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ILAR Journal V35(3/4) 1993 [FORMERLY ILAR NEWS]
Issues for Institutional Animal Care and Use Committees (IACUCs)

IRAC Recommendation on LD₅₀ Testing
Interagency Research Animal Committee (IRAC)

INTRODUCTION

The Interagency Research Animal Committee (IRAC) recommendations on LD₅₀ testing presented below was adopted by IRAC on June 23, 1993. IRAC is a focal point for federal agencies to discuss issues involving all animal species needed for biomedical research and testing, especially their care, use, and conservation. Responsibilities include promoting information exchange, contributing to developing unified federal policies, coordinating animal programs among agencies, and representing the U.S. government on international issues. Agencies represented on the committee are the Veterans Administration, Department of Energy, National Aeronautics and Space Administration, Environmental Protection Agency, Department of the Interior, Department of State, Department of Defense, National Science Foundation, U.S. Department of Agriculture, Consumer Product Safety Commission, and Department of Health and Human Services, including the National Institutes of Health (NIH), Fogarty International Center, Centers for Disease Control and Prevention, Office of International Health, the Health Research Services Administration, and the Food and Drug Administration. The NIH serves as the lead agency and Dr. James F. Taylor, Office of Animal Care and Use, NIH, is the chairman. These recommendations were prepared by a subcommittee as a revision of recommendations adopted in October 1989. The subcommittee included Drs. Kallash Gupta, Consumer Product Safety Commission; Helene Guttman, U.S. Department of Agriculture; Louis Sibal, NIH; William Stokes, National Institute of Environmental Health Sciences; and James Vickers, Food and Drug Administration.

RECOMMENDATION

The LD₅₀ test evaluates acute lethality from exposure to a substance or product. An LD₅₀ value is the dose at which 50 percent of the test animals can be expected to die. The test is used to (1) classify substances or products for regulatory purposes, including safe transportation and labeling; (2) provide information for treatment of acute intoxications; (3) standardize certain biological products; (4) set dose levels for subsequent toxicity studies; (5) provide comparative information on the chemical's dose response curve; and (6) provide data for evaluation and validation of alternative test methods. The LD₅₀ tests have become controversial among toxicologists, animal welfare organizations, legislators, and the public primarily due to the ethics of using a large number of animals and evaluating only mortality. The Interagency Research Animal Committee (IRAC) studied this issue in depth and recommends the following:

1. The Classical LD₅₀ test should only be conducted when specifically justified for reasons of scientific necessity and approved by the institutional animal care and use committee (IACUC).
2. Toxicity testing procedures based on the principles of reduction and refinement (such as the Limit test) should be used until alternative test methods become validated.

BACKGROUND

IRAC has reviewed the policies and recommendations of federal agencies and international organizations relevant to the LD₅₀ test. These organizations include the Environmental Protection Agency, the Food and Drug Administration, the Consumer Product Safety Commission, the Department of Transportation, the National Toxicology Program¹, the Organization for Economic Cooperation and Development, the British Toxicology Society, and the European Chemical Industry Ecology and Toxicology Center. There is consistency among the policies and recommendations on the following points:

1. The use of the Classical LD₅₀ test, which may require the use of 100 or more animals to establish the desired statistical confidence limits and evaluates only mortality, is unnecessary for the determination of acute oral toxicity.
2. Tests involving animals are currently essential in the determination of acute oral toxicity, which is a step in the assessment of the potential hazard of a chemical or product.
3. There are recommended alternatives to the Classical LD₅₀ test using the principles of reduction and refinement (see Summary of Current Policies).
4. A validated in vitro test or battery of tests is presently not available that can be used as a replacement for tests on animals in the determination of the LD₅₀ dose. There are tests in various stages of development and validation. Instead of waiting for a validated test (or tests) for the universe of chemicals or products, the process of development and validation may be accelerated using a group of related chemicals or products. For that group, a validated test (or tests) could be accepted as a screen or as a replacement of the LD₅₀ test on animals.

DEFINITIONS

Classical LD₅₀
The Classical LD₅₀ test is used to determine the lethal dose (LD) of a substance that will kill 50 percent of test animals. Typically, this method can use 100 or more animals. The test material is administered in increasing doses, usually five or more, to groups of 10 male and 10 female animals. Mortalities are recorded within a given period, and the LD₅₀ is determined with the aid of statistical calculations.

