|
Items in cart [1] |
Questions? Call 888-624-8373 |
PREPUB + PDF PREPUB PAPERBACK PDF BOOK PREPUB PDF CHAPTER PREPUB Free Resources |
||||||||||||||||
|
||||||||||||||||
|
|
|||||||||||||||
| ||||||||||||||||||||||||||||||
|
||||||||||||||||||||||||||||||
| Copyright © 2009. National Academy of Sciences. All rights reserved. Terms of Use and Privacy Statement |
Below are the first 10 and last 10 pages of uncorrected machine-read text (when available) of this chapter, followed by the top 30 algorithmically extracted key phrases from the chapter as a whole.
Intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text on the opening pages of each chapter.
Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.
Do not use for reproduction, copying, pasting, or reading; exclusively for search engines.
OCR for page 125
CHAPTER 5. Humane endpoints for animals in
pain
This chapter presents an overview of the concept of humane endpoints
as it pertains to studies that cause pain in research subjects. It sets the stage
with pertinent guidance documents and guidelines, focusing specifically on the
Organization for Cooperation and Development (OECD) 2000 Guidance on
Humane Endpoints for Experimental Animals Used in Safety Evaluation. It
discusses the usefulness of pilot studies as a refinement and potentially a
replacement tool. Further, it presents humane endpoints in relation to specific
research fields - toxicology, infectious diseases, cancer, and pain. It concludes
with a discussion of euthanasia.
Introduction
Moral and ethical obligations towards the research subjects are inherent
in all aspects of research, testing, and teaching in which animals play a central
role. The question of when a study 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. Defining 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 detrimental to hitherto
unknown study objectives. However, the absence of specifics in this report
does not relieve investigators, study personnel, veterinary staff, and
Institutional Animal Care and Use Committees (IACUCs) from the obligation of
thoroughly researching and incorporating humane endpoints in every study or
use involving laboratory animals.
The Office of Laboratory Animal Welfare (OLAW) provides guidance to
Institutional Animal Care and Use Committees (IACUC) on the evaluation of
humane endpoints. The OLAW guidebook defines this 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
Prepublication copy 125
OCR for page 126
126 Recognition and Alleviation of Pain in Laboratory Animals
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 a document
to assist programs and researchers with guidance on humane endpoints. The
general guideline from this organization states that “experiments involving
animals, any actual or potential pain, distress, or discomfort should be
minimized or alleviated by choosing the earliest endpoint that is compatible
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). This is an excellent
document for providing general recommendations on potential endpoints in
animal studies.
OECD invested considerable time and effort addressing and defining
potential endpoints in safety assessment studies (see Note at the end of this
chapter for a more complete description of the OECD definition). According to
their definition, humane endpoints “can be defined 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
toxicology 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
toxicology 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 minimised 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 recommendation.
While the predictive parameters must be established as reliable, reproducible,
and objective, they would allow study objectives and goals to be met and
appropriate methodologies to be employed at the earliest point to alleviate or
avoid pain. Incorporating validated endpoints in research results the
minimization, alleviation, or avoidance of pain (Morton 1999, 2000; Stokes
2002; NRC 2008, p. 61), but scientific identification and validation, which are
crucial for their credibility, can be accomplished using a small number of
animals and conducting pilot studies as discussed later in this chapter.
In 1998, an international conference was convened in Zeist, The
Netherlands to discuss the issue. 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 concluded that the establishment of humane endpoints is, and
should be, in a constant state of flux as societal mores, attitudes, regulations,
Prepublication Copy
OCR for page 127
CHAPTER 5: HUMANE ENDPOINTS FOR ANIMALS IN PAIN 127
and technologies change over time. 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.).
Guidance documents and guidelines
Many articles, recommendations, and guidance documents that discuss
humane endpoints target 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 sepsis and shock animal models provide an excellent
overview of the methodologies employed to determine humane endpoints yet
still achieve study objectives (Nemzek 2004, 2008). At the same time, the
Institute for Laboratory Animal Research (ILAR) bases its reports on its mission
statement promoting “high-quality science and humane care and use of
research animals based upon the principles of refinement, replacement, and
reduction (the 3Rs) and high ethical standards” (ILAR 2007). The Institute’s
Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral
Research (NRC 2003) provide criteria for evaluating levels of pain that help in
the development of endpoints for studies in neuroscience and behavioral
research. The 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 or inadvertent outcome,
including infectious disease and cancer research (Olfert and Godson 2000;
Wallace 2000); and vaccine potency and and acute toxicity testing (Hendriksen
and Steen 2000; Sass 2000). ILAR published the proceedings of a symposium on
Regulatory Testing and Animal Welfare detailing best practices for humane
conduct of animal testing for regulatory purposes (NRC 2002). It is not possible
to cover all aspects of humane endpoints in this report; other sources of
information and guidance for establishing humane endpoints, some of which
have already been discussed, include the Office of Laboratory Animal Welfare
(OLAW), the Animal Welfare Information Center (AWIC), and the Canadian
Council for Animal Care (CCAC). Scientists, IACUCs, veterinarians, and study
personnel should consider these and other references to address specific issues
in their research projects and institutions.
