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5
Humane Endpoints for
Animals in Pain
T
his chapter presents an overview of the concept of humane endpoints
and their application in studies that cause pain in research animals.
It sets the stage with a review of pertinent guidance documents,
focusing on the Organization for Economic Cooperation and Development
(OECD) 2000 Guidance on Humane Endpoints for Experimental Animals
Used in Safety Evaluation. It provides a discussion of the usefulness of pilot
studies as a refinement and potential replacement tool. Further, it presents
humane endpoints in relation to specific research fields—toxicology, infec-
tious diseases, vaccine safety, cancer, and pain. It concludes with a discus-
sion of euthanasia.
GUIDELINES AND REFERENCE DOCUMENTS
Moral and ethical obligations are inherent in all aspects of research,
testing, and teaching that use research subjects. The question of when a
study using animal models should end or the study design be changed due
to animal pain, distress, or welfare considerations has been the subject of
many publications, symposia, guidance documents, and regulations. Defin-
ing a humane endpoint can vary widely depending on a number of factors,
of which study design and research objectives are but two. Consequently,
attempting to provide specific endpoint criteria for all study designs and
other factors cannot be adequately addressed in this one report (Morton
1999, 2000). Not only would such a list be inadequate, it could prove det-
rimental to hitherto unknown study objectives. This report does not go into
specifics but rather presents selected pertinent guidelines and documents.
11
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120 RECOGNITION AND ALLEVIATION OF PAIN IN LABORATORY ANIMALS
Investigators, study personnel, veterinary staff, and institutional animal care
and use committees (IACUCs) are obligated to thoroughly research and
incorporate humane endpoints in every study or use involving laboratory
animals.
National and International Guidelines
A number of national and international guidelines are available to assist
researchers in determining humane endpoints for research animals. The
Office of Laboratory Animal Welfare (OLAW) defines these as “[c]riteria
used to end experimental studies earlier in order to avoid or terminate
unrelieved pain and/or distress are referred to as humane endpoints. An
important feature of humane endpoints is that they should ensure that study
objectives will still be met even though the study is ended at an earlier
point. Ideally, humane endpoints are sought that can be used to end studies
before the onset of pain and distress” (OLAW/ARENA 2002, p. 103).
The Canadian Council for Animal Care (CCAC) has published an excel-
lent document with general recommendations on humane endpoints in
animal studies. According to the CCAC guidelines, in “experiments involv-
ing animals, any actual or potential pain, distress, or discomfort should be
minimized or alleviated by choosing the earliest endpoint that is compat-
ible with the scientific objectives of the research. Selection of this endpoint
by the investigator should involve consultation with the laboratory animal
veterinarian and the animal care committee” (CCAC 1998, p. 5).
In 1994, the OECD recognized that while ambiguous test guidelines
may be necessary, such ambiguity fosters an overbroad interpretation of
what constitutes a humane endpoint in toxicology studies. The organiza-
tion therefore created a working group to develop a guidance document
using clinical signs as humane endpoints in safety evaluation studies (OECD
2000; Box 5-1). The resulting document put forth criteria based on the
principles of the 3Rs as well as descriptions of clinical signs to assist study
personnel in determining when death may be imminent or when severe pain
may be present after an animal’s exposure to a test substance. The criteria
are broad enough to apply to a wide range of study types, test substances,
species, and strains of animals. The reader is encouraged to examine this
resource when developing internal guidance documents to assess humane
endpoints.
OECD invested considerable time and effort in addressing and defin-
ing potential endpoints in safety assessment studies (see the Addendum
at the end of this chapter for the OECD definition). The OECD Guidance
Document defines humane endpoints “as the earliest indicator in an animal
experiment of severe pain, severe distress, suffering, or impending death.
The ultimate purpose of the application of humane endpoints to toxicol-
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HUMANE ENDPOINTS FOR ANIMALS IN PAIN
BOX 5-1
OECD Guidance Document on the Recognition, Assessment,
and Use of Clinical Signs as Humane Endpoints for
Experimental Animals Used in Safety Evaluation (OECD 2000)
• A humane endpoint can be defined as the earliest indicator in an animal ex-
periment of severe pain, severe distress, suffering, or impending death.
• The ultimate purpose of the application of humane endpoints to toxicology
studies is to be able to accurately predict severe pain, severe distress, suffer-
ing, or impending death, before the animal experiences these effects. However,
the science of toxicology is not yet to the point where such accurate predic-
tions can be made prior to the onset of severe pain and distress. It is possible
at this time to identify pain, distress, or suffering, very early after their onset
by careful clinical examination of animals on test using well-defined endpoints
and criteria. Humane endpoints for use in research and testing have been
addressed in a number of publications. . . . These adverse conditions, once
identified, should be minimized or eliminated, either by humanely killing the
animal or, in long-term studies, by (temporary) termination of exposure, or by
reduction of the test substance dose.
