Pain, Discomfort or Distress Classifications

Painful Procedures

In laboratory animal research, a painful procedure is defined as any procedure that would reasonably be expected to cause more than slight or momentary pain and/or distress in a human being. The PI must examine all procedures to be used in animals and make a preliminary determination as to the associated level of pain, discomfort, and distress (see Category Descriptions below). Investigators uncertain about the pain category should consult the Attending Veterinarian. The preliminary category must be validated by the Attending Veterinarian prior to the IACUC review of the protocol.

UNC Charlotte’s Institutional Animal Care and Use Committee (IACUC) is responsible for ensuring that animals receive appropriate care and attention to their well-being. Federal regulations require scientific justification(s) for not administering analgesics when procedures are expected to cause more than momentary pain or distress.

Consideration of Alternatives

The Animal Welfare Regulations require consideration of alternatives to procedures that may cause more than momentary or slight pain or distress to the animals. As part of the protocol application, PIs planning Category D or E procedures must provide a written description of the methods used and sources consulted to determine the availability of less painful / distressful alternatives (including refinements, reductions, and replacements) that would accomplish project goals.

Use the following table to determine the category or categories applicable to your protocol. Within each category description, a number of example procedures and considerations are included.

If you propose Category E procedures, you will be required to scientifically justify these procedures. If only a subset of experiments described in a protocol fall into Category E, this should be clarified within the protocol text. The protocol, however, will be classified for review at the highest pain and distress category level. Special reporting requirements apply to Category E studies.

CategoryDefinitions, Examples, & Considerations


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


  • Breeding colonies of any animal species (USDA does not require listing of rats, mice, birds) that are held in legal sized caging and handled in accordance with the Guide and other applicable regulations. Breeding colony includes parents and offspring.
  • Young breeders or young that cannot be used because of improper genotype or gender, and any other animals that will not have research performed on them or participate in research studies.
  • 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.


  • If ear punch biopsies or tail snips are necessary for genotyping, this category is not appropriate.


Animals upon which teaching, research, experiments, or tests will be conducted involving no pain or only very brief pain or distress with no need for or use of pain-relieving drugs.


  • Observational studies;
  • Procedures performed correctly by trained personnel such as the administration of electrolytes/fluids, administration of oral medication, percutaneous blood collection from a peripheral vein using needle stick or using IACUC-approved nicking of the superficial vein with a hypodermic needle, standard radiography, parenteral injections of non-irritating substances.
  • Ear punch biopsies or tail snips necessary for genotyping;
  • Collection of cells and/or tissues from animals after euthanasia has been performed in accordance with the recommendations of the most recent AVMA Panel on Euthanasia. Euthanasia must utilize 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 non human primate


  • If any of the procedures in an approved C category protocol cause pain, distress or discomfort, the attending veterinarian and facility director must be contacted immediately to assure prompt and appropriate action is taken to alleviate the pain, distress or discomfort.


Animals that will undergo procedures involving potential pain or distress that is relieved by appropriate anesthetics, sedatives, or analgesics and/or humane endpoints are used to avoid pain, distress or discomfort.


  • Nonsurvival surgery (e.g. tissue or organ collections prior to euthanasia in which animals are maintained at a surgical plane of anesthesia for the duration of the study following which the animals are euthanized without regaining consciousness) and
  • Minor survival surgical procedures ( e.g. cannulation of peripheral veins and arteries or subcutaneous implants) in which the appropriate anesthetics and analgesics are used and methods of euthanasia are those recommended by the AVMA Panel on Euthanasia.
  • Infectious disease and toxicity studies in which appropriate humane endpoints are included to assure animals are removed from the study and euthanized at the first sign of morbidity, for example: >15% weight loss, inactivity or any behavioral or clinical sign of disease pain or distress.

Also, in Category D, there are procedures which will require specific humane endpoints to be included to minimize pain, distress and discomfort and/or analgesics must be used as directed to prevent and minimize pain.

Examples requiring specific end points

  • Animals that will undergo major survival surgery (e.g. penetrates and exposes a body cavity or produces substantial impairment of physical or physiologic function such as laparotomy, thoracotomy, craniotomy and limb amputation.
  • Studies that involve multiple survival surgical procedures.
  • Prolonged restraint. (e.g. > 1 hour)
  • Studies that involve special housing requests which are exceptions to The Guide such as: housing rodents on wire bottom cages; cages that do not conform to space and height recommendations; requests to house animals outside recommended temperatures.
  • Infectious disease or toxicity studies that have a high probability of producing substantial disease and impairment of physical or physiologic function (e.g., injection of endotoxins, infectious disease models, etc.)
  • Induction of nutritional deficiencies and disease models that do not involve chronic clinical signs of disease and/or impairment of motor function.


  • If animal will experience more than momentary or slight pain, appropriate anesthetics/analgesics must be used, OR
  • Humane endpoints must be implemented and animal(s) must be immediately euthanized.


