Protocol Development Resources

Pain & Distress

Humane Endpoints

Alternatives & Non-duplication

Justifying Animal Numbers

Recognizing, Defining, and Categorizing Animal Pain and Distress

USDA Pain & Distress Categories 

The requirement 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 in 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 sound scientific practices, is imperative. Unless the contrary is established, investigators should consider that procedures that cause pain or distress in human beings 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 – 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 = animals 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.

Many animals 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 handled (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
  • LD 50 determinations
  • Electrical shocks, including shock reinforcement
  • Intercardiac or periobital 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 than 12 hours.

* These procedures would be listed as (1) Category D if alleviated with appropriate anesthetics and/or analgesics or (2) Category E if they 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 alterative procedures in order to avoid second guessing of reviewing agencies and/or regulatory compliance concerns, and to avoid potential delays in protocol approval.

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

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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

Animals upon which teaching, research, experiments, or tests 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

Animals upon which experiments, teaching, research, surgery, or tests 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 animals 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 chemicals 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

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USDA Policy #12 Consideration of Alternatives to Painful/Distressful Procedures

References: AWA Section 13(a)(3)(B), 9 CFR, Part 2, Section 2.31 (d)(1)(ii)and (e), 9 CFR, Part 2, Section 2.32 (c)(2) and (5)(ii), Animal Welfare Information Center

History: Provides guidance on the requirement to provide a written narrative of the consideration of alternatives to painful and distressful procedures. Replaces Policy #12 dated April 14, 1997.

Justification: The Animal Welfare Act (AWA) regulations require principal investigators to consider alternatives to procedures that may cause more than momentary or slight pain or distress to the animals and provide a written narrative of the methods used and sources consulted to determine the availability of alternatives, including refinements, reductions, and replacements.

Policy: Alternatives or alternative methods are generally regarded as those that incorporate some aspect of replacement, reduction, or refinement of animal use in pursuit of the minimization of animal pain and distress consistent with the goals of the research. These include methods that use non-animal systems or less sentient animal species to partially or fully replace animals (for example, the use of an in vitro or insect model to replace a mammalian model), methods that reduce the number of animals to the minimum required to obtain scientifically valid data, and methods that refine animal use by lessening or eliminating pain or distress and, thereby, enhancing animal well-being. Potential alternatives that do not allow the attainment of the goals of the research are not, by definition, alternatives.

A fundamental goal of the AWA and the accompanying regulations is the minimization of animal pain and distress via the consideration of alternatives and alternative methods. Toward this end, the regulations state that any proposed animal activity, or significant changes to an ongoing animal activity, must include:

  • a rationale for involving animals, the appropriateness of the species, and the number of animals to be used;
  • a description of procedures or methods designed to assure that discomfort and pain to animals will be limited to that which is unavoidable in the conduct of scientifically valuable research, and that analgesic, anesthetic, and tranquilizing drugs will be used where indicated and appropriate to minimize discomfort and pain to animals;
  • a written narrative description of the methods and sources used to consider alternatives to procedures that may cause more than momentary or slight pain or distress to the animals; and
  • the written assurance that the activities do not unnecessarily duplicate previous experiments

We believe that the performance of a database search remains the most effective and efficient method for demonstrating compliance with the requirement to consider alternatives to painful/distressful procedures. However, in some circumstances (as in highly specialized fields of study), conferences, colloquia, subject expert consultants, or other sources may provide relevant and up-to-date information regarding alternatives in lieu of, or in addition to, a database search. When other sources are the primary means of considering alternatives, the Institutional Animal Care and Use Committee (IACUC) and the inspecting Veterinary Medical Officer should closely scrutinize the results. Sufficient documentation, such as the consultant's name and qualifications and the date and content of the consult, should be provided to the IACUC to demonstrate the expert's knowledge of the availability of alternatives in the specific field of study. For example, an immunologist cited as a subject expert may or may not possess expertise concerning alternatives to in vivo antibody production.

When a database search is the primary means of meeting this requirement, the narrative must, at a minimum, include:

  • the names of the databases searched
  • the date the search was performed
  • the period covered by the search
  • the key words and/or the search strategy used

The Animal Welfare Information Center (AWIC) is an information service of the National Agricultural Library specifically established to provide information about alternatives. AWIC offers expertise in formulation of the search strategy and selection of key words and databases, access to unique databases, on- and off-site training of institute personnel in conducting effective alternatives searches, and is able to perform no-cost or low-cost electronic database searches. AWIC can be contacted at (301) 504-6212, via E-mail at, or via its website. Other excellent resources for assistance with alternative searches are available and may be equally acceptable.

Regardless of the alternatives sources(s) used, the written narrative should include adequate information for the IACUC to assess that a reasonable and good faith effort was made to determine the availability of alternatives or alternative methods. If a database search or other source identifies a bona fide alternative method (one that could be used to accomplish the goals of theanimal use proposal), the written narrative should justify why this alternative was not used.

