Ethical Considerations and Regulatory Issues PDF

C H A P T E R 1 Ethical Considerations and Regulatory Issues Marilyn J. Brown1 and Kathleen L. Smiler2 1 Charles River Laboratories, East Thetford, Vermont, USA; 2Lakeville, Michigan, USA O U T L I N E Introduction 3 Institutional Animal Welfare Oversight 20 Ethical Concepts Moral Theories De...

C H A P T E R 1 Ethical Considerations and Regulatory Issues Marilyn J. Brown1 and Kathleen L. Smiler2 1 Charles River Laboratories, East Thetford, Vermont, USA; 2Lakeville, Michigan, USA O U T L I N E Introduction 3 Institutional Animal Welfare Oversight 20 Ethical Concepts Moral Theories Descriptive Laboratory Animal Use Ethics 4 4 5 Regulations and Non-regulatory Considerations United States Regulatory Considerations 21 21 Ethical Principles Respect for Life Societal Benefit Non-Maleficence The Three Rs (Replacement, Reduction, and Refinement) 8 Ethical Challenges Breeding Colonies Genetically Modified Rodents Cancer Research Perinatal Animal Use Neuroscience and Behavioral Research 14 14 15 16 17 18 Food and Fluid Restriction Neuroanatomic Studies Neural Injury and Disease Behavioral Studies 18 18 19 19 Prolonged Restraint and Anesthesia Restraint of Awake Animals Prolonged Studies in Anesthetized Animals United States Animal Welfare Act Public Health Service Policy on Humane Care and Use of Animals Food and Drug Administration Good Laboratory Practices Interagency Cooperation Environmental Protection Agency Good Laboratory Practices 5 5 5 6 Non-Regulatory Considerations 22 22 23 23 23 Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals (Guide) 23 International Regulations, Policies, and Standards 24 AAALAC, International Canada European Union Pacific Rim 24 24 24 24 25 19 Conclusion 27 19 20 References 27 INTRODUCTION “… by now it is widely recognized that the [most humane] possible treatments of experimental animals, far from being an obstacle, is actually a prerequisite for successful animal experiments.” Russell and Burch, 1959 The Principles of Humane Experimental Technique The Laboratory Rabbit, Guinea Pig, Hamster, and Other Rodents DOI: 10.1016/B978-0-12-380920-9.00001-8 21 Like many aspects of life, involvement with animal research presents ethical challenges – areas where competing interests require use of an ethical decision-making 3 © 2012 Elsevier Inc. 4 1. Ethical Considerations and Regulatory Issues process, to provide guidance. Individuals in the laboratory may face basic competing interests between scientists, technicians, veterinary colleagues, an employing institution, the public, and concern for the animals themselves (e.g., individual health versus health of the colony). According to Tannenbaum, normative veterinary ethics refers to the search for correct principles of good and bad, right and wrong, and looks for the correct norms for veterinary professional behavior and attitudes (Tannenbaum, 1995). The intent of this chapter is to provide information which will be useful to all individuals involved in animal-based research, testing and teaching: scientists; technicians; laboratory animal veterinarians; and Institutional Animal Care and Use Committee (IACUC) members. This process might be considered an effort to define normative laboratory animal use ethics. The search for an appropriate ethical solution rarely leads to a complete and absolute answer, as science is a very dynamic field and new issues and new insights influence the outcome. This look at normative laboratory animal use ethics will examine some general ethical concepts within the context of Tannebaum’s definition of descriptive [laboratory animal use] ethics, where descriptive ethics is the study of the actual values or standards of a profession; that is, what members of a profession consider to be right and wrong regarding professional behavior and attitudes (Tannenbaum, 1995). In this chapter, reference will be made to relevant values and standards found in various principles and guidelines developed for use by individuals involved in animal research. Potential ethical challenges which might confront laboratory animal professionals using rodents and rabbits (e.g., challenges related to breeding colonies, genetically modified rodents, use of animals in cancer research, perinatal animal use, and use of animals in neuroscience and behavioral research) will be used as examples. Related ethical questions, and the appropriate principles which may pertain to the situation are discussed. The reader is challenged to test the general rules and principles against his or her own moral experience and intuition and thus create his or her own descriptive laboratory animal ethos. Further, the reader is encouraged to recognize that this is an ongoing process, that one’s professional ethos will likely grow and mature as new challenges are encountered. It is recognized that animal welfare is a “core concern of veterinary ethics … [and] this subject has gained increased importance as society and the profession endorse ever more strongly the moral imperative to treat animals decently.” When discussing laboratory animal ethics, one often uses the term “humane” (Tannenbaum, 1995). The Guide for the Care and Use of Laboratory Animals (Guide) states “Humane care means those actions taken to assure that laboratory animals are treated according to high ethical and scientific standards” (National Research Council, 2011). The Guide uses the words ‘ethics’ or ‘ethical’ 59 times in either the text or references. The first place ‘ethics’ is mentioned in the Guide is the Preface: “The Guide is also intended to assist investigators in fulfilling their obligation to plan and conduct animal experiments in accord with the highest scientific, humane, and ethical principles” (National Research Council, 2011). Humane care, ethics, and animal welfare are closely linked and, for the purposes of this chapter, may be used interchangeably. ETHICAL CONCEPTS Moral Theories Moral theory is an expansive field of study with its own vocabulary and competing points of view. It is beyond the scope of this chapter to provide a comprehensive discussion of the many theories that discuss the humane use of animals. Instead, focus is made on some principles which the authors have found helpful. Consideration of ethics with respect to use of animals in research begins with the basic idea of where one feels animals (versus people) fit in the moral spectrum. In other words, what is the moral value of an animal relative to a human? Moral status or standing represents “the position or rank of an entity along a moral continuum from minimal to maximal moral significance” (Kraus and Renquist, 2000). Because animals lack moral agency, defined as a uniquely human capacity for making moral judgments (Kraus and Renquist, 2000), it may be concluded that animals, while having some moral status, fall below that accorded to humans. Even when examining the moral status of animals, there are many who accord different levels of moral status for different species. Such a continuum of moral status is essentially a sliding scale where moral status is based on a combination of cognitive and sensory capacities. This concept has been coined “speciesism” by some philosophers who then compare it to other concepts which foster differential treatment based upon a given trait, similar to racism or sexism (Singer, 1975). Regardless of where a species is placed on this Darwinian