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ILAR Journal V41(2) 2000
Humane Endpoints for Animals Used in Biomedical Research and Testing
David B. Morton
| David B. Morton, Ph.D., B.V.Sc., M.R.C.V.S., F.I.Biol., is Head of the Centre for Biomedical Ethics, Division of Primary Care, Public and Occupational Health, School of Medicine, University of Birmingham, Edgbaston, Birmingham, United Kingdom. |
Assessing pain in animals, which is similar to assessing pain in human babies, relies on the observations of care staff and owners. However, one of the notable drawbacks in both humans and animals is observer variability (in animal studies, e.g., Beynen et al. 1987; Holton et al. 1998) as well as the interpretation of observations in terms of whether an animal is distressed. Clinical signs (by definition, those observable and not reported by a patient) potentially provide an objective, rather than a subjective, judgment of animal pain and distress. However, for such signs to be effective, observers must have good powers of observation, knowledge of what is normal for that individual animal and strain and species, and good clinical skills. Ideally, they should also have an empathetic attitude. ("There is none so blind as those who will not see.") The best strategy for assessment is to involve all those in animal research work because each will bring a different focus to bear: caretakers "know" their animals, veterinarians possess clinical skills, and researchers know the potential side effects of their scientific procedures.
Several systems have been used for assessing pain in humans, but they all rely on some form of "patient reporting,'' which is obviously not possible with animals. Such schemes include the following: visual analogue scales (the observer indicates by a mark on a line which indicates normal to severe pain), numerical rating (same as visual analogue scales but using a scale from 0-10), and simple description (e.g., no pain, mild-moderate-substantial-severe pain, and distress). Systems of scoring clinical signs for seriousness of ill health (which often involve signs of pain and distress) have been described for humans to help mothers know when to call the doctor (Bouwmeester et al. 1999; Morley et al. 1991) and for assessing pain and distress in animals (Baumans et al. 1994; Morton and Griffiths 1985). More recently, the scoring scheme for animals has been considerably strengthened by being made more rigorous and with less room for observer interpretation as observers are able to record only the presence or absence of specified clinical signs (Morton 1990, 1995, 1997a,b, 1998a,b, 1999). In addition, the newer scheme addresses husbandry and scientific needs and provides explicit guidance on scoring the clinical signs.
In animal research, unlike clinical practice, healthy animals have standardized scientific procedures carried out on them. Therefore, any assessment system should be broadly applicable to all animals undergoing that procedure, as well as across different laboratories. Moreover, some animal protocols involve repetitive scientific procedures and so become very amenable to the use of scoring systems and particularly to the introduction of humane endpoints (e.g., vaccine potency testing). In such cases, the clinical signs are very reproducible from one experiment to another, and it is possible to develop a standard score sheet. It may also be possible to validate early clinical signs that indicate impending or predictable death, or severe pain or severe distress, regardless of the experiment. On other occasions, the input of the researcher can be essential to determine the earliest stage when the scientific objective has been achieved, or when the animal has become physiologically or psychologically altered such that it is no longer useful scientifically. In fact, the use of score sheets has sometimes enhanced the science by providing information that would otherwise have been unrecorded (see below).
Adverse effects experienced by animals during research are more than just pain; they include other conscious emotions like fear, discomfort, dystress (original spelling for stress under which an animal fails to thrive or cope [Morton 1998c]), and mental distress (e.g., frustration, boredom, separation anxiety). However, before any of these states can be alleviated or assessed, or experiments refined in any way, one first must be able to recognize when the welfare of animals is being adversely affected. Animal well-being, defined as the welfare of an individual animal, is usually affected negatively; and it is therefore important to recognize states of normal well-being. Like many laboratories, we use clinical signs as a way to determine the degree to which an animal's physiology and mental state have deviated from normal, and we then use the magnitude of these perturbations for an assessment of severity. This approach is applicable not only to mammals but also to other vertebrates and even nonvertebrates, provided there is a good knowledge of their normal ethology and physiology.
