3/5/2020 CITI - Collaborative Institutional Training Initiative

Vanessa Zuleta Quintero

ID 9011046

Using Animal Subjects in Research

(RCR-Basic)
Universidad de Antioquia - Researchers RCR

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Using Animal Subjects in Research

(RCR-Basic)
Content Author

Paul Braunschweiger, PhD

CITI Program

Introduction

Please review the video below before you begin reading the
module. It is approximately three minutes long.

Animal Care & Use

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The use of animals in research is a complex issue. Many are working to develop non-
animal alternatives to the use of animals in research, but the research community is
not entirely there yet. A dilemma we face when we use animals in research is that
unlike research with humans, animals cannot give informed consent for procedures
that might cause them pain, distress, or even death. As a result, some argue that
there is no acceptable justi cation for the use of live animals in a research setting.
Others take a more utilitarian approach and point to the many medical advances that
have been possible because of animal research. They argue that animal research
bene ts all animals (human and nonhuman), and society in general, if the research
has scienti c justi cation and utilizes procedures that minimize pain and distress to
the extent possible.

In the U.S., animal research is illegal unless it is in full compliance with the:

Ethical and scienti c principles in the U.S. Government Principles for the
Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training
(OSTP 1985)
Health Research Extension Act of 1985
Animal Welfare Act of 1966 (updated in 1985, 2002, and 2008)

This module introduces you to the major ethical and regulatory concepts related to
laboratory animal research, particularly as they t the broader focus of the
responsible conduct of research. This module does not replace CITI Program's Animal
Care and Use courses. Those courses provide a comprehensive treatment of the use
of animals in research with the speci c intent to meet the federal mandate for
investigator training in laboratory animal welfare.

Learning Objectives

By the end of this module, you should be able to:

Identify the ethical perspectives in animal research.

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Discuss the U.S. regulations governing animal research.

Describe the roles of the Institutional O cial (IO), the Institutional Animal Care
and Use Committee (IACUC), and the Attending Veterinarian (AV) in animal care
and use programs.
Recognize the ethical responsibilities of the principal investigator (PI) and
members of the research team who work with laboratory animals.
Describe how to report incidents of animal abuse and regulatory noncompliance
at your organization.

Perspectives on the Use of Animals in Research

Although not speci cally targeted to research activities, the rst laws to address
animal welfare were animal anticruelty laws (such as Martin’s Act 1822 [Ortiz-Robles
2016]). In some cases, convicted individuals received substantial nes and even
prison time. In 1856, Marshall Hall authored the earliest publication on the humane
treatment of animal subjects in a biomedical research context. Hall (1856) recognized
that pain and su ering often accompanies animal experimentation. He argued that
animal experimentation is ethical only when it meets these ve principles:

1. Alternative approaches are not available.

2. The experiment has a clearly stated objective.
3. The work is not unduly repetitious.
4. The investigators have a commitment to minimize pain and distress.
5. The study’s published results are clear and concise.

The rst laws to address the animal research principles set by Hall appeared in the
Cruelty to Animals Act passed by the British Parliament in 1876 (Hamilton 2013).

The "3Rs" of Animal Research

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About 100 years after Hall's paper appeared, William Russell and Rex Burch (1959)
published their landmark work entitled The Principles of Humane Experimental
Technique. In addition to experimental procedures of the day, this important work
described key ethical guidelines for scientists. Russell and Burch described
approaches to reduce pain or distress in research animals through the principles of:

These three principles and the attendant implementation strategies are known as
the “3Rs” (Russell and Burch 1959).

Reduction

Reduction is a process employed by the investigator that decreases the number of

animals used in a study to the minimum number su cient to obtain reliably
meaningful results with the desired precision and level of statistical signi cance. To
minimize the number of animal research subjects, investigators must pay attention
to the research study’s design and to their skills with the procedures. Some common
reduction strategies you can use include:

Improvements in experimental techniques with advanced technology and/or

better training of personnel. This will improve the precision of the data (reduced
type 1 error, reduced variance).
Implementation of a rigorous quantitative study design to include a scienti c
determination of the most appropriate sample size (n) based on the variances in
the expected data (“power analysis”).
The use of a pilot study with just a few animals to establish variances in data and
reliably determine the sample size.

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Implementation of animal or tissue sharing programs (within or outside the

organization) when possible.
Rationalization for the number of animals used with the potential harms from
performing multiple experiments or procedures in a single animal.

