experimental endpoint definition

Wayne State University

Division of research & innovation, iacuc institutional animal care and use committee, defining humane endpoints.

Animals used in biomedical research may experience pain or distress as part of the experimental protocol. Researchers have a moral, ethical, and legal obligation to minimize pain and distress in pursuit of reliable, reproducible data. This is best achieved by the use of humane endpoints, or criteria used to define when an animal is to be removed from a study. Appropriate humane endpoints provide opportunities for refinement and reduce confounding systemic effects which may increase the precision of the data. 11 All studies must describe humane endpoints in their animal use protocol.

Definitions

Experimental Endpoints -  Endpoint of a study which occurs when the scientific aims and objectives have been reached (Guide p. 27).

Humane Endpoints - Predetermined physiological or behavioral signs that define the point at which an experimental animal's pain and/or distress is reduced or terminated through actions such as halting a procedure, providing appropriate treatment or analgesia, or humane euthanasia.

Morbidity - A condition of being unhealthy or diseased.

Moribund - A severely debilitated clinical state that precedes imminent death.

IACUC Guidelines

Humane endpoints must be clearly defined and based on objective criteria (See table below for examples).  They are study-specific and should be formulated through discussion with the PI, IACUC, and veterinarian while considering the following:

• Animal model and normal species-specific behaviors
• Scientific goals and experimental endpoints
• Expected and/or potential adverse effects (pain, distress, illness, death, etc.)
• Probable progression and timeline of adverse effects
• Earliest most predicative indicators of present or impending adverse effects

Monitoring for humane endpoints must be described in the protocol, including specific assessment criteria and the frequency of animal observation and assessment.

The action(s) to be taken when an animal reaches a humane endpoint must be described in the protocol and include either:

• Institution of appropriate treatment and either pausing study participation or permanent removal from study; or
• Humane euthanasia

Pilot studies may be necessary to determine humane endpoints, particularly when the effects of a treatment are unknown. A pilot study may help determine the morbidity, time course of effects, and frequency of monitoring necessary to establish endpoints.

The IACUC considers the following clinical signs as humane endpoints for animals exhibiting study adverse effects and/or spontaneous disease.

For an animal to continue on study while exhibiting these clinical signs, it must be described and approved in the IACUC protocol with alternative humane endpoints and appropriate monitoring plans; or undergoing an appropriate treatment prescribed by a DLAR veterinarian.

Failure to respond to treatment is considered an endpoint and the animal must be humanely euthanized.

*Treatment of any of the above conditions must be directed by a DLAR veterinarian.

Death or Moribundity as an Experimental Endpoint

Every attempt should be made to recognize and humanely euthanize an animal prior to moribundity or death. However, there are some studies that require moribundity or mortality as an endpoint.

Signs of a moribund animal include, but are not limited to:

• Lateral recumbency or immobility
• Lack of response to stimulation
• Inability to eat or drink
• Hypothermia 13

Studies that require death or moribundity as an endpoint must be classified as a pain and distress category E.

The protocol must include the following information:

• Specific alternatives or refinements that were considered.
• Why earlier humane endpoints cannot be employed?
• Whether animals will be humanely euthanized if moribund, and if not, what additional information can be gained from the interval between moribundity and death.
• Frequency of monitoring should increase with morbidity

Tools for humane endpoint evaluation:

• Mouse body condition score

2. Pain scoring systems

a) Grimace scales

i) Mouse, Rat & Rabbit Grimace scales available at https://www.nc3rs.org.uk/3rs-resources/grimace-scales

3. Humane endpoints scoring systems

Scoring templates should be adapted to  individual studies and based on the scientific needs and expected outcomes in the animal model.

• Example Rubric for Humane Endpoints as adapted for a murine sepsis model 8

Mice were euthanized at a score of ≥ 21 or if respiration rate or quality were ≥ 3

• 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 116:431-43
• Canadian Council on Animal Care (1998), Humane Endpoints in Animal Experiments for Biomedical Research, teaching, and testing. Ottowa, Canada.
• Ullman-Cullere 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.
•  Kort WJ, Hekking-Weijma JM, TenKate MT, Sorm V, R. V. A microchip implant system as a method to determine body temperature of terminally ill rats and mice. Lab Animal. 1998;32:260 269.
• Love JA, Booth CD, Boyd J. Remote temperature sensing for determining humane endpoints. In: 2 nd World Congress on Alternatives and Animal Use in the Life Sciences. Utrecht, The Netherlands; 1996
• United Kingdom Coordinating Committee on Cancer Research.  Guidelines for the welfare of animals in experimental neoplasia . London, UK; 1997.
• Wallace J.  Humane Endpoints and Cancer Research.  ILAR Journal 2000; 41(2).
• Shrum et al. BMC Research Notes 2014, 7:233
• IACUC Guideline Humane Endpoints for Laboratory Animals, University of Pennsylvania
• Guidelines for Endpoints in Animal Study Proposals. NIH OACU  27 April 2022. https://oacu.oir.nih.gov/system/files/media/file/2022-04/b13_endpoints_guidelines.pdf
• Workman P, Aboagye E, Balkwill F et al. (2010) Guidelines for the welfare and use of animals in cancer research. Br J Cancer 102, 15551577. https://doi.org/10.1038/sj.bjc.6605642 .
• National Centre for the Replacement Refinement & Reduction of Animals in Research (NC3R s ) Humane Endpoints 02 October 2015. < https://nc3rs.org.uk/3rs-resources/humane-endpoints >.
• Toth LA. (2000) Defining the Moribund Condition as an Experimental Endpoint for Animal Research. ILAR J. 41:72-79.   https://academic.oup.com/ilarjournal/article/41/2/72/747769.
• Toth LA. (2018) Identifying and Implementing Endpoints for Geriatric Mice. Comp. Med. 68: 439-451.

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Revisions Approved: 12/2012, 5/2018, 9/2022

Reviewed: 10/2017

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• Published: 18 December 2015

How to determine humane endpoints for research animals

Lab Animal volume  45 ,  page 19 ( 2016 ) Cite this article

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The website “Humane endpoints in laboratory animal experimentation” is a rich resource with information to help researchers define and implement humane endpoints when planning research with laboratory animals. Now, as a project of the 3Rs-Centre Utrecht Life Sciences, the site is updated with photo illustrations, short videos, links to other websites and downloadable sample datasheets.

The website is divided into public pages that are free for all to view and restricted-access pages that require login credentials, once a member has been approved by the owner of the site. The public portion of the website contains information on how humane endpoints are used to plan experiments, alongside information on mouse and rat behavior with external links. The restricted-access portion has more in-depth information on behavior, physiology, pathology, physical exams and how to determine when an animal should be euthanized. There are training modules in the restricted-access portion of the website as well.

The homepage, with a large photo of a mouse, centers on a link, 'What are humane endpoints', which leads readers to a subpage on definitions for humane endpoints. Below that, there are more links with information about the website and how to register for access to the restricted sections and training modules. The left-hand side of the homepage, which contains links to the bulk of the website's content, expands to show several additional topics and related subtopics. Some are only accessible after registration and are clearly marked. The links to publicly accessible topics lead to well organized pages featuring key concepts, definitions, pictures and videos to help guide the reader. Additionally, each page has terms highlighted in grey that link to definitions in an internal glossary.

The overall purpose of the public portion of the website is to inform readers about what humane endpoints are and how they can be used in animal research. This public material also explains some of the important areas of research wherein animal models are used—cancer, toxicity studies, vaccine potency, infectious disease and autoimmune disease—and the various levels of pain and distress that animals can experience. The material further points out that there are moral, scientific and legal considerations in determining the humane endpoint for an animal in research.

The restricted portion of the website features information and training modules on several topics, all intended to help researchers, veterinarians and animal caregivers in identifying pain and distress in animals and making informed and humane decisions regarding euthanasia. Specific topics range from 'pain and distress' to 'deviant spontaneous behavior'. A subsection on 'types of analgesia' gives examples of when analgesia is necessary, the specific types of analgesics typically used (opioids, local analgesics and non-steroidal anti-inflammatory drugs) and includes tables detailing dosages of different types of opioids and non-steroidal anti-inflammatory drugs for mice and rats. There are also links to multiple score sheets for assessing the health status or monitoring the welfare of rodents. Scoring health parameters on a regular basis gives caregivers and researchers a tool to determine if an animal is in distress or pain, which enables more accurate and consistent decisions concerning humane endpoints and euthanasia.

This is a superb site with material that can be used as a quick reference, a long read or study material as part of a class. It is available in English, Dutch and French, with a Spanish version expected by the end of 2015 and a German version by July of 2016. At the time Lab Animal visited, the training modules were fully functional only with Internet Explorer or Google Chrome and were provided only in English. As this site continues to grow it will no doubt become a useful and ubiquitous source of information for lab animal scientists throughout the world.

https://www.humane-endpoints.info/en

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How to determine humane endpoints for research animals. Lab Anim 45 , 19 (2016). https://doi.org/10.1038/laban.908

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What are humane endpoints?

A humane endpoint can be defined as:

‘the earliest indicator in an animal experiment of (potential) pain and/or distress that, within the context of moral justification and scientific endpoints to be met, can be used to avoid or limit pain and/or distress by taking actions such as humane killing or terminating or alleviating the pain and distress’ (Hendriksen and Morton, 1999).

Some elements of this definition can be further explained as follows:

• ‘…. potential pain ….’ (Hendriksen): indicates that also non-clinical endpoints might be used e.g. pre-clinical parameters such as hormone level changes, biochemical parameters or gene up/down regulations as an indicator for pain/distress later on in the disease process or even physiological parameters such as induction of antibody titres.
• ‘… within the context of the scientific endpoints ….” (Wallace, Hendriksen): applying humane endpoints should always be balanced against the scientific endpoint(s).
• ‘…. taking actions such as …’ (CCAC, Hendriksen): alleviation of pain/distress or terminating the painful/stressful procedure are also considered to be a humane endpoint .

The following conclusions can be drawn from this definition. A humane endpoint :

• Not necessarily means the humane killing of the animal, but could also result in interventions to alleviate the stressful/painful experimental procedure (e.g. performing surgery) or providing analgesics .
• Is not necessarily based on clinical signs but could also start from pre-clinical signs or from physiological or molecular biomarkers predictive of pain/distress later on in the disease process.
• Should be balanced against the scientific endpoints to be met. Thus, pain and distress might be intrinsic to a certain experimental model (e.g. arthritis ). However, in this case the humane endpoint should never be beyond the scientific endpoint.
• Should never be beyond the level of moral justification.

A humane endpoint can be considered as a possible refinement alternative for those experiments that involve pain and discomfort to the animals. In the Netherlands annually about 2,7 percent of all animals used in research experience more than ‘moderate/severe’ ( Zo Doende 201 4 , an annual report of the Netherlands Food and Consumer Product Safety Authority, only in Dutch). 

Biomedical research areas with relatively high percentages of pain and distress are cancer research, toxicity studies, vaccine potency studies, infectious disease studies and autoimmune disease studies. 

Applying humane endpoints should seriously be considered when animal experiments involve severe pain and suffering .

Above the mentioned definition, several other definitions are being used to describe a humane endpoint , like for example the following glostest of the OECD, the CCAC and Wallace: 

• The earliest indicator in an animal of pain , distress , suffering , or impending death on the basis of which an animal is killed (definition OECD);
• “The point at which an experimental animal's pain and/or distress is terminated, minimized or reduced, by taking actions such as killing the animal humanely, terminating a painful procedure, or giving treatment to relieve pain and/or distress” (definition CCAC).
• 'The limits placed on the amount of pain and distress any laboratory animal will be allowed to experience within the context of the scientific endpoints to be met’ (Wallace 2000)

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Rethinking Clinical Trials

A living textbook of pragmatic clinical trials.

• Introduction

CHAPTER SECTIONS

Choosing and Specifying Endpoints and Outcomes

Lesley Curtis, PhD Adrian F. Hernandez, MD, MHS Kevin P. Weinfurt, PhD

Contributing Editor Karen Staman, MS

For an explanatory trial, investigators can specify any outcome or endpoint, define the endpoint, and then measure it. The term outcome usually refers to the measured variable (e.g., peak volume of oxygen or PROMIS Fatigue score), whereas an endpoint refers to the analyzed parameter (e.g., change from baseline at 6 weeks in mean PROMIS Fatigue score). Even after a specific outcome is selected, it may be challenging to determine the best way to measure the effect of an intervention in terms of an analyzable endpoint, especially with pragmatic research where data are collected as part of routine care.

With pragmatic research, the endpoints and outcomes need to be available as part of routine care. Although the research question regarding the relative risks, benefits, and burdens of a specific intervention or activity will drive the selection of endpoints and outcomes, in a PCT, the selection must be balanced with an understanding of what is available in the electronic health record (EHR) or claims data and what additional resources will be needed to capture information not found in these sources.

Watch the video module: What do Endpoints and Outcomes Look Like in Pragmatic Clinical Trials

Some conditions can be objectively defined with a lab test, are straightforward to diagnose, and/or have International Classification of Diseases (ICD) codes . Some conditions, such as a broken leg, almost certainly require medical intervention and are likely to be captured in an EHR. Other conditions, however, are more ambiguous or less severe, and patients might not go to providers for treatment. Events or conditions that are not medically attended are unlikely to be captured in an EHR.

Defining endpoints and outcomes for some health phenomena is relatively easy for things like

• acute myocardial infarction
• broken bone
• hospitalization

However, many outcomes are not routinely recorded as part of healthcare delivery. For example:

• suicide attempt
• silent myocardial infarction
• early miscarriage

To detect these types of outcomes in pragmatic research, some additional work may be necessary, making the trial "less pragmatic"; however, this does not mean a pragmatic trial is not feasible. Key questions include:

• What challenges do you anticipate in trying to ascertain the endpoint?
• How might you address the challenges?

In this chapter, we will discuss endpoints and outcomes in pragmatic trials.

• Meaningful endpoints
• Outcomes measured via the electronic health record
• Outcomes measured via direct patient report
• Mobile health technology
• Meaningful Endpoints
• Outcomes Measured via the Electronic Health Record
• Inpatient Endpoints in Pragmatic Clinical Trials
• Using Death as an Endpoint
• Outcomes Measured via Mobile Devices
• Outcomes Measured via Direct Patient Report
• Additional Resources

Version History

February 22, 2024: Updated video module (changes made by K. Staman)

September 30, 2022: Made minor nonsubstantive text edits (changes made by K. Staman and L. Stewart)

January 22, 2021: Added embedded video (change made by G. Uhlenbrauck).

July 2, 2020: Minor corrections to layout and formatting (changes made by D. Seils).

December 4, 2018: Added key questions (changes made by K. Staman).

Published August 25, 2017

current section : Introduction

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Humane Endpoints Policy

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I.     Purpose

The purpose of this guideline is to provide criteria for identifying and utilizing the earliest endpoints that are compatible with the scientific objective of research studies while preventing, minimizing, or alleviating any actual or potential pain, distress, or discomfort to study animals.

II.     Scope

This applies to all animal users working under an AACUC approved protocol. Principal Investigators are responsible for ensuring staff are trained on the humane endpoints listed in the protocol.

III.     Definitions

Humane Endpoint: Humane endpoints refer to one or more predetermined physiological or behavioral signs that define the point at which a research or teaching animal’s pain and/or distress is terminated, minimized, or reduced by taking actions such as euthanizing the animal, terminating a painful procedure or giving treatment to relieve pain and/or distress.

Experimental Endpoint: When the scientific aims and objectives are reached. 

Anorexia: Loss of Appetite

IV.     Guidance

Certain animal use protocols include procedures or approaches that require special consideration during the AACUC review process due to their potential for unrelieved pain or distress or other animal welfare concerns.

The AACUC is obliged to weigh the objectives of the study against potential animal welfare concerns. By considering opportunities for refinement, the use of appropriate non-animal alternatives, and the use of fewer animals, both the institution and the principal investigator (PI) can begin to address their shared obligations for humane animal care and use.

Use of humane endpoints contributes to refinement by providing an alternative to experimental endpoints that result in unrelieved or severe animal pain or distress, including death.

The PI should identify, explain, and include in the animal use protocol an experimental endpoint that is both humane and scientifically sound.

Critical information that should be included in defining experimental endpoints includes precise definition of the humane endpoint (including assessment criteria), the frequency of animal observation, training of personnel responsible for assessment and recognition of the humane endpoint, and the response required upon reaching the humane endpoint.

The earliest possible humane endpoints that meet the scientific aims and objectives must be used for studies in which it is anticipated that animals may experience more than mild pain or distress.

The number of animals that may experience more than momentary pain or distress must be clearly described and scientifically justified. If humane endpoints that allow for greater degrees of pain and/or distress per experimental group are used, the number of animals for each endpoint must be clearly described and justified.

Animals must be monitored at a frequency acceptable by the AACUC and described in the protocol by personnel trained and experienced in recognizing signs of illness, injury, or abnormal behavior.  The frequency of observation of the entire group must be increased when one animal or more animals in a group are observed to be in unrelieved or severe pain or distress, including death.

Personnel with training to determine humane end points must make an assessment as soon as possible once notified of an adverse health event, including Program Veterinarian and facility manager.

Weight loss exceeding 15% of body weight compared to the pre-study weight or to age-matched controls.

Body Condition Score (BCS). With some species, disease processes or in growing animals, body weight is a poor indicator, thus body condition scoring (e.g., muscle atrophy or emaciation) may be more useful. No animal should be allowed to fall into poor body condition (less than 2 out of 5 or 3 out of 9), based on the species-specific reference scales defined in the protocol, unless scientifically justified and approved by the AACUC,

In specific models, animals may be allowed to persist in poor body condition or body weight loss may be allowed to exceed 15%. In these instances, a request for exemption must be written in the AACUC protocol and approved.

Anorexia. Complete anorexia for up to 5 days, OR partial anorexia (less than 50% of caloric requirement) for up to 7 days. Anorexia may be “normal” for immediate post-surgical patients.

Inability to obtain food/water. Inability to ambulate to reach food or water; lesions that interfere with eating or drinking or reluctance to stand.

Infection. Infection involving any organ system (either clinical or as indicated by laboratory testing) which fails to respond to antibiotic therapy and is accompanied by systemic signs of illness.

Marked change in behavior/depression. Lethargy, abnormal vocalization, aggression, recumbency, rough hair coat/hunched posture.