• Different animal species, and animals at different stages of development,
may respond differently to test conditions, and exhibit different indications of
distress. The clinical signs described here should be evaluated in consider-
ation of these potential differences. If relevant humane endpoints have been
identified, they should be described when an experiment is being planned, and
incorporated into the experimental protocol and all related standard operating
procedures (SOPs).
ogy studies is to be able to accurately predict severe pain, severe distress,
suffering, or impending death, before the animal experiences these effects”
(OECD 2000, p. 10). While the OECD indicated that the science of toxi-
cology cannot accurately predict pain prior to onset, careful observations
can “identify pain, distress, or suffering, very early after their onset . . .
using well-defined endpoints and criteria.” The OECD further advises that
suffering “should be minimized or eliminated, either by humanely killing
the animal or, in long-term studies, by (temporary) termination of exposure,
or by reduction of the test substance dose. Different animal species, and
animals at different stages of development, may respond differently to test
conditions, and exhibit different indications of distress” (ibid.).
These guidance documents are consistent in their recommendations.
Predictive parameters must be reliable, reproducible, and objective, and
allow both the achievement of study objectives and goals and the use of
appropriate methodologies at the earliest point to alleviate or avoid pain. As
discussed below, pilot studies are an effective means to identify and validate
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122 RECOGNITION AND ALLEVIATION OF PAIN IN LABORATORY ANIMALS
humane endpoints, which can then be incorporated in research methods to
minimize, alleviate, or avoid pain for the animal subjects (also see Morton
1999, 2000; Stokes 2002; NRC 2008, p. 61).
Humane endpoints were the focus of a 1998 international confer-
ence in Ziest, The Netherlands. The editors of the conference proceedings
determined that humane endpoints are specific to individual studies or a
particular testing paradigm (Hendriksen and Morton 1999, pp. v-vi), based
on study design and intent, regulatory requirements, personnel connected to
the study, and the animals themselves, whether as individuals or as a group.
The conference participants concluded that the establishment of humane
endpoints is, and should be, subject to adaptation as societal mores, atti-
tudes, regulations, and technologies change. The conference report further
stated that for ethical reasons, the formulation of endpoints to avoid or
alleviate pain in laboratory animals must be a high ethical priority in every
facility that conducts any form of animal experimentation (ibid.).
Beyond Formal Guidelines
Many of the articles and recommendations that address humane end-
points focus on very specific study or research types that can cause pain
to laboratory animals; for example, studies on the identification and use of
humane endpoints in animal models of sepsis and shock provide an excel-
lent overview of the methodologies to determine humane endpoints yet still
achieve study objectives (Nemzek et al. 2004, 2008). More generally, the
Institute for Laboratory Animal Research (ILAR) bases its reports on its mis-
sion statement promoting “high-quality science and humane care and use
of research animals based on the principles of refinement, replacement, and
reduction (the 3Rs) and high ethical standards” (ILAR 2009). The Institute’s
Guidelines for the Care and Use of Mammals in Neuroscience and Beha-
ioral Research (NRC 2003) provide criteria for evaluating levels of pain
that help in the development of endpoints for studies in neuroscience and
behavioral research. An ILAR Journal issue dedicated to Humane Endpoints
for Animals Used in Biomedical Research and Testing (ILAR 2000) provides
an overview of several research areas where pain is a potential outcome,
including infectious disease and cancer research (Olfert and Godson 2000;
Wallace 2000) and vaccine potency and and acute toxicity testing (Hen-
driksen and Steen 2000; Sass 2000). ILAR also published the proceedings
of a symposium on Regulatory Testing and Animal Welfare, detailing best
practices for the humane conduct of animal testing for regulatory purposes
(NRC 2004).
While these references are extremely valuable, it is important to view
them in accordance with their intent: they are guidance documents only
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HUMANE ENDPOINTS FOR ANIMALS IN PAIN
and as such have limitations. No single document could cover all poten-
tially painful study types, all animal species used in research, or all clinical
signs associated with all research projects. In the absence of comprehensive
guidance, the scientific community has an ethical responsibility to develop
a general humane endpoint policy at each institution to provide guidance
and a basis for dialogue between scientists and IACUCs about individual
protocols.
Caution is advisable, however, in efforts to develop a policy on humane
endpoints. While the ideal is to avoid pain, personnel also need to ensure
that the study objectives are attained before a procedure or animal is termi-
nated (OLAW/ARENA 2002, p. 103). If a full study, or aspect of a study, is
ended before the objectives have been met, one can argue that the animals
used have been wasted. Moreover, if the purpose of a study is to meet the
requirements for the safety assessment of a substance, a regulatory agency
may reject the submitted data as insufficient and require that the study be
repeated. On the other hand, if researchers are reluctant to intervene, study
animals may unnecessarily experience pain, distress, or severely diminished
welfare. Further, without adequate guidance, death is likely to be selected
as a convenient endpoint that is reproducible and objective. If regulatory
guidelines do not specify an endpoint, as in vaccine potency studies (CFR
Title 9, 2006), regulated entities can and will use lethality.