scale, a commonality shared by all vertebrates is that of sentience. Sentience is the capacity to perceive and process sensory input and thus the ability to feel pain and distress. Moral agents (humans) have obligations or are bound to do certain things out of a sense of duty, custom, or law and have responsibility toward other beings. It is from this obligation that most humans feel it is right to minimize the pain and distress felt by other sentient beings. This obligation may be called non-maleficence and is addressed in greater detail later in this chapter. I. GENERAL Ethical Concepts In the most general sense, there are two approaches to ethics: utilitarianism and deontology. Utilitarian theories look at the consequences of actions to determine which actions are good and which are bad. The goal is to maximize good consequences and minimize bad ones. In its most basic form, this is similar to what an IACUC protocol review does when it performs a cost or harm/ benefit analysis. However, there are different views of the “good” that should be maximized. Utilitarianism is an example of action-oriented ethical theories, because it examines the consequences of actions. These theories tend to stress the concepts of duty and obligation. In contrast, deontological theories are also action-oriented ethical theories; however, there are some moral imperatives which are independent of how much “good” results from an action. Some deontologists advocate a set of “obligatory” moral principles but allow some compromise when different moral principles are conflicting (e.g., one principle trumps another). There may also be non-obligatory principles which are desirable but not mandatory to follow. Other ethical approaches include those based on values and ethics. Value-based ethics center around basic values to be sought. These values tend to be hierarchical. There are also virtue-oriented ethical theories. A virtue contributes to a good moral life (e.g., honesty, kindness, generosity). These approaches tend to instill “attitudes, feelings, and states of mind central to the virtuous disposition” (Tannenbaum, 1995). It has been suggested that a satisfactory approach to normative ethics must include actions, values, and virtues (Tannenbaum, 1995). Descriptive Laboratory Animal Use Ethics Ethical Principles Descriptive laboratory animal ethics represents an approach for determining appropriate moral behavior and attitude (Tannenbaum, 1995). Principles can be defined as “accepted generalizations about a topic that are frequently endorsed by many and diverse organizations” (National Research Council, 2011); and several sets of principles with relevance to ethical use of laboratory animals will be discussed. It is hoped that application of these principles in the discussion of example ethical challenges later in this chapter will serve as a basis for how other ethical challenges may be approached. One set of principles, the concept of the “Five Freedoms,” was originally created by the United Kingdom Farm Animal Welfare Advisory Council (FAWC) in 1979 specifically to address issues related to the use of animals in agriculture. Today, the “Five Freedoms” are also often mentioned within the context of animals used in research. The “Five Freedoms” include: (1) freedom from malnutrition; (2) freedom 5 from thermal or physical discomfort; (3) freedom from injury and disease; (4) freedom to express normal social behavior; and (5) freedom from fear. The Five Freedoms were revised in 1993 to include: (1 ) freedom from hunger and thirst, by assuring ready access to fresh water and a diet sufficient to maintain full health and vigor; (2) freedom from discomfort, by providing an environment including shelter and a comfortable resting area; (3) freedom from pain, injury and disease, by preventive means or rapid diagnosis and treatment; (4) freedom from fear and distress, by ensuring conditions that avoid mental suffering; and (5) freedom to express normal behavior, by providing sufficient space, proper facilities, and company of the animal’s own kind (Webster, 2001). In 1996, the United States National Aeronautics and Space Administration (NASA) developed basic principles, referred to as the Sundowner Principles (NASA, 1996). These principles were based upon the Belmont Report which had been written for the protection of human research subjects (National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research, 1979). These principles of bioethics offer a simple, yet elegant framework for looking at ethical questions: “The use of animals in research involves responsibility – not only for the stewardship of the animals but to the scientific community and society as well. Stewardship is a universal responsibility that goes beyond the immediate research needs to include acquisition, care and disposition of the animals, while responsibility to the scientific community and society requires an appropriate understanding of, and sensitivity to scientific needs and community attitudes toward the use of animals. Among the basic principles generally accepted in our culture, three are particularly relevant to the ethics of research using animals: respect for life, societal benefit, and non-maleficence” (NASA, 1996). Respect for Life Living creatures deserve respect. This principle requires that animals used in research should be of an appropriate species and health status, and should involve the minimum number required to obtain valid scientific results. It also recognizes that the use of different species may raise different ethical concerns. Selection of appropriate species should consider cognitive capacity and other morally relevant factors. Additionally, methods such as mathematical models, computer simulation, and in vitro systems should be considered and used whenever possible. Societal Benefit The advancement of biological knowledge and improvements in the protection of the health and wellbeing of both humans and other animals provide strong justification for biomedical and behavioral research. I. GENERAL 6 1. Ethical Considerations and Regulatory Issues This principle entails that when animals are used, the assessment of the overall ethical value of such use should include consideration of the full range of potential societal goods, the populations affected, and the burdens that are expected to be borne by the subjects of the research. Non-Maleficence Based upon the idea that vertebrate animals are sentient, this principle holds that the minimization of distress, pain, and suffering is a moral imperative. Unless the contrary is established, investigators should consider that procedures that cause pain or distress in humans may cause pain or distress in other sentient animals (Interagency Research Animal Committee, 1985). The International Guiding Principles for Biomedical Research Involving Animals (Table 1.1) were developed by the Council for International Organizations of Medical Sciences (CIOMS) as a result of extensive international and interdisciplinary consultations spanning 1982–1984 (Bankowski, 1985; Council for International Organizations of Medical Sciences, 1985). These principles have a considerable measure of acceptance internationally. European Medical Research Councils (EMRC), an international association that includes all the West European medical research councils, fully endorsed the CIOMS Guiding Principles in 1984. In the same year, the CIOMS Guiding