6
New Approaches

The committee’s review of current toxicity-testing strategies revealed a system that is approaching a turning point. Most toxicity-testing frameworks were developed decades ago and may not adequately reflect today’s science, let alone the emerging challenges and new approaches of the future. Agencies have responded by altering individual tests and by adding tests to the existing regimens to incorporate end points and mechanistic evaluations newly recognized to be of potential importance. Those patches have not provided a fully satisfactory solution of the fundamental problem.

The core of the problem appears to be tension among four objectives of regulatory testing schemes that are difficult to meet simultaneously: depth, providing the most accurate, relevant information possible for hazard identification and dose-response assessment; breadth, providing data on the broadest possible universe of chemicals, end points, and life stages; animal welfare, causing the least animal suffering possible and using the fewest possible animals; and conservation, minimizing the expenditure of money and time on testing and regulatory review (see Figure 6-1). The committee initially noted that decreasing animal use and decreasing costs may pull the testing programs in similar directions, such as toward the use of in vitro methods. However, those two objectives may not always be aligned; initial efforts to reduce animal suffering and animal use may increase costs in some situations. Thus, approaches designed to move toxicity testing toward one of the objectives frequently move it away from one or more of the others. The Environmental Protection Agency (EPA) and other agencies that perform or require toxicity



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Toxicity Testing for Assessment of Environmental Agents: Interim Report 6 New Approaches The committee’s review of current toxicity-testing strategies revealed a system that is approaching a turning point. Most toxicity-testing frameworks were developed decades ago and may not adequately reflect today’s science, let alone the emerging challenges and new approaches of the future. Agencies have responded by altering individual tests and by adding tests to the existing regimens to incorporate end points and mechanistic evaluations newly recognized to be of potential importance. Those patches have not provided a fully satisfactory solution of the fundamental problem. The core of the problem appears to be tension among four objectives of regulatory testing schemes that are difficult to meet simultaneously: depth, providing the most accurate, relevant information possible for hazard identification and dose-response assessment; breadth, providing data on the broadest possible universe of chemicals, end points, and life stages; animal welfare, causing the least animal suffering possible and using the fewest possible animals; and conservation, minimizing the expenditure of money and time on testing and regulatory review (see Figure 6-1). The committee initially noted that decreasing animal use and decreasing costs may pull the testing programs in similar directions, such as toward the use of in vitro methods. However, those two objectives may not always be aligned; initial efforts to reduce animal suffering and animal use may increase costs in some situations. Thus, approaches designed to move toxicity testing toward one of the objectives frequently move it away from one or more of the others. The Environmental Protection Agency (EPA) and other agencies that perform or require toxicity

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Toxicity Testing for Assessment of Environmental Agents: Interim Report FIGURE 6-1 The four objectives of toxicity testing. testing are constantly being challenged to meet all four objectives and are often caught between competing priorities. Setting priorities among the competing objectives is more than a scientific issue. Individuals and organizations can have different, sometimes strongly held beliefs about which of the four is most important, and trying to satisfy the objectives has driven different efforts to reform toxicity testing. For example, legislation requiring creation of an endocrine-disruptor screening program was driven by an effort to increase breadth, whereas the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) was created in an effort to conserve animals. Rather than attempting to set priorities among the objectives, this committee recognized that all four are important. The committee acknowledges, however, that embracing all four poses a difficult challenge. This chapter and the next review some selected approaches that may ultimately help to move toxicity testing toward one or more of the objectives. In this chapter, the committee summarizes and comments on some strategies proposed by others for near-term improvements in existing toxicity-testing approaches, including the EPA review of toxicity data available for establishing reference doses (RfDs) and reference concen-

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Toxicity Testing for Assessment of Environmental Agents: Interim Report trations (RfCs), the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) draft reports proposing modifications of EPA’s approach for pesticide testing, the National Toxicology Program (NTP) Roadmap for the Future, and the European Union (EU) strategy in the proposed REACH (Registration, Evaluation and Authorisation of Chemicals) testing scheme for existing industrial chemicals. APPROACHES FOR IMPROVING EXISTING TOXICITY-TESTING STRATEGIES Toxicity-testing guidelines and strategies described in the previous chapters are the results of the gradual evolution of testing requirements and risk-assessment approaches that took place as the field of toxicology advanced. However, agencies have struggled to incorporate recent scientific and technologic advances in toxicology, basic human biology, molecular biology, pharmacokinetics, dose-response modeling, imaging, computation, and other relevant fields, so many of the current requirements are still based on approaches that originated more than 40 years ago. In addition, more sophisticated exposure assessments have identified different durations and routes of exposure for various populations, such as residential exposures of toddlers, that require more toxicology data for risk assessment. Proposed strategies for improving toxicity testing can be difficult to compare directly. Some strategies aim at meeting regulatory mandates and therefore focus on specific needs. For example, the ILSI-HESI documents described below focus on toxicity-testing strategies for pesticides, whereas the REACH program attempts to address the numerous industrial chemicals that have been inadequately studied. The different purposes of those testing strategies contribute to major differences between them. However, there can also be important differences between testing strategies and approaches of initiatives and proposals that try to fulfill the same risk-management needs. The committee elected to focus primarily on the major aspects of the reports reviewed, rather than critiquing the details. Most of the reviewed reports describe initiatives or proposals that are still under development, some of which are sometimes presented with few details; some reports were available to the committee only as drafts. The committee

