2nd Edition 2002 arena/olaw institutional Animal Care and Use Committee Guidebook

minimum pain, distress, anxiety, or apprehension

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minimum pain, distress, anxiety, or apprehension;

  • compatibility with requirement and purpose, including subsequent use of tissue;

  • compatibility with species, age, and health status; and

  • drug availability and human abuse potential.

Recommended Methods

The 2000 Report of the AVMA Panel on Euthanasia categorizes methods as acceptable, conditionally acceptable, or unacceptable under specific circumstances.


  1. Barbiturates (most species)

  2. Carbon dioxide (CO2)- bottled gas only (most species)

  3. Inhalant anesthetics (most species)

  4. Microwave irradiation (mice and rats)

  5. Tricaine methane sulfate (TMS, MS222) (fish, amphibians)

  6. Benzocaine hydrochloride (fish, amphibians)

  7. Captive penetrating bolt (horse, ruminant, swine)

  8. Ether and carbon monoxide are acceptable for many species, but relatively dangerous to personnel.

Conditionally Acceptable (Requires IACUC Approval of Scientific Justification)

  1. Cervical dislocation (birds, small rodents and rabbits)

  2. Decapitation (birds, rodents, some other species)

  3. Pithing (some ectotherms)

  4. Various pharmacological and physical methods


  1. Chloral hydrate, chloroform and cyanide

  2. Decompression

  3. Neuromuscular blockers

  4. Various pharmacological and physical methods

  5. Dry ice-generated Co2

Methods described as conditionally acceptable are considered acceptable when used in deeply anesthetized animals. Some euthanasia methods (e.g., KCl or formalin by intracardiac injection, or exsanguination) are acceptable only under deep general anesthesia.

For more information on methods of euthanasia, see Appendix D.
C.2.c. Humane Endpoints
Animals used in research and testing may experience pain from induced diseases, procedures, and toxicity. The PHS Policy and AWRs state that procedures that cause more than momentary or slight pain or distress should be performed with appropriate sedation, analgesia, or anesthesia. However, research and testing studies sometimes involve pain that cannot be relieved with such agents because they would interfere with the scientific objectives of the study. Accordingly, federal regulations require that IACUCs determine that discomfort to animals will be limited to that which is unavoidable for the conduct of scientifically valuable research, and that unrelieved pain and distress will only continue for the duration necessary to accomplish the scientific objectives.
The PHS Policy and AWRs further state that animals that would otherwise suffer severe or chronic pain and distress that cannot be relieved should be painlessly killed at the end of the procedure, or if appropriate, during the procedure.

Developing Humane Endpoints

Criteria used to end experimental studies earlier in order to avoid or termi-nate unrelieved pain and/or distress are referred to as humane endpoints. An important feature of humane endpoints is that they should ensure that study objectives will still be met even though the study is ended at an earlier point. Ideally, humane endpoints are sought that can be used to end studies before the onset of pain and distress.

It is important to understand that stress may lead to distress when major shifts in biologic function, to which the animal cannot adapt, threaten the animal's well-being. If pain and distress are anticipated, a detailed plan for when and how these will be alleviated should be provided in the protocol.

The plan should include detailed written criteria for the humane endpoints that will be used to determine when animals can be removed from the study, treated, or euthanized. There should be clear directions concerning who can make the decision to euthanize or treat animals, including procedures to follow if a situation arises on weekends, holidays, or in the absence of the responsible study director.

Even if pain or distress are not anticipated, every protocol should contain a contingency plan for dealing with unexpected situations that may arise.
The development and use of humane endpoints can reduce the severity and duration of unrelieved pain and distress. Clinical score sheets can be developed and used to establish humane endpoints for experimental studies. Score sheets are used to record and identify clinical signs and conditions associated with a particular experimental model. Single or multiple clinical signs that are predictive of the current experimental endpoint can then be used to allow for earlier and more humane endpoints.
Establishing and implementing humane endpoints is best achieved by a collaborative effort on the part of investigators, veterinarians, and animal care staff.

Moribund Condition as a Humane Endpoint

Moribund has been defined as "in the state of dying," or "at the point of death." A moribund condition may be an appropriate humane experimental endpoint for some studies where there is the induction of severe disease states and high rates of mortality. Pre-emptive euthanasia of moribund ani-mals can prevent further pain and distress.

Objective data-based criteria that are predictive of impending death can be used to implement timely euthanasia to avoid spontaneous deaths. FDA regulatory testing guidelines allow for humane killing of animals that are moribund. However, it is important to recognize that euthanasia of a mori-bund animal does not eliminate pain and distress that may be experienced during progression to a moribund condition. It should also be noted that while death is not a required endpoint for routine toxicity testing, animals are often found dead during studies. Establishing procedures to detect and humanely euthanize moribund or pre-moribund animals can reduce the number of animals that die spontaneously. In addition to reducing animal pain and distress, euthanasia of moribund animals allows for the collection of tissues and other biologic specimens that may otherwise be lost or rendered unusable when an animal is found dead.
Various clinical signs are indicative of a moribund condition in laboratory animals. These typically include one or more of the following:
 impaired ambulation which prevents animals from reaching food or water,

  • excessive weight loss and emaciation,

  • lack of physical or mental alertness,

  • difficult labored breathing, and

  • inability to remain upright.

Animals should be observed frequently enough to detect signs of impending death so they can be euthanized in a timely manner. When increased morbidity or mortality is expected, a minimum of twice daily observation is recommended. Animals not likely to survive until the next scheduled observation should normally be euthanized. In situations where animals are often found dead, closer and more frequent observation for moribund animals should be considered to reduce spontaneous deaths. Euthanasia of animals that are moribund or experiencing severe pain and distress should always be done in a manner that produces the least possible amount of additional pain and distress.

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