Safety assessment guidelines
In addition to the above-mentioned references, regulatory guidelines for
safety or toxicology studies are beginning to address the concept of endpoints
when animals are observed in severe pain or distress even when regulatory
guidance is vague in describing potential endpoints. The U.S. Environmental
Protection Agency (EPA) Health Effects Test Guidelines for Acute Oral Toxicity
(EPA OPPTS 2002) provide instruction for following the OECD Guidance
Prepublication Copy
OCR for page 128
128 Recognition and Alleviation of Pain in Laboratory Animals
Document on…Humane Endpoints (2000) to reduce the suffering of animals in
toxicity studies. Euthanasia of animals that are either moribund or in severe
pain is also encouraged. However, vague statements such as “animals showing
severe and enduring signs of distress and pain may need to be humanely killed”
are common in regulatory guidelines (USEPA OPPTS 1998). In fairness to test
guidelines, these documents cover a wide range of test materials and cannot
address all potential endpoints. Unfortunately, because of the vague language,
laboratories conducting such safety studies may be reluctant to terminate a
study or the exposure of an animal 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,
but potential reality nonetheless.
In 1994, the OECD recognized that while it may be necessary to create
ambiguous test guidelines, such ambiguity fosters an overbroad interpretation
of what constitutes a humane endpoint in toxicology studies. The organization,
therefore, created a working group to develop a guidance document using
clinical signs as humane endpoints in safety evaluation studies (OECD 2000).
The resulting document put forth criteria based on the principles of the 3Rs to
assist researchers in determining when animals should be considered moribund
or in severe distress or pain, and these criteria be broad enough to apply to a
wide range of study types, test substances, species, and strains of animals.
The guidance document provides descriptions of clinical signs to assist study
personnel in determining when death may be imminent or when severe pain
may be present followin exposure to a test substance. The reader is
encouraged to examine this resource when developing internal guidance
documents to assess humane endpoints.
While these references are extremely valuable, it is important to view
them in accordance with their intent: they are guidance documents only and as
such have limitations. No single document could cover all potentially painful
study types, all animal species used in such 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 on individual protocols.
However, caution is advisable in developing a policy on humane endpoints.
While the ideal is to avoid pain, personnel also need to assure that the study
objectives are attained before a procedure or animal is terminated
(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 a study is being conducted to meet the
requirements for the safety assessment of a substance, a regulatory agency
may elect to 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
Prepublication Copy
OCR for page 129
CHAPTER 5: HUMANE ENDPOINTS FOR ANIMALS IN PAIN 129
objective. If regulatory guidelines do not specify an endpoint, as in vaccine
potency studies (CFR Title 9, 2006), lethality can and will be used by regulated
entities.
For all these reasons identification of humane endpoints should take into
account the following factors: the role regulatory agencies should play in the
overall process; the need for scientifically appropriate endpoints; and the
reliability of the clinical observations of the animals (i.e., the observations
must be accurate 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 judgments about an animal’s well-being.
The Office of Laboratory Animal Welfare approached the subject of
humane endpoints in the Institutional Animal Care and Use Committee
Guidebook (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 accomplish 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.
Pilot studies
An effective way to lessen negative impacts on laboratory animals is the
use of pilot studies (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
complement of animals necessary to conduct a statistically valid study. The
small sample size would not be statistically relevant, but could be critical to
the success of a larger study. Pilot studies have a number of advantages:
help identify potential interactions between proposed analgesic and
anesthetic treatments and specific research goals
identify potentially useful means of assessing pain in a specific research
model
help identify humane endpoint criteria that are specific to an individual
project.
If problems occur in the pilot study, strategies can be discussed and
devised to address an animal’s deteriorating conditions. Such strategies may
include, but certainly not be limited to, adjustment of dose levels, changes in
Prepublication Copy
OCR for page 130
130 Recognition and Alleviation of Pain in Laboratory Animals
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. Information learned in
a pilot study can avoid unnecessary pain for a larger number of animals in a
definitive study.
Caution needs to be exercised when conducting pilot studies in
laboratory animals, as the risk of causing significant pain 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 between all involved can ensure both collection of the
maximum amount of data and appropriate interventions on behalf of the
animals (NRC 2003, p. 14).
Humane endpoints
Regulations and guidelines
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). These assessments were
developed and strengthened in response to unpredicted adverse effects of
drugs or chemicals on humans, animals, and the environment. For example,
the use of thalidomide by pregnant women in the 1960s caused severe birth
defects in the long bones of their fetuses. U.S. legislation subsequently
required adequate testing of drugs in animals before human exposure (Gallo
2001; Nies 2001). Similar legislative actions followed environmental disasters
like the Love Canal contamination (Merrill 2001).