Principles were endorsed by the World Health Organization (WHO) Advisory Committee on Medical Research. It should be noted that, at the time of writing this chapter, the CIOMS were undergoing revision. During 1984–1985, the U.S. National Institutes of Health (NIH) convened the U.S. Interagency Research Animal Committee (IRAC) which created a similar set of principles, the U.S. Government Principles for the Utilization and Care of Vertebrate Animals used in Testing, Research and Training (Table 1.2) for research funded by the U.S. Public Health Service (PHS) (Interagency Research Animal Committee, 1985). These principles were, to a considerable extent, based on the CIOMS Guiding Principles. As an indication of the wide acceptance of both the IRAC and CIOMS Principles, discussion of the IRAC Principles can be found under the heading “Ethics and Animal Use” in the Guide (National Research Council, 2011). NASA and CIOMS are also listed in Appendix A of the Guide under the heading “Ethics and Welfare” TABLE 1.1 Council for International Organizations of Medical Sciences (CIOMS) Basic Principles (1985) I. The advancement of biological knowledge and the development of improved means for the protection of the health and well-being both of man and of animals require recourse to experimentation on intact live animals of a wide variety of species II. Methods such as mathematical models, computer simulation and in vitro biological systems should be used wherever appropriate III. Animal experiments should be undertaken only after due consideration of their relevance for human or animal health and the advancement of biological knowledge IV. The animals selected for an experiment should be of an appropriate species and quality, and the minimum number required to obtain scientifically valid results V. Investigators and other personnel should never fail to treat animals as sentient, and should regard their proper care and use and the avoidance or minimization of discomfort, distress, or pain as ethical imperatives VI. Investigators should assume that procedures that would cause pain in human beings cause pain in other vertebrate species, although more needs to be known about the perception of pain in animals VII. Procedures with animals that may cause more than momentary or minimal pain or distress should be performed with appropriate sedation, analgesia, or anesthesia in accordance with accepted veterinary practice. Surgical or other painful procedures should not be performed on unanesthetized animals paralyzed by chemical agents VIII. Where waivers are required in relation to the provisions of article VII, the decisions should not rest solely with the investigators directly concerned but should be made, with due regard to the provisions of articles IV, V, and VI, by a suitably constituted review body. Such waivers should not be made solely for the purposes of teaching or demonstration IX. At the end of, or, when appropriate, during an experiment, animals that would otherwise suffer severe or chronic pain, distress, discomfort, or disablement that cannot be relieved should be painlessly killed X. The best possible living conditions should be maintained for animals kept for biomedical purposes. Normally the care of animals should be under the supervision of veterinarians having experience in laboratory animal science. In any case, veterinary care should be available as required XI. It is the responsibility of the director of an institute or department using animals to ensure that investigators and personnel have appropriate qualifications or experience for conducting procedures on animals. Adequate opportunities shall be provided for in-service training, including the proper and humane concern for the animals under their care I. GENERAL Ethical Concepts (National Research Council, 2011). The complete IRAC Principles are found on the cover of the PHS Policy (Office of Laboratory Animal Welfare, 2002) and Appendix B of the Guide (National Research Council, 2011). There are really no points of conflict and, in fact, there are many points of consensus between the Sundowner, CIOMS, and IRAC principles all of which offer that animal-based research should: l l l Acknowledge the importance of research with relevance to human or animal health, advancement of knowledge, or the good of society; Stress consideration of alternatives to reduce or replace the use of animals; Require avoiding or minimizing discomfort, distress, and pain. Some items which are addressed in greater detail in the CIOMS and IRAC principles but are more generally under the concept of non-maleficence in the Sundowner Principles include: l l Use of appropriate sedation, analgesia, and anesthesia; Establishment of humane endpoints; l l 7 Provision of adequate veterinary care; Assurance of appropriate training and qualifications of personnel using and caring for animals. As previously mentioned, descriptive ethics is the study of actual values or standards of a profession. With respect to ethics governing laboratory animal use, the Sundowner, CIOMS, and IRAC principles could be considered major components. Commonly accepted ethical principles result in development of professional guidelines. For example, in 1831 in the United Kingdom (U.K.), Marshall Hall, a leading British physiologist, developed guidelines for animal experimentation (Zurlo et al., 1993). The British Association for the Advancement of Science further refined these principles in 1871, 5 years before the first legislation in the U.K. In 1909, Walter B. Cannon developed guidelines for animal experimentation for the American Physiological Association. Many other scientific organizations have also created similar guidelines. Scientists are urged to seek those within their own professional societies. (e.g., Federation of American Societies for Experimental Biology at http://www.faseb. TABLE 1.2 Interagency Research Animal Committee (IRAC) U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training (Interagency Research Animal Committee, 1985) The development of knowledge necessary for the improvement of the health and well-being of humans as well as other animals requires in vivo experimentation with a wide variety of animal species. Whenever U.S. Government agencies develop requirements for testing, research, or training procedures involving the use of vertebrate animals, the following principles shall be considered; and whenever these agencies actually perform or sponsor such procedures, the responsible Institutional Official shall ensure that these principles are adhered to: I. The transportation, care, and use of animals should be in accordance with the Animal Welfare Act (7 U.S.C. 2131 et. seq.) and other applicable Federal laws, guidelines, and policies. II. Procedures involving animals should be designed and performed with due consideration of their relevance to human or animal health, the advancement of knowledge, or the good of society. III. The animals selected for a procedure should be of an appropriate species and quality and the minimum number required to obtain valid results. Methods such as mathematical models, computer simulation, and in vitro biological systems should be considered. IV. Proper use of animals, including the avoidance or minimization of discomfort, distress, and pain when consistent with sound scientific practices, is imperative. Unless the contrary is established, investigators should consider