Replacement

Russell and Burch (1959) described replacement as the substitution of a lower

phylum species for a higher one. Some examples of how you can do this include:

Replace the test animals with a non-animal alternative such as computer

simulations, in vitro models (cell cultures), or in silico (“Organ on a Chip”
technology) models to predict the potential toxicity of experimental medications
or environmental pollutants.
Replace the test animals (such as dogs or nonhuman primates) with those from a
less sentient species (such as sh, frogs, or mice). This approach is ethical only if
the replacement model is appropriate for the study question and the substitution
could provide a scienti cally meaningful result.

Refinement

According to Russell and Burch (1959), refinement refers to e orts to minimize the
occurrence or severity of painful procedures that the investigators determined are
necessary for a study. Re nement of procedures can also result in greater precision

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and, therefore, a potential reduction of test animals. Some re nement e orts you
can utilize include:

Use non-invasive imaging procedures that are less painful or stressful than
invasive measurements to assess disease progression in animal models.
Implement procedures such as randomization and blinding strategies in the
experimental design to decrease unintended bias and increase the study’s
signi cance.
Use more e ective analgesics or anesthetics or more e ective treatment
schedules or administration strategies.
Substitute one experimental endpoint for another less painful endpoint.
Modify an animal's habitat to make it more interesting, more stimulating, or less
stressful for the animal under study.

Animal Welfare and Animal Rights

The ethical guidelines, laws, and regulations use the term animal welfare to
describe the appropriate care and use of animals as research subjects. The term
animal rights is often used by individuals and organizations who do not recognize
the concept of animal welfare, but instead, they believe that animals have the right to
not be used in research, education, testing, and even as food.

Early activists in the animal rights movement included Ruth Harrison (1964), Stanley
Godlovitch et al. (1972), and Richard Ryder. In the 1970s, Ryder coined the term
speciesism to promote the notion that discrimination against other (nonhuman)
species is similar to being racist or sexist.

The utilitarian philosopher Peter Singer, in his 1975 book Animal Liberation, did not
embrace the term animal rights. Instead, Singer argued that because animals can
su er, humans have a moral obligation to consider their interests. He contended
that individuals ought to apply the utilitarian principle of the "greatest good for the
greatest number" to nonhuman animals and that failure to give moral consideration

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to the interests of both humans and nonhumans is ethically indefensible (Singer

1975). However, Singer did not suggest that we literally treat human and nonhuman
animals equally. For example, a nonhuman animal cannot be granted the right to
vote, but Singer and his colleagues attempted to extend basic legal rights to great
apes, chimpanzees, bonobos, gorillas, and orangutans through the Great Apes
Project (Cavalieri and Singer 1993).

Other scholars, including Thomas Regan (2004), maintained that nonhuman animals
have inherent value as a "subject-of-a-life," and thus, the use of animals for research
purposes is an invasion of that life and not morally acceptable.

Regulations Governing Animal Research in the United

States

The U.S. government has regulations that govern the use of vertebrate animals in
research. The U.S. Department of Agriculture (USDA) and the U.S. Public Health
Service (PHS) enforce these regulations.

The U.S. Department of Agriculture (USDA)

U.S. Congress gave the USDA broad authority to regulate animal research when it
passed the Animal Welfare Act (AWA) in 1966. The USDA then established the Animal
Welfare Regulations (AWR) to enforce the AWA (USDA, Animal and Plant Health
Inspection Service [APHIS] 2017). The AWA and AWR regulate all activities with “any
live or dead dog, cat, nonhuman primate, guinea pig, hamster, rabbit, or any warm-
blooded animal used for research, teaching, testing, experimentation, or exhibition
purposes, or as a pet” (USDA, APHIS 2008).

The USDA, through the AWA, speci cally excludes rats, mice, and birds that are
“purpose bred” for use in research from its regulatory purview. Wild rats, mice, and
birds used in research still fall under the USDA’s purview.

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The U.S. Public Health Service (PHS)

In 1985, the U.S. Congress passed the Health Research Extension Act, which directed
the PHS to develop speci c guidelines for animal research.

The O ce of Laboratory Animal Welfare (OLAW) monitors institutional compliance

with PHS Policy and guidelines. To assess institutional compliance, OLAW primarily
relies on these two documents:

The PHS Policy on Humane Care and Use of Laboratory Animals (PHS Policy)
(OLAW 2015)
The Guide for the Care and Use of Laboratory Animals (the Guide) (National Research
Council [NRC] 2011)

The PHS Policy di ers from the AWR because PHS Policy covers all live vertebrate
species used for research, teaching, and testing activities funded by the National
Institutes of Health (NIH) or other branches of the PHS.