Signs of severe organ system dysfunction. Non-responsive to treatment or with a poor prognosis as determined by a consulting veterinarian in consultation with the Program Veterinarian.

Veterinary care, analgesia, and/or supportive care to the animal

Modification of housing or husbandry practices

Increasing the frequency of animal observations

Modification of experimental procedures

Termination of painful procedures

Removal of animals from the study

Humane euthanasia of the animal

When novel studies are proposed and information on a procedure’s effect on animals is limited or unavailable, or humane endpoints cannot be identified or defined, a pilot study may be recommended or required by the IACUC.

When such pilot studies are approved by the IACUC, the IACUC must be informed of outcomes (e.g., morbidity/mortality) as described in the approved protocol, and the protocol must be amended to include requirements related to animal monitoring and humane endpoints determined by the pilot study.

Regular observations of the animals throughout a pilot study are required to identify critical periods during the experiment when the animals’ well-being will be especially at risk.

AACUC Approval Date:  8/22/23                                                                                    

Review Date:  8/22/23

Issue Date:  9/1/23

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Article Contents

Introduction, pain and distress in the moribund state, data-based definition of the moribund state, human outcome research: perspectives for animal experimentation, predicting death in experimental animals.

• < Previous

Defining the Moribund Condition as an Experimental Endpoint for Animal Research

Linda A. Toth, D.V.M., Ph.D., is Director of Laboratory Animal Medicine and Professor of Pharmacology at Southern Illinois University School of Medicine, Springfield, Illinois.

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Linda A. Toth, Defining the Moribund Condition as an Experimental Endpoint for Animal Research, ILAR Journal , Volume 41, Issue 2, 2000, Pages 72–79, https://doi.org/10.1093/ilar.41.2.72

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The obligation to alleviate unnecessary pain and distress is an important correlate of the responsible use of animals in biomedical research. Many strategies have been used to meet this challenge, including administering analgesics and anesthetics when appropriate, reducing the numbers of animals tested, and avoiding the use of death as an experimental endpoint. However, many types of research protocols are associated with high mortality rates or require the production of progressive and severe disease states that clearly could cause the deaths of experimental animals. These types of protocols generally specify conditions under which preemptive euthanasia will be performed and may state that animals will be euthanized when they become “moribund.” However, persons with different biomedical backgrounds may have varying concepts of the term’s implications, rendering it poorly defined and arbitrarily interpreted. Dictionary definitions of moribund include words and phrases such as “dying,” “at the point of death,” “in the state of dying,” or “approaching death.” However, these definitions are severely limited for laboratory animal research because they do not describe the moribund state in behavioral or physiologic terms. Developing a sound approach to identifying the moribund state is crucial to its effective use as an experimental endpoint.

The moribund condition typically implies a severely debilitated state that precedes imminent death. The following discussion describes a general data-based approach for predicting imminent death and defining specific moribund conditions in objective terms that are relevant to specific experimental models ( Toth 1997 ). This process depends on the investigator’s ability to identify objective data-based criteria that forecast the death of an experimental animal. Several measurable conditions appear to have good predictive value in specific experimental models. An objective data-based approach to predicting death in the context of specific experiments could provide an unambiguous signal that the experimental endpoint has been reached, thereby facilitating the implementation of timely euthanasia and reducing unnecessary pain and distress.

The moribund state is preferred to death as an experimental endpoint because of the assumption that euthanizing a moribund animal will avoid or reduce terminal distress. However, the image triggered by the term moribund is often one of a prostrate, unresponsive, and perhaps seemingly comatose animal. From that perspective, one might seriously question whether such severely debilitated animals continue to experience pain or distress. If moribund animals are physiologically debilitated beyond the capacity for cognitive awareness of aversive sensations, euthanasia to avoid “spontaneous” death would not significantly reduce terminal distress.

A review of the literature on humans can provide insights into the relationship between behavioral responsiveness and awareness or consciousness. Clearly, in humans, as in animals, the absence of behavioral responses to painful stimuli does not prove lack of consciousness ( Ashwal et al. 1994b ; McQuillen 1991 ). A state of unresponsiveness may be similar under some circumstances to the condition of neuromuscular blockade, in which persons cannot generate motor responses but nonetheless maintain cognitive and sensory awareness. In human medicine, this condition is referred to as a “locked-in” state ( Ashwal et al. 1994a ; Giacino 1997 ).

A surprisingly large proportion of behaviorally unresponsive persons experience some type of awareness, ranging from clear sensory perception to “out-of-body” or paranormal experiences ( Lawrence 1995 ; Schnaper 1975 ; Tosch 1988 ). Coma and persistent vegetative states in human patients are generally associated with severe brain damage, brain hypometabolism to a level consistent with that of general anesthesia, and an apparent unconsciousness as assessed by neurologic examination ( Ashwal et al. 1994a ). These findings suggest that such patients do not experience pain or suffering, although their eyes may be open, they may appear to be awake, and they may occasionally demonstrate apparent semipurposeful behavior mediated by residual islands of cortical function ( Ashwal et al. 1994a ; Plum et al. 1998 ; Schiff 1999 ). However, states of consciousness obviously have gradations. Patients may experience states of minimal awareness or minimal unconsciousness, particularly if brain function is adversely influenced by metabolic derangements or infectious agents whose effects could fluctuate or resolve ( Ashwal et al. 1994b ; Bernat 1992 ). Inferences about awareness of pain are less reliable under such conditions. In fact, many patients in vegetative states eventually experience the return of some degree of awareness ( Beresford 1997 ; Childs and Mercer 1996 ). Accurate assessment of consciousness and awareness is a significant clinical issue in human medicine ( Andrews et al. 1996 ; Giacino 1997 ).

The difficulty inherent in assessing the cognitive state of unresponsive individuals is apparent from the literature on humans. Similarly, in animal research, an unresponsive apparently moribund animal cannot necessarily be considered unaware, and one cannot necessarily assume that an unresponsive animal can no longer experience pain. The possibility that even unresponsive subjects might experience pain or distress reinforces the need for a clear definition of moribundity and for a method of predicting death so that the potential for suffering can be minimized by timely euthanasia.

The ability to predict death with high probability and accuracy could have several advantages to the research process. Animals would clearly benefit because unnecessary terminal distress could be eliminated or significantly reduced. Moreover, the research effort itself might benefit directly because experimental goals could be met more consistently. If one could accurately predict the time of death, euthanasia could be scheduled to permit timely collection of samples that would be lost if the animal died unexpectedly. For example, a recent study of rats with leptomeningeal tumors used hindlimb paralysis, rather than death, as an endpoint, thereby allowing the collection of tissues necessary for histopathologic documentation of the extent of the tumor and the possible response to therapy ( Janczewski et al. 1998 ). In addition, imminent death might modify important physiologic variables, rendering data collected under those conditions unusually variable or even uninterpretable within the context of the study. For example, microbially infected mice that are near death develop both marked hypothermia ( Soothill et al. 1992 ; Toth et al. 1995 ; Wong et al. 1997 ) and abnormal EEG findings ( Gourmelon et al. 1986 , 1991 ; Toth et al. 1995 ) that could render them physiologically unsuitable for some studies.

Thus, preemptive euthanasia could have several advantages for research: Data collected after severe physiologic derangements develop may not be useful or may be misleading for some purposes, and tissues that might otherwise be lost can be collected for postmortem analysis. If the research team recognizes the advantages of timely euthanasia when developing study endpoints, compliance with established endpoint criteria will undoubtedly be easier to achieve. Finally, a clear definition of the moribund state could also improve the ability of veterinarians and animal care personnel to promote animal well-being and the efficient collection of high-quality data.

The moribund state can be defined by identifying the values of various variables that precede imminent death and can serve as signals for preemptive euthanasia. To be most useful for routine application, these values should be derived from and tailored to specific experimental models and should be among the values evaluated as part of the normal process of data acquisition. Research data are generally subjected to close scrutiny by the research team, which increases the likelihood that predictive changes in values of key variables may be recognized. Furthermore, this strategy facilitates the routine collection of information relevant to moribundity as part of the experiment, without requiring extra work. Monitoring many diverse clinical signs may be labor intensive, and researchers using a generalized evaluation system may be required to evaluate variables that might appear arbitrary or even unrelated to the severity of the animal’s condition. Conscientious compliance with that approach may be difficult to achieve. However, researchers’ compliance may be increased if the measurement of the variables is related to research goals. Furthermore, the consideration of numerous variables that may be irrelevant can obscure the impact of pertinent factors, as occurred in a system for evaluating pain ( Beynen et al. 1988 ). To be most useful, specific variables should be identified and weighted in terms of their predictive value. It is important to know which factors matter most in particular situations.

Comparing data collected from animals that die with data from animals that survive may reveal experimental variables that change before imminent death and could be valid predictors of death or moribundity. Information relating the frequency at which critical observations or measurements should be made, the time when specific conditions change, and the time of death is also useful. This type of evaluation can often be conducted during initial or pilot experiments, but long-term familiarity with the model may be necessary before useful but subtle indicators of imminent death are recognized. The identification and statistical validation of variables that correlate with and predict imminent death establishes indices that can be applied in subsequent studies using the same model. The validation data should be sound enough to convince the research team either that appearance of the predictor indicates imminent death or, alternatively, that data collected after the appearance of the predictor would be invalid. Because subjective evaluations require constant vigilance to avoid bias, objective or quantifiable variables may be preferable for routine use in standardized protocols.

Objective definition of the moribund state requires the differentiation of dying from illness, pain, and distress. An assessment of clinical and behavioral signs can certainly indicate that euthanasia is warranted for humane reasons, but many clinical signs, despite their severity, may not predict imminent or even eventual death. For example, seizures may indicate illness that is severe enough to warrant euthanasia for humane reasons, but obviously in many situations animals can live for a long time after having a seizure, and indeed they may never have another seizure. Scoring systems based on the severity of multiple behavioral or physiologic abnormalities ( Morton and Griffiths 1985 ; Olfert 1996 ) can provide useful benchmarks for animal evaluation, and they can be used to support veterinary determinations that euthanasia is warranted for humane reasons. However, even severe illness or distress may not forecast imminent death.

Several other considerations influence the selection of an indicator for preemptive euthanasia. First, an indicator that is appropriate for one model may not predict imminent death in another. The variables discussed below as useful for predicting death are examples that undoubtedly are not applicable in all situations. Administering certain drugs, for example, could induce hypothermia and an unresponsive state from which the animal will eventually recover. In this situation, these conditions obviously do not indicate a moribund state because death is not the ultimate outcome. Second, the experimental endpoint can be modified as more is learned about the model. For example, a group studying mice inoculated with a myeloma cell line observed that the animals developed hind-leg paralysis, indicative of metastatic compression of the spinal cord, approximately 2 to 7 days before death ( Huang et al. 1993 ). In a subsequent study, the experimental endpoint was redefined, and hind leg paralysis was used as an indicator for preemptive euthanasia ( Huang et al. 1995 ). As experience with and data collected from a specific model accrue, more information will be available for developing endpoint refinements.

The choice of euthanasia indicators and the implementation of euthanasia must be compatible with the experimental design and maintain the scientific integrity of the experiment because the advancement of knowledge and the value of the research will depend on maintaining the animal long enough to collect crucial data. Furthermore, some studies may require prolonged clinical maintenance of animals before euthanasia, despite the occurrence of signs that forecast eventual death, or may have valid justification for using spontaneous death as the experimental endpoint. Finally, despite conscientious efforts by the research team, objective data-based criteria that predict imminent death and provide a signal for preemptive euthanasia yet allow the completion of experimental objectives may be difficult or impossible to identify in the context of some experimental paradigms. The establishment of requirements to identify criteria for and to implement preemptive euthanasia rests with institutional animal care and use committees, based on the assessment of specific protocols.

Subjective predictions of death may be prone to bias ( Dawes et al. 1989 ; Forster and Lynn 1988 ; Heyse-Moore and Johnson-Bell 1987 ; Knaus et al. 1991 ; Parkes 1972 ; Seneff and Knaus 1990 ). For example, in one study, predictions of patients’ life expectancy by experienced physicians and nurses were overwhelmingly optimistic rather than random ( Parkes 1972 ). Such bias could be due to a variety of factors. Because patients are likely to receive supportive care and medication to relieve symptoms, those who are near death may appear less seriously ill than they actually are. Alternatively, they may remain in a relatively stable condition until death is imminent, or they may die unexpectedly. Another possible reason for the preponderance of optimistic predictions is the potential psychologic difficulty associated with stating definitively that a person will die very soon. Research personnel may be similarly influenced by a pragmatic view that if death cannot be predicted with relative certainty, an experiment may as well continue. Statistically based systems of outcome prediction are often more reliable and more accurate than is human subjective judgment ( Dawes et al. 1989 ; Knaus et al. 1991 ; Seneff and Knaus 1990 ).

Objective approaches to predicting clinical outcomes in human medicine are frequently used to assess the effectiveness of medical or surgical treatments, evaluate the quality of hospital services, and develop changes in public health policy ( Allard et al. 1995 ; Barriere and Lowry 1995 ; den Daas, 1995 ; Knaus et al. 1991 ; Le Gall et al. 1995 ; Lemeshow et al. 1987 ; Seneff and Knaus 1990 ). Studies of outcome prediction offer some insights into the potential problems and benefits associated with using similar approaches in animal research. In general, two basic strategies have been applied in human studies. The first involves the use of disease severity scores, which are typically based on the deviation from normal of various physiologic measurements, such as serum glucose concentrations and urine output. In some systems, these variables are weighted and integrated with patient factors such as age and other preexisting disease to arrive at a score that can then be correlated with outcome and that may be predictive or prognostic. Classification systems developed for the assessment of disease severity and the prediction of patients’ clinical outcomes include the acute physiology and chronic health evaluation (“APACHE”) ( Knaus et al. 1985 ), simplified acute physiology score (“SAPS”) ( Le Gall et al. 1993 ), mortality probability model (“MPM”) ( Lemeshow et al. 1985 ), and sickness impact profile (“SIP”) ( Bergner et al. 1976 ).

As opposed to using broad-based scoring systems, numerous studies have attempted to relate patients’ specific symptoms to survival or death. For example, several studies report that the incidence of dyspnea and other breathing problems increases before death ( Coyle et al. 1990 ; Higginson and McCarthy 1989 ; Reuben and Mor 1986 ; Reuben et al. 1988 ; Ventafridda et al. 1990 ). A correlational study of nearly 1600 cancer patients showed that indicators of poor nutritive status (e.g., difficulty swallowing, recent weight loss, anorexia, and dry mouth) were also predictive of death, but pain (even when severe), gastrointestinal signs, and signs of central nervous system involvement were not ( Reuben et al. 1988 ). Nutritional status as estimated by the change in midarm circumference was a predictive indicator of mortality in a series of hospitalized geriatric patients ( Incalzi et al. 1998 ). That variable exemplifies several advantageous features, including ease of measurement, reproducibility, availability for all patients, marginal alteration due to hydrational status or edema, and incorporation of both muscle and fat mass ( Incalzi et al. 1998 ).

Problems or signs associated with poor clinical outcomes are often assessed in patients with specific medical conditions. for example, for patients in ischemic coma, a poor prognosis (defined to include either survival in a vegetative state or death) is associated with the bilateral absence of somatosensory evoked potentials during the first week of coma ( Zandbergen et al. 1998 ) and with high serum levels of a glial calcium-binding protein ( Martens et al. 1998 ). Documented seizures or loss of consciousness is associated with a poor prognosis for children with shigellosis ( Khan et al. 1999 ), as is the need for ventilation or hemodynamic support for patients who have received bone marrow transplants ( Jackson et al. 1998 ). Similarly, the development of hypothermia in septic patients is significantly correlated with death due to septic shock ( Clemmer et al. 1992 ).

The prognostic significance of clinicopathologic variables has also been evaluated in humans. For example, in some groups of patients with malaria, factors significantly correlated with death include impaired consciousness and respiratory distress, creatinemia, bilirubinemia, hyperlactemia, hypoglycemia, and proportion of pigment-containing neutrophils ( Krishna et al. 1994 ; Marsh et al. 1995 ; Phu et al. 1995 ; Waller et al. 1996 ). Levels of inflammatory cytokines are correlated with mortality in patients with burns ( Marano et al. 1990 ), sepsis ( Barriere and Lowry 1995 ; Debets et al. 1989 ; Girardin et al. 1988 ), adult respiratory distress syndrome ( Meduri et al. 1995 ), and malaria ( Krishna et al. 1994 ).

As strategies for predicting patient outcomes, broadbased evaluations and analysis of specific variables each have strengths and weaknesses. Broad-based scoring systems can be useful for statistical assessment of population tendencies, but they may be less accurate in predicting clinical outcomes for individual patients ( Barriere and Lowry 1995 ; Dellinger 1988 ; Deyo and Inui 1984 ; Jackson et al. 1998 ; Jones 1998 ). for example, scores on the Karnovsky index, which evaluates overall performance capabilities of patients ( Yates et al. 1980 ), generally correlate very well with the length of survival of populations of patients ( Evans and McCarthy 1985 ; Mor et al. 1984 ; Reuben et al. 1988 ; Yates et al. 1980 ), but when the index is applied to individual patients, scores associated with a specific survival interval can span the entire scale ( Evans and McCarthy 1985 ). Such broad-based scoring systems may accurately predict mortality rates if scores reflect very poor health, but overestimate or underestimate mortality rates for patients with more moderate disease ( Jackson et al. 1998 ; Prytherch et al. 1998 ). Several factors probably contribute to the inaccuracy of broad-based scales in assessing the imminence or likelihood of death for individual humans or animals. First are issues of sensitivity. These scales may detect large changes in patients’ conditions (e.g., the progression from inpatient to outpatient status), but some clinically significant changes in health status that are obvious to both clinician and patient may only modestly affect the overall score (e.g., symptom progression in ambulatory patients with chronic disease) ( Barriere and Lowry 1995 ; Deyo and Inui 1984 ; Fitzpatrick et al. 1992 ). Related to the issue of sensitivity is the so-called “floor phenomenon,” which refers to the inability of health status measures to detect clinically important changes in patients whose baseline health status is poor ( Baker et al. 1997 ; Bindman et al. 1990 ). Another factor that can influence the predictive accuracy of broad-based scoring systems is the possibility that considering irrelevant variables can reduce the impact of pertinent factors.

