Animal Welfare Information Center Newsletter, Spring 1995, Vol. 6 No. 1

Defining an Acceptable Endpoint in Invasive Experiments

by
Ernest D. Olfert, B.A., D.V.M., M.Sc., Director, Animal Resources Centre
University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 0W0

The following article is from an address presented at the annual conference of the Canadian Association for Laboratory Animal Science/ L'association Canadienne pour la Technologie des Animaux de Laboratoire (CALAS/ACTAL). The meeting was held in Montreal, Quebec, on June 15, 1993. It originally appeared in the October 1993 issue of the CALAS/ACTAL Newsletter (Vol. 27, #5).

Abstract

Animal Care Committees reviewing research protocols often demand an earlier endpoint to an experiment for humane reasons. Scientists support the efforts to reduce potential pain and suffering, but may be reluctant to adopt earlier endpoints unless these can be objectively determined and will not invalidate the experimental data being collected. In this presentation, a framework for objectively setting endpoints that may be scientifically supported will be presented. Crucial to this exercise is the involvement of all persons directly working with the animals, and their ability to observe the animals and make accurate assessments of their condition.

Introduction

We have an obligation to minimize the potential pain and suffering experienced by an animal in the course of biomedical research. That sentiment is explicitly stated in ethical position statements of a number of organizations concerned with experimental animal use. For example, the Canadian Council on Animal Care (CCAC) Ethics of Animal Investigation (5) document says:

"Animals must not be subjected to unnecessary pain or distress. The experimental design must offer them every practicable safeguard, whether in research, in teaching, or in testing procedures; ..."

Further, with respect to humane endpoints, the CCAC document says:

"An animal observed to be experiencing severe, unrelievable pain or discomfort should be immediately killed, using a method providing initial rapid unconsciousness."; and, " Studies such as toxicological and biological testing, cancer research and infectious disease investigation may, in the past, have required continuation until the death of the animal. However, in the face of distinct signs that such processes are causing irreversible pain or distress, alternative endpoints should be sought to satisfy both the requirements of the study and the needs of the animal."

One of the general ethical statements that has long been cited as guiding us to a more humane use of animals in research is the now famous 3Rs tenet of Russell and Burch (15)--Replacement (of animals with other, non-sentient material), Reduction (of numbers of animals used), and Refinement (of technique "to reduce to an absolute minimum the amount of distress imposed on those animals that are still used."). Their book-- The Principles of Humane Experimental Technique (15)--was a landmark publication, and still deserves to be consulted for its comments on the humane use of animals.

Although our ethical responsibilities seem quite clearly stated, these pronouncements do not answer the very important questions:

How do we choose the endpoint that satisfies these principles?
Where do we draw the line?
How do we "refine" our experiments through establishing earlier, more humane endpoints to invasive animal experiments, particularly those that may have death as an endpoint?

This presentation will attempt to draw a framework for selecting an endpoint that reduces the potential for animal pain and suffering, and that hopefully will satisfy the experimental design requirements for objective evaluation.

Types of Studies Where Death of the Animal May be the Endpoint

There are several types of studies where the death of the animal may be the endpoint as part of the experimental design. These would include: regulatory toxicology, diagnostic toxicology, acute toxicity studies in research, infectious disease studies, microorganism virulence challenge studies, vaccine efficacy trials, cancer research, cancer treatment evaluation, etc.

In some research investigations pain and suffering may unavoidably be part of the disease or condition being studied (e.g., some models of human diseases such as arthritis or cancer, and studies on pain, etc.).

Also, in some experimental animal uses, any pain and suffering is an unwelcome accompaniment to the animal use (e.g., monoclonal antibody production, Freund's adjuvant use in antibody production, etc.). In these latter cases humane endpoints are relatively easy to define (e.g., limiting the volume and number of times a mouse with ascites is collected), and guidelines pertaining to these procedures already exist (6).

In all of these research endeavors, our responsibilities include the prevention and minimization of any unnecessary pain and distress for the animals. As the CCAC noted in Ethics of Animal Investigation, in the past the death of the animal may have been the endpoint in some experiments. Although quite conservative in tone, this CCAC statement acknowledges that pain and suffering may occur well before the animal is moribund.

In fact, the animal in a moribund state may be past suffering (and actually comatose). The observations that suggest an animal is "moribund" are quite clear. Before the animal gets to the point of being "moribund," however, our best judgements, based on the accuracy of our observations of the animal, will help set the earlier endpoint and thereby reduce the potential pain and suffering the animal is experiencing. Thus it is incumbent on us to continually refine our skills at seeking earlier endpoints for such experiments.

