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Policy#: 529-226

Title: AUP PROCEDURES: Alternatives to Painful Procedures

Effective: 9/14/2023


I: Background and Purpose

Federal regulations stipulate eliminating or reducing unnecessary pain/distress must be considered when preparing research applications. These ideas are commonly referred to as the “3 R’s.”

Replacement is simply replacing the use of animals with non-animal techniques. A practical example of “replacement” is the use of cell culture techniques to replace animals as incubators for cell lines.

Reduction refers to strategies that reduce the number of animals used. Methods would include a statistical analysis to determine the correct sample size for valid results. An example of “reduction” would be designing experiments so animals serve as their controls.

Refinement refers to changing experiments or procedures to reduce pain or distress in those animals that must be used. An example of “refinement” would be the improvement of surgical techniques that result in fewer model failures.

The 3 Rs were initially articulated by Russell and Burch (1959) and are essential considerations for all animal research studies. This policy aims to help writers and reviewers of animal protocols assign procedures and animals to the United States Department of Agriculture (USDA) pain and distress categories and help PIs understand how to search for alternatives to category D and E procedures.

II: Related policies

·         529-224 IACUC Review Procedures for Animal Use Protocols (AUPs) and Related Amendments

·         529-271: Authority of the Attending Veterinarian

·         529-201 Reporting the Mistreatment of Animals and Deficiencies in Their Care at UCR



III: Policy


1.       When completing an AUP, all investigators should review the definitions and examples of USDA pain and distress categories provided below and in the Experimental Groups and Numbers section in Kuali:


Category C – Animal use activities that involve no more than momentary or slight pain or distress (no greater than an injection) where there is no need for pain-relieving drugs.

Category D – Animal use activities that involve accompanying pain or distress to the animals and for which appropriate anesthetics, analgesics, tranquilizing drugs, and/or humane endpoints are used to avoid pain, distress, or discomfort.

Category E – Animal use activities that involve accompanying pain or distress to the animals and for which appropriate anesthetic, analgesic, tranquilizing drugs, or other methods for relieving pain or distress are not used. Category E procedures require strong scientific justification for why pain-relieving medicines or other methods for relieving pain cannot be used. This includes citation(s) to published studies if applicable — describing the alternatives that were considered and how options will be used whenever possible, clarifying whether animals will be euthanized when moribund, and if not, what information is to be gained in the interval between moribundity and death.


2.       Investigators must then assign each procedure in the protocol to the appropriate category. They must also assign each animal or group of animals to the highest pain and/or distress category (E > D > C) it will be subjected to. Furthermore, they are obligated to:

a.       avoid or minimize discomfort, distress, and pain to the animals in the study;

b.       consider appropriate alternatives to any procedure that may cause more than slight or momentary pain or distress (categories D and E); and

c.       consult with the Attending Veterinarian in planning procedures for USDA-regulated species.



1.       The Animal Welfare Act and Regulations require PIs to:

a.       Consider alternatives to category D and E procedures, including refinements, reductions, and replacements.

b.       Provide a written narrative that describes the search for alternatives to painful procedures. This should include:

                                                               i.      The databases searched (Index, Medicus, Medline, Current Contents, Animal Welfare Information Center (AWIC), etc.) or other sources used, such as colleagues at scientific meetings, journal articles read, and presentations attended.

                                                             ii.      The date that any database searches were performed.

                                                           iii.      The years of citations covered by database searches.

                                                           iv.      The keywords and/or search strategy used when searching a database.

                                                             v.      A search for reduction and refinement, not just replacement of animals.

2.       Kuali includes a specific section to provide the information in detail. 

3.       A search for alternatives is required for any AUP that uses USDA-covered species and includes procedures categorized as D and E.


IV: References

USDA Policy #11: Painful and Distressful Procedures. Issue Date: March 25, 2011 References:

AWA Section 2143, 9 CFR, Part 2, Sections 2.31(d)(1)(i,ii,iv), 2.31(e)(4), 2.36(b)(5,6,7)


Guide for the Care and Use of Laboratory Animals 8th edition. 2011. Institute for Laboratory

Animal Research. Washington DC: The National Academies Press.

Originally adopted: 10/6/98; Updated: 12/9/04, 10/4/10, 8/14/23