Classification of Animal Pain and Stress

Classification of Animal Pain and Distress Levels

For the purpose of this categorization, assume that any procedure known to cause pain or distress to humans will have a similar effect on other vertebrate animals. The Animal Welfare Act requires placing animal usage into categories according to potential pain and distress and the reporting of such usage annually to the U. S. Department of Agriculture.


Classification B: Animals being bred, conditioned, or held for use in teaching, testing, experiments, research, or surgery, but not yet used for such purposes. Examples:

  • Breeding colonies of any animal species that are held in legal sized caging and handled in accordance with the Guide for the Care and Use of Laboratory Animals and other applicable regulations. Breeding colony includes parents and offspring.
  • Newly acquired animals that are held in proper caging and handled in accordance with applicable regulations.
  • Animals held under proper captive conditions or wild animals that are being observed.

Classification C: Animals upon which teaching, research, experiments, or tests will be conducted involving no pain, distress, or use of pain-relieving drugs. Examples:

  • Procedures performed by trained personnel such as the administration of electrolytes/fluids, administration of oral medication, blood collection from a common peripheral vein per standard veterinary practice (dog cephalic, cat jugular) or catheterization of same, standard radiography, parenteral injections of non-irritating substances.
  • Euthanasia performed in accordance with the recommendations of the most recent AVMA Panel on Euthanasia, utilizing procedures that produce rapid unconsciousness and subsequent humane death.
  • Manual restraint that is no longer than would be required for a simple exam; short period of chair restraint for an adapted nonhuman primate.

Classification D: Animals upon which experiments, teaching, research, surgery, or tests will be conducted involving accompanying pain or distress to the animals and for which appropriate anesthetic, analgesic, or tranquilizing drugs will be used. Examples:

  • Surgical procedures conducted by trained personnel in accordance with standard veterinary practice such as biopsies, gonadectomy, exposure of blood vessels, chronic catheter implantation, laparotomy or laparoscopy.
  • Blood collection by more invasive routes such as intracardiac or periorbital collection from species without a true orbital sinus such as rats and guinea pigs.
  • Administration of drugs, chemicals, toxins, or organisms that would be expected to produce pain or distress but which will be alleviated by analgesics.

Classification E: Animals upon which teaching, experiments, research, surgery, or tests will be conducted involving accompanying pain or distress to the animals and for which the use of appropriate anesthetic, analgesic, or tranquilizing drugs will adversely affect the procedures, results, or interpretation of the teaching, research, experiments, surgery, or tests. Examples:

  • Procedures producing pain or distress unrrelieved by analgesics such as toxicity studies, microbial virulence testing, radiation sickness, and research on stress, shock, or pain.
  • Surgical and postsurgical sequella from invasion of body cavities, orthopedic procedures, dentistry or other hard or soft tissue damage that produces unrelieved pain or distress.
  • Negative conditioning via electric shocks that would cause pain in humans.
  • Chairing of nonhuman primates not conditioned to the procedure for the time period used.

Note regarding classification E: An explanation of the procedures producing pain or distress in these animals and the justification for not using appropriate anesthetic, analgesic or tranquilizing drugs must be provided in Kuali. This information is required to be reported to the USDA, will be available from USDA under the Freedom of Information Act, and may be publicly available through the Internet via USDA's website.

Consideration of Alternatives

If any procedures fall into USDA's Classification D or E, causing more than momentary or slight pain or distress to the animals, describe your consideration of alternatives and your determination that alternatives are not available. Delineate the methods and sources used in the search. Database references must include databases searched, the date of the search, period covered, and the keywords used. Alternatives include methods that

  • refine existing tests by minimizing animal distress
  • reduce the number of animals necessary for an experiment, or
  • replace whole-animal use with in vitro or other tests

When ascites production is used to produce antibodies, justification needs to be given as to why in vitro systems cannot be used. Note that you must certify in Section Q.5. that no valid alternative was identified to any described procedures which may cause more than momentary pain or distress, whether relieved or not.

