Introduction: Reducing unrelieved pain and distress in laboratory animals using humane endpoints. ILAR 41 (2): 059.
[Reviewer's Note - Any parts of this introductory article which recapped specific articles published in this ILAR issue were not reviewed as other LABSG members will be summarizing these articles for the LABSG list.] The importance of managing pain in laboratory animals has led to the establishment of policies (PHS), guidelines, and regulations (USDA) for lab animal pain management. Both human and animal standards seek to minimize the occurrence of unrelieved pain. However, in some animal studies, management of unrelieved pain is problematic as pain-reducing agent cannot be used. Research animals may experience pain from induced disease, procedures, and toxicity. Animal welfare regulations state that procedures that cause more than momentary or slight pain and distress should be performed with appropriate sedation, analgesia, or anesthesia (USDA). However, for some research, such agents could interfere with the scientific objectives of the study, thus placing the animal in a situation where pain would not be relieved. Federal regulations and policies mandate that discomfort to animals must be limited to what is unavoidable for the conduct of scientifically valuable research. Unrelieved pain and distress should continue only for the duration necessary to accomplish the scientific objectives (USDA). Animals that would suffer unrelieved severe or chronic distress should be painlessly killed at the end of the procedure, or if appropriate, during the procedure (PHS). Researchers are required to report unrelieved pain and distress in certain species of animals to USDA. There are no USDA reporting requirements for rats and mice. Thus, the total number of lab animals experiencing unrelieved pain and distress likely exceeds one million each year. One way it might be possible to reduce the number of animals that experience unrelieved pain and distress would be to end studies earlier to avoid or terminate unrelieved pain and/or distress. Criteria or humane endpoints must be identified and validated to ensure study objectives will still be met. Criteria that can be used to end studies before the onset of pain and distress are ultimately the ideal humane endpoints. Recent establishment of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) now provides a mechanism by which new testing methods that incorporate more humane endpoints can be evaluated and adopted. Establishing and implementing humane endpoints requires commitment and innovation on the part of scientists, veterinarians, and animal care staff. NIH is conducting and supporting research into methods of medical research and experimentation that produce less pain and distress in animals. In The Principles of Humane Experimental Technique, Russell and Burch presented the concept of the replacement, reduction, and refinement of animal use. Refinement is any development leading to a decrease in the incidence or severity of inhumane procedures applied to those animals that have to be used. The development and use of humane endpoints epitomizes this concept of refinement as a means to achieve more humane use of animals.
1. What are the three "Rs"? Who defined what the three "Rs" are?
2. Match the appropriate USDA pain and distress category (C, D, E) to its description.
i. Procedures which cause no pain or significant distress in the absence of anesthesia (including approved euthanasia methods).
ii. Procedures involve pain or distress which is not alleviated.
iii. Procedures are potentially painful, but anesthetics, tranquilizers, etc. are given to alleviate pain and distress.
3. The Animal Welfare Act requires a search of the literature for alternatives when covered animals are placed into which of these USDA
pain and distress categories?
4. The annual report for research facilities to the USDA AC (Animal Care) Regional Office is due on or before which date of each year?
A. June 1
B. December 1
C. January 1
D. March 1
5. What period does the annual report for research facilities to the USDA AC Regional Office cover?
A. USDA FY
B. October 1 through September 30
C. December 1 through October 31
D. January 1 through December 31
E. A and B
F. A and C
1. Replacement, reduction, and refinement of animal use. William Russell and Rex Burch.
2. i. C; ii. E, iii. D
3. Categories D and E.
4. B. December 1 of each year
5. E. A and B. The USDA FY (October 1 through September 30)

Recognizing pain and distress in laboratory animals. ILAR 41 (2): 062.