For all these reasons identification of humane endpoints should take
into account the following factors: the role of regulatory agencies in the
overall process; the need for scientifically appropriate endpoints; and the
reliability of clinical observations of the animals to ensure a proper outcome
for both the animals and the study. As a corollary, it is worth emphasizing
that investigators, technicians, and other staff responsible for the care of
research animals should be well trained and able to make impartial judg-
ments about an animal’s well-being.
OLAW approached the subject of humane endpoints in its Institutional
Animal Care and Use Committee Guidebook (OLAW/ARENA 2002, p. 103),
advising internal oversight committees to review protocols to determine
whether “discomfort to animals will be limited to that which is unavoidable
for the conduct of scientifically valuable research, and [whether] unrelieved
pain and distress will only continue for the duration necessary to accom-
plish the scientific objectives.” The OLAW reference is careful to state that
potential pain or distress should be relieved with appropriate medication
or with euthanasia, although the study objectives should still be met. The
intent is to end a study before the development of pain or distress, as is
emphasized in the OECD document.
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124 RECOGNITION AND ALLEVIATION OF PAIN IN LABORATORY ANIMALS
PILOT STUDIES
An effective way to reduce negative impacts on laboratory animals is
the use of a pilot study, which can be critical to the success of a larger study
(DeHaven 2002; Morton et al. 1990; NRC 2003, p. 14; NRC 2008, pp. 61-
62; OECD 2000, p. 14). The premise behind this concept is to conduct the
proposed study on a small number of animals rather than the full comple-
ment necessary for a statistically valid study and thus prevent unnecessary
pain for a larger number of animals.
Pilot studies are advantageous because they help researchers to
identify:
• potential interactions between proposed analgesic and anesthetic
treatments and specific research goals,
• potentially useful means of assessing pain in a specific research
model, and
• humane endpoint criteria specific to an individual project.
Problems that occur in the pilot study can inform the discussion and
development of strategies to address an animal’s deteriorating condition.
Such strategies may include (but certainly not be limited to) the adjustment
of dose levels, changes in sample size, identification of adverse effects,
incorporation of refinements (e.g., use of analgesics, procedural changes),
or alteration to the duration of exposure to minimize negative impacts on
the animals.
Caution is essential in the design and conduct of pilot studies as the risk
of causing significant pain to the animals in such studies can be high. This
risk necessitates close oversight by the IACUC and careful monitoring of
the animals by study personnel and veterinary staff. Good communication
among all involved can ensure both the collection of the maximum amount
of useful data and appropriate interventions on behalf of the animals (NRC
2003, p. 14).
INTERNATIONAL REGULATIONS AND
GUIDELINES FOR SAFETY ASSESSMENT
Regulatory bodies in most countries have developed standards and
guidelines to ensure the conduct of appropriate safety assessments on test
substances (Hicks 1997; Merrill 2001; USEPA 2008). For example, after the
use of thalidomide by pregnant women in the 1960s caused severe birth
defects in the long bones of the fetuses, US legislation required adequate
testing of drugs in animals before human exposure (Gallo 2001; Nies 2001).
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HUMANE ENDPOINTS FOR ANIMALS IN PAIN
Similar legislative actions followed environmental disasters like the Love
Canal contamination (Merrill 2001).
The purpose of testing requirements for pharmaceutical, consumer, and
industrial products is to ensure the safety of the environment and of the
human and animal populations. However, these requirements have tended
to focus on the safety of the user and do not necessarily consider humane
endpoints for the animals used in the safety assessment, although such
consideration is becoming a more prominent component of some newer
regulatory requirements.
In June 2007, the European Commission established a regulation to
evaluate the hazards and risks of chemicals (Regulation (EC) No. 1907/2006
of the European Parliament and of the Council of 18 December 2006); the
mission of REACH (Registration, Evaluation, and Authorization of Chemi-
cals) is to improve the assessment of chemicals in order to better protect
human health and the environment. Because the range of chemicals cov-
ered by REACH is enormous, there is great potential for increased use of
animals in corresponding toxicity and safety testing. But the regulation
ensures the authorization of animal testing only when necessitated by iden-
tification of data gaps (ECHA 2008). Furthermore, the regulation requires
industry to share data on similar chemicals to avoid duplicative animal
testing; allows for the submission of data using nonanimal tests; strongly
encourages the use of Quantitative Structure-Activity Relationship (QSAR)
or other computer-generated information; and invites the grouping of sub-
mitted data for similar chemicals that may result in similar hazards and risks
(the so-called “read-across” principle). While these efforts do not define
humane endpoints, the authors of the regulation are commended for the
consideration of responsible animal use in safety assessment.
Also useful in the toxicology regulatory arena is a February 2008 Mem-
orandum of Understanding (MOU) that lays the foundation and framework
for the US Environmental Protection Agency (EPA) and two NIH agencies to
collaborate in sharing data, resources, and expertise in efforts to replace ani-
mal testing for chemical toxicity assessment (Collins et al. 2008; NIH/USEPA
2008; NIH 2008). The MOU calls for the evaluation of in vitro assays, such
as those used for identification of toxicity pathways and high-throughput
screening (as described in NRC 2007), to better predict potential health and
environmental hazards from chemicals. The ambitious goals of the MOU
are the development of more accurate assays and changes in regulatory
guidelines, both of which are likely to be a long-term process. Similar goals
should be encouraged on a global scale to effect change in regulatory agen-
cies and eliminate potentially painful animal testing.