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Toxicity Testing for Assessment of Environmental Agents: Interim Report reviewed the documents primarily in an effort to compare various testing strategies proposed and to evaluate their potential to move toward the overall objectives of broadening coverage, increasing depth, addressing animal-welfare issues, and conserving resources. In general, the committee did not engage in detailed assessments of individual bioassays, although a few observations related to the alternatives presented by EPA for discussion are included. Environmental Protection Agency Review of Data Needs for Risk Assessment A Review of the Reference Dose and Reference Concentration Processes (EPA 2002), written by the EPA Risk Assessment Forum’s RfD/RfC Technical Panel, provides recommendations for improvements in deriving RfDs and RfCs (see Chapter 5 of the present report for a discussion of RfD and RfC derivation). The committee focused on aspects of the EPA document related to toxicity testing, particularly its Chapter 3, which reviews the adequacy of tests in EPA guidelines (EPA 2005) for deriving RfDs and RfCs for chronic, acute, and other less-than-lifetime exposures. EPA reviewed information generated from currently required acute, short-term, chronic, and specialized toxicity studies. It then identified data gaps with regard to the assessment of life stages, end points, route and duration of exposure, and latency of response and made recommendations to fill the gaps. Options for alternative testing systems were also presented. EPA (2002) was careful to point out that the intent of this review is not to suggest that additional testing be conducted for each and every chemical in order to fill in the information gaps identified for those organ systems evaluated. Nor is it suggested that the alternative testing protocols discussed in this chapter be conducted for every chemical or become part of current toxicology testing requirements or that these alternative protocols are the only options available. Rather, it is the goal of this document to provide a basis for the development of innovative alternative testing approaches and the use of such data in risk assessment.

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Toxicity Testing for Assessment of Environmental Agents: Interim Report Specifically, EPA made a number of observations about the generation of toxicity data under the test guidelines for exposures at different life stages. Most of the standard adult toxicity-testing guidelines for acute, subchronic, chronic, and carcinogenicity testing were not designed to evaluate different life stages, and that has led to substantial gaps (see Figure 6-2). Acute and short-term testing is done only in prenatally exposed animals and in young adults, not in postweaning young animals or aged animals. In addition, only a few toxicologic end points are evaluated in the EPA-required acute toxicity (lethality) studies. EPA discusses how data typically collected in subchronic studies—such as hematologic, clinical, and histologic data—could augment acute studies so that acute RfDs could be developed on the basis of end points other than lethality. Subchronic and chronic toxicity studies are conducted in young adult animals, and exposure in the rodent chronic–carcinogenicity studies continues to the age of 2 years, considered by EPA to be into old age. FIGURE 6-2 Guideline study designs used to derive the oral reference dose. Life stages during which exposure occurs (gray), times at which observations are made, and end points evaluated are indicated. Source: EPA 2002.

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Toxicity Testing for Assessment of Environmental Agents: Interim Report No subchronic or chronic toxicity studies include exposures beginning in the prenatal period, in the early postnatal period (before weaning), or in animals younger than 6-8 weeks old. Tests for reproductive effects provide data on subchronic exposures of animals that are exposed from before birth up to mating of the F1 males and females and through pregnancy of the F1 young adult females. No subchronic-toxicity evaluations are conducted in aged animals. EPA emphasized the need to collect pharmacokinetic data that would help define the internal dose of the active agent to the target site. That information could be used to improve study design, study interpretation, dose scaling, and route extrapolation. EPA also notes that there are no guideline protocols for pharmacokinetic evaluations related to exposures and outcomes during infant development or later in old age. The pharmacokinetic and pharmacodynamic datasets are described as useful in determining the interspecies and intraspecies uncertainty factors and in calculating human equivalent exposure concentrations and doses. As a result of its review, EPA identified several important gaps in the current toxicity-testing framework and concluded that there was minimal evaluation of the following: aged animals in general, but especially after early exposures; some systems and end points (for example, cardiovascular and immunologic) in terms of both structure and function; latency and reversibility; effects of acute and short-term exposure, which are needed to determine acute and short-term RfDs and RfCs; pharmacokinetics; and portal-of-entry effects, especially for substantial dermal exposure. EPA’s recommendations address two main objectives for testing strategies—evaluating a broader array of end points and life stages and increasing information on mechanism or mode of action to improve the human relevance of risk assessment—and include the following: Develop a strategy for alternative approaches to toxicity testing, with guidance on how and when to use existing and newly recommended guidelines. Develop guidelines or guideline study protocols that will provide more systematic information on pharmacokinetics and pharmacodynamics (that is, mechanism or mode of action), which includes information at different life stages. Develop protocols for acute and short-term studies that provide more comprehensive data for setting reference values.

