TOXICITY-PATHWAY-BASED RISK ASSESSMENT


PREPARING FOR PARADIGM CHANGE


A Symposium Summary



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TOXICITY-PATHWAY-BASED RISK ASSESSMENT PREPARING FOR PARADIGM CHANGE A Symposium Summary

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Summary of the Symposium In 2007, a committee of the National Research Council (NRC) proposed a vision that embraced recent scientific advances and set a new course for toxicity testing (NRC 2007a). The committee envisioned a new paradigm in which bio- logically important perturbations in key toxicity pathways would be evaluated with new methods in molecular biology, bioinformatics, computational toxicol- ogy, and a comprehensive array of in vitro tests based primarily on human biol- ogy. Although some view the vision as too optimistic with respect to the prom- ise of the new science and debate the time required to implement the vision, no one can deny that a revolution in toxicity testing is under way. New approaches are being developed, and data are being generated. As a result, the U.S. Envi- ronmental Protection Agency (EPA) expects a large influx of data that will need to be evaluated. EPA also is faced with tens of thousands of chemicals on which toxicity information is incomplete and emerging chemicals and substances that will need risk assessment and possible regulation. Therefore, the agency asked the NRC Standing Committee on Risk Analysis Issues and Reviews to convene a symposium to stimulate discussion on the application of the new approaches and data in risk assessment. The standing committee was established in 2006 at the request of EPA to plan and conduct a series of public workshops that could serve as a venue for discussion of issues critical for the development and review of objective, realis- tic, and scientifically based human health risk assessment. An ad hoc planning committee was formally appointed under the oversight of the standing commit- tee to organize and conduct the symposium. The biographies of the standing committee and planning committee members are provided in Appendixes A and B, respectively. The symposium was held on May 11-13, 2009, in Washington, DC, and included presentations and discussion sessions on pathway-based approaches for hazard identification, applications of new approaches to mode-of-action analy- ses, the challenges to and opportunities for risk assessment in the changing paradigm, and future directions. The symposium agenda, speaker and panelist biographies, and presentations are provided in Appendixes C, D, and E, respec- tively. The symposium also included a poster session to showcase examples of 3

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4 Toxicity-Pathway-Based Risk Assessment: A Symposium Summary how new technologies might be applied to quantitative and qualitative aspects of risk assessment. The poster abstracts are provided in Appendix F. This summary provides the highlights of the presentations and discussions at the symposium. Any views expressed here are those of the individual committee members, pre- senters, or other symposium participants and do not reflect any findings or con- clusions of the National Academies. A PARADIGM CHANGE ON THE HORIZON Warren Muir, of the National Academies, welcomed the audience to the symposium and stated that the environmental-management paradigm of the 1970s is starting to break down with recent scientific advances and the exponen- tial growth of information and that the symposium should be seen as the first of many discussions on the impact of advances in toxicology on risk assessment. He introduced Bernard Goldstein, of the University of Pittsburgh, chair of the Standing Committee on Risk Analysis Issues and Reviews, who stated that al- though the standing committee does not make recommendations, symposium participants should feel free to suggest how to move the field forward and to make research recommendations. Peter Preuss, of EPA, concluded the opening remarks and emphasized that substantial changes are on the horizon for risk as- sessment. The agency will soon be confronted with enormous quantities of data from high-throughput testing and as a result of the regulatory requirements of the REACH (Registration, Evaluation, Authorisation and Restriction of Chemi- cals) program in Europe that requires testing of thousands of chemicals. He urged the audience to consider the question, What is the future of risk assess- ment? Making Risk Assessment More Useful in an Era of Paradigm Change E. Donald Elliott, of Yale Law School and Willkie Farr & Gallagher LLP, addressed issues associated with acceptance and implementation of the new pathway approaches that will usher in the paradigm change. He emphasized that simply building a better mousetrap does not ensure its use, and he provided sev- eral examples in which innovations, such as movable type and the wheel, were not adopted until centuries later. He felt that ultimately innovations must win the support of a user community to be successful, so the new tools and approaches should be applied to problems that the current paradigm has difficulty in ad- dressing. Elliott stated that the advocates of pathway-based toxicity testing should illustrate how it can address the needs of a user community, such as satis- fying data requirements for REACH; providing valuable information on sensi- tive populations; evaluating materials, such as nanomaterials, that are not easily evaluated in typical animal models; and demonstrating that fewer animal tests are needed if the approaches are applied. He warned, however, that the new ap-