Animals that will undergo procedures for teaching, experiments, research, surgery, or tests which pain or distress is NOT relieved with the use of anesthetics, analgesics, tranquilizers, or by euthanasia where the use of such pain relievers will adversely affect the procedures, results, or interpretation of the teaching, research, experiments, surgery, or tests.


  • Procedures producing pain or distress unrelieved by analgesics such as toxicity studies, microbial virulence testing, radiation sickness, and research on stress, shock, or pain.
  • Toxicity testing where death of an animal is an endpoint.
  • Survival surgeries where no post-operative analgesics are provided.
  • Disease and cancer models that involve chronic clinical signs of disease and impairment of motor function (e.g. EAE, adjuvant arthritis, etc.)
  • Long-term restraint.
  • Food and water restriction.
  • Surgical and post surgical 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.
  • Investigator must consider humane endpoints to minimize or eliminate unnecessary pain and distress.
  • Humane endpoints can be defined as points at which an experimental animal’s pain and/or distress can be terminated, minimized or reduced by treatment to relieve pain and/or distress, or terminating the painful/distressful procedure or euthanasia.

The fact that no literature exists to define (or even begin to explore) an alternative for a particular Category E protocol is not a sufficient justification for performing such studies. Stated simply, just because a particular procedure has been performed in the past may not be an adequate justification for performing this study now.

For example, if withholding analgesics is to be justified by the possibility that they may “theoretically” affect a physiological response being measured, documentation of previous publications to support such a claim should be given. If no such studies have been done, then it is the investigator’s burden to justify any possible negative affects of analgesics on that particular physiological response.

Another example exists for pathogen-induced LD50 studies. Previous publications demonstrating that weight loss, body temperature, microbial burden, etc. are not acceptable endpoints would be an acceptable justification. In the absence of such documentation, the burden remains with the investigator to justify why death is the only acceptable endpoint.

If there is no information regarding the effects of different analgesics, alternatives for LD50 studies, etc., the investigator, in consultation, with the IACUC and Attending Veterinarian should consider a pilot study to evaluate such alternatives.

For those Category E studies for which alternative protocols are deemed necessary, a pilot study should be outlined and described in detail in the core experimental protocol form detailing the study. Typically, such studies will be performed and completed prior to beginning the official proposed Category E studies. The results of such pilot studies could then be evaluated and used to justify the Category E studies, or to alter the protocols in the Category E studies.

Information Needed in Category E Section of Animal Care Application

1. Explanation of Procedures: An explanation of the procedures producing pain or distress in the study animals and the justification for not using appropriate anesthetic, analgesic, or tranquilizing drugs must be provided. 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.

2. Consideration of Alternatives: If any procedures fall into USDA’s Classification 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 database sources used in the search.

3. Database References: Database references must include databases searched, the date of the search, period covered, and the keywords used. Alternatives include methods that:

(1) existing tests by minimizing animal distress,
(2) reduce the number of animals necessary for an experiment, or
(3) 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 whyin vitro systems cannot be used.

NOTE: You must certify that no valid alternative was identified to any described procedures which may cause more than momentary pain or distress, whether relieved or not.

4. Method of Euthanasia or Disposition of Animal at End of Study: Indicate the proposed method of euthanasia. If a chemical agent is used, specify the dosage and route of administration. If the method(s) of euthanasia include those which are not recommended by the 2020 AVMA Euthanasia Guidelines, (e.g., decapitation or cervical dislocation without anesthesia), provide scientific justification why such methods must be used.

Additional Reporting Requirements for Category E Studies

Additional reporting requirements will apply for all animal care/use protocols which involve designation of pain/distress Category E (for both designation for an entire study, or for a specific experiment/phase of a study).The purpose of the report will be to inform the IACUC periodically of experimental results and any possible intended/unintended consequences affecting animal health/well-being. It will serve as a basis for dialog between the PI and Committee on how to address and resolve animal health or well-being issues as they arise during experimentation.

The ORPI will request from investigators reports at intervals of 90 calendar days until the study/study phase is complete. Reports will be due no later than the 90-day mark. Information about reporting requirements and timelines will be noted on the initial protocol approval certification letter. The initial 90-day reporting period will be set by the initial approval date; subsequent reports will follow every 90 days thereafter.

Content to be Included in a 90-Day Report

A report must briefly cover the following:

  1. The total number of animals used to date;
  2. A summary of experimental results/outcomes thus far;
  3. A summary of animal health outcomes, particularly any unexpected or adverse events compromising animal health/well-being beyond what was anticipated; and
  4. The investigator’s own interpretation of results/outcomes.

Since animal health information is being requested, it is strongly recommended that PIs consult with the Attending Veterinarian during report preparation to ensure that reported animal health outcomes are in line with veterinary and animal care staff observations.

If experimentation has not yet begun during a reporting period, a report is still required. The PI should simply state that experimentation has not begun and provide the estimated timeframe for initiation of experimentation.

All reports are to be submitted to the ORPI, which will forward the report to the IACUC for review. For more information, refer to the policy “Reporting Requirements: Protocols Using Pilot Studies, Categorized as USDA Pain/Distress Category E.

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