Plan Overview	Preface
Executive Summary	Introduction
CHAPTER 1 - Research, Development, Translation, and Validation Activities for Priority Test Methods to Reduce, Refine, and Replace Animals in Regulatory Testing
CHAPTER 2 - Incorporating New Science and Technology
CHAPTER 3 - Fostering Acceptance and Appropriate Use of Alternative Test Methods
CHAPTER 4 - Developing Partnerships and Strengthening Interactions with ICCVAM Stakeholders
References - Acronyms and Abbreviations - Glossary	Appendices
Acknowledgements - ICCVAM Roster - About NICEATM and ICCVAM

Chapter 2: Incorporating New Science and Technology

NICEATM and ICCVAM will identify and promote research incorporating new technologies that can be expected to support the future development of new test methods and approaches to reduce and eliminate the need for animals. While many of these approaches will require several years of development and validation, some may be ready for use more quickly. To maximize the efficiency of this process, NICEATM and ICCVAM are working with Federal agencies and other stakeholders to link research and development activities to the standardization and validation of alternative test methods that may be used in regulatory testing.

High Throughput Screening

The NTP promotes improvements in toxicology test methods that will enhance its ability to efficiently evaluate large numbers of substances in the environment for which there is little or no information about potential adverse effects. In this regard, NTP is working to identify and develop rapid biochemical or cell-based tests that can be used to screen large numbers of environmental substances for their potential biological activity (that is, high throughput screening [HTS]). The results of HTS experiments provide a starting point for understanding the potential human and animal toxicity of the substances to be tested, and might be useful in setting priorities for more comprehensive testing. The NTP HTS activities are coordinated with similar activities being conducted by the EPA and organizations such as ECVAM. One goal of these studies is to identify batteries of HTS assays that ultimately may reduce or replace the use of animals in toxicological tests. This approach follows the National Research Council’s recently published vision and strategy of toxicity testing in the 21st century, which emphasizes the development of predictive high throughput assays to evaluate alterations to key toxicity pathways. Furthermore, such activities and assays that are developed under the NTP Roadmap will be conducted in cooperation and consultation with ICCVAM to maximize their value to regulatory agencies. In this regard, NICEATM and ICCVAM will facilitate reviews of the usefulness and limitations of defined HTS approaches, and also assist in the identification of assays and endpoints that are relevant for alternative test methods that have already been adopted.

Other Animal Systems Both the NIEHS and the FDA are evaluating the roundworm (Caenorhabditis elegans) for its usefulness as a more rapid method to provide information about potential adverse human health effects of chemicals (see pages at the NIEHS and NIH websites). A short life cycle, easy and inexpensive maintenance and culturing, and detailed knowledge of its biology has allowed for the development of rapid low-cost assays that provide information potentially relevant to various types of toxicity. Because many of the C. elegans genes are the same as those of more complex animals (including humans), it is possible that many of the responses elicited in C. elegans can be related to other species. NICEATM and ICCVAM will evaluate the validation status of future tests with this model system that have utility for regulatory testing. Through the U.S. National Coordinator for the OECD Test Guidelines Program, EPA is working in concert with OECD member countries to develop assays to evaluate various toxicity endpoints in fish and amphibians. This includes a multi-phased project to validate and peer review an amphibian metamorphosis (tadpole) assay, which will help to assess the potential of chemicals to interact with the thyroid system. This work will help assess the utility of these tests for predicting mammalian and non-mammalian effects. NICEATM and ICCVAM will closely follow these efforts, and if considered appropriate, will facilitate evaluation of the validation status of these types of test methods.

The roundworm C. elegans is being evaluated as an alternative species for toxicity testing. Because the genes involved in many biological processes (for example, the stress response) have remained essentially unchanged throughout evolution, responses elicited in C. elegans may be applicable to understanding similar processes in higher organisms, including humans. Testing using this organism can be adapted to automated laboratory systems, which allow for increased throughput.

Computational Approaches

Using data generated from a collection of high throughput bioassays that measure interactions with proteins or genes (e.g. microarrays), EPA is developing computer models for prioritizing chemicals for toxicology testing. This will result in a "toolbox" (referred to as ToxCast™) that will be used for prioritizing chemicals for toxicology evaluation. If the preliminary phases are successful, the EPA will proceed to an implementation phase where profiles of chemicals in need of toxicological evaluation will be obtained and used to develop recommendations for testing priorities.