Although harmonization of regulatory guidelines has significantly
reduced discrepancies between cooperating countries, efforts for the global
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126 RECOGNITION AND ALLEVIATION OF PAIN IN LABORATORY ANIMALS
harmonization of safety guidelines are neither consistent nor well coordi-
nated. As a result, tests must comply with all the requirements of each coun-
try where a product is to be marketed for a particular use. For example, the
regulatory agency of one country may require an additional group of ani-
mals to assess recovery from exposure, while other countries may not have
this requirement or may even reject the study depending on their review
process. Or one country’s regulatory agency may accept an alternative that
has been validated as scientifically reliable and relevant (NIH 1997), such
as the local lymph node assay in mice, whereas agencies in other countries
may not accept the data in lieu of the guinea pig dermal sensitization test.
While a comparison of all safety assessment guidelines is well beyond
the scope of this report, differences in regulatory-driven studies can have
a negative impact on the prevention and alleviation of pain in laboratory
animals. An example of a safety assessment test that may cause pain is the
acute eye irritation study, the purpose of which is to evaluate the potential
hazards of ocular exposure to a substance. Although requirements for this
procedure are generally in agreement across international regulatory bod-
ies and national agencies (JMAFF 2000; OECD 1987, #405; USEPA OPPTS
1998, #870.2400), the same is not true for the reversibility of ocular lesions,
an additional requirement of this test in order to more fully assess the risk of
human exposure. The procedures for this component of the toxicity evalua-
tion vary considerably with respect to animal welfare. The OECD guidelines
recommend a step-wise evaluation paradigm that starts with assessment
of structurally related substances and other in vitro tests prior to any ani-
mal use. The guidelines also identify ocular lesions that are considered
irreversible and thereby meet OECD criteria for terminating the study and
euthanizing the animal. But while guidelines in various countries reference
the OECD guidance document for humane endpoints and recommend the
use of local anesthetics in cases of extreme pain, they do not recognize the
OECD criterion for early termination of the study (identification of irrevers-
ible lesions).
HUMANE ENDPOINTS IN TOXICOLOGY STUDIES
In recognition of the pain and distress inflicted on animals in many
safety and toxicology studies, regulatory guidelines have begun to address
the concept of humane endpoints, although sometimes in vague terms. The
EPA Health Effects Test Guidelines for Acute Oral Toxicity (USEPA OPPTS
2002) provide instruction for following the OECD Guidance Document
(OECD 2000) to reduce the suffering of animals in toxicity studies. Eutha-
nasia of animals that are either moribund or in severe pain is also encour-
aged. Regrettably, vague statements such as “animals showing severe and
enduring signs of distress and pain may need to be humanely killed,” which
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HUMANE ENDPOINTS FOR ANIMALS IN PAIN
are common in regulatory guidelines (USEPA OPPTS 1998), may promote
a reluctance to terminate a study or an animal’s exposure to the testing
substance because a regulatory body may consider the action premature
and mandate a repeat study. This is not a good situation for researchers,
laboratories, or animals.
Not all test substances cause ocular (or other) pain or injury, but the
potential exists. As pointed out by Durham and colleagues (1992), there is
a gap in the data for analgesia appropriate for use in ocular toxicity tests
and that gap persists, as evidenced in a US Federal Register notice (Federal
Register 2007) requesting data on analgesic use in ocular irritancy tests
to alleviate pain without affecting test results. Current guidelines include
neither justification for withholding analgesic agents nor guidance for the
use of analgesic agents to alleviate ongoing pain. As a result, testing entities
may be reluctant to provide analgesia beyond initial local anesthetics, to
avoid the possibility of interference with the test substance (Stokes 2005).
Yet numerous published studies demonstrate that the use of analgesics to
alleviate pain from ocular irritancy tests does not interfere with the scien-
tific objectives of this safety test (Patrone et al. 1999; Peyman et al. 1994;
Stiles et al. 2003). Such evidence can be used to avoid or alleviate pain
as well as to provide scientific rationale for the use of analgesics in ocular
irritancy tests.
Chronic toxicity and carcinogenicity testing are currently required to
assess effects after long-term, repeated exposure to a test substance (JMAFF
2000; OECD 1987, #405; USEPA OPPTS 1998, #870.2400). The incidence
of tumor burden, geriatric changes, and premature death can be signifi-
cant near the scheduled termination of these studies. Guidelines generally
specify the survival rates necessary to provide meaningful interpretation
of a chronic study, but the OECD document is the only one to discuss
humane endpoints and provide guidance for the early termination of a
study if survival rates fall below a specified percentage. In order to achieve
the required survival rate at the end of the mandated study, animals often
are not euthanized until very close to death, an outcome that may entail
needless pain for the animals. True harmonization of guideline safety assess-
ment tests and global adoption of the OECD humane endpoints document
would be an important step toward the alleviation and avoidance of pain
in laboratory animals.