In contrast to broad-based evaluation systems, analysis of specific factors may provide greater accuracy in specific situations. However, the weakness of the targeted approach is the difficulty in selecting the optimal variables from many possible options. Thus, the initial consideration of numerous variables that are relevant to the clinical condition is likely to be informative ( Goldhill and Withington 1996 ), and in animal studies a broad-based system may be useful during the initial development of a model. When used to define moribundity, this type of analysis can help to identify those variables with the strongest prognostic implications, so that the assessment can ultimately be tailored to specific condition or situation. In general, accurate prediction of outcomes requires at the very least a standardized approach to data collection ( Goldhill and Withington 1996 ).

Other factors also complicate the evaluation of the moribund state in human populations. The precise time of disease onset may be unknown, and various clinical therapies are likely to have been applied. Even the diagnosis or the cause of the medical condition may be uncertain. Another complicating factor is “lead-time bias,” or the inaccuracy introduced into risk prediction if treatment is initiated before physiologic measurements relevant to scoring have been completed ( Tunnell et al. 1998 ). This situation arises, for example, if patients receive supportive medical treatment (e.g., the administration of intravenous fluids) in an ambulance, but physiologic data used for outcome assessment (e.g., hematology values) are not collected until the patient arrives in the emergency room or intensive care unit. Moreover, in humans, preexisting or secondary medical conditions are common, and their importance may vary depending on the patient population ( Jackson et al. 1998 ; Jones 1998 ; Tunnell et al. 1998 ). Demographic factors such as age, history, life style, and genetic background also vary widely in patient populations. Finally, terminally ill patients generally receive intensive supportive care that may delay, modify, or blunt the clinical signs predictive of dying. Such issues complicate assessment of the moribund state in human patients.

The evaluation of the moribund state may be somewhat easier in animal populations than in humans. In experimental models, the precise nature, time, and magnitude of challenges and subsequent interventions are known and are generally standardized. Furthermore, animal populations may be very homogeneous in terms of age, history, environment, and genetic background. Thus, accurate prediction of imminent death may be more feasible in experimental animal models than in human populations.

Hypothermia is perhaps the most commonly reported predictor of experimental animals' imminent death ( Gordon et al. 1990 ; Kort et al. 1997 ; Soothill et al. 1992 ; Stiles et al. 1999 ; Wong et al. 1997 ). Its use as an endpoint requires determination of a specific index temperature that is invariably associated with imminent death. Preemptive euthanasia is then performed if an animal’s temperature drops below the predetermined value. In one study, for example, mice with acute experimental bacterial infections developed rectal temperatures of less than 34°C before the onset of clinically overt illness that eventually warranted euthanasia ( Soothill et al. 1992 ). In studies of influenza-infected mice, rectal temperatures of less than 32°C were inevitably associated with death in one study ( Wong et al. 1997 ), whereas another found that mice recovered after even more profound hypothermia and used a core temperature of 28°C as the indication for euthanasia ( Toth et al. 1995 ). A toxicity study found a linear relationship between the 50% lethal dose and the dose of metallic salts that reduced body temperature to 35°C ( Gordon et al. 1990 ). Premorbid variability in temperature, uninterrupted wheel-running for 3 hr, and hypothermia below 30°C were sequential temporal markers of moribundity and death in rats studied in an activity-stress model ( Morrow et al. 1997 ).

Several considerations are relevant to the use of temperature as a sign of imminent death: (1) Body temperature can be significantly influenced by ambient temperature and by other aspects of the environment, such as the type of bedding or the presence of cage mates. This consideration is particularly important for mice. Thus, index values developed under one experimental situation may not be applicable in all situations. (2) Different strains or species of animals may react differently to the same challenge. For example, BALB/c mice are more sensitive than C57BL/6 mice to influenza challenge, demonstrating higher mortality rates ( Toth et al. 1995 ) and perhaps a different critical index temperature. In contrast, staphylococcal enterotoxin A elicits more severe hypothermia and greater rates of mortality in C57BL/6 mice than in BALB/c mice ( Stiles et al. 1999 ). (3) The method of temperature measurement may influence the interpretation of specific values. Intraperitoneal transmitters that provide a continuous record of core temperatures can be used to evaluate the duration of hypothermia ( Toth et al. 1995 ), but data collection with this type of system requires expensive equipment and surgical manipulation of the animal. Implanted microchips ( Kort et al. 1997 ; Stiles et al. 1999 ) and rectal measurements using hand-held thermometers or probes ( Gordon et al. 1990 ; Soothill et al. 1992 ; Wong et al. 1997 ) are less expensive and less invasive, but they provide only snapshot evaluations of temperature. In some cases, prolonged hypothermia may reflect imminent death, whereas transient or short-term hypothermia may resolve and be associated with eventual recovery. Furthermore, rectal measurement of temperature requires significant animal manipulation and may be associated with stress.

Another simple but specific endpoint marker that is visually obvious, objective, and easy to assess is the inability to rise or ambulate. This condition was a good predictor of imminent death in guinea pigs with Pseudomonas -induced sepsis ( Louie et al. 1997 ) and in mice with endotoxemia ( Krarup et al. 1999 ). Moreover, mice with severe or palpable hypothermia are also likely to be recumbent and unresponsive to handling or other stimuli ( Krarup et al. 1999 ). Although a moribund state is commonly interpreted to mean a prostrate, unresponsive condition, substitution of the specific physical definition of "unable to walk" illustrates how the use of precise terminology can limit ambiguity in the decision-making process for euthanasia.

A study of the survival of rats with central nervous system tumors provides another example of defining morbidity predictors according to the experimental model ( Redgate et al. 1991 ). When evaluating animals after tumor inoculation, these researchers noticed that all animals underwent three phases of weight change: an initial period of weight loss associated with irradiation and tumor implantation, a period of weight gain when the animals appeared to be in a clinically stable condition, and a terminal period of weight loss that preceded death. Total survival time varied primarily because of the length of the first two phases, but statistical analysis showed that imminent death could be accurately predicted for animals that lost weight for 7 to 8 consecutive days during the terminal period. This criterion could be used in future studies to permit timely euthanasia of animals without biasing experimental outcomes. Interestingly, related variables (e.g., percentage of weight loss or reduction in food intake) were not predictive of imminent death in this model, indicating that careful analysis of the data may be needed to find the best variable for accurate prediction of moribundity.

Other experimental variables that may be useful for predicting moribundity can be developed based on specific experimental models. For example, experiments that monitor EEG patterns have shown a terminal flattening of EEG tracings in rabbits with infectious diseases ( Toth et al. 1993 ), rats experiencing chronic sleep deprivation ( Rechtschaffen et al. 1983 ), and mice that die after infectious challenge ( Gourmelon et al. 1986 , 1991 ) or spontaneously from agerelated conditions ( Welsh et al. 1986 ). Studies of rats and mice have shown that hind-limb paralysis, rather than death, can sometimes be used as the endpoint in tumor models ( Huang et al. 1993 , 1995 ; Janczewski et al. 1998 ).

Biochemical variables can also provide useful prognostic markers in animals. For example, plasma lactate concentrations were a good predictor of clinical outcome in dogs with gastric dilatation-volvulus ( de Papp et al. 1999 ). However, in some research situations, timely biochemical measurements may be difficult to obtain because serum or other samples collected periodically over the course of a study may not be analyzed until a minimum number of samples have been accrued, or until the experiment has ended. Nonetheless, experience with the experimental model can sometimes provide clues about an animal’s condition even before precise biochemical or physiological analyses are completed. For example, marked hypeitriglyceridemia and distinct sleep patterns develop in rabbits that succumb to bacterial infections compared with those that survive ( Toth et al. 1993 ). Although plasma triglyceride assays and quantitative evaluation of sleep may not be performed until days or weeks after the end of the experiment, the severe hypertriglyceridemia causes a marked milky opacity of the plasma that is visually obvious after centrifugation. Similarly, qualitative changes in the EEG are visible on polygraph tracings even before the EEG amplitudes are accurately assessed. Thus, experience with the physiologic responses of terminally ill animals can teach the investigative team to recognize signs of imminent death so that timely euthanasia can be performed.

Criteria that are used experimentally to define moribundity and that have been validated as predictive markers for imminent death should be reported ( Clarke 1997 ). Unfortunately, these types of observations are often published only in methods sections of research articles, and therefore many researchers and veterinarians may not encounter or be aware of such refinements. Furthermore, many researchers may not have a practical appreciation of humane, as opposed to scientific, refinement of an experimental model. Providing simple but practical examples and encouraging dissemination of information about refinements are important facets of training of research personnel to use experimental animals humanely.

Our obligation to alleviate the unnecessary pain and distress of experimental animals mandates the implementation of timely euthanasia. Subjective clinical judgments are essential for the evaluation of the animal’s well-being and support the veterinary prerogative of euthanasia for humane reasons. However, subjective evaluations may be biased when used to predict imminent death. Objective data-based approaches to predicting imminent death developed for specific experimental models could facilitate the implementation of timely euthanasia before the onset of clinically overt signs of moribundity and could thereby reduce pain and distress experienced by experimental animals.

The author thanks Flo Witte for editorial review of this manuscript. This work was supported in part by National Institutes of Health grants NS-26429 and CA-21765 and by the American Lebanese Syrian Associated Charities at St. Jude Children’s Research Hospital, Memphis, Tennessee.

Allard P Donne A Potvin D 1995 Factors associated with length of survival among 1081 terminally ill cancer patients . J Palliat Care 11 : 20 – 24 .

Google Scholar

Andrews K Murphy L Munday R Littlewood C 1996 Misdiagnosis of the vegetative state: Retrospective study in a rehabilitation unit Br Med J 313 : 13 – 16

Ashwal S Cranford R Bernat JL Celesia G Coulter D Eisenberg H Myer E Plum F Walker M Watts C Rogstad T 1994 . Medical aspects of the persistent vegetative state (first of two parts) . N Engl J Med 330 : 1499 – 1508 .

Ashwal S Cranford R Bernat JL Celesia G Coulter D Eisenberg H Myer E Plum F Walker M Watts C Rogstad T 1994 . Medical aspects of the persistent vegetative state (second of two parts) . N Engl J Med 330 : 1572 – 1579 .

Baker DW Hays RD Brook RH 1997 . Understanding changes in health status: Is the floor phenomenon merely the last step of the staircase? Med Care 35 : 1 – 15 .

Barriere SL Lowry SF 1995 An overview of mortality risk prediction in sepsis Crit Care Med 23 : 376 – 393 .

Beresford HR 1997 The persistent vegetative state: A view across the legal divide Ann NY Acad Sci 835 : 386 – 394 .

Bergner M Bobbitt RA Pollard WE Martin DP Gilson BS 1976 The sickness impact profile: Validation of a health status measure Med Care 14 : 57 – 67 .

Bernat JL 1992 The boundaries of the persistent vegetative state . J Clin Ethics 3 : 176 – 180 .

Beynen AC Baumans V Bertens APMG Haas JWM Van Hellemond KK Van Herck H Peters MAW Stafleu FR Van Tintelen G 1988 Assessment of discomfort in rats with hepatomegaly . Lab Anim 22 : 320 – 325 .

Bindman AB Keane D Lurie N 1990 Measuring health changes among severely ill patients: The floor phenomenon Med Care 28 : 1142 – 1152 .

Childs NL Mercer WN 1996 Late improvement in consciousness after post-traumatic vegetative state N Engl J Med 334 : 24 – 25 .

Clarke R 1997 Issues in experimental design and endpoint analysis in the study of experimental cytotoxic agents in vivo in breast cancer and other models Breast Cancer ResTreat 46 : 255 – 278 .

Clemmer TP Fisher CJ Bone RC Slotman GJ Metz CA Thomas FO 1992 Hypothermia in the sepsis syndrome and clinical outcome Crit Care Med 20 : 1395 – 1401 .

Coyle N Adelhardt J Foley KM Portenoy RK 1990 Character of terminal illness in the advanced cancer patient: Pain and other symptoms during the last four weeks of life J Pain Symptom Manage 5 : 83 – 93 .

Dawes RM Faust D Meehl PE 1989 Clinical versus actuarial judgment . Science 243 : 1668 – 1674 .

Debets JMP Kampmeijer R van der Linden MPMH Buurman WA van der Linden CJ 1989 Plasma tumor necrosis factor and mortality in critically ill septic patients Crit Care Med 17 : 489 – 494 .

Dellinger EP 1988 Use of scoring systems to assess patients with surgical sepsis Surg Clin N Am 68 : 123 – 145 .

den Daas N 1995 Estimating length of survival in end-stage cancer: A review of the literature J Pain Symptom Manage 10 : 548 – 555 .

de Papp E Drobatz KJ Hughes D 1999 Plasma lactate concentrations as a predictor of gastric necrosis and survival among dogs with gastric dilatation-volvulus: 102 cases (1995-1998) J Am Vet Med Assoc 215 : 49 – 52 .

Deyo RA Inui TS 1984 Toward clinical applications of health status measures: Sensitivity of scales to clinically important changes . Health Serv Res 19 : 275 – 289 .

Evans C McCarthy M 1985 Prognostic uncertainty in terminal care: Can the Karnovsky index help? Lancet 1 : 1204 – 1206 .

Fitzpatrick R Ziebland S Jenkinson C Mowat A Mowat A 1992 Importance of sensitivity to change as a criterion for selecting health status measures . Qual Health Care 1 : 89 – 93 .

Forster LE Lynn J 1988 Predicting life span for applicants to inpatient hospice Arch Intern Med 148 : 2540 – 2543 .

Giacino JT 1997 Disorders of consciousness: Differential diagnosis and neuropathologic features Semin Neurol 17 : 105 – 111 .

Girardin E Grau GE Dayer JM Roux-Lombard P Lambert PH 1988 Tumor necrosis factor and interleukin-1 in the serum of children with severe infectious purpura . N Engl J Med 319 : 397 – 400 .

Goldhill DR Withington PS 1996 Mortality predicted by APACHE II: The effect of changes in physiological values and post-ICU hospital mortality . Anaesthesia 51 : 719 – 723 .

Gordon CJ Fogelson L Highfill JW 1990 Hypothermia and hypometabolism: Sensitive indices of whole-body toxicity following exposure to metallic salts in the mouse . J Toxicol Environ Health 29 : 185 – 200 .

Gourmelon P Briet D Clarencon D Court L Tsiang H 1991 . Sleep alterations in experimental street rabies virus infection occur in the absence of major EEG abnormalities Brain Res 554 : 159 – 165 .

Gourmelon P Briet D Court L Tsiang H 1986 . Electrophysiological and sleep alterations in experimental mouse rabies . Brain Res 398 : 128 – 140 .

Heyse-Moore LH Johnson-Bell VE 1987 Can doctors accurately predict the life expectancy of patients with terminal cancer? Palliat Med 1 : 165 – 166 .

Higginson I McCarthy M 1989 . Measuring symptoms in terminal cancer: Are pain and dyspnea controlled? J R Soc Med 82 : 264 – 267 .

Huang Y-W Richardson JA Tong AW Zhang B-Q Stone MJ Vitetta ES 1993 Disseminated growth of a human multiple myeloma cell line in mice with severe combined immunodeficiency disease . Cancer Res 53 : 1392 – 1396 .

Huang Y-W Richardson JA Vitetta ES 1995 Anti-CD54 (ICAM-1) has antitumor activity in SCID mice with human myeloma cells . Cancer Res 55 : 610 – 616 .

Incalzi RA Landi F Pagano F Capparella O Gemma A Carbonin PU 1998 . Changes in nutritional status during the hospital stay: A predictor of long-term survival . Aging Clin Exp Res 10 : 490 – 496 .

Jackson SR Tweeddale MG Barnett MJ Spinelli JJ Sutherland HJ Reece DE Klingemann H-G Nantel SH Fung HC Toze CL Phillips GL Shepherd JD 1998 . Admission of bone marrow transplant recipients to the intensive care unit: Outcome, survival and prognostic factors . Bone Marrow Transplant 21 : 697 – 704 .

Janczewski KH Chalk CL Pyles RB Parysek LM Unger LW Warnick RE 1998 . A simple, reproducible technique for establishing leptomeningeal tumors in nude rats . J Neurosci Meth 85 : 45 – 49 .

Jones GR 1998 Assessment criteria in identifying the sick sepsis patient . J Infect 37 (Suppl l) : 24 – 29 .

Khan WA Dhar U Salam MA Griffiths JK Rand W Bennish ML 1999 Central nervous system manifestations of childhood shigellosis: Prevalence, risk factors, and outcome . Pediatrics 103 : 1 – 8 .

Knaus WA Draper EA Wagner DP Zimmerman JE 1985 APACHE II: A severity of disease classification system . Crit Care Med 13 : 818 – 829 .

Knaus WA Wagner DP Lynn J 1991 Short-term mortality predictions for critically ill hospitalized adults: Science and ethics . Science 254 : 389 – 394 .

Kort WJ Hekking-Weijma JM TenKate MT Sorm V 1997 A microchip implant system as a method to determine body temperature of terminally ill rats and mice . Lab Anim 32 : 260 – 269 .

Krarup A Chattopadhyay P Bhattacharjee AK Burge JR Ruble GR 1999 . Evaluation of surrogate markers of inpending death in the galactosamine-sensitized murine model of bacterial endotoxemia . Lab Anim Sci 49 : 545 – 550 .

Krishna S Waller DW ter Kuile F Kwiatkowski D Crawley J Craddock CFC Nosten F Chapman D Brewster D Holloway PA White NJ 1994 . Lactic acidosis and hypoglycaemia in children with severe malaria: Pathophysiological and prognostic significance . Trans R Soc Trop Med Hyg 88 : 67 – 73 .

Lawrence M 1995 The unconscious experience . Am J Crit Care 4 : 227 – 232 .

Le Gall J-R Lemeshow S Leleu G Klar J Huillard J Rué M Teres D Artigas A 1995 Customized probability models for early severe sepsis in adult intensive care patients . JAMA 273 : 644 – 650 .

Le Gall J-R Lemeshow S Saulnier F 1993 A new simplified acute physiology score (SAPS II) based on a European/North American multicenter study . JAMA 270 : 2957 – 2963 .

Lemeshow S Teres D Pastides H Avrunin JS Steingrub JS 1985 . A method for predicting survival and mortality of ICU patients using objectively derived weights . Crit Care Med 13 : 519 – 525 .

Lemeshow S Teres D Pastides H Avrunin JS Pastides H 1987 A comparison of methods to predict mortality of intensive care unit patients . Crit Care Med 15 : 715 – 722 .