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5 Summary of the Symposium proaches will not be as influential if they are defined as merely less expensive screening techniques. Elliott continued by saying that the next steps needed to effect the para- digm change will be model evaluation and judicial acceptance. NRC (2007b) and Beck (2002) set forth a number of questions to consider in evaluating a model, such as whether the results are accurate and represent the system being modeled? The standards for judicial acceptance in agency reviews and private damage cases are different. The standards for agency reviews are much more lenient than those in private cases in which a judge must determine whether an expert’s testimony is scientifically valid and applicable. Accordingly, the best approach for judicial acceptance would be to have a record established on the basis of judicial review of agency decisions, in which a court generally defers to the agency when decisions involve determinations at the frontiers of science. Elliott stated that the key issue is to create a record showing that the new ap- proach works as well as or better than existing methods in a particular regulatory application. He concluded, however, that the best way to establish acceptance might be for EPA to use its broad rule-making authority under Section 4 of the Toxic Substances Control Act to establish what constitutes a valid testing method in particular applications. Emerging Science and Public Health Lynn Goldman, of Johns Hopkins Bloomberg School of Public Health, a member of the standing committee and the planning committee, discussed the public-health aspects of the emerging science and potential challenges. She agreed with Elliott that a crisis is looming, given the number of chemicals that need to be evaluated and the perception that the process for ensuring that com- mercial chemicals are safe is broken and needs to be re-evaluated. The emerging public-health issues are compounding the sense of urgency in that society will not be able to take 20 years to make decisions. Given the uncertainties surround- ing species extrapolation, dose extrapolation, and evaluation of sensitive popula- tions today, the vision provided in the NRC report Toxicity Testing in the 21st Century: A Vision and a Strategy offers tremendous promise. However, Gold- man used the example of EPA’s Endocrine Disruptor Screening Program as a cautionary tale. In 1996, Congress passed two laws, the Food Quality Protection Act and the Safe Drinking Water Act, that directed EPA to develop a process for screening and testing chemicals for endocrine-disruptor potential. Over 13 years, while three advisory committees have been formed, six policy statements have been issued, and screening tests have been modified four times, no tier 2 proto- cols have been approved, and only one list of 67 pesticides to be screened has been generated. One of the most troubling aspects is that most of the science is now more than 15 years old. EPA lacked adequate funding, appropriate exper- tise, enforceable expectations by Congress, and the political will to push the

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6 Toxicity-Pathway-Based Risk Assessment: A Symposium Summary program forward. The fear that a chemical would be blacklisted on the basis of a screening test and the “fatigue factor,” in which supporters eventually tire and move on to other issues, compounded the problems. Goldman suggested that the following lessons should be learned from the foregoing example: support is needed from stakeholders, administration, and Congress for long-term invest- ments in people, time, and resources to develop and implement new toxicity- testing approaches and technologies; strong partnerships within the agency and with other agencies, such as the National Institutes of Health (NIH), are valu- able; new paradigms will not be supported unless there are convincing proof-of- concept and verification studies; and new processes are needed to move science into regulatory science more rapidly. Goldman concluded that the new ap- proaches and technologies have many potential benefits, including improvement in the ability to identify chemicals that have the greatest potential for risk, the generation of more scientifically relevant data on which to base decisions, and improved strategies of hazard and risk management. However, she warned that resources are required to implement the changes: not only funding but highly trained scientists will be needed, and the pipeline of scientists who will be quali- fied and capable of doing the work needs to be addressed. Toxicity Testing in the 21st Century Kim Boekelheide, of Brown University, who was a member of the com- mittee responsible for the report Toxicity Testing in the 21st Century: a Vision and a Strategy reviewed the report and posed several questions to consider throughout the discussion in the present symposium. The committee was formed when frustration with toxicity-testing approaches was increasing. Boekelheide cited various problems with toxicity-testing approaches, including low through- put, high cost, questionable relevance to actual human risks, use of conservative defaults, and reliance on animals. Thus, the committee was motivated by the following design criteria for its vision: to provide the broadest possible coverage of chemicals, end points, and life stages; to reduce the cost and time of testing; to minimize animal use and suffering; and to develop detailed mechanistic and dose-response information for human health risk assessment. The committee considered several options, which are summarized in Table 1. Option I was es- sentially the status quo, option II was a tiered approach, and options III and IV were fundamental shifts in the current approaches. Although the committee ac- knowledged option IV as the ultimate goal for toxicity testing, it chose option III to represent the vision for the next 10-20 years. That approach is a fundamental shift—one that is based primarily on human biology, covers a broad range of doses, is mostly high-throughput, is less expensive and time-consuming, uses substantially fewer animals, and focuses on perturbations of critical cellular re- sponses.