ATSDR is also developing and applying computational methods to prioritize testing of chemicals of concern and to direct targeted research. Through these activities, ATSDR provides guidance for efficient experimental design, including the determination of appropriate doses for testing chemicals and mixtures.

The DOE is developing computer models for studying the biological effects of radiation. These models will help estimate the minimum number of animals that are needed in experiments dealing with low-dose radiation exposure. They may also help make decisions regarding the possible use of in vitro models instead of live animals.

Biomarkers of Toxicity

The NIEHS and the FDA are evaluating biomarkers that could be used in current toxicology tests to predict damage to a specific organ. Such biomarkers may be used as the basis for early humane euthanasia to reduce or relieve the pain and distress experienced by animals with tumors or chronic disease. They will also support the development of predictive in vitro screening tests. ATSDR and the National Center for Environmental Health, in collaboration with NIEHS, EPA, NCI, the Armed Forces Institute of Pathology, and the International Commission for Occupational Health, recently organized an international conference on Biomarkers for Toxicology and Molecular Epidemiology (Fowler et al., 2005). This conference evaluated advances in biomonitoring technologies and the translation of biomarker endpoints for human epidemiological studies to a number of adverse health outcomes including target organ system toxicity and cancer.

NTP subsequently organized a workshop on Biomarkers for Toxicology Studies to identify biomarkers related to injury or altered function of heart, lung, and lipid/carbohydrate metabolism. These biomarkers could be included in toxicology tests to better understand the development of environmentally induced diseases. As a result, the NTP has begun including serum cholesterol and triglycerides in their clinical pathology panel as routine measures in toxicity tests. Assays for several other biomarkers are undergoing standardization and validation. The NIEHS and the FDA are also exploring gene chip technologies that may allow for the identification of sets of biomarkers that are more predictive of risks or benefits than a single biomarker. NICEATM and ICCVAM will follow progress in these areas.

Toxicology Databases

NIEHS is developing searchable databases of toxicological information that will be made available to the general public via the Internet. These databases will be a source of high quality animal test data that can be used as reference data for comparison to new non-animal test methods. For example, NICEATM will be making a database available that contains rabbit eye test data from ocular toxicity studies. The database will provide the user with detailed protocol information, test substance information, and animal response information. As part of the NICEATM and ICCVAM priority to encourage the development of new test methods, this database will also incorporate other types of toxicity data that can be used for the development/validation of other types of non-animal test methods (for example, dermal toxicity, in vitro cytotoxicity).

The Chemical Effects in Biological Systems (CEBS) Knowledgebase is being developed by NIEHS to promote a systems biology approach to understanding the biological effects of environmental stressors. CEBS will house data derived from studies on the effects of environmental chemicals on genes, proteins, and metabolism. Specifics for each study, including study design details, treatment protocols, animal characteristics and toxic endpoints, will be available. All of these data types can be integrated to enable data query and analysis in a biologically meaningful manner. CEBS contains data from both in vivo and in vitro studies, primarily in rodents, but can house data from other species (for example, humans). This integration of data should improve the understanding of how in vitro endpoints could be used to predict in vivo effects, and aid in overcoming a critical barrier to the replacement of animals in testing. NICEATM and ICCVAM will promote the availability of data from CEBS for use in the development of alternative test methods.

Emerging Needs: Nanomaterials Testing

Nanotechnology is the control of matter at dimensions of roughly 1 to 100 nanometers (a nanometer is one-billionth of a meter; a sheet of paper is about 100,000 nanometers thick), and is being applied in many fields in the physical and biological sciences to create improved materials, devices, and systems. The unique characteristics of nanomaterials can affect their toxicity. Because hazards associated with these types of materials have yet to be characterized, the applicability of current toxicity tests to nanomaterials will have to be evaluated, and new tests may be needed for regulatory use. The number of tests needed to characterize potential hazards of nanomaterials could be very large, as could the number of animals required for such testing. NICEATM and ICCVAM will work with regulators and stakeholders to identify tests that might be useful, while also addressing the 3Rs.

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