Pain & Distress Guidance


The categories below reflect the potential levels of pain, discomfort, and/or distress in common lab procedures. The examples are not exhaustive and are only to guide PIs and the IACUC. They cannot cover the range of severity and duration of P & D in specific study procedures, which must be considered in assigning categories.

Where appropriate, PIs should assign research groups undergoing different procedures to separate categories. The IACUC determines the final pain category. PIs’ Annual Updates should include any category changes that occurred during the course of the study.



Pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage or described in terms of such damage. ‘Momentary or slight pain’ is no greater than the severity and duration of common injection pain.


Distress is the state associated with conditions which significantly compromise the welfare of an animal, which may or may not be associated with pain, and where the animal must devote substantial effort or resources to the adaptive response to environmental challenges.


Discomfort is viewed as a mild form of distress.


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

C - The research involves procedures that cause no more than momentary or slight pain or discomfort and do not require pain-relieving drugs.

  • Injection
  • Venipuncture (blood sampling)
  • Collection of tissues preceded by approved methods of euthanasia
  • Behavioral testing with no more than momentary stress
  • Toxicity testing in which no pain is expected
  • Naturalistic observations
  • Short-term physical restraint of animals
  • Animal identification procedures that do not require anesthesia
  • Chemical restraint
  • Live trapping studies where animals are not in traps longer than 12 hours
  • Chronic maintenance of animals with a disease or functional deficit that involves no more than slight pain or distress

D - The research involves procedures that involve pain or distress which will be treated with appropriate anesthetics/analgesics.

  • Major and minor surgical procedures with anesthesia (both survival and terminal)
  • Inducement of a functional deficit (e.g., inflammation, infection, and tumor disease states)
  • Toxicological, microbiological, or infectious disease research that requires continuation until significant clinical signs are evident and death is not the endpoint
  • Prolonged physical restraint of animals with appropriate conditioning
  • Prolonged food or water restriction with appropriate monitoring to ensure animal well being
  • Induction of more than minor behavioral stress
  • Terminal perfusion, cardiac blood collection, exsanguination under anesthesia
  • Ocular blood collection in mice
  • Implantation of devices

E - The research involves procedures that involve pain or distress which cannot or will not be alleviated through the administration of appropriate anesthetic, analgesic, or tranquilizer drugs.

  • Toxicity testing – infectious agent challenge where death is the endpoint
  • Chronic maintenance of a disease or functional deficit where death is the endpoint
  • Studies causing inflammation, tissue damage, or neoplasia that result in morbidity or mortality which is not the result of euthanasia
  • Application of noxious stimuli from which escape is impossible

Category E protocols require consultation with the AV and written justification (which goes in the annual report to USDA).

In trying to justify precluding analgesics (because of their real or perceived potential effects they may have on the animal's immune system), the researcher should consider the effects that unalleviated pain may have on the animal and the experimental results.  The investigator should consider that pain itself affects the animal's overall physiology and immune system.  Such effects can in many cases be more significant than the effect that analgesics themselves may have on the research results.  Some of the effects of unalleviated pain may include:

1.     Activation of the sympathetic nervous system

2.     Increased cardiac output

3.     Increased systemic vascular resistance

4.     Increased blood pressure

5.     Increased oxygen demand

6.     Vasoconstriction of coronary arteries

7.     Spleen ischemia

8.     Adverse renal effects

9.     Increased aldosterone output

10.  Increased sodium and water retention

11. Release of endogenous glucocorticoids, which may be immunosuppressive

12. Higher rate of metastasis

13. Self-mutilation

14.  Stop grooming

15.  Anorexia

16.  Weight loss

17.  Catabolic state

18.  Increased clotting

19.  Increased blood viscosity

20.  Increased platelet aggregation

21.  Altered mental status (anxiety)

22.  Interference with normal activity

23.  Lastly, pain itself constitutes an uncontrollable research variable