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Toxicity Testing for Assessment of Environmental Agents: Interim Report Modify existing guideline study protocols to provide more comprehensive coverage of life stages for both exposure and outcomes. Collect more information on less-than-lifetime exposures to evaluate latency and reversibility of effect. Develop guidelines or guideline study protocols to assess immunotoxicity, carcinogenicity, and cardiovascular toxicity at different life stages. Explore the feasibility of setting dermal reference values for direct toxicity, including sensitization, at the portal of entry. EPA explored, but did not endorse, different testing protocols for acute, subchronic, and chronic toxicity testing to address gaps pertaining to life stage, duration of exposure, and latency. Specifically, EPA described an alternative acute-toxicity testing protocol and two alternative chronic toxicity-testing protocols. The purpose of the alternative acute-toxicity testing protocol is to provide hazard and dose-response information after a single acute exposure. That protocol uses a control group and at least three dose groups with 10 animals per sex per group. Clinical signs of toxicity are recorded daily, and food consumption and body weights are recorded on days 1-4, 8, and 14. Five animals per sex per group are killed 3 days after dosing, and the remaining animals are killed 2 weeks after dosing. At both times, urinalysis and hematologic and clinical-chemistry analyses are conducted to address potential reversibility and latency of effects within 14 days of dosing. In addition, the animals are necropsied, organ weights are recorded, and the organs are examined histologically. On the basis of other toxicologic data, this study may be conducted with animals at different life stages and include other end points. EPA described two chronic protocols with continuous exposure through all life stages. The first is essentially the in utero carcinogenicity evaluation used by the Food and Drug Administration (FDA) for food additives but with yearly interim kills and study termination when the animals reach the age of 3 years rather than 2 years. It involves exposure before mating and then continuous exposure of offspring. In addition to routine clinical pathology and histopathologic evaluations, unspecified neurotoxicity and immunotoxicity testing would be conducted yearly. The second protocol is a unified screening study that has at least four segments: a two-generation reproduction and fertility study, an expanded chronic–carcinogenicity study (Figure 6-3), a developmental-toxicity

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Toxicity Testing for Assessment of Environmental Agents: Interim Report FIGURE 6-3 Expanded chronic-carcinogenicity study. Source: EPA 2002. study, a developmental-neurotoxicity study, and an optional continuous-breeding study. EPA identified important challenges to the chronic protocols, including the total number of animals needed and the feasibility of conducting studies as complex and large as the two chronic studies without increasing experimental error. EPA presented the chronic protocols “to demonstrate the advantages (and disadvantages) of exploring nontraditional testing paradigms” and noted that using them in a regulatory setting would require thorough discussion between EPA and registrant scientists. Committee’s Evaluation of Environmental Protection Agency Review of Data Needs for Risk Assessment Overall, the committee agrees with EPA’s analysis and conclusions regarding data gaps. For the most part, EPA’s recommendations, if implemented, would improve RfD and RfC development. They encourage the development of innovative toxicity-testing protocols, and following EPA’s recommendations for any one chemical would enhance the depth of information on pharmacokinetics, life stages, and end points available for hazard identification and dose-response assessment. However, such an intensive toxicity-testing approach would probably be applied to only a small fraction of chemicals in commerce, would increase the number of animals used to study one chemical, and might even reduce the numbers of chemicals tested. The EPA analysis and recommendations do not address the overall goals of increasing the breadth of coverage of chemicals, conserving animals, and reducing costs and other expenditures. Ultimately, the EPA recommendations need to be evaluated within a lar-

