Standard on Tumor Production and Cancer Research In Mice and Rats


University of North Carolina at Chapel Hill Standard on Tumor Production and Cancer Research in Mice and Rats



The standards and procedures described below provide guidance to all researchers and animal handlers for requirements for humane endpoints for mice and rats in tumor studies.


This Standard applies to all personnel engaged in research involving tumor burden in mice and rats. The standards described in this document apply to all research involving spontaneous and experimentally induced tumor studies in mice and rats.

The University of North Carolina at Chapel Hill (UNC-Chapel Hill) Institutional Animal Care and Use Committee (IACUC) expects that anyone involved in animal work at UNC-Chapel Hill will comply with this Standard. Requests for exceptions to this Standard must be reviewed and approved by the IACUC.


The purpose of this Standard is to provide investigators the requirements and humane endpoints for conducting spontaneous and experimentally-induced tumor studies in mice and rats. Consideration when designing these studies should include tumor size, potential clinical complications of tumor burden, metastatic and internal tumors, monitoring of animals, and criteria for intervention and/or euthanasia. Investigators producing or passaging tumors in mice or rats should use the information contained in this document as a reference when preparing their IACUC protocol.

Careful consideration should be given to the following items:

  • scientific requirements of the study
  • methods of reducing pain and distress
  • tumor size
  • clinical signs associated with tumor development
  • frequency of monitoring
  • potential clinical complications of tumor burden
  • criteria for intervention, humane endpoints, and the overall health of the animal
  • documentation

1. Pathogen testing of tumor cell lines

All cells/tumors implanted or injected into rodents should be tested for infectious agents and determined to be pathogen free. Inoculation with human cell lines or tumors must be included in the IACUC protocol, the laboratory safety plan and in a Schedule F form. Note that most companies selling human cell lines require that UNC-Chapel Hill’s Biosafety Officer or the Institutional Official approve the order. Please contact DCM (919-962-5335) for further information regarding pathogen screening.

2. Tumor size, volume, anatomical location, and number

Tumor size or volume

The humane endpoint (euthanasia required) for a single tumor is 2.0 cm (mouse) and 4.0 cm (rat) in any direction. The tumor size may not exceed 2.0 cm (mouse) and 4.0 cm (rat) in any direction without an approved Exception request (Section 8).

As tumors located within the cranium (bones that surround and protect the brain), thoracic cavity, mouth, or behind the eyes may interfere with vital functions and result in morbidity or mortality, the maximum tumor size should be considerably smaller with assessment of overall health status taking priority. As the use of imaging or biomarkers may detect tumors before they are visible or can be palpated, consideration should also be given to the use of imaging in studies where the tumors are located at sites not readily observable. Please note that size may not be a useful indicator for metastatic or internal tumors.

When tumor volume rather than size is more appropriate as an endpoint, internal tumor volume can be measured using 3D imaging (CT, MRI, ultrasound, etc.). In such cases, volume endpoints, rather than sizes, should be clearly defined in the protocol. Alternatively, a common method of estimating volume is the formula d2 x D/2, where d is the shortest diameter and D is the longest diameter. The humane endpoint for mice is 2000 mm3 and for rats is 5000 mm3. Multiple tumors should have a total volume that is under the limit of a single tumor. See examples in Table 1.

Tumor volume = d2 x D/2 (d=shortest diameter; D=longest diameter)
Mouse humane endpoint = 2000 mm3, Rat humane endpoint = 5000 mm3

TABLE 1. Sample volume calculations in determining humane endpoints in mice
Sample tumor dimensions Volume of tumor
= d2 x D/2
Humane endpoint?
Sample tumor: d=20mm, D=20mmd=20mm

= 202 x 20/2 = 400 x 10
= 4000mm3


  • Volume > 2000mm3
  • d, D = 2cm
Sample tumor: d=15mm, D=20mmd=15mm

= 152 x 20/2 = 225 x 10
= 2250mm


  • Volume > 2000mm3
  • D = 2cm
Sample tumor: d=15mm, D=15mmd=15mm

= 152 x 15/2 = 225 x 7.5
= 1687.5mm3


  • Volume < 2000mm3
  • d, D < 2cm
Sample tumor: d=15mm, D=18mmd=15mm

= 152 x 18/2 = 225 x 9
= 2025mm3


  • Volume > 2000mm3
    (despite d,D < 2cm)
Sample tumor: d=18mm, D=18mmd=18mm

= 182 x 18/2 = 324 x 9
= 2916mm3


  • Volume >2000mm3
    (despite d,D < 2cm)

Tumor location

Tumor implantation sites should be chosen to minimize the impact on the animal’s health status. However, removal of a primary tumor may result in metastatic disease elsewhere, necessitating the monitoring of general well-being. The animal’s back or flank is the preferred site for tumor implantation and is thought to cause the least pain and distress. Sites involving the face, limbs and perineum should be avoided as even minimal tumor growth in these areas can interfere with basic bodily functions, including eating, drinking, and urinating. In many studies, however, the implantation site is selected based on clinical relevance. If locations other than the back or flank are selected, they should be scientifically justified in the protocol and the humane endpoints clearly defined. Since cancer cells can metastasize to unexpected locations and form secondary tumors, it is important to monitor the general well-being of the animal and not just the primary site.