that procedures that cause pain or distress in human beings may cause pain or distress in other animals. V. Procedures with animals that may cause more than momentary or slight pain or distress should be performed with appropriate sedation, analgesia, or anesthesia. Surgical or other painful procedures should not be performed on unanesthetized animals paralyzed by chemical agents. VI. Animals that would otherwise suffer severe or chronic pain or distress that cannot be relieved should be painlessly killed at the end of the procedure or, if appropriate, during the procedure. VII. The living conditions of animals should be appropriate for their species and contribute to their health and comfort. Normally, the housing, feeding, and care of all animals used for biomedical purposes must be directed by a veterinarian or other scientist trained and experienced in the proper care, handling, and use of the species being maintained or studied. In any case, veterinary care shall be provided as indicated. VIII. Investigators and other personnel shall be appropriately qualified and experienced for conducting procedures on living animals. Adequate arrangements shall be made for their in-service training, including the proper and humane care and use of laboratory animals. IX. Where exceptions are required in relation to the provisions of these Principles, the decisions should not rest with the investigators directly concerned but should be made, with due regard to Principle II, by an appropriate review group such as an institutional animal care and use committee. Such exceptions should not be made solely for the purposes of teaching or demonstration (Interagency Research Animal Committee, 1985). I. GENERAL 8 1. Ethical Considerations and Regulatory Issues org/Policy-and-Government-Affairs/Science-PolicyIssues/Animals-in-Research-and-Education/Statementof-Principles.aspx; American Physiological Society at http://www.the-aps.org/pa/resources/policyStmnts/ paPolicyStmnts_Guide.htm). Examples of professional guidelines for laboratory animal veterinarians include the Principles of Veterinary Medical Ethics and Veterinary Oath of the American Veterinary Medical Association (at http://www.avma .org/issues/policy/ethics.asp, http://www.avma.org/ about_avma/whoweare/oath.asp). Further, many laboratory animal science professionals refer to the Position Statements of the American Association for Laboratory Animal Science (AALAS; http://www.aalas.org/ association/position_statements.aspx). Central to these codes of conduct, principles, statements of ethics, and position statements is the commitment to the humane care and use of research animals. The Three Rs (Replacement, Reduction, and Refinement) Several sets of ethical principles and guidelines covering the use of animals in research, testing, and teaching have been mentioned but perhaps the simplest and the one with the greatest impact on animal research today is the ethical concept called “The 3Rs”, a call to apply whenever possible the alternatives of replacement of animals, reduction in the number of animals used, and the refinement in procedures used on animals in research (Russell and Burch, 1959). The Guide was originally published in 1963 and has undergone numerous revisions, with the most recent edition being published in 2011. The Statement of Task of the latest revision committee begins with “The use of laboratory animals for biomedical research, testing and education is guided by the principles of the Three Rs… .” (National Research Council, 2011). The 3Rs are a common theme in the Guide which states “Throughout the Guide, scientists and institutions are encouraged to give careful and deliberate thought to the decision to use animals taking into consideration the contribution that such use will make to new knowledge, ethical concerns, and the availability of alternatives to animal use. A practical strategy for decision making, [is] the “Three Rs” (Replacement, Reduction, and Refinement) approach, …”(National Research Council, 2011). The concept of the 3Rs is also infused in U.S. regulations covering research using animals. Although the United States Department of Agriculture (USDA) Animal Welfare Act (AWA) regulations do not include the word “alternatives” in its section of definitions, the term is used several times in the regulations themselves. For example, in the section on IACUC review of protocols the regulations state protocols must indicate that “(i) Procedures involving animals will avoid or minimize discomfort, distress, and pain to the animals; (ii) The principle investigator has considered alternatives to procedures that may cause more than momentary or slight pain or distress to the animals, and has provided a written narrative description of the methods and sources, e.g., The Animal Welfare Information Center, used to determine that alternatives were not available ….” (Office of the Federal Register, 2002). The focus of USDA inspectors on adherence to this section of the regulations can be appreciated when looking at the USDA Research Facility Inspection Guide which instructs inspectors several times to evaluate institutional compliance in this area (USDA, 2009). In addition, the requirement for a search for alternatives is the subject of a specific Animal Care Policy – Policy 12 (Animal and Plant Health Inspection Service, 2000). Strategies to enhance electronic search efficiency using a search filter for PubMed have been published (Hooijmans et al., 2010b). A “Gold Standard Publication Checklist” has been proposed to help fully integrate the 3Rs into systematic reviews of the literature (Hooijmans et al., 2010a). In a section on personnel qualifications, the AWA Regulations state that the institution should assure adequate training and qualifications and that this is fulfilled in part through the provision of training and instruction on the “concept, availability, and use of research and testing methods that limit the use of animals [Reduction] or minimize animal distress [Refinement]” (Office of the Federal Register, 2002). The AWA Regulations further indicate that research staff should be trained on the “utilization of services (e.g., National Agriculture Library of Medicine) available to find information: … (ii) On alternatives to the use of live animals in research [Replacement]; …” (Office of the Federal Register, 2002). In addition to the specific references above, the AWA Regulations also refer to the use of anesthetics, analgesics, and sedatives, the availability of appropriate veterinary care, the use of appropriate housing; and timely, appropriate euthanasia, all of which demonstrate the practice of “refinement”. The United States Public Health Service (PHS) Policy contains similar language regarding minimizing discomfort, distress pain, use of appropriate anesthesia, and the use of humane endpoints as examples of refinement. In addition, the PHS Policy refers to the IRAC Principles, requiring institutions receiving PHS funds to use the Guide as a basis for their animal care and use programs. National and international agencies and organizations such as the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) (http://iccvam.niehs.nih.gov/), the European Centre for the Validation of Alternative Methods (ECVAM) (http://ecvam.jrc.ec.europa.eu/), and the National I. GENERAL Ethical Concepts 9 Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) (http://www.nc3rs .org.uk/) are charged with helping to find and promote the use of alternatives. With so much official emphasis on the 3Rs, there is sometimes a perception that the concept is not being adequately implemented in practice. Indeed, it has been suggested that scientists and IACUCs do not fully understand the concepts of the 3Rs (Graham, 2002; Schuppli and Fraser, 2005). In addition to incomplete understanding of the concepts, factors believed to negatively influence the full implementation of the 3Rs by IACUCs might include: (1) a belief that the scientists themselves would implement the 3Rs; (2) an assumption that funding agencies have reviewed the use of the 3Rs during proposal review; (3) confidence that sample size, rather than study design, is the sole criterion for reduction; and (4) focus upon potential harm from procedures without consideration for potential distress that animals might experience from husbandry and housing. Although these conclusions were based upon a relatively small number of IACUCs, these are troubling observations and indicate the need for greater emphasis on the 3Rs in training programs for scientists and IACUCs. The authors hope that this chapter can be a resource for that process. It would be disingenuous to imply that, although well accepted, the concept of the 3Rs is universally accepted. In an article titled “Time to Abandon the Three Rs”, Derbyshire wrote that the 3Rs “draw attention away from the value of experimentation and toward the importance of animal welfare” (Derbyshire, 2006). Although the article supports the concept of reducing animal stress for the sake of science, the authors do not clearly recognize the opportunity to balance facilitation of science and application of the 3Rs. animals commonly kept as pets such as horses, dogs, and cats are often regarded differently than rats and mice, which in turn are regarded differently from fruit flies and worms, and so on. Development of fully validated and accepted replacement alternatives can be a frustratingly slow process. However, there are significant examples of successful replacement of live animals. For example, one of the most criticized uses of animals for toxicity testing is the Draize test in rabbits. This test was developed to determine ocular toxicity and irritancy caused by products and chemicals. Ocular toxicity tests represent one of the four most commonly conducted product safety tests (Interagency Coordinating Committee on the Validation of Alternative Methods, 2010). The 3Rs were implemented by the development of three validated and accepted replacements for screening products for ocular toxicity: the bovine corneal opacity and permeability test using a cow eye or the isolated chicken eye test (both by-products of the meat industry); and the Cytosensor® microphysiometer (Molecular Devices, Inc., Sunnyvale, CA). A balanced preemptive pain management plan for rabbit Draize test studies, when the test is still required, has also been validated and accepted as a refinement (Interagency Coordinating Committee on the Validation of Alternative Methods, 2010). A second example of implementation of the 3Rs involves the replacement of rabbits in the testing of pharmaceuticals and medical devices for pyrogens by use of an in vitro alternative, the Limulus Ameobecyte Lysate (LAL) Test. In the LAL, blood of horseshoe crabs is collected and the animals are returned, unharmed, back to the ocean. Previous tests required the injection of drugs, biologics, medical devices, or raw materials into rabbits to look for a febrile response as an indication of contamination with endotoxins. REPLACEMENT REDUCTION Replacement refers to methods that avoid using animals. The term includes absolute replacements (i.e., replacing animals with inanimate systems such as computer programs) as well as relative replacements (i.e., replacing animals, such as vertebrates, with animals that are lower on the phylogenic scale) (National Research Council, 2011). Relative replacement may be controversial to some people as it implies “speciesism”, the idea that one species has greater moral standing than another (Singer, 1975). Like many of the ethical considerations relating to animal use, relative replacement is essentially a continuum of moral standing. Society, in general, often differentiates between humans and non-human animals; however, with respect to the animal world, different opinions exist regarding our obligations to some species versus others. For example, non-human primates and “Reduction includes strategies for obtaining comparable levels of information from the use of fewer animals or for maximizing the information obtained from any given number of animals (without increasing pain or distress) so to ultimately require fewer animals to acquire the same scientific information. This approach relies on an analysis of experimental design, applications of newer technologies, the use of appropriate statistical methods, and control of environmentally related variability in animal housing and study areas” (National Research Council, 2011). Strategies to reduce the numbers of animals needed include improved statistical design of a study (Dell et al., 2002) and improved selection of an animal model, including selection of animals with the most appropriate health and genetic status. Control of the genetic status is an advantage of using rats and mice. The use I. GENERAL 10 1. Ethical Considerations and Regulatory Issues of inbred strains of rats and mice allows scientists to control and investigate genetic variation, and to evaluate responses to treatments on specific areas of interest (Festing, 2004). The use of animals without confounding disease or genetic variation results in less variation, thus requiring fewer animals to determine a treatment effect. Individuals involved with study design, study review, or those participating as a member of the research team, have the ethical imperative to ensure studies use the minimum number of animals necessary to achieve the scientific objective of the study. Scientists should design studies with particular attention to methodology, statistics, and choice of model. Veterinarians and facility staff should collaborate to minimize nonexperimental variables in animal care. IACUCs should be diligent during review of the protocol, semiannual program and facility evaluations, and review of postapproval monitoring to assure that the appropriate number of animals have been used. Having a statistician on the IACUC is one strategy that may be helpful. REFINEMENT “Refinement refers to modifications of husbandry or experimental procedures to enhance animal well-being and minimize or eliminate pain and distress” (National Research Council, 2011). In the authors’ opinion, refinement is commonly employed by scientists in ongoing efforts to improve their science; that is, better animal welfare leads to higher-quality science. Many scientists do not recognize this as utilization of “alternatives”, even though it clearly falls within the 3Rs. However, this is also an area where scientists, veterinarians, and IACUCs can make significant strides to enhance animal welfare. Use of less invasive procedures (e. g., use of a blood pressure cuff instead of an implanted catheter for blood pressure monitoring) is one method of refinement. However, there are also situations where an invasive procedure, such as implantation of telemetry sensors to allow