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7 Summary of the Symposium TABLE 1 Options for Future Toxicity-Testing Strategies Considered by the NRC Committee on Toxicity Testing and Assessment of Environmental Agents Option I Option II Option III Option IV In Vivo Tiered In Vivo In Vitro and In Vivo In Vitro Animal Animal Primarily human Primarily human biology biology biology biology High doses High doses Broad range of Broad range of doses doses Low throughput Improved throughput High and medium High throughput throughput Expensive Les expensive Less expensive Less expensive Time-consuming Less time- Less time- Less time-consuming consuming consuming Relatively large Fewer animals Substantially Virtually no animals number of animals fewer animals Apical end points Apical end points Perturbations of Perturbations of toxicity pathways toxicity pathways Some in silico and in In silico screens In silico screens vitro screens possible Source: Modified from NRC 2007a. K. Boekelheide, Brown University, presented at the symposium. Boekelheide described the components of the vision, which are illustrated in Figure 1. The core component is toxicity-testing, in which toxicity-pathway assays play a dominant role. The committee defined a toxicity pathway as a cel- lular-response pathway that, when sufficiently perturbed, is expected to result in an adverse health effect (see Figure 2), and it envisioned a toxicity-testing sys- tem that evaluates biologically important perturbations in key toxicity pathways by using new methods in computational biology and a comprehensive array of in vitro tests based on human biology. Boekelheide noted that since release of the report, rapid progress in human stem-cell biology, better accessibility to human cells, and development of bioengineered tissues have made the committee’s vi- sion more attainable. He also noted that the toxicity-pathway approach moves away from extrapolation from high dose to low dose and from animals to hu- mans but involves extrapolation from in vitro to in vivo and between levels of biologic organization. Thus, there will be a need to build computational sys- tems-biology models of toxicity-pathway circuitry and pharmacokinetic models that can predict human blood and tissue concentrations under specific exposure conditions.

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8 Toxicity-Pathway-Based Risk Assessment: A Symposium Summary FIGURE 1 Components of the vision described in the report, Toxicity Testing in the 21st Century: A Vision and a Strategy. Source: NRC 2007a. K. Boekelheide, Brown Univer- sity, presented at the symposium. Source Fate/Transport Exposure Tissue Dose Toxicity Pathways: Cellular response pathways that, when Biologic Interaction sufficiently perturbed, are expected to result in adverse Perturbation health effects. Biologic Normal Biologic Inputs Function Early Cellular Significance of perturbation Changes will depend not only on magnitude but on underlying Adaptive Stress Cell nutritional, genetic, disease, Injury and life-stage status of host. Responses Morbidity and Mortality FIGURE 2 Perturbation of cellular response pathway, leading to adverse effects. Source: Modified from NRC 2007a. K. Boekelheide, Brown University, modified from sympo- sium presentation.

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9 Summary of the Symposium Boekelheide stated that the vision offers a toxicity-testing system more fo- cused on human biology with more dose-relevant testing and the possibility of addressing many of the frustrating problems in the current system. He listed some challenges with the proposed vision, including development of assays for the toxicity pathways, identification and testing of metabolites, use of the results to establish safe levels of exposure, and training of scientists and regulators to use the new science. Boekelheide concluded by asking several questions for consideration throughout the symposium program: How long will it take to im- plement the new toxicity-testing paradigm? How will adaptive responses be distinguished from adverse responses? Is the proposed approach a screening tool or a stand-alone system? How will the new paradigm be validated? How will new science be incorporated? How will regulators handle the transition in test- ing? Symposium Issues and Questions Lorenz Rhomberg, of Gradient Corporation, a member of the standing committee and chair of the planning committee, closed the first session by pro- viding an overview of issues and questions to consider throughout the sympo- sium. Rhomberg stated that the new tools will enable and require new ap- proaches. Massive quantities of multivariate data are being generated, and this poses challenges for data handling and interpretation. The focus is on “normal” biologic control and processes and the effects of perturbations on those proc- esses, and a substantial investment will be required to improve understanding in fundamental biology. More important, our frame of reference has shifted dra- matically: traditional toxicology starts with the whole organism, observes apical effects, and then tries to explain the effects by looking at changes at lower levels of biologic organization, whereas the new paradigm looks at molecular and cel- lular processes and tries to explain what the effects on the whole organism will be if the processes are perturbed. People have different views on the purposes and applications of the new tools. For example, some want to use them to screen out problematic chemicals in drug, pesticide, or product development; to identify chemicals for testing and the in vivo testing that needs to be conducted; to establish testing priorities for data-poor chemicals; to identify biomarkers or early indicators of exposure or toxicity in the traditional paradigm; or to conduct pathway-based evaluations of causal processes of toxicity. Using the new tools will pose challenges, such as distinguishing between causes and effects, dissecting complicated networks of pathways to determine how they interact, and determining which changes are adverse effects rather than adaptive responses. However, the new tools hold great promise, particularly for examining how variations in the population affect how people react to various exposures.