The written narrative for federally-mandated animal testing (for example, testing product safety/efficacy/potency) needs only to include a citation of the appropriate government agency’s regulation and guidance documents. Mandating agency guidelines should be consulted since they may provide alternatives (for example, refinements such as humane endpoints or replacements such as the Murine Local Lymph Node Assay) that are not included in the Code of Federal Regulations. If a mandating agency-accepted alternative is not used, the principal investigator should explain the reason in the written narrative.

Alternatives should be considered in the planning phase of the animal use proposal. When a proposal is modified during its performance, significant changes are subject to prior review by the IACUC, including the review of the implications of those changes concerning the availability of alternatives. Although additional attempts to identify alternatives or alternative methods are not required by Animal Care at the time of each annual review of the animal protocol, Animal Care would normally expect the principal investigator to reconsider alternatives at least once every 3 years, consistent with the triennial review requirements of the Public Health Service Policy (IV,C,5).

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Additional Resources for Recognizing and Categorizing Pain and Distress

  • Recognizing Pain and Distress in Laboratory Animals An article by E. Carstens and Gary P. Moberg “The recognition of pain, stress, and distress is critical in maintaining the well-being of laboratory animals. However, this important task is difficult because of a lack of agreed-upon definitions of these terms, as well as the absence of absolute, objective measures. Because animals cannot verbalize what they are experiencing, investigators and caretakers must deduce the animal's condition based on appearance and behavior. [...]In this article, we discuss the biologic meaning of pain, stress, and distress, with the aid of a model, and review and update guidelines for the recognition and assessment of pain and distress in laboratory animals.”  ILAR Journal V41(2) 2000
  • Definition of Pain and Distress And Reporting Requirements for Laboratory Animals Proceedings of the Institute for Laboratory Animal Research (ILAR) Workshop held June 22, 2000 (online book) “This workshop provides an opportunity for the speakers and members of the audience to engage in a discussion of the definitions of pain, distress, and how one can recognize and alleviate the pain and distress that can occur in the course of using animals in biomedical research as well as in education and testing. The purpose of the workshop is to focus on the proposed wording for the USDA to use in writing regulations that will implement the Animal Welfare Act. Because the Animal Welfare Act contains the phrase “pain and distress,” the USDA must define those terms to implement the act.”
  • A Reference Source for the Recognition & Alleviation of Pain & Distress in Animals Compiled by Dr. Richard L. Crawford, USDA Animal Welfare Information Center This publication provides a list of published reference sources on the recognition and alleviation of pain and distress in animals. It has been developed to provide a source of information and guidance to those using animals in research so that they may better recognize when an animal is suffering pain or distress, or may be likely to suffer pain or distress, and how that pain or distress may be minimized. This publication is not meant to be a complete resource on this subject but to provide a starting point for those concerned about the welfare and humane care of animals used in research.
  • Pain, Distress and Endpoints A training module from the Canadian Council on Animal Care
  • Distress in Animals: Is it Fear, Pain or Physical Stress Temple Grandin and Mark Deesing Colorado State University, Department of Animal Science This paper refutes claims that feeling pain is dependent on brain size and complexity. Instead, it suggests that a reasonable criterion for assessing pain-induced suffering is whether or not the animal actively seeks pain relief.
  • Refinement of Animal Use – Assessment and Alleviation of Pain and Distress International Journal of Laboratory Animal Science and Welfare P.A. Flecknell, Medical School, University of Newcastle A review article on methods of clinical pain assessment in animals, including a discussion of pain alleviation techniques
  • Pain – Assessment, Alleviation and Avoidance in Laboratory Animals ANZCCART Facts Sheet Paul Flecknell, University of Newcastle Based on material from Pain Management in Animals, edited by Flecknell and Waterman-Pearson.
  • Use of Behavior Analysis to Recognize Pain in Small Mammals Lab Animal – 36,6 (2007) Jörg Mayer, Dr. Med.Vet, MSc Cummings School of Veterinary Medicine, Tufts University, North Grafton, MA The minimization of pain in laboratory animals is a gold standard with implications for improvements in both animal welfare and research quality. Changes in behavioral parameters may indicate that an animal is in pain, but in order to effectively use behavioral change to assess pain, the observer must be familiar with normal behaviors. The author discusses normal and pain-related behaviors exhibited by rodents, rabbits, and ferrets.