Pain and stress can cause distress and the biological effects can compromise experimental results. That is why we work to control the effects of pain and stress on lab animals. Pain normally serves as a protective function to warn of impending danger. Pain and stress are not inherently bad for animals, unless they fail to protect the animal or the cost of coping takes too great a toll on the animal. The question is when does an animal cross over from nonthreatening stress to threatening distress? Recognition of pain and distress is critical to maintaining the well-being of an animal. there is a lack of agreement on terms as well as absence of absolute, objective measurements. The authors discuss the biologic meaning of pain, stress and distress and review guideline for the recognition and assessment of pain and distress. Pain- (For humans) an unpleasant sensory and emotional experience with potential or actual tissue damage. Animals can not tell us what hurts. so we assess "pain-like" (nocifensive) behavior. Animal react to avoid noxious stimuli suggesting they perceive pain. Animal have nervous systems that receive and process noxious stimuli similar to humans at the subcortical level. Because cerebral cortices are smaller and less developed than man, animals may not comprehend the meaning of pain or an awareness of impending doom. It is arguable whether animals suffer in the same sense as humans, but they should be treated as if they do Possible indicators of pain, stress and distress are; stereotypic withdrawal reflex, vocalization, orientation toward the stimulus, escape or attack, rubbing, scratching, biting or licking , and autonomic reactions such as increased blood pressure or heart rate, piloerection, and pupillary dilatation. Withdrawal reflex is elicited generally at the same threshold as humans and the magnitude of response is similar. The maximum stimulus intensity tolerated by humans is called the tolerance limit. The tolerance limit seems to vary widely across species. Hyperalgesia is characterized by a heightened intensity of pain elicited by a noxious stimuli at a site or previous injury. This is sometimes accompanied by allodynia which is pain elicited by a noxious stimuli (e.g. touch). These are adaptive in that animals are motivated to reduce movement to minimize re-injury. Since it is difficult to objectively measure pain, scaling procedures may be helpful to assess animals. Chronic pain has no adaptive value but instead causes distress. It is more difficult to recognize than acute pain, but is a crucial indication for treatment to minimize or prevent distress. Neural basis of pain Acute pain is stimulation of high-threshold receptors called nociceptors. There are varieties of nociceptors that selectively respond to mechanical stimuli or non-selectively to thermal or chemical stimuli (polymodal). Some nociceptors (sleeping) respond to inflammation rather than other stimuli. Afferent fibers travel in peripheral nerves to the spinal cord where they contact 2nd order neurons of the dorsal horn. The neurotransmitter glutamate, and peptides substance P and neurokinin A are release from the presynaptic terminals. The signal stimulates the dorsal horn neurons which either send axons to the ascending sensory pathways or to neurons involved in the reflex pathways. Ascending pathways include the spinothalamic, spinocervicothalamic, spinohypothalamic, sinoreticular, spinobrachial amygalar, the postsynaptic dorsal column or the newly discovered pathway for visceral pain in the dorsal columns. Pain may be transmitted rostrally by multisynaptic connections. Hyperalgesia has 2 possible explanations. Sensitization of nociceptors causing a greater response to noxious stimuli or sensitization of 2nd order spinal neurons. The hyperexcited spinal neurons send a larger pain signal to the higher centers. It is likely both of these 4 mechanisms are at work it peripheral injuries. Pain-transmitting spinal neurons are under powerful descending control from the brainstem. Descending pathways can either excite or inhibit spinal neurons. The dual types of descending modulation play an important role in the effects that environmental stressors can have in increasing or reducing pain sensitivity. Chronic pain likely involves central sensitization and remodeling of synapses within the spinal cord. Recognition of pain in Lab animals "unless the contrary is established, investigators should consider that procedures that cause pain or distress in human beings may cause pain or distress in other animals" (IRAC 1985) Proposed observations for assessing pain include change in body weight, physical appearance, clinical signs, changes in unprovoked behavior, and changes to external stimuli. Some indications may be; signs of spontaneous pain and/or hyperalgesia, altered posture or gait, limb guarding, change in muscle tone or limb temperature, vocalization, exaggerated limb withdrawal, shaking, biting, licking or scratching the affected area. (See table 1 page 64 for specific indicators by species). An animal may self mutilate a denervated limb, it is not known it this is a pain reaction or failure to recognize the limb as "self" due to lack of sensation. Signs of chronic pain develop slowly, vary in magnitude over time and may only be characterized by change in body weight, unkempt appearance, reduced activity, "hunched" posture, porphyrin secretions ( rats) spontaneous vocalization when handled, failure to exhibit normal curiosity or social interactions, withdrawal from handler, or increased aggression. Influence of stress on pain Environmental or psychological stress can increase or decrease pain perception. Stress induced analgesia is mediated in part by the descending pathways from the brain that can modulate the spinal transmission of pain signals. Examples include inescapable footshock, centrifugal rotation, noxious stimuli, fear and aggression. Thus level of pain may be influenced by other stresses. Ill animals may experience hyperalgesia (i.e. due to fever). Animal stress and distress The term stress has very broad and different connotations to different people. For the authors purpose they define stress as the biological responses an animal exhibits in an attempt to cope with a threat to homeostasis. No stress occurs unless an animal perceives a threat either consciously or unconsciously. (Tumor growth may cause a threat to homeostasis without the animal being consciously aware of the threat.) Stress may or may not be distressful. (See diagram of model of stress pg 65). All stress results in one of four general biological defense responses: behavioral, autonomic nervous system, neuroedocrine, or immune. A brief stress may have no lasting significant effect. When stress is severe, of long duration, or the accumulation of multiple stressors that require the animal to divert energy from other critical life functions in order to cope with the stress the animal enters a prepathologic state. At this point the animal is in danger of pathology and is in distress. Pathologic changes include not only disease, but such things as decreased reproduction, abnormal behaviors, or failure to grow. (See table 2 pg 69 for indicators of animal well being. Deviations from "normal" characteristics may indicate distress) The goals is to recognize distress and deal with it before the animal reaches a pathologic state. Nonpain distress There are 2 general categories: stressors associated with experimental manipulation and stressors resulting from routine husbandry practices. The most reasonable strategy for measuring stress would be to monitor the responses of the four major defense systems (behavior, autonomic nervous, neuroendocrine and immune systems). None have proved to be a reliable measure of stress. No one system response to all stressors, and there is intraanimal variations in responses to the same stressors. Measuring glucocorticoids is a popular way to determine stress levels, however not all animals show increases in glucocorticoids when stressed. Animals may show the same level of "stress hormones when mating as when restrained or exercised. Intraanimal variations in the stress response may be due to early experience, genetics, age, and physiologic state. Stress induced by sampling procedures (i.e. blood collection, heart rate and blood pressure monitoring) may make it difficult to differentiate the source of the stress. Samples via canula or telemetric methods may reduce this problem, but are not always practical. Behavior may be the most unobtrusive method of detecting distress. However our lack of understanding of animal behavior as it relates to stress in limiting. In absence of simple, definitive measures of distress our best approach is to use our intuition and sensitivity to reduce potential distress in laboratory colonies. By being sensitive to the potential biologic costs of individual stressors it is possible to develop husbandry and experimental protocols that ameliorate distress even in the absence of definitive measures of stress or distress.