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Toxicity Testing for Assessment of Environmental Agents: Interim Report ger strategy that considers the uses of toxicity testing for purposes other than to support quantitative risk assessments (for example, chemical-class testing to guide broader screening and coverage). EPA also made recommendations for research on uncertainty factors used in dose-response assessment. The committee agrees with EPA’s conclusion that it is important to research the basis of uncertainty factors. For example, research on the intraspecies uncertainty factor is needed to evaluate whether it adequately addresses the full range of variability of response due to different life stages, genetic susceptibility, and other factors. However, the committee is reserving its detailed comments on the issues surrounding uncertainty factors for its second report. Specifically, the committee identified five major issues raised in the EPA review that require evaluation and comment: (1) the presence of data gaps in current toxicity-testing approaches, (2) a possible need to refine acute-toxicity testing protocols to support short-term risk assessments, (3) concerns about methods to incorporate pharmacokinetic and pharmacodynamic data into toxicity-testing approaches, (4) questions regarding incorporation of data on direct dermal toxicity into RfD development, and (5) a need to reconsider current toxicity-testing strategies systematically with an eye to improving efficiency and effectiveness. In addition to comment on those major issues, the committee offers some general observations on the acute and chronic toxicity-testing protocols that are explored in Section 3.3 of the EPA report as alternatives to guideline studies. Presence of Data Gaps The committee agrees with EPA that there are numerous data gaps in life stages and end points covered in current testing approaches and in functional assessments of some organ systems. However, there are insufficient data to determine the degree to which those data gaps have practical significance in risk assessment or whether they are primarily of theoretical or academic concern. Depending on how the data are used in risk assessment, some of the data gaps may have little effect on the final outcome, whereas others may be very important. The committee cautions against adding testing requirements only for the sake of thoroughness, because such an approach can result in a substantial waste of animals and resources with little gain. The challenge is to try to cover a broader range

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Toxicity Testing for Assessment of Environmental Agents: Interim Report of end points and life stages with improved sensitivity to subtle or functional effects without indiscriminately expanding current toxicity-testing approaches. The committee favors a two-pronged approach in the near term. Such an approach includes relatively modest enhancements of current protocols designed to improve screening for a somewhat wider array of end points in a somewhat broader array of life stages. Such enhanced protocols could trigger more in-depth testing. Adequate second-tier evaluation of such end points as immunotoxicity and cardiovascular toxicity may require the creation of new test guidelines with indications of what would trigger such testing. Research is needed to determine the practical effect of filling or not filling some of the data gaps. In the meantime, it is presumed that current uncertainty and adjustment factors applied by EPA to derive reference values are sufficiently large, although that conclusion has not yet been established. The magnitude of uncertainty factors used in deriving reference values can be reassessed once the enhanced protocols are developed, implemented, and validated and a comprehensive review of outcomes is conducted for a set of test chemicals. It is also important to look beyond animal toxicity data for help in resolving some of the questions. Epidemiologic studies with reliable exposure assessment can shed some light on the likelihood that current toxicity-testing data are missing important end points or are insufficiently sensitive to be applied to life stages not studied. For example, the prospective National Children’s Study1 or other prospective cohort studies that include children and the elderly may be helpful in ascertaining differences in susceptibility. Studies of workers who handle various agents can be of great help in assessing effects on the immune system, cardiovascular system, and functional end points that are not now well assessed with animal studies. However, human data will be available on only chemicals that are already in use and, because of problems with exposure assessment and other difficulties in epidemiologic studies, can be collected only on a small subset of existing chemicals. In addition, legal and ethical issues limit the design of human studies that can be conducted and integrated into toxicity-testing strategies. 1   The National Children's Study can be found at http://nationalchildrensstudy.gov/.

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Toxicity Testing for Assessment of Environmental Agents: Interim Report Refinement of Acute-Toxicity Testing Protocols The committee agrees with EPA that the current protocols for acute toxicity testing focus on lethal effects and gross observations and generally do not provide adequate information for acute and short-term RfDs and RfCs. EPA has identified a regulatory need for improved data for setting acute RfDs and RfCs, and such data are within reach of current toxicity-testing approaches. Improved acute-toxicity assessment may be particularly important in situations where there is high worker exposure and for the development of acute-exposure guideline levels (AEGLs) used to design emergency response plans for accidental releases of chemicals. However, conducting acute protocols that address latency, reversibility, and differential susceptibility for all toxicity end points currently required in subchronic and chronic protocols (such as hematology, clinical chemistry, pathology, functional tests, and detailed clinical observations) will lead to very complex animal studies. Before such complex protocols are conducted, acute LD50 studies, repeated-dose toxicity studies, and human data should be evaluated to determine the need for these studies and ultimately to guide the design of such studies. Pharmacokinetics2 and Pharmacodynamics The committee agrees with EPA that generally little information is available on pharmacokinetics, including possible differences across life stages. Although there is a need to develop more information on pharmacokinetics and pharmacodynamics, it is critical to define the purpose of such studies to avoid the creation of data that are unlikely to be used and that constitute a waste of animals, time, and resources. Data on absorption, distribution, metabolism, and excretion (ADME) should be used in guiding toxicity testing, including identifying the most relevant routes of exposure. Beyond the ADME studies, additional data should not be required routinely but instead case by case. It is important to use caution in drawing major conclusions on the basis of relatively few pharmacokinetic and pharmacodynamic data. For example, such data may not always be sufficiently explanatory to support major conclusions about 2   Pharmacokinetics as used in this chapter encompasses both the quantitative and qualitative aspects of absorption, distribution, biotransformation, and excretion of chemicals.

