The Laboratory Rabbit, Guinea Pig, Hamster, and Other Rodents

1236 Pages·2012·48.642 MB·English

by  Mark A. Suckow, Karla A. Stevens and Ronald P. Wilson (Eds.)

C H A P T E R
1
Ethical Considerations and
Regulatory Issues
Marilyn J. Brown1 and Kathleen L. Smiler2
1Charles River Laboratories, East Thetford, Vermont, USA; 2Lakeville, Michigan, USA
O U T L I N E
Introduction 3 Institutional Animal Welfare Oversight 20
Ethical Concepts 4 Regulations and Non-regulatory Considerations 21
Moral Theories 4 United States Regulatory Considerations 21
Descriptive Laboratory Animal Use Ethics 5 United States Animal Welfare Act 21
Ethical Principles 5 Public Health Service Policy on Humane
Respect for Life 5 Care and Use of Animals 22
Societal Benefit 5 Food and Drug Administration Good Laboratory
Non-Maleficence 6 Practices 22
The Three Rs (Replacement, Reduction, and Interagency Cooperation 23
Refinement) 8 Environmental Protection Agency Good
Laboratory Practices 23
Ethical Challenges 14
Non-Regulatory Considerations 23
Breeding Colonies 14
Institute for Laboratory Animal Research 23
Genetically Modified Rodents 15
Guide for the Care and Use of Laboratory
Cancer Research 16
Animals (Guide) 24
Perinatal Animal Use 17
International Regulations, Policies, and Standards 24
Neuroscience and Behavioral Research 18
AAALAC, International 24
Food and Fluid Restriction 18
Canada 24
Neuroanatomic Studies 18
European Union 24
Neural Injury and Disease 19
Pacific Rim 25
Behavioral Studies 19
Prolonged Restraint and Anesthesia 19 Conclusion 27
Restraint of Awake Animals 19
References 27
Prolonged Studies in Anesthetized Animals 20
“… by now it is widely recognized that the [most INTRODUCTION
humane] possible treatments of experimental animals, far
from being an obstacle, is actually a prerequisite for suc- Like many aspects of life, involvement with animal
cessful animal experiments.” Russell and Burch, 1959 research presents ethical challenges – areas where com-
The Principles of Humane Experimental Technique peting interests require use of an ethical decision-making
The Laboratory Rabbit, Guinea Pig, Hamster, and Other Rodents
3
DOI: 10.1016/B978-0-12-380920-9.00001-8 © 22001122 Elsevier Inc.
4 1. ETHICAL CONSIDERATIONS AND REGULATORy ISSUES
process, to provide guidance. Individuals in the labora- standards” (National Research Council, 2011). The
tory may face basic competing interests between scien- Guide uses the words ‘ethics’ or ‘ethical’ 59 times in
tists, technicians, veterinary colleagues, an employing either the text or references. The first place ‘ethics’ is
institution, the public, and concern for the animals mentioned in the Guide is the Preface: “The Guide is
themselves (e.g., individual health versus health of the also intended to assist investigators in fulfilling their
colony). According to Tannenbaum, normative veterinary obligation to plan and conduct animal experiments in
ethics refers to the search for correct principles of good accord with the highest scientific, humane, and ethical
and bad, right and wrong, and looks for the correct principles” (National Research Council, 2011). Humane
norms for veterinary professional behavior and attitudes care, ethics, and animal welfare are closely linked
(Tannenbaum, 1995). The intent of this chapter is to pro- and, for the purposes of this chapter, may be used
vide information which will be useful to all individuals interchangeably.
involved in animal-based research, testing and teach-
ing: scientists; technicians; laboratory animal veterinar-
ians; and Institutional Animal Care and Use Committee
ETHICAL CONCEPTS
(IACUC) members. This process might be considered an
effort to define normative laboratory animal use ethics. The
Moral Theories
search for an appropriate ethical solution rarely leads
to a complete and absolute answer, as science is a very Moral theory is an expansive field of study with its
dynamic field and new issues and new insights influence own vocabulary and competing points of view. It is
the outcome. This look at normative laboratory animal use beyond the scope of this chapter to provide a compre-
ethics will examine some general ethical concepts within hensive discussion of the many theories that discuss the
the context of Tannebaum’s definition of descriptive [lab- humane use of animals. Instead, focus is made on some
oratory animal use] ethics, where descriptive ethics is the principles which the authors have found helpful.
study of the actual values or standards of a profession; Consideration of ethics with respect to use of animals
that is, what members of a profession consider to be in research begins with the basic idea of where one feels
right and wrong regarding professional behavior and animals (versus people) fit in the moral spectrum. In
attitudes (Tannenbaum, 1995). In this chapter, reference other words, what is the moral value of an animal rela-
will be made to relevant values and standards found tive to a human? Moral status or standing represents
in various principles and guidelines developed for use “the position or rank of an entity along a moral con-
by individuals involved in animal research. Potential tinuum from minimal to maximal moral significance”
ethical challenges which might confront laboratory ani- (Kraus and Renquist, 2000). Because animals lack moral
mal professionals using rodents and rabbits (e.g., chal- agency, defined as a uniquely human capacity for mak-
lenges related to breeding colonies, genetically modified ing moral judgments (Kraus and Renquist, 2000), it may
rodents, use of animals in cancer research, perinatal ani- be concluded that animals, while having some moral
mal use, and use of animals in neuroscience and behav- status, fall below that accorded to humans. Even when
ioral research) will be used as examples. Related ethical examining the moral status of animals, there are many
questions, and the appropriate principles which may who accord different levels of moral status for different
pertain to the situation are discussed. The reader is chal- species. Such a continuum of moral status is essentially
lenged to test the general rules and principles against a sliding scale where moral status is based on a combi-
his or her own moral experience and intuition and thus nation of cognitive and sensory capacities. This concept
create his or her own descriptive laboratory animal has been coined “speciesism” by some philosophers
ethos. Further, the reader is encouraged to recognize who then compare it to other concepts which foster dif-
that this is an ongoing process, that one’s professional ferential treatment based upon a given trait, similar to
ethos will likely grow and mature as new challenges are racism or sexism (Singer, 1975). Regardless of where
encountered. a species is placed on this Darwinian scale, a com-
It is recognized that animal welfare is a “core con- monality shared by all vertebrates is that of sentience.