The purpose of testing requirements on pharmaceutical, consumer, and
industrial products is to ensure the safety of the environment and of
human/animal populations. However, these requirements have tended to
focus on the safety of the user; they do not necessarily consider humane
endpoints for animals used in 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 Authorisation of Chemicals) is to
improve the assessment of chemicals in order to better protect human health
and the environment. Because the range of chemicals covered by REACH is
enormous, there is great potential for increased use of animals in
corresponding toxicity and safety testing. Embedded in the regulation,
however, are components that ensure animal testing only when necessitated by
Prepublication Copy
OCR for page 131
CHAPTER 5: HUMANE ENDPOINTS FOR ANIMALS IN PAIN 131
identification of data gaps. The regulation requires industry to share data on
similar chemicals to avoid duplicative animal testing; further allows for
submission of data using non-animal tests; strongly encourages the use of
Quantitative Structure-Activity Relationship (QSAR) or other computer-
generated information; and invites the grouping of submitted data for similar
chemicals that may result in similar hazard and risk (the so-called “read-
across” principle). REACH authorizes animal testing only after this information
has been collected and data gaps identified (ECHA 2008). While these efforts
do not define humane endpoints, the authors of the regulation should be
commended for the consideration of responsible animal use in safety
assessment.
Also useful in the toxicology regulatory arena is a Memorandum of
Understanding signed in February 2008, laying the foundation and framework
for the U.S. Environmental Protection Agency (USEPA) and two agencies of the
U.S. National Institutes of Health to collaborate in sharing data, resources, and
expertise in efforts to replace animal testing for chemical toxicity assessment
(Collins et al. 2008; MOU 2008; NIH 2008). The ambitious goal is to evaluate in
vitro assays, such as those used for identification of toxicity pathways and high-
throughput screening (as described in the NRC report Toxicity Testing in the
Twenty-first Century: A Vision and a Strategy; NRC 2007) to better predict
potential health and environmental hazards from chemicals. The dual goal of
this collaboration to develop more accurate assays and to change regulatory
guidelines is likely to be a long-term process. Similar steps forward should be
encouraged on a global scale to effect change in regulatory agencies and
eliminate potentially painful animal testing schemes.
Despite the promotion of responsible animal use in the REACH
regulation, many safety assessment guidelines still differ between countries.
As a result, tests must comply with all the requirements of each country where
a product is to be marketed for a particular use. The regulatory agency of one
country could require an additional group of animals to assess recovery from
exposure, while other countries may not have this requirement and may even
reject the study depending on their review process. While 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, agencies in other countries may not accept the data in lieu of
the guinea pig dermal sensitization test.
Although harmonization of regulatory guidelines has significantly
reduced discrepancies between cooperating countries, efforts for the global
harmonization of safety guidelines are in a constant state of flux. Such efforts
should continue to support and promote responsible animal use, establishment
of humane endpoints in safety studies, and acceptance of scientifically
validated alternatives to eliminate unnecessary animal pain.
A comparison of all safety assessment guidelines is well beyond the
scope of this report, but examples illustrate how differences in regulatory-
driven studies can have a negative impact on the prevention and alleviation of
pain in laboratory animals. For example, a safety assessment test that may
Prepublication Copy
OCR for page 132
132 Recognition and Alleviation of Pain in Laboratory Animals
cause pain is the acute eye irritation study, the purpose of which is to evaluate
the potential hazards of ocular exposure to a substance; requirements for this
procedure are generally in agreement across international regulatory bodies
and national agencies (JMAFF 2000; OECD 1987 #405; USEPA OPPTS 1998 #
870.2400). Also of concern is the reversibility of ocular lesions as an additional
requirement of this test in order to more fully assess the risk of human
exposure. But the procedures for this component of the toxicity evaluation can
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 animal use.
These guidelines also identify ocular lesions that are considered irreversible
and thereby meet OECD criteria for terminating the study and euthanizing the
animal. While current 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 (i.e., identification of irreversible lesions).
Humane endpoints and toxicology studies
Not all test substances will cause ocular 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 test and that gap
persists as evidenced in a U.S. Federal Register notice (Federal Register 2007)
requesting data on analgesic use in ocular irritancy tests that alleviates pain
without affecting test results. Current guidelines include no 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 affected animals, to avoid the
possibility of interference with the test substance (Stokes 2005). However,
numerous published studies demonstrate that the use of analgesics to alleviate
pain from ocular irritancy tests does not interfere with the scientific objectives
of this safety test (Patrone 1999; Peyman 1994; Stiles 2003). Such evidence
can be utilized 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 significant near
the scheduled termination of these studies. Guidelines generally specify the
survival rates necessary to provide meaningful interpretation of the chronic
study, but the OECD document is the only one to discuss humane endpoints and
provide guidance for early termination of a study if survival rates fall below the
specified percentage. In order to achieve the required survival rate at the end
of the mandated study, animals may not be euthanized until very close to
death. Situations of this nature are not in the best interest of the animals.
True harmonization of guideline safety assessment tests and global adoption of
Prepublication Copy
OCR for page 133
CHAPTER 5: HUMANE ENDPOINTS FOR ANIMALS IN PAIN 133
the OECD humane endpoints document would be an important step in
alleviation and avoidance of pain in laboratory animals.
The future of toxicology
The NRC report Toxicity Testing in the Twenty-first Century: A Vision
and a Strategy (NRC 2007) reviewed and 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 pharmacokinetics) and emerging technologies, such as bioinformatics.