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Toxicity Testing for Assessment of Environmental Agents: Interim Report NTP work in pharmacokinetics by establishing a minimal pharmacokinetic dataset on agents evaluated by the NTP, developing and validating PBPK models for agents of concern, building a multidisciplinary capacity within the NTP to do the work, and developing training and user-friendly tools. Continue to explore nonmammalian in vivo alternatives to toxicity testing as tools for medium-throughput screening (MTS) of specific toxicity end points. Expand the use of imaging technologies for detecting and quantifying molecular and cellular lesions and for improving the speed and precision of pathology reviews. This approach could increase the statistical power of NTP studies and reduce the number of animals used in the cancer bioassay and other studies. In the near term, the NTP plans to capture digital images from all pathology slides from the cancer bioassay routinely and to develop new image-analyzing techniques to guide the review and evaluation of lesions. A long-term goal is to provide a digital archive of such images from NTP studies that is accessible via the NTP’s website. Most of the above actions are intended to refine and increase the efficiency of the NTP’s toxicity-testing strategy and study portfolio. The NTP Roadmap notes, however, the time-consuming and resource-intensive nature of traditional toxicity testing and the consequently large volume of newly introduced and existing chemicals in commerce that have been inadequately assessed for toxicity. The Roadmap outlines the NTP’s long-range research strategy to develop rapid screening systems for providing toxicity information on large numbers of chemicals. The NTP plans to develop further MTS systems, such as in vivo nonmammalian systems, including C. elegans. High-throughput screening (HTS) may enable the evaluation of thousands of agents rapidly with in vitro biologic systems and robotics technology. The NTP’s emphasis is on cellular targets known to influence disease or data interpretation. The NTP expects initially to target genotoxicity, cytotoxicity, cell proliferation, apoptosis, and some receptor-mediated activities and to run all relevant agents previously tested by the NTP through HTS with a set of other identified chemicals. The databases created with the HTS and MTS can be used to determine the predictive value of the higher-throughput screens. In the short term, the NTP expects HTS to help to set priorities among agents for more extensive testing. In the longer term, the NTP hopes that such

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Toxicity Testing for Assessment of Environmental Agents: Interim Report screens will become sufficiently developed and validated to be usable more directly in public-health decision-making. The NTP has staged its work in this field in phases, as outlined in Box 6-1. Committee Evaluation of the NTP Roadmap The NTP Roadmap is designed both to refine existing toxicity-testing approaches and to develop new approaches for eventual incorporation into toxicity testing. Although the NTP strategy has been presented publicly in bold brushstrokes rather than in detail, the general approaches are of great interest. The NTP’s near-term efforts to refine and extend its toxicity tests and to improve the generation and use of pharmacokinetic and mechanistic data promise to increase the depth of toxicity information on chemicals assayed and to provide greater insight in applying test findings to humans. Furthermore, the NTP’s stated objective of reducing animal use and increasing efficiency may be partly realized. However, as acknowledged by the NTP, the result- BOX 6-1 Roadmap Activities: High-Throughput Screening (HTS) Short-Term Activities Catalog available assays Convene working groups to provide advice on selection of assays Develop assays Identify initial set of chemicals for testing Medium-Term Activities Continue assay development Validate individual assays Develop methods for analysis of data Develop HTS database Review effectiveness Long-Term Activities Develop mechanisms to make chemical sets and tissue banks available for external researchers Evaluate HTS data for predictability of toxicity Develop a communication plan Review effectiveness