Louie A Liu W Liu Q-F Sucke AC Miller DA Battles A Miller MH 1997 Predictive values of several signs of infection as surrogate markers for mortality in a neutropenic guinea pig model of Pseudomonas aeruginosa sepsis . Lab Anim Sci 47 : 617 – 623 .

Marano MA Fong Y Moldawer LL Wei H Calvano SE Tracey KJ Barie PS Manogue K Cerami A Shires GT Lowry SF 1990 Serum cachectin/tumor necrosis factor in critically ill patients with burns correlates with infection and mortality . Surg Gynecol Obstet 170 : 32 – 38 .

Marsh K Forster D Waruiru C Mwangi I Winstanley M Marsh V Newton C Winstanley P Warn P Peshu N Pasvol G Snow R 1995 . Indicators of life-threatening malaria in African children . N Engl J Med 332 : 1399 – 1404 .

Martens P Raabe A Johnsson P 1998 Serum S-100 and neuron-specific enolase for prediction of regaining consciousness after global cerebral ischemia . Stroke 29 : 2363 – 2366 .

McQuillen MP 1991 Can people who are unconscious or in the “vegetative state” perceive pain? Issues Law Med 6 : 373 – 383 .

Meduri GU Headley S Tolley E Shelby M Stentz F Postlethwaite A 1995 . Plasma and BAL cytokine response to corticosteroid rescue treatment in late ARDS . Chest 108 : 1315 – 1325 .

Mor V Laliberte L Morris JN Wiemann M 1984 . The Karnovsky performance status scale: An examination of its reliability and validity in a research setting . Cancer 53 : 2002 – 2007 .

Morrow NS Schall M Grijalva CV Geiselman PJ Garrick T Nuccion S Novin D 1997 . Body temperature and wheel running predict survival times in rats exposed to activity-stress . Physiol Behav 62 : 815 – 825 .

Morton DB Griffiths PHM 1985 . Guidelines on the recognition of pain, distress and discomfort in experimental animals and an hypothesis for assessment . Vet Rec 116 : 431 – 436 .

Olfert ED 1996 . Considerations for defining an acceptable endpoint in toxicological experiments . Lab Anim 25 : 38 – 43 .

Parkes CM 1972 . Accuracy of predictions of survival in later stages of cancer Br Med J 2 : 29 – 31 .

Phu NH Day N Diep PT Ferguson DJP White NJ 1995 . Intraleucocytic malaria pigment and prognosis in severe malaria . Trans R Soc Trop Med Hyg 89 : 200 – 204 .

Plum F Schiff N Ribary U Llinas R 1998 . Coordinated expression in chronically unconscious persons . Philos Trans R Soc Lond 353 : 1929 – 1933 .

Prytherch DR Whiteley MS Higgins B Weaver PC Prout WG Powell SJ 1998 . POSSUM and Portsmouth POSSUM for predicting mortality . Br J Surg 85 : 1217 – 1220 .

Rechtschaffen A Gilliland MA Bergmann BM Winter JB 1983 . Physiological correlates of prolonged sleep deprivation in rats . Science 221 : 182 – 184 .

Redgate ES Deutsch M Boggs SS 1991 . Time of death of CNS tumorbearing rats can be reliably predicted by body weight-loss patterns . Lab Anim Sci 41 : 269 – 273 .

Reuben DB Mor V 1986 . Dyspnea in terminally ill cancer patients . Chest 89 : 234 – 236 .

Reuben DB Mor V Hiris J 1988 . Clinical symptoms and length of survival in patients with terminal cancer . Arch Intern Med 148 : 1586 – 1591 .

Schiff ND 1999 . Commentary: Neurobiology, suffering, and unconscious brain states . J Pain Symptom Manage 17 : 303 – 304 .

Schnaper N 1975 . The psychological implications of severe trauma: Emotional sequelae to unconsciousness . J Trauma 15 : 94 – 98 .

Seneff M Knaus WA 1990 . Predicting patient outcome from intensive care: A guide to APACHE, MPM, SAPS, PRISM, and other prognostic scoring systems . J Intensive Care Med 5 : 33 – 52 .

Soothill JS Morton DB Ahmad A 1992 . The HJX>50 (hypothermia-inducing dose 50): An alternative to the LD50 for measurement of bacterial virulence . Int J Exp Path 73 : 95 – 98 .

Stiles BG Campbell YG Castle RM Grove SA 1999 . Correlation of temperature and toxicity in murine studies of staphylococcal enterotoxins and toxic shock syndrome toxin 1 . Infect Immun 67 : 1521 – 1525 .

Tosch P 1988 . Patients’ recollections of their posttraumatic coma . J Neurosci Nurs 20 : 223 – 228 .

Toth LA 1997 . The moribund state as an experimental endpoint . Contemp Top Lab Anim Sci 36 : 44 – 48 .

Toth LA Rehg JE Webster RG 1995 . Strain differences in sleep and other pathophysiological sequelae of influenza virus infection in naive and immunized mice . J Neuroimmunol 58 : 89 – 99 .

Toth LA Tolley EA Krueger JM 1993 . Sleep as a prognostic indicator during infectious disease in rabbits . Proc Soc Exp Biol Med 203 : 179 – 192 .

Tunnell RD Millar BW Smith GB 1998 . The effect of lead time bias on severity of illness scoring, mortality prediction and standardised mortality ratio in intensive care—A pilot study . Anaesthesia 53 : 1045 – 1053 .

Ventafridda V Ripamonti C De Conno F Tamburini M 1990 . Symptom prevalence and control during cancer patients’ last days of life . J Palliat Care 6 : 7 – 11 .

Waller D Krishna S Crawley J Miller K Nosten F Chapman D ter Kuile FO Craddock C Berry C Holloway PAH Brewster D Greenwood BM White NJ 1996 . Clinical features and outcome of severe malaria in Gambian children . Clin Infect Dis 21 : 577 – 587 .

Welsh DK Richardson GS Dement WC 1986 . Effect of age on the circadian pattern of sleep and wakefulness in the mouse . J Gerontol 41 : 579 – 586 .

Wong M-L Bongiorno PB Rettori V McCann SM Licinio J 1997 . Interleukin (IL) 1β, IL-1 receptor antagonist, IL-10, and IL-13 gene expression in the central nervous system and anterior pituitary during systemic inflammation: Pathophysiological implications . Proc Natl Acad Sci U S A 94 : 227 – 232 .

Yates JW Chalmers B McKegney P 1980 . Evaluation of patients with advanced cancer using the Karnovsky performance status . Cancer 45 : 2220 – 2224 .

Zandbergen EGJ de Haan RJ Stoutenbeek CP Koelman JHTM Hikdra A 1998 . Systematic review of early prediction of poor outcome in anoxicischaemic coma . Lancet 352 : 1808 – 1812 .

Abbreviation used in this article: EEG, electroencephalogram.

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How “Humane” Is Your Endpoint?—Refining the Science-Driven Approach for Termination of Animal Studies of Chronic Infection

* E-mail: [email protected]

Affiliation IBMC - Institute for Molecular and Cell Biology (Laboratory Animal Science Group), University of Porto, Porto, Portugal

Affiliations Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal, ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal

• Nuno H. Franco, 
• Margarida Correia-Neves, 
• I. Anna S. Olsson

Published: January 19, 2012

• https://doi.org/10.1371/journal.ppat.1002399
• Reader Comments

Citation: Franco NH, Correia-Neves M, Olsson IAS (2012) How “Humane” Is Your Endpoint?—Refining the Science-Driven Approach for Termination of Animal Studies of Chronic Infection. PLoS Pathog 8(1): e1002399. https://doi.org/10.1371/journal.ppat.1002399

Editor: Glenn F. Rall, The Fox Chase Cancer Center, United States of America

Copyright: © 2012 Franco et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: Nuno H. Franco is funded by Fundação para a Ciência e Tecnologia (SFRH/BD/38337/2007). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Public concern on issues such as animal welfare or the scientific validity and clinical value of animal research is growing, resulting in increasing regulatory demands for animal research. Abiding to the most stringent animal welfare standards, while having scientific objectives as the main priority, is often challenging. To do so, endpoints of studies involving severe, progressive diseases need to be established considering how early in the disease process the scientific objectives can be achieved. We present here experimental studies of tuberculosis (TB) in mice as a case study for an analysis of present practice and a discussion of how more refined science-based endpoints can be developed. A considerable proportion of studies in this field involve lethal stages, and the establishment of earlier, reliable indicators of disease severity will have a significant impact on animal welfare. While there is an increasing interest from scientists and industry in moving research in this direction, this is still far from being reflected in actual practice. We argue that a major limiting factor is the absence of data on biomarkers that can be used as indicators of disease severity. We discuss the possibility of complementing the widely used weight loss with other relevant biomarkers and the need for validation of these parameters as endpoints. Promotion of ethical guidelines needs to be coupled with systematic research in order to develop humane endpoints beyond the present euthanasia of moribund animals. Such research, as we propose here for chronic infection, can show the way for the development and promotion of welfare policies in other fields of research.

Research on chronic infection relies heavily on the use of animals, as only the integral animal body can model the full aspect of an infection. That animals are generally made to develop a disease in infection studies exacerbates the tension between human benefit and animal well-being, which characterizes all biomedical research with animals. Scientists typically justify animal research with reference to potential human benefits, but if accepting the assumption that human benefits can offset animal suffering, it still needs to be argued that the same benefits could not be achieved with less negative effects on animal welfare. Reducing the animal welfare problems associated with research (“refinement” [1] ) is therefore crucial in order to render animal-based research less of an ethical problem and to assure public trust in research.

Studies that are designed to measure time of death or survival percentages present a particularly challenging situation in which at least some of the animals are made to die from the disease. These studies are frequent in experimental research on severe infections. The scientific community, industry, and regulatory authorities have responded to the ethical concerns over studies in which animals die from severe disease by developing new policies and guidelines for the implementation of humane endpoints as a key refinement measure (e.g., [2] – [4] ). The most widely used definition considers a humane endpoint to be the earliest indicator in an animal experiment of severe pain, severe distress, suffering, or impending death [5] , underlining that ideally such indicators should be identified before the onset of the most severe effects.

Euthanizing animals, rather than awaiting their “spontaneous” death, is important to avoid unnecessary suffering in studies in which data on survival is thought to be required for scientific or legal reasons. However, several questions remain open regarding how humane endpoints are to be applied to address real animal welfare problems. We used TB experiments in mice as a case study to highlight the potential to establish biomarkers of disease progress that can replace survival time as a measure of disease severity.

Humane Endpoints Applied to Murine TB Experiments—State of the Arts

To illustrate the state of the art in the implementation of humane endpoints, we present data from a systematic review of articles published on murine TB in 2009. Papers were selected by performing an advanced search on the ISI Web of Science database (search performed in June 2011, using the previous 4.1 version, accessible at http://webofknowledgev4.com ) with the terms “TS = ((mouse OR mice) SAME tuberculosis) AND PY = (2009)” and refined to exclude reviews, proceedings, articles in languages other than English, in vitro studies, and studies on other animal species and/or with other infectious agents than Mycobacterium tuberculosis , which resulted in a total of 80 papers reviewed. The severity of disease of the animals used in each study was analyzed in terms of the disease stage animals were allowed to reach, the carrying out of invasive procedures and the implementation of relevant refinement measures, and humane endpoints in particular. All studies in which animals were allowed to die spontaneously or to reach a moribund state were considered among the highest level of severity. Also, depending on whether studies were terminated before animals reached advanced stages of disease (which would rapidly progress towards spontaneous death if no other endpoints were applied) or not, these were classified as “lethal” or “non-lethal”, respectively. Lethal studies comprised those conducted in very susceptible strains as well as in more resistant mouse strains in which disease was allowed to progress to very advanced stages. Nearly half (47%) of the studies fit the lethal category, of which 66% were classified within the highest severity level. Although 26% of lethal studies explicitly reported having applied humane endpoints to avoid spontaneous death, in most of these studies (eight out of ten) humane endpoints were applied when animals were moribund and severely cachectic or when other animals in the same group began to die. Thus, most such endpoints consisted of merely replacing spontaneous death with euthanasia of moribund animals and may therefore fail to have a significant impact on the degree of poor animal well-being. Of the 25 studies considered within the highest severity, 24 had reported some sort of regulatory compliance, of which 17 explicitly stated that the study had been ethically approved.

Are Near-Death Endpoints Really Humane?

The attention to humane endpoints is reflected in an increase in their implementation. This is clearly visible when reviewing research on murine TB: while the use of humane endpoints is reported in 26% of the terminal infection studies published in 2009, the same figure for 1999 is 15% (three of 20 papers reporting studies on lethal infections). However, a careful analysis reveals a significant limitation of humane endpoints as they are often presently applied. In fact, from both scientific and ethical perspectives, it is questionable if euthanasia of moribund animals is the most effective measure to prevent excessive animal suffering. It is admittedly difficult to estimate how much awareness of aversive sensations an individual retains in such a debilitated, unresponsive, and seemingly comatose state as the moribund animal is in [6] . But considering that the moribund state constitutes a terminal stage of a progressively distressing disease, the main animal welfare problem consists in the unrelieved suffering that precedes it. Furthermore, there is also the risk that researchers (as well as journal editors and animal ethics / animal care and use committees) may see euthanasia of moribund animals as a sufficient measure to guarantee the “ethicality” of their studies, thus hindering the pursuit for more welfare-relevant endpoints. An additional scientific concern pertains to the definition of the “moribund state” in itself; it is seldom defined in the material and methods section of the published articles. Therefore, it is unclear which clinical signs are used as indicators of imminent or pending death, and how soon before the estimated time of death they are detectable.

The Potential for Scientific Refinement of Endpoints in Studies on Chronic Infection—Murine TB as a Case Study

We focus here on mice models for TB as a case study to discuss the potential for refining endpoints from both animal welfare and scientific viewpoints. TB is representative of chronic infection research in which, typically, adult animals are studied in experiments that last several weeks. The long duration means a greater potential — as well as a stronger animal welfare reason — for developing more refined early endpoints than in infections of a more acute nature. Mice experimentally infected with M. tuberculosis develop TB as a progressive disease. Although mouse strains vary in their susceptibility to infection [7] , all mice eventually succumb to experimental TB [8] , [9] and die before their natural average life-span if no measures are undertaken to treat them. Even the more TB-resistant C57BL/6 mice have a median survival time of fewer than 300 days [8] , while uninfected animals live over 800 days [10] , irrespective of the number of viable bacteria with which they are infected.

Time to death, when animals are found dead in the cage, is a frequently used outcome measure in experimental TB studies. Of studies published in 2009, 31% (25 out of 80) used death (18 out of 25) or the moribund stage (7 out of 25) as endpoints. Of notice and certainly as a consequence of awareness of animal welfare issues, an increasing number of studies are defining death as the time point when moribund animals are euthanized, rather than the time when they are found dead. This approach improves scientific accuracy both in that the exact time of death is known and in that biological samples can be collected immediately post-mortem. The informative value of time to death for understanding disease physiopathology is, however, limited by the fact that the actual cause of death may vary between animals: mice infected with M. tuberculosis may die from different lung pathologies in response to experimental infection [8] , [11] – [13] , as well as from causes only indirectly related to infection such as sepsis [14] , hunger, or dehydration [15] .

While euthanasia of moribund animals is an improvement compared to using death as an endpoint, as we have argued above, it is still a rather inefficient measure to safeguard animal welfare. Nevertheless, in the absence of validated predictors of death, this may presently be the best researchers can do if they need to establish whether animals reached a state from which they will deteriorate and die or might still recover. Identification of the turning point towards terminal disease, with greater understanding of the underlying host and pathogen factors leading to this stage, may provide endpoint measures that reliably reflect disease progression. Research on early predictors of a terminal disease stage (sometimes termed “surrogate endpoints” [16] ), at times in which scientifically relevant data can be obtained before animals reach pronounced levels of distress, should therefore be encouraged.

Humane endpoint protocols are usually based on a combination of clinical signs; however, objectively and numerically measurable parameters are sometimes included [17] . Amidst these, percentage of body weight loss is the most commonly used as a cut-off parameter for euthanasia in experimental TB studies in mice, since it can be used as an objective measure of infection-related morbidity in this animal model (e.g., [18] ). While non-infected mice tend to maintain or gain body weight, infected and untreated mice start to lose weight on account of active disease, in an irreversible and fairly linear manner [19] , [20] – [23] . A consistent correlation between weight loss and survival has been reported specifically for backcrossed animals derived from A/Sn and I/St mice [23] – [25] , which led Nikonenko and coworkers [25] to propose that drug efficacy can be evaluated through the systematic assessment of body weight changes in rapidly progressive TB in C3H mice, an immunocompetent but TB-susceptible strain.

Although this parameter is in itself quantitative and objectively measurable, the definition of the upper boundary values for euthanasia is potentially subjective, unless previously validated against data on disease progress and survival. Threshold weight losses vary considerably across studies, ranging from 15% of pre-infection weight [26] or of the average weight of control mice [27] to more severe 20% [28] , 25% [29] , or even 30% weight loss [30] , with no scientific justification for choosing different cut-off points in different studies.

Systematic studies, in which data on biomarkers are collected repeatedly as the disease progresses and biomarker data is correlated with survival, are essential to develop new potential predictors of death/survival. Such studies need to be carried out specifically for distinct combinations of mouse and bacterial strains, with the aim of detecting selected biomarkers that indicate the point of no return after which animals do not recover. In addition to body weight, non-transient hypothermia has been suggested as a potentially useful predictor of death in some models of infection [31] – [36] . Hypothermia typically settles when disease reaches a near fatal stage and body temperature (which can be measured without handling stress by either infrared thermometers [35] or telemetric monitoring [36] ) may constitute an important refinement of current moribund endpoints, particularly if scored with other clinical parameters [16] . A non-invasive method for the assessment of blood oxygen saturation in a murine model of viral pulmonary disease has also been shown to be a more reliable indicator of lung pathology and predictor of disease outcome than body weight loss [37] . This indicator of lung function has the potential to be used as a measure of morbidity in other pulmonary diseases if this can be validated in further research. Animals dying from distinct prolonged chronic infections have been shown to present other pathophysiological alterations that could be carefully studied as potential blood biomarkers of approaching death. These include anaemia and/or erythropoiesis and the metabolic perturbations in the glycolytic enzyme pathway with consequent altered blood glucose homeostasis, parameters that might be easily analyzed in blood samples and have been shown to correlate with the degree of disease severity [38] , [39] . Biomarkers related to the immune response to infection are also described as potential indicators, reflecting the animals’ capacity to control the infection. In addition to the most widely studied cytokines associated with susceptibility/resistance to TB, like IFN-γ, TNF, IL-1, and IL-10 analyzed individually or in combinations [40] – [42] , others have been suggested. A set of transcriptional biomarkers was recently shown to serve as diagnostic and prognostic tools, by the identification of a TB-specific 86-gene transcriptome signature in whole-blood samples, with the transcriptional signature being shown to correlate with extent of disease in patients with active TB and to also reflect changes at the site of disease [43] . However, while potentially clinically interesting, its measurement might be cumbersome unless a smaller number of transcripts proves to be still valid as molecular signature.