Although the report acknowledges that implementation of the strategy will
require much effort on the part of scientists, regulators, and lawmakers to
develop workable testing schemes, the concepts envisioned could significant
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 above-mentioned document was the
ACSA 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 multi-faceted approach redesigns traditional toxicology tests to
integrate several sciences, such as metabolism/kinetics and life stages, in a
single study so that the requirement for separate studies to evaluate each
parameter is eliminated and the number of animals used is reduced
(Carmichael et al. 2006; Cooper et al. 2006; Doe et al. 2006; ILSI 2008).
Further, the metabolism/kinetics 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,
which is based on a step-wise, or tiered, testing, is expected to reduce animal
numbers, minimize potential pain to laboratory animals because it would avoid
exposure levels that produce clinical signs of toxicity in animals, and improve
the quality of data for risk assessments 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 culminates in clinical
disease, including death. The animals may experience significant pain during
these experiments, but identifying and validating earlier endpoints to
safeguard animal welfare can be difficult, as an inappropriate endpoint may
Prepublication Copy
OCR for page 134
134 Recognition and Alleviation of Pain in Laboratory Animals
not adequately identify the full course of a disease or the efficacy of a
potential medication (Olfert and Godson 2000). It is imperative, therefore, to
examine and validate endpoints within a solid scientific foundation that
includes, among others, immunological parameters, biochemical and endocrine
changes, and other pathophysiologic changes as, for example, 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 fresh tissues as
opposed to autolyzed tissues from animals that have been allowed to die
(Copps 2005).
Vaccine safety and potency testing
Another area of research that frequently results in the death of a study
animal is vaccine testing for regulatory agencies. Since vaccines are biological
products and one batch may not be as potent as the next or may contain
harmful byproducts, both efficacy and safety must be tested (Castle 1999;
Cussler et al. 1999; Hendriksen 2002). To ensure quality control and the safety
of each batch, regulatory agencies such as the U.S. Food and Drug
Administration (FDA), the U.S. Department of Agriculture (USDA), the European
Pharmacopoeia, and the World Health Organization (WHO), require potency
testing during which animals are vaccinated and subsequently 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 others are based on survival of 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 seven day test period in which
case subsequent repeat safety tests over a larger test population are then
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 vaccinated vs. control animals, as, for
example, in the USDA safety test for Marek’s disease vaccine ((CFR 2006,
113.330). In this type of testing a more humane endpoint would specify that
the onset of clinical signs in unvaccinated controls in itself constitutes a valid
test: the potency test for tetanus antitoxin states that the test is met when
unvaccinated control guinea pigs are unable to stand within 24 hours post-
challenge at which point animals may be euthanized (CFR 2006, 113.451).
Regulations may also encourage the use of in vitro methods. The canine
distemper killed virus vaccine potency test for the USDA (CFR 2006, 113.201)
accepts serum titer levels in vaccinated animals for potency data; if, however,
those tests are inconclusive, a viral challenge test is required utilizing both
vaccinated and unvaccinated controls. The agency identifies the survival of all
Prepublication Copy
OCR for page 135
CHAPTER 5: HUMANE ENDPOINTS FOR ANIMALS IN PAIN 135
vaccinated animals and the death of all controls as a satisfactory indicator 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 a worthwhile endeavor to identify reliable markers of
predictive or impending mortality to serve as alternative and more humane
endpoints. No purpose is served when the administration of a vaccine itself
results in harm rather than protection, but, as with all research studies and
testing guidelines, a balance between effective safety evaluation and humane
endpoints should be achieved for the sake of the laboratory animal.
Humane endpoints in cancer research
Identification of humane endpoints in cancer research can be
challenging. Although the wide range of tumor types and scientific objectives
associated with this research prohibits standardization of humane endpoints in
this field (Wallace 1999; Wallace 2000), the United Kingdom Coordinating
Committee on Cancer Research (UKCCCR) has developed a document to guide
researchers working with animal models (UKCCCR 1988). Tumor size, tumor
appearance, and animal condition can be evaluated in order to identify reliable
indicators that may permit earlier termination of a study. Researchers are
encouraged to establish and validate endpoints that retain scientific objectives
and yet can avoid, minimize, or alleviate potential pain in the laboratory
animals. Avoiding death or excessive tumor burden, particularly when coupled
with clinical signs of pain or distress, should be considered a desirable goal in
cancer research studies.
Humane endpoints in pain research
Of critical importance to this report, as well as to improvements in
quality of life for both humans and animals, is research on pain itself including
the mechanisms of the 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 a specific 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 that animal welfare
and study objectives are not adversely compromised in these research
programs (Mench 1999).
Prepublication Copy
OCR for page 136
136 Recognition and Alleviation of Pain in Laboratory Animals
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, p. 102-104). A humane death, or endpoint, is a fundamental
tenent of the U.S. Principles for the Utilization and Care of Vertebrate Animals
Used in Testing, Research, and Training (2001), as Principle VI states that
“[a]nimals that would otherwise suffer severe or chronic pain that cannot be
relieved shold be painlessly killed at the end of the procedure or, if
appropriate, during the procedure”. The humane death of an animal is defined
as 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. The point
at which euthanasia should be performed for humane reasons cannot be rigidly
defined in one document, as it is not possible to apply a single set of
euthanasia criteria across all study designs, animal models, and experimental
goals. Body condition scores, as described in Chapter 3, can be used to
determine when to consider euthanasia for humane reasons. This decision
should involve a team approach among veterinarians, study directors, and
animal care personnel using all available information on the affected animal(s).