ongoing collection of real-time data, can result in much less stressful data collection (Stephens et al., 2002). Examples of other refinements include accurate recognition of pain and the use of analgesics and supportive care; implementation of humane endpoints; and enhanced housing and husbandry. Carbone and Garnett (2008), state, “… the prime ethical concerns in laboratory animal welfare is what animals consciously experience: their pain, distress, fear, boredom, happiness and psychological well being.” The emotional dimension of pain, a characteristic of suffering, requires pain pathways to extend to higher levels of the cortex unique to humans and some other primates (such as apes) (Nuffield Council on Bioethics, 2005), but it has been stated that “… the absence of analogous structures cannot necessarily be taken to mean that they [animals] are incapable of experiencing pain, suffering or distress or any other higher order states of conscious experience” (Nuffield Council on Bioethics, 2005). Basic to minimization of pain is the ability to recognize the signs of pain in specific species. It has been suggested that some animals, particularly prey species, may try to mask pain to avoid displaying abnormal activity that might increase their risk of predation (Roughan and Flecknell, 2000). Further, many animals are most active during the dark cycle, when observations are more difficult. Since clinical indices of pain may be very subtle, it is important to be able to recognize a departure from normal behavior and appearance (Table 1.3; National Research Council, 2003). A short list of general signs and measurements that might indicate pain or distress includes: (1) vigorous attempt to escape; (2) changes in biological characteristics such as food and water consumption and body weight; (3) changes in blood levels of hormones and glucose; (4) increased adrenal gland mass; and (5) appearance, posture, and behavior (Moberg, 1985, 2000). Behavioral indicators of pain in mice and rats have also been described (Flecknell, 1999; Kohn et al., 2007; Roughan and Flecknell, 2000, 2001, 2003). In addition, guidelines for the assessment and management of pain in rodents and rabbits have been published by the American College of Laboratory Animal Medicine (ACLAM, 2006). Scientists sometimes have concerns about the effect of perioperative analgesics on the research. Many studies have been done investigating analgesic effect on a wide variety of parameters (e.g., litter size, body weight, TABLE 1.3 Indicators of Pain in Rodents and Rabbits Species General Behavior Appearance Other Rodents Decreased activity; excessive licking and scratching; selfmutilation; may be unusually aggressive; abnormal locomotion (stumbling, falling); writhing; does not make nest; hiding Piloerection; rough/ stained haircoat; abnormal stance or arched back; porphyrin staining (rats) Rapid, shallow respiration; decreased food/water consumption; tremors Rabbit Head pressing; teeth grinding; may become more aggressive; increased vocalizations; excessive licking and scratching; reluctant to locomote Excessive salivation; hunched posture Rapid, shallow respiration; decreased food/water consumption No single observation is sufficiently reliable to indicate pain; rather several signs, taken in the context of the animal’s situation should be evaluated. The signs of pain may vary with the type of procedure (e.g., orthopedic versus abdominal pain) (National Research Council, 2003) I. GENERAL Ethical Concepts behavior, and hemodynamic parameters) (Bourque et al., 2010; Goulding et al., 2010; Lamon et al., 2008; McBrier et al., 2009; Valentim et al., 2008). These studies have demonstrated varying effects on parameters of interest, including no effect. Therefore, rather than assuming that analgesics cannot be given, it is recommended that a literature search be conducted to determine if studies have been done to validate the existence of an effect of analgesia on a given experimental parameter. If data do not exist, consideration should be given for conducting and publishing an appropriate study to indicate which analgesics are, or are not, a viable scientific option for future experiments. Investigators should be required to provide scientific justification for withholding analgesia when potentially painful procedures are to be conducted. Defining distress in animals has proven to be a difficult task. The ILAR Committee on the Recognition and Alleviation of Distress in Laboratory Animals stated “Although most definitions of distress characterize it as an aversive, negative state in which coping and adaptation processes in response to stressors fail to return an organism to physiological and/or psychological homeostasis, philosophical differences center on the inclusion of emotions and feelings affected by this state of being.” (Committee on Recognition and Alleviation of Distress in Laboratory Animals, 2008). Stress has been defined as “the biological response an animal exhibits in an attempt to cope with threats to its homeostasis” (Stokes, 2000). This response can involve immunologic, metabolic, autonomic, neuroendocrine, and behavioral changes (Moberg, 2000). The type, pattern, and level of the response depend upon the strength, severity, intensity, and duration of the stressor(s). The aversive state of distress results when an animal is unable to adapt. By limiting the frequency, strength, severity, intensity, and/or duration of the stressor(s), it may be possible to limit the level of distress in the animal. One method is to reduce the cumulative stress an animal experiences (allowing recovery or adaptation to a given stressful situation before adding additional stressors) or refining practices and procedures to make the individual stressors less severe or shorter. The use of humane endpoints contributes to refinement by providing an alternative to experimental endpoints that result in more severe animal pain and distress. Scientific or experimental endpoints are defined as occurring when the objectives of the study have been reached. Humane endpoints occur at the point at which pain or distress is prevented, terminated, or relieved in an experimental animal. In most studies, the scientific and humane endpoints occur at the same time, in other words, the scientific endpoint occurs prior to the development of pain or distress. Studies that may result in severe or chronic pain or significant alterations in the animal’s ability to maintain normal physiology, or 11 adequately respond to stressors, should contain descriptions of appropriate humane endpoints or provide science-based justification as to why a particular, accepted humane endpoint cannot be employed. Veterinary consultation must occur when pain or distress is beyond the level anticipated in the protocol description or when interventional control is not possible (National Research Council, 2011). Most of the ethical principles guiding humane animal research mention the use of humane endpoints (Bankowski, 1985; Council for International Organizations of Medical Sciences, 1985; Interagency Research Animal Committee, 1985). Stokes (2000, 2002) provides an overview and reviews specific situations where endpoints are appropriately utilized. The need, criteria, and timing for humane endpoints should be part of pre-study planning and are often best done as a research team including scientists, technicians, and veterinarians (Canadian