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Toxicity Testing for Assessment of Environmental Agents: Interim Report ing test portfolio would still be resource-intensive and incapable of addressing large numbers of chemicals that require some level of toxicity assessment. In the relative near term, the alternative—in vivo nonmammalian MTS—holds promise for improvements in cost, animal use, resources, and throughput, but it generally received little attention in the Roadmap. The extent of the NTP MTS effort is unclear. The NTP’s development of such systems to the point where they may be used in public-health decision-making (for example, NTP identifications of chemicals as reproductive toxicants) would be a major advance. In the long term, HTS has the potential to increase the breadth of chemical assessment substantially while preserving the goal of limiting animal testing. Development of improved biomarkers of effect with new -omics approaches would contribute to all four goals of increasing breadth, increasing depth (if biomarkers provide valid mechanistic information), conserving animals, and reducing costs. A specific focus on developing nonanimal models by an agency, such as the NTP, is needed if such approaches are to become a useful alternative to traditional toxicity testing in animals. COMMITTEE EVALUATION OF SUGGESTED IMPROVEMENTS IN TESTING STRATEGIES Several government agencies and nongovernment organizations have recognized important gaps and inefficiencies in toxicity testing. EPA, ILSI-HESI, the NTP, and the EU have assessed or undertaken initiatives to build on and improve testing strategies. There is much to learn from their varied assessments and strategies. EPA evaluated its testing requirements for pesticides and toxic substances in the context of establishing RfDs and RfCs, found substantial data gaps in life stages studied and end points assessed, and made recommendations for improvements in the near term by using existing toxicity-testing methods and techniques. ILSI-HESI convened a panel of scientists to evaluate EPA’s approaches to toxicity testing for pesticides and developed its own set of recommendations. EPA made ambitious recommendations that would increase the breadth of toxicity testing in two key ways: coverage of different life stages and improved coverage of different end points, such as cardiovascular effects and immunotoxicity. In doing so, EPA explored alternative test protocols that would increase the complexity of some tests but ulti-

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Toxicity Testing for Assessment of Environmental Agents: Interim Report mately conserve animal use while increasing critical toxicity information. ILSI-HESI emphasized potential test redundancies and test elimination. It recommended eliminating the mouse carcinogenicity study and 1-year dog study and making the rat acute toxicity study optional, thereby reducing animal use, the robustness of the database, and the potential to confirm findings. It also included proposals to increase the breadth of testing by modifying tests to evaluate more end points. It did not propose specific protocols to address effects of exposures in the elderly, although it did suggest that special studies could be triggered by findings in first-tier studies in young adult animals. Both EPA and ILSI-HESI recommended increased efforts to generate pharmacokinetic data, a practice already in place at the NTP, which increases the depth of available toxicity data. Both proposals also suggested reductions in group sizes in toxicity testing. Although in some cases the evaluation of end point or life stage may be more detailed, the small group size (for example, 20 vs 50 in the unified study and six vs eight dogs in the systemic-toxicity evaluations) can significantly reduce the statistical power of a study to detect effects. The EU is engaged in a bold effort to restructure its approach to toxicity testing to screen the backlog of tens of thousands of industrial chemicals that have not been adequately assessed. It has taken the approach of attempting to broaden the screening of chemicals maximally while minimizing animal use in existing protocols. It is unclear whether the EU approach will provide adequate depth of information for dose-response assessment, especially with respect to the breadth of coverage of relevant end points and life stages. The approach to obtaining test data for detailed risk assessment is still under development, although for some chemicals (pesticides) schemes similar to those used in the United States are in place. It is therefore difficult to compare the REACH approach directly with the EPA and ILSI-HESI approaches because they are designed primarily to focus on different subsets of chemicals (tens of thousands of existing industrial chemicals vs hundreds of pesticides). The REACH approach includes greater depth of information and coverage of end points and life stages than the current EPA approach to testing of existing or new industrial chemicals under the Toxic Substances Control Act. The NTP has not proposed specific changes in toxicity tests but has initiated a process, involving scientific workshops and public input, that could in the near term lead to fundamental changes in the design and conduct of the bioassays of cancer, reproductive and developmen-

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Toxicity Testing for Assessment of Environmental Agents: Interim Report tal toxicity, and other end points. The NTP is beginning by evaluating and considering changes in cancer-bioassay design. Also in the near term, the NTP seeks to improve testing by capitalizing on recent advances in imaging technologies, devising strategies for routine incorporation of toxicogenomic analyses, further developing nonmammalian test systems, and using transgenic animals. This effort goes considerably beyond the proposals and recommendations of ILSI-HESI and EPA. That is in part understandable in that some of the NTP effort involves technology transfer and the development and validation of new approaches; the changes are not yet sufficiently well defined to be incorporated into mandated test requirements, such as the EPA pesticide test guidelines. However, specific study designs and protocols should be given consideration for such purposes as they emerge from the NTP program. The NTP also is taking a longer view through its initiative to develop rapid screening systems for providing toxicity information on large numbers of chemicals. The NTP approach is the only one that incorporates strategies that have potential for satisfying all four key goals of increasing breadth and depth and decreasing animal use and costs. Yet the NTP HTS of large numbers of chemicals relies in the long term on the development of new methods that are as yet unproved and will have to be evaluated. The NTP HTS approach is many years away from being practical for adoption in testing requirements by a regulatory agency, such as EPA. In the meantime, some of the near-term initiatives at the NTP, as well as strategies proposed in the EPA review and perhaps in the ILSI-HESI papers and in the REACH proposal, may be of use for improving current regulatory toxicity testing. The committee identified several recurring themes and questions in the various reports that it was asked to review: Which environmental agents should be tested? All new and existing environmental agents should be evaluated; however, the intensity and depth of testing should be decided according to practical needs, including the use of the chemical, the likelihood of human exposure, and the scientific questions that such testing must answer to support a reasonable science-policy decision. Fundamentally, the design and scope of a toxicity-testing approach needs to reflect risk-management needs. Regulatory agencies are pursuing different testing strategies depending on whether their goal is generating detailed data for optimal dose-response assessment of pesticides or screening exist-