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 AVMA (AVMA
2007), although objective information on laboratory animals is sparse,
particularly concerning the evaluation of potential pain and distress that may
be caused by a specific euthanasia technique. The controversy that may result
from this lack of data is illustrated by 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 for the
foreseeable future, 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 age groups and laboratory animal species are needed and
recommended. The assessment tools and measures to considered for such
studies include electroencephalograms, electrocardiograms, electromyograms,
arterial blood pressure, respiration and heart rates, serum biochemical
parameters, pupil diameter, and behavioral changes. In particular, studies
that provide measures of nociception, pain, distress, and the relation of these
to loss of consciousness are urgently required.
Conclusions and recommendations
Avoiding or minimizing pain in animal research is a fundamental
obligation of all researchers for moral and ethical reasons. The criteria used
Prepublication Copy
OCR for page 137
CHAPTER 5: HUMANE ENDPOINTS FOR ANIMALS IN PAIN 137
for earlier termination of a research project or alteration to a study design for
the purpose of alleviating of avoiding pain in an animal are defined as humane
endpoints. Identification and validation of earlier humane endpoints should be
considered for studies involving pain, but this is neither an easy nor a simple
process.
1. An important part of the process is to ensure that the endpoints are
validated and based on sound science. Pilot studies are invaluable for
determination of earlier and more humane endpoints.
2. Given the wide scope of procedures and goals of animal research, no one
reference can document every humane endpoint for every research
project. Therefore, more effort must be made to identify appropriate
humane endpoints for each protocol. 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 to develop and validate alternative procedures
that can be incorporated into 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 scientists,
managers, technicians, oversight committees, and regulators involved with
animal experimentation where pain is a potential component should participate
in productive dialogue 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 sustained pursuit of these
directed efforts can, and will, result in more humane animal use.
Prepublication Copy
OCR for page 138
138 Recognition and Alleviation of Pain in Laboratory Animals
Additional information
OECD: Guidance Document on the Recognition, Assessment, and Use of
Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety
Evaluation December 2000
A humane endpoint can be defined 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 toxicology
studies is to be able to accurately predict severe pain, severe distress, suffering, or
impending death, before the animal experiences these effects. However, the science
of toxicology is not yet to the point where such accurate predictions 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 minimised 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 consideration 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).
As stated in this chapter, establishing surrogate or humane endpoints as
part of the experimental protocol and before experiments commence, is one of
the ways to minimize and alleviate pain and safeguard the well-being and
welfare of laboratory animals. The following two examples are: 1. A score
sheet to assess animals in cancer studies based on a behavioral and tumor
scoring system. The recorded symptomatology will determine the diagnosis
and subsequent alleviatory actions. They can be adapted to any protocol or
animal care facility system as long as the behavioral definitions are uniform
across the same facility; 2. A model for developing guidelines for humane
endpoints that may be applied to any protocol within a facility.
Prepublication Copy
OCR for page 139
CHAPTER 5: HUMANE ENDPOINTS FOR ANIMALS IN PAIN 139
Chapter 5-Example Tumor Scoring Sheet. Developed by Fraser Darling, The Institute of
Cancer Research, London, UK.
Guidelines for Humane Endpoints in Animal Studies
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 Principle Investigator/Study Director to
define humane endpoints and to explore alternatives to death as an endpoint. If no
alternative exists, the PI/SD should 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 should be made to minimize pain and distress experienced by animals used in
research. This policy letter is to provide investigators with guidelines for determining
humane endpoints in compliance with the XXXXXX policy. To this end, the use of
death as an endpoint to experimental studies, rather than performing euthanasia to
humanely terminate an animal, is discouraged and should 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
Prepublication Copy
OCR for page 140
140 Recognition and Alleviation of Pain in Laboratory Animals
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 responsible 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 should 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.
There should 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 should 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. PI/SD should 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 expiration); abnormal behavior (reduced
mobility/unconsciousness, unsolicited vocalizations, self mutilation); severe
depression or abnormal/exaggerated responses to external stimuli.
Prepublication Copy
OCR for page 141
CHAPTER 5: HUMANE ENDPOINTS FOR ANIMALS IN PAIN 141
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 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 experience and good
judgement 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 should 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 necessary suspend any
activity which violates applicable laws, regulations, standards, guidelines, policies and
procedures.
REFERENCES
Montgomery CA. 1990. Oncologic and toxicologic research: Alleviation and control of
pain and distress in laboratory animals. Canc Bull 42(4):230-237.
Stokes WS. 2002. Humane endpoints for laboratory animals used in regulatory testing.
ILAR J. 43(Suppl):S31-S38.
References
ACLAM [American College of Laboratory Animal Medicine]. 2005. Public
Statements: Report of the ACLAM Task Force on Rodent Euthanasia.
