Guidelines for Endpoints in Animal Study Proposals
Introduction:
Endpoints are a critical component of every Animal Study Proposal (ASP) which must be reviewed
by the Animal Care and Use Committee (ACUC). It is ideal when the scientific aims and the study
objectives can be accomplished without adverse effects, pain, or distress to the animal.
However, this is not always possible and careful consideration must be given to the:••••Scientific requirements of the study;
Expected and/or possible adverse effects that the research animals may experience(pain,distress, illness, etc.);
Probable time course and progression of those adverse effects;
Earliest most predictive indicators of present or impending adverse effects.

Where pain or distress is a necessary part of the study, a humane endpoint must be used andapprovedby the ACUC.1 A humane endpoint is the earliest scientifically justified point at whichpain or distress in an experimental animal can be prevented, terminated, or relieved, while
meeting the scientific aims andobjectives of the study. Such endpoints are preferable to deathor moribundity since they minimize painand distress and may be considered refinements.
The effective implementation of endpoints requires properly trained and qualified individuals toperformboth general and study-specific observations on research animals at appropriate timepoints. The assessment criteria and required response from the PI or designee must be clearly
defined within the ASP, and the use of study-specific animal assessment records should beconsidered. Studies must be designed to minimize pain and/or distress.
When initiating a new set of experiments, the potential for pain and/or distress may be unknown
and/orthe nature and extent of resulting morbidity and/or moribundity and/or mortality cannot
be anticipated.Therefore, smaller pilot studies may be useful as they can be instrumental to thedevelopment of an appropriate endpoint and determining animal numbers.
Finally, investigators performing studies that include pain or distress should re-assess thenecessity for any morbidity, moribundity, or mortality throughout the studies and refine theendpoints whenever possible. Post-approval monitoring is another tool the ACUC can use todetermine if endpoints are being used as intended and if further refinements are possible.
Morbidity:
ASPs that include morbidity as an endpoint or that include animal procedures that have thepotential to cause adverse sequela should address the following as they relate to the expected
outcomes:
1. Criteria to identify the humane endpoint:a. There are several examples in the literature that might be considered1

for most species,including:i. Evaluation of five aspects of an animal's condition as described by Mortonand Griffiths6. These are: body weight, physical appearance, measurable
clinical signs, unprovoked behaviorand response to external stimuli.
ii. Clinical observations used in cancer research and toxicological studies
include changes in general appearance, skin and hair, eyes, nose, mouthand head, respiration, urine, feces and locomotion 12 (Table 1).
iii. Objective endpoints such as weight loss or body condition (BC) scoring
can be applied to most species. Body condition scoring may be useful inyounger animals that are actively growing or oncology studies wheretumor growth increases weight concurrent with catabolism. 2 , 3, 14b. The clinical signs, depending on severity, duration and response to appropriate
therapy, that may constitute an endpoint in most species include, but are not limited to:••••••••••••••••

Rapid or progressive weight loss. Young or growing animals should haveweight assessed by using growth charts typical for their species/strain, body
condition scoring or comparison to untreated age & sex matched conspecifics.
Anorexia (lack or loss of appetite) or failure to drink
Debilitating diarrhea
Dehydration/reduced skin turgor EdemaSizable abdominal enlargement or ascites
Dermatitis or other conditions not responsive to treatment
Rough hair coat/unkempt appearanceHunched posture
Lethargy or persistent recumbency
Loss of righting reflex or failure to maintain equilibrium
Coughing, labored breathing, nasal discharge, gasping
Jaundice, cyanosis, and/or pallor/anemia
Neurological signs (seizures, paralysis, paresis, circling/head tilt, blindness)
Bleeding from any orificeSelf-induced trauma with exposure of underlying muscle
Any condition interfering with daily activities (e.g., eating or drinking, nestbuilding,ambulation, elimination or normal postural movements)
Excessive or prolonged hyperthermia or hypothermiac. Additional signs in neoplasia studies that may constitute an endpoint
include, but are notlimited to:i. A tumor burden greater than 10% body weight. In an adult mouse, a tumor
should not exceed 20 mm in any one dimension; in an adult rat, a tumor
should not exceed 40 mm in any one dimension. Formulas for calculatingtumor size can be found in the literature18-20-24.
ii. Tumors that ulcerate and/or become necrotic and/or infected.
iii. Tumors that interfere with eating or impair ambulation.