The NRC report Toxicity Testing in the Twenty-first Century: A Vision
and a Strategy (NRC 2007) evaluated current toxicity testing schemes and
developed a long-term strategy for the direction of safety assessments based
on state-of-the-art sciences (e.g., genomics, proteomics, and pharmacoki-
netics) and emerging technologies (e.g., bioinformatics). Although the report
acknowledges that implementation of the strategy will require much effort
on the part of scientists, regulators, and law makers to develop workable
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128 RECOGNITION AND ALLEVIATION OF PAIN IN LABORATORY ANIMALS
testing schemes, the concepts envisioned could significantly improve the
science of toxicology, assessment of risk to human safety, alleviation of pain
in laboratory animals, and reduction or replacement of animals in toxicity
testing (ibid.).
One of the sources reviewed for the NRC report was the approach
developed by the Health and Environmental Sciences Institute (HESI) of
the International Life Sciences Institute (ILSI). In 2000, this organization
convened an Agricultural Chemical Safety Assessment (ACSA) committee
to redesign safety testing schemes for agricultural chemicals. The resulting
multifaceted approach redesigns traditional toxicology tests to integrate sev-
eral sciences, such as metabolism/kinetics and life stages, in a single study
to eliminate the requirement for separate studies to evaluate each parameter
and reduce the number of animals used (Carmichael et al. 2006; Cooper
et al. 2006; Doe et al. 2006; ILSI-HESI 2008). Further, the metabolism/kinet-
ics component of the strategy is particularly relevant to the alleviation of
pain in laboratory animals: based on the metabolism of a test substance in
the animal model, a saturation point can be determined and used as the
high dose level in subsequent studies because it is considered more relevant
to actual human exposure levels. This approach, based on step-wise, or
tiered, testing, is expected to reduce animal numbers, minimize potential
pain to laboratory animals by avoiding exposure levels that produce clinical
signs of toxicity, and improve the quality of data for assessments of risk to
humans (Carmichael et al. 2006).
HUMANE ENDPOINTS IN INFECTIOUS DISEASE RESEARCH
There has been an increase in infectious disease research as a result of
bioterrorism threats and anthrax attacks since September 11, 2001 (Copps
2005; Jaax 2005). Whether the disease agent is of interest for bioterrorism
or for human or animal welfare, the study of a targeted disease typically
involves exposing healthy research animals to a disease agent that culmi-
nates in clinical disease and death. The animals may experience significant
pain during these experiments, but identification and validation of earlier
endpoints to safeguard animal welfare can be difficult, as an inappropriate
endpoint may not adequately identify the full course of a disease or the
efficacy of a potential medication (Olfert and Godson 2000). It is impera-
tive, therefore, to examine and validate endpoints within a solid scientific
framework that includes, among others, immunological parameters, bio-
chemical and endocrine changes, and other pathophysiologic changes (e.g.,
decreased body temperature). Moreover, eliminating death as the endpoint
for infectious disease research can benefit not only the laboratory animals
but the research itself because pathological changes are easier to identify in
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fresh tissues as opposed to autolyzed tissues from animals that have been
allowed to die (Copps 2005).
HUMANE ENDPOINTS IN VACCINE SAFETY AND POTENCY TESTING
Another area of research that frequently results in the death of study
animals is vaccine testing for regulatory agencies. Because vaccines are
biological products and one batch may not be as potent as the next or may
contain harmful byproducts, it is important to test both their efficacy and
safety (Castle 1999; Cussler et al. 1999; Hendriksen 2002). To ensure qual-
ity control and the safety of each batch, regulatory agencies such as the US
Food and Drug Administration (FDA), the US Department of Agriculture
(USDA), the European Pharmacopoeia, and the World Health Organiza-
tion (WHO) require potency testing during which animals are vaccinated
and then exposed to the virulent disease agent. However, the endpoint for
each potency test is not consistent across disease agents. In some instances,
regulations require that a certain percentage of control animals die before
a test is considered valid, while other tests are based on the survival of
the vaccinated animals. For example, the FDA-administered safety test for
general biological products requires vaccination of healthy guinea pigs
and mice with a small dose of the final product from each vaccine lot (CFR
2008, 610.11). A safety test is considered unsatisfactory if the animals do
not survive the 7-day test period, in which case additional safety tests over
a larger test population are required. The USDA-mandated potency testing
for Leptospira pomona bacterin (CFR 2006, 113.101) requires that at least
eight of ten unvaccinated control animals die in order to validate the test.
Other potency testing may require a comparison of death rates in the vac-
cinated versus control animals, as, for example, in the USDA safety test
for Marek’s disease vaccine (CFR 2006, 113.330). For this type of testing
a more humane endpoint would be the onset of clinical signs in unvac-
cinated controls; thus for example the potency test for tetanus antitoxin is
met when unvaccinated control guinea pigs are unable to stand within 24
hours postchallenge, at which point the animals may be euthanized (CFR
2006, 113.451).