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Toxicity Testing for Assessment of Environmental Agents: Interim Report ing industrial chemicals. Hence, neither the EPA testing recommendations in its RfD-RfC review nor the EU REACH program is “correct” or “incorrect,” because they are designed for different purposes. How should priorities for testing chemicals be set? Priority-setting should be a key component of any testing strategy that is designed to address a large number of chemicals, and a well-designed scheme is essential for systematic testing of industrial chemicals on which there are few data. It makes sense to consider exposure potential in designing test strategies. Chemicals to which people are more likely to be exposed or to which some populations may receive relatively high exposures—whether they are pesticides or industrial chemicals—should undergo more in-depth testing. This concept is embedded in several existing and proposed strategies. In some strategies, production volume is the primary measure of potential human exposure. But production volume alone may not be the best surrogate of human exposure. Other important factors to consider are use, exposure patterns, and a chemical’s environmental persistence and bioaccumulation, which is important because of the potential for increasing exposure over time and continuing exposure even after use has ceased. Indicators of potential toxicity from existing toxicity data or structural analogues and computational approaches, such as structure-activity relationships, may help to refine priorities further. In addition, there has been some investigation of high-throughput in vitro methods as a possible priority-setting tool. The committee will discuss possible priority-setting approaches in greater depth in its second report. What strategies for toxicity testing are the most useful and effective? Existing test strategies include test batteries, tiered-testing strategies, tailored approaches, and strategies that combine various approaches. The committee finds that there are pros and cons of various approaches but leans toward tiered testing with the goal of focusing resources on the evaluation of the more sensitive adverse effects of exposures of greatest concern rather than full characterization of all adverse effects irrespective of relevance for risk-assessment needs. A tiered-testing approach would require that EPA have clear regulatory authority to require additional testing of pesticides or industrial chemicals beyond the first tier when it is indicated. Such a strategy would also require a priori stopping rules to prevent all chemicals from going through all tiers. For example, some observers have expressed the concern that some potentially “positive” result may often pop up in tiered testing and trigger the next tier of testing of nearly all chemicals, which

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Toxicity Testing for Assessment of Environmental Agents: Interim Report would erase the benefit of tiering. Other observers have expressed the concern that in tiered strategies, strong positive results may simply trigger moving to ever-higher and more time-consuming tiers of testing while delaying risk-management action based on the findings. Both concerns are related to the need to have clear rules that move environmental agents out of the tiered-testing strategy into either a “stop” category or a preliminary risk-management category. One important point regarding the rules is that one should determine whether additional testing is likely to make a difference in human risk assessment. For example, the margin of exposure may be sufficiently large that further testing is not needed to make a decision or the end point in question is unlikely to be more sensitive than other end points of concern, in which case no further testing regarding that end point would be needed. How can toxicity testing generate data that are more useful for human-relevant dose-response assessment? Many observers have criticized existing approaches to toxicity testing on the grounds that it is difficult to use their results in risk assessment. In particular, observed results are often difficult to extrapolate with confidence to humans at environmentally relevant doses. As a result, such extrapolations are often made with little scientific justification, and conventional uncertainty factors are used to bridge the gaps. Current approaches to toxicity testing could be enhanced in some cases by the use of pharmacokinetic data from basic ADME studies to derive dose information before embarking on toxicity testing and by the judicious use of some pharmacokinetic data to aid in extrapolation. In addition, adding lower doses to some protocols could help with obtaining more environmentally relevant information, although to maintain adequate statistical power increased group size at lower doses may be needed, with attendant increases in cost. Using benchmark dose extrapolations in place of NOAELs is another strategy that has been shown to be useful in maximizing the use of the existing dataset. However, the current menu of approaches to toxicity testing is unlikely to solve the fundamental problem. The committee cautions against indiscriminately generating large amounts of data on single chemicals in an effort to generate the optimal dose-response dataset. Such an approach entails the substantial use of animals and often comes at the expense of broader screening that may also be useful. Newer approaches to toxicity testing may help to address this problem and will be discussed in