Score Sheet System
It is not possible to have a generalized list of all clinical signs that will occur in all experiments (e.g., the predictable side effects for a failed skin graft differ significantly from the clinical effects from a failed kidney or heterotopic heart graft). Therefore, score sheets must be prepared specifically for each scientific procedure. Moreover, as clinical signs can vary between species and sometimes even strains, or the method of recording can differ, the score sheets may have to be specific for each of those characteristics----experiment, species, and strain. The use of pilot studies as recommended by the Canadian Council on Animal Care (CCAC 1998) cannot be overemphasized.
The score sheets list the cardinal clinical signs that are observable and measurable, and the key clinical signs are identified through the experience of those involved in the research. The animal caretakers are crucial in this identification because they are most likely to know when an animal is "not right" and will pick up changes in behavior, posture, appearance, or even the feel or smell of an animal. The veterinarian should be skilled in identifying objective clinical signs and should have a knowledge of the biology of the species, including the range of its relevant behavioral and physiologic responses (Kuijpers and Walvoort 1991; Morton and Townsend, 1995; Schlede et al. 1992, 1999; van den Heuvel et al. 1990). The scientists should be conversant with the perturbations that might be expected during an experiment due to the scientific paradigm. All of these factors taken as a whole will be important guides in the assessment of the effects of a scientific procedure on an animal. By detailing the cardinal signs of any particular protocol and regularly observing animals at critical periods during the experiment, an objective assessment of animal well-being can be made throughout the experimental period.
Types of clinical signs and conditions that may be observed vary from those that are measurable (parametric or analogue) to those that are more qualitative in nature (non-parametric or binary signs). However, both types are crucially important for the assessment of well-being. For example, body weight can be measured, whereas the appearance of an animal with a ruffled coat or hunched posture may simply have to be recorded as present or absent. It may be possible to score some qualitative signs such as lameness more precisely with some form of grading: Animals may be unable to bear weight, show an obvious limp through partial weight bearing, or exhibit barely discernible lameness.
Lists of clinical signs are developed by very closely observing the first few animals undergoing a new scientific procedure. The list is modified with experience until a set of signs is established that most animals will show during that experiment and that are relevant to the assessment of pain and distress. These cardinal clinical signs are set out against time in the score sheet (an example is given in Table 1). Crucially, all clinical signs have to be described in a way that reduces the variation in observer interpretation and recorded only as present (+), absent (-), or "+/-" if an observer is unsure). The convention is that negative signs indicate normality (i.e., within the normal range), and positive signs indicate that the animal is outside the normal range. In this way, it is possible to scan a score sheet to gain an overall impression of animal well-being: The more plus signs, the more an animal has deviated from normality with the inference that it is experiencing more pain and distress than before. Animals should be scored during critical periods when they could predictably give rise to concern (e.g., in the immediate postoperative period; in a study on infection after the incubation period; and at the predicted time of tumor growth or organ graft failure).
Practically, it is important to develop a disciplined strategy to recognize adverse effects in animals. Whenever possible at the beginning of an assessment, the animal should be viewed from a distance and its natural undisturbed behavior and its appearance noted. This observation avoids disturbing an animal to any great extent. Next, as the observer approaches the pen or removes the cage lid, the animal will inevitably begin to interact with the observer, who can determine whether that interaction is normal or abnormal. Finally, a detailed clinical examination can be carried out by handling and restraining the animal in some way, observing its appearance carefully, and making any relevant clinical measurements (e.g., bodyweight and temperature) in addition to noting any change in its behavior (it may have become more aggressive or fearful, or even vocalize). Some habituation of animals to such examinations may be necessary to avoid misinterpretation of their responses.
At the bottom of the score sheet are guidance notes for animal caretakers and veterinary technicians regarding what they should provide in terms of husbandry and care for animals undergoing the particular scientific procedure. There are also guidelines on how to record qualitative clinical signs (e.g., lameness, diarrhea, and respiration) as well as criteria by which to implement humane endpoints (see Hendriksen and Morton 1999). Finally, for cases in which an animal must be killed, instructions are provided regarding what other actions should be taken, such as tissues to be retrieved or placed in a mixture of 10% formaldehyde in saline; these instructions help ensure that the maximum information is always obtained from any animal in a study.
Although these score sheets take time to complete, it is not difficult for an experienced person to assess whether an animal is normal, in which case the Nothing Abnormal Detected ("NAD") box is simply checked. However, if an animal is not normal, the completed score sheet facilitates the evaluation and decisions regarding additional action.
