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Research Animals - Reduction, Refinement, Andreplacement (the 3rs)

pain distress corrosive iccvam

In 1959 scientists William Russell and Rex Burch published a book entitled Principles of Humane Experimental Technique that advocated three principles for the animal research industry: reduction, refinement, and replacement. The authors called these principles "the three R's for the removal of inhumanity" in the scientific community.

The book was largely ignored until the 1980s, when public protest against the use of animals in laboratory testing became more widespread. Scientists and animal welfare organizations then embraced the 3R's as scientifically reasonable and humane goals for the industry. The 3R's, however, are guiding principles, not legal requirements.

The 3R's are defined as follows:

  • Reduction is a goal to reduce the number of animals used in research overall by reducing the number required for individual experiments or areas of study without sacrificing the statistical validity of the results. In other words, researchers are urged to use statistics to determine the minimum number of animals that can be used in an experiment and still provide valid data. Another goal is to reduce the number of procedures that require whole animals. For example, tissues from an animal used in one experiment could be used in other experiments in place of live whole animals.
  • Refinement is a goal to refine experimental and care practices to reduce animal suffering and distress and encourage well-being. Such practices include the use of painkillers during and after experiments, the use of humane euthanasia techniques, and improvements in animals' living environments.
  • Replacement is a goal to replace live laboratory animals with suitable alternatives (for example, computer simulations) and to replace higher animal species with lower species.

The Search for Alternatives to Animal Tests

In 1993 the National Institutes of Health Revitalization Act was passed, requiring formation of an agency to oversee validation of alternatives to toxicological animal testing. The result was the Interagency Coordinating Committee for the Validation of Alternative Methods (ICCVAM) and the National Toxicology Program Inter-agency Center for the Evaluation of Alternative Toxicological Methods (NICEATM).

The ICCVAM is responsible for establishing validation criteria and for encouraging government agencies that regulate toxicity testing to accept validated methods. The NICEATM facilitates information sharing among all the parties involved.

The ICCVAM and NICEATM work with fifteen federal agencies:

  • Agency for Toxic Substances and Disease Registry
  • Consumer Product Safety Commission
  • Department of Agriculture
  • Department of Defense
  • Department of Energy
  • Department of the Interior
  • Department of Transportation
  • Environmental Protection Agency
  • Food and Drug Administration
  • National Institutes of Health
  • National Cancer Institute
  • National Institute of Environmental Health Sciences
  • National Institute for Occupational Safety and Health
  • National Library of Medicine
  • Occupational Safety and Health Administration

The ICCVAM also works with the European Centre for the Validation of Alternative Methods (ECVAM) to coordinate international validation efforts.

Between 1999 and 2005 the ICCVAM has recommended two alternative tests to regulatory agencies: the local lymph node assay (LLNA) and Corrositex. LLNA is a mouse-based test for determining if new chemicals cause allergic contact dermatitis (skin reactions). The traditional test for this condition used guinea pigs. The LLNA is reported to use fewer animals and cause much less pain and distress than the traditional test. It is also much faster.

Corrositex is an in vitro test in which synthetic skin is used to test chemical irritancy. In vitro is a Latin phrase that means "in glass." In vitro tests are commonly conducted in test tubes. The traditional test for skin irritancy relied on rabbits and could take several weeks. The new one takes just a few minutes or hours.

In May 2004 the ICCVAM published recommended performance standards for two additional in vitro skin corrosion tests: EPISKIN and EpiDerm (EPI-200). These are proprietary tests developed by commercial companies. The ICCVAM notes that these tests could be used to satisfy corrosivity testing required by the U.S. Environmental Protection Agency, U.S. Department of Transportation, and the United Nations (which has a system for classifying and labeling chemicals transported internationally). The new tests would replace the current testing protocol in which corrosive substances are placed on the skin of living animals (usually rabbits) for specific lengths of time, as shown in Table 5.7. The extent of tissue damage in the animals is assessed after the exposure time to determine the corrosivity of the chemicals. The ICCVAM report states that use of the new tests could "avoid pain and distress that may result from the application of corrosive substances to animals."