Regulations may also encourage the use of in vitro methods. The USDA
canine distemper killed virus vaccine potency test (CFR 2006, 113.201)
accepts serum titer levels in vaccinated animals for potency data; if, how-
ever, the tests are inconclusive, a viral challenge test is required, using both
vaccinated and unvaccinated controls. The agency identifies the survival of
all vaccinated animals and the death of all controls as a satisfactory indica-
tor of both the safety and efficacy of a canine distemper vaccine batch.
While lethality may be the easier endpoint because of its objectivity and
simplicity (Cussler et al. 1999), it is always worthwhile to identify reliable
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130 RECOGNITION AND ALLEVIATION OF PAIN IN LABORATORY ANIMALS
markers of predictive or impending mortality to serve as alternative and
more humane endpoints. No purpose is served when the administration of a
vaccine results in harm rather than protection but, as with all research stud-
ies and testing guidelines, there must be a balance between effective safety
evaluation and humane endpoints for the sake of the laboratory animal.
HUMANE ENDPOINTS IN CANCER RESEARCH
Identification of humane endpoints in cancer research can be chal-
lenging. Although the wide range of tumor types and scientific objectives
associated with this research prohibits standardization of humane endpoints
(Wallace 1999, 2000), the United Kingdom Coordinating Committee on
Cancer Research (UKCCCR) has developed a document to guide researchers
working with animal models (UKCCCR 1988). Investigators should evalu-
ate tumor size, tumor appearance, and animal condition to identify reliable
indicators that may permit earlier termination of a study, and establish and
validate endpoints that retain scientific objectives and avoid, minimize, or
alleviate potential pain in the laboratory animals. Avoiding death or exces-
sive tumor burden, particularly when coupled with clinical signs of pain or
distress, should be a desirable goal in cancer research studies.
HUMANE ENDPOINTS IN PAIN RESEARCH
Of critical importance to this report, as well as to improvements in qual-
ity of life for both humans and animals, is research on pain itself, including
the mechanisms of pain and methods of pain alleviation. Complicating the
ethical issues inherent in producing pain in research subjects is the ability
to accurately predict and measure pain responses in animals (Le Bars et al.
2001; Meyerson and Linderoth 2006; Walker et al. 1999). It is imperative for
pain investigators to establish endpoints in each study design to minimize
the duration and intensity of the pain and to validate those endpoints for the
integrity, objectivity, and reproducibility of the study. Productive dialogue
between the IACUC and researcher is critical for ensuring the best outcome
for both the animals’ welfare and the study objectives in these research
programs (Mench 1999).
EUTHANASIA
Euthanasia, the act of inducing death without pain, is an acceptable
method for relieving or alleviating pain that cannot be controlled by other
means (NRC 1992, pp. 102-104). The humane death of an animal is one
in which the animal is first rendered unconscious, and thus insensitive to
pain, as rapidly as possible and with a minimum of fear and anxiety. A
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HUMANE ENDPOINTS FOR ANIMALS IN PAIN
humane death, or endpoint, is a fundamental tenet of the US Principles for
the Utilization and Care of Vertebrate Animals Used in Testing, Research,
and Training (IRAC 2001), as Principle VI states that “[a]nimals that would
otherwise suffer severe or chronic pain that cannot be relieved should be
painlessly killed at the end of the procedure or, if appropriate, during the
procedure.”
There is no rigidly defined point at which euthanasia should be per-
formed for humane reasons, as it is not possible to apply a single set of
euthanasia criteria across all study designs, animal models, and experimen-
tal goals. The decision should involve a team approach among veterinarians,
study directors, and animal care personnel using all available information
about the affected animal(s). Body condition scores, as described in Chap-
ter 3, can be used to determine when to consider euthanasia for humane
reasons. The earliest possible indicators for euthanasia should be clearly
identified so as to avoid pain and yet still achieve study objectives.
Methods of euthanasia have recently been updated by the American
Veterinary Medical Association (AVMA 2007), although objective informa-
tion on laboratory animals is sparse, particularly concerning the evaluation
of potential pain and distress that may be caused by a particular euthanasia
technique. The controversy that may result from this lack of data is evi-
dent in the recent discussions about the use of carbon dioxide on rodents
(ACLAM 2005; AVMA 2007; Conlee et al. 2005; Hawkins et al. 2006;
Kirkden et al. 2008; Niel et al. 2008; NRC 2003; Stephens et al. 2002). As
conversations on this subject will likely continue, the reader is encouraged
to follow the published literature for the most up-to-date information.
For all these reasons, well-designed objective studies of euthanasia
across all laboratory animal species and age groups are needed and rec-
ommended. The assessment tools and measures to consider for such stud-
ies include electroencephalograms, electrocardiograms, electromyograms,
arterial blood pressure, respiration and heart rates, serum biochemical
parameters, pupil diameter, and behavioral changes. In particular, there
is an urgent need for studies that provide measures of nociception, pain,
distress, and the relation of these to loss of consciousness.