Animal Care Coordinator
To promote continuity of good care, staff in the University of Birmingham research animal facility allocate a senior animal technician/caretaker or veterinary technician to be responsible for liaising with scientists and other technical staff and maintaining and updating the score sheets. The roles of the Animal Care Coordinator are as follows:
- to check that the appropriate licenses and approved protocols are in order and to cross-check them with what the scientist actually intends to do with the animal(s) that day;
- to check the score sheet before an experiment begins;
- to know the purpose of the experiment and the scientific objectives and to become familiar with the scientific procedures to be carried out on the animals and the clinical signs that may occur;
- to ensure that all personnel (caretakers, technicians, scientists, veterinarians) know how to use score sheets, can recognize the clinical signs, and can interpret the signs clearly into scientific and humane endpoints;
- to verify that caretakers and technicians not familiar with that scientific procedure (e.g., doing a weekend or holiday rotation) are informed about the animals;
- to liaise with scientists over the experiment (e.g., timing, numbers of animals, equipment, endpoints);
- to update the score sheets and endpoints based on new signs or combinations of signs observed;
- to report to the responsible persons (e.g., the veterinarian or principal investigator) any concerns over the animals or personnel involved.
Interpreting the Score Sheet: Example 1
A sample score sheet developed to record clinical signs for rats with streptozotocin-induced diabetes is provided in Table 1. It can be seen from this sample score sheet that there are more plus signs on the righthand side. Several other points should be noted: As the animal started to show clinical signs, it was scored more frequently. During day 0 (the day of the injection of streptozotocin to induce diabetes), the animal lost body weight due to the restricted food intake the previous night. Thereafter it recovered well and so the NAD box was checked. Over the next 2 days, it lost body weight although it was normal in all other respects. By the next day (4), the coat had become starey (ruffled), the body temperature had dropped significantly, and the breathing had become more rapid and labored. Furthermore, there was a significant body weight loss (22%)--a strong indication that the animal had not eaten or drank much or that it was not maintaining its fluid balance; and tip-toe walking indicated some degree of abdominal pain. Thus, the rapid weight loss and dehydration, labored breathing, abnormal posture and movement, lack of a red light response (this test is carried out by turning out the normal white lights and observing the animal in the dark or under a red light, when it will carry out its nocturnal patterns of behavior characterized by an increase in activities such as investigation, climbing, and play within 5 min), among other signs, all confirmed that the animal was becoming severely physiologically compromised and was not going to yield valid results in relation to the scientific objective. Even more significantly, its temperature was now at 32°C--a very poor sign. The animal was given fluids, placed in a warm environment, and observed 3 hr later. It was not responding adequately, and in our experience from following such animals through to death in earlier studies, this animal would have died that night if not sooner. Consequently, we decided to kill the animal on humane as well as on scientific grounds before the end of the experiment. Moreover, in the United Kingdom, where an ethical balance is struck between the anticipated benefits of a research project and the degree of animal distress (as indicated by the humane endpoints), the severity limit had been exceeded. Even if the animal might not have died, the level of pain and distress was agreed to be a sufficient reason to kill the animal on humane grounds alone.
Rabies Vaccine Potency Test: Example 2
In Table 2 is a second example of the regulatory requirement for a mouse lethality test with a 14-day survival period to be performed on each batch of rabies vaccine (Cussler et al. 1998). In these experiments, a surrogate endpoint was established for death (Hendriksen and Morton 1999). Animals were closely observed during three vaccine potency tests, when the clinical signs were noted. These clinical signs were then analyzed for a single sign or a combination of signs that could be reliably used to indicate that death was an inevitable outcome, that is, a surrogate endpoint. Although variable in terms of time of onset (depending on vaccine dilution and viral dose), the clinical signs were relatively predictable in that their sequence and duration were nearly always the same (although the incubation period varied between 7.7 and 13 days). Combinations of possible clinical signs were given numerical codes for ease of reference: 1 = ruffled fur and hunched back; 2 = slow and circular movements; 3 = trembling and shaky movements and convulsions; 4 = paresis and paralysis; 5 = prostration and permanent recumbency (leading to dehydration, starvation, and death). Body weight loss was also helpful as an early indicator of the adverse effects; in fact, it decreased before clinical signs were observed. Surprisingly, in some animals, weight loss reached 30 to 50% shortly before death. It was also found that body temperature (measured by transponders) always decreased; however, a significant change was observed only in the terminal stages of infection and so, unlike other studies (Acred et al. 1994; Soothill et al. 1992), body temperature was not a useful early discriminating parameter. It was important that staff observing the animals could reliably score their condition. Independent studies showed that accurate scores could be made with a minimum of training. Finally, to discriminate between faulty intracerebral injection technique (challenge route) and "true" rabies infection, clinical signs were used as data for the potency test only after 48 hr.