Traditional skin corrosivity testing performed on the skin of living animals
SOURCE: "Table 1-1. Skin Corrosive Category and Subcategories," in Recommended Performance Standards for In Vitro Test Methods for Skin Corrosion, Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM), May 2004, (accessed March 17, 2005)

Corrosive category (category 1) (applies to authorities not using subcategories) Potential corrosive subclassesa (UN packing group classificationb) Corrosive in at least 1 of 3 animals
Exposure Observation
Corrosive Corrosive subcategory 1A (I) ≤3 minutes ≤1 hour
Corrosive subcategory 1B (II) >3 minutes/≤1 hour ≤14 days
Corrosive subcategory 1C (III) >1 hour/≤4 hours ≤14 days
aClassifications designated by the United Nations (UN) Globally Harmonised System for the Classification and Labelling of Chemical Substances and Mixtures (GHS).
bCorresponding UN packing group classifications to be used for the transport of dangerous goods.

Pain and Distress

One of the goals of refinement is to relieve animal pain and distress. The USDA tracks the occurrence of pain and distress in regulated animals, as shown in Table 5.8. These numbers are based on reports by research institutions to the USDA for fiscal year 2002. During that year, 57% of the regulated animals (646,886) experienced no pain or distress, 34% experienced pain or distress but were administered drugs for relief (387,560), and 9% suffered pain and distress but were not given drugs for relief (103,272). Hamsters and guinea pigs were the species most involved in experiments in which pain and distress were not relieved. More than 80,000 of them fell into this category during 2002. In addition, nearly 2,500 dogs, cats, and primates also suffered pain and distress that was not relieved.

Animal welfare groups have expressed doubts about the validity of USDA pain and distress numbers, saying that these numbers are greatly underreported by research institutions. In 1998 the HSUS launched a Pain and Distress Initiative to focus attention on issues involved in assessing and relieving pain in laboratory animals. The HSUS publishes a quarterly newsletter entitled Pain and Distress Report to publicize these issues. The goal of the initiative is to eliminate pain and distress in research animals by the year 2020.

The HSUS acknowledges that animal rights advocates want to eliminate animal testing, not reform it. The organization states, "The HSUS would like to see the day when animals are no longer used in harmful research. However, we believe the most urgent public priority is eliminating pain and distress among laboratory animals."

Scientists recognize that eliminating pain and distress in laboratory animals is not only humane but good scientific practice. The animal use policy at Vanderbilt University, for example, acknowledges that experimental results can be compromised by a physical or mental state of distress in the subject and recommends relieving pain and distress in animal subjects.

One concept embraced by the HSUS is the use of humane endpoints. This means that test animals can be humanely euthanized after exhibiting specific symptoms of a disease rather than dying of the disease itself. The October 2002 issue of Pain and Distress Report described a study performed to determine a humane endpoint for the L1210 model of murine leukemia. Researchers injected mice with lymphocytic leukemia cells and recorded their symptoms and appearance every six TABLE 5.8
Animals used at USDA-registered facilities, categorized by pain and/or distress and relief measures, fiscal year 2002
SOURCE: Adapted from "Animals Used in Research," in Animal Care Report: Annual Report of Enforcement by Fiscal Year, 2002, U.S. Department of Agriculture, Animal and Plant Health Inspection Service, 2003, (accessed March 17, 2005)

No pain and/or distress––no drugs were needed for relief Pain and/or distress––drugs were used for relief Pain and/or distress––no drugs could be used for relief Total animals used
Guinea pigs 148,220 61,848 35,508 245,576
Rabbits 140,560 96,954 6,324 243,838
All other covered species 120,900 48,442 11,146 180,488
Hamsters 86,607 48,161 45,232 180,000
Pigs 23,491 42,794 2,204 68,489
Dogs 32,615 34,214 1,424 68,253
Non-human primates 25,554 25,937 788 52,279
Other farm animals 37,968 10,642 278 48,888
Sheep 16,462 9,129 94 25,685
Cats 14,509 9,439 274 24,222
    Total number of animals 646,886 387,560 103,272 1,137,718
    Percent of total 57% 34% 9%
Note: This excludes rats, mice, birds, and cold-blooded animals.

hours until all of the mice died. They concluded that a humane endpoint was the appearance of hunched posture, decreased activity, or abdominal swelling in two consecutive examinations or the appearance of two specific symptoms during a single examination. According to the HSUS, the researchers concluded that early euthanasia at these humane endpoints would not compromise the research results but would substantially reduce the pain and distress experienced by the mice.

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