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10 Toxicity-Pathway-Based Risk Assessment: A Symposium Summary Rhomberg concluded with some overarching questions to be considered throughout the symposium: What are the possibilities of the new tools, and how do we realize them? What are the pitfalls, and how can we avoid them? How is the short-term use of the new tools different from the ultimate vision? When should the focus be on particular pathways rather than on interactions, variabil- ity, and complexity? How is regulatory and public acceptance of the new para- digm to be accomplished? THE NEW SCIENCE An Overview John Groopman, of Johns Hopkins Bloomberg School of Public Health, began the discussion of the new science by providing several examples of how it has been used. He first discussed the Keap1-Nrf2 signaling pathway, which is sensitive to a variety of environmental stressors. Keap1-Nrf2 signaling pathways have been investigated by using knockout animal models, and the investigations have provided insight into how the pathways modulate disease outcomes. Re- search has shown that different stressors in Nrf2 knockout mice affect different organs; that is, one stressor might lead to a liver effect, and another to a lung effect. Use of knockout animals has allowed scientists to tease apart some of the pathway integration and has shown that the signaling pathways can have large dose-response curves—in the 20,000-fold range—in response to activation. Groopman stated, however, that some of the research has provided cau- tionary tales. For example, when scientists evaluated the value of an aflatoxin- albumin biomarker to predict which rats were at risk for hepatocellular carci- noma, they found that the biomarker concentration tracked with the disease at the population level but not in the individual animals. Thus, one may need to be wary of the predictive value of a single biomarker for a complex disease. In an- other case, scientists thought that overexpression of a particular enzyme in a signaling pathway would lead to risk reduction, but they found that transgene overexpression had no effect on tumor burden. Overall, the research suggests that a reductionist approach might not work for complex diseases. Groopman acknowledged substantial increases in the sensitivity of mass spectrometry over the last 10 years but noted that the throughput in many cases has not increased, and this is often an underappreciated and underdiscussed aspect of the new paradigm. Groopman concluded by discussing the recent data on cancer genomes. Sequence analysis of cancer genomes has shown that different types of cancer, such as breast cancer and colon cancer, are not the same disease, and although there are common mutations within the same cancer type, the disease differs among individuals. Through sequence analysis, the number of confirmed genetic contributors to common human diseases has increased dramatically since 2000. Genome-wide association studies have shown that many alleles have modest

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11 Summary of the Symposium effects in disease outcomes, that many genes are involved in each disease, that most genes that have been shown to be involved in human disease were not pre- dicted on the basis of current biologic understanding, and that many risk factors are in noncoding regions of the genome. Sequencing methods and technology have improved dramatically, and researchers who once dreamed of sequencing the human genome in a matter of years can now complete the task in a matter of days (see Figure 3). Groopman concluded by stating that the sequencing tech- nology needs to be extended to experimental models so that questions about the concordance between effects observed in people and those observed in experi- mental models can be answered. Gene-Environment Interactions George Leikauf, of the University of Pittsburgh, discussed the current un- derstanding of gene-environment interactions. In risk assessment, human vari- ability and susceptibility are considered, and an uncertainty factor of 10 has tra- ditionally been used to account for these factors. However, new tools available today are helping scientists to elucidate gene-environment interactions, and this research may provide a more scientific basis for evaluating human variability and susceptibility in the context of risk assessment. Leikauf noted that genetic disorders, such as sickle-cell anemia and cystic fibrosis, and environmental dis- orders, such as asbestosis and pneumoconiosis, cause relatively few deaths com- pared with complex diseases that are influenced by many genetic and environ- mental factors. Accordingly, it is the interaction between genome and envi- ronment that needs to be elucidated in the case of complex diseases. 1,000,000,000 100,000,000 Massively parallel sequencing Single 10,000,000 Kilobases per day per machine molecule? 1,000,000 Short-read 100,000 Capillary sequencers sequencing 10,000 Microwell Gel-based systems pyrosequencing 200 Second-generation Automated capillary sequencer 100 Manual slab gel First-generation capillary slab gel 10 1980 1980 1985 1990 1995 2000 2005 2010 Future Year FIGURE 3 DNA sequencing output. Current output is 1-2 billion bases per machine per day. The human genome contains 3 billion bases. Source: Stratton et al. 2009. Reprinted with permission; copyright 2009, Nature. J. Groopman, Johns Hopkins Bloomberg School of Public Health, presented at the symposium.