cern of veterinary ethics … [and] this subject has Sentience is the capacity to perceive and process sen-
gained increased importance as society and the profes- sory input and thus the ability to feel pain and distress.
sion endorse ever more strongly the moral imperative Moral agents (humans) have obligations or are bound
to treat animals decently.” When discussing labora- to do certain things out of a sense of duty, custom, or
tory animal ethics, one often uses the term “humane” law and have responsibility toward other beings. It is
(Tannenbaum, 1995). The Guide for the Care and Use of from this obligation that most humans feel it is right
Laboratory Animals (Guide) states “Humane care means to minimize the pain and distress felt by other sentient
those actions taken to assure that laboratory animals beings. This obligation may be called non-maleficence
are treated according to high ethical and scientific and is addressed in greater detail later in this chapter.
I. GENERAL
ETHICAL CONCEPTS 5
In the most general sense, there are two approaches from thermal or physical discomfort; (3) freedom from
to ethics: utilitarianism and deontology. Utilitarian theo- injury and disease; (4) freedom to express normal social
ries look at the consequences of actions to determine behavior; and (5) freedom from fear. The Five Freedoms
which actions are good and which are bad. The goal is to were revised in 1993 to include: (1 ) freedom from hun-
maximize good consequences and minimize bad ones. ger and thirst, by assuring ready access to fresh water
In its most basic form, this is similar to what an IACUC and a diet sufficient to maintain full health and vigor;
protocol review does when it performs a cost or harm/ (2) freedom from discomfort, by providing an envi-
benefit analysis. However, there are different views of the ronment including shelter and a comfortable resting
“good” that should be maximized. Utilitarianism is an area; (3) freedom from pain, injury and disease, by
example of action-oriented ethical theories, because it exam- preventive means or rapid diagnosis and treatment;
ines the consequences of actions. These theories tend to (4) freedom from fear and distress, by ensuring condi-
stress the concepts of duty and obligation. In contrast, tions that avoid mental suffering; and (5) freedom to
deontological theories are also action-oriented ethical the- express normal behavior, by providing sufficient space,
ories; however, there are some moral imperatives which proper facilities, and company of the animal’s own kind
are independent of how much “good” results from an (Webster, 2001).
action. Some deontologists advocate a set of “obligatory” In 1996, the United States National Aeronautics and
moral principles but allow some compromise when dif- Space Administration (NASA) developed basic prin-
ferent moral principles are conflicting (e.g., one principle ciples, referred to as the Sundowner Principles (NASA,
trumps another). There may also be non-obligatory prin- 1996). These principles were based upon the Belmont
ciples which are desirable but not mandatory to follow. Report which had been written for the protection of
Other ethical approaches include those based on val- human research subjects (National Commission for
ues and ethics. Value-based ethics center around basic the Protection of Human Subjects of Biomedical and
values to be sought. These values tend to be hierarchi- Behavioral Research, 1979). These principles of bioeth-
cal. There are also virtue-oriented ethical theories. A virtue ics offer a simple, yet elegant framework for looking at
contributes to a good moral life (e.g., honesty, kindness, ethical questions:
generosity). These approaches tend to instill “attitudes,
feelings, and states of mind central to the virtuous dispo- “The use of animals in research involves responsibility – not only
sition” (Tannenbaum, 1995). It has been suggested that a for the stewardship of the animals but to the scientific community
and society as well. Stewardship is a universal responsibility that
satisfactory approach to normative ethics must include
goes beyond the immediate research needs to include acquisition, care
actions, values, and virtues (Tannenbaum, 1995).
and disposition of the animals, while responsibility to the scientific
community and society requires an appropriate understanding of,
and sensitivity to scientific needs and community attitudes toward
the use of animals.
Descriptive Laboratory Animal Use Ethics
Among the basic principles generally accepted in our culture,
three are particularly relevant to the ethics of research using animals:
Ethical Principles
respect for life, societal benefit, and non-maleficence” (NASA, 1996).
Descriptive laboratory animal ethics represents an
approach for determining appropriate moral behav-
Respect for Life
ior and attitude (Tannenbaum, 1995). Principles can be
Living creatures deserve respect. This principle
defined as “accepted generalizations about a topic that
requires that animals used in research should be of
are frequently endorsed by many and diverse organi-
an appropriate species and health status, and should
zations” (National Research Council, 2011); and sev-
involve the minimum number required to obtain valid
eral sets of principles with relevance to ethical use
scientific results. It also recognizes that the use of dif-
of laboratory animals will be discussed. It is hoped
ferent species may raise different ethical concerns.
that application of these principles in the discussion
Selection of appropriate species should consider cog-
of example ethical challenges later in this chapter will
nitive capacity and other morally relevant factors.
serve as a basis for how other ethical challenges may be
Additionally, methods such as mathematical models,
approached.
computer simulation, and in vitro systems should be
One set of principles, the concept of the “Five
considered and used whenever possible.