1. Why is pain "good"?
2. What is the difference between stress and distress?
3. What are the four general areas of response to pain?
4. When should an investigator assume that a procedure is painful?
5. Beside experimental manipulation, what is the other major stressor in laboratory animals?
T or F
6. High glucocorticoid levels are the best indicator we have that an animal is in distress.
7. Our own intuition and sensitivity to the potential distress experienced my laboratory animals may be the best approach to reducing problems.
8. Animals will acclimate to husbandry practices that occur at the same time every day and will not show signs of stress.
9. Since changing cages causes stress is best to do it only once a week.
10. Both peripheral and central nervous system sensitization very likely contribute to increased pain sensitivity after peripheral injury
1. Pain serves a normal protective function to warn of impending danger.
2. Stress occurs as the biological response an animal exhibits to cope with a threat to it's homeostasis. Stress does not become distress until those changes alter normal biological function and the possibility of pathology exists.
3. Behavioral, autonomic nervous system, neuroendocrine system, or immune.
4. "unless the contrary is established, investigators should consider that procedures that cause pain or distress in human beings may cause pain or distress in animals. (IRAC, 1995)
5. Husbandry practices.
6. False. Not all stressor elicit a hypothalmic-pituitary-adrenal response, so an animal could be in distress without increased levels. There is often comparable HPA response to both potentially threatening and non-threatening stimuli.
7.T
8. False. Duke et al; CT 40(1) 17-20. Showed SD rats showed an equal stress response to cage changes the 1st and 4 weeks of the study. Rats changed every 2 weeks showed a slightly higher response then those changed every week.
9. False. All aspects of stress must be considered when scheduling cage change frequency. An animal may be more stressed by high ammonia levels in the dirty cage than by the husbandry activity.
10. T

Defining the moribund condition as an experimental endpoint for animal research. ILAR 41 (2): 072.
In many research protocols involving animals, especially those protocols associated with a high mortality rate or severe disease states, the deciding point for determining when to euthanize an animal is when it becomes "moribund". This article describes how to define that term to achieve better data collection, increased compliance by the investigative group, and promote animal well-being.
Moribund is the term often used to describe the state of an animal who is severely debilitated and close to death. This subjective definition may differ from one individual or study to the next. By only vaguely defining moribund, many animals may die or become comatose before they can be euthanized. If the criteria for euthanasia were clearly defined before a study began, not only would animal suffering decrease, but the research group would have a greater chance to collect data. The definition of moribund may change with the species involved and the nature of the study. The ultimate goal of defining the term moribund is to predict death with high probability and accuracy. An example given in this article is a study of rats with leptomenigeal tumors using hind limb paralysis as an endpoint instead of death. This allowed the group to collect tissues for histopathology to document the extent of the tumor in all animals, with minimal animal pain and distress, and without any complicating factors due to dying or death.
Predicting the moribund state may be easier in an animal population than in a human one due to the homogeneity of a research group and standardized experimental procedures. Some common predictors include: hypothermia, inability to rise or ambulate, weight loss, biochemical variables, or experimental variables (e.g. EEG, hind limb paralysis). This article concludes by stating:
Objective data-bases approaches to predicting imminent death developed for specific experimental models could facilitate the implementation of timely euthanasia before the onset of clinically overt signs of moribundity and could thereby reduce pain and distress experienced by experimental animals.
Questions:
1. List 3 benefits that occur when the term "moribund" is defined for a specific research protocol.
2. Give 3 objective indicators that may be used in determining moribund.
Answers:
1. Increased quantity and quality of data collected, increased compliance by the research group in euthanizing animals, and increased animal well-being.
2. Hypothermia, weight loss, biochemical changes in the serum.

A Systematic Approach for Establishing Humane Endpoints. ILAR 41 (2): 080.
This paper addresses assessing pain and distress in animals via using score sheets to decrease observer variablity and interpretation. The scoring scheme proposed is an improved version of previously reported systems. This newer scheme allows the observer to record only the presence or absence of specific clinical signs. Additionally this scheme addresses husbandry and scientific needs and provides explicit guidance on scoring clinical signs. This article presents guidelines for developing an assessment system tailored to a given experiment that will be applicable to all animals undergoing a particular procedure within that experiment as well as across different laboratories performing the same experiment.
Because it is not possible to have a generalized list of all clinical signs that will occur in all experiments to all species, score sheets must be specifically tailored to each scientific procedure. Additionally, score sheets may have to be specific for each species or strain undergoing a procedure. Pilot studies are useful in refining the procedure and species specific score sheets.
