GUIDELINES FOR TUMOR INOCULATION IN RODENTS

STUDY CONSIDERATIONS

The following should be considered when developing a protocol using tumors in vivo:

1. Tumor kinetics – a detailed knowledge of the biology of the tumor model aids in the selection of appropriate humane endpoints
2. Viral status – cell lines or tumor tissues may be contaminated with viruses which may serve as a source of infection for the animals in the study, as well as other colonies in the facility. Viral infections may confound research results. (Ref: Transplantable Tumor SOP)
3. Solid tumor inoculation – solid tumors should be minced into fine pieces or dispersed prior to transplantation to minimize trauma to the animal. For mice, transplantation of tumor fragments less than 1mm is preferred. Larger fragments may require anesthesia and a surgical procedure to implant.
4. Transplantation site – whenever possible the tumor should be placed such that it can grow with minimal impact on the animal’s ability to ambulate and perform normal bodily functions.

ENDPOINTS IN TUMOR STUDIES

All animal care and use proposals involving tumorigenesis must include criteria that establish the clinical endpoints at which animals are to be euthanized. Tumors may occur spontaneously, or be experimentally induced through transplantation or administration of carcinogens.

1. Notify Laboratory Animal Services each and every time a tumor study is started.

2. Comply with the Use of Transplantable Tumors and Cell Lines in Animals SOP.

3. The following information must be recorded on the Transplantable Tumor Tracking cage card provided by LAS:

Date of injection Animal weight on day of injection

Cell line Number of cells injected

Site of Injection

General criteria for consideration of euthanasia:

1. Maximum tumor size of 10% of body weight at day of injection, calculated using the formula:

Mass (mg) =Tumor volume (mm³) =d²x D/2 where d and D are the shortest and longest diameter in mm, respectively

2. Mean tumor diameter exceeding 20mm in mice, 40mm in rats (Mean = (d + D)/2)
3. Ulceration of tumor regardless of size
4. Where the tumor is interfering with movement or function of vital organs. When an animal is seen to be in distress, such as labored breathing, it should be euthanized regardless of tumor size
5. Animal is unable to eat or drink

Animals in which tumors are growing should be checked at least three times weekly, at intervals no greater than three days apart, by the PI or designee.

In the case of rapidly growing tumors or situations where the progress of clinical signs is likely to be rapid, animals should be checked daily. Animals will be evaluated in consultation with veterinarians from Laboratory Animal Services when the tumor size approaches 10% of body weight, or any of the above criteria are observed. Any time a veterinarian determines that an animal is experiencing pain or distress, he or she has the authority to require euthanasia of that animal.

NIH Recommendations

Guidelines for Endpoints in Animal Study Proposals

Introduction

Experimental studies may involve procedures that cause clinical symptoms or morbidity in animals. The Animal Care and Use Committee must consider the selection of the most appropriate endpoint(s). This requires careful consideration of the scientific requirements of the study, the expected and possible adverse effects the research animals may experience (pain, distress, illness, etc.), the most likely time course and progression of those adverse effects, and the earliest most predictive indicators of present or impending adverse effects. The effective use of endpoints requires that properly qualified individuals perform both general and study-specific observations of the research animals at appropriate time points. Optimally, studies are terminated when animals begin to exhibit clinical signs of disease if this endpoint is compatible with meeting the research objectives. Such endpoints are preferable to death or moribundity since they minimize pain and distress. Efforts must be made to minimize pain and distress experienced by animals used in research.

Morbidity

Animal Study Proposals that include morbidity as an endpoint or include animal procedures that have the potential to cause adverse sequellae should address the following:

1. Criteria that establish when the endpoint has been reached.

a. There are several examples in the literature that might be considered, including:

1) Evaluation of five aspects of an animal's condition as described by Morton and Griffiths. These are body weight, physical appearance, measurable clinical signs, unprovoked behavior and response to external stimuli.

2) Clinical observations used in cancer research and toxicological studies as described by Montgomery. Parameters include changes in general appearance, skin and hair, eyes, nose, mouth and head, respiration, urine, feces and locomotion (Table 1).