Meeting Ethical Standards by a Science-Based Approach to Endpoints

It is praiseworthy that researchers, institutions, the public, and the authorities are all more aware of the need to establish ethical guidelines and rules in animal research to minimize distress. With that purpose, euthanasia is nowadays commonly established in most animal facilities when animals reach distress levels considered to be beyond an “acceptable” threshold [44] . There are, however, several problems with such a practice being imposed on research from external entities. First, the level found acceptable is bound to be somewhat arbitrary, depending on factors such as the research institution guidelines and the opinions of the appointed veterinarian, animal welfare officer, and members of the ethics committee. Second, from a scientific viewpoint, it is crucial to make sure that experimental objectives are not compromised by loss of experimental data due to unexpected deaths or compulsory euthanasia. Therefore, the establishment of earlier endpoints should ideally be centered on the earliest time point allowing the collection of adequate scientific information and valuable biological material, rather than on welfare-centered criteria.

As discussed above, in many cases additional research will be necessary to establish the correspondence of early clinical signs and final disease outcome. This calls for a new type of refinement research, in which measures to reduce animal welfare problems are developed specifically for the various fields of research and in close collaboration between laboratory animal specialists and researchers using animal models. Some such initiatives have emerged on the national level (e.g., National Centre for the 3Rs in the United Kingdom), but major international funding is unfortunately still limited to either the actual biomedical research itself or alternative methods to replace animal experiments, thus excluding studies on how to refine actual animal use.

Surrogate endpoints in the true sense constitute a crucial refinement of studies where a reliable measure of lethality is assumed to be necessary, in particular when these studies are long-term. We emphasize, however, the collection of informative data in less severe phases as the ideal approach for basic and applied research, allowing for a science-based, rather than welfare-centered justification for the termination of such experiments.

With respect to interventional euthanasia, although presently necessary to avoid further suffering of animals reaching unacceptably severe pain or other distress, it must be emphasized that this intervention ought to be more of an exception than the rule, and that studies should be planned so that such interventions are unnecessary.

Animal research is valuable for identifying mechanisms of disease and novel therapies. The increased awareness of the ethical issues pertaining to animal experimentation requires that relevant scientific information and biological materials are obtained as early as possible, avoiding that animals have to reach severe stages of disease. In research on progressive diseases, it is pertinent to identify early surrogate endpoints of death as well as reliable biomarkers of disease progression. Changes in body weight and temperature, along with biomarkers easily measured in blood samples, should be carefully investigated as potential early predictors of death/survival. A science-driven approach for the termination of animal studies may not only prevent unnecessary and avoidable suffering, but also contribute to optimizing financial and human resources, enhancing the scientific output and speeding up the scientific process. The real humane endpoint challenge is to extend this approach into situations where presently the scientific outcome measure requires that animals reach more severe stages. While acknowledging current efforts by funding bodies and charities, regulators, industry, and academia in promoting and developing new standards for animal welfare, we stress that further collaborative research as well as funding for such research is essential to achieve more meaningful refinement of studies of chronic infections.

Protein Accession Numbers/IDs

• Ifng interferon gamma [ Mus musculus ]; Gene ID: 15978; Protein ID: NP_032363
• Tnf tumor necrosis factor [ Mus musculus ]; Gene ID: 21926; Protein ID: NP_038721
• Il1b interleukin 1 beta [ Mus musculus ]; Gene ID: 16176; Protein ID: NP_032387
• Il10 interleukin 10 [ Mus musculus ]; Gene ID: 16153; Protein ID: NP_034678

Acknowledgments

The authors would like to thank David B. Morton and Joana Palha for reading earlier versions of this manuscript and making suggestions, and Coenraad Hendriksen for input on humane endpoint development.

• 1. Russell WMS, Burch RL (1959) The principles of humane experimental technique London: Methuen & Co. Ltd.
• View Article
• Google Scholar
• 3. NRC (2009) Recognition and alleviation of pain in laboratory animals. Washington (D.C.): National Academies Press.
• 4. Guittin P, Decelle T (2002) Future improvements and implementation of animal care practices within the animal testing regulatory environment. ILAR J 43 Suppl. pp. S80–S84.
• 5. OECD (2000) Guidance document on the recognition, assessment and use of clinical signs as humane endpoints for experiemental animal used in safety evaluation. Paris: Organisation for Economic Co-operation and Development.
• 15. Mench J (1999) Defining endpoints: the role of animal care committees. In: Morton CFMHaDB, editor. Humane endpoints in animals experiments for biomedical research. London: Royal Society of Medicine Press. pp. 133–138.
• 16. Morton DB (2006) Ethical issues in the use of animal models of infection and some practical refinements. In: Friedman H, Specter S, Bendinelli M, editors. In vivo models of HIV disease and control. pp. 405–424. Springer US.

Humane Endpoints and End of Life in Primates Used in Laboratories

• First Online: 02 February 2023

Cite this chapter

• Sarah Wolfensohn 3  

Deciding when and how to end an animal’s life are critical when managing its welfare. This chapter gives an overview of the use of nonhuman primates in science, and of the European regulations and the ethical perspective of justifying such use with a harm:benefit assessment. It reviews the definitions of a humane endpoint and considers how to set that endpoint to limit the harms by objectively assessing the animal’s welfare. The setting of humane endpoints should be incorporated into management systems with other considerations around the end of life of nonhuman primates, and applied to their use in other contexts, such as in zoos and sanctuaries.

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American Veterinary Medical Association (2001) American Veterinary Medical Association report of the AMVA panel on euthanasia. J Am Vet Med Assoc 218(5):669–696. https://doi.org/10.2460/javma.2001.218.669

Article   Google Scholar  

Animal Science Committee (2017) Review of harm-benefit analysis in the use of animals in research. Home Office

Google Scholar  

Bailey J (2022) Arguments against using nonhuman primates in research. In: Robinson LM, Weiss A (eds) Nonhuman primate welfare: from history, science, and ethics to practice. Springer, Cham, pp 547–574

Bayne K, Hau J, Morris T (2022) The welfare impact of regulations, policies, guidelines and directives and nonhuman primate welfare. In: Robinson LM, Weiss A (eds) Nonhuman primate welfare: from history, science, and ethics to practice. Springer, Cham, pp 629–646

Bloomsmith M, Perlman J, Franklin A, Martin AL (2022) Training research primates. In: Robinson LM, Weiss A (eds) Nonhuman primate welfare: from history, science, and ethics to practice. Springer, Cham, pp 517–546

Boly M, Seth AK, Wilke M et al (2013) Consciousness in humans and non-human animals: recent advances and future directions. Front Psychol 4:625. https://doi.org/10.3389/fpsyg.2013.00625

British Veterinary Association (2016) British Veterinary Association Euthanasia of animals guidelines. https://www.bva.co.uk/media/2981/bva_guide_to_euthanasia_2016.pdf . Accessed 16 June 2021

Brønstad A, Newcomer CE, Decelle T et al (2016) Current concepts of harm–benefit analysis of animal experiments–report from the AALAS–FELASA working group on harm–benefit analysis, part 1. Lab Anim 50(1 Suppl):1–20. https://doi.org/10.1177/0023677216642398

Canadian Council on Animal Care (1998) Canadian Council on Animal Care Guidelines on choosing an appropriate endpoint in experiments using animals for research and testing. https://ccac.ca/Documents/Standards/Guidelines/Appropriate_endpoint.pdf . Accessed 16 June 2021

Carstens E, Moberg GP (2000) Recognizing pain and distress in laboratory animals. ILAR J 41(2):62–71. https://doi.org/10.1093/ilar.41.2.62

Article   CAS   Google Scholar  

Chatfield K, Morton D (2018) The use of non-human primates in research. In: Schroeder D, Cook J, Hirsch F, Fenet S, Muthuswamy V (eds) Ethics dumping. SpringerBriefs in research and innovation governance. Springer, Cham. https://doi.org/10.1007/978-3-319-64731-9_10 . Accessed 16 June 2021

Close B, Banister K, Baumans V et al (1996) Recommendations for euthanasia of experimental animals: part 1. Lab Anim 30(4):293–316. https://doi.org/10.1258/002367796780739871

Close B, Banister K, Baumans V et al (1997) Recommendations for euthanasia of experimental animals: part 2. Lab Anim 31(1):1–32. https://doi.org/10.1258/002367797780600297

Davies G, Golledge H, Hawkins P, Rowland A, Wolfensohn S, Smith J, Wells D (2017) Review of harm-benefit analysis in the use of animals in research. Report of the Animals in Science Committee Harm-Benefit Analysis Sub-group. https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/675002/Review_of_harm_benefit_analysis_in_use_of_animals_18Jan18.pdf . Accessed 16 June 2021

European Commission (2010) Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes. https://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2010:276:0033:0079:en:PDF . Accessed 16 June 2021

Gilbert C, Wolfensohn S (2011) Veterinary ethics and the use of animals in research: are they compatible? In: Wathes C, Corr S, May S et al (eds) Veterinary and animal ethics: Proceedings of the first International Conference on Veterinary and Animal Ethics. Wiley-Blackwell, Oxford, pp 155–173

Gleerup KB, Forkman B, Lindegaard C, Andersen PH (2015) An equine pain face. Vet Anaesth Analg 42(1):103–114. https://doi.org/10.1111/vaa.12212

Halpern-Lewis JG (1996) Understanding the emotional experiences of animal research personnel. Contemp Top Lab Anim Sci 35(6):58–60

CAS   Google Scholar  

Hendriksen C, Cussler K, Morton D (2011) Use of humane endpoints to minimize suffering. In: Howard B, Nevalainen T, Peratta G (eds) The COST manual of laboratory animal care and use. CRC Press, Boca Raton, FL, pp 333–354

Home Office (2014) Guidance on the operation of the animals (Scientific Procedures) Act 1986. https://www.gov.uk/guidance/research-and-testing-using-animals . Accessed 16 June 2021

Home Office (2015) The harm-benefit analysis process: New project licence applications. Advice Note: 05/2015. Animals in Science Regulation Unit, Home Office, London. https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/487914/Harm_Benefit_Analysis__2_.pdf . Accessed 16 June 2021

Honess PE, Wolfensohn S (2010a) The extended welfare assessment grid: a matrix for the assessment of welfare and cumulative suffering in experimental animals. Altern Lab Anim 38(3):205–212. https://doi.org/10.1177/026119291003800304

Honess PE, Wolfensohn S (2010b) Welfare of exotic animals in captivity. In: Tynes VV (ed) Behavior of exotic pets. Wiley-Blackwell, Oxford, p 215

Honess PE, Marin C, Brown AP, Wolfensohn SE (2005) Assessment of stress in non-human primates: application of the neutrophil activation test. Anim Welf 14(4):291–295

Jennings M, Prescott MJ (2009) Refinements in husbandry, care and common procedures for non-human primates. Lab Anim 43(Suppl 1):1–47. https://doi.org/10.1258/la.2008.007143

Justice WSM, O’Brien MF, Szyszka O et al (2017) Adaptation of the animal welfare assessment grid (AWAG) for monitoring animal welfare in zoological collections. Vet Rec 181(6):143–143. https://doi.org/10.1136/vr.104309

Langford DJ, Bailey AL, Chanda ML et al (2010) Coding of facial expressions of pain in the laboratory mouse. Nat Methods 7:447–449. https://doi.org/10.1038/nmeth.1455

Lloyd MH, Foden BW, Wolfensohn SE (2008) Refinement: promoting the 3Rs in practice. Lab Anim 42(3):284–293. https://doi.org/10.1258/la.2007.007045

Luttrell L, Acker L, Urben M, Reinhardt V (1994) Training a large troop of rhesus macaques to co-operate during catching: analysis of time investment. Anim Welf 3:135–140

Lutz CK, Baker KC (2022) Using behavior to assess primate welfare. In: Robinson LM, Weiss A (eds) Nonhuman primate welfare: from history, science, and ethics to practice. Springer, Cham, pp 171–206

Main DCJ, Whay HR, Green LE, Webster AJF (2003) Preliminary investigation into the use of expert opinion to compare the overall welfare of dairy cattle farms in different farm assurance schemes. Anim Welf 12(4):565–569

Mellor DJ (2012) Affective states and the assessment of laboratory-induced animal welfare impacts. ALTEX 444–449

Morton DB (2000) A systematic approach for establishing humane endpoints. ILAR J 41(2):80–86. https://doi.org/10.1093/ilar.41.2.80

Morton DB, Griffiths PH (1985) Guidelines on the recognition of pain, distress and discomfort in experimental animals and an hypothesis for assessment. Vet Rec 116(16):431–436. https://doi.org/10.1136/vr.116.16.431

Nuffield Council on BioEthics (2005) The ethics of research involving animals. https://www.nuffieldbioethics.org/publications/animal-research . Accessed 16 June 2021

Organisation for Economic Cooperation and Development (2000) Guidance document on the recognition, assessment, and use of clinical signs as humane endpoints for experimental animals used in safety evaluation. https://www.ncbi.nlm.nih.gov/books/NBK32660/ . Accessed 16 June 2021

Pekow CA (1994) Suggestions from research workers for coping with research animal death. Lab Anim (NY) 23:28–29

Reinhardt V (2003) Working with rather than against macaques during blood collection. J Appl Anim Welf Sci 6(3):189–197. https://doi.org/10.1207/s15327604jaws0603_04

Reinhardt V, Liss C, Stevens C (1995) Restraint methods of laboratory non-human primates: a critical review. Anim Welf 4(3):221–238

Rennie AE, Buchanan-Smith HM (2006) Refinement of the use of non-human primates in scientific research. Part III: refinement of procedures. Anim Welf 15(3):239–261

Russell W, Burch R (1959) The principles of humane experimental technique. Methuen & Co, London

Sauceda R, Schmidt MG (2000) Refining macaque handling and restraint techniques. Lab Anim 29:47–49

Sebel PS, Bowdle TA, Ghoneim MM et al (2004) The incidence of awareness during anesthesia: a multicenter United States study. Anesth Analg 99(3):833–839. https://doi.org/10.1213/01.ane.0000130261.90896.6c

Smith E, Delargy M (2005) Locked-in syndrome. BMJ 330(7488):406–409. https://doi.org/10.1136/bmj.330.7488.406

Sotocina SG, Sorge RE, Zaloum A et al (2011) The rat grimace scale: a partially automated method for quantifying pain in the laboratory rat via facial expressions. Mol Pain 7:1–10. https://doi.org/10.1186/1744-8069-7-55

t’Hart BA, Laman JD, Kap YS (2022) An unexpected symbiosis of animal welfare and clinical relevance in a refined nonhuman primate model of human autoimmune disease. In: Robinson LM, Weiss A (eds) Nonhuman primate welfare: from history, science, and ethics to practice. Springer, Cham, pp 591–612

Wallace J (2000) Humane endpoints and cancer research. ILAR J 41(2):87–93. https://doi.org/10.1093/ilar.41.2.87

Wolfensohn S (2010a) Euthanasia and other fates for laboratory animals. In: Hubrecht RC, Kirkwood J (eds) The UFAW handbook on the care and management of laboratory and other research animals, 8th edn. Wiley-Blackwell, Oxford, pp 219–226

Chapter   Google Scholar  

Wolfensohn SE (2010b) Old World monkeys. In: Hubrecht RC, Kirkwood J (eds) The UFAW handbook on the care and management of laboratory and other research animals, 8th edn. Wiley-Blackwell, Oxford, pp 592–617

Wolfensohn S (2020) Too cute to kill? The need for objective measurements of quality of life. Animals 10(6):1054. https://doi.org/10.3390/ani10061054

Wolfensohn S, Finnemore P (2006) Refinements in primate husbandry: a DVD training resource

Wolfensohn S, Honess PE (2007) Laboratory animal, pet animal, farm animal, wild animal: which gets the best deal? Anim Welf 16:117–123

Wolfensohn SE, Lloyd MH (2013) Handbook of laboratory animal management and welfare, 4th edn. Wiley-Blackwell, Oxford

Wolfensohn S, Sharpe S, Hall I, Lawrence S, Kitchen S, Dennis M (2015) Refinement of welfare through development of a quantitative system for assessment of lifetime experience. Anim Welf 24:139–149. https://doi.org/10.7120/09627286.24.2.139

Wolfensohn S, Shotton J, Bowley H, Davies S, Thompson S, Justice W (2018) Assessment of welfare in zoo animals: towards optimum quality of life. Animals 8(7):110. https://doi.org/10.3390/ani8070110

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Wolfensohn, S. (2023). Humane Endpoints and End of Life in Primates Used in Laboratories. In: Robinson, L.M., Weiss, A. (eds) Nonhuman Primate Welfare. Springer, Cham. https://doi.org/10.1007/978-3-030-82708-3_16

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Humane Endpoints

How to Select Humane Endpoints  | Body Condition Scoring   | Welfare Assessment Scoring Systems  

Evaluating Health and Welfare in Research Rodents

The accuracy and translatability of animal-based research is directly dependent on the health and welfare of the animals used. Animals that are distressed, painful or excessively stressed are not normal and data from these animals are likely to be inaccurate. Investigators are responsible for recognizing when situations and procedures may adversely affect their animals. Sources of adverse effects can include:

• Genetic background and manipulation
• Experimental procedures (e.g., substance administration, blood collection, surgery, etc.)
• Husbandry (e.g., housing, breeding, diet, etc.)
• Handling (including routine)
• Transport (including within the research facility)

For each experimental study, the investigator should develop a welfare assessment plan that identifies all procedures and situations may cause distress, pain and/or excessive stress in their animals and indicate any physiological or behavioral signs expected as a result. These signs will be used as indicators when evaluating each animal’s health and welfare. Monitoring frequency and humane endpoints must also be specified for each identified procedure and situation. As the study progresses, welfare indicators may be reviewed and updated as needed to improve effectiveness.