Twenty-seven of the 38 mice that lived had no observable clinical signs (Table 2). Ten of the surviving mice showed general clinical signs of malaise (code 1), and so those signs could not be used as a surrogate endpoint. Of the 59 mice that died, a spectrum of the five score codes was observed. Clinical signs in code 2 were appropriate to use as a surrogate endpoint inasmuch as all mice except one showing those signs progressed to death. Because 10 mice are required for each test group, this result would not have changed the decision for the test group. However, body weight measurement could have been used to predict correctly the outcome of the one false-positive mouse that showed signs of code 2. Body weight in mice showing code 2 was always marked, with an average of 14% being lost; but in this one mouse, it did not exceed 10%. Consequently, by recording its body weight as less than 10%, it could have been eliminated as a false code 2 positive.
From these data, and taking into account the overall test design and objective, a surrogate endpoint of slow circular movements (or above if that stage was passed through too quickly to have been observed) could be successfully used as a humane endpoint for the rabies vaccine potency test. Finally, from the data, it can be calculated that the use of this surrogate endpoint would shorten the experiment an average of 77.7 hr for each animal that eventually died (Cussler et al. 1998).
Advantages of the Score Sheet System
The score sheet system has been shown to have the following advantages:
- There is closer observation of animals by all involved at critical times in the experiment inasmuch as the score sheets indicate the times when animals find their circumstances most aversive.
- Subjective assessments of pain and distress by staff and scientists are avoided, thereby promoting more fruitful dialogue, as evidence-based opinion becomes possible based on the clinical signs. In a sense, they empower the care staff and scientists by helping them illustrate to less experienced persons why an animal is "not right."
- Consistency of scoring is increased because the guidance is clear and the scoring options are limited.
- Single signs or combination of signs can be used to indicate overall severity of the procedure.
- Single signs or combination of signs can be used to indicate scientific sampling points during an experiment (e.g., blood sampling).
- The score sheets help determine the effectiveness of any therapy intended to relieve adverse effects.
- The score sheets can be used to determine which experimental models cause least pain and distress (e.g., by comparing alternative animal models), thus helping to refine scientific procedures.
- The score sheets help in training those inexperienced in the assessment of adverse effects or in that particular scientific procedure.
- The score sheets can be used to analyze retrospectively the adverse effects of any scientific procedure and its level of severity.
- The score sheets have been found to add to the scientific value of an experiment as a more careful observation of animals is carded out.
In the United Kingdom, where severity limits are imposed on every scientific procedure, the sheet can be used to indicate when such limits have been breached, are about to be breached (see Table 1 where the humane endpoint had been set at 20% body weight loss), or may have to be reviewed, by the precise observation of the clinical signs.
The scoring system has proved to be especially useful with new procedures or when users are not always sure of what effects a procedure will have. In my experience, the literature rarely records adverse effects on the animals, or how to avoid or measure them, and scientists have a moral obligation to do so (Morton 1992). We now look more closely at ways of improving our monitoring of animals. For example, our system of perioperative care requires that we attempt to operate early in the day so animals have maximum time under close observation and can be given more support such as fluid therapy or special diets (e.g., jelly, fruit, vegetables). This has proven to save animals' lives as well as well as improve the speed of recovery, although on rare occasions it could theoretically interfere with the experiment. This change in diet is unlikely to be more of a confounding factor than an animal becoming dehydrated and physiologically perturbed as it tries to regain its homeostasis.