The score sheets list the cardinal clinical signs that are observable and measurable, and the key clinical signs that are identified through the experience of the animal caretakers and researchers. The clinical signs may be measurable (eg weight) or qualitative (eg hunched posture), however all qualitative signs are recorded on the score sheet as present (+), absent (-), or +/-. By convention, negative signs indicate the animal's normal state, positive signs indicate observations outside the normal range. Thus, by scanning a score sheet, the animal's general well-being can be assessed: The more plus signs, the more the animal has deviated from normality, and it is inferred that the animal is experiencing more pain and distess. The score sheets allow for multiple measurements over time, thus an animal's condition may be followed. Animals should be scored during critical periods, such as postoperatively, at the end of an infection incubation period, or at the predicted time of graft failure.
When filling out the score sheet, a standard routine is advised. Everyone who uses the score sheet must follow the same routine to decrease variablity. Whenever possible, the animal must first be viewed at a distance, then its response assessed as its cage or pen is opened, then the animal is thoroughly examined. Because the full sheet takes some time to fill out, a short cut is provided for the event where the animal is entirely normal. There is a "Nothing Abnormal Detected" (NAD) box to check.
The score sheet also provides guidance notes on husbandry, how to record qualitative signs, and criteria for implementing humane endpoints. Instructions are also provided for what to do when an animal must be euthanized, such as what tissues need to be collected and how to preserve those tissues.
The paper gives an example score sheet for reference. That score sheet will not be reproduced here.

The score sheet system advantages:
--Closer observation of animals during critical times in the experiment
--Subjective assessments of pain and distress avoided
--Consistency of scoring is increased because the guidance is clear and the scoring options are limited
--Single signs or combinations of signs can be used to indicate overall severity of the procedure
--Single signs or combinations of signs can be used to indicate scientific sampling points during an experiment (eg blood sampling).
--Score sheets help determine the effectiveness of any therapy intended to relieve adverse effects
--Score sheets can be used to determine which experimental modes cause least pain and distress
--Score sheets help in training those inexperienced in assessment of adverse effects
--Score sheets can be used to retrospectively analyze adverse effects and level of severity
--Score sheets add to the scientific value of an experiment as more careful and consistent observation of animals is carried out.
--Signs of poor animal well-being can be identified early, thus humane endpoints can be reached sooner
Questions:
1. Under the proposed scoring system, an animal with a large number of "+" signs on its score sheet is:
a) Exhibiting a large number of abnormal signs and is likely distressed
b) In normal condition
c) Responding as expected to the experiment and no further assessment is needed.
2. The "NAD" box in the proposed scoring system is:
a) An unfortunate acronym when dealing with people with 8th grade humor levels
b) Used to indicate that no abnormalities are detected in the animal under observation
c) Useful as a time-saving device so the entire score sheet isn't filled out when the animal is healthy
d) All of the above
3. T or F: A single type of score sheet can be constructed that will cover all species or strains under all experiments.
Answers:
1. a
2. d
3. F

Humane Endpoints and Cancer Research. ILAR 41 (2): 087.
This article discusses how to develop humane endpoints for cancer research studies. A variety of experimental tumor systems have been developed using laboratory rodents as hosts to study cancer biology and treatments. However, laboratory animals may experience significant adverse affects from experimental cancers and/or treatment regimes. The routine or intentional use of either death or significant pain/distress as an experimental endpoint should be discouraged.
A useful starting point for development of endpoints is to seek the least traumatic techniques feasible that will allow the scientific questions to be answered. Since scientific and humane endpoints are linked, they should be developed concurrently. Clearly defined experimental objectives are essential in this process. The experimental objective will determine the scientific approach, tumor system, animal model, and group size.
Tumor growth or excision assays should replace survival endpoints. Examples of this include tumor growth delay, tumor doubling time (Td), tumor regression and clonogenic survival of tumor cells (in vivo, in other animals or in vitro- examples of excision assays). The potential problems with tumor growth studies include tumors getting too large, tumor associated disease, and death. Detailed knowledge of the proposed model, particularly the nature of an adverse effects on the animal, is crucial to establishing humane endpoints. Pilot experiments, using a small number of animals, can help determine the response to an novel experimental challenge and help develop humane endpoints for future studies.
Experimental tumor systems in animals are characterized by short latent period and rapid tumor growth. It should be possible to predict the timing of tumor development and associated disease. However, tumor cell lines may be genetically unstable and subject to constant selection in the host or laboratory. Serial transplantation of a tumor line is associated with reduced latent period, increase in tumor incidence, increased malignancy, and changes in sensitivity to therapeutic agents. Therefore, experimental tumor propagation models should be followed to yield reliable and reproducible results.
Animals that have developed tumors should be monitored frequently. UKCCCR (United Kingdom Coordinating Committee on Cancer Research) guidelines suggest limiting solid tumor size to a maximum of 10% of host's body weight. This may be difficult if the tumor is growing while the host is losing weight. Measuring tumor growth with calipers is a more reliable way to monitot tumor growth. Tissue necrosis or skin ulceration may result in loss of body weight and/or infection and will alter the growth pattern of the tumor.