Available at: http://www.aclam.org/print/report_rodent_euth.pdf.
Accessed January 5 2008.
Prepublication Copy
OCR for page 142
142 Recognition and Alleviation of Pain in Laboratory Animals
AVMA [American Veterinary Medical Association]. 2007. AVMA Guidelines on
Euthanasia. Available at:
http://www.avma.org/issues/animal_welfare/euthanasia.pdf
AWIC [Animal Welfare Information Center]. Humane Endpoints. Available at:
http://awic.nal.usda.gov/nal. Accessed January 5 2008.
Castle P, 1999. The European Pharmacopoeia and Humane Endpoints. In
Humane Endpoints in Animal Experiments for Biomedical Research,
Proceedings of the International Conference, 22-25 November 1998,
Ziest, The Netherlands, Hendriksen CFM, Morton DB, eds. The Royal
Society Medical Press, p 15-19.
CCAC [Canadian Council on Animal Care]. 1993. Guide to the Care and Use of
Experimental Animals, Vol. 1, Ontario, Canada: Canadian Council on
Animal Care. Available at:
http://www.ccac.ca/en/CCAC_Programs/Guidelines_Policies/GUIDES/E
NGLISH/toc_v1.htm.
CCAC 1998. Guidelines on choosing an appropriate endpoint in experiments
using animals for research, teaching and testing. Available at:
http://www.ccac.ca/en/CCAC_Programs/Guidelines_Policies/GDLINES/E
NDPTS/g_endpoints.pdf.). Accessed January 5 2008.
CFR [Code of Federal Regulations]. 2006. Regulations Title 9, Chapter 1, Part
113, Section 113.33. Available at:
http://www.access.gpo.gov/nara/cfr/waisidx_00/9cfrv1_00.html.
Accessed January 5 2008.
CFR. 2008. Regulations Title 21, Chapter 1, Subchapter F, Part 610.11.
Available at:
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cf
m?fr=610.11. Accessed October 26 2008
Collins FS, Gray GM, Bucher JR. 2008. Transforming environmental health
protection. Science 319:906-907.
Conlee KM, Stephens ML, Rowan AN, King LA. 2005. Carbon dioxide for
euthanasia: concerns regarding pain and distress, with special reference
to mice and rats. Lab Anim 39:137-161.
Cooper RL, Lamb JC, Barlow SM, Bentley K, Brady AM, Doerrer NG, Eisenbrandt
DL, Fenner-Crisp PA, Hines RN, Irvine LF, Kimmel CA, Koeter H, Li AA,
Makris, SL, Sheets LP, Speijers G, Whitby KE. 2006. A tiered approach to
life stages testing for agricultural chemical safety assessment. Crit Rev
Toxicol 36(1):69-98.
Copps J. 2005. Issues related to the use of animals in biocontainment research
facilities. ILAR J 46(1):34-43.
Cussler K., Morton DB, Hendriksen CFM. 1999. Humane endpoints in vaccine
research and quality control. In Humane Endpoints in Animal
Experiments for Biomedical Research, Proceedings of the International
Conference, 22-25 November 1998, Ziest, The Netherlands, Hendriksen
CFM, Morton DB, eds. The Royal Society Medical Press, p 95-101.
DeHaven WR. 2002. Best practices for animal care committees and animal
oversight. ILAR J 43(Suppl):S59-62.
Prepublication Copy
OCR for page 143
CHAPTER 5: HUMANE ENDPOINTS FOR ANIMALS IN PAIN 143
Doe JE, Boobis AR, Blacker A, Dellarco V, Doerrer NG, Franklin C, Goodman JI,
Kronenberg JM, Lewis R, McConnell EE, Mercier T, Moretto A, Nolan C,
Padilla S, Phang W, Solecki R, Tillbury L, van Ravenzwaay B, Wolf DC.
2006. A tiered approach to systemic toxicity testing for agricultural
chemical safety assessment. Crit Rev Toxicol 36(1):37-68.
Durham RA, Sawyer DC, Keller WF, Wheeler CA. 1992. Topical ocular
anesthetics in ocular irritancy testing: A review. Lab Anim Sci 42(6):535-
541.
European Chemicals Agency (ECHA). 2008. REACH Guidance. Available at:
http://guidance.echa.europa.eu/. Accessed January 2, 2009.
Federal Register Notice, Volume 72, Number 89, May 9, 2007. Department of
Health and Human Services, National Toxicology Program (NTP), NTP
Interagency Center for the Evaluation of Alternative Toxicological
Methods (NICEATM): Request for Data on the Use of Topical Anesthetics
and Systemic Analgesics for In Vivo Eye Irritation Testing. Available at:
http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/FR/FR_E7_8898.pdf.
Gallo MA. 2001. History and scope of toxicology, Klaassen CD, ed. Casarett and
Doull’s Toxicology: The Basic Science of Poisons, Sixth Edition, New
York, McGraw Hill Medical Publishing Division, p. 3-10.