CONCLUSIONS AND RECOMMENDATIONS
Avoiding or minimizing pain in animal research is a fundamental obli-
gation of all researchers for moral and ethical reasons. The criteria for early
termination of a research project or alteration to a study design for the
purpose of alleviating or avoiding pain in an animal are defined as humane
endpoints. Identification and validation of humane endpoints should be
considered for studies involving pain, but this is neither an easy nor a simple
process.
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132 RECOGNITION AND ALLEVIATION OF PAIN IN LABORATORY ANIMALS
1. It is important to ensure that endpoints are validated and based on
sound science. Pilot studies are invaluable for the determination of
earlier and more humane endpoints.
2. Given the wide scope of procedures and goals of animal research,
no single reference can document every humane endpoint for every
research protocol. Therefore, more effort must be made to identify
appropriate humane endpoints for each. Good communication
between researchers, veterinary staff, animal care staff, and the
IACUC is crucial.
3. Productive strides have been made in the harmonization of safety
assessment guidelines between countries but global harmonization
is not yet complete. For global acceptance of humane endpoints
in safety assessment test guidelines, dialogue should continue
between all countries and agencies responsible for animal welfare,
the environment, and human safety.
4. Efforts should continue in the development and validation of alter-
native procedures for incorporation in research projects and safety
assessment tests to avoid or alleviate pain in laboratory animals.
Hendriksen and Morton (1999) observed that the goal of developing
humane endpoints in animal experiments is constantly shifting. All scien-
tists, managers, technicians, oversight committees, and regulators involved
with animal experimentation where pain is a potential component should
participate in regular communication and creative problem solving. The
criteria for determining the humane end to a study should be frequently
reevaluated and revised as new information becomes available. The sus-
tained pursuit of these directed efforts can, and will, result in more humane
animal use.
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133
HUMANE ENDPOINTS FOR ANIMALS IN PAIN
ADDENDUM
As stated in this chapter, the establishment of surrogate or humane
endpoints as part of the experimental protocol and before experiments com-
mence is one of the ways to minimize and alleviate pain and safeguard the
well-being and welfare of laboratory animals. In support of this goal, two
sample resources are provided for adaptation and use. The first is a score
sheet to assess animals in cancer studies based on a behavioral and tumor
scoring system (Table 5A-1). The recorded symptomatology will determine
the diagnosis and measures for alleviation. The sheet can be adapted to any
protocol or animal care facility system as long as the behavioral definitions
are uniform across the same facility. The second resource is a model for
developing guidelines for humane endpoints that may be suitable for any
protocol within a facility (Box 5A-1).
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TABLE 5A-1 Sample Tumor Scoring Sheet
134
Calendar days
SECTION A LESION/TUMOR CHARACTERISTICS Score MOUSE ID Study No. Group No.
Dr y scab/crust forming coherent covering with skin 0
Acute burst releasing fluid/pus OR acute split at border 1
Chronically wet/weeping scab/crust OR solid yellow matter exposed 2
Bleeding or raw tissue exposed or white basal layer exposed 3
BEHAVIORAL CHANGES
0
No changes (i.e., normal)
Repeated grooming (tumor may not be easily visible or quite small) 1
Abnormal gait (tumor may not be easily visible or quite small) 1
Locomotion impeded (tumor is pronounced) 2
Recurrent scratching/biting of tumor 3
Nociception (struggling/sqeaking) on touching tumor 3
IF YOU HAVE SCORED ANY 3s IN SECTION A, CULL TODAY.
IF YOU HAVE SCORED ANY 2s IN SECTION A, REFER TO SECTION B, ELSE GO TO SECTION C.
# D1/D2**
SECTION B SIZE/PROGRESSION OF LESION (expressed in orthogonal diameters**)
0
Shrinking
1
Static
2
2-3 mm growing
3
3-5 mm growing
4
5+ mm growing
SIZE/PROGRESSION OF TUMOR
0
Shrinking
1
Static
2
10-12 mm growing
4
12-14 mm growing
6
14+ mm growing (mean D1/D2 or D1 or either > 17 mm)
TOTAL FOR SECTION A + SECTION B
**# D1 and D2 are or thogonal diameters
IF THE TOTAL FOR SECTIONS A + B IS 6, CULL WITHIN 1 DAY. IF 4-5 MONITOR DAILY, CULL WITHIN 1 WEEK IF NO IMPROVEMENT.
ELSE GO TO SECTION C
SECTION C KEEP MONITORING AS REQUIRED (DAILY)
Date
Adapted with permission from a scoring sheet developed by Fraser Darling, The Institute of Cancer Research, London, UK.