Tumors growing internally are more difficult to monitor. There is increasing interest in growing tumors orthotopically, in the tissue or site of origin. Methods to detect or profile these types of tumors in live animals include serial termination of animals, investigative surgery, diagnostic imaging and palpation for tumor development. Surrogate markers of tumor development may include consistent weight loss and behavioral changes. All tumor bearing animals should be watched for signs of cachexia, and weight loss should be limited to 25% of starting body weight.
Developing scoring systems for cancer studies focuses attention on the animals condition, and can lead to refinements of humane endpoints, but may not provide a reliable prognosis of long term survival. Continuing weight loss, emaciation, dehydration, anorexia, hypothermia and leathery, if all present together, indicate a life threatening situation. Other signs associated with sick animals (piloerection, hunched posture), do not indicate the seriousness of the animals illness.
Perturbed hematalogic, physiologic, metabolic or biochemical values may precede the onset of clinical signs and may provide more objective and reproducible experimental and humane endpoints than clinical observations alone. Laboratory assays utilizing small quantities of body fluids or tissues, and telemetry now make it possible to monitor metabolic and physiologic values in rodents.
Questions:
1. What does UKCCCR stand for?
2. When a tumor is transplanted orthotopically, what is the location of the tumor?
3. What changes in tumor biology are associated with serial transplatation of tumor cells?
Answers
1. United Kingdom Coordinating Committee on Cancer Research
2. The tumor is transplanted into the site of orgin. For example, a hepatic tumor would be transplanted to the liver.
3. Reduced latent period, increase in tumor incidence, reduced tumor doubling time, increased malignancy.

Humane endpoints for genetically engineered animal models. ILAR 41 (2): 094.
Introduction: Before transgenic (tg) technology, methods for modifying animal genomes included spontaneous and
induced mutagenesis, hybridization, and selective breeding. Advocates of tg technology assert that tg models are more specific and
reliable than spontaneous disease models. Opponents believe that tg technology is wasteful due to large #s of animals (donors,
recipients, breeder, non-tg offspring) which are euthanized and don't produce usable data. There is concern that genetically engineered
animals may experience significant pain and distress due to genetic manipulations. Genetically engineered animals often have decreased
disease resistance, increased tumor production, or compromised basic bodily functions. Genetic engineering involves DNA insertion,
deletion, or alteration. Methodologies include (1) pronuclear microinjection producing gene overstatement; (2) targeted mutagenesis in
embryonic stem cells to "knock out" genes; and (3) introduction of genetic material via various vectors. Establishing humane endpoints
for genetically engineered animals can present challenges, depending on the phenotype. The unpredictability of genetic manipulations
and the variety of possible phenotypes further complicate the situation.
IACUC Review of Genetic Engineering Studies: In the US, the IACUC has animal use oversight. USDA has published regulations
that currently apply only to studies involving species other than rats, mice, and birds. The Guide for the Care and Use of Laboratory
Animals applies to all vertebrate animals. Guidelines for Research Involving Recombinant DNA provides direction for oversight of
genetic engineering experiments. Most studies in which the animal's genome is altered by the stable introduction of recombinant DNA
into the germline require approval by both the institutional biosafety committee and the IACUC.
Unanticipated Outcomes: A significant problem in establishing humane endpoints for genetic engineering studies is the occurrence of
unanticipated adverse outcomes. In order for the IACUC and the PI to make informed decisions regarding genetically engineered
animals, there must be ongoing monitoring of the progeny and of succeeding generations. In some lines, problems may not become
apparent until the gene of interest is homozygous. Sometimes an unanticipated outcome may produce a useful model for a different
application. Full characterization of genetically altered animals may require continued breeding of compromised animals. In principle
IX of the US Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training,
it is stated that such decisions for continued breeding should not rest with the PIs but with a review group (i.e. IACUC). However, the
PI must describe how new conditions involving pain and distress will be handled, including appropriate and humane endpoints.
Protocol Oversight: Welfare issues can be readily anticipated in well-defined tg and knockout animals. In incompletely characterized
animals, the PI may be asked to predict outcomes based on what is known about the gene(s) of interest or related genes. The IACUC
can use surveillance of ongoing studies to ensure adequate oversight of welfare considerations. Such surveillance can be accomplished
by using a morbidity and mortality reporting program with feedback to the IACUC proceeding through the attending veterinarian. As
problems are identified, IACUC and veterinary personnel can meet with PIs to devise strategies to improve welfare or establish
endpoints. Rereview of animal use protocols is required annually by the USDA and every 3 years by the PHS policy. It seems prudent
to require PIs to return to the IACUC for approval of continued breeding of each genetically altered line as soon as the phenotype is
known. The IACUC can then review the phenotyping data and grant or withhold approval for the continued breeding of the line.
Whether to allow continued breeding of a line with well-being problems may be more important than establishing humane endpoints for
individual animals. In some cases, embryo or embryonic stem cell cyropreservation may be a useful alternative to continued breeding of
animals with problems. Consistency of phenotypic statement within a particular line can be helpful in establishing endpoints based on
age. Treatment of a condition that compromises the welfare of the animals can also be a useful solution. Another strategy to potentially
improve welfare is to maintain the line by breeding phenotypically normal heterozygotes. However, this system may require a larger #
of animals, depending on the # of breeders necessary to perpetuate the line and whether the wild type offspring produced could be
used. Background strain is an important consideration in phenotypic statement of a gene. Classic example is the diabetic (db) mutation
in mice. On a C57BL/6J background, db/db mice are obese and have insulin resistance but do not develop diabetes. On the closely
related C57BL/KsJ background, mice are severely diabetic. Such background differences could be used to maintain a valuable mutant
allele without producing the severely debilitating phenotype. The age of mice at the time a gene is expressed is another factor that can
vary according to strain background. Inducible promoters can be used to regulate gene statement, enabling limitation of gene effects to
a particular time period and allowing control over severity of statement of the induced phenotype.