Hawkins P, Playle L, Golledge H, Leach M, Banzett R, Coenen A, Cooper J,
Danneman P, Flecknell P, Kirkden R, Niel L, Raj M. 2006. Newcastle
Consensus Meeting on Carbon Dioxide Euthanasia of Laboratory Animals.
Available at:
http://www.nc3rs.org.uk/downloaddoc.asp?id=416&page=292&skin=0.
Hendriksen CFM. 2002. Refinement, reduction, and replacement of animal use
for regulatory testing: Current best scientific practices for the
evaluation of safety and potency of biologicals. ILAR J 43(Supl):S43-S48.
Hendriksen CFM, Morton DB, eds. 1999. Humane Endpoints in Animal
Experiments for Biomedical Research. Proceedings of the International
Conference, 22-25 November 1998, Ziest, The Netherlands. London: The
Royal Society Medical Press.
Hicks JM. 1997. Animal welfare and toxicology/safety studies: Making sense of
the regulatory environment. Cont Topics 36(3):49-54.
ILAR (Institute for Laboratory Animal Research). 2007. Mission and Core Values.
Available at: http://del.nas.edu/ilar_n/ilarhome/mission.shtml.
ILAR J. 2000. Humane Endpoints for Animals Used in Biomedical Research and
Testing. 41(2). Available at:
http://dels.nas.edu/ilar_n/ilarjournal/41_2/.
ILAR J. 2002. Regulatory Testing and Animal Welfare. 43(Supplement).
Available at: http://dels.nas.edu/ilar_n/ilarjournal/43_suppl/.
ILSI-HESI (International Life Sciences Institute– Health and Environmental
Sciences Institute. Agricultursal Chemical Safety Assessment factsheet.
2008. Available at:
(http://www.hesiglobal.org/committees/TechnicalCommittees/ACSA/).
Accessed January 5 2008.
Prepublication Copy
OCR for page 144
144 Recognition and Alleviation of Pain in Laboratory Animals
IRAC [Interagency Research Animal Committee]. 1985. The U.S. Government
Principles for the Utilization and Care of Vertebrate Animals Used in
Testing, Research, and Training. Federal Register Vol. 50, No. 97 (May
20, 1985). Available online at:
http://grants.nih.gov/grants/olaw/references/phspol.htm#USGovPrincip
les. Accessed June 9 2008.
Jaax J. 2005. Administrative issues related to infectious disease research in the
age of Bioterrorism. ILAR J 46(1):34-43.
JMAFF (Japanese Ministry of Agriculture, Forestry and Fisheries). 2000. Testing
Guidelines for Toxicology Studies.
Kirkden RD, Niel L, Stewart SA, Weary DM. 2008. Gas killing of rats: The effect
of supplemental oxygen on aversion to carbon dioxide. Anim Welf 17:79-
87.
Le Bars D, Gozariu M, Cadden SW. 2001. Animal models of nociception.
Pharmacol Rev 53:597-652.
Mench J. 1999. Defining endpoints: The role of the animal care committee. In:
Humane Endpoints in Animal Experiments for Biomedical Research.
London, The Royal Society Medical Press. p. 133-138.
Merrill RA. 2001. Regulatory toxicology. In Casarett and Doull’s Toxicology: The
Basic Science of Poisons, Sixth Ed, Klaassen CD, ed. New York: McGraw
Hill Medical Publishing Division. p. 1141-1153.
Meyerson BA, Linderoth B. 2006. Mode of action of spinal cord stimulation in
neuropathic pain. J Pain Sympt Manag 31(4S):S6-S12.
Morton DB. 1999. Humane endpoints in animal experimentation for biomedical
research: Ethical, legal and practical aspects. In Humane Endpoints in
Animal Experiments for Biomedical Research. London: The Royal Society
Medical Press. p. 5-12.
Morton DB. 2000. A systematic approach for establishing humane endpoints.
ILAR J 41(2):80-86.
Morton DB, Burghardt GM, Smith JA. 1990. Critical anthropomorphism, animal
suffering and the ecological context. Hastings Cent Rep 20(3):13-19.
MOU (Memorandum of Understanding) on High Throughput Screening, Toxicity
Pathway Profiling, and Biological Interpretation of Findings. 2008.
Available at:
http://www.niehs.nih.gov/news/releases/2008/docs/ntpncgcepamou.p
df.
Nemzek JA, Hugunin KM, Opp MR. 2008. Modeling sepsis in the laboratory:
Merging sound science with animal well-being. Comp Med 58(2):120-128.
Nemzek JA, Xiao H-Y, Minard AE, Bolgos GL, Remick DG. 2004. Humane
Endpoints in Shock Research. Shock 21(1):17-25.
Niel L, Stewart SA, Weary DM. 2008. Effect of flow rate on aversion to gradual-
fill carbon dioxide exposure in rats. Appl Animl Behav Sci 109:77-84.
Nies AS. 2001. Principles of Therapeutics. In Goodman and Gilman’s The
Pharmacological Basis of Therapeutics, Tenth Ed, Hardman JG, Limbird
LE, eds. New York: McGraw-Hill. p 45-66.