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Table 1. Examples of Clinical Observation-Based Endpoints

ParameterObservations

General Appearance

Dehydration, decreased body weight, missing anatomy, abnormal
posture,hypothermia, fractured appendage, swelling, tissue masses,
prolapse, paraphimosis

Skin and furDiscoloration, urine stain, pallor, redness, cyanosis, icterus, wound,sore,abscess, ulcer, alopecia, ruffled furEyes

Exophthalmos, microphthalmia, ptosis, reddened eye, lacrimation,discharge,opacity

Nose, Mouth, andHead
Respiration

Head tilted, nasal discharge, malocclusion, salivation
Sneezing, dyspnea, tachypnea, rales

Urine

Discoloration, blood in urine, polyuria, anuria

Feces

Discoloration, blood in the feces, softness/diarrhea

Locomotor

Hyperactivity, coma, ataxia, circling, muscle, tremors,
Montgomery, C.A. Jr. (1990), Cancer Bulletin 42:230-237

d. Considerations for aging studies:i. Senescent animals may naturally exhibit several clinical signs that wouldindicate significant morbidity in younger animals. Aging animals may also
experience certain benign ailments at an increased incidence. Genotype,background strain, chronological age, and sex should therefore beconsidered in the process of developing endpoints in these studies.
ii. For lifespan studies, where clinical signs of morbidity associated withaging are expected and necessary for the scientific aims and objectives ofthe study, the endpoint of the studyshould be as objectively described as
possible by the investigators. Recent information suggests that in aging
rodents there are changes in serial measurements of temperature, andbody weight that correlate with imminent death 12,15,17,18. However, thesechanges may not be predicative at the individual animal level. Where
more subjective endpoints such as deterioration in general health orquality of life are used, the assessment will rely on a veterinarian’s
observation and judgment in consultation with the Principal Investigatoras to when the endpoint has been reached. Additional parameters toconsider in evaluation of larger species could include complete bloodcounts, blood chemistries, urinalysis and other minimally invasive
techniques to evaluate organ function.

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e. Unexpected/spontaneous non-experimentally related conditions: Conditions

may arise in breeders or other “normal” animals or during the conduct of
research that are unexpected and unrelated to the research being conducted.
Conditions may be specific, such as a spontaneous tumor, or may be more of ageneral deterioration of health/quality of life. These conditions can still have asignificant impact on animal welfare and experimental results and must beaddressed appropriately. Any animal found unexpectedly to be moribund,cachectic, or unable to obtain food or water must be euthanized. In less severecases that may include pain or distress, the unexpected/unrelated condition
should be assessed for the impact on animal welfare and experimental results. If
the condition impacts experimental results, the animal should be euthanized. If
it does not affect the experimental results (this would also be the case with abreeder or a normal untreated animal), standard veterinary treatment must beprovided. If standard veterinary treatment would affect the experimental results,
the animals should be euthanized unless withholding treatment for thecondition(s) is specifically approved in the ASP.If the condition worsens following
treatment, the animal should be euthanized, dependent on veterinary judgment
and with Principal Investigator consultation. In some animal models, such as
specific phenotypes in Genetically Modified Animals (GMAs), conditions that
impact animal welfare can be expected to continue or reoccur. If not alreadypresent, these should be included in a modification to the ASP as “expectedresultant affects” to alert animal care staff and specify appropriate additional
animal care and/or euthanasia.
2. Monitoring for endpoints:
a. A plan for monitoring the animals both before and after a change in any of theabove aspects, providing care if appropriate, and increasing the level ofmonitoring as necessary, should be in place. Monitoring or clinical care on
weekends and holidays may require involvement of the investigative staff tosupplement that provided by the animal care and veterinary staff. Unlessotherwise specified in the ASP, monitoring and taking the appropriate actions is
the responsibility of the investigative staff, including on weekends and holidays.
b. Identify the personnel responsible for evaluation, record keeping, notificationof 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 requirementsof 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.
Commonly used signs of moribundity include, but are not limited to:• lack of responsiveness to manual stimulation;

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•
•

immobility; and/oran inability to eat or drink.

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 Careand Use Committees in reviewing proposals with death or moribundity as endpoints.
Required Information for ASPs Utilizing Death or Moribundity as an Endpoint:
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 instead of death, and how alternatives will be used whenever possible.
b. Why measures to relieve pain and/or distress cannot be utilized.
c. The number of animals that will be allowed to reach moribundity/death andjustification for it being the minimum necessary to achieve the scientificobjectives.
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 bemonitored at least daily by personnel experienced in recognizing signs ofmorbidity (illness, injury, or abnormal behavior) for at least the following:
abnormal posture, rough hair coat, head tucked into abdomen, exudates aroundeyes and/ or nose, skin lesions; abnormal breathing, difficulty with ambulation,
decreased food or water intake, lack of response to stimulation, or selfmutilation.
b. The frequency of observation will be increased when animals exhibit the above
described or other signs of morbidity. Monitoring on weekends and holidays may
require involvement of the investigative staff to supplement that provided by theanimal care and veterinary staff. A systemshould be in place where designatedpersonnel, including a veterinarian, are notified when animals show signs ofdisease. 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 theircondition deteriorates to the point that injury from other animals is likely. Deadanimals must be promptlyremoved.
d. Written records will be kept of monitoring frequency and observations.
Imaging/Biomarkers:
The use of serial imaging or biomarkers may permit the detection of experimental endpoints
that precede the development of significant clinical signs. Consideration should be given to
their use especially in studies that could result in morbidity, moribundity, or mortality.