Phenotyping Protocols: IACUC members can request or require submission of phenotype data to conduct its review of a particular
line. Table 2 on p. 97 gives a sample phenotyping protocol. Some unique safety issues the IACUC should consider when reviewing
protocols involving breeding and housing animals with altered germlines include (1) proper animal containment to preclude inadvertent
sexual contact with other animals and to prevent escape; (2) excess genetically altered animals should not be used as food source for
other animals; (3) minimization of theoretical danger of horizontal transfer of viruses and genetic material to other animals when
replication-deficient viruses are used for a vector; and (4) potential danger to humans from accidental needle sticks. Successful
resolution of welfare concerns may be achieved when the PI, attending veterinarian, and IACUC cooperate.
1. This definition of an "animal" is from which document? "Any live, vertebrate animal used or intended for use in research, research
training, experimentation, or biological testing or for related purposes."
a. Animal Welfare Act
b. The "Guide"
c. PHS policy
d. All of the above
2. Regarding IACUC membership, to which regulation can this membership composition be attributed: Attending DVM, at least one
practicing scientist, and at least one public member.
a. Animal Welfare Act
b. The "Guide"
c. PHS policy
d. All of the above
3. Which of the following committees must give approval in most studies involving an animal with a genome altered by the stable
introduction of recombinant DNA into the germline?
a. IACUC
b. Environmental health and safety committee
c. Institutional biosafety committee
d. Radiation safety committee
e. A and B
f. A and C
4. Rereview of animal use protocols is required by the USDA and every by the PHS policy.
a. annually, annually
b. annually, 3 years
c. 3 years, annually
d. 3 years, 3 years
e. biannually, annually
1. c. PHS policy; 2. b. The "Guide"; 3. f. A and C; 4. b. annually, 3 years

Humane endpoints for infectious disease animal models. ILAR 41 (2): 099.
The Canadian Council on Animal Care (CCAC) guidelines states that the earliest endpoint compatible with the scientific
research objectives should be chosen in order to minimize any actual/potential pain, distress, or discomfort to experimental animals.
The same principle also applies to infectious disease research. The process of establishing more humane endpoints in infectious disease
research must involve the scientist, laboratory animal veterinarian, IACUC, and technical staff. Animals used in infectious disease
research may experience significant pain and/or distress as part of the disease process. Seeking earlier, more humane endpoints can
reduce potential pain and distress, but these earlier endpoints should be based on scientifically valid values of variables and accurately
predict experimental outcome. Activation of immune system-mediated defense mechanisms is called the acute phase response (Fig 1,
p. 100). This response involves a complex cascade of biochemical, endocrinologic, physiologic, behavioral, and pathologic changes.
Early responses include increases in cytokines and acute phase proteins (APP), while behavioral and physiologic changes occur later.
Biochemical changes can also serve as infectious disease indicators and as predictors of the severity and outcome of disease.
Initial bodily response to infectious agents is usually local and involves neutrophils, macrophages, and other immune cells. Response
also includes cytokine release from macrophages and neutrophils. Cytokines act as intercellular messengers to coordinate the
development and regulation of the immune response. Cytokines produce a # of rapidly occurring systemic effects if infection becomes
generalized or prolonged (i.e. liver - APP production and release, brain and nervous system - fever, lethargy/sleep, inappetence,
hyperalgesia). Behavioral effects produced by cytokines acting in brain (lethargy, sleep, inappetence/anorexia) are termed "sickness
behavior." Deviations from normal physiologic and behavioral states can be evaluated through observations/checklists in order to
establish endpoints that are predictive of eventual experimental outcome. Use of immunodeficient or genetically modified rodents
presents additional problems such as (1) signs of disease differing from "normal" rodents, and (2) isolator housing making frequent,
thorough observations of these rodents more difficult. Telemetry may help reduce some of these problems while decreasing the risk of
contamination.
Some specific endpoints in infectious disease animal models discussed included body temperature (BT) change, weight loss, and
other behavioral/physiological changes. Initial BT response to infection is fever but this may be transient. In specific disease/toxic states
(sepsis), lowered BT can be an indicator of animal deterioration. Body weight can be monitored by a # of scoring systems. Total
amount of weight loss, as well as duration and consistency, should be used to determine endpoints. Changes in activity level and
alertness as well as secondary signs should be monitored. A condition/observational scoring checklist should be developed with a
veterinarian for each infectious disease model since models can vary in the # of observations needed to determine endpoints.
The transient nature of elevated cytokine levels and the rapid production of binding and regulatory factors during infection limit the
use of cytokines as a means to determine endpoints. However, the consequences of increased cytokines such as increased APP levels
may be more practical to use in setting earlier endpoints. In normal healthy animals, APP are barely detectable but rise rapidly in
plasma during the acute phase response. Based on the magnitude of the increase, APP are grouped as major, moderate, or minor.