A Framework for Selecting an Earlier Endpoint

There are several considerations in arriving at the objective assessment of pain and suffering, and translating that into the appropriate endpoint in a given experiment. Firstly, we must improve our skills at observing the animals and assigning some objective values to the observations we make (of animal behavior and physiology). Secondly, we need to know, in any given study, which observations are the most significant indicators of animal pain and suffering. Thirdly, we must have scientific acceptance of these measurements.

Objectively Assessing Signs and Symptoms

With respect to the first point, the work of Morton and Griffiths in 1985 can be considered a landmark publication. They presented a set of criteria for assessing pain, distress, and discomfort in laboratory animals based on evaluating five aspects of an animal's condition. Those five aspects are: a) changes in body weight (including levels of food and water intake); b) external appearance; c) measurable clinical signs (e.g., changes in heart rate, in respiratory rate and nature); d) unprovoked behaviour; and e) behavioural responses to external stimuli. In each of these categories, a rating of 0 (normal or mild) to 3 (severe changes from normal) is made, the cumulative rating indicating increasing deviation from the normal in the animal. The cumulative rating is interpreted as an indication of increasing pain, distress, and suffering. Table 1 presents this proposed scoring system in a checklist format.

Table 1. Qualifying Pain / Distress / Suffering

The British Association of Veterinary Teachers and Research Workers (AVTRW) (16) has developed a set of guidelines for the recognition and assessment of pain in animals to assist scientists in their compliance with the British Animals (Scientific Procedures) Act of 1985. Specific information on the behavioral and physiological changes in the various animal species that may indicate the presence of pain are published (16, 19). The report of a committee of the British Laboratory Animal Science Association (19) includes an assessment of the severity of a wide variety of procedures performed on animals in the course of biomedical research. A number of other publications are also available to help identify the signs and symptoms of experimental animal pain, distress, and suffering (1, 2, 3, 4, 9, 18).

The publications of Morton and Griffiths (13) and the British Association of Veterinary Teachers and Research Workers (16) focus on an important matter--that of trying to make more objective assessments of the pain, distress, and suffering that may occur in an animal in the course of biomedical research.

In addition to general signs of pain and distress, there are the specific signs and symptoms related to the condition being studied. For most animal models of disease, information on the organ system(s) affected, the specific symptoms, the progression of symptoms, the time course of the disease condition, and the expected lesions, is available from the general veterinary and laboratory animal science literature. Such specific signs and symptoms must also be used in the overall evaluation of the animal's condition on which selection of the endpoint will be based.

Identifying Significant Indicators of Pain and Suffering

The next problem is deciding, in any given animal model, which of the many possible observations and measurements are the most important or significant indicators of the condition of the animal, or perhaps more importantly from the scientist's perspective, which are indicators of an irreversible deteriorating condition of the animal. This is not an easy task, since a large number of different behavioral observations and physiological measurements are possible. Two papers dealing with the adjuvant-induced arthritis model in the rat provide insight into the difficulties in finding / choosing the right observations (4, 9).

To determine which behaviors in arthritic rats correlated with progression of the induced arthritis, Butler et al. (4) conducted detailed behavioral studies. This was done by measuring the frequency of a wide range of specific behaviors with the use of videotape computer analysis. The time arthritic rats spent performing 12 specific behaviours was measured. These behaviours include: rearing, sniffing, food-hoarding, grooming, scratching, freezing (arresting), resting, sleeping, running, climbing, eating and drinking. Although changes in the frequency of several behavior patterns were found (decreased rearing, running, eating, drinking and climbing; increased resting, freezing, scratching), the conclusion was that of all these changes in behavior, increased scratching was the most significant behavioral change that tied in with developing arthritis, indicating chronic pain.

Such behavioral evaluations (4, 9) are research projects in themselves, involving many hours of technical time, with expensive monitoring and analytical equipment. It may be unrealistic to demand a similar degree of preliminary behavioral evaluation each time an animal-based research program is begun where the potential for pain and distress are high (mice in a liver cancer research program, for example). Nevertheless, conducting a pilot study to establish the observational criteria to be used to set the endpoint may be a very useful exercise, particularly at the onset of a research program that may be ongoing.

Scoring of Significant Behavioral and Physiological Observations to Set Endpoints

As noted above, information on the general signs of pain, distress and suffering for the various animal species commonly used in biomedical research are readily available (1, 2, 3, 4, 9, 16, 18). Of these, significant weight loss may be one of the more important signs of deterioration in the animal's condition (reflecting a change in food and water consumption).

