FACTSHEET:
NIEHS and the Use of Alternative Methods
in Toxicological Research and Testing

Q. Why is animal testing important, and why are scientists pursuing alternative methods?

A. To protect the public from harmful effects of toxic substances, and to ensure that medications and consumer products are safe for public use, scientists conduct toxicological testing of these substances in laboratory animals. Without laboratory animals, scientists would lose a fundamental method for obtaining the data needed to make wise decisions about potential health risks to humans. Scientists also want to improve their understanding of the mechanisms by which toxic substances cause harmful effects, which in turn will improve their ability to predict toxicity. Alternative methods, such as in vitro and computer technologies, and the use of nonmammalian organisms, are playing an increasingly important role in this process. Furthermore, scientists continue to develop alternative methods which will improve animal well-being and further minimize the number of animals needed for testing.

Q. What is meant by the term "animal alternatives"?

A. To researchers and others concerned with animal welfare, the term describes approaches to animal testing that incorporate the "three R's" -reduction, refinement, and replacement - while maintaining the quality and validity of scientific information obtained. It is a common misconception that "alternatives" implies the complete elimination of animals for research and testing purposes.

Q. How are scientists applying the "three R's" to animal testing?

A. Reduction refers to strategies, such as sharing of research animals or the modification of experimental designs, to decrease the number of animals needed for a particular study. Refinement is the modification of existing procedures to further enhance the well-being of laboratory animals. The use of new, more effective analgesics, and the development of improved methods to monitor animals and ensure their health, are examples of refinements. Replacement refers to the use of alternative research methods that do not require the use of whole animals, or their replacement with a lower species such as cold-blooded animals or invertebrates. These alternatives will be described in greater detail.

Q. What are in vitro methods, and how are they used?

A. The term "in vitro" refers to experiments performed in laboratory containers, such as test tubes or petri dishes, with living tissues, organs, or cells obtained from animals or people. In vitro testing encompasses a wide range of methods, from the study of sperm cells and frog eggs to the use of ultrasensitive probes to test for toxic effects in human blood and urine. Cell cultures and microorganisms allow researchers to study a single effect or action of a substance in an isolated environment, thereby eliminating interference from other biological phenomena, such as hormones or immune responses. These models are most effective during the early and intermediate stages of the laboratory research process. They are used by researchers to obtain a preliminary indication of how and why a specific chemical may affect a living system. However, the fact that these tests are conducted in isolated systems, independent of other complex biological systems, creates limitations in their interpretation. In the end, the validity of such tests must be estblished by comparing their performance with the results of testing conducted in appropriate mammalian model systems.

Q. What other kinds of alternative testing methods are available?

A. Single-cell organisms such as bacteria and protozoa are increasingly useful in screening chemicals for genetic toxicity. One example is the "Ames" test, in which a compound is tested for its ability to produce mutations in selected strains of bacteria. Positive test results signal the possibility that the compound could cause cancer. Other invertebrate species have also played important roles in the study of biological function. The Nobel-winning experiments that demonstrated the importance of the action potential in nerve cell transmission could not have been done without the giant axons of the squid. The sea urchin is an excellent model for screening some types of chemicals for reproductive toxicity and mutations. Although not often thought of as an alternative, another way in which researchers assess the health effects of chemical exposure is through human epidemiological studies. For example, in one epidemiological approach, a group of people with a known exposure to an environmental agent is compared against a control group on such factors as symptomatology and hospital admissions.

Q. How applicable are mathematical and computer models?

A. Mathematical and computer models, which have a long tradition of use in the biological sciences, can effectively complement animal experimentation. In fact, the use of computers in biomedicine will increase as more biological processes are understood and converted into mathematical forms. Computer models enable scientists to predict the way in which an organism may respond to varying levels of exposure to a certain chemical, and to design better experiments to actually determine the response in a living animal. For the most part, data in computer-based predictive systems are derived from the results of animal studies. Thus, the predictability is only as good as the animal data on which it is based. When our knowledge bases become reasonably complete, it should be possible to use these predictive systems instead of animal screening. In certain cases, confirmative studies in animals may still be necessary.

Q. In what ways have scientists benefitted from computer technology?

A. Although computers do not create scientific data, they permit researchers to view data in more manageable ways. Computer-assisted data banks allow greater accessibility of experimental results to scientists in laboratories all over the world, thus reducing the need for test duplication. More recently, new technologies powered by computers, such as nuclear magnetic resonance spectroscopy, make it possible to observe biological phenomena that previously could only be inferred.

