1 .1 . | Before assessing the severity of any regulated procedure on the well-being of an animal, it is essential that the observer is familiar with the normally accepted behaviour, anatomy, physiology and environmental requirements of the species used, for example, growth rate, dietary intake and microbial status. |
l .2. | Particular attention should be paid to those body systems most likely to be affected by the procedure. With solid tumours this will include ulceration, distension of covering tissues and cachexia. In the case of ascitic tumours, abdominal distension, anaemia and cachexia will be important. Lymphatic involvement from lymphoma and neurological disturbance from intracerebral tumours are examples of special complications arising in specific situations. |
1 .3. | Certain deviations from normal well-being may be difficult to observe, for example, induction of anaemia or the development of metastases, and special investigations may be required to detect them. |
1.4. | Appropriate control animals should always be included, so that the individual effects of the tumour and of any treatment can be distinguished . |
2. I . | Due consideration should be given to the known biology of the tumour. For spontaneous and transplanted tumours, important features will include growth rate, invasion, distension, ulceration, metastases, site, and production of cachectic factors. |
2.2. | In the case of tumours induced by carcinogens, viruses or genetic manipulation, factors such as method of induction may affect the nature and location of resulting tumours. |
2.3. | Contamination of tumour cell lines with viruses and other microorganisms may compromise experimental results, as well as causing an outbreak of disease among laboratory animals. Screening of cell lines for rodent viruses is strongly recommended. For example, Sendai virus is often used to induce cell fusion in vitro and is pathogenic to mice and rats. A potential hazard exists for research workers from immune-compromised animals receiving human tumour xenografts which may be contaminated with human pathogens. In such cases, special facilities should be considered for both tissue preparation and animal containment (e.g. flexible film isolators) (see UKCCCR Guidelines for the Xenografting of Human Tumours, 1980). |
3.1. | Considerable care should be given to the judicious choice of end point for tumour growth. This should take into account predictable indications of pain, distress or significant deviation from normal behaviour. Unless specified otherwise on the Project Licence, animals should be killed before:
(i) | predictable death occurs; |
(ii) | they get into poor condition; |
(iii) | the tumour mass becomes over-large, likely to ulcerate or unacceptably limits normal behaviour. |
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3.2. | In the case of local solid tumours, the required information on response to therapy may be obtained by tumour regrowth delay or clonogenic assay, rather than by tumour weight at a given time. Difficulties may arise with this last method because optimum shrinkage of treated tumours may not be achieved before control tumours become excessively large and/or distressing to the host animal. Where such an assay has to be used, the tumour burden should be regulated as indicated in section 3 . 1 . |
3.3 | The choice of site for solid tumours also requires considerable care, and particular attention should be given to avoidance of sites involving the special senses or where the capacity for tumour growth is limited. Subcutaneous or intradermal growth on the back or in the flank are considered to cause the least distress, while tumours implarited in the footpad, tail, brain and eye will require much greater justification. Distension of musculature is generally painful and this should be considered with intramuscular implants. Extra attention must be paid if multiple sites are used. |
3.4 | The survival end point should be avoided wherever possible, and its use will require special justification. Where this end point has to be used for animals with ascites or disseminated tumours, particular care should be taken. It is, in general, unacceptable to wait for predictable death. Animals expected shortly to become moribund should be killed, unless specified otherwise in the Project Licence. |
3.5 | Difficulties may occur where the effects of anticancer agents on tumour growth are being evaluated. The high toxicity of these agents may combine with the adverse effects of the tumour, but this might be justified by the prospect of a therapeutic remission (as occurs in man). Thus, the outcome of such experiments may be uncertain, and uncritical culling would frustrate the purpose of the study. However, where the outcome can be reasonably predicted, animals about to become moribund should be killed. |
3.6 | No precise quantitative guide can be given as to the acceptable upper limit of tumour burden, since the adverse effects on the host will depend on the biology of the tumour, the site and mode of growth, and the nature of associated treatments. However, tumour burden should not usually exceed 10% of the host animal's normal body weight. It should be emphasized, however, that problems may arise with much smaller tumour burdens. |