5References - General Endpoints:
1. Canadian Council on Animal Care. 1998. Guidelines on: Choosing an appropriate
endpoint in experiments using animals for research, teaching and testing. Ottawa,Canada.
2. Clark, TS, LM Pandolfo, CM Marshall, AK Mitra, and JM Schech, (2018). Body Condition
Scoring forAdult Zebrafish. JAALAS 57(6):698-702.
3. Clingerman K, and Summers L.2012. Validation of a Body Condition Scoring System in
RhesusMacaques (Macaca mulatta) J Am Assoc Lab Anim Sci. 51(1): 31-36.
4. Hendriksen CFM and Morton DB, ed. 1998. Humane Endpoints in AnimalExperiments 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,
5. Institute for Laboratory Animal Research Journal. 2000. Humane Endpoints forAnimals Used inBiomedical Research and Testing. 41: No. 2.
6. Morton DB and Griffiths PHM. 1985. Guidelines on the recognition of pain, distress anddiscomfort in experimental animals and a hypothesis for assessment. Veterinary Record
116:431-43.
7. OECD Guidance Document on the Recognition, Assessment, and Use of Clinical Signs as
Humane Endpoints forExperimental
Animals
Used inSafety Evaluation. 2000.
8. Ray MA, et al. 2010. Identification of Markers for Imminent Death in Mice Used inLongevity andAging Research. JAALAS 48(3):282-288
9. Recognition andAlleviationofPainIn
Laboratory
Animals;
NRC 2009
http://www.nap.edu/catalog/12526/recognition-andalleviation-of-pain-in-laboratory-animals10. Spangenberg, Elin MF, and Linda J Keeling. 2016. Assessing the welfare of laboratory
mice in their home environment using animal-based measures –a benchmarking tool.
Laboratory Animals, 50(1) 30–38.
11. Stokes, WS. Humane endpoints for laboratory animals used in regulatory testing.
Lab Anim Sc 49:319-323.
12. Toth LA .1997. The moribund state as an experimental endpoint. Lab Anim Sc 49:319323.
13. Toth, LA. 2018. Identifying and Implementing Endpoints for Geriatric Mice. Comp. Med.
8(6): 439
14. Ullman-Culleré MH and Foltz CJ 1999. Body condition scoring: a rapid and accurate
method for assessing health status of mice. ILAR Journal, Volume 41, Issue 2: 87-93
15. United Kingdom Co-ordinating Committee on Cancer Research. 1997. UKCCCR
Guidelines for the Welfare of Animals in Experimental Neoplasia, 2nd ed. London,England
16. Yorke A, Kane AE, Hancock Friesen CL, Howlett SE, O'Blenes S. Development of a Rat
Clinical Frailty Index. J Gerontol A Biol Sci Med Sci. 2017 Jul 1;72(7):897-903.
17. Jocelyne C. Whitehead, Barbara A. Hildebrand, Michael Sun, Michael R. Rockwood,Robert A. Rose, Kenneth Rockwood and Susan E. Howlet. 2014. A Clinical Frailty Index inAging Mice: Comparisons With Frailty Index Data in Humans. . Lab Anim Sc 49:319-323.

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References - Tumor Size:
18. 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 40:41019. 427.
20. Hamm. 1995. Proposed institutional animal care and use committee guidelines fordeath as anendpoint in rodent studies. Contemp Top Lab Anim Sc 34:69-71.
21. 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.
22. Tomayko MM and Reynolds CP (1989), Determination of subcutaneous tumor size inathymic (nude) mice. Cancer Chemother Pharmacol 24:148-154.
23. Wallace, J. (2000) Humane Endpoints and Cancer Research. ILAR Journal, Volume 41, Issue
2: 87-93.
24. 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.
25. Workman P, et al; Guidelines for the welfare and use of animals in cancer research.
British Journalof Cancer (2010) 102,1555-1577
26. Montgomery, C.A. Jr. (1990) Oncologic and Toxicologic Research: Alleviation and Controlof Pain and Distress in Laboratory Animals. The Cancer Bulletin 42:230-237

Approved by ARAC 10/09/1996
Reapproved - 02/10/1999
Revised - 03/08/2000; 01/12/2005; 11/14/2007; 05/11/2011; 04/10/2013; 03/04/2016;04/24/2019; 4/27/20227