In addition to identifying welfare indicators, investigators must specify what treatments or actions will occur when indicator(s) are noticed. These may include intervention points and/or humane endpoints. Intervention points are specific therapies or treatments provided to an animal when certain clinical signs or indicators appear. For example, fluid therapy may be provided if signs of dehydration are found, or especially palatable food provided if weight loss is >/= ten percent. Humane endpoints (the time at which an animal is removed from the study; usually via euthanasia in rodents) must also be defined.

Selecting Appropriate Welfare Indicators

To be effective, welfare indicators must be objective, easily and reliably recognized, relevant to the project and species and practical to use. Indicators may be behavioral or physiological and can be generally applicable or specific for an individual model or project. Rather than relying on a single indicator, using multiple behavioral and physiological indicators, both general and specific, will provide a more detailed and complete picture of an animal’s welfare and prevent interpretation errors 1 .

Potential indicators of poor animal welfare can include:

• Changes in physical condition such as loss of body weight, abnormal coat condition or posture; lameness; excessive licking or scratching.
• Changes in heart rate, respiratory rate and character, blood pressure, or stress hormone levels.
• Deviations from “normal” behavior (e.g., apathy or withdrawal, increased aggression, stereotypic behavior). These may also include changes in the use of enrichment or behavioral time budgets (e.g., decreased nest building or excessive sleeping).

Welfare Assessment Scoring Systems

It is often helpful to use a numerical scoring system to assess the health and welfare of animals on study. A form called a score sheet is used to record the health status of individual animals at regular, predetermined intervals. The welfare indicators monitored on a score sheet must be objective, easy to evaluate and relevant to the clinical signs expected for that procedure or situation. Scores from multiple indicators are often added up and the result used to determine whether action is needed according to a predetermined key. Scoring systems must be adapted for each study/model so that appropriate indicators are used.

Investigators are expected to provide training to staff and students who will be using a scoring system to ensure they are competent and able to recognize and deal with welfare issues. Outside observers such as veterinary or IACUC personnel should be able to easily read and understand how scores are determined and decisions made based on the scores received. Analysis of an animal's score sheet must clearly show the effect of the procedure/experiment on the animal's health including a pattern of either recovery or deterioration over time. It is also recommended that a description of the welfare assessment protocol be included in the methods section of peer-reviewed journal papers as part of the scientific method 1 . 

Examples of Welfare Assessment Scoring Systems

Detailed examples of scoring systems for various rodent and rabbit models may be found in the online “ European Commission Expert Working Group: Examples to illustrate the process of severity classification, day-to-day assessment and actual severity assessment ” (2013) document - scroll down the page to " Severity assessment – illustrative examples pdf" . These examples are meant to illustrate the development and use of welfare assessment protocols for specific laboratory animal studies. Please refer to this document for detailed information on how scoring systems may be used.

• Hawkins P, Morton DB, Burman O, Dennison N, Honess P, Jennings M, Lane S, Middleton V, Roughan JV, Wells S, Westwood K; UK Joint Working Group on Refinement BVAAWF/FRAME/RSPCA/UFAW. June 2010. A guide to defining and implementing protocols for the welfare assessment of laboratory animals: eleventh .

Humane endpoints refer to one or more predetermined physiological or behavioral signs that define the point at which an experimental animal’s pain and/or distress is terminated, minimized or reduced by taking actions such as euthanizing the animal, terminating a painful procedure or giving treatment to relieve pain and/or distress ( CCAC ). Humane endpoints function as an alternative to experimental endpoints and provide investigators with an effective way to refine their research. The establishment of humane endpoints prior to the start of an experiment allows the investigator to prevent unnecessary animal pain and distress while ensuring accurate and timely data collection.

To be effective, humane endpoints must be clearly defined and based on objective criteria. Non-specific signs of illness such as inactivity, hunched posture or a rough coat are an indication that an animal should be examined more closely. By themselves these signs do not often constitute an endpoint. Familiarity with the animal model in use is necessary to select endpoints that are both humane and scientifically sound. As experience with and data collected from a specific animal model accrue, endpoints can be refined or modified. Further information on humane endpoints may be found on these and other websites:

Humane Endpoints in Laboratory Animal Experimentation UC Davis Center for Animal Alternatives USDA National Agricultural Library Canadian Council on Animal Care

Investigators should include a precise definition of the humane endpoint(s), including specific assessment criteria, when describing how humane endpoints will be used in their IACUC protocols. The frequency of animal observation and assessment must also be clearly stated. Note:  Normal, healthy experimental animals must be observed at least once a day. Animals in studies involving pain and/or distress will often require more frequent observations to effectively determine the time at which a specific endpoint has been reached. An appropriate monitoring schedule must be specified in the IACUC protocol for each study. In addition, the IACUC protocol must describe the training provided for personnel responsible for observation and assessment and the action(s) to be taken when an animal reaches a humane endpoint.

Type of Humane Endpoints

Humane endpoints are often based on the following:

• Clinical signs
• Pathophysiological changes
• Behavioral changes
• Biochemical changes
• Hormonal changes

The exact time of the endpoint (the point at which an animal is removed from study) will depend on the objective of the experiment but should occur before the onset of distress (i.e., unable to adapt completely to a stressor) or as soon as possible thereafter.

Moribund Animals and Death as an Endpoint

The term moribund refers to an animal that is near death or in the process of dying. Animals in this state are often comatose (unresponsive and unaware of stimuli) and so beyond awareness of suffering. However, an animal may have experienced much pain and distress prior to reaching a moribund state. Stating that animals will be euthanized when they become moribund is not an appropriate humane endpoint as this may not reduce or alleviate any suffering that the animal will experience. The purpose of identifying endpoints is to prevent or minimize animal pain and distress. While certain types of studies have historically used death of the animal as a scientific endpoint, this is now rarely accepted and investigators must present conclusive evidence to support the use of such an endpoint.

How to select and use humane endpoints:

Choose appropriate endpoints that are objective and relevant for the assessment of pain/distress in the species. This may include:

• Body weight changes
• External physical appearance
• Physiological changes (e.g., body temperature, hormonal fluctuations, clinical pathology, etc…)

Research personnel responsible for observing and evaluating animals must be adequately trained and experienced in the recognition of these signs for the species being used. Especially when using behavioral assessment, personnel must be familiar with “normal” before they can be expected to recognize “abnormal”. Investigators are responsible for ensuring these students and employees are appropriately trained and have the skill and authority to treat or euthanize animals who have reached an endpoint.

Pilot studies (experiments) can be useful in determining endpoints, especially when the effects of an experimental treatment in animals are not well known. They may also function to refine experimental studies by allowing for the establishment of earlier endpoints and provide training for personnel in the recognition of endpoints.

Examples of humane endpoints:

Deteriorating body condition score

• Objective and easy to use for assessing the condition of animals used in research, especially studies where animals may experience some degree of debilitation as the study progresses.
• Scoring methods have been developed for many species including mice.
• Weight loss
• Rapid weight loss of 15-20 percent within a few days. This requires frequent monitoring of body weight.
• Gradual weight loss - over an extended period of time leading to emaciation. The degree of weight loss should be specified in terms of % or quantity (grams, pounds, kg).
• Note: Certain debilitating conditions such as tumor growth and ascites may mask true weight loss.

Changes in nest building behavior in mice

• Studies have shown that use of nesting material and nest quality decrease with increasing pain/distress in mice.
• Mice must be housed individually and provided a sufficient amount of material to construct a nest. In addition, the mouse must build a new nest each day  for accurate evaluation to occur (e.g., either replacing the nest with new, unconstructed material or disrupt the nest structure and evaluate the mouse's ability to reconstruct using the used material).

The inability to rise or ambulate

• Correlates with inability to access food or water.
• Visually obvious, objective and easy to assess.
• Usually measured as mean diameter of the mass or tumor volume as a percentage of body weight (i.e. greater than 1.5 cm diameter in mice or greater than 10% of body weight)

The presence of labored respiration

• The animal shows increased respiratory rate and/or effort. Labored respiration is often accompanied by a strong abdominal component to breathing.
• Dehydration
• The skin looses its elasticity. In a hydrated animal, skin pinched over the back quickly returns to its normal position after it is released. In a dehydrated animal the skin is slow to return to normal or remains tented. Please note, dehydration must be moderate to severe before skin tenting will be noticable. Elderly, very young, obese or thin animals may show altered responses.

Ulcerated, necrotic or infected tumors.

• The presence of open wounds.

Suggestions for the use of humane endpoints in selected studies:

Chronic studies where some degree of debilitation is expected.

Body Condition Scoring

• Weight Loss
• Loss of ability to ambulate (inability to access food or water).
• Labored respiration may be associated with lung pathology or abdominal enlargement placing pressure on the diaphragm.

Experimental Neoplasia

• Tumor size - tumor volume or mean diameter.
• Tumor ulceration, infection, or necrosis.
• For internal tumors body condition scoring may be more useful than weight loss.

Acute Studies

Experimental Surgery

Many of the same signs as listed above in addition to conditions specific to post-surgical infections or other complications including:

• Pain, swelling, redness or discharge from surgical incisions.
• Dehiscense (splitting apart) of surgical incisions.

These signs may not by themselves be endpoints but are medical issues that suggest there are deficiencies in surgical techniques or care that require attention.

Body condition scoring is a health assessment method useful for many species and can be used as a humane endpoint. Body condition scoring systems were originally designed to evaluate fat and muscle development in livestock. Scoring techniques for many species, including rodents, have since been published and are widely used to assess health and fitness in animals. Body condition scoring offers an objective and easy to use assessment method that can be incorporated into humane endpoints. Use of body condition scoring can provide a more accurate determination of health and fitness than body weight measurements, especially in studies where animals may develop tumor masses or fluid accumulation that obscures true weight loss. In addition, body condition scoring is useful in chronic studies where animals may lose muscle mass and fat over time.

A body condition scoring method for mice was developed by Ullman-Cullere, MH and CJ Foltz. Body condition scoring: A rapid and accurate method for assessing health status in mice. Laboratory Animal Science 1999, 49 (3):319-323.

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National Research Council (US) Committee on Recognition and Alleviation of Pain in Laboratory Animals. Recognition and Alleviation of Pain in Laboratory Animals. Washington (DC): National Academies Press (US); 2009.

Recognition and Alleviation of Pain in Laboratory Animals.

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5 Humane Endpoints for Animals in Pain

This chapter presents an overview of the concept of humane endpoints and their application in studies that cause pain in research animals. It sets the stage with a review of pertinent guidance documents, focusing on the Organization for Economic Cooperation and Development (OECD) 2000 Guidance on Humane Endpoints for Experimental Animals Used in Safety Evaluation. It provides a discussion of the usefulness of pilot studies as a refinement and potential replacement tool. Further, it presents humane endpoints in relation to specific research fields—toxicology, infectious diseases, vaccine safety, cancer, and pain. It concludes with a discussion of euthanasia.

• GUIDELINES AND REFERENCE DOCUMENTS

Moral and ethical obligations are inherent in all aspects of research, testing, and teaching that use research subjects. The question of when a study using animal models should end or the study design be changed due to animal pain, distress, or welfare considerations has been the subject of many publications, symposia, guidance documents, and regulations. Defining a humane endpoint can vary widely depending on a number of factors, of which study design and research objectives are but two. Consequently, attempting to provide specific endpoint criteria for all study designs and other factors cannot be adequately addressed in this one report ( Morton 1999 , 2000 ). Not only would such a list be inadequate, it could prove detrimental to hitherto unknown study objectives. This report does not go into specifics but rather presents selected pertinent guidelines and documents. Investigators, study personnel, veterinary staff, and institutional animal care and use committees (IACUCs) are obligated to thoroughly research and incorporate humane endpoints in every study or use involving laboratory animals.

National and International Guidelines

A number of national and international guidelines are available to assist researchers in determining humane endpoints for research animals. The Office of Laboratory Animal Welfare (OLAW) defines these as “[c]riteria used to end experimental studies earlier in order to avoid or terminate unrelieved pain and/or distress are referred to as humane endpoints. An important feature of humane endpoints is that they should ensure that study objectives will still be met even though the study is ended at an earlier point. Ideally, humane endpoints are sought that can be used to end studies before the onset of pain and distress” ( OLAW/ARENA 2002 , p. 103).

The Canadian Council for Animal Care (CCAC) has published an excellent document with general recommendations on humane endpoints in animal studies. According to the CCAC guidelines, in “experiments involving animals, any actual or potential pain, distress, or discomfort should be minimized or alleviated by choosing the earliest endpoint that is compatible with the scientific objectives of the research. Selection of this endpoint by the investigator should involve consultation with the laboratory animal veterinarian and the animal care committee” ( CCAC 1998 , p. 5).

In 1994, the OECD recognized that while ambiguous test guidelines may be necessary, such ambiguity fosters an overbroad interpretation of what constitutes a humane endpoint in toxicology studies. The organization therefore created a working group to develop a guidance document using clinical signs as humane endpoints in safety evaluation studies ( OECD 2000 ; Box 5-1 ). The resulting document put forth criteria based on the principles of the 3Rs as well as descriptions of clinical signs to assist study personnel in determining when death may be imminent or when severe pain may be present after an animal’s exposure to a test substance. The criteria are broad enough to apply to a wide range of study types, test substances, species, and strains of animals. The reader is encouraged to examine this resource when developing internal guidance documents to assess humane endpoints.

OECD Guidance Document on the Recognition, Assessment, and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation (OECD 2000). A humane endpoint can be defined as the earliest indicator in an animal experiment of (more...)

OECD invested considerable time and effort in addressing and defining potential endpoints in safety assessment studies (see the Addendum at the end of this chapter for the OECD definition). The OECD Guidance Document defines humane endpoints “as the earliest indicator in an animal experiment of severe pain, severe distress, suffering, or impending death. The ultimate purpose of the application of humane endpoints to toxicology studies is to be able to accurately predict severe pain, severe distress, suffering, or impending death, before the animal experiences these effects” ( OECD 2000 , p. 10). While the OECD indicated that the science of toxicology cannot accurately predict pain prior to onset, careful observations can “identify pain, distress, or suffering, very early after their onset . . . using well-defined endpoints and criteria.” The OECD further advises that suffering “should be minimized or eliminated, either by humanely killing the animal or, in long-term studies, by (temporary) termination of exposure, or by reduction of the test substance dose. Different animal species, and animals at different stages of development, may respond differently to test conditions, and exhibit different indications of distress” (ibid.).

These guidance documents are consistent in their recommendations. Predictive parameters must be reliable, reproducible, and objective, and allow both the achievement of study objectives and goals and the use of appropriate methodologies at the earliest point to alleviate or avoid pain. As discussed below, pilot studies are an effective means to identify and validate humane endpoints, which can then be incorporated in research methods to minimize, alleviate, or avoid pain for the animal subjects (also see Morton 1999 , 2000 ; Stokes 2002 ; NRC 2008 , p. 61).

Humane endpoints were the focus of a 1998 international conference in Ziest, The Netherlands. The editors of the conference proceedings determined that humane endpoints are specific to individual studies or a particular testing paradigm ( Hendriksen and Morton 1999 , pp. v–vi), based on study design and intent, regulatory requirements, personnel connected to the study, and the animals themselves, whether as individuals or as a group. The conference participants concluded that the establishment of humane endpoints is, and should be, subject to adaptation as societal mores, attitudes, regulations, and technologies change. The conference report further stated that for ethical reasons, the formulation of endpoints to avoid or alleviate pain in laboratory animals must be a high ethical priority in every facility that conducts any form of animal experimentation (ibid.).

Beyond Formal Guidelines

Many of the articles and recommendations that address humane endpoints focus on very specific study or research types that can cause pain to laboratory animals; for example, studies on the identification and use of humane endpoints in animal models of sepsis and shock provide an excellent overview of the methodologies to determine humane endpoints yet still achieve study objectives ( Nemzek et al. 2004 , 2008 ). More generally, the Institute for Laboratory Animal Research (ILAR) bases its reports on its mission statement promoting “high-quality science and humane care and use of research animals based on the principles of refinement, replacement, and reduction (the 3Rs) and high ethical standards” ( ILAR 2009 ). The Institute’s Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research ( NRC 2003 ) provide criteria for evaluating levels of pain that help in the development of endpoints for studies in neuroscience and behavioral research. An ILAR Journal issue dedicated to Humane Endpoints for Animals Used in Biomedical Research and Testing ( ILAR 2000 ) provides an overview of several research areas where pain is a potential outcome, including infectious disease and cancer research ( Olfert and Godson 2000 ; Wallace 2000 ) and vaccine potency and and acute toxicity testing ( Hendriksen and Steen 2000 ; Sass 2000 ). ILAR also published the proceedings of a symposium on Regulatory Testing and Animal Welfare, detailing best practices for the humane conduct of animal testing for regulatory purposes ( NRC 2004 ).

While these references are extremely valuable, it is important to view them in accordance with their intent: they are guidance documents only and as such have limitations. No single document could cover all potentially painful study types, all animal species used in research, or all clinical signs associated with all research projects. In the absence of comprehensive guidance, the scientific community has an ethical responsibility to develop a general humane endpoint policy at each institution to provide guidance and a basis for dialogue between scientists and IACUCs about individual protocols.

Caution is advisable, however, in efforts to develop a policy on humane endpoints. While the ideal is to avoid pain, personnel also need to ensure that the study objectives are attained before a procedure or animal is terminated ( OLAW/ARENA 2002 , p. 103). If a full study, or aspect of a study, is ended before the objectives have been met, one can argue that the animals used have been wasted. Moreover, if the purpose of a study is to meet the requirements for the safety assessment of a substance, a regulatory agency may reject the submitted data as insufficient and require that the study be repeated. On the other hand, if researchers are reluctant to intervene, study animals may unnecessarily experience pain, distress, or severely diminished welfare. Further, without adequate guidance, death is likely to be selected as a convenient endpoint that is reproducible and objective. If regulatory guidelines do not specify an endpoint, as in vaccine potency studies ( CFR Title 9, 2006 ), regulated entities can and will use lethality.

