Date: Revised June 2009
Reference:   Administrative Panel on Laboratory Animal Care

DIRECTIONS: Review the following material. Keep copies of guidelines with applicable protocols. You may find it helpful to post a copy of these guidelines in your laboratory. Questions should be forwarded to the APLAC office, 723-4550.

TRAINING: Training in these techniques and the humane treatment of laboratory animals during the procedures is taught by the Veterinary Service Center (VSC) staff. All new personnel who will be performing these techniques should contact VSC staff for training (725-9901).

In order to comply with guidelines governing the use of animals in experimentation, performing humane euthanasia to terminate an animal that is being used in a study is more appropriate than allowing death as an endpoint. Preemptive euthanasia of moribund animals can prevent further pain and distress, as well as provide for the collection of more appropriate tissues and biological specimens for future studies. Protocol Directors (PDs) must euthanize all moribund experimental animals unless there is scientific justification that euthanasia would invalidate the study.

Moribund is defined as "a clinically irreversible condition leading inevitably to death." Animals are considered to be moribund if they manifest any of the following clinical signs:

• Any condition interfering with eating or drinking (e.g. difficulty with ambulation)
• Inability to maintain itself in an upright/sternal position
• Prolonged (> 48 hours) inappetence and/or clinical dehydration
• Signs of severe organ or system involvement such as agonal breathing, cyanosis, paralysis, shock, hemorrhage, debilitating diarrhea or constipation, blindness, and seizures
• Progressive hypothermia (decrease of 4-6ºC in body temperature of rodents)
• Hematologic or biochemical parameters that indicate organ failure
• Unresponsive to noxious external stimuli such as a toe-pinch withdrawal test

If humanely euthanizing a moribund animal would invalidate the study, then scientific justification for using death as an endpoint must be provided in writing as part of the animal care protocol and must be approved by the Administrative Panel on Laboratory Animal Care (APLAC) prior to initiating this procedure. Protocol Directors who receive approval from the APLAC to use death as an experimental endpoint must also agree to the following:

1. To use the minimum number of animals necessary to achieve statistical significance and to use the earliest, most predictive indicator of present or impending adverse effects, other than death, whenever possible.
2. At a minimum, animals must be monitored twice daily (in the early morning and late afternoon, including weekends and holidays) and any animals displaying clinically abnormal behavior must be removed from group housing situations and housed individually with easy access to food and water. Supportive care should also be provided in the form of water-soaked pellets placed on the cage floor, administration of subcutaneous fluids, and provision of a high calorie oral supplement (e.g., Diet Gel®, Nutri-Gel™, Nutrical®).
3. Written records of all monitoring sessions, indicating the time of the observations, the person observing the animals, form(s) of supportive care that were administered, and any observations such as the number of animals displaying clinically abnormal behavior and the number of animals found dead, must be maintained and made available to VSC or APLAC on request.

The development and use of humane endpoints can reduce the duration and severity of an animal’s pain and distress. Clinical score sheets may be helpful in ensuring appropriate observations are made, consistently interpreted, and properly documented. Score sheets are used to document clinical signs and conditions associated with a particular experimental animal model. Single or multiple clinical signs that are predictive of the experimental endpoint can be used to determine earlier and more humane endpoints. (A sample score sheet can be found at reference 2 below).

PROTOCOL DIRECTORS SHOULD NOTE THAT ANY APPROVED USE OF DEATH AS AN EXPERIMENTAL ENDPOINT WILL BE NOTED ON ALL PROTOCOL FORMS AND REGULATORY PAPERS AS BEING IN PAIN LEVEL CATEGORY "E," UNLESS ANALGESICS OR ANESTHETICS ARE PROVIDED TO ALLEVIATE PAIN OR DISTRESS IN THE EXPERIMENTAL ANIMALS.

References

1. Pitts M, et al. Institutional Animal Care and Use Committee Guidebook. 2nd ed. Bethesda: OLAW, 2002.
2. Aldred AJ, Ming CC, Meckling-Gill KA. 2002. Determination of a humane endpoint in the L1210 model of murine leukemia. Contemp Top Lab Anim Sci. 41(2):24-27.
3. Guidelines for Endpoints in Animal Study Proposals, 2005 http://oacu.od.nih.gov/ARAC/Endpoints.pdf
4. Stokes WS. 2000. Reducing unrelieved pain and distress in laboratory animals using humane endpoints. ILAR J 41:59-61.

Contact: APLAC Administrator
Last updated: June 2009