Protocol Development Resources

Recognizing, Defining,  and Categorizing Animal Pain and Distress

USDA Pain & Distress Categories

The requirements to specify levels of pain and/or distress experienced by animals used in research, testing, or teaching comes from a number of federal mandates requiring the minimization of pain and distress. This requirement to minimize pain and distress is most clearly described int the U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training.

IV. Proper use of animals, including the avoidance or minimization of discomfort, distress, and pain when consistent with should scientific practices, is imperative. Unless the contrary is established, investigators should consider that procedures that cause pain or distress in human being  may cause pain or distress in other animals.

In order to assure that efforts at minimizing pain and distress are thorough, federal regulations require a "written narrative of the methods used and sources consulted to determine the availability of alternatives, including refinements, reductions, and replacements". This requirement originates from one of the central ethical frameworks of using animals in research as presented in The Principles of Humane Experimental Technique and the 100 Year Celebration three "Rs". That is, every study involving animal subjects should make real efforts to replace the use of animals with non-animal techniques or systems; to reduce the number of animals to be used, and to refine all experiments or procedures to reduce pain or distress in those animals that must be used.

Consequently, the requirement to categorize levels of pain and distress satisfies three requirements: (1) the IACUC's responsibility to distinguish which protocols involve painful and/or distressful procedures; (2) the need to identify when the PI's must provide a "Written narrative of the methods used and sources consulted" to determine appropriate pain relieving measures for the specific protocol, and assure that procedures are not being unnecessarily duplicated; and (3) UNC's regulatory responsibility to report all animal use to the USDA annually.

Assessing Pain and Distress

Pain/distress categories are designated B-E by the United States Department of Agriculture (USDA) and summarized as follows:

• B=animal being bred, held or observed;
• C=animals undergoing procedures involving minimal or no pain;
• D=animals undergoing painful procedures that are relieved by appropriate measures;
• E=animals undergoing painful procedures where pain is unrelieved.

Evaluation of pain/distress related to a procedure is complex and requires consideration of several variables. The critical difference between Category C and Category D procedures is whether the pain or distress associated with the procedure is momentary or non-existent (Category C) or whether the procedure causes pain or distress and necessitates the use of appropriate pain relieving measures (Category D). However, in some instances, it can be difficult to determine what is more than momentary or slight type of pain or distress. Therefore, each protocol submitted to the IACUC must undergo individual scrutiny to assure that there is a suitable assessment of the pain/distress level of the proposed procedures.

May animal exhibit behaviors that are indicative of their experiencing pain or distress. Listed below are some more commonly observed behavioral indicators that an animal may be experiencing pain or distress. Although there is considerable interspecies and individual variability in pain responses, it is important for the PI or others responsible for the care of the animals to be vigilant when observing any changes in behavior.

Examples of Behavioral Indicators of Pain

• Vocalization
• Lethargy, loss of appetite
• Excess salivation
• Lack of grooming
• Assuming unusual positions
• Shivering
• Abnormal or labored breathing
• Change in behavior, acting "anxious"
• Looking at, licking, chewing, smelling or guarding a painful area
• Biting or resistance to being handles (in adapted animals)
• Reluctance to mobilize or otherwise bear weight, limping

The Animal Welfare Act (as amended July 1993) defines a painful procedure as "any procedure that would reasonably be expected to cause more than slight or momentary pain or distress in a human being to which that procedure was applied, that is, pain in excess of that caused by injections or other minor procedures." Several experimental manipulations are thought to be significantly painful and/or distressful to animals. Although not an exhaustive list, the box below contains examples of procedures that fall into that grouping.

It is important to note that there is no penalty for identifying procedures in a protocol as USDA Pain/Distress Category D, because there are no laws, regulations, or accreditation requirements that limit the number of procedures that fall under Categories D. Rather, the goal is to have a truthful reflection of the assessment of the pain and/or distress animals being used for research, teaching, or training will experience. From the perspective of submitting a protocol or amendment for IACUC review, the essential difference  with procedures identified under Category D is that a search for alternative procedures must be provided (usually by, but not limited to, performance of a database search).