Freedoms,” was originally created by the United
Kingdom Farm Animal Welfare Advisory Council
Societal Benefit
(FAWC) in 1979 specifically to address issues related
to the use of animals in agriculture. Today, the “Five The advancement of biological knowledge and
Freedoms” are also often mentioned within the con- improvements in the protection of the health and well-
text of animals used in research. The “Five Freedoms” being of both humans and other animals provide strong
include: (1) freedom from malnutrition; (2) freedom justification for biomedical and behavioral research.
I. GENERAL
6 1. ETHICAL CONSIDERATIONS AND REGULATORy ISSUES
This principle entails that when animals are used, an international association that includes all the West
the assessment of the overall ethical value of such use European medical research councils, fully endorsed
should include consideration of the full range of poten- the CIOMS Guiding Principles in 1984. In the same
tial societal goods, the populations affected, and the year, the CIOMS Guiding Principles were endorsed
burdens that are expected to be borne by the subjects of by the World Health Organization (WHO) Advisory
the research. Committee on Medical Research. It should be noted
that, at the time of writing this chapter, the CIOMS
Non-Maleficence
were undergoing revision.
Based upon the idea that vertebrate animals are sen- During 1984–1985, the U.S. National Institutes of
tient, this principle holds that the minimization of dis- Health (NIH) convened the U.S. Interagency Research
tress, pain, and suffering is a moral imperative. Unless Animal Committee (IRAC) which created a simi-
the contrary is established, investigators should consider lar set of principles, the U.S. Government Principles
that procedures that cause pain or distress in humans for the Utilization and Care of Vertebrate Animals
may cause pain or distress in other sentient animals used in Testing, Research and Training (Table 1.2) for
(Interagency Research Animal Committee, 1985). research funded by the U.S. Public Health Service (PHS)
The International Guiding Principles for Biomedical (Interagency Research Animal Committee, 1985). These
Research Involving Animals (Table 1.1) were devel- principles were, to a considerable extent, based on the
oped by the Council for International Organizations of CIOMS Guiding Principles.
Medical Sciences (CIOMS) as a result of extensive inter- As an indication of the wide acceptance of both the
national and interdisciplinary consultations spanning IRAC and CIOMS Principles, discussion of the IRAC
1982–1984 (Bankowski, 1985; Council for International Principles can be found under the heading “Ethics and
Organizations of Medical Sciences, 1985). These princi- Animal Use” in the Guide (National Research Council,
ples have a considerable measure of acceptance interna- 2011). NASA and CIOMS are also listed in Appendix
tionally. European Medical Research Councils (EMRC), A of the Guide under the heading “Ethics and Welfare”
TABLE 1.1 Council for International Organizations of Medical Sciences (CIOMS) Basic Principles (1985)
I. The advancement of biological knowledge and the development of improved means for the protection of the health and well-being both
of man and of animals require recourse to experimentation on intact live animals of a wide variety of species
II. Methods such as mathematical models, computer simulation and in vitro biological systems should be used wherever appropriate
III. Animal experiments should be undertaken only after due consideration of their relevance for human or animal health and the
advancement of biological knowledge
IV. The animals selected for an experiment should be of an appropriate species and quality, and the minimum number required to obtain
scientifically valid results
V. Investigators and other personnel should never fail to treat animals as sentient, and should regard their proper care and use and the
avoidance or minimization of discomfort, distress, or pain as ethical imperatives
VI. Investigators should assume that procedures that would cause pain in human beings cause pain in other vertebrate species, although
more needs to be known about the perception of pain in animals
VII. Procedures with animals that may cause more than momentary or minimal pain or distress should be performed with appropriate
sedation, analgesia, or anesthesia in accordance with accepted veterinary practice. Surgical or other painful procedures should not be
performed on unanesthetized animals paralyzed by chemical agents
VIII. Where waivers are required in relation to the provisions of article VII, the decisions should not rest solely with the investigators directly
concerned but should be made, with due regard to the provisions of articles IV, V, and VI, by a suitably constituted review body. Such
waivers should not be made solely for the purposes of teaching or demonstration
IX. At the end of, or, when appropriate, during an experiment, animals that would otherwise suffer severe or chronic pain, distress,
discomfort, or disablement that cannot be relieved should be painlessly killed
X. The best possible living conditions should be maintained for animals kept for biomedical purposes. Normally the care of animals should
be under the supervision of veterinarians having experience in laboratory animal science. In any case, veterinary care should be available
as required
XI. It is the responsibility of the director of an institute or department using animals to ensure that investigators and personnel have
appropriate qualifications or experience for conducting procedures on animals. Adequate opportunities shall be provided for in-service
training, including the proper and humane concern for the animals under their care
I. GENERAL
ETHICAL CONCEPTS 7
(National Research Council, 2011). The complete IRAC l Provision of adequate veterinary care;
Principles are found on the cover of the PHS Policy (Office l Assurance of appropriate training and qualifications
of Laboratory Animal Welfare, 2002) and Appendix B of of personnel using and caring for animals.
the Guide (National Research Council, 2011).