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40 Toxicity-Pathway-Based Risk Assessment: A Symposium Summary Panel Discussion The afternoon session closed with a panel discussion that focused on the definition of toxicity pathway, an issue raised during several presentations. Faustman noted that defining a toxicity pathway is context-dependent. For ex- ample, apoptosis is generally seen as an adverse effect, but if apoptosis did not occur during development, humans would have fins. So time, place, and re- sponse make a difference. Goodsaid added that he would be hesitant to label a pathway as a toxicity pathway in isolation. For FDA, pathway information helps the agency to make regulatory decisions; mechanistic data allow the agency to interpret other test data. Waters stated that ideally what should be identified are pathways that are consistently and measurably changed within 2 weeks—or possibly even 4 weeks—of exposure and that are indicative and predictive of some outcome downstream that is recognized as a toxicity end point. She noted, however, that her definition raises the controversy about distinguishing an adverse response from an adaptive response; until gene expression, protein abundance, or some other measure has been evaluated over the dynamic cycle, one cannot distin- guish whether it is a time-dependent expression of some adaptive response or truly a dose-dependent change indicative of toxicity. Thomas stated that his work has not focused on defining a toxicity path- way itself, but on grouping pathways according to common biologic function to make predictions. Faustman noted that there are differences between the various analytic tools and approaches that are being used to define pathways and that the differences could affect how one defines a pathway. The tools and approaches are only as good as the data that go into them, and the scientific community has not yet developed an unbiased approach for integrating pathway information. Rhomberg stated that toxicity pathway might not be the best descriptive term. Normal biologic processes need to be understood, as does how the proc- esses are changed by various agents and result in toxicity. Furthermore, the processes are not linear sequences of events but networks of interactions. Thus, Rhomberg concluded that the focus should be on discovering the key pathway components and how they are integrated to make the functional modules dis- cussed by Waters. One participant questioned, however, whether one needed to understand what a toxicity pathway was; perhaps one could use the models de- scribed during the symposium to derive a benchmark dose for a response, calcu- late a tissue dose, and then extrapolate to a human exposure that would drive the given response. Thomas and Kedderis agreed, but Kedderis noted that it is im- portant to evaluate mechanisms when dealing with unknowns, agents on which few, if any, data are available. Thomas countered that one can provide guidance on reference doses or risk-specific doses by using genomic approaches and thus bridge the gap between making decisions on the basis of reasonable scientific data and not making decisions because of lack of data and simply ignoring the possible risks posed by exposure to the unknowns. He added that if genomic

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41 Summary of the Symposium profiles are obtained on six specific tissues, one can predict about 90% of all positive responses by using rat and mouse bioassays and thus obtain valuable information for risk assessment. WHAT THE FUTURE HOLDS Where Are We Going? or, Are We There Yet, and Where Is There The final session of the symposium was devoted to presentations and dis- cussions on visions for the future and the path forward. Preuss began the session by providing a perspective on the changing landscape of risk assessment and the need for modernization. He stated that advances in understanding of the gene environment and the pending test data from the European REACH program are driving the need for change. He described two paradigms for understanding the effects of toxic chemicals. One is the human-disease model in which genetic profiles of people with and without a disease are compared to yield fingerprints of disease and susceptibility, and the other is the current animal-testing model in which chemically induced events are matched to rodent test results and rodent modes of action. Preuss noted that the new science and technologies should al- low movement between paradigms, although the two approaches will probably progress in tandem for many years. However, the question now is how to move from assessing a few chemicals each year to assessing thousands of chemicals each year as the REACH program anticipates. Dossiers on 40,000 chemicals are expected by 2012, and the U.S. government is ill prepared to use the volume and complexity of information resulting from that or a similar program. Anticipating the need for change, EPA sponsored several NRC reports over the last few years that focused on toxicity testing and risk assessment (for example, NRC 2007a, 2008, 2009). Overall, those reports concluded that risk-assessment data needs cannot be met with the current testing methods, that scientists need to determine how to use the new data being generated for risk assessment, and that the trans- formation of risk assessment has to occur with stakeholder input. Preuss described EPA’s dual approach to developing the next generation of risk assessments. First, EPA is considering creating a high-priority list of chemicals and streamlining the process for assessment by narrowing the scope, using off-the-shelf risk approaches, and focusing and coordinating stakeholder reviews. Second, EPA is considering broadening the scope of some assessments to synthesize more information into each assessment, such as assessments on cumulative effects of agents that cause the same effect or on families of chemi- cals that are physically similar. EPA intends to explore the new science, meth- ods, and policies that could be incorporated into emerging and future risk as- sessments with the primary goal of mapping a path forward. Preuss listed many questions that will need to be addressed, including, How can the new informa- tion best be incorporated into risk assessment and used to inform risk managers?