Council for Animal Care, 1998; NRC, 2003; Organisation for Economic Co-operation and Development, 2000). Endpoints are an important element of IACUC protocol review. Therefore, it is essential that a protocol contain all appropriate information regarding the criteria for humane endpoints, observation schedules, and training of personnel to adequately observe for the agreed-upon criteria. Pilot studies may be useful for gathering this information if it is not known at the time of protocol submission. In addition, the criteria and other details may need to be modified when unexpected adverse events occur. The IACUC should be notified when this happens and the protocol amended as needed. Development of criteria for humane endpoints may be general and applied to any study. Institutions often adopt standards or policies that cover situations when study-specific endpoints have not been determined. These documents often appear as Standard Operating Procedures (SOPs) or guidelines, and may be developed and instituted by the IACUC, the veterinary staff, the institutional administration, or any collaboration of the above. The policies should encompass generic clinical or behavioral conditions that potentially are associated with pain and distress, and should be widely recognized and accepted by the research staff. General clinical signs which may be monitored include: weight loss, inability to ambulate adequately to obtain food and/or water, and body condition scores (Figure 1.1) (Hickman and Swan, 2010). Anorexia or lack of appetite is a significant observation since parenteral supplementation in rodents is not commonly used. In some cases in these species, clinical abnormalities are only obvious when advanced illness, toxicity, or impending death (e.g., a moribund state) are reached (Toth, 2000). In these situations, decisions to initiate humane endpoints must be made promptly. I. GENERAL 12 1. Ethical Considerations and Regulatory Issues BC 1 Rat is emaciated • Segmentation of vertebral column prominent if not visible. • Little or no flesh cover over dorsal pelvis. Pins prominent if not visible. • Segmentation of caudal vertebrae prominent. BC 2 Rat is under conditioned • Segmentation of vertebral column prominent. • Thin flesh cover over dorsal pelvis, little subcutaneous fat. Pins easily palpable. • Thin flesh cover over caudal vertebrae, segmentation palpable with slight pressure. BC 3 Rat is well-conditioned • Segmentation of vertebral column easily palpable. • Moderate subcutaneous fat store over pelvis. Pins easily palpable with slight pressure. • Moderate fat store around tail base, caudal vertebrae may be palpable but not segmented. BC 4 Rat is overconditioned • Segmentation of vertebral column palpable with slight pressure. • Thick subcutaneous fat store over dorsal pelvis. Pins of pelvis palpable with firm pressure. • Thick fat store over tail base, caudal vertebrae not palpable. BC 5 Rat is obese • Segmentation of vertebral column palpable with firm pressure; may be a continuous column. • Thick subcutaneous fat store over dorsal pelvis. Pins of pelvis not palpable with firm pressure. • Thick fat store over tail base, caudal vertebrae not palpable FIGURE 1.1 System for body condition scoring (reproduced with permission from Hickman and Swan, 2010). In some cases, humane endpoints may be developed for a specific type of study (Dennis, 2000; Montgomery, 1987; Olfert, 1996; Olfert and Godson, 2000; Sass, 2000; Workman et al., 2010) or a specific individual study (Hickman and Swan, 2010; Singh et al., 2010). The use of scoring systems has been described and usually utilizes multiple observations which, in total, identify the humane endpoint (LLoyd and Wolfensohn, 1998; Medina, 2004; Morton, 2000). Other systematic approaches to determining humane endpoints have also been reported (Medina, 2004; Morton, 2000). There are excellent references available that review the establishment and use of humane endpoints to avoid death as an endpoint (National Research Council, 2011). To be prepared for situations when unanticipated pain and distress occur, the institution should have sufficient veterinary oversight in place to advise when alleviation of negative consequences from experimental procedures should be addressed. This may result in animals receiving veterinary medical care, removal from the experiment, or euthanasia to best align with the scientific objectives of the research. These decisions are ideally made through a collaborative discussion including animal care, scientific, and veterinary staff. However, in cases where the animals are significantly compromised, the veterinarian is obligated to take whatever actions are necessary for animal welfare. When multiple animals receiving the same treatment exhibit severe adverse effects, consideration should be given to adjusting the endpoint in the remaining animals or euthanizing an entire treatment group if experimental objectives can no longer be achieved. Refinements in husbandry and handling (e.g., provision of species-appropriate enrichment, positive humane interaction and operant training to minimize stress in handling and sample collection), are also ways in which distress and other negative impacts can be minimized. Refinements of handling that may seem minor (e.g., picking up a mouse by using a plastic tunnel or cupped hand instead of the traditional method of grasping the base of the tail) (Hurst and West, 2010) can have significant impact, particularly when applied to a large number of animals. Many refinements in husbandry rely on a good working knowledge of the ethology (study of animal behavior, generally under natural conditions) of the animal species used. The knowledge of normal species-specific behavior is necessary to recognize abnormal behavior and to create an appropriate environment. But consider an animal’s natural environment – what is natural for mice and rats that live in forests, rural areas, and urban environments? Some species are highly adaptable, thus attempting to recreate a natural environment in the laboratory may not only be impractical, but difficult to even define. In addition, most of the rodents and rabbits used in research today are purpose-bred and have been for many generations. The ethograms for such animals would be expected to be different from their wild counterparts (e.g, it would be expected and desirable that rodents and rabbits habituated to humans would be less fearful and easier to handle). However, animals bred for research may retain, and attempt to exhibit, certain intrinsic behaviors. Knowledge of those behaviors can help the laboratory animal scientist develop practical strategies to maximize those behaviors and minimize stress. When considering the stressors in the laboratory animals’ environment, one must consider not only I. GENERAL Ethical Concepts individual stressors, but also the potential cumulative effect of multiple stressors and plan activities accordingly (Committee on Recognition and Alleviation of Distress in Laboratory Animals, 2008). For example, a laboratory mouse will have been exposed to multiple stressors, such as weaning, transport, and a new environment before it even reaches the laboratory. Allowing adequate time for animals to acclimate to their new environment and recover from these stressors is not only good for animal welfare but good for science (Conour et al., 2006). Additional stressors may include