As previously mentioned, descriptive ethics is the
There are really no points of conflict and, in fact,
study of actual values or standards of a profession.
there are many points of consensus between the
With respect to ethics governing laboratory animal use,
Sundowner, CIOMS, and IRAC principles all of which
the Sundowner, CIOMS, and IRAC principles could be
offer that animal-based research should:
considered major components.
l Acknowledge the importance of research with Commonly accepted ethical principles result in devel-
relevance to human or animal health, advancement opment of professional guidelines. For example, in 1831
of knowledge, or the good of society; in the United Kingdom (U.K.), Marshall Hall, a lead-
l Stress consideration of alternatives to reduce or ing British physiologist, developed guidelines for ani-
replace the use of animals; mal experimentation (Zurlo et al., 1993). The British
l Require avoiding or minimizing discomfort, distress, Association for the Advancement of Science further
and pain. refined these principles in 1871, 5 years before the first
legislation in the U.K. In 1909, Walter B. Cannon devel-
Some items which are addressed in greater detail in
oped guidelines for animal experimentation for the
the CIOMS and IRAC principles but are more generally
American Physiological Association. Many other sci-
under the concept of non-maleficence in the Sundowner
entific organizations have also created similar guide-
Principles include:
lines. Scientists are urged to seek those within their own
l Use of appropriate sedation, analgesia, and anesthesia; professional societies. (e.g., Federation of American
l Establishment of humane endpoints; Societies for Experimental Biology at http://www.faseb.
TABLE 1.2 Interagency Research Animal Committee (IRAC) U.S. Government Principles for the Utilization and Care of Vertebrate
Animals Used in Testing, Research, and Training (Interagency Research Animal Committee, 1985)
The development of knowledge necessary for the improvement of the health and well-being of humans as well as other animals requires in
vivo experimentation with a wide variety of animal species. Whenever U.S. Government agencies develop requirements for testing, research, or
training procedures involving the use of vertebrate animals, the following principles shall be considered; and whenever these agencies actually
perform or sponsor such procedures, the responsible Institutional Official shall ensure that these principles are adhered to:
I. The transportation, care, and use of animals should be in accordance with the Animal Welfare Act (7 U.S.C. 2131 et. seq.) and other
applicable Federal laws, guidelines, and policies.
II. Procedures involving animals should be designed and performed with due consideration of their relevance to human or animal health,
the advancement of knowledge, or the good of society.
III. The animals selected for a procedure should be of an appropriate species and quality and the minimum number required to obtain valid
results. Methods such as mathematical models, computer simulation, and in vitro biological systems should be considered.
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.
V. Procedures with animals that may cause more than momentary or slight pain or distress should be performed with appropriate sedation,
analgesia, or anesthesia. Surgical or other painful procedures should not be performed on unanesthetized animals paralyzed by chemical
agents.
VI. Animals that would otherwise suffer severe or chronic pain or distress that cannot be relieved should be painlessly killed at the end of the
procedure or, if appropriate, during the procedure.
VII. The living conditions of animals should be appropriate for their species and contribute to their health and comfort. Normally, the
housing, feeding, and care of all animals used for biomedical purposes must be directed by a veterinarian or other scientist trained
and experienced in the proper care, handling, and use of the species being maintained or studied. In any case, veterinary care shall be
provided as indicated.
VIII. Investigators and other personnel shall be appropriately qualified and experienced for conducting procedures on living animals.
Adequate arrangements shall be made for their in-service training, including the proper and humane care and use of laboratory animals.
IX. Where exceptions are required in relation to the provisions of these Principles, the decisions should not rest with the investigators directly
concerned but should be made, with due regard to Principle II, by an appropriate review group such as an institutional animal care and
use committee. Such exceptions should not be made solely for the purposes of teaching or demonstration (Interagency Research Animal
Committee, 1985).
I. GENERAL
8 1. ETHICAL CONSIDERATIONS AND REGULATORy ISSUES
org/Policy-and-Government-Affairs/Science-Policy- “(i) Procedures involving animals will avoid or minimize
Issues/Animals-in-Research-and-Education/Statement- discomfort, distress, and pain to the animals; (ii) The
of-Principles.aspx; American Physiological Society at principle investigator has considered alternatives to pro-
http://www.the-aps.org/pa/resources/policyStmnts/ cedures that may cause more than momentary or slight
paPolicyStmnts_Guide.htm). pain or distress to the animals, and has provided a writ-
Examples of professional guidelines for laboratory ten narrative description of the methods and sources,
animal veterinarians include the Principles of Veterinary e.g., The Animal Welfare Information Center, used to
Medical Ethics and Veterinary Oath of the American determine that alternatives were not available ….”
Veterinary Medical Association (at http://www.avma (Office of the Federal Register, 2002). The focus of USDA
.org/issues/policy/ethics.asp, http://www.avma.org/ inspectors on adherence to this section of the regulations
about_avma/whoweare/oath.asp). Further, many labo- can be appreciated when looking at the USDA Research
ratory animal science professionals refer to the Position Facility Inspection Guide which instructs inspectors
Statements of the American Association for Laboratory several times to evaluate institutional compliance in
Animal Science (AALAS; http://www.aalas.org/ this area (USDA, 2009). In addition, the requirement
association/position_statements.aspx). for a search for alternatives is the subject of a specific
Central to these codes of conduct, principles, state- Animal Care Policy – Policy 12 (Animal and Plant Health
ments of ethics, and position statements is the commit- Inspection Service, 2000). Strategies to enhance elec-
ment to the humane care and use of research animals. tronic search efficiency using a search filter for PubMed
have been published (Hooijmans et al., 2010b). A “Gold
The Three Rs (Replacement, Reduction, and
Standard Publication Checklist” has been proposed to
Refinement)
help fully integrate the 3Rs into systematic reviews of the
Several sets of ethical principles and guidelines cov- literature (Hooijmans et al., 2010a).