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42 Toxicity-Pathway-Based Risk Assessment: A Symposium Summary What new policies and procedures will be needed? How can EPA ensure that decision-makers, the courts, and Congress see the new approach as an accept- able way to proceed? EPA’s strategy for the next generation of risk assessment is to implement the framework presented in the NRC report Science and Deci- sions (NRC 2009); to develop an operational knowledge of bioinformatics, data mining, and gene-environment databases to support risk-assessment work; and to develop prototype examples of increasingly complex assessments that are responsive to risk context and refined through discussions with scientists, risk managers, and stakeholders. Preuss concluded that EPA estimates that it may take a decade before risk assessment can rely primarily on the new advances in science, but it is necessary to begin now to address the needed changes. Bucher continued the discussion by providing a perspective from the Na- tional Institute of Environmental Health Sciences (NIEHS) and noted that cur- rent NTP research includes many projects that are amenable to high-throughput screening and high-content data analysis. Furthermore, NTP has made a com- mitment to the development and use of high-throughput screening that could be used to set priorities among chemicals for further in-depth toxicologic evalua- tion, to identify mechanisms of toxicity, and to develop predictive models of in vivo biologic responses in humans. NTP’s commitment is consistent with its vision, articulated 5 years ago, of supporting the evolution of toxicology from an observational science to a predictive one that is based on a broad array of target- specific, mechanism-based biologic observations. Bucher noted that several people had commented during the symposium that tools need to be developed to handle the enormous volume of data being generated. He described a program at NIEHS led by Christopher Portier that is attempting to create such a tool. The approach is to use knowledge about genes associated with diseases to determine pathways linked to the genes and thus link the pathways to the diseases (that is, to elucidate “disease” pathways). The next step is to use toxicogenomic and proteomic databases on well-studied chemicals to link chemicals to diseases through pathways and then to analyze the toxicity pathways to find the best points for screening, such as critical nodes or connec- tion points. –Omics and other molecular tools can then be used to validate the choices. What is ultimately created is an interaction network (see Figure 14). Bucher concluded that NTP expectations for the 21st century are to continue to refine traditional methods and to develop new methods to generate information on mechanisms, exposure-response relationships, and life-stage and genetic sus- ceptibility that will allow better prediction of toxicity to humans and ultimately better protection of public health; to reconcile results from new data-rich tech- niques—such as genomics, proteomics, and high-throughput screens—with ex- isting testing information for conceptual validation; and to develop approaches to accomplish formal validation of new methods for human hazard and risk es- timations. Tina Bahadori, of the American Chemistry Council Long-Range Research Initiative, continued the discussion of the future vision and path forward by pro-

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43 Summary of the Symposium FIGURE 14 Interaction network that can be used to associate environmental factors with toxicity pathways and associated human diseases. Source: Gohlke et al. 2009. Reprinted with permission; copyright 2009, BMC Systems Biology. J. Bucher, National Institute of Environmental Health Sciences, presented at the symposium. viding an industry perspective. She stated that an unprecedented opportunity exists to improve the scientific understanding of the potential effects of chemi- cals on human health and the environment. New technologies to test the effects of chemicals have the potential to revolutionize risk assessment, and if it is done in a scientifically robust way, risks could be understood better, faster, and less expensively. The concern, however, is that the technology is advancing faster than the means to interpret the data accurately. Although investments are made to generate volumes of data, comparable investments to interpret the data are lacking; without investment in the “science of interpretation,” the tendency will be to rely on high-throughput hazard data as a surrogate for risk assessment. Bahadori stated that scientists need to determine how information is going to be translated into a real-world context to protect public health and the envi- ronment. Information on host susceptibility and background exposures will be needed for interpretation and extrapolation of in vitro test results. Furthermore, information on actual human exposure will be needed for selection of doses for toxicity testing so that hazard information can be developed on environmentally relevant effects and for determination of whether concentrations that perturb