husbandry and care. For example, most rodent and rabbit species are highly olfactory and loss of their olfactory cues when given a completely clean cage is a stressor (Duke et al., 2001, Sharp et al., 2002). Implementing methods that minimize this impact, such as transfer of the animal along with some nesting material, enrichment device, or at least the same cage mates has been suggested as a means to help mitigate the stress. Most rodents are nocturnal, yet husbandry and experimental activities are usually performed during the sleep cycle. Reverse light cycles may be an alternative that could reduce the stress to animals of having procedures conducted during the sleep cycle, but most husbandry and research procedures are difficult to perform in a red light environment. Laboratory rodents and rabbits originated as prey species and thus are not, by nature, comfortable with handling and restraint. Some degree of habituation may be desirable in some species (Moynihan et al., 1992) Positive reinforcement after such handling and restraint has been shown to aid tractability in some species, however there is little published about the use of this technique in rodents and rabbits, and so its use is based upon extrapolation and empirical observations. The environment in which an animal is housed also impacts on its welfare, and assuring that the environment addresses the physical and behavioral needs of the animal can minimize negative impacts on welfare. Failure of the environment to meet the animal’s needs can have a negative impact on both animal welfare and can affect scientific validity of the research (Garner, 2005; National Research Council, 2011; van Praag et al., 2000; Wurbel, 2001). An animal’s environment consists of the physical environment and the social environment. Within the physical environment there are many variables, such as temperature, lighting, noise, and primary housing. Discussion here will be limited to that aspect of primary housing considered environmental enrichment. Within the animal’s social environment the primary consideration is individual versus social housing. The goal of environmental enrichment is to provide an animal with some choice, some degree of control over 13 its environment, and to promote species-specific normal behaviors (National Research Council, 2011). Logically, one must understand the specific normal behaviors and how important those behaviors are to the animal. Examples of environmental enrichment for rodents and rabbits include: (1) elevated shelves (Stauffacher, 1992); (2) shelters for guinea pigs, hamsters, and rats (Baumans, 2005); (3) nesting materials for rats, mice, hamsters, and gerbils (Baumans, 2005); and (4) gnawing material for rats, gerbils, hamsters, and rabbits (Baumans, 2005; Patterson-Kane, 2003). It is important to note that enrichment can, under some circumstances, lead to negative consequences such as increased aggression (Marashi et al., 2003). Further, the response to enrichment may be strain-specific (Nevison et al., 1999; van de Weerd et al., 1994). To determine the most appropriate social environment for an animal, the species-specific social behavior must be known and the needs of the experiment must be accommodated. It has been demonstrated that group housing of social species such as most rodents and some rabbits is beneficial to the animal and minimizes stress (Hall, 1998; Spani et al., 2003; Van Loo et al., 2001, 2007). Some species and/or genders have a higher risk of incompatibility, such as with male mice of some strains (Jacoby et al., 2002), and adult male rabbits (Suckow et al., 2002). Risk of incompatibility can be reduced if animals are grouped and raised together from a young age, if group composition remains stable, and if the environment is designed to allow escape and minimize competition for resources (National Research Council, 2011). In addition, socially housed groups should be monitored for early signs of aggression and separated if necessary (National Research Council, 2011). Some potential impacts of an enriched environment on research have been known for decades. For example, enhanced performance on complex learning was reported in rats housed in a more “stimulating” environment (Bingham and Griffiths, 1952; Forgays and Read, 1962). The effect of enrichment on variability of research results has been argued as both increasing variability (Mering et al., 2001; Tsai et al., 2002, 2003) and not affecting variability (Wurbel, 2000). Positive human interaction costs little and it is hard to imagine a negative effect on research outcomes. Therefore, this is only an ethical issue if it does not happen. Operant conditioning does require staff time and thus may only be appropriate for those studies in which procedures are going to be done frequently, or are potentially harmful enough to make the time investment worthwhile. When examining environmental enrichment, in addition to investigating real or potential effects on research, the impact on data obtained from an animal that is more highly stressed if unable to perform normal behaviors must also be considered. I. GENERAL 14 1. Ethical Considerations and Regulatory Issues Instituting changes in the environment may affect the ability to compare research results with historical data. In that case, more animals may be required; and this must be balanced against continuing to house animals in environments which do not maximize animal welfare. Who decides what is practical? Literature searches and pilot studies may be helpful in this decision. In summary, enrichment and social housing is not a “one size fits all” and it is important to balance the science with the well-being of the animals (Bayne, 2005; Weed and Raber, 2005). Decisions of where to balance such potentially conflicting interests benefit from consideration of the ethical principles discussed elsewhere in this chapter. Once various forms of refinement have been applied, how can it be determined if animal welfare has been enhanced? In other words, how can it be determined if the refinement is successful? Assessment of animal welfare can involve examination of physiology, behavior, and gross appearance of the animals. Some physiologic measurements relevant to animal welfare involve indicators of stress, such as levels of corticosteroids and glucose. Blood sampling can cause elevations in stress indicators making interpretation difficult. Instead, the use of fecal corticoid has been reported as a non-invasive, less distressful alternative (Bamberg et al., 2001; Cavigelli et al., 2005). Assessment of reproduction parameters such as number of litters, pups per litter, and number and weight of pups weaned may provide indicators of the level of animal welfare for animals maintained as part of a breeding colony. Behavior and gross appearance are often assessed using scoring systems which, after appropriate training of the observers, can provide readily accessible and reproducible assessments of animal well-being (Lloyd and Wolfensohn, 1998; Lloyd et al., 2000; Morton, 2000). It is worth noting that body weight alone as a measure of animal wellbeing can be deceiving if the animal is young and in a growth phase, or bears an experimental tumor burden, as these situations often are characterized by changes in body weight that may mask those associated with wellbeing. Body condition scoring has been advocated as a non-invasive technique to assess h