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Toxicity Testing for Assessment of Environmental Agents: Interim Report the committee’s second report. In the interim, uncertainty factors must still be used to address scientific uncertainties. How can toxicity testing be applied to a broader universe of chemicals, life stages, and end points? It is clear that there are major gaps in current toxicity-testing approaches. The real importance of the gaps is a matter of debate and depends on whether effects of public-health importance are being missed by current approaches. Historically, an important end point or life-stage susceptibility has in many cases been missed in initial toxicity testing. However, it is neither practical nor desirable to attempt to test every chemical (or mixture) against every end point during a wide range of life stages. The committee recommends toxicity screening of every agent to which there is a strong potential for human exposure. A well-designed tiered strategy could help to set priorities among environmental agents for screening and could identify end points or mechanisms of action that would trigger more in-depth testing for various end points or in various life stages. Newer methods of screening that incorporate such tools as -omics or computational toxicology may help to screen more chemicals rapidly and trigger appropriate further testing where necessary. Those approaches are discussed briefly in Chapter 7 of this report and will be discussed in more depth in the committee’s second report. How can environmental agents be screened with the minimal use of animals and minimal expenditure of time and other resources? One strategy that is useful to reduce animal use is the grouping of chemicals of similar structural class and the in-depth testing of only one or a few representative chemicals; risk assessments of all chemicals in the class would then be based on the resulting data. In grouping chemicals, known modes of action (for example, nicotinic agonists) should be emphasized. Such strategies should address any data needed to support application of study findings on the representative chemicals to other chemicals in the group. Newer approaches also have great promise. Chapter 7 discusses current developments in reduction, refinement, and replacement of animals in toxicity testing and newer technologies that hold promise for greatly reducing the reliance on animal testing. How should tests and testing strategies be evaluated? An important consideration in evaluating test strategies is the risk-management context in which they are being applied. For example, intensive study of untested industrial chemicals is of little use if it means that few chemicals can be addressed. However, further explora-

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Toxicity Testing for Assessment of Environmental Agents: Interim Report tory and intensive study of a relatively well-tested chemical can be of great value if costs of controlling exposure are high, there is widespread and relatively high exposure, and study is likely to refine the risk assessment substantially. Test strategies may be evaluated in terms of the value of information they provide in light of the cost of the testing and the animal resources expended. That should be kept in mind in considering testing strategies in terms of the four testing objectives—increasing depth of knowledge for more accurate risk assessment; increasing coverage of chemicals, life stages, and end points; preserving animal welfare; and minimizing cost. In evaluating new tests and test strategies, there remains the difficult question of what is to serve as a “gold standard” for performance. Simply comparing the outcomes of new tests with the outcomes of current tests may not be the best approach; whether it is will depend on the reliability and relevance of the current tests. Another consideration is how test results will be used in the assessment. Even if a test strategy provides robust and informative data, the risk assessor may be unable, because of legal constraints or risk-assessment guidelines, to use the data. Ideally, regulations and risk-assessment guidelines will evolve with testing capabilities and scientific understanding. That issue will increase in importance with greater use of screening approaches that produce indirect evidence (in vitro tests, gene arrays, and mode-of-action screens), for both cancer and noncancer end points. REFERENCES Cooper, V.L., J.C. Lamb, S.M. Barlow, K. Bentley, A.M. Brady, N.G. Doerrer, D.L. Eisenbrandt, P.A. Fenner-Crisp, R.N. Hines, L.F.H. Irvine, C.A. Kimmel, H. Koeter, A.A. Li, S.L. Makris, L.P. Sheets, G.J.A. Speijers, and K.E. Whitby. 2006. A tiered approach to life stages testing for agricultural chemical safety assessment. Crit. Rev. Toxicol. 36 (1):69-98. Doe, J.E., A.R., Boobis, A. Blacker, V.L. Dellarco, N.G. Doerrer, C. Franklin, J.I. Goodman, J.M. Kronenberg, R. Lewis, E.E. McConnell, T. Mercier, A. Moretto, C. Nolan, S. Padilla, W. Phang, R. Solecki, L. Tilbury, B. van Ravenswaay, and D.C. Wolf. 2006. A tiered approach to systemic toxicity testing for agricultural chemical safety assessment. Crit. Rev. Toxicol. 36 (1):37-68. EPA (U.S. Environmental Protection Agency). 1998a. Health Effects Test Guidelines OPPTS 870.1100. Acute Oral Toxicity. EPA 712-C-98-190. Office of Prevention, Pesticides, and Toxic Substances, U.S. Envi-

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