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44 Toxicity-Pathway-Based Risk Assessment: A Symposium Summary toxicity pathways are biologically relevant. Scientists also will need to under- stand the progression from exposure to effect. Exposure science will need to predict and link exposure among all levels of biologic organization and to use the new technologies to characterize exposure. Bahadori emphasized the need to invest in the new tools and technologies and to stay committed to the long-range vision, recognizing that it may be years before the new science can be used to advance risk assessment. She concluded by saying that her organization has de- veloped a research strategy to support the vision and is investing in research on interpreting the new data being generated, on developing innovative approaches to characterize biologically relevant exposures and their relation to health risk, and on determining the genetic influences and gene-environment interactions of susceptible populations. Roger Ulrich, of Calistoga Pharmaceuticals, provided a perspective from the pharmaceutical industry on what the future holds. The key difficulties that the pharmaceutical industry faces are late-stage failures and product withdraw- als, which are extremely expensive and reduce the ability to reinvest in research; the erosion of consumer confidence in and the increased consumer expectations for product safety; the paucity of new products; and the shift of risk and product development from large pharmaceutical companies to small ones. To overcome the difficulties, Ulrich stated, the industry must focus on the pipeline and do a better job of assessing products, and this requires more thorough preclinical as- sessment of toxicity and more research on mechanisms and affected biologic processes or pathways. The goal is to identify prognostic biomarkers—markers that tell what might happen rather than markers that tell what has happened (di- agnostic biomarkers). He also stated that the industry must focus on patients and identify at-risk people or populations in parallel with clinical development. Ulrich stated that the new technologies can help to improve the processes of drug discovery and development. They can help to identify molecular path- ways involved in pharmacologic and toxicologic mechanisms; this will help in making decisions as to whether to pursue specific compounds earlier in the process. The new technologies can also help to identify potential biomarkers and to detect adverse drug effects in animals that do not necessarily result in the ex- pression of an adverse outcome in an animal model. Regarding the patient, the new technologies can be used to understand the idiosyncrasies that may increase the risk or decrease the benefit for individual patients. For example, they can be used to identify genetic defects or susceptibilities that can lead to adverse events in response to specific drugs. Thus, the vision for the drug industry is to use con- temporary tools to understand the full spectrum of drug effects on biologic pathways in the preclinical phase before formal development, to overlay drug- response networks on patient phenotype and genetic networks to understand individual patient risk, and to identify benefits, as well as risks, and apply the knowledge in prescription practices. In the next 5 years, Ulrich stated, scientists will continue to develop and explore the potential of the new tools and tech- nologies, but the excitement will be in the development of in silico models and

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45 Summary of the Symposium application of new discoveries to the clinical setting. He concluded by listing several resources needed, including an open-source genetic and functional ge- nomic data platform, most likely funded by government, and training for the next generation of scientists. Helmut Zarbl, of the University of Medicine and Dentistry of New Jersey, continued the discussion with an academic perspective on the future vision and discussed the NRC report Applications of Toxicogenomic Technologies to Pre- dictive Toxicology and Risk Assessment (NRC 2007c), which was released shortly after Toxicity Testing in the 21st Century. The committee that produced the report was asked to evaluate the status of toxicogenomics and to discuss po- tential applications, including applications to risk assessment. It concluded that toxicogenomic technologies have strong potential to affect decision-making but are not ready to replace existing testing regimens in risk assessment and regula- tory toxicology; that toxicogenomics can provide information to be added to the weight of evidence for refining risk judgments; and that ultimately the technolo- gies are envisioned as more sensitive and informative than existing technologies and have the potential to replace some current approaches or tests. That commit- tee recommended a human toxicogenomics initiative to accomplish the follow- ing tasks: create and manage a large public database for storing and integrating the results of toxicogenomic analysis with conventional toxicity-testing data; assemble toxicogenomic and conventional toxicologic data on hundreds of com- pounds into a single database; create a centralized national biorepository for human clinical and epidemiologic samples; develop bioinformatic tools—such as software, analysis, and statistical tools—further; consider ethical, legal, and social implications of collecting and using toxicogenomic data and samples; and coordinate subinitiatives to evaluate the application of toxicogenomic technolo- gies to the assessment of risks associated with chemical exposures. Zarbl concluded by discussing the path forward and stated that improve- ments in technology and science often build on previous knowledge and that scientists should not abandon the tools and knowledge of classical toxicology and risk assessment. He continued, saying that the paradigm shift will require a reduction in reliance on apical end points, and the challenge will be to validate toxicogenomic data to ensure that they are predictive of outcomes that occur much further downstream. Thus, the path for the next several years will be to develop in vitro assays, tools, and strategies; to continue to populate public da- tabases of curated data; to invest in systems toxicology and computational tools for pathway-based risk assessment; to incorporate toxicogenomic data into the weight of evidence for risk assessment; and to continue to explore and validate the utility of the pathway-based approach for risk assessment. Further in the future, pathway-based approaches may be used for routine hazard screening of both new and legacy compounds. Zarbl again highlighted, however, the need for validation of the new approaches before they become stand-alone processes, and he cautioned that if the assays yield negative results, we need to proceed with care to ensure that nothing was missed.