For some specific areas of biomedical research, particularly in cancer research, more detailed criteria for selecting the endpoint have been proposed by Montgomery (11, 12), Redgate, Deutsch and Boggs (14), and the United Kingdom Coordinating Committee on Cancer Research (UKCCCR) (20).

Table 2. Selected Clinical Observations Used in Cancer Research and Toxicological Studies
Table 3. Selected Criteria For Euthanasia of Moribund Animals

Table 2 presents some specific clinical abnormalities that are useful indicators in cancer research and toxicologic studies (11, 12). Table 3 presents some of the endpoint criteria, physiological, behavioral and pathological, that Montgomery (12) identified for euthanasia of moribund animals.

One of the scientific concerns about arbitrarily establishing an early endpoint, particularly in cancer therapy studies, is that early euthanasia may alter longevity or survival data which are an important indicator of "successful" treatment. For example, the "successful" treatment of cancer in a group of rats which resulted in them living a month longer, might be masked by early euthanasia based only on clinical observations. In such cases, finding the signs of disease and distress that point to an irreversible deterioration in the animal is important. Redgate, Deutsch and Boggs (14), in their examination of a brain tumor model in a rat (9L gliosarcoma in Fischer 344 rats), concluded that a weight-loss period of more than 6 days had a high correlation with irreversible progression to death, regardless of which treatment group was studied. In this model then, an endpoint that satisfied the scientific concerns could be established at the end of a 6-day period of consecutive weight loss (which in this case was about 10 days before death of the animals).

The UKCCCR Guidelines for the Welfare of Animals in Experimental Neoplasia (20) contain some general recommendations for endpoints, e.g., when tumor size exceeds 10 percent of body weight, or if weight loss exceeds 20 percent (these would be in the maximum score category in the Morton and Griffiths proposal, indicating severe negative effect on the animal).

Siems and Allen (17) recommended that the endpoint in a disease model (chronic infection with systemic Candida albicans) be set at (among other measurements) the point when the animals lose more than 20 percent of body weight, or when the body temperature drops more than 4^o C (both of which are easily monitored). The magnitude of these changes from normal would also give a maximum score on the Morton and Griffiths proposal, indicating severe negative effects on the animal.

Thus, there is a good body of information developing on selecting more humane endpoints based on clinical observations of the animals in a variety of biomedical research areas. The development and use of observational checklists for scoring the animal's condition in a study provides for an objective basis on which decisions about endpoints can be made. The advantages of checklists are the same here as for airline pilots; nothing is overlooked or taken for granted. The other real advantage is that such checklists help us to improve our observational capabilities, particularly with the smaller laboratory animals where some of the conventional clinical observations made on larger animals are not so useful (e.g., temperature, heart rate, respiratory rate).

Another matter that must be addressed is the frequency with which the observations should be made. It is generally accepted that normal healthy animals should be observed at least once a day (8). However, once an animal is in a potentially critical period with respect to impairment, more frequent observations must be demanded. But what is adequate? Montgomery (12) suggests that at least two observations daily should be made; more often during critical times. The sensitivity and judgement of the animal care committee review process will help determine what is acceptable.

The Role of the Institution's Animal Care Committee

The role of the animal care committee is vital in establishing the structure that will ensure earlier endpoints are used. With respect to setting and determining humane endpoints, each individual's responsibilities should be clearly defined, and a clear chain of consultation established. This is particularly important for dealing with unanticipated negative effects on the animals in an invasive study.

Some of the questions that might help a protocol review committee ensure that an acceptable, humane endpoint will be in place include:

What are the scientific justifications for using death or "moribund" as the proposed endpoint?
What is the expected time course for the animals, from initial treatment to first signs of pain/distress to the death of the animal, based on previous information with the specific model under study?
When are the effects to the animal expected to be the most severe?
Has a "checklist of observations," on which the endpoint will be based, been established?
If the course of the disease and expected signs of the negative effects are unknown, could an initial (pilot) study, under close observation by the laboratory animal veterinary staff, answer these questions?
Who will monitor the animals (identify all responsible)?
What will be the frequency of animal observations: a) during the course of the study, and b) during critical times for the animals?
Do the animal care and technical staff have the training and expertise to monitor the animals adequately?
What provisions have been made to deal with any animals that show unexpectedly severe signs and symptoms?

Summary

All of us involved in biomedical research, from the scientist and the animal care staff, to the laboratory animal veterinarians and the animal care committees, have responsibilities for the humane care and use of experimental animals. In establishing humane endpoints, the institutional animal care committee should ensure that acceptable criteria are used by the principal investigator to determine the endpoint. Through the use of observational checklists and animal condition scoring systems, objective, humane endpoints can be identified. Responsibilities for observing and monitoring the animal's condition must be clearly delineated. Persons involved in establishing and effecting humane endpoints in invasive experiments are encouraged to present and publish this data to support our efforts at continually refining the animal use practices that occur in biomedical research.