3) Body condition scoring as described by Ullman-Culleré and Foltz.

b. The clinical signs, depending on severity and duration, that may constitute an endpoint include, but are not limited to:

Rapid weight loss.

Diarrhea, if debilitating.

Progressive dermatitis.

Rough hair coat, hunched posture, lethargy or persistent recumbency.

Coughing, labored breathing, nasal discharge.

Jaundice and/or anemia.

Neurological signs.

Bleeding from any orifice.

Self-induced trauma.

Any condition interfering with eating or drinking (e.g. difficulty with ambulation).

Excessive or prolonged hyperthermia or hypothermia.

c. Additional signs in neoplasia studies that may constitute an endpoint include, but are not limited to:

1) A tumor burden greater than 10% body weight, and in an adult mouse, a mean tumor diameter exceeding 20 mm or in an adult rat, a mean tumor diameter exceeding 40 mm. Formulas for calculating tumor size can be found in the literature (see tumor size references).

2) Tumors that ulcerate, become necrotic or infected.

d. Any animal found unexpectedly to be moribund, cachectic, or unable to obtain food or water.

2. A plan for monitoring the animals both before and after a change in any of the above aspects, providing care if appropriate, and increasing the level of monitoring.
Monitoring or clinical care on weekends and holidays may require involvement of the investigative staff to supplement that provided by the animal care and veterinary staff.

3. Identification of personnel responsible for evaluation, record keeping, notification of the investigator and/or veterinarian and persons responsible for euthanasia.
Checklists or score sheets may be helpful in ensuring appropriate observations are made, consistently interpreted, and properly documented.

Death or Moribundity

While it is preferable to use the earliest endpoints compatible with the scientific

requirements of each study, there are studies that require moribundity or mortality as an endpoint. The moribund condition is defined as a clinically irreversible condition leading inevitably to death. In these studies, animals are permitted to die or become moribund, as a result of experimental procedures. In some cases, pain relieving measures are not used because such measures may compromise the experimental integrity of the study.

Examples of research proposals that may have death or moribundity as an endpoint include: infectious disease studies, drug and toxicity studies, and cancer research. The following guidelines are suggested to assist the Animal Care and Use Committees in reviewing proposals with death or moribundity as endpoints.

Animal Study Proposals utilizing death or moribundity as an endpoint should contain the following information:

1. The scientific rationale for death or moribundity as an endpoint, including:

a. What alternatives were considered, why morbidity as an endpoint cannot be used, and how alternatives will be used whenever possible.

b. Why pain relieving measures cannot be utilized.

c. Number of animals to be used and why this is the minimal number of animals required.

d. Whether animals will be euthanized when moribund and if not, what information is to be gained in the interval between moribundity and death.

2. A plan for the following animal care and monitoring procedures:

a. Animals involved in experiments that may lead to moribundity or death will be monitored daily by personnel experienced in recognizing signs of morbidity (illness, injury, or abnormal behavior) for at least the following: abnormal posture, rough hair coat, head tucked into abdomen, exudate around eyes and/ or nose, skin lesions, or abnormal breathing, difficulty with ambulation, decreased food or water intake, or self mutilation.

b. The frequency of observation will be increased when animals exhibit the above or other signs of moribundity. Monitoring on weekends and holidays may require involvement of the investigative staff to supplement that provided by the animal care and veterinary staff. Designated personnel, including a veterinarian, should be notified as soon as animals show signs of disease. An assessment of the animals' condition should be made as soon as possible and a plan of action established.

c. Consideration will be given to moving animals to individual cages when their condition deteriorates to the point that injury from other animals is likely. Dead animals must be promptly removed.

d. Written records will be kept of monitoring.

General endpoint references:

1. Alternatives to Animal Testing on the Web (2004), Humane Endpoints Database. (http://apps1.jhsph.edu/altweb/humane/) Johns Hopkins Center for Alternatives to Animal Testing. Baltimore.

2. Canadian Council on Animal Care (1998), Guidelines on: Choosing an appropriate endpoint in experiments using animals for research, teaching and testing. Ottawa, Canada.