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46 Toxicity-Pathway-Based Risk Assessment: A Symposium Summary Gina Solomon, of the Natural Resources Defense Council, closed with a perspective from an environmental-advocacy group and stated that she is en- couraged by the research presented at the symposium. However, she noted that many people have concerns about the new direction, and some effort will be needed to persuade the broader community that the new approach has merit. The NRC report Toxicity Testing in the 21st Century offered the hope of rapid, cost- effective, animal-sparing testing of thousands of chemicals and a chance to in- tervene to prevent pathway perturbations before the occurrence of disease. Many data have been generated since publication of that report, but few of them have been incorporated or used in risk assessment, and the backlog of 80,000 chemi- cals remains. Solomon noted that the situation is reminiscent of EPA’s Endo- crine Disruptor Screening Program (EDSP), which was originally designed to screen thousands of chemicals but was delayed for more than a decade by an overcumbersome validation process. She stated that the EDSP has not lived up to its promise, and the scientific community needs to work hard not to repeat the history of that program. She acknowledged that some effort will be required to bring the new science into the process of regulatory decision-making in a timely fashion. However, the revision and reauthorization of the Toxic Substances Con- trol Act now under way may help to facilitate the adoption of new scientific tools. Solomon concluded that at the end of the day, if the pathway-based assays are concerned only with generating more in-depth, detailed mode-of-action data on the same subset of old chemicals, the new paradigm will fail. However, if it can deal with the backlog of chemicals and foster faster, health-protective haz- ard identification and risk assessment, it will be heralded as an important and valuable advance. Panel Discussion The symposium closed with a panel discussion focused on questions about and hopes for advancing the new paradigm. Preuss expressed concern that the field is quickly becoming, if it is not already, extremely complex and that distin- guishing important signals from ones that are not important is going to be chal- lenging. Furthermore, validating the sophisticated, complex models and ap- proaches is going to present another challenge. The new paradigm will not be successful if scientists create models that are intelligible only to a select few. The fear is that the research community is far outpacing the ability of the regula- tory community to use the information that is generated and to make intelligent decisions about it. If that happens, much will fall by the wayside. The problem is that the EPA’s research budgets have decreased each year to the point where EPA now has 50% of the purchasing power that it had 10 years ago. The avail- able resources are not commensurate with the kind of effort needed. Zarbl noted that pathway predictions at this point may be premature given that a majority of gene expression is still not understood and that huge gaps must be addressed before the data should be used in risk assessment. Ulrich agreed

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47 Summary of the Symposium that huge gaps exist but stated that the gaps are being systematically filled. His concern was that realistic expectations be set. For example, it is unrealistic to expect that in 10 years scientists will be able to screen 200,000 chemicals in cell cultures and know the human risks on the basis of the resulting data. Bahadori added that the paradigm will fail if perfection of the science is required. Infor- mation that can inform and improve risk assessment will be available soon, if it is not already available. The scientific community needs to determine the level of knowledge needed for different purposes, that is, what is good enough for a given task. Bahadori expressed concern, however, about funding and empha- sized the need to articulate the value of the current effort to ensure that resources are available. The scientific community needs to create a compelling case for requesting resources. Bucher remarked on the need expressed throughout the symposium for creating a large, publicly accessible data platform and the need for a concerted effort to overcome the inertia that might exist in creating such a platform. He stated that expectations for the new science and approaches will not be fulfilled if the necessary computational and analytic tools are not made available. Zarbl clarified that what is needed is a platform where data are curated; standards would have to be met to enter data so that they are standardized or uniform. What is being proposed is not simply a data repository. The question arose about what must be accomplished in the near term to illustrate the value of the new science. Bucher commented that the results of high-throughput screening should be used to design and guide current toxicity testing. The resulting data can help to move standard toxicology models for- ward. Zarbl stated that studies that demonstrate that pathway-based risk assess- ment can produce results at least as good as standard approaches (such as the studies described by Thomas earlier in the symposium) are the milestones needed. Ulrich noted that high priority should be given to a concerted effort to pull together the existing knowledge to create a cohesive and comprehensive output. Kenny Crump, of Environ, concurred: the key is to start applying and using the data and comparing the results with those of the current path. That would illuminate the gaps and help to differentiate between data that EPA might need and data that some other application would need. Hal Zenick, of EPA, commented that application of the new approaches may depend on what information is needed. One size does not fit all risk as- sessments. For example, the information needed to make a clean-up decision will be quite different from that needed to set exposure guidelines on a nation- ally pervasive bioaccumulative chemical. The nature of the question that needs to be answered may determine how soon the new approaches can be applied to risk assessment and risk management. Whenever the new science is finally used to make risk-assessment and risk-management decisions, several noted, a major challenge will be risk communication, that is, explaining the new approaches to policy-makers and the public.

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