Q. What are the general advantages of using alternative species?

A. Non-mammalian species share a high degree of structural and physiologic similarities with other vertebrate species that enhances their usefulness in the laboratory. Furthermore, the amount of time required to obtain information from toxicity testing is often less with non-mammalian species due to shorter lifespans and developmental periods. In addition, lower species are often easier to handle and less expensive than most mammals. Finally, the knowledge gained from using alternative species may help scientists in their efforts to conserve threatened, endangered, or declining populations of related species. However, these advantages are sometimes the basis for limitations in the use of alternative species.

Q. What are some of these limitations?

A. The results of toxicity tests with lower species may, in some cases,have limited applicability to higher-order animals. For example, some non-mammalian species may metabolize chemicals differently than humans and other mammals. Further studies are needed to understand how the processes of metabolic activation and deactivation are similar or different from one species to another. In the Ames test, not all substances that cause mutations in bacteria are found to cause cancer in animals or man. There are also significant anatomic and physiologic differences between mammals and lower species that must be taken into consideration, and may even limit the range of uses for these species. For example, the evaluation of inhalation toxicity cannot be conducted on fish.

Q. What is the role of NIEHS in advancing these technologies?

A. The National Institutes of Health Revitalization Act, signed by the President in June, 1993, directs the National Institute of Environmental Health Sciences to develop and validate assays and protocols for safety testing, including alternative methods. This act also required the development of specific criteria that will enhance the likelihood that scientifically validated methods will be acceptable to regulatory agencies. A report has been prepared that describes criteria and processes for the validations and regulatory acceptance of new toxicological methods (Validation and Regulatory Acceptance of Toxicological Test Methods: A Report of the ad hoc Interagency Coordinating Committee on the Validation of Alternative Methods).

Q. What is the NIEHS doing to develop alternative methods?

A. The NIEHS, as one of the research agencies of the NATIONAL TOXICOLOGY PROGRAM, will continue to develop and validate new testing methods that will improve our ability to predict the harmful effects of new and existing chemicals. As basic and applied toxicologic research reveal the molecular mechanisms by which chemicals exert their toxic effects, there will be significant opportunities to develop improved alternative test methods and models. These alternative methods and models will include computerized modelling and predictive systems, tissue cultures, transgenic cells and animals (See Transgenic Model Studies at NIEHS), invertebrate species, and non-mammalian species such as fish. NIEHS will also continue to support the Marine and Freshwater Biomedical Sciences Centers in encouraging the use of aquatic organisms as experimental models, and in fostering a better understanding of how environmental chemicals and other factors lead to adverse health effects in humans. The NIH Small Business Innovative Research Grant Program will be used to promote the technology transfer of new knowledge about the mechanisms of toxicity into applied testing methodologies. The following examples illustrate the contributions of Institute researchers to the advancement of alternative methods and models:

• NIEHS scientists are evaluating specific strains of small fish to determine their usefulness in screening chemicals for potential toxicity and carcinogenicity. Because of their genetic uniformity and low spontaneous tumor rates, these strains can minimize the experimental variability normally associated with other species. To find out whether this model can accurately predict cancer risk in higher animals, the fish will be exposed to three compounds that have already been identified as carcinogens in rodents.
  
  
• Institute researchers are also working with crustaceans and fish to study the elimination of foreign chemicals from the body. They are using isolated kidney preparations to explore the biochemical processes within an intact renal cell during excretion. These studies are among the first to demonstrate how some toxic compounds are sequestered within specific intracellular sites, and what effect this process has on the movement of these toxicants through the cell.
  
  

• NIEHS toxicologists are validating a model system known as the Frog Embryo Teratogenesis Assay-Xenopus (FETAX) that can be used to screen chemicals for their potential to cause birth defects. Because FETAX embryos undergo all of the cellular events that characterize mammalian development, this system can potentially detect growth retardation, structural malformation, and behavioral and functional deficits. The U.S. Army's Biomedical Research Laboratory at Fort Detrick, Maryland, is currently evaluating FETAX as a method to detect hazardous agents in contaminated groundwater.
  
  
• Institute scientists, grantees, and contractors are using various genetic strains of the fruitfly Drosophila as a model system for studying spontaneous and induced mutations and the mechanisms of DNA repair and replication in ways that are directly applicable to mammals, including humans. The Drosophila system is also ideal for identifying specific genes, molecularly cloning them, and engineering modified versions of the genes that enable researchers to determine how gene function contributes to the developmental process.
  
  

Q. Where can additional information be obtained?

A. The Center for Alternatives to Animal Testing at Johns Hopkins University has information about programs, publications, resources, symposia and meetings relating to alternatives.

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NTP liaison office: 919/541-0530 | Website: NTP Home Page

 Additional Information:

• NIEHS Health Topic: Alternative Methods and Models