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.

BOX 5-1

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.).

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

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.

1. 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.
2. 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.
3. 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.
4. 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.

ADDENDUM

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).

TABLE 5A-1

Sample Tumor Scoring Sheet.

BOX 5A-1

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...)

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