AAALAC International (AAALAC)

AAALAC International (AAALAC) is a nonpro t organization that accredits animal care

and use programs. An organization's participation in the accreditation program is

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strictly voluntary, but it has become a recognized standard for high-quality animal
research programs.

The Institutional Animal Care and Use Committee (IACUC)

Both the PHS Policy and the AWR speci cally mandate an Institutional Animal Care
and Use Committee (IACUC) for independent oversight of your organization’s care
and use of animals in research, teaching, and testing. At your organization, the Chief
Executive O cer (CEO) or the CEO’s written designee (the Institutional O cial [IO])
appoints members to the IACUC. The IO is a senior member of the administration
who has the authority to allocate the resources needed to ensure the overall
e ectiveness of the organization's animal care and use program. The IO bears
ultimate responsibility for the animal program, and has responsibilities for resource
planning and for the alignment of program goals with the organization's mission.

The Animal Research Project

IACUC Membership Requirements

Per the AWR at 9 CFR 2.31 (Institutional 2004), the IACUC must consist of an IACUC
chair and at least two additional members. Among these members:

i. At least one shall be a Doctor of Veterinary Medicine, with training or experience

in laboratory animal science and medicine, who has direct or delegated program
responsibility for activities involving animals at the research facility.
ii. At least one shall not be a liated in any way with the facility other than as a
member of the Committee, and shall not be a member of the immediate family of
a person who is a liated with the facility.

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The PHS Policy has similar membership requirements, except that the IACUC must
have at least ve members, including a nonscientist and a practicing scientist
experienced in animal research. At organizations with larger animal use programs,
the number of members is usually larger than what the regulations require.

IACUC Responsibilities

The IACUC’s oversight responsibilities include the implementation of policies and

procedures to:

Ensure that the organization and its employees are in, and remain in, compliance
with all federal regulations and institutional policies related to the use of animals
in research, teaching, and testing.
Review and approve all animal use activities at the organization. The common
term for the written, detailed statement of these activities is the “animal protocol.”
Ensure that all investigators, sta , and students have proper training in animal use
principles and in the procedures that they will use in their animal activities.
Conduct semiannual inspections of all facilities where investigators house or use
animals in research, teaching, and testing.
Conduct semiannual reviews of the entire animal care and use program at the
organization. This includes a review of the quality of the veterinary care program,
the personnel training programs, the occupational health and safety program, and
the conduct of the IACUC itself.
Identify and investigate instances of noncompliance.
Provide a semiannual report to the Institutional O cial on the status of the
organization's animal care and use program.
Maintain written records associated with the review of animal care and use
activities, including the IACUC meeting minutes, for at least three years after the
conclusion of a research project.

The Role of the Attending Veterinarian (AV)

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The Attending Veterinarian (AV) is responsible for the health and care of all animals
at the organization's facilities. The AV is the ultimate authority for the medical
condition, treatment, and husbandry of your organization’s animals. The AV may
intervene immediately in any animal-related procedure and has the authority to halt
the procedure, if investigators are conducting the procedures incorrectly or if any
study animal is experiencing undue pain and distress during the procedure.

The Veterinary Consultation

The veterinary consultation is another important role for your organization’s AV. The
AWA stipulates that if the proposed procedures on animals have the potential to
cause more than momentary or slight pain or distress, the investigators must consult
with a veterinarian who has training or experience in laboratory animal medicine
before conducting those procedures. Many organizations require a veterinary
consultation during the planning stages for all projects involving animals.

The Responsibilities of Investigators

The AWR and the PHS Policy both describe many responsibilities of the principal
investigator (PI) and the research team. The research process begins when the PI
submits an animal protocol to the IACUC. The animal protocol describes, among
other things, the study’s purpose, what will happen to the animals, and the study’s
scienti c endpoints.

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All members of the research team must ensure that they maintain the appropriate
and humane treatment of animal subjects. It is the PI’s speci c responsibility to
provide access to proper training for team members before they receive permission
to conduct procedures with animal subjects. The PI, PI's designee(s), or AV usually
provides the training.

In the animal protocol, the PI must speci cally describe the items listed below.

The rationale and purpose for the use of animals.

The justi cation for the use of the species and the number of animals in the study.
The PI must provide scienti c justi cation, often using statistical methods, for the
number of animals in the study.
An assurance that the proposed studies do not unnecessarily duplicate prior
research.
Each procedure that the research team will conduct with animals.
A justi cation for any procedures that deviate from the AWR or the PHS Policy (for
example, withholding antibiotics from an animal after surgery because they are
known to interfere with the study of some scienti c endpoints).
The availability or appropriateness of alternatives, such as the use of less-invasive
procedures, other species, cell or tissue cultures, or computer simulations.
The methods to relieve more than momentary pain and distress.
The methods of euthanasia.