3.7 | With ascitic tumours, including hybridomas, care should be taken to ensure that the volume of ascitic fluid does not become excessive, causing gross abdominal distension, and that solid deposits and cachexia are not allowed to become clinically significant. Ascitic volumes should not usually exceed 20% of normal body weight in mice and rats. Retired breeders are advantageous for monoclonal antibody production, since their abdominal musculature more readily allows larger ascites volumes to be tolerated without discomfort. Ascitic tumours should normally be drained only once. This will minimize the development of solid tumour deposits, bleeding into the peritoneal cavity and cachexia, as well as reducing the risk of introducing an infection. General anaesthesia should always be employed. |
3.8 | In tumour therapy experiments with adult rodents, it is recommended that weight loss should not normally exceed 20% of the host body weight at the commencement of the experiment. For younger animals, failure to maintain the weight gain seen in untreated control animals should be considered as an indication of toxicity. |
3.9 | Care should be taken that general housing conditions are appropriate to the known or anticipated condition of the tumour-bearing animal, for example, in terms of appropriate bedding, cage structure and accessibility of food and water. |
3.10 | Humane end points and other procedures should be refined in the light of experience. |
4. I . | The frequency with which animals must be inspected for signs of pain or distress and the extent of each examination will be dictated by: (i) the known biology of the tumour and/or the effects of the inducing agent ; (ii) the effects of any associated techniques; (iii) the changing clinical status of the animal . |
4.2. | Rapidly growing or invasive tumours will require more frequent attention, and greater care will be required as the tumour burden increases . |
4.3. | As a minimum, every tumour-bearing animal should be inspected daily and additional more detailed examinations undertaken as appropriate. The frequency of the latter should be increased during critical periods where the potential for animal suffering may be anticipated. The experimental design should ensure that these do not occur when staff are absent. Particular attention should be given to animals in poor health. |
4.4. | Appropriate assessment techniques will include: evaluation of overall clinical condition, including appearance, posture, body temperature, behaviour and physiological responses; assessment of food and water intake; weighing to determine changes in body weight (both positive and negative changes compared to controls can be associated with increasing tumour burden); caliper measurements to determine tumour volume or mass; and inspection and palpation to locate the sites of tumour growth, as well as to assess distension, ulceration and compromised mobility. |
4.5. | Other special examination techniques will be more valuable for specific sites, e.g. breathing rate for lung deposits, neurological disturbances for brain neoplasms, and blood cell counts for leukaemias. Laparotomy or endoscopy may be appro-priate in some instances. Estimation of circulating tumour marker substances may also be of value. Autopsy of animals may expose adverse effects undetected by external examinations. |
5.1. | Researchers are strongly urged, for each tumour model in use in their laboratory, to document the expected behaviour of the tumour and host animal under various experimental conditions, including therapy. They should also document humane end points to limit severity with regard to acute and delayed toxicity and maximal tumour burden, and indicate any particular problems which may be encountered in the use of each model. The appropriate response to such problems should be described and the chain of consultation and responsibility clearly defined. Consideration should be given to the inclusion of a numerical scoring system to facilitate decision-making, e.g. when to contact senior staff or to kill an animal. The guidelines for specific tumour models should be readily available to and agreed between all research and animal husbandry staff involved with that model. Particular care should be taken that all procedures are understood by junior and occasional staff. Researchers are also encouraged to share this information with other groups using the same system, for example when providing a tumour cell line to another laboratory. |
5.2. | Researchers are encouraged to publish improvements in humane end points in appropriate journals, so as to ensure wide dissemination of the information. |
5.3. | Encouragement is given to incorporate animal welfare statements into experimental protocols, and in addition to report compliance with these and other appropriate guidelines (including any local ones) when publishing results. Certain journals require this (e.g. British Journal of Radiology, Cancer Research, and Journal of the National Cancer Institute) . |