Multiple tumors

Individual tumors that are smaller than the single tumor maximum size may not have the same adverse health consequences as a single, large tumor. Nevertheless, even some relatively small tumors can interfere with basic bodily functions, especially when located in the face or perineum. When multiple tumors are present, the assessment of overall health status should take priority. The humane endpoint (euthanasia required) for tumor size when there are multiple tumors is 1.5 cm (mouse) and 3.0 cm (rat) in any direction. No tumor may exceed 1.5 cm (mouse) or 3.0 cm (rat) in any direction without an approved Exception request. Cases involving multiple tumors that alter the health status of the animal, or its ability to ambulate normally or obtain food and water should be euthanized.

3. Humane endpoints

Under conditions in which pain or distress is an unavoidable component of the study, humane endpoints must be identified and clearly defined. The humane endpoint is defined as the earliest point at which pain or distress in an experimental animal is prevented, terminated, or relieved. Monitoring of humane endpoints in an approved protocol as well as an animal’s general well-being is especially important with metastatic or internal tumors that cannot be easily measured externally.

Table 2. Selected Clinical Observations
Parameter What to look for
General appearance Dehydration, weight loss, abnormal posture, hypothermia, abnormal appearance of limbs, abdominal distension, swelling, tissue masses, vocalization
Skin and fur Discoloration, urine stain, pallor, redness, blueness, jaundice, wound, abscess, ulceration, bald spot, ruffled fur, bloody or purulent discharge
Eyes Enlarged eyes, small eyes, droopy lids, red-eye, tears, discharge, opacity
Nose, mouth, and head Head tilted, nasal discharge, malocclusion, drooling
Respiration Sneezing, rattle, abnormal breathing
Urine Discoloration of back/ventral fur, blood in urine, excessive or no urination
Feces Discoloration of back/ventral fur, blood in the feces, softness/diarrhea
Genital area Prolapses, paraphimosis (constriction of penis)
Locomotion Hyperactivity, hypoactivity, impaired movement, poor coordination, circling, tremors

Adapted from Montgomery, C.A. Jr. (1990), Cancer Bulletin 42:230-237 and AWIC Newsletter, Spring 1995

Body Condition Score (BCS)

Assessment of the overall health of the animal should take priority over assessment of tumor size or loss of body weight. In some cases, tumor growth may result in an increase in body weight and a decrease in lean body mass.  Close assessment of tumor size and body weight is especially important for younger, growing animals as failure to maintain weight gain comparable to untreated control animals may indicate adverse tumor effects. The Body Condition Scoring system (BCS), developed by Ullman-Cullere and Foltz (Body Condition Scoring: A Rapid and Accurate Method for Assessing Health Status in Mice. Lab. Animal Science; 49, 319-323, 1999), has proven to be a reliable indicator of general health and utilizes a scoring system that ranges from “1” (emaciated/wasted) to “5” (obese). The BCS offers a useful, rapid, and objective assessment of an animal’s health, especially in cases where treatments designed to affect tumor growth (e.g., radiation, chemotherapy) lead to poor body condition. BCS is also useful for assessing metastatic that affect other organs or tissues or internal tumors that are difficult to monitor accurately. This system quantitatively assesses several indicators of health status, including body weight, physical appearance, measurable clinical signs, unprovoked behavior, and response to external stimuli. However, the use of this method does not preclude other criteria for euthanasia prior to study endpoint including, but not limited to, the ability to eat or drink, labored breathing, ulceration (i.e., a breakdown of the skin cells resulting in exposure of the underlying tissue) and necrosis (i.e., death of cells in an organ or tissue due to disease, injury, or failure of the blood supply) at the tumor site.