ering the use of animals in research, testing, and teach- In a section on personnel qualifications, the AWA
ing have been mentioned but perhaps the simplest and Regulations state that the institution should assure ade-
the one with the greatest impact on animal research quate training and qualifications and that this is fulfilled
today is the ethical concept called “The 3Rs”, a call to in part through the provision of training and instruction
apply whenever possible the alternatives of replace- on the “concept, availability, and use of research and test-
ment of animals, reduction in the number of animals ing methods that limit the use of animals [Reduction]
used, and the refinement in procedures used on animals or minimize animal distress [Refinement]” (Office of
in research (Russell and Burch, 1959). the Federal Register, 2002). The AWA Regulations fur-
The Guide was originally published in 1963 and has ther indicate that research staff should be trained on
undergone numerous revisions, with the most recent the “utilization of services (e.g., National Agriculture
edition being published in 2011. The Statement of Task Library of Medicine) available to find information: …
of the latest revision committee begins with “The use (ii) On alternatives to the use of live animals in research
of laboratory animals for biomedical research, test- [Replacement]; …” (Office of the Federal Register, 2002).
ing and education is guided by the principles of the In addition to the specific references above, the AWA
Three Rs… .” (National Research Council, 2011). The Regulations also refer to the use of anesthetics, analge-
3Rs are a common theme in the Guide which states sics, and sedatives, the availability of appropriate vet-
“Throughout the Guide, scientists and institutions are erinary care, the use of appropriate housing; and timely,
encouraged to give careful and deliberate thought appropriate euthanasia, all of which demonstrate the
to the decision to use animals taking into consider- practice of “refinement”.
ation the contribution that such use will make to new The United States Public Health Service (PHS) Policy
knowledge, ethical concerns, and the availability of contains similar language regarding minimizing dis-
alternatives to animal use. A practical strategy for comfort, distress pain, use of appropriate anesthesia,
decision making, [is] the “Three Rs” (Replacement, and the use of humane endpoints as examples of refine-
Reduction, and Refinement) approach, …”(National ment. In addition, the PHS Policy refers to the IRAC
Research Council, 2011). Principles, requiring institutions receiving PHS funds
The concept of the 3Rs is also infused in U.S. regu- to use the Guide as a basis for their animal care and use
lations covering research using animals. Although programs.
the United States Department of Agriculture (USDA) National and international agencies and organiza-
Animal Welfare Act (AWA) regulations do not include tions such as the Interagency Coordinating Committee
the word “alternatives” in its section of definitions, the on the Validation of Alternative Methods (ICCVAM)
term is used several times in the regulations themselves. (http://iccvam.niehs.nih.gov/), the European Centre
For example, in the section on IACUC review of proto- for the Validation of Alternative Methods (ECVAM)
cols the regulations state protocols must indicate that (http://ecvam.jrc.ec.europa.eu/), and the National
I. GENERAL
ETHICAL CONCEPTS 9
Centre for the Replacement, Refinement and Reduction animals commonly kept as pets such as horses, dogs,
of Animals in Research (NC3Rs) (http://www.nc3rs and cats are often regarded differently than rats and
.org.uk/) are charged with helping to find and promote mice, which in turn are regarded differently from fruit
the use of alternatives. flies and worms, and so on.
With so much official emphasis on the 3Rs, there is Development of fully validated and accepted replace-
sometimes a perception that the concept is not being ment alternatives can be a frustratingly slow process.
adequately implemented in practice. Indeed, it has However, there are significant examples of successful
been suggested that scientists and IACUCs do not fully replacement of live animals. For example, one of the
understand the concepts of the 3Rs (Graham, 2002; most criticized uses of animals for toxicity testing is the
Schuppli and Fraser, 2005). In addition to incomplete Draize test in rabbits. This test was developed to deter-
understanding of the concepts, factors believed to neg- mine ocular toxicity and irritancy caused by products
atively influence the full implementation of the 3Rs by and chemicals. Ocular toxicity tests represent one of the
IACUCs might include: (1) a belief that the scientists four most commonly conducted product safety tests
themselves would implement the 3Rs; (2) an assump- (Interagency Coordinating Committee on the Validation
tion that funding agencies have reviewed the use of the of Alternative Methods, 2010). The 3Rs were imple-
3Rs during proposal review; (3) confidence that sample mented by the development of three validated and
size, rather than study design, is the sole criterion for accepted replacements for screening products for ocu-
reduction; and (4) focus upon potential harm from pro- lar toxicity: the bovine corneal opacity and permeability
cedures without consideration for potential distress that test using a cow eye or the isolated chicken eye test (both
animals might experience from husbandry and hous- by-products of the meat industry); and the Cytosensor®
ing. Although these conclusions were based upon a microphysiometer (Molecular Devices, Inc., Sunnyvale,
relatively small number of IACUCs, these are troubling CA). A balanced preemptive pain management plan for
observations and indicate the need for greater empha- rabbit Draize test studies, when the test is still required,
sis on the 3Rs in training programs for scientists and has also been validated and accepted as a refinement
IACUCs. The authors hope that this chapter can be a (Interagency Coordinating Committee on the Validation
resource for that process. of Alternative Methods, 2010).