References

American Veterinary Medical Association (1987). Colloquium on recognition and alleviation of animal pain and distress. Journal of the American Veterinary Medical Association 191(10): 1184-1296.
Barclay, R.J., W.J. Herbert, and T.B. Poole, eds. (1988). The disturbance index: a behavioural method of assessing the severity of common laboratory procedures on rodents. UFAW Animal Welfare Research Report No. 2. Universities Federation for Animal Welfare, 8 Hamilton Close, South Mimms, Potters Bar, Hertfordshire EN6 3QD, England, 35 pp.
British Veterinary Association (1985). Proceedings of a symposium: the detection and relief of pain in animals. BVA, Animal Welfare Foundation, 7 Mansfield Street, London WlM OAT England, 101 pp.
Butler, S.H., J. Weil-Fugazza, F. Godefroy, and J-M. Besson (1985). Reduction of arthritis and pain behaviour following chronic administration of amitriptyline or imipramine in rats with adjuvant-induced arthritis. Pain 23: 159-175.
CCAC (1989). Ethics of Animal Investigation. Canadian Council on Animal Care, Constitution Square, Tower II, 315-350 Albert Street, Ottawa, Ontario, CANADA K1R 1B1.
CCAC (1991). CCAC Guidelines on Acceptable Immunological Procedures. Canadian Council on Animal Care, Constitution Square, Tower II, 315-350 Albert Street, Ottawa, Ontario, CANADA K1R 1B1.
CCAC (1991). Categories of Invasiveness in Animal Experiments. Canadian Council on Animal Care, Constitution Square, Tower II, 315-350 Albert Street, Ottawa, Ontario, CANADA K1R 1B1.
CCAC (1993). Guide to the Care and Use of Experimental Animals Volume I (2nd Edition). Canadian Council on Animal Care, Constitution Square, Tower II, 315-350 Albert Street, Ottawa, Ontario, CANADA K1R 1B1.
De Castro Costa, M., P. De Sutter, J. Gybels, and J. Van Hees (1981). Adjuvant-induced arthritis in rats: A possible model of chronic pain. Pain 10: 173-185.
Keefe, F.J., R.B. Fillingim, and D.A. Williams (1991). Behavioral assessment of pain: nonverbal measures in animals and humans. ILAR News 33(1-2): 3-13.
Montgomery, C.A. Jr. (1987). Control of animal pain and distress in cancer and toxicological research. Journal of the American Veterinary Medical Association 191(10): 1277-1281.
Montgomery, C.A. Jr. (1990). Oncological and toxicological research: alleviation and control of pain and distress in laboratory animals. Cancer Bulletin 42(4): 230-237.
Morton, D.B. and P.H.M. Griffiths (1985). Guidelines on the recognition of pain and discomfort in experimental animals and an hypothesis for assessment. Veterinary Record 116: 431-436.
Redgate, E.S., M. Deutsch, and S.S. Boggs (1991). Time of death of CNS tumor-bearing rats can be reliably predicted by body weight-loss patterns. Laboratory Animal Science 41(3): 269-273.
Russell, W.M.S. and R.L. Burch (1959). The Principles of Humane Experimental Technique. Methuen: London, 238 pp. (Reprinted in a Special Edition, 1992, by Universities Federation for Animal Welfare, 8 Hamilton Close, South Mimms, Potters Bar, Herts EN6 3QD, England.)
Sanford, J., R. Ewbank, V. Molony, W.D. Tavernor, and O. Uvarov (1986). Guidelines for the recognition and assessment of pain in animals. Veterinary Record 118: 334-338.
Siems, J.J. and S.D. Allen (1989). Early euthanasia as an alternative to death in chronic infectious disease studies using a systemic Candida albicans model. Abstracts of the 89th Annual Meeting of the American Society for Microbiology, 1989
Soma, L.R. (1987). Assessment of animal pain in experimental animals. Laboratory Animal Science 37: 71-74.
Wallace, J., J. Sanford, M.W. Smith, and K.V. Spencer (1990). The assessment and control of the severity of scientific procedures on laboratory animals. Lab Animal 29: 97-130.
Workman, P., A. Balmain, J.A. Hickman, et al. (1988). UKCCCR guidelines for the welfare of animals in experimental neoplasia. Lab Animal 22: 195-201.

This article appeared in the Animal Welfare Information Center Newsletter, Volume 6, Number 1, Spring 1995

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