For all these reasons identification of humane endpoints should take into account the following factors: the role of regulatory agencies in the overall process; the need for scientifically appropriate endpoints; and the reliability of clinical observations of the animals to ensure a proper outcome for both the animals and the study. As a corollary, it is worth emphasizing that investigators, technicians, and other staff responsible for the care of research animals should be well trained and able to make impartial judgments about an animal’s well-being.

OLAW approached the subject of humane endpoints in its Institutional Animal Care and Use Committee Guidebook ( OLAW/ARENA 2002 , p. 103), advising internal oversight committees to review protocols to determine whether “discomfort to animals will be limited to that which is unavoidable for the conduct of scientifically valuable research, and [whether] unrelieved pain and distress will only continue for the duration necessary to accomplish the scientific objectives.” The OLAW reference is careful to state that potential pain or distress should be relieved with appropriate medication or with euthanasia, although the study objectives should still be met. The intent is to end a study before the development of pain or distress, as is emphasized in the OECD document.

• PILOT STUDIES

An effective way to reduce negative impacts on laboratory animals is the use of a pilot study, which can be critical to the success of a larger study ( DeHaven 2002 ; Morton et al. 1990 ; NRC 2003 , p. 14; NRC 2008 , pp. 61–62; OECD 2000 , p. 14). The premise behind this concept is to conduct the proposed study on a small number of animals rather than the full complement necessary for a statistically valid study and thus prevent unnecessary pain for a larger number of animals.

Pilot studies are advantageous because they help researchers to identify:

• potential interactions between proposed analgesic and anesthetic treatments and specific research goals,
• potentially useful means of assessing pain in a specific research model, and
• humane endpoint criteria specific to an individual project.

Problems that occur in the pilot study can inform the discussion and development of strategies to address an animal’s deteriorating condition. Such strategies may include (but certainly not be limited to) the adjustment of dose levels, changes in sample size, identification of adverse effects, incorporation of refinements (e.g., use of analgesics, procedural changes), or alteration to the duration of exposure to minimize negative impacts on the animals.

Caution is essential in the design and conduct of pilot studies as the risk of causing significant pain to the animals in such studies can be high. This risk necessitates close oversight by the IACUC and careful monitoring of the animals by study personnel and veterinary staff. Good communication among all involved can ensure both the collection of the maximum amount of useful data and appropriate interventions on behalf of the animals ( NRC 2003 , p. 14).

• INTERNATIONAL REGULATIONS AND GUIDELINES FOR SAFETY ASSESSMENT

Regulatory bodies in most countries have developed standards and guidelines to ensure the conduct of appropriate safety assessments on test substances ( Hicks 1997 ; Merrill 2001 ; USEPA 2008 ). For example, after the use of thalidomide by pregnant women in the 1960s caused severe birth defects in the long bones of the fetuses, US legislation required adequate testing of drugs in animals before human exposure ( Gallo 2001 ; Nies 2001 ). Similar legislative actions followed environmental disasters like the Love Canal contamination ( Merrill 2001 ).

The purpose of testing requirements for pharmaceutical, consumer, and industrial products is to ensure the safety of the environment and of the human and animal populations. However, these requirements have tended to focus on the safety of the user and do not necessarily consider humane endpoints for the animals used in the safety assessment, although such consideration is becoming a more prominent component of some newer regulatory requirements.

In June 2007, the European Commission established a regulation to evaluate the hazards and risks of chemicals (Regulation (EC) No. 1907/2006 of the European Parliament and of the Council of 18 December 2006); the mission of REACH (Registration, Evaluation, and Authorization of Chemicals) is to improve the assessment of chemicals in order to better protect human health and the environment. Because the range of chemicals covered by REACH is enormous, there is great potential for increased use of animals in corresponding toxicity and safety testing. But the regulation ensures the authorization of animal testing only when necessitated by identification of data gaps ( ECHA 2008 ). Furthermore, the regulation requires industry to share data on similar chemicals to avoid duplicative animal testing; allows for the submission of data using nonanimal tests; strongly encourages the use of Quantitative Structure-Activity Relationship (QSAR) or other computer-generated information; and invites the grouping of submitted data for similar chemicals that may result in similar hazards and risks (the so-called “read-across” principle). While these efforts do not define humane endpoints, the authors of the regulation are commended for the consideration of responsible animal use in safety assessment.

Also useful in the toxicology regulatory arena is a February 2008 Memorandum of Understanding (MOU) that lays the foundation and framework for the US Environmental Protection Agency (EPA) and two NIH agencies to collaborate in sharing data, resources, and expertise in efforts to replace animal testing for chemical toxicity assessment ( Collins et al. 2008 ; NIH/USEPA 2008 ; NIH 2008 ). The MOU calls for the evaluation of in vitro assays, such as those used for identification of toxicity pathways and high-throughput screening (as described in NRC 2007 ), to better predict potential health and environmental hazards from chemicals. The ambitious goals of the MOU are the development of more accurate assays and changes in regulatory guidelines, both of which are likely to be a long-term process. Similar goals should be encouraged on a global scale to effect change in regulatory agencies and eliminate potentially painful animal testing.

Although harmonization of regulatory guidelines has significantly reduced discrepancies between cooperating countries, efforts for the global harmonization of safety guidelines are neither consistent nor well coordinated. As a result, tests must comply with all the requirements of each country where a product is to be marketed for a particular use. For example, the regulatory agency of one country may require an additional group of animals to assess recovery from exposure, while other countries may not have this requirement or may even reject the study depending on their review process. Or one country’s regulatory agency may accept an alternative that has been validated as scientifically reliable and relevant ( NIH 1997 ), such as the local lymph node assay in mice, whereas agencies in other countries may not accept the data in lieu of the guinea pig dermal sensitization test.

While a comparison of all safety assessment guidelines is well beyond the scope of this report, differences in regulatory-driven studies can have a negative impact on the prevention and alleviation of pain in laboratory animals. An example of a safety assessment test that may cause pain is the acute eye irritation study, the purpose of which is to evaluate the potential hazards of ocular exposure to a substance. Although requirements for this procedure are generally in agreement across international regulatory bodies and national agencies ( JMAFF 2000 ; OECD 1987 , #405; USEPA OPPTS 1998 , #870.2400), the same is not true for the reversibility of ocular lesions, an additional requirement of this test in order to more fully assess the risk of human exposure. The procedures for this component of the toxicity evaluation vary considerably with respect to animal welfare. The OECD guidelines recommend a step-wise evaluation paradigm that starts with assessment of structurally related substances and other in vitro tests prior to any animal use. The guidelines also identify ocular lesions that are considered irreversible and thereby meet OECD criteria for terminating the study and euthanizing the animal. But while guidelines in various countries reference the OECD guidance document for humane endpoints and recommend the use of local anesthetics in cases of extreme pain, they do not recognize the OECD criterion for early termination of the study (identification of irreversible lesions).

• HUMANE ENDPOINTS IN TOXICOLOGY STUDIES

In recognition of the pain and distress inflicted on animals in many safety and toxicology studies, regulatory guidelines have begun to address the concept of humane endpoints, although sometimes in vague terms. The EPA Health Effects Test Guidelines for Acute Oral Toxicity ( USEPA OPPTS 2002 ) provide instruction for following the OECD Guidance Document ( OECD 2000 ) to reduce the suffering of animals in toxicity studies. Euthanasia of animals that are either moribund or in severe pain is also encouraged. Regrettably, vague statements such as “animals showing severe and enduring signs of distress and pain may need to be humanely killed,” which are common in regulatory guidelines ( USEPA OPPTS 1998 ), may promote a reluctance to terminate a study or an animal’s exposure to the testing substance because a regulatory body may consider the action premature and mandate a repeat study. This is not a good situation for researchers, laboratories, or animals.

Not all test substances cause ocular (or other) pain or injury, but the potential exists. As pointed out by Durham and colleagues (1992) , there is a gap in the data for analgesia appropriate for use in ocular toxicity tests and that gap persists, as evidenced in a US Federal Register notice ( Federal Register 2007 ) requesting data on analgesic use in ocular irritancy tests to alleviate pain without affecting test results. Current guidelines include neither justification for withholding analgesic agents nor guidance for the use of analgesic agents to alleviate ongoing pain. As a result, testing entities may be reluctant to provide analgesia beyond initial local anesthetics, to avoid the possibility of interference with the test substance ( Stokes 2005 ). Yet numerous published studies demonstrate that the use of analgesics to alleviate pain from ocular irritancy tests does not interfere with the scientific objectives of this safety test ( Patrone et al. 1999 ; Peyman et al. 1994 ; Stiles et al. 2003 ). Such evidence can be used to avoid or alleviate pain as well as to provide scientific rationale for the use of analgesics in ocular irritancy tests.

Chronic toxicity and carcinogenicity testing are currently required to assess effects after long-term, repeated exposure to a test substance ( JMAFF 2000 ; OECD 1987 , #405; USEPA OPPTS 1998 , #870.2400). The incidence of tumor burden, geriatric changes, and premature death can be significant near the scheduled termination of these studies. Guidelines generally specify the survival rates necessary to provide meaningful interpretation of a chronic study, but the OECD document is the only one to discuss humane endpoints and provide guidance for the early termination of a study if survival rates fall below a specified percentage. In order to achieve the required survival rate at the end of the mandated study, animals often are not euthanized until very close to death, an outcome that may entail needless pain for the animals. True harmonization of guideline safety assessment tests and global adoption of the OECD humane endpoints document would be an important step toward the alleviation and avoidance of pain in laboratory animals.

The NRC report Toxicity Testing in the Twenty-first Century: A Vision and a Strategy ( NRC 2007 ) evaluated current toxicity testing schemes and developed a long-term strategy for the direction of safety assessments based on state-of-the-art sciences (e.g., genomics, proteomics, and pharmacokinetics) and emerging technologies (e.g., bioinformatics). Although the report acknowledges that implementation of the strategy will require much effort on the part of scientists, regulators, and law makers to develop workable testing schemes, the concepts envisioned could significantly improve the science of toxicology, assessment of risk to human safety, alleviation of pain in laboratory animals, and reduction or replacement of animals in toxicity testing (ibid.).

One of the sources reviewed for the NRC report was the approach developed by the Health and Environmental Sciences Institute (HESI) of the International Life Sciences Institute (ILSI). In 2000, this organization convened an Agricultural Chemical Safety Assessment (ACSA) committee to redesign safety testing schemes for agricultural chemicals. The resulting multifaceted approach redesigns traditional toxicology tests to integrate several sciences, such as metabolism/kinetics and life stages, in a single study to eliminate the requirement for separate studies to evaluate each parameter and reduce the number of animals used ( Carmichael et al. 2006 ; Cooper et al. 2006 ; Doe et al. 2006 ; ILSI-HESI 2008 ). Further, the metabolism/kinetics component of the strategy is particularly relevant to the alleviation of pain in laboratory animals: based on the metabolism of a test substance in the animal model, a saturation point can be determined and used as the high dose level in subsequent studies because it is considered more relevant to actual human exposure levels. This approach, based on step-wise, or tiered, testing, is expected to reduce animal numbers, minimize potential pain to laboratory animals by avoiding exposure levels that produce clinical signs of toxicity, and improve the quality of data for assessments of risk to humans ( Carmichael et al. 2006 ).

• HUMANE ENDPOINTS IN INFECTIOUS DISEASE RESEARCH

There has been an increase in infectious disease research as a result of bioterrorism threats and anthrax attacks since September 11, 2001 ( Copps 2005 ; Jaax 2005 ). Whether the disease agent is of interest for bioterrorism or for human or animal welfare, the study of a targeted disease typically involves exposing healthy research animals to a disease agent that culminates in clinical disease and death. The animals may experience significant pain during these experiments, but identification and validation of earlier endpoints to safeguard animal welfare can be difficult, as an inappropriate endpoint may not adequately identify the full course of a disease or the efficacy of a potential medication ( Olfert and Godson 2000 ). It is imperative, therefore, to examine and validate endpoints within a solid scientific framework that includes, among others, immunological parameters, biochemical and endocrine changes, and other pathophysiologic changes (e.g., decreased body temperature). Moreover, eliminating death as the endpoint for infectious disease research can benefit not only the laboratory animals but the research itself because pathological changes are easier to identify in fresh tissues as opposed to autolyzed tissues from animals that have been allowed to die ( Copps 2005 ).

• HUMANE ENDPOINTS IN VACCINE SAFETY AND POTENCY TESTING

Another area of research that frequently results in the death of study animals is vaccine testing for regulatory agencies. Because vaccines are biological products and one batch may not be as potent as the next or may contain harmful byproducts, it is important to test both their efficacy and safety ( Castle 1999 ; Cussler et al. 1999 ; Hendriksen 2002 ). To ensure quality control and the safety of each batch, regulatory agencies such as the US Food and Drug Administration (FDA), the US Department of Agriculture (USDA), the European Pharmacopoeia, and the World Health Organization (WHO) require potency testing during which animals are vaccinated and then exposed to the virulent disease agent. However, the endpoint for each potency test is not consistent across disease agents. In some instances, regulations require that a certain percentage of control animals die before a test is considered valid, while other tests are based on the survival of the vaccinated animals. For example, the FDA-administered safety test for general biological products requires vaccination of healthy guinea pigs and mice with a small dose of the final product from each vaccine lot ( CFR 2008 , 610.11). A safety test is considered unsatisfactory if the animals do not survive the 7-day test period, in which case additional safety tests over a larger test population are required. The USDA-mandated potency testing for Leptospira pomona bacterin ( CFR 2006 , 113.101) requires that at least eight of ten unvaccinated control animals die in order to validate the test. Other potency testing may require a comparison of death rates in the vaccinated versus control animals, as, for example, in the USDA safety test for Marek’s disease vaccine ( CFR 2006 , 113.330). For this type of testing a more humane endpoint would be the onset of clinical signs in unvaccinated controls; thus for example the potency test for tetanus antitoxin is met when unvaccinated control guinea pigs are unable to stand within 24 hours postchallenge, at which point the animals may be euthanized ( CFR 2006 , 113.451).

Regulations may also encourage the use of in vitro methods. The USDA canine distemper killed virus vaccine potency test ( CFR 2006 , 113.201) accepts serum titer levels in vaccinated animals for potency data; if, however, the tests are inconclusive, a viral challenge test is required, using both vaccinated and unvaccinated controls. The agency identifies the survival of all vaccinated animals and the death of all controls as a satisfactory indicator of both the safety and efficacy of a canine distemper vaccine batch.

While lethality may be the easier endpoint because of its objectivity and simplicity ( Cussler et al. 1999 ), it is always worthwhile to identify reliable markers of predictive or impending mortality to serve as alternative and more humane endpoints. No purpose is served when the administration of a vaccine results in harm rather than protection but, as with all research studies and testing guidelines, there must be a balance between effective safety evaluation and humane endpoints for the sake of the laboratory animal.

• HUMANE ENDPOINTS IN CANCER RESEARCH

Identification of humane endpoints in cancer research can be challenging. Although the wide range of tumor types and scientific objectives associated with this research prohibits standardization of humane endpoints ( Wallace 1999 , 2000 ), the United Kingdom Coordinating Committee on Cancer Research (UKCCCR) has developed a document to guide researchers working with animal models ( UKCCCR 1988 ). Investigators should evaluate tumor size, tumor appearance, and animal condition to identify reliable indicators that may permit earlier termination of a study, and establish and validate endpoints that retain scientific objectives and avoid, minimize, or alleviate potential pain in the laboratory animals. Avoiding death or excessive tumor burden, particularly when coupled with clinical signs of pain or distress, should be a desirable goal in cancer research studies.

• HUMANE ENDPOINTS IN PAIN RESEARCH

Of critical importance to this report, as well as to improvements in quality of life for both humans and animals, is research on pain itself, including the mechanisms of pain and methods of pain alleviation. Complicating the ethical issues inherent in producing pain in research subjects is the ability to accurately predict and measure pain responses in animals ( Le Bars et al. 2001 ; Meyerson and Linderoth 2006 ; Walker et al. 1999 ). It is imperative for pain investigators to establish endpoints in each study design to minimize the duration and intensity of the pain and to validate those endpoints for the integrity, objectivity, and reproducibility of the study. Productive dialogue between the IACUC and researcher is critical for ensuring the best outcome for both the animals’ welfare and the study objectives in these research programs ( Mench 1999 ).

Euthanasia, the act of inducing death without pain, is an acceptable method for relieving or alleviating pain that cannot be controlled by other means ( NRC 1992 , pp. 102–104). The humane death of an animal is one in which the animal is first rendered unconscious, and thus insensitive to pain, as rapidly as possible and with a minimum of fear and anxiety. A humane death, or endpoint, is a fundamental tenet of the US Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training (IRAC 2001), as Principle VI states that “[a]nimals that would otherwise suffer severe or chronic pain that cannot be relieved should be painlessly killed at the end of the procedure or, if appropriate, during the procedure.”

There is no rigidly defined point at which euthanasia should be performed for humane reasons, as it is not possible to apply a single set of euthanasia criteria across all study designs, animal models, and experimental goals. The decision should involve a team approach among veterinarians, study directors, and animal care personnel using all available information about the affected animal(s). Body condition scores, as described in Chapter 3 , can be used to determine when to consider euthanasia for humane reasons. The earliest possible indicators for euthanasia should be clearly identified so as to avoid pain and yet still achieve study objectives.

Methods of euthanasia have recently been updated by the American Veterinary Medical Association ( AVMA 2007 ), although objective information on laboratory animals is sparse, particularly concerning the evaluation of potential pain and distress that may be caused by a particular euthanasia technique. The controversy that may result from this lack of data is evident in the recent discussions about the use of carbon dioxide on rodents ( ACLAM 2005 ; AVMA 2007 ; Conlee et al. 2005 ; Hawkins et al. 2006 ; Kirkden et al. 2008 ; Niel et al. 2008 ; NRC 2003 ; Stephens et al. 2002 ). As conversations on this subject will likely continue, the reader is encouraged to follow the published literature for the most up-to-date information.

For all these reasons, well-designed objective studies of euthanasia across all laboratory animal species and age groups are needed and recommended. The assessment tools and measures to consider for such studies include electroencephalograms, electrocardiograms, electromyograms, arterial blood pressure, respiration and heart rates, serum biochemical parameters, pupil diameter, and behavioral changes. In particular, there is an urgent need for studies that provide measures of nociception, pain, distress, and the relation of these to loss of consciousness.