It would be disingenuous to imply that, although well A second example of implementation of the 3Rs
accepted, the concept of the 3Rs is universally accepted. involves the replacement of rabbits in the testing of
In an article titled “Time to Abandon the Three Rs”, pharmaceuticals and medical devices for pyrogens by
Derbyshire wrote that the 3Rs “draw attention away from use of an in vitro alternative, the Limulus Ameobecyte
the value of experimentation and toward the importance Lysate (LAL) Test. In the LAL, blood of horseshoe crabs
of animal welfare” (Derbyshire, 2006). Although the article is collected and the animals are returned, unharmed,
supports the concept of reducing animal stress for the sake back to the ocean. Previous tests required the injection
of science, the authors do not clearly recognize the oppor- of drugs, biologics, medical devices, or raw materials
tunity to balance facilitation of science and application of into rabbits to look for a febrile response as an indica-
the 3Rs. tion of contamination with endotoxins.
REPLACEMENT REDUCTION
Replacement refers to methods that avoid using ani- “Reduction includes strategies for obtaining compara-
mals. The term includes absolute replacements (i.e., ble levels of information from the use of fewer animals
replacing animals with inanimate systems such as or for maximizing the information obtained from any
computer programs) as well as relative replacements given number of animals (without increasing pain or dis-
(i.e., replacing animals, such as vertebrates, with ani- tress) so to ultimately require fewer animals to acquire
mals that are lower on the phylogenic scale) (National the same scientific information. This approach relies on
Research Council, 2011). Relative replacement may be an analysis of experimental design, applications of newer
controversial to some people as it implies “speciesism”, technologies, the use of appropriate statistical meth-
the idea that one species has greater moral standing ods, and control of environmentally related variability
than another (Singer, 1975). in animal housing and study areas” (National Research
Like many of the ethical considerations relating to Council, 2011).
animal use, relative replacement is essentially a con- Strategies to reduce the numbers of animals needed
tinuum of moral standing. Society, in general, often dif- include improved statistical design of a study (Dell
ferentiates between humans and non-human animals; et al., 2002) and improved selection of an animal model,
however, with respect to the animal world, different including selection of animals with the most appropri-
opinions exist regarding our obligations to some species ate health and genetic status. Control of the genetic
versus others. For example, non-human primates and status is an advantage of using rats and mice. The use
I. GENERAL
10 1. ETHICAL CONSIDERATIONS AND REGULATORy ISSUES
of inbred strains of rats and mice allows scientists to mean that they [animals] are incapable of experiencing
control and investigate genetic variation, and to evalu- pain, suffering or distress or any other higher order
ate responses to treatments on specific areas of interest states of conscious experience” (Nuffield Council on
(Festing, 2004). The use of animals without confound- Bioethics, 2005).
ing disease or genetic variation results in less variation, Basic to minimization of pain is the ability to recog-
thus requiring fewer animals to determine a treatment nize the signs of pain in specific species. It has been sug-
effect. gested that some animals, particularly prey species, may
Individuals involved with study design, study try to mask pain to avoid displaying abnormal activity
review, or those participating as a member of the that might increase their risk of predation (Roughan and
research team, have the ethical imperative to ensure Flecknell, 2000). Further, many animals are most active
studies use the minimum number of animals necessary during the dark cycle, when observations are more dif-
to achieve the scientific objective of the study. Scientists ficult. Since clinical indices of pain may be very subtle,
should design studies with particular attention to meth- it is important to be able to recognize a departure from
odology, statistics, and choice of model. Veterinarians normal behavior and appearance (Table 1.3; National
and facility staff should collaborate to minimize non- Research Council, 2003). A short list of general signs
experimental variables in animal care. IACUCs should and measurements that might indicate pain or distress
be diligent during review of the protocol, semiannual includes: (1) vigorous attempt to escape; (2) changes in
program and facility evaluations, and review of post- biological characteristics such as food and water con-
approval monitoring to assure that the appropriate sumption and body weight; (3) changes in blood levels of
number of animals have been used. Having a statisti- hormones and glucose; (4) increased adrenal gland mass;
cian on the IACUC is one strategy that may be helpful. and (5) appearance, posture, and behavior (Moberg,
1985, 2000). Behavioral indicators of pain in mice and
REFINEMENT rats have also been described (Flecknell, 1999; Kohn
“Refinement refers to modifications of husbandry or et al., 2007; Roughan and Flecknell, 2000, 2001, 2003).
experimental procedures to enhance animal well-being In addition, guidelines for the assessment and manage-
and minimize or eliminate pain and distress” (National ment of pain in rodents and rabbits have been published
Research Council, 2011). In the authors’ opinion, refine- by the American College of Laboratory Animal Medicine
ment is commonly employed by scientists in ongoing (ACLAM, 2006).
efforts to improve their science; that is, better animal Scientists sometimes have concerns about the effect
welfare leads to higher-quality science. Many scientists of perioperative analgesics on the research. Many stud-
do not recognize this as utilization of “alternatives”, ies have been done investigating analgesic effect on a
even though it clearly falls within the 3Rs. However, wide variety of parameters (e.g., litter size, body weight,
this is also an area where scientists, veterinarians, and
IACUCs can make significant strides to enhance ani-
TABLE 1.3 Indicators of Pain in Rodents and Rabbits
mal welfare. Use of less invasive procedures (e. g., use
of a blood pressure cuff instead of an implanted cath- Species General Behavior Appearance Other
eter for blood pressure monitoring) is one method of
Rodents Decreased activity; Piloerection; Rapid, shallow
refinement. However, there are also situations where
excessive licking rough/ respiration;
an invasive procedure, such as implantation of telem-
and scratching; self- stained decreased
etry sensors to allow ongoing collection of real-time mutilation; may be haircoat; food/water
data, can result in much less stressful data collection unusually aggressive; abnormal consumption;
(Stephens et al., 2002). Examples of other refinements abnormal locomotion stance or tremors
(stumbling, falling); arched back;
include accurate recognition of pain and the use of anal-
writhing; does not porphyrin
gesics and supportive care; implementation of humane
make nest; hiding staining (rats)
endpoints; and enhanced housing and husbandry.