Appendix I: Body Condition Scoring (BCS) Guide

Body Condition Scoring (BCS) Guide

Any tumor-bearing animal must be humanely euthanized when the following criteria are met (unless scientifically justified in the protocol and specifically approved by the IACUC):

  • The humane endpoint (euthanasia required) for BCS is 2.0. An approved protocol Exception is required for animals to remain in the colony once the BCS has reached 2.0.
  • the tumor meets the tumor size limitations*
  • the tumor becomes ulcerated or necrotic
  • the animal’s ability to eat or drink is impaired
  • the animal’s ability to ambulate normally is impaired
  • the animal’s breathing is labored

Investigators should consult with a DCM veterinarian regarding appropriate endpoints and must have a plan for euthanasia or treatment that is based on clearly defined endpoints.

* Possible exceptions to this Standard: There are cases in which the tumor reaches the maximum size limit and there are no observable health issues. In these instances, if continued use of the animal is critical for meeting the study’s objective, the PI must submit an Exception request amendment. The Exception request must include scientific justification and rationale for the exception and describe the revised criteria for euthanasia (e.g., maximum tumor size, changes in BCS, changes in body weight, etc.) as well as a revised frequency of monitoring.

4. Monitoring

Frequency of monitoring

All animals involved in tumor studies must be monitored for tumor size, health status, especially for metastatic or internal tumors, and pain/distress by qualified laboratory personnel. The exact monitoring and documentation frequency is based on the IACUC-approved protocol. The guidelines listed below apply to weekdays, weekends as well as holidays.

  • After implantation of tumor cells or in animals being utilized for the development of spontaneous tumors, animals may need to be monitored at least twice per week at intervals no greater than three days apart.
  • After a visible or palpable tumor is evident, the animals may need to be monitored at least three times weekly at intervals no greater than three days apart.
  • If tumor growth is expected to be rapid, up to twice daily monitoring of the general health of the animals may be necessary. More frequent observations are necessary when the general health of the animal shows signs of deterioration. Animals displaying general health concerns (e.g., not eating, drinking, reduced mobility) or approaching 50% of the tumor size humane endpoints may need to be monitored at least daily and monitoring must occur at the cage level using the purple Investigator Health Monitoring Card.

5. Documentation

The Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals requires proper documentation of animal care and use to assess compliance with research protocols and clinical care procedures.

Animal ID, tumor size, BCS, body weight, surgical procedures, post-surgical evaluations, euthanasia, and administration of anesthesia and pain medications must be recorded per the approved protocol and UNC Standards. All records must be available for review at any time by IACUC and external regulatory or accreditation officials.

6. Assignment to USDA pain categories

Most subcutaneous implanted tumors are thought to cause no pain or discomfort and thus should be classified as USDA Pain Category C. Other types of tumors (e.g., spontaneously developing, virus-induced, metastatic) may have a different pain profile and thus alternate pain categories should be considered.

The animals should be classified as USDA Pain Category D when the tumor burden is expected to cause pain/distress or disruption of normal activity. Examples include situations where palliative treatments are used to relieve pain and discomfort [e.g., anesthetics, analgesics] and when animals are euthanized once humane endpoints are reached.

In cases where the tumor burden is expected to cause pain/distress or disruption of normal activity; yet, scientific justification is provided for withholding palliative treatments (e.g., anesthetics, analgesics) or exceeding humane endpoints, the animals should be classified as USDA Pain Category E.


Requests for exceptions to this Standard must be reviewed and approved by the IACUC.


University Standard: The minimum acceptable limits or rules used to achieve Policy implementation, enforceable by the IACUC.

Humane Endpoint: The earliest point at which pain or distress in an experimental animal is prevented, terminated, or relieved.

Related Requirements

External Regulations and Consequences

University Policies, Standards, and Procedures

Contact Information

Primary Contacts

Contact Information Table
Subject Contact Telephone Email
Division of Comparative Medicine (DCM) Division of Comparative Medicine (DCM) 919-962-5335  
IACUC Protocol Office of Animal Care and Use (OACM) 919-966-5569

Important Dates

  • Effective Date and title of Approver: 07/26/2002; UNC-Chapel Hill IACUC
  • Revision and Review Dates, Change notes, title of Reviewer or Approver: Revised 07/26/2002; 04/26/2006, 11/14/2008, 02/13/2009, 06/12/2009, 11/09/2012, 04/2014, 11/14/2014, 10/2015, May 2018; UNC-Chapel Hill IACUC; June 2019: Updated: multiple tumors limitations, amendment required for greater than approved endpoint, included ambulation ability as indicator of animal wellbeing.
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Article ID: 132214
Thu 4/8/21 9:26 PM
Wed 2/14/24 11:54 AM
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07/12/2022 12:00 AM
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Vice Chancellor
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10/18/2023 12:00 AM
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07/12/2022 12:00 AM
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10/18/2024 12:00 AM
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09/23/2019 12:00 AM
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Research-Institutional Animal Care & Use Committee