The Lay Summary of the Animal Protocol

The lay summary of the animal protocol should summarize the proposed study in
terms that will allow a person without a science background to understand what the
investigators hope to accomplish. The summary should include an explanation of:

The study’s purpose

The experimental approach (how the investigators will conduct the study)
The study endpoints (the types of data the investigators will collect)

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What will happen to the animals when the study is over

The signi cance of the research to the scienti c community and to the public

An IACUC will likely require the PI to revise the lay summary if it fails to address these
issues in a way that a layperson would understand.

Training and Experience

The Animal Welfare Regulations and the PHS Policy both require that all personnel
who use animals in research, testing, and teaching complete basic training in the
humane care and use of animals in a research environment. The PHS Policy also
delineates the relevant content to include in an adequate training program.

The PI must show that the team has su cient experience with the chosen animal
model and the proposed procedures to complete the project humanely. Research
team members who lack experience in the procedures must receive additional
training from the PI or the veterinary sta , or from other experts in the eld outside
their organization. The IACUC will review the quali cations of the research team prior
to the approval of the animal study.

Avoiding Pain and Distress

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The PI must provide a rigorous scienti c justi cation to the IACUC for studies that
may involve unrelieved pain and distress to research animals. The general guideline
that IACUCs use to determine if a procedure is painful is: “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” (OSTP 1985).

Many organizations require that the research protocol describe the potential for and
severity of the pain, distress, and discomfort that the animals will experience during
the proposed research procedures. It is common to use the pain category system
provided by the USDA, APHIS (2016).

The AWR require that the IACUC submit an annual report to the USDA that details the
number of each species used and the degree of discomfort/pain experienced by the
animals according to speci cally de ned categories. Each year, the USDA publishes a
compilation of the number of animals used in research nationally according to the
pain category.

Alternatives

The PHS Policy and the AWR require that investigators provide written assurance, in
the animal protocol, that they have searched the pertinent literature for alternatives
to any painful procedures. The written assurance should provide the details
(databases, keywords, inclusive dates) of the search. If the required literature search
indicates that less painful alternatives exist, investigators must use those alternatives
unless they can provide an acceptable justi cation for why the alternatives are not
appropriate for the proposed study.

Analgesia

If investigators cannot identify alternatives to the painful procedures, the animals

must receive relief from the painful procedures or conditions through appropriate
pain-relieving drugs and comfort care. The pain-relieving strategies must be

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appropriate to the species, conform to veterinary best practices, and receive

approval from the IACUC. The AV should provide guidance to investigators on the
species-appropriate use of analgesia and anesthesia for the speci c procedures.

At times, the use of pain-relieving drugs may interfere with study endpoints and the
research project’s aims. When the condition under study is pain itself, the IACUC
must decide whether to waive the requirement for analgesia/anesthesia, ask the
investigator to modify the study to have a less painful endpoint, or disapprove the
study. If the IACUC permits the exemption, there would likely be a requirement for
special monitoring strategies to use with the study animals.

Euthanasia

In many circumstances, euthanasia is a necessary study endpoint. When euthanasia

is necessary, PIs must assure the IACUC that they will skillfully perform the
euthanasia and use methods that are appropriate for the species and consistent with
the study’s stated endpoints and goals. PIs should discuss the euthanasia strategy
and methodology during the veterinary consultation, prior to the start of the study.
Further, all members of the research team must have proper training to perform the
approved euthanasia procedure.

The PHS Policy and the Guide state that the means of euthanasia should be
consistent with the recommendations in the American Veterinary Medical Association
Guidelines for the Euthanasia of Animals.

Making Changes to the Animal Protocol After Initial

Approval

Once the IACUC approves the animal protocol, neither the PI nor the research sta
can make changes to the methods or procedures unless the IACUC rst reviews and
approves those new methods, reagents, or species. Federal regulations make it clear

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that the IACUC must review and approve all signi cant changes to the procedures
described in an approved animal protocol before the research team can implement
such changes. NIH and OLAW provide guidance on what constitutes a “signi cant
change.”

Investigators must not conduct unapproved procedures with their animal subjects.
The conduct of unapproved procedures is noncompliance with IACUC policy and
federal regulations. The use of an unapproved procedure can result in the
suspension of the study until the incident of noncompliance undergoes a full
investigation and an approved corrective action plan begins. Resolution of the issues
could take months and put the organization in a position where it would need to
return grant funds to the funding agency.