The specific APP to be measured depends on the species and the type of infection/inflammatory response being studied (Table 1, p.
102). For example, the major APP in mouse is serum amyloid A, whereas in rat it is alpha2-macroglobulin. High major APP levels
correlate well with the presence and severity of infectious disease and can be used for diagnosis/prognosis as well as predictors of
experimental outcome. Research is still needed to determine a specific APP level beyond which an animal never recovers as well as to
develop standardized, rapid APP tests. Haptoglobin levels in a bovine respiratory disease model (Pasteurella haemolytica) was
discussed as an example of how an APP useful for diagnostic, prognostic, and treatment evaluation can also serve as an early indicator
for determining an earlier, more humane endpoint in an infectious disease model. Haptoglobin in cattle increases in response to
experimental inflammation, many clinical diseases, surgery, and stress. Methods for measuring serum haptoglobin levels include
hemoglobin binding assay, radial immunodiffusion assay (RIA), fluorometric competitive immunoassay, and ELISA.
1. Acronym quiz - What do the following acronyms stand for? CCAC, IACUC, APP, ACLAD, NIAID, ELISA
2. Which of the following IS NOT an APP?
a. Haptoglobin
b. Serum albumin
c. Serum amyloid A
d. C-reactive protein
e. Fibrinogen
3. Which of the following IS NOT a cytokine?
a. Interleukin-1 (IL-1)
b. Interleukin-6 (IL-6)
c. Alpha1 acid glycoprotein (AGP)
d. Tumor necrosis factor (TNF)
e. All of the above are cytokines
4. Serum amyloid A (SAA) IS NOT a major APP in which of the following species?
a. Dog
b. Cow
c. Mouse
d. Rat
e. Horse
f. Human
5. Which of the following IS NOT a method for measuring serum haptoglobin levels?
a. Hemoglobin binding assay
b. Hemagglutination inhibition assay
c. Radial immunodiffusion assay
d. Fluorometric competitive immunoassay
e. ELISA
1. CCAC = Canadian Council on Animal Care, IACUC = Institutional Animal Care and Use Committee, APP = acute
phase proteins, ACLAD = American Committee on Laboratory Animal Diseases, NIAID = National Institute of Allergy and Infectious
Disease, ELISA = enzyme-linked immunosorbent assay.
2. b. Serum albumin.
3. c. Alpha1 acid glycoprotein (it is an APP).
4. d. Rat (FYI - pig also does not produce SAA).
5. b. Hemagglutination inhibition (HAI) assay.

Refinement of vaccine potency testing with the use of humane endpoints. ILAR 41 (2): 105.
This article covers the use of humane endpoints to replace lethality in vaccine potency testing.
The first half of the article covers a review of regulatory policies defining procedures for vaccine quality control testing and how the use of humane endpoints can minimize pain and distress in animals by replacing lethality as the endpoint.
For vaccine quality control testing, animals are used either for safety testing or potency testing. Potency testing of inactivated vaccines involves immunization and challenge procedures or modifications of this procedure such as quantitative multidilution tests, qualitative single dilution tests, and individual protection test where serum samples of immunized animals are titrated on in vivo neutralization test. Each of these tests generally requires large numbers of animals, up to 100. Regulatory agency documents such as the European Parmacopoeia specify lethal endpoints for some vaccines such as pertussis, tetanus, diphtheria, rabies, leptospira, and clostridial vaccines. Death is specified as the endpoint because the goal of vaccine potency testing is to demonstrate that a vaccine will protect against a lethal dose infection. In addition, death is an objective parameter that is easy to standardize and requires little training for personnel to monitor. However, some of these vaccines, such as tetanus and diphtheria allow the possibility of using clinical signs as endpoint.
Some regulatory bodies, such as the WHO and USDA, have written guidelines for humane treatment and care of research animals with recommendations to minimize pain and distress. However, based on the wording of the recommendations, validation of the predictive power of humane endpoints will be needed before the regulatory bodies will accepted.
Applying the principles of the 3Rs to vaccine potency research, there are some in vitro testing methods described for some vaccines such as rabies and leptospira. These methods include tests based on the physiochemical and immunochemical characterizations of the vaccine batches and tests that focus on specific immunologic parameters such as cytokine responses as an indication of the vaccine's immunogenicity or to study its functional characteristics. However, since most inactivated vaccines still require immunization challenge testing to determine the biologic activity of new batches, reduction and refinement appear to be the best methods for improving animal welfare. Refinement includes use of humane endpoints which is defined as the point at which an experimental animal's pain and/or distress can be terminated, minimized, or reduced by actions such as euthanasia, termination of the procedure causing pain/distress, or providing treatment to relieve the pain/distress. One approach that can be considered a humane endpoints for vaccine potency testing includes replacing the challenge procedure after immunization with an in vitro serologic test to assess antibody responses after immunization. This method is being validated for the toxoid vaccines. Another advantage to this approach is the potential reduction in animal numbers since more data per animal is obtained. Another approach is to use the clearance rate of microorganisms after challenge as an outcome for evaluating the potency of a vaccine. Measurement of hematologic parameters have also been proposed. Use of clinical signs as criteria for euthanasia has been emphasized. However, there is reluctance to use this as a recommendation in regulatory documents due to objectivity, cost, and lack of information on the extent to which a clinical sign is a prognostic indicator of death. If euthanasia can't be used as an humane endpoint, then provision of supportive therapy and analgesia might be considered.