Death as an Endpoint

In order to comply with guidelines governing the use of animals in experimentation, the use of death as an endpoint to experimental manipulation, rather than performing euthanasia to humanely terminate an animal, is strongly discouraged. Investigators must perform euthanasia on all moribund experimental animals unless there is scientific justification that euthanasia would invalidate experimental data collection. Moribund is defined as "in a dying state". Animals are considered to be moribund if the following criteria are met:

  1. They manifest one or more of the following clinical signs:
  2. Inability to ambulate that prevents the animal's easy access to food and/or water.
  3. Inability to maintain itself in an upright position.
  4. Prolonged (greater than 48 hours) inappetance and/or clinical dehydration.
  5. Agonal breathing and cyanosis; chronic diarrhea or constipation.
  6. Hematologic or biochemical parameters that indicate organ failure incompatible with life.
  7. Unconsciousness with no response to external stimuli such as toe-pinch withdrawal test.

The above symptoms are not a temporary consequence of an experimental treatment (e.g., a surgical manipulation or drug treatment) that has been described and justified in an approved protocol.

If an experimental animal appears to be moribund, the Animal Care Office should be notified promptly so that the attending veterinarian can examine the animal, discuss the animal's condition with the researcher, and determine if euthanasia is warranted.

If killing a moribund animal would invalidate the study, the 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 IACUC prior to initiating this procedure. Investigators who receive approval from the IACUC 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 alternative endpoints other than death whenever possible.
  2. Animals must be monitored twice daily (in the early morning and late afternoon including weekends and holidays) and any animals evidencing clinically abnormal behavior must be removed from group housing situations and housed individually with easy access to food and water.
  3. Written records of all monitoring sessions, indicating the time of the observations, the person observing the animals, and any observations such as the number of animals evidencing clinically abnormal behavior and the number of animals found dead, must be maintained and made available to the Animal Care Office or IACUC on request.

Investigators 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 the highest USDA pain level category "E" unless analgesics or anesthetics are provided to alleviate pain or distress in the experimental animals.

Euthanasia Endpoint Criteria

Category I

  • ruffled hair coat
  • hunched posture
  • lethargy
  • ocular discharge/eyes closed
  • diarrhea

Category II

  • obvious weight loss or dehydration
  • inability to eat or drink
  • failure or delay to right when placed on back
  • dyspnea
  • bleeding from gastrointestinal or respiratory tract
  • neurological signs, such as circling, head tilt, partial paresis, seizures

Any animal exhibiting any two of Category I clinical signs or any one of Category II clinical signs will be humanely euthanized using the method of euthanasia listed on the original animal subjects approval form.

Hazardous Agents and Animals

Use of any hazardous materials (biohazards, toxic chemicals or drugs and radiation hazards) in animals is limited to activities which can be safely carried out in the available facilities and personnel are trained appropriately.  Investigators using hazardous agents in animals must be guided by the Material Safety Data Sheet (MSDS), federal and state regulations, and University policies.  Hazardous Materials and Right to Know training is required by Massachusetts law before working with hazardous materials.  Contact EHS for a training schedule. Before the project involving hazardous materials is approved by the IACUC the project must also be reviewed and approved by the appropriate oversight committee and/or Environmental Health and Safety personnel.

Biohazardous Agents

Use of any biohazardous agents in animals, including recombinant DNA, is limited to activities which can be safely carried out in the available facilities.  The Biosafety Officer and the Institutional Biosafety Committee(IBC) review and approve projects using biohazardous agents including Select Agents

Radioactive Agents

The Radiation Use Committee oversees use of radioactive materials and radiation sources. RUC review and review by the Radiation Safety Officer (RSO) in EHS proceeds concurrently with IACUC review. The RSO ensures that all investigators using radioactive materials in animals have appropriate training and that the use, storage and disposal of radioactive materials will be in compliance with federal and state regulations before the research begins.

Use of Animal Blood and Tissues

If animals are euthanized for a research project for the purpose of obtaining blood or tissue an IACUC protocol is required. IACUC approval is not required if tissue collection takes place postmortem as a by-product of a commercial enterprise like a slaughterhouse. IACUC approval is not required for the harvest of tissues from dead animals from other research projects as long as the collection of tissues does not alter the approved procedures in any way and the animals were used exclusively for the other research project. 

PIs should be aware that if the “use of vertebrate animals” box is not checked on the face page of a PHS grant application form references to use of animals in the grant proposal may trigger questions about IACUC approval. To avoid delays in peer review of the proposal the PI is advised to explain in the grant application the source of the tissues.