises, and todayâs predictions about the promise of lines of research might prove to be too pes- simistic or too optimistic in some details. Therefore, the commit- tee recommends that an independent scientific assessment of the
16 Toxicity Testing in the 21st Century research program supporting implementation of the vision be conducted every 3-5 years to provide advice for midcourse correc- tions. The interim assessments would weigh progress, evaluate the promise of new methods on the research horizon, and refine the committeeâs vision in light of the many scientific advances that are expected to occur in the near future. Regulatory acceptance of the new toxicity-testing strategy will depend on several factors. New testing requirements will be expected to reflect the state of the science and be founded on peer- reviewed research, established test protocols, validated models, and case studies. Other factors affecting regulatory acceptance stem from administrative procedures associated with rule- making, such as documenting scientific sources; providing oppor- tunities for scientific experts, stakeholders, and the interested pub- lic to participate; and consulting with sister agencies and interna- tional organizations. Implementing the vision will require improvements and focused effort over a period of decades. How- ever, given the political will and the availability of funds to adapt the current regulatory system to take advantage of the best possi- ble scientific approaches to toxicity testing in the future, the com- mittee foresees no insurmountable obstacles to implementing the vision presented here. Resources are always limited, and current toxicity-testing practices are long established and deeply ingrained in some sec- tors. Thus, some resistance to the vision proposed by this commit- tee is expected. However, the vision takes full advantage of cur- rent and expected scientific advances to enhance our understanding of how environmental agents can affect human health. It has the potential to greatly reduce the cost and time of testing and to lead to much broader coverage of the universe of environmental agents. Moreover, the vision will lead to a marked reduction in animal use and focus on doses that are more relevant to those experienced by human populations. The vision for toxic- ity testing in the twenty-first century articulated here is a para-
Summary 17 digm shift that will not only improve the current system but trans- form it into one capable of overcoming current limitations and meeting future challenges.