The second half of the article describes a strategy for establishing humane endpoints and a case study which applies this to potency testing for the whole cell pertussis vaccine. The study evaluated body weight, temperature, and clinical signs as humane end point alternative to death. The clinical signs evaluated included reduced activity, lack of grooming, ruffled fur, crouching, loss of appetite and reduced weight, dehydration, sunken eyes, apathy, loss of locomotor control, and finally convulsions leading to death. The study found that weight loss was not a good criterion to use as a humane endpoint as many animals died before substantial weight loss was seen and some animals that had marked weight loss survived. A body temperature of <34.5 degree C was a good criterion to use as a humane endpoint as it was a good predictor of the lethal progress of pertussis infection. The study also found that the clinical sign that was an accurate criterion for humane endpoint was loss of coordination with almost all animals dying within 1-3 days of onset of incoordination. Euthanasia at the onset of this clinical sign can substantially reduce distress to the animals.
The authors have chosen to validate only the selected clinical sign of loss of muscle coordination for the whole cell pertussis vaccines. They chose not to validate body temperature as an endpoint due to the cost associated with transponder technology necessary to utilize this method as an alternative endpoint.
Questions:
1) Animals are used in vaccine quality control testing for
a- Humane endpoint testing
b- Safety testing
c- Toxicity testing
d- Taste testing
2) T or F
Refinement includes use of humane endpoints which is defined as the point at which an experimental animal's pain and/or distress can be terminated, minimized, or reduced by actions such as euthanasia, termination of the procedure causing pain/distress, or providing treatment to relieve the pain/distress.
3) T or F
Marked weight loss, a decrease in body temperature to <34.5 C, and loss of motor coordination are good criteria for humane endpoint alternatives to lethality in pertussis vaccine potency testing.
Answers:
1) b
2) T
3) F

Humane endpoints and acute toxicity testing. ILAR 41 (2): 114.
Acute toxicity testing is conducte to determine the effects of a single exposure to a substance. Subchronic and chronic toxicity tesing is conducted to determine the effects of an exposure of an extended duration. These effects need to be identified so that practices can be established to prevent injury or regulatory disease.
The first topic discussed was acute lethal toxicity testing. The goals of this testing include: defining the degree of hazard, determining susceptible population, identifying target organs/systems, developing risk evaluations, and providing information to clinicians to predict, diagnose, and/or provide treatment for acute exposures.
LD50 is one of the most infamous acute lethality test. The Organization for Economic Cooperation and Development (OECD) has established four methods: (1) acute oral toxicity [moribund condition], (2) fixed dose procedure [toxic signs, estimated within a dose range from toxic effects], (3) up and down procedure [moribund condition], and (4) acute toxic class [moribund condition]. In all methods, fewer animals are needed and more humane endpoints are employed.
Acute dermal irritation testing is another acute lethality test. Up to four compounds can be tested on one rabbit, three rabbits are tested. Alternatives include use of hairless guinea pig or fuzzy rats (replace lower on phylogenetic scale); measurement of changes in cutaneous blood flow (using Doppler flowmetery), Corrositex [collagen-based biolayer with liquid chemical color change detection system], Episkin and epiderm [tissue culture-based alternative], and transcutaneous electrical resistance test.
Occular irritation testing (Draize testing) is another infamous acute lethality test. Approximately 0.1ml or 0.1 gram of material in placed in the conjunctival sac of one eye and scored for injury. Alternatives include reduction in number of animals used [use computer models to predict response as much as possible, look at dermal data, too] and low volume ocular irritation assays have been proposed. Organotropic models employ eye tissue from multiple animals, but a drawback is that the repair mechanisms are not available. Cell cytotoxicity, using chorioallantoic membrane (CAM)'s from embryonated chicken eggs.
Validation is the process by which reliability and relevance of the method are established. Existing guidance is meant to be flexible and change with complete or improved knowledge. Pilot studies can help with the identification of viable alternatives and to identify the earliest decision point for successful completion of an experiment (endpoints).
Humane killing and use of analgesics should always be considered to ensure animal welfare consideration. The IACUC must also remember that the principal investigator must ensure appropriate training for the study execution.
"The public is alert to any actions taken by scientists. All measures must be taken to assure the public that (1) their safety is being considered and (2) no animlas are suffering from needless pain, stress or distress in efforts to assure this safety." page 122.
1) What does OECD stand for?
2) List three alternatives to the use of an LD50.
3) List two alternatives to the use of the acute dermal irritation test.
4) List two alternatives to the use of the Draize
test.
1) Organization for Economic Cooperation and Development
2) Acute oral toxicity, fixed dose procedure, up and down procedure, acute toxic class
3) Fewer animals, Corrositex, Episkin and EpiDerm, transcutaneous electrical resistance test
4) Low dose ocular irritation assay; extrapolation of skin testing; In vitro assays; and CAM based assays.