3. Hendriksen CFM and Morton DB, ed. (1998), Humane Endpoints in Animal Experiments for Biomedical Research. Proceedings of the International Conference, 22-25 November 1998, Zeist, The Netherlands. Laboratory Animals Ltd, by Royal Society of Medicine Press Limited, London, England.

4. Institute for Laboratory Animal Research Journal (2000), Humane Endpoints for Animals Used in Biomedical Research and Testing. 41: No. 2

5. Montgomery CA (1990), Oncological and toxicological research: Alleviation and control of pain and distress in laboratory animals. Cancer Bulletin 42:230-237.

6. Morton DB and Griffiths PHM (1985), Guidelines on the recognition of pain, distress and discomfort in experimental animals and an hypothesis for assessment. Veterinary Record 16:431-43.

7. OECD Guidance Document on the Recognition, Assessment, and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation (2000)
http://www.olis.oecd.org/olis/2000doc.nsf/4f7adc214b91a685c12569fa005d0ee7/c125692700623b74c12569bb005aa3d5/$FILE/00087372.pdf

8. Stokes WS (1999), Humane Endpoints in Animal Experiments for Laboratory Animals Used in Toxicity Testing Proceedings of the 3rd World Congress on Alternatives and Animal use in the Life Sciences, 31 August - 2 September 1999, Toth (1997), The moribund state as an experimental endpoint. Contemp Top Lab Anim Sc 36:44-48.

10. Ullman-Culleré MH and Foltz CJ (1999), Body condition scoring: a rapid and accurate method for assessing health status of mice. Lab Anim Sc 49:319-323.

11. United Kingdom Co-ordinating Committee on Cancer Research (1997), UKCCCR Guidelines for the Welfare of Animals in Experimental Neoplasia, 2nd ed. London, England.

Tumor size references:

1. Bullard DE, Schold SC Jr, Bigner SH, Bigner DD (1981), Growth and chemotherapeutic response in athymic mice of tumors arising from human glioma-derived cell lines. J Neuropath Exp Neurol 40:410-427.

2. Hamm (1995), Proposed institutional animal care and use committee guidelines for death as an endpoint in rodent studies. Contemp Top Lab Anim Sc 34:69-71.

3. Sung C, Dedrick RL, Hall WA, Johnson PA, Youle RJ (1993), The spatial distribution of immunotoxins in solid tumors: assessment by quantitative autoradiography. Cancer Research 53: 2092-2099.

4. Tomayko MM and Reynolds CP (1989), Determination of subcutaneous tumor size in athymic (nude) mice. Cancer Chemother Pharmacol 24:148-154.

5. Welch DR, Chen P, Miele ME, McGary CT, Bower JM, Stanbridge EJ, Weissman BE (1994), Microcell-mediated transfer of chromosome 6 into metastatic human C8161 melanoma cells suppresses metastasis but does not inhibit tumorigenicity. Oncogene 9: 255-262.

Table 1. Selected Clinical Observations Used in Cancer Research and Toxicological Studies

Parameter	What to look for
General Appearance	Dehydration, decreased body weight, missing anatomy or fractured appendages. Abnormal posture, swelling of tissues or masses. Prolapses or paraphimosis, hypothermia
Skin and Fur	Discoloration, urine stain, pallor, redness, cyanosis, icterus, wound, sore, abscess, ulcer. Alopecia or ruffled fur.
Eyes	Exophthalmos, microphthalmia, ptosis, reddened eye, increased lacrimation or colored discharge. Opacity to the eye or cellular or blood accumulation in the eye.
Nose, Mouth, and Head	Head tilted, nasal discharge, malocclusion of teeth or jaw. Salivation or malodor associated with any orifice.
Respiration	Sneezing, dyspnea, tachypnea, rales
Urine	Discoloration, blood in urine, polyuria, anuria
Feces	Discoloration, blood in the feces, softness/diarrhea
Locomotor	Hyperactivity, hyperactivity, coma, ataxia, circling, muscle, tremors,

Montgomery, C.A. Jr. (1990), Cancer Bulletin 42:230-237 and appeared in AWIC Newsletter, Spring 1995 6:4

Approved by the Rutgers Animal Care and Facilities Committee March 22, 2006