Another consequence is that data collected during intervals of noncompliance will

likely not be publishable. Most journals will reject papers reporting animal studies
that an IACUC has not approved. If the author is deceptive about IACUC approval, a
misconduct allegation could follow.

Reporting Concerns About Animal Use

You must immediately report concerns about the treatment of animals owned by
your organization or the use of procedures that do not comply with federal
regulations or institutional guidelines. You can report these concerns to any of the
following:

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The AWR and the PHS Policy require the IACUC to review and, if warranted,
investigate allegations of mistreatment, misuse, or noncompliance.

The IACUC has the authority to suspend an animal study to protect the animals and
the investigators. Only the IACUC can lift a decision to suspend a study or animal
activity. The organization's administrators or the IO cannot overturn the decision.
However, the IO does have the responsibility to review the case, review the actions
taken by the IACUC, and determine whether to take additional action. The IO then
must report the suspension and the corrective action plan to the appropriate
regulatory agencies.

Summary

Animal research is a privilege that to do ethically, requires integrity and

professionalism. Improper research practices can put animal subjects at risk, waste
valuable resources, jeopardize careers, and delay the development of new medicines
and technologies. Accountability and ethical behavior by investigators and
organizations are critical and non-negotiable requirements.

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The rules for using animals in research may seem lengthy and complex, but
investigators engaged in research with animals must strive to understand the ethical
principles inherent in laboratory animal welfare. All members of the research team
must also understand and comply with all federal regulations, guidelines, and
institutional policies that govern animal research activities. This high-level of
professionalism and integrity in the scienti c community is crucial to the
preservation of the public's trust in science and in the global research enterprise.

References

Cavalieri, Paola, and Peter Singer, eds. 1993. The Great Ape Project: Equality Beyond
Humanity. London: Fourth Estate.
Godlovitch, Stanley, Roslind Godlovitch, and John Harris, eds. 1972. Animals, Men
and Morals; An Enquiry into the Maltreatment of Non-Humans. New York: Taplinger
Publishing Company.
Hall, Marshall. 1856. "Of the Principles of Investigation in Physiology." Lancet
1:393-4.
Hamilton, Susan. 2013. “On the Cruelty to Animals Act, 15 August 1876.” Branch:
Britain, Representation and Nineteenth-Century History, February. Accessed May 7,
2019.
Harrison, Ruth. 1964. Animal Machines: The New Factory Farming Industry. London:
Vincent Stuart Publishers Ltd.
Health Research Extension Act of 1985, Pub. L. No. 99-158, 99 Stat. 820-86 (1985).
Institutional Animal Care and Use Committee (IACUC), 9 CFR § 2.31 (2004).
National Research Council (NRC). 2011. Guide for the Care and Use of Laboratory
Animals: Eighth Edition. Washington, DC: The National Academies Press.
O ce of Laboratory Animal Welfare (OLAW). 2015. "Public Health Service Policy
on Humane Care and Use of Laboratory Animals." Accessed May 7, 2019.
O ce of Science and Technology Policy (OSTP). 1985. "U.S. Government
Principles for the Utilization and Care of Vertebrate Animals Used in Testing,
Research, and Training." Federal Register 50(97). Accessed May 7, 2019.

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Ortiz-Robles, Mario. 2016. “Animal Acts: 1822, 1835, 1849, 1850, 1854, 1876,
1900.” Accessed May 23, 2019.
Regan, Tom. 2004. The Case for Animal Rights. Berkeley, CA: University of California
Press.
Russell, William Moy Stratton, and Rex Leonard Burch. 1959. The Principles of
Humane Experimental Technique. London: Methuen. Accessed May 7, 2019.
Singer, Peter. 1975. Animal Liberation. London: Pimlico.
U.S. Department of Agriculture (USDA), Animal and Plant Health Inspection Service
(APHIS). 2008. “The Animal Welfare Act: An Overview.” Accessed May 7, 2019.
U.S. Department of Agriculture (USDA), Animal and Plant Health Inspection Service
(APHIS). 2016. "Annual Report of Research Facility." Accessed May 7, 2019.
U.S. Department of Agriculture (USDA), Animal and Plant Health Inspection Service
(APHIS). 2017. “Animal Welfare Act and Animal Welfare Regulations.” Accessed
May 7, 2019.

Original Release: February 2009

Last Updated: July 2019

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