This policy establishes the ARC expectation for application of humane interventions to minimize pain and distress, and sets out criteria that warrant prompt premature euthanasia.
Humane Interventions
Humane interventions are actions designed to prevent or relieve unnecessary pain or distress including, but not limited to, the following:
- Increasing the frequency of animal observations.
- Modification to the housing and husbandry practices to improve the comfort of the animal(s).
- Veterinary treatment, including analgesia and/or supportive therapy.
- Modification of the experimental procedures to minimize discomfort to the animal(s)
- Short-term termination of painful/stressful procedures.
- Permanent termination of painful/stressful procedures i.e. removal of the animal(s) from the study.
- Euthanasia.
Required Endpoints
Unless otherwise described in an approved protocol, the ARC expects that animals will be promptly euthanized when one or more of the following general conditions are observed:
- Weight loss exceeding 20% of baseline bodyweight. For growing animals, failure to maintain normal weight gain within 15% of age-matched control animals.
- Body Condition Score (BCS) of less than 2.
- Grimace Scale[1] > 2.
- Uncontrollable seizures, incoordination, or paralysis.
- Hunched posture combined with lethargy and lack of grooming.
- Decreased mobility that interferes with normal eating, drinking or grooming.
- No or weak response to external stimuli.
- Respiratory distress, as indicated by increased or decreased breathing rate, labored breathing, or cyanosis.
- Pale eyes, ears and/or extremities.
- Uncontrolled bleeding.
- Self-mutilation.
- Specific organ failure.
- Mass that is ulcerated, necrotic, and/or impairing normal function.
Frequency of observations
Normal, healthy experimental animals must be observed at least once a day, as described in the ARC Policy on Monitoring Laboratory Animals.
Animals in studies involving pain and/or distress will often require more frequent observations to effectively determine the time at which a specific endpoint has been reached. An appropriate monitoring schedule must be specified in the ARC protocol for each study.
Moribund animals
“Moribund” refers to an animal that is near death or in the process of dying. Animals in this state are often comatose (unresponsive and unaware of stimuli) and beyond awareness of suffering. The moribund condition is defined as a clinically irreversible condition leading inevitably to death.
Commonly used signs of moribundity include, but are not limited to:
- Lack of responsiveness to manual stimulation;
- Immobility; and/or
- Inability to eat or drink.
An animal may experience pain and/or distress prior to reaching a moribund state; therefore, stating that animals will be euthanized when they become moribund is not appropriate, as euthanasia at this late stage will not reduce or alleviate suffering that the animal will likely experience prior to reaching this state. The purpose of identifying humane endpoints is to prevent or minimize animal pain and distress.
Death as an Endpoint [2]
With the goal of minimizing animal pain and distress, investigators are required to administer euthanasia in death endpoint experiments, rather than allowing the animal to expire. If euthanasia will compromise experimental validity, this must be justified in an approved protocol. Moreover, animals in these experiments must be monitored at least daily (including weekends and holidays) by personnel trained and experienced in recognizing signs of morbidity. More frequent monitoring may be indicated for some experimental protocols. For experiments where scientific validity requires allowing an animal to expire as an endpoint, the experimental design must reflect a rigorous effort to minimize the number of animals used.
LD50 Experiments and Alternative Approaches
The traditional LD50 (lethal dose 50) experiment to measure toxicity is widely criticized as inhumane and wasteful of animals. This procedure involves groups of animals that are exposed to increasing doses of a toxic substance or infectious agent to determine the dose required to kill 50% of the animals. As reviewed in the references below, alternative methods have been developed that reduce the number of vertebrate animals used in LD50 experiments without compromising experimental reliability. One approach that has been used to derive median values with relatively few samples is the up-and-down procedure. Whereas conventional methods using a dose-response experimental design generally employ 24 to 40 subjects, LD50 values calculated through the up-and-down procedure can use as few as six animals. The up-and-down procedure yields essentially identical LD50 values as the conventional dose-response experiments. Those investigators whose work requires death as an endpoint must review current statistical approaches to reducing animal numbers and apply these methods whenever possible.
References
- Erhirhie E.O., Ihekwereme C.P., Ilodigwe E.E. 2018. Advances in acute toxicity testing: strengths, weaknesses and regulatory acceptance. Interdiscip. Toxicol. 11(1):5-12.
- Saganuwan S.A., 2017. Toxicity studies of drugs and chemicals in animals: An overview. Bulg. J. Vet. Med. 20 (4): 291–318.
- Parasuraman, S. 2011. Toxocological screening. J. Pharmacol. Pharmacother. 2(2):74-9.
- Aldred A.J., Cha M.C., Meckling-Gill K.A. 2002. Determination of a humane endpoint in the L1210 model of murine leukemia. Contemporary Topics 41(2):24-27.
- Rispin A., Farrar D., Margosches E., Gupta K., Stitzel K., Carr G., Greene M., Meyer W., McCall D. 2002. Alternative methods for the medial lethal dose (LD50) test: The up-and-down procedure for acute oral toxicity. ILAR J. 43(4):233-43.
- Warn P.A., Brampton M.W., Sharp A., Morissey G., Steel N., Denning D.W., Priest T. 2002. Laboratory Animals 37:126-131.
- Sass N. 2000. Humane endpoints and acute toxicity testing. ILAR J. 41(2):114-23.
- Lichtman A.H. 1998. The up-and-down method substantially reduces the number of animals required to determine anti-nociceptive ED50 values. J. Pharmacol. Toxicol. Methods 40:81-5.
- Hamm T.E. 1995. Proposed Institutional Animal Care and Use Committee guidelines for death as an endpoint in rodent studies. Contemporary Topics 34(3):69-71.
- Laboratory Animal Welfare Bibliography, prepared by Scientists Center for Animal Welfare for the USDA National Agriculture Library, December 1989.
- Tomasovic S.P., et. al. 1988. IACUC evaluation of experiments requiring death as an endpoint. Lab Animal 17:31-34.
- UKCCCR Guidelines for the Welfare of Animals in Experimental Neoplasia. 1988. Laboratory Animals 22:195-201. Reprinted in ILAR News 31:16-23 (1989) with Commentary.
- Spinelli J.S., Morrish R.H. 1987. Pain and discomfort: How to recognize and manage pain associated with animal research. Invest. Radiol. 22:348-352.
- Morton D.B., Griffiths P.H.M. 1985. Guidelines on the recognition of pain, distress, and discomfort in experimental animals and an hypothesis for assessment. The Veterinary Record 116:431-436
[1] NC3Rs: Grimace Scales
[2] This section of the Policy was previously a standalone Policy on Death as an Endpoint, initially approved in 1990 and last updated in 2009.
Approved 7/8/2019