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HUMANE ENDPOINTS FOR ANIMALS IN PAIN
BOX 5A-1
Guidelines for Humane Endpoints in Animal Studiesa
PURPOSE: To assure compliance with the Animal Welfare Act (AWA), the Guide
for the Care and Use of Laboratory Animals (the “Guide”) and (institutionally
relevant) policies, as well as to promote good research. This policy describes the
responsibilities and procedures that investigators and veterinary staff must follow
when determining appropriate, humane endpoints.
PRINCIPLES: It is the responsibility of the Principal Investigator/Study Director
(PI/SD) to define humane endpoints and to explore alternatives to death as an
endpoint. If no alternative exists, the PI/SD must scientifically justify the use of
death as an endpoint, and outline procedures that will be taken to minimize pain
and distress to the animal.
Efforts must be made to minimize pain and distress experienced by animals used
in research. This policy letter is to provide investigators with guidelines for deter-
mining humane endpoints in compliance with the XXXXXX policy. To this end, the
use of death as an endpoint to experimental studies, rather than performing eutha-
nasia to humanely terminate an animal, is discouraged and must be justified.
Each Animal Use Protocol (AUP), especially those that are anticipated to result in
severe or chronic pain, should describe endpoint(s) and specify a plan and criteria
for removal/euthanasia of animals from the study, or the disposition of animals at
the termination of the study. For many studies, the endpoint will be euthanasia
upon study completion, euthanasia at certain time points, or the return of animals
to stock. For studies where moderate to severe clinical signs can be anticipated,
the endpoint description in the AUP shall include identification of personnel re-
sponsible for decision making, specific criteria (body weight, mass size, appetite,
etc.) that will be monitored at prescribed frequencies (daily, weekly, etc), and a
disposition (treatment, euthanasia, early removal from study, etc.) once those
criteria have been met or exceeded.
SCOPE: This policy covers any animal used for research.
POLICY STATEMENT: Animal studies may involve procedures that cause severe
clinical signs or morbidity, and investigators must consider the selection of the
most appropriate endpoint(s) for their study. This requires careful consideration
of the scientific objectives of the study, the expected and possible adverse effects
the research animals may experience, the most likely time course and progression
of those adverse effects, and the earliest most predictive indicators of present or
impending adverse effects. Prior to the initiation of the study, the PI/SD should
determine the criteria that would lead to termination of the study for any animal,
when appropriate, and the method of euthanasia to be employed. A clear chain
of command for the decision-making process should be documented, including
contingency plans if said individuals are unavailable for consultation. Optimally,
studies are terminated when animals begin to exhibit severe clinical signs if this
endpoint is compatible with meeting the research objectives. Such endpoints are
preferable to death or moribundity (defined by the IACUC as imminent death) as
endpoints since they minimize pain and distress.
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136 RECOGNITION AND ALLEVIATION OF PAIN IN LABORATORY ANIMALS
There must be scientific justification in the AUP for allowing an animal to die
without intervention if the goals of a study can be accomplished by euthanizing
animals before they become moribund.
Animals involved in experiments that may lead to moribundity or death must be
monitored daily (including weekends) by personnel experienced in recognizing
signs of morbidity. Once severe clinical signs develop, more frequent observation
(2-3 times daily) may be required.
The following conditions usually necessitate euthanasia. The PI/SD must provide
scientific justification for exemptions:
Rapid weight loss of ≥20% of body weight.
•
• Extended period of weight loss, progressing to emaciated state.
• Surgical complications unresponsive to medical intervention.
• Combination of the following: poor physical appearance (very rough hair coat,
abnormal posture, grunting on exhalation); abnormal behavior (reduced mobil-
ity/unconsciousness, unsolicited vocalizations, self-mutilation); severe depres-
sion or abnormal/exaggerated responses to external stimuli.
• Severe respiratory distress, which is unresponsive to treatment.
• Occurrence of a serious injury or trauma from which recovery is unlikely.
• Neurological signs (e.g., persistent convulsions, persistent circling, paresis/
paralysis) that interfere with eating and drinking and from which recovery is
unlikely.
• Frank bleeding from any orifice, which is unresponsive to treatment.
• One or more skin ulcers that do not heal, depending upon the species and
severity of the ulcers.
• Mass size or location that interferes with normal function or ulcerates with no
evidence of healing.
• A mass that is greater than 15% of normal body weight. For chronic toxicology
studies (e.g., 2-year carcinogenicity studies), it is necessary to rely on experi-
ence and good judgment when deciding when to euthanize an animal as a
result of one or more masses. Many of these masses grow slowly and do not
compromise the animal.
RESPONSIBILITY: The PI/SD is responsible for ensuring that this IACUC policy
is followed. Exceptions to this policy must be scientifically justified and approved
by the IACUC before they can be implemented.
The IACUC has the authority, mandated by law (7 U.S. Code Section 2131 et
seq.), to act on behalf of the head of the institution to investigate and if neces-
sary suspend any activity which violates applicable laws, regulations, standards,
guidelines, policies and procedures.
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aExample of an IACUC Humane Endpoints Policy Letter, developed by Maryfrances Lutz,
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HUMANE ENDPOINTS FOR ANIMALS IN PAIN
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