Examples of Significantly Painful and/or Distressful Procedures*

• Surgery
• Fracturing of bones
• Drug or radiation toxicity
• Moderate to severe malnutrition
• Intracerebral or Intracardiac inoculations
• Neurophysiological preparations
• Burning or freezing
• LC 50 determinations
• Electrical Shocks, including shock reinforcement
• Intercardiac or perobital blood collection
• Diseases that result in tissue destruction or death
• Application of noxious stimuli without escape
• Imposition of abnormal environmental conditions
• Agents that cause excessive inflammation or necrosis
• Chair or stock restraint of unadapted animals or restraint of any animal for more that 12 hours

* These procedures would be listed as (1) Category D if alleviated with appropriate anesthetics and/or analgesics or (2) Category E if the were not alleviated

In the end, the IACUC is responsible for providing a comprehensive assessment of the potential pain and distress of the proposed use of animals in teaching, training, and research, and upholding that responsibility is an essential part of maintaining public trust in the research enterprise. An additional part of that responsibility is the ongoing compliance of Cornell's research community with university policy and federal regulations. So in instances where procedures do not clearly fall into Category C or D, it is generally best to list animals in Category D and provide the search for alternative procedures in order to avoid second guessing of reviewing agencies and/or regulatory compliance concerns, and to avoid potential delay in protocol approval.

Animal Care & Use Newsletter Volume 1, Issue 2 Cornell University Office of Research and Sponsored Programs Integrity and Assurance.

USDA Pain & Distress Categories

Category B

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

• Standard husbandry procedures not for research, teaching or testing
• Standard animal health programs: i.e., routine physical examinations, vaccinations, etc.
• Pre-weaning methods of identification (toe or tail clipping, tattooing, wing banding, ear notching, etc.)

Category C

Animal upon which teaching, research, experiments, or test were conducted involving no pain, distress, or use of pain-relieving drugs.

• Holding or weighing animals
• Injections, blood collection or catheter implantation via superficial vessels
• Routine physical examinations
• Observation of animal behavior
• Humane euthanasia procedures
• Live trapping with minimal stress and little potential for injury
• Chemical immobilization or restraint
• Studies involving clinical signs with not more than slight pain or distress

Category D

Animal upon which experiments, teaching, research, surgery, or test were conducted involving accompanying pain or distress to the animals and for which appropriate anesthetic, analgesic, or tranquilizing drugs were used.

• Diagnostic procedures such as laparoscopy or needle biopsies
• Non-survival surgical procedures
• Survival surgical procedures, including biopsies and cut-downs for catheter placement when postoperative pain or distress is alleviated.
• Ocular blood collection in mice.
• Exsanguinations under anesthesia
• Induced infections with appropriate anesthesia and post-op/post-procedure analgesia when necessary.

Category E

Animals upon which teaching, experiments, research, surgery or tests were conducted involving accompanying pain or distress to the animal and for which the use of appropriate anesthetic, analgesic, or tranquilizing drugs would have adversely affected the procedures, results, or interpretation of the teaching, research, experiments, surgery or tests.

• Research or procedures that require continuation until death occurs.
• Application of noxious chemical or stimuli (e.g., electrical shock) if the animal cannot avoid/escape the stimuli, and/or it is severe enough to cause pain or distress.
• Novel prolonged restraint
• Exposure to extreme environmental conditions
• Prolonged withholding of food and water
• Infectious disease studies involving unrelieved pain or distress

Additional Resources for Recognizing and Categorizing Pain and Distress

• Recognizing Pain and Distress in Laboratory Animals
• Definition of Pain and Distress and Reporting Requirements for Laboratory Animals
• A Reference Source for the Recognition & Alleviation of Pain & Distress in Animals
• Pain, Distress and Endpoints
• Distress in Animals: Is it Fear, Pain or Physical Stress
• Refinement of Animal Use-Assessment and Alleviation of Pain and Distress
• Pain-Assessment, Alleviation and Avoidance in Laboratory Animals
• Recognition and Alleviation of Pain in Laboratory Animals
• Use of Behavior Analysis to Recognize Pain in Small Mammals