Rabbit Head pressing; Excessive Rapid, shallow
Carbone and Garnett (2008), state, “… the prime ethi-
teeth grinding; salivation; respiration;
cal concerns in laboratory animal welfare is what ani-
may become more hunched decreased
mals consciously experience: their pain, distress, fear, aggressive; increased posture food/water
boredom, happiness and psychological well being.” vocalizations; consumption
The emotional dimension of pain, a characteristic of excessive licking and
scratching; reluctant
suffering, requires pain pathways to extend to higher
to locomote
levels of the cortex unique to humans and some other
primates (such as apes) (Nuffield Council on Bioethics, No single observation is sufficiently reliable to indicate pain; rather several signs, taken
in the context of the animal’s situation should be evaluated. The signs of pain may vary
2005), but it has been stated that “… the absence of
with the type of procedure (e.g., orthopedic versus abdominal pain) (National Research
analogous structures cannot necessarily be taken to Council, 2003)
I. GENERAL
ETHICAL CONCEPTS 11
behavior, and hemodynamic parameters) (Bourque et al., adequately respond to stressors, should contain descrip-
2010; Goulding et al., 2010; Lamon et al., 2008; McBrier tions of appropriate humane endpoints or provide
et al., 2009; Valentim et al., 2008). These studies have science-based justification as to why a particular,
demonstrated varying effects on parameters of interest, accepted humane endpoint cannot be employed.
including no effect. Therefore, rather than assuming that Veterinary consultation must occur when pain or dis-
analgesics cannot be given, it is recommended that a lit- tress is beyond the level anticipated in the protocol
erature search be conducted to determine if studies have description or when interventional control is not pos-
been done to validate the existence of an effect of anal- sible (National Research Council, 2011). Most of the ethi-
gesia on a given experimental parameter. If data do not cal principles guiding humane animal research mention
exist, consideration should be given for conducting and the use of humane endpoints (Bankowski, 1985; Council
publishing an appropriate study to indicate which anal- for International Organizations of Medical Sciences,
gesics are, or are not, a viable scientific option for future 1985; Interagency Research Animal Committee, 1985).
experiments. Investigators should be required to provide Stokes (2000, 2002) provides an overview and reviews
scientific justification for withholding analgesia when specific situations where endpoints are appropriately
potentially painful procedures are to be conducted. utilized.
Defining distress in animals has proven to be a diffi- The need, criteria, and timing for humane endpoints
cult task. The ILAR Committee on the Recognition and should be part of pre-study planning and are often best
Alleviation of Distress in Laboratory Animals stated done as a research team including scientists, techni-
“Although most definitions of distress characterize it as cians, and veterinarians (Canadian Council for Animal
an aversive, negative state in which coping and adap- Care, 1998; NRC, 2003; Organisation for Economic
tation processes in response to stressors fail to return Co-operation and Development, 2000). Endpoints are an
an organism to physiological and/or psychological important element of IACUC protocol review. Therefore,
homeostasis, philosophical differences center on the it is essential that a protocol contain all appropriate infor-
inclusion of emotions and feelings affected by this state mation regarding the criteria for humane endpoints,
of being.” (Committee on Recognition and Alleviation observation schedules, and training of personnel to ade-
of Distress in Laboratory Animals, 2008). quately observe for the agreed-upon criteria. Pilot stud-
Stress has been defined as “the biological response an ies may be useful for gathering this information if it is
animal exhibits in an attempt to cope with threats to its not known at the time of protocol submission. In addi-
homeostasis” (Stokes, 2000). This response can involve tion, the criteria and other details may need to be modi-
immunologic, metabolic, autonomic, neuroendocrine, fied when unexpected adverse events occur. The IACUC
and behavioral changes (Moberg, 2000). The type, pat- should be notified when this happens and the protocol
tern, and level of the response depend upon the strength, amended as needed.
severity, intensity, and duration of the stressor(s). The Development of criteria for humane endpoints may
aversive state of distress results when an animal is be general and applied to any study. Institutions often
unable to adapt. By limiting the frequency, strength, adopt standards or policies that cover situations when
severity, intensity, and/or duration of the stressor(s), it study-specific endpoints have not been determined.