• CONCLUSIONS AND RECOMMENDATIONS

Avoiding or minimizing pain in animal research is a fundamental obligation of all researchers for moral and ethical reasons. The criteria for early termination of a research project or alteration to a study design for the purpose of alleviating or avoiding pain in an animal are defined as humane endpoints. Identification and validation of humane endpoints should be considered for studies involving pain, but this is neither an easy nor a simple process.

• It is important to ensure that endpoints are validated and based on sound science. Pilot studies are invaluable for the determination of earlier and more humane endpoints.
• Given the wide scope of procedures and goals of animal research, no single reference can document every humane endpoint for every research protocol. Therefore, more effort must be made to identify appropriate humane endpoints for each. Good communication between researchers, veterinary staff, animal care staff, and the IACUC is crucial.
• Productive strides have been made in the harmonization of safety assessment guidelines between countries but global harmonization is not yet complete. For global acceptance of humane endpoints in safety assessment test guidelines, dialogue should continue between all countries and agencies responsible for animal welfare, the environment, and human safety.
• Efforts should continue in the development and validation of alternative procedures for incorporation in research projects and safety assessment tests to avoid or alleviate pain in laboratory animals.

Hendriksen and Morton (1999) observed that the goal of developing humane endpoints in animal experiments is constantly shifting. All scientists, managers, technicians, oversight committees, and regulators involved with animal experimentation where pain is a potential component should participate in regular communication and creative problem solving. The criteria for determining the humane end to a study should be frequently reevaluated and revised as new information becomes available. The sustained pursuit of these directed efforts can, and will, result in more humane animal use.

As stated in this chapter, the establishment of surrogate or humane endpoints as part of the experimental protocol and before experiments commence is one of the ways to minimize and alleviate pain and safeguard the well-being and welfare of laboratory animals. In support of this goal, two sample resources are provided for adaptation and use. The first is a score sheet to assess animals in cancer studies based on a behavioral and tumor scoring system ( Table 5A-1 ). The recorded symptomatology will determine the diagnosis and measures for alleviation. The sheet can be adapted to any protocol or animal care facility system as long as the behavioral definitions are uniform across the same facility. The second resource is a model for developing guidelines for humane endpoints that may be suitable for any protocol within a facility ( Box 5A-1 ).

Sample Tumor Scoring Sheet.

Guidelines for Humane Endpoints in Animal Studies. PURPOSE : To assure compliance with the Animal Welfare Act (AWA), the Guide for the Care and Use of Laboratory Animals (the “Guide”) and (institutionally relevant) policies, as well as (more...)

• ACLAM (American College of Laboratory Animal Medicine). Public Statements: Report of the ACLAM Task Force on Rodent Euthanasia. 2005. [Accessed January 5, 2008]. Available at www ​.aclam.org/print/report_rodent_euth ​.pdf .
• AVMA (American Veterinary Medical Association). AVMA Guidelines on Euthanasia. 2007. Available at www ​.avma.org/issues/animal_welfare ​/euthanasia.pdf .
• AWIC (Animal Welfare Information Center). Humane Endpoints. [Accessed January 5, 2008]. Available at http://awic ​.nal.usda.gov/nal .
• Carmichael NG, Barton HA, Boobis AR, Cooper RL, Dellarco VL, Doerrer NG, Fenner-Crisp PA, Doe JE, Lamb JC 4th, Pastoor TP. Agricultural chemical safety assessment: A multisector approach to the modernization of human safety requirements. Crit Rev Toxicol. 2006; 36 (1):1–7. [ PubMed : 16708692 ]
• Castle P. The European pharmacopoeia and humane endpoints. In: Hendriksen CFM, Morton DB, editors. Humane Endpoints in Animal Experiments for Biomedical Research; Proceedings of the International Conference; 22–25 November 1998; Ziest, The Netherlands. The Royal Society Medical Press; 1999. pp. 15–19.
• CCAC (Canadian Council on Animal Care). Guidelines on choosing an appropriate endpoint in experiments using animals for research, teaching and testing. 1998. [Accessed January 5. 2008]. Available at www ​.ccac.ca/en/CCAC_Programs ​/Guidelines_Policies ​/GDLINES/ENDPTS/g_endpoints.pdf .
• CFR (Code of Federal Regulations). 2006. [Accessed January 5 2008]. 9 CFR 113.33. Available at www ​.access.gpo.gov/nara ​/cfr/waisidx_00/9cfrv1_00.html .
• CFR. 2008. [Accessed October, 26 2008]. Regulations 21 CFR 610.11. Available at www ​.accessdata.fda.gov ​/scripts/cdrh/cf-docs/cfcfr/CFRSearch ​.cfm?fr=610.11 .
• Collins FS, Gray GM, Bucher JR. Transforming environmental health protection. Science. 2008; 319 :906–907. [ PMC free article : PMC2679521 ] [ PubMed : 18276874 ]
• Conlee KM, Stephens ML, Rowan AN, King LA. Carbon dioxide for euthanasia: Concerns regarding pain and distress, with special reference to mice and rats. Lab Anim. 2005; 39 :137–161. [ PubMed : 15901358 ]
• Cooper RL, Lamb JC, Barlow SM, Bentley K, Brady AM, Doerrer NG, Eisenbrandt DL, Fenner-Crisp PA, Hines RN, Irvine LF, Kimmel CA, Koeter H, Li AA, Makris SL, Sheets LP, Speijers G, Whitby KE. A tiered approach to life stages testing for agricultural chemical safety assessment. Crit Rev Toxicol. 2006; 36 (1):69–98. [ PubMed : 16708695 ]
• Copps J. Issues related to the use of animals in biocontainment research facilities. ILAR J. 2005; 46 (1):34–43. [ PMC free article : PMC7108559 ] [ PubMed : 15644562 ]
• Cussler K, Morton DB, Hendriksen CFM. Humane endpoints in vaccine research and quality control. In: Hendriksen CFM, Morton DB, editors. Humane Endpoints in Animal Experiments for Biomedical Research; Proceedings of the International Conference; 22–25 November 1998; Ziest, The Netherlands. The Royal Society Medical Press; 1999. pp. 95–101.
• DeHaven WR. Best practices for animal care committees and animal oversight. ILAR J. 2002; 43 (Suppl):S59–S62. [ PubMed : 12388853 ]
• Doe JE, Boobis AR, Blacker A, Dellarco V, Doerrer NG, Franklin C, Goodman JI, Kronenberg JM, Lewis R, McConnell EE, Mercier T, Moretto A, Nolan C, Padilla S, Phang W, Solecki R, Tillbury L, van Ravenzwaay B, Wolf DC. A tiered approach to systemic toxicity testing for agricultural chemical safety assessment. Crit Rev Toxicol. 2006; 36 (1):37–68. [ PubMed : 16708694 ]
• Durham RA, Sawyer DC, Keller WF, Wheeler CA. Topical ocular anesthetics in ocular irritancy testing: A review. Lab Anim Sci. 1992; 42 (6):535–541. [ PubMed : 1479802 ]
• ECHA (European Chemicals Agency). REACH Guidance. 2008. [Accessed January 2, 2009]. Available at http://guidance ​.echa.europa.eu/
• Department of Health and Human Services, National Toxicology Program (NTP), NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM). Request for Data on the Use of Topical Anesthetics and Systemic Analgesics for In Vivo Eye Irritation Testing. Federal Register Notice. May 9, 2007. Available at http://iccvam ​.niehs.nih ​.gov/SuppDocs/FedDocs/FR/FR_E7_8898 ​.pdf .
• Gallo MA. History and scope of toxicology. In: Klaassen CD, editor. Casarett and Doull’s Toxicology: The Basic Science of Poisons. 6th Edition. New York: McGraw Hill Medical Publishing Division; 2001. pp. 3–10.
• Hawkins P, Playle L, Golledge H, Leach M, Banzett R, Coenen A, Cooper J, Danneman P, Flecknell P, Kirkden R, Niel L, Raj M. Newcastle Consensus Meeting on Carbon Dioxide Euthanasia of Laboratory Animals. 2006. Available at www ​.nc3rs.org.uk/downloaddoc ​.asp?id=416&page ​=292&skin=0 .
• Hendriksen CFM. Refinement, reduction, and replacement of animal use for regulatory testing: Current best scientific practices for the evaluation of safety and potency of biologicals. ILAR J. 2002; 43 (Suppl):S43–S48. [ PubMed : 12388851 ]
• Hendriksen CFM, Morton DB, editors. Humane Endpoints in Animal Experiments for Biomedical Research. Proceedings of the International Conference; 22–25 November 1998; Ziest, The Netherlands. London: The Royal Society Medical Press; 1999.
• Hendriksen CFM, Steen B. Refinement of vaccine potency testing with the use of humane endpoints. ILAR J. 2000; 41 :105–113. [ PubMed : 11417495 ]
• Hicks JM. Animal welfare and toxicology/safety studies: Making sense of the regulatory environment. Cont Topics. 1997; 36 (3):49–54. [ PubMed : 16450954 ]
• ILAR (Institute for Laboratory Animal Research). Humane Endpoints for Animals Used in Biomedical Research and Testing. ILAR J. 2000. Available at http://dels ​.nas.edu/ilar_n ​/ilarjournal/41_2/
• ILAR. Mission and Core Values. 2009. Available at http://del ​.nas.edu/ilar_n ​/ilarhome/mission.shtml .
• ILSI-HESI (International Life Sciences Institute–Health and Environmental Sciences Institute). Agricultural Chemical Safety Assessment factsheet. 2008. [Accessed July 14, 2009]. Available at http://www. hesiglobal.org/files/public/Factsheets/AgriculturalChemicalSafetyAssessment.pdf .
• IRAC (Interagency Research Animal Committee). The U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training. Federal Register. May 20, 1985. 1985. [Accessed June 9, 2008]. Available at http://grants ​.nih.gov ​/grants/olaw/references/phspol ​.htm#USGovPrinciples . [ PubMed : 11655791 ]
• Jaax J. Administrative issues related to infectious disease research in the age of bioterrorism. ILAR J. 2005; 46 (1):34–43. [ PubMed : 15644559 ]
• JMAFF (Japanese Ministry of Agriculture, Forestry and Fisheries). Testing Guidelines for Toxicology Studies. 2000
• Kirkden RD, Niel L, Stewart SA, Weary DM. Gas killing of rats: The effect of supplemental oxygen on aversion to carbon dioxide. Anim Welf. 2008; 17 :79–87.
• Le Bars D, Gozariu M, Cadden SW. Animal models of nociception. Pharmacol Rev. 2001; 53 :597–652. [ PubMed : 11734620 ]
• Mench J. Humane Endpoints in Animal Experiments for Biomedical Research. London: The Royal Society Medical Press; 1999. Defining endpoints: The role of the animal care committee; pp. 133–138.
• Merrill RA. Regulatory toxicology. In: Klaassen CD, editor. Casarett and Doull’s Toxicology: The Basic Science of Poisons. 6th ed. New York: McGraw Hill Medical Publishing Division; 2001. pp. 1141–1153.
• Meyerson BA, Linderoth B. Mode of action of spinal cord stimulation in neuropathic pain. J Pain Sympt Manag. 2006; 31 (4S):S6–S12. [ PubMed : 16647596 ]
• Morton DB. Humane Endpoints in Animal Experiments for Biomedical Research. London: The Royal Society Medical Press; 1999. Humane endpoints in animal experimentation for biomedical research: Ethical, legal and practical aspects; pp. 5–12.
• Morton DB. A systematic approach for establishing humane endpoints. ILAR J. 2000; 41 (2):80–86. [ PubMed : 11406701 ]
• Morton DB, Burghardt GM, Smith JA. Critical anthropomorphism, animal suffering and the ecological context. Hastings Cent Rep. 1990; 20 (3):13–19. [ PubMed : 11650362 ]
• Nemzek JA, Xiao H-Y, Minard AE, Bolgos GL, Remick DG. Humane endpoints in shock research. Shock. 2004; 21 (1):17–25. [ PubMed : 14676679 ]
• Nemzek JA, Hugunin KM, Opp MR. Modeling sepsis in the laboratory: Merging sound science with animal well-being. Comp Med. 2008; 58 (2):120–128. [ PMC free article : PMC2703167 ] [ PubMed : 18524169 ]
• Niel L, Stewart SA, Weary DM. Effect of flow rate on aversion to gradual-fill carbon dioxide exposure in rats. Appl Animl Behav Sci. 2008; 109 :77–84.
• Nies AS. Principles of Therapeutics. In: Hardman JG, Limbird LE, editors. Goodman and Gilman’s The Pharmacological Basis of Therapeutics. 10th ed. New York: McGraw-Hill; 2001. pp. 45–66.
• NIH (National Institutes of Health). Validation and Regulatory Acceptance of Toxicological Test Methods: A Report of the ad hoc Interagency Coordinating Committee on the Validation of Alternative Methods. Research Triangle Park, NC: 1997. NIH Publication No. 97-3981.
• NIH. NIH Collaborates with EPA to Improve the Safety Testing of Chemicals. News Press Release. 2008. Available at www ​.nih.gov/news/health ​/feb2008/nhgri-14.htm .
• NIH/USEPA (National Institutes of Health/US Environmental Protection Agency). MOU (Memorandum of Understanding) on High Throughput Screening, Toxicity Pathway Profiling, and Biological Interpretation of Findings. 2008. Available at www ​.niehs.nih.gov/news ​/releases/2008/docs/ntpncgcepamou.pdf .
• NRC (National Research Council). Recognition and Alleviation of Pain and Distress in Laboratory Animals. Washington: National Academy Press; 1992. [ PubMed : 25144086 ]
• NRC. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington: National Academies Press; 2003. [ PubMed : 20669478 ]
• NRC. The Development of Science-based Guidelines for Laboratory Animal Care: Proceedings of the November 2003 International Workshop. Washington: National Academies Press; 2004. [ PubMed : 20669462 ]
• NRC. Toxicity Testing in the Twenty-first Century: A Vision and a Strategy. Washington: National Academies Press; 2007.
• NRC. Recognition and Alleviation of Distress in Laboratory Animals. Washington: National Academies Press; 2008. [ PubMed : 20669418 ]
• OECD (Organization for Economic Cooperation and Development). Guidelines for Testing of Chemicals, Section 4: Health Effects. Paris: OECD; 1987.
• OECD. Guidance Document on the Recognition, Assessment, and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation. Paris: OECD; 2000.
• OLAW/ARENA (Office of Laboratory Animal Welfare/Applied Research Ethics National Association). Institutional Animal Care and Use Committee Guidebook. 2nd ed. Bethesda: National Institutes of Health; 2002.
• Olfert ED, Godson DL. Humane endpoints for infectious disease animal models. ILAR J. 2000; 41 (2):99–104. [ PubMed : 11406703 ]
• Patrone G, Sacca SC, Macri A, Rolando M. Evaluation of the analgesic effect of 0.1% indomethacin solution on corneal abrasions. Ophthalmologica. 1999; 213 :350–354. [ PubMed : 10567865 ]
• Peyman GA, Rahimy MH, Fernandes ML. Effects of morphine on corneal sensitivity and epithelial wound healing: Implications for topical ophthalmic analgesia. Br J Ophthalmol. 1994; 78 :138–141. [ PMC free article : PMC504718 ] [ PubMed : 8123623 ]
• Sass N. Humane endpoints and acute toxicity testing. ILAR J. 2000. Available at http://dels ​.nas.edu/ilar_n ​/ilarjournal/41_2/AcuteToxicity ​.shtml . [ PubMed : 11406704 ]
• Stephens ML, Conlee K, Alvino G, Rowan AN. Possibilities for refinement and reduction: Future improvements within regulatory testing. ILAR J. 2002; 43 (Suppl):S74–S79. [ PubMed : 12388856 ]
• Stiles J, Honda CN, Krohne SG, Kazacos EA. Effect of topical administration of 1% morphine sulfate solution on signs of pain and corneal wound healing in dogs. Am J Vet Res. 2003; 64 (7):813–818. [ PubMed : 12856763 ]
• Stokes WS. Humane endpoints for laboratory animals used in regulatory testing. ILAR J. 2002; 43 (Suppl):S31–S38. [ PubMed : 12388849 ]
• Stokes WS. Summary of ICCVAM-NICEATM-ECVAM Ocular Toxicity Scientific Symposia. Presentation. 2005. [Accessed July 14, 2009]. Available at http://iccvam ​.niehs.nih ​.gov/meetings/SACpresent/SACocular ​.pdf .
• UKCCCR (United Kingdom Coordinating Committee on Cancer Research). Guidelines for the welfare of animals in experimental neoplasia. Lab Anim. 1988; 22 :195–201. [ PubMed : 3172698 ]
• USEPA (United States Environmental Protection Agency). Summary of the Toxic Substances Control Act, 15 USC 2601 et seq. (1976). 2008. [Accessed January 5 2008]. Available at www ​.epa.gov/lawsregs/laws/tsca.html .
• USEPA OPPTS (Office of Prevention, Pesticides, and Toxic Substances). 870 Series Final Guidelines: Health Effect Test Guidelines. 1998. [Accessed January 5, 2008]. Available at www ​.epa.gov/opptsfrs ​/publications/OPPTS_Harmonized ​/870_Health ​_Effects_Test_Guidelines/Series/
• USEPA OPPTS. 870 Series Final Guidelines: Health Effect Test Guidelines, OPPTS 870.1100. Acute Oral Toxicity. 2002. [Accessed July 14, 2009]. Available at www ​.epa.gov/opptsfrs ​/publications/OPPTS_Harmonized ​/870_Health ​_Effects_Test_Guidelines/Series/
• Walker K, Fox AJ, Urban LA. Animal models of pain. Mol Med Today. 1999; 5 :319–321. [ PubMed : 10498437 ]
• Wallace J. Humane endpoints in cancer research. In: Hendriksen CFM, Morton DB, editors. Humane Endpoints in Animal Experiments for Biomedical Research; Proceedings of the International Conference; 22–25 November 1998; Ziest: The Netherlands: The Royal Society Medical Press; 1999. pp. 79–84.
• Wallace J. Humane endpoints in cancer research. ILAR J. 2000; 41 (2):87–93. [ PubMed : 11417496 ]
• Cite this Page National Research Council (US) Committee on Recognition and Alleviation of Pain in Laboratory Animals. Recognition and Alleviation of Pain in Laboratory Animals. Washington (DC): National Academies Press (US); 2009. 5, Humane Endpoints for Animals in Pain.
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