Prepublication Copy
OCR for page 145
CHAPTER 5: HUMANE ENDPOINTS FOR ANIMALS IN PAIN 145
NIH [National Institutes of Health]. 1997. Validation and Regulatory Acceptance
of Toxicological Test Methods: A Report of the ad hoc Interagency
Coordinating Committee on the Validation of Alternative Methods. NIH
Publication No. 97-3981, Research Triangle Park, NC.
NIH. 2008. NIH Collaborates with EPA to Improve the Safety Testing of
Chemicals. News Press Release. Available at:
http://www.nih.gov/news/health/feb2008/nhgri-14.htm.
NRC [National Research Council]. 1992. Recognition and Alleviation of Pain and
Distress in Laboratory Animals. Washington DC. National Academy Press.
NRC. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and
Behavioral Research. Washington DC. The National Academies Press.
NRC. 2007. Toxicity Testing in the Twenty-first Century: A Vision and a
Strategy. Washington DC. The National Academies Press.
NRC. 2008. Recognition and Alleviation of Distress in Laboratory Animals.
Washington DC. The National Academies Press.
OECD [Organization for Economic Co-operation and Development]. 1987.
Guidelines for Testing of Chemicals, Section 4: Health Effects.
Paris:OECD.
OECD. 1996. Guidelines for Testing of Chemicals, Section 4: Health Effects.
Paris: OECD.
OECD. 2000. Guidance Document on the Recognition, Assessment, and Use of
Clinical Signs as Humane Endpoints for Experimental Animals Used in
Safety Evaluation. Paris: OECD.
OLAW/ARENA (Office of Laboratory Animal Welfare/Applied Research Ethics
National Association). 2002. Institutional Animal Care and Use
Committee Guidebook, 2nd Ed. Bethesda: National Institutes of Health.
Olfert ED, Godson DL. 2000. Humane endpoints for infectious disease animal
models. ILAR J 41(2):99-104.
Patrone G, Sacca SC, Macri A, Rolando M. 1999. Evaluation of the analgesic
effect of 0.1% indomethacin solution on corneal abrasions.
Ophthalmologica 213:350-354.
Peyman GA, Rahimy MH, Fernandes ML. 1994. Effects of morphine on corneal
sensitivity and epithelial wound healing:Implications for topical
ophthalmic analgesia. Br J Ophthalmol 78:138-141.
Sass N. 2000. Humane endpoints and acute toxicity testing. ILAR J 41(2).
Available at:
http://dels.nas.edu/ilar_n/ilarjournal/41_2/AcuteToxicity.shtml.
Stephens ML, Conlee K, Alvino G, Rowan AN. 2002. Possibilities for refinement
and reduction: Future improvements within regulatory testing. ILAR J
43(Suppl):S74-79.
Stiles J, Honda CN, Krohne SG, Kazacos EA. 2003. Effect of topical
administration of 1% morphine sulfate solution on signs of pain and
corneal wound healing in dogs. Am J Vet Res 64 (7):813-818.
Stokes WS. 2002. Humane endpoints for laboratory animals used in regulatory
testing. ILAR J 43(Suppl):S31-S38.
Prepublication Copy
OCR for page 146
146 Recognition and Alleviation of Pain in Laboratory Animals
Stokes WS. 2005. Summary of ICCVAM-NICEATM-ECVAM Ocular Toxicity
Scientific Symposia, Presentation. Available at:
(http://iccvam.niehs.nih.gov/meeting/SACpresents/SACocular.pdf),
1/5/08.
UKCCCR (United Kingdom Coordinating Committee on Cancer Research). 1988.
Guidelines for the welfare of animals in experimental neoplasia. Lab
Anim 22:195-201.
USEPA (United States Environmental Protection Agency). 1998. Series 870
Health Effects Test Guidelines – Final Guidelines. Available at:
http://www.epa.gov/opptsfrs/publications/OPPTS_Harmonized/870_He
alth_Effects_Test_Guidelines/Series/.
USEPA. 2008. Summary of the Toxic Substances Control Act, 15 U.S.C. s/s 2601
et seq 1976. Available at:
http://www.epa.gov/lawsregs/laws/tsca.html. Accessed January 5
2008.
USEPA Office of Prevention, Pesticides, and Toxic Substances. 1998. 870 Series
Final Guidelines: Health Effect Test Guidelines. Available at:
http://www.epa.gov/opptsfrs/publications/OPPTS_Harmonized/870_He
alth_Effects_Test_Guidelines/Series/. Accessed January 5 2008.
USEPA Office of Prevention, Pesticides, and Toxic Substances. 2002. 870 Series
Final Guidelines: Health Effect Test Guidelines, OPPTS 870.1100. Acute
Oral Toxicity.
Walker K, Fox AJ, Urban LA. 1999. Animal models of pain. Mol Med Today
5:319-321.
Wallace J. 1999. Humane endpoints in cancer research. In: Humane Endpoints
in Animal Experiments for Biomedical Research. Proceedings of the
International Conference, 22-25 November 1998, Hendriksen CFM,
Morton DB, eds. Ziest: The Netherlands. The Royal Society Medical Press.
p 79-84.
Wallace J. 2000. Humane endpoints in cancer research. ILAR J 41(2):87-93.
Prepublication Copy