Limit Test
The Limit test is used to determine if the toxicity of a test substance is above or below a specified dose. Five to 10 animals of each sex or 10 animals of the susceptible sex are administered a dose specified by regulations. Toxic responses occurring within a given period are recorded. Based on the results, a regulatory action or additional testing may be required.

SUMMARY OF CURRENT POLICIES

U.S. Organizations

Consumer Product Safety Commission:

• Strongly discourages Classical LD₅₀ test.
• Recommends other alternatives--existing animal data, prior human experience, expert opinion.
• Recommends the Limit test.

• Discourages the use of the Classical LD₅₀ test.
• Recommends the use of existing animal data and human experience.
• Recommends the Limit test.

• Discourages the use of the Classical LD₅₀ test.
• Uses Structure Activity Relationships (SAR) to obviate the need for testing on animals.
• Recommends the Limit test.
• When testing beyond the Limit test is conducted, recommends use of abbreviated test methods such as the approximate lethal dose, the moving average, and the up-and-down methods. Stresses the need for multiple endpoint evaluations, including onset, nature, reversibility of effects, and gross necropsy.

• Does not require the use of the Classical LD₅₀ test.
• Accepts altematives
• Refers to the Limit test.

• Does not use Classical LD₅₀ test.
• Uses in-depth toxicology with many endpoints.

• Discourages the use of the Classical LD₅₀ test.
• Recommends the Limit test (2 g/kg dose).
• When compound related mortality occurs in the Limit test, recommends five animals per dose and recommends using at least three dose levels to produce a range of toxic effects and mortality rates. Clinical observations and pathological investigations should be conducted.
• Also recommends a fixed dose procedure, which uses morbidity instead of mortality as the endpoint.

• Recommends that the LD₅₀ test only be determined with any accuracy where scientifically and ethically justified. Such cases are relatively rare.
• Recommends examination of few animals in detail rather than many for statistical purposes.
• Allows that Limit tests could be used, provided animals in distress are killed humanely unless this would interfere with the study objectives.
• For classification of substances and preparations, suggests using a fixed-dose procedure targeted to acute signs to replace the current practice of LD₅₀ determination.

• Recognizes importance of acute toxicity profiles.
• Maintains that the Classical LD₅₀ test is seldom necessary.
• Maintains that protocols exist for estimating lethal dose.
• Believes that the LD₅₀ above 2 g/kg is irrelevant.
• Recommends that regulations not specify a minimum number of animals.
• Encourages procedures for the selection of a toxicity class.
• States that acute toxicity data from some products may be unnecessary for the protection of human health.
• Encourages predictions based on alternatives as an aid to dose selection.

¹ The National Toxicology Program includes the National Institute of Environmental Health Sciences of the National Institutes of Health, the National Center for Toxicological Research of the Food and Drug Administration, and the National Institute for Occupational Health and Safety of the Center for Disease Control.

Adopted by IRAC--September 22, 1993.

BIBLIOGRAPHY

Alternative Methods in Toxicology. 1984. Vol. 2. Acute Toxicity Testing: Alternative Approaches. A. M. Goldberg, ed. New York: Mary Ann Liebert, Inc. Publishers.

British Toxicology Society. 1984. A new approach to the classification of substances and preparations on the basis of acute toxicity. Human Toxicology. 3:85-92.

Consumer Product Safety Commission. 1984. Animal Testing Policy. Fed. Reg. 49:22522-22523.

Department of Transportation. 1992. Poisonous material. Class 6, Division 6. l-Definitions. 49 CFR Section 173.132.

European Chemical Industry Ecology and Toxicology Center. 1985. Acute toxicity tests, LD₅₀, (LC₅₀) determinations and alternatives. Monograph 6, Brussels, Belgium.

Environmental Protection Agency. 1988. Revised policy for acute toxicity testing. Office of Pesticides and Toxic Substances. September 22, 1988.

Food and Drug Administration. 1988. LD₅₀ Test Policy. Federal Register 53:39650-39651.

Organization for Economic Cooperation and Development. 1987. Guidelines for testing of chemicals. Section 4: Health Effects. Addendum to Test Guideline 401. Fixed Dose Procedure. Paris, France.

SOT Position Paper -- Comments on the LD₅₀ and Acute Eye and Skin Irritation Tests. 1989. Fundam. Appl. Toxicol. 13:621-623.

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