References
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Table 1 Streptozotocin-induced diabetes in Wistar rats
| RAT No: 3 | DATE ON STUDY: 5/9/99 | ISSUE No: 8978 |
| ON-STUDY WEIGHT: 214 | PRE-ON-STUDY WEIGHT: 219 |
| DATE | 10-May | 11-May | 12-May | 13-May | 14-May | 14-May |
| DAY | 0 | 1 | 2 | 3 | 4 | 4 |
| TIME | 8:40 | 9:00 | 8:50 | 8:55 | 8:05 | 11:00 |
| FROM A DISTANCE | ||||||
| Fed mash/jelly/grain | Y | Y | Y | Y | Y | Y |
| Inactive | - | - | - | + | + | |
| Isolated | - | - | - | - | - | |
| Walking on tiptoe | - | - | - | + | + | |
| Hunched posture | - | - | - | + | + | |
| Pinched face | - | - | - | + | + | |
| Starey coat | - | - | - | + | +/- | |
| Type of breathing | N | N | N | 120 L | 70L | |
| Negative red light response | nd# | nd | nd | nd | + | + |
| ON HANDLING | ||||||
| Not inquisitive and alert | - | - | - | + | + | |
| Not eating | - | - | - | +/- | + | |
| Not drinking | - | - | - | ? | + | |
| Vocalization on gentle palpation | - | - | - | - | - | |
| Volume water drunk (average of rats in cage) mL | 50 | 113 | 133 | 140 av | 0 | |
| Body weight (g) | 204 | 209 | 203 | 192 | 170 | 168 |
| % change from prestarved weight | 7 | 5 | 7 | 12 | 22 | |
| Body temperature (°C) | 37.5 | 37.4 | 37.6 | 32.4 | 34.7 | |
| Pale or sunken eyes | - | - | - | + | + | |
| Dehydration | - | - | - | + | + | |
| Distended abdomen/swollen | - | - | - | +/- | +/- | |
| ** Diarrhea 0 to 3 (+m or +b) | - | - | - | - | - | |
| Cage wet | - | +/- | + | + | - | |
| Condition score 4 to 1* | 4 | 4 | 3 | 2+ | 2 | |
| Saline given s/c - volume/sites? | - | - | - | 2 mL/x2 | - | |
| Blood sugar level | nd | nd | nd | nd | nd | |
| Nothing abnormal detected | - | - | - | - | - | |
| OTHER | Day 0 streptozotocin @65 mg/kg | Animal killed | ||||
| SIGNATURE: | KL | KL | KL | KL | ||
Special Husbandry Requirements
Feed irradiated diet and adapt animals to it 2-3 days before diabetes induction.
Animals should be cleaned out twice daily.
Two bottles of UV water should be provided for each cage and filled twice daily.
Deprivation of water over night may be sufficient to cause death by dehydration.
Scoring Details
* Breathing: R = rapid; S = shallow; L = labored; N = normal
**0 = normal; 1 = loose feces on floor; 2 = pools of feces on floor; 3 = running out on handling +m = mucus; b = blood
Refer to condition chart: 4: normal to 1: emaciated
#nd = not determined
Humane Endpoints and Actions
1. Any animal showing signs of coma within the first 24-48 hr will be killed.
2. Any amimals weighing less than the starting weight after 7 days will be killed, or losing more than 20% than start weight at any time will be killed.
3. Any animal showing tiptoe or slow ponderous gait will be killed.
4. Inform veterinarian and principal investigator if more than one clinical sign occurs.
Scientific Measures
Tissues to be kept--Kidney into a mixture of 10% formaldehyde in saline.
Table 2 Incidence of clinical signs shown by mice during three rabies vaccine potency testsa
Mice that died during the test (N = 59)
| Clinical score | 1 | 2 | 3 | 4 | 5 | |
| No. of mice showing those clinical signs | 25 | 49 | 37 | 50 | 42 |
Mice that lived for the test period (N = 38)
| Clinical score | 1 | 2 | 3 | 4 | 5 | |
| No. of mice showing those clinical signs | 10 | 1 | 0 | 0 | 0 |
aCombinations of possible clinical signs were given numerical codes for ease of reference: 1 = ruffled fur and hunched back; 2 = slow and circular movements; 3 = trembling and shaky movements and convulsions; 4 = paresis and paralysis; 5 = prostration and permanent recumbency (leading to dehydration, starvation, and death).
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