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Establishing Humane Endpoints

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Searching for Alternatives & Documenting Non-Duplication

  • University of California Center for Animal Alternatives Information The UCDavis Center for Animal Alternatives places special emphasis on disseminating up-to-date information concerning animal alternatives through every level of public and private education. It also seeks to provide investigators who use animals with information on the most current methods for improving all aspects of animal care during their work and offers help with the alternatives search, including search strategy, database selection, and general guidance, and performs low-cost searches (530-754-9122 or
  • Tips for Searching for Alternatives to Animal Research & Testing - Animal Welfare Information Center (AWIC) These guidelines were developed to assist researchers, information specialists, and Institutional Animal Care and Use Committee (IACUC) members, when conducting literature searches to determine if alternatives to the use of animals exist and whether a protocol unnecessary duplicates previous research. AWIC offers expertise in formulation of the search strategy and selection of key words an databases, access to unique databases, on and off-site training in conducting effective alternative searches, and is able to perform no-cost or low-cost electronic database searches. (301-504-6212 or
  • Alternatives and the Animal Welfare Act - AWIC Brochure
  • National Centre for the Replacement Refinement and Reduction of Animals in Research The English Information Portal is an area of the website containing annotated links to online databases, websites, journal articles, legislation and other publications. These resources provide information to help you apply the 3Rs and ensure the best possible standards in animal welfare.
  • Animal Welfare Information Center Alternatives Website This site provides information on methods and sources available to reduce, refine, or replace animals used in teaching, testing, and research. A fundamental goal of the Animal Welfare Act and the accompanying regulations is the minimization of animal pain and distress via the consideration of alternatives and alternative methods.
  • Searching Bibliographic Databases for Alternatives AWIC Bulletin, Summer 2006, Vo. 12, No.3-4 Tim Allen and D’Anna Jensen This article describes in detail how to conduct a search and provides examples.
  • Animal Use Alternatives Thesaurus A listing of terms to be used when conducting alternatives searches provided by the USDA National Agricultural Library
  • Why conduct Literature Searches for Alternatives? Article from the American Society of Laboratory Animal Practitioners (ASLAP) Newsletter
  • A free online course provides a broad overview of diverse topics in the practice of and approaches to humane animal experimentation. It addresses such issues as experimental design (including statics and sample size determination), humane endpoints, environmental enrichment, post-surgical care, pain management, and the impact of stress on the quality of data.
  • European Centre for the Validation of Alternative Methods ECVAM promotes the scientific and regulatory acceptance of non-animal tests which are important to biomedical sciences through research, test development and validation and the establishment of a specialized database service.
  • Fund for the Replacement of Animals in Medical Experiments (FRAME) FRAME advocates the Three Rs approach. While its long-term goal is the total elimination of laboratory animal use, through the development, validation and acceptance of replacement alternative methods, until that goal is reached, they support efforts to reduce the numbers of animals used through better science and better experimental design, and to refine procedures so that the suffering of any animals necessarily used is minimized.

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Determining and Justifying Animal Numbers

  • Estimating Animal NumbersNational Academies Press Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research Appendix B – Estimating Animal Numbers
  • Reducing Animal Numbers: Sequential Sampling Animal Welfare Information Center One way of reducing the number of animals needed in an experiment is to use more sophisticated statistics, not a more difficult method, in fact less difficult once you know how. I'll describe a technique enabling you to shave the number of animals used by about 30-40 percent or more and to do your statistical analyses faster and easier.
    • To have all these advantages you must have some estimate of three things: your control/baseline mean, control/baseline variance, and the effect size. You will most likely have all of these already, but you will need to use them in a slightly different way than you have in the past.
  • Web Pages that Perform Statistical Calculations The web pages listed here comprise a powerful, conveniently-accessible, multi-platform statistical software package. There are also links to online statistics books, tutorials, downloadable software, and related resources. All of these resources are freely accessible, once you can get onto the Internet.
  • Sample Size Determination From the book, Guidelines for the Care & Use of Mammals in Neuroscience and Behavioral Research Available online from the National Academies Press
  • Experimental Design & Statistics in Biomedical Research ILAR Journal V43 (4) 2002 This edition of the ILAR Journal is dedicated to articles related to the use of statistical analysis and improved experimental design.
  • Brief Tutorial on Sample Size & Statistical Power Calculations for Animal Experiments By Phillip Chapman, Department of Statistics, Colorado State University Provides an overview of basic ideas of power and sample size calculations in the context of simple examples; introduces easily available tools for power and sample size, including interactive software available for free on the web. (Russell Lenth, U. of Iowa, and UCLA Online Power Calculator.); and gives some practical advice about how to proceed in some common situations.
  • Lecture Notes from Sample Size and Statistical Power Taught by Dr. Eric Rexstad at the University of Alaska Department of Biology and Wildlife, 1993-1999 A brief overview on how to evaluate sample size during the design phase of a research project. From the viewpoint of an IACUC review process, it is important to address the number of animals used. One of the 3 R's is reduction, so the IACUC wants to be assured that no more animals than necessary are used to achieve a project’s stated objectives. However, the IACUC is equally concerned that adequate numbers are used to achieve the objectives. As painful as it might be, this invariably requires some thought about statistics, sample size, power, and type I and type II errors.

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