Pain, Discomfort, & Distress

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’s 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.

For more information, please review the following resources, as well as the content provided below.

Pain / Distress Category Descriptions

Use the table (below) 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 will have animals in multiple pain categories, they should be listed separately in the appropriate section of the protocol proposal in Niner Research. The protocol will be classified for review at the highest pain and distress category level listed (i.e., if you are using both Category B and Category E animals, the overall protocol would be considered Category E).

If only a subset of experiments described in a protocol fall into Category E, this should be clarified within the protocol text, too.

Category Definitions, Examples, & Considerations

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 (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.

Considerations

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

C

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.

Examples

  • 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

Considerations

  • 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.

D

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.

Examples

  • Non-survival 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.

Considerations

  • 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.

E

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.

Examples

  • 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.

Considerations

  • 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.
LITERATURE SEARCHES FOR CATEGORY D & E PROTOCOLS

If any procedures fall into USDA Category D or E, causing more than momentary or slight pain or distress to the animals, the protocol must clearly describe your consideration of alternatives and your determination that alternatives are not available or a justification for why alternatives will not work for your study. This requirement is fulfilled through a literature search.

  • Your literature review should include articles that discuss the 3 R’s (replacement, reduction, refinement) in terms of the specific project.
  • Delineate the methods and database sources used in the search. A minimum of two (2) databases must be searched (the Animal Welfare Information Center at the National Agricultural Library recommends a minimum of three). Include your database references, the date of the search, period covered, and the keywords (search strings) used. 
  • You must certify that no valid alternative was identified for procedures which may cause more than momentary pain or distress, whether relieved or not.

Detailed guidance on the 3 R’s and Literature Searches (including resources to help you fulfill these requirements) is provided on the Protocols, Amendments, & Renewals page.

NOTES: 

  • The literature search must be updated with each continuing protocol submission.
  • Justification must be provided for using ascites antibody production instead of in vitro methods.
Justification for Category E Procedures

A protocol is classified as “Category E” when animals will undergo procedures causing pain or distress that will not be relieved by anesthetics, analgesics, tranquilizers, or euthanasia. For Category E protocols, researchers must attempt to minimize and mitigate pain and distress by other means. These efforts include using timely endpoints and using the minimum number of animals necessary (possibly through a pilot study). If you propose Category E procedures, you will be required to provide a detailed scientific justification in your protocol application.

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 effects 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 one justification. In the absence of such documentation, the burden remains with the investigator to justify why death is the only acceptable endpoint.

The fact that no literature exists to define (or even begin to explore) an alternate and potentially less painful procedure for a particular Category E protocol is not a sufficient justification for performing such studies. Stated simply, the fact that a particular procedure has been performed in the past is not an adequate justification for performing the procedure now.

If there is no information regarding the effects of different analgesics or anesthetics, 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 more information on pilot studies, see the Protocols, Amendments, and Renewals page.

ADDITIONAL REPORTING REQUIREMENTS FOR CATEGORY E STUDIES

In addition to Annual Renewals, Adverse Event Reports, possible PAMs, etc., required for all protocols, 90-day reporting requirements apply for IACUC protocols that involve Category E procedures. The purpose of the 90-day report is to inform the IACUC of experimental results and any possible intended/unintended consequences affecting animal health or well-being. The 90-day reports serves as a basis for dialog between the PI and Committee on how to address and resolve issues as they arise during experimentation.

90-day reports are to be submitted by email to the ORPI/IACUC Office for subsequent IACUC review. The Principal Investigator must submit the initial report 90 calendar days following the start of work on the protocol. Subsequent reports may be requested by the IACUC, depending on the review of initial findings.

It is the responsibility of the Principal Investigator to notify the IACUC when research activity is about to begin. This will begin the 90-day clock for reporting.

Information to be Included in the 90-day Report

  1. The original goal of the study (cut/paste from the protocol).
  2. The total number of animals used to date.
  3. A summary of experimental results/outcomes thus far.
  4. A summary of animal health outcomes, including unexpected or adverse events.
  5. The investigator’s own interpretation of results and outcomes.
  6. A discussion addressing pertinent changes (if any) in:
    • future experiments
    • goals of the study
  7. Documentation regarding whether the study is complete/finished.

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.

For further information regarding 90-day reports, please see the IACUC’s policy on Reporting Requirements for Pilot Studies and Category E (USDA Pain/Distress) Protocols.