may be possible to limit the level of distress in the ani- These documents often appear as Standard Operating
mal. One method is to reduce the cumulative stress an Procedures (SOPs) or guidelines, and may be devel-
animal experiences (allowing recovery or adaptation to a oped and instituted by the IACUC, the veterinary staff,
given stressful situation before adding additional stress- the institutional administration, or any collaboration of
ors) or refining practices and procedures to make the the above. The policies should encompass generic clini-
individual stressors less severe or shorter. cal or behavioral conditions that potentially are asso-
The use of humane endpoints contributes to refine- ciated with pain and distress, and should be widely
ment by providing an alternative to experimental recognized and accepted by the research staff. General
endpoints that result in more severe animal pain and clinical signs which may be monitored include: weight
distress. Scientific or experimental endpoints are defined loss, inability to ambulate adequately to obtain food
as occurring when the objectives of the study have been and/or water, and body condition scores (Figure 1.1)
reached. Humane endpoints occur at the point at which (Hickman and Swan, 2010). Anorexia or lack of appetite
pain or distress is prevented, terminated, or relieved is a significant observation since parenteral supplemen-
in an experimental animal. In most studies, the scien- tation in rodents is not commonly used. In some cases
tific and humane endpoints occur at the same time, in in these species, clinical abnormalities are only obvious
other words, the scientific endpoint occurs prior to the when advanced illness, toxicity, or impending death
development of pain or distress. Studies that may result (e.g., a moribund state) are reached (Toth, 2000). In
in severe or chronic pain or significant alterations in these situations, decisions to initiate humane endpoints
the animal’s ability to maintain normal physiology, or must be made promptly.
I. GENERAL
12 1. ETHICAL CONSIDERATIONS AND REGULATORy ISSUES
BC 1 endpoints to avoid death as an endpoint (National
Rat is emaciated Research Council, 2011).
• Segmentation of vertebral column prominent To be prepared for situations when unanticipated
if not visible.
pain and distress occur, the institution should have suf-
• Little or no flesh cover over dorsal pelvis. Pins
prominent if not visible. ficient veterinary oversight in place to advise when
• Segmentation of caudal vertebrae prominent. alleviation of negative consequences from experimen-
tal procedures should be addressed. This may result
BC 2 in animals receiving veterinary medical care, removal
Rat is under conditioned from the experiment, or euthanasia to best align with the
• Segmentation of vertebral column prominent.
scientific objectives of the research. These decisions are
• Thin flesh cover over dorsal pelvis, little
subcutaneous fat. Pins easily palpable. ideally made through a collaborative discussion includ-
• Thin flesh cover over caudal vertebrae, ing animal care, scientific, and veterinary staff. However,
segmentation palpable with slight pressure.
in cases where the animals are significantly compro-
mised, the veterinarian is obligated to take whatever
BC 3
actions are necessary for animal welfare. When multi-
Rat is well-conditioned
ple animals receiving the same treatment exhibit severe
• Segmentation of vertebral column easily
palpable. adverse effects, consideration should be given to adjust-
• Moderate subcutaneous fat store over pelvis. ing the endpoint in the remaining animals or euthanizing
Pins easily palpable with slight pressure.
an entire treatment group if experimental objectives can
• Moderate fat store around tail base, caudal
vertebrae may be palpable but not segmented. no longer be achieved.
Refinements in husbandry and handling (e.g., pro-
BC 4 vision of species-appropriate enrichment, positive
Rat is overconditioned humane interaction and operant training to minimize
• Segmentation of vertebral column palpable stress in handling and sample collection), are also ways
with slight pressure.
in which distress and other negative impacts can be
• Thick subcutaneous fat store over dorsal
pelvis. Pins of pelvis palpable with firm minimized. Refinements of handling that may seem
pressure. minor (e.g., picking up a mouse by using a plastic tun-
• Thick fat store over tail base, caudal vertebrae
nel or cupped hand instead of the traditional method of
not palpable.
grasping the base of the tail) (Hurst and West, 2010) can
have significant impact, particularly when applied to a
BC 5
large number of animals.
Rat is obese
Many refinements in husbandry rely on a good work-
• Segmentation of vertebral column palpable
with firm pressure; may be a continuous ing knowledge of the ethology (study of animal behavior,
column. generally under natural conditions) of the animal species
• Thick subcutaneous fat store over dorsal
used. The knowledge of normal species-specific behavior
pelvis. Pins of pelvis not palpable with firm
pressure. is necessary to recognize abnormal behavior and to create
• Thick fat store over tail base, caudal vertebrae an appropriate environment. But consider an animal’s nat-
not palpable
ural environment – what is natural for mice and rats that
FIGURE 1.1 System for body condition scoring (reproduced with live in forests, rural areas, and urban environments? Some
permission from Hickman and Swan, 2010). species are highly adaptable, thus attempting to recre-
ate a natural environment in the laboratory may not only
be impractical, but difficult to even define. In addition,
most of the rodents and rabbits used in research today
In some cases, humane endpoints may be developed are purpose-bred and have been for many generations.
for a specific type of study (Dennis, 2000; Montgomery, The ethograms for such animals would be expected to be
1987; Olfert, 1996; Olfert and Godson, 2000; Sass, 2000; different from their wild counterparts (e.g, it would be
Workman et al., 2010) or a specific individual study expected and desirable that rodents and rabbits habitu-
(Hickman and Swan, 2010; Singh et al., 2010). The use of ated to humans would be less fearful and easier to han-
scoring systems has been described and usually utilizes dle). However, animals bred for research may retain, and
multiple observations which, in total, identify the humane attempt to exhibit, certain intrinsic behaviors. Knowledge
endpoint (LLoyd and Wolfensohn, 1998; Medina, 2004; of those behaviors can help the laboratory animal scientist
Morton, 2000). Other systematic approaches to determin- develop practical strategies to maximize those behaviors
ing humane endpoints have also been reported (Medina, and minimize stress.
2004; Morton, 2000). There are excellent references avail- When considering the stressors in the laboratory
able that review the establishment and use of humane animals’ environment, one must consider not only
I. GENERAL