Critical points to Consider During Preparation of the Application

The following are the criteria employed by the IACUC during review of the application. Investigators are encouraged to consider these criteria during both the design and conduct phases of their research projects. The IACUC application should be written with careful attention paid to the requirements outlined below and contained in the Policy for Review and Approval.

  1. Potential Value of the Study. When live animals are used in research or biological testing, there must be a reasonable expectation that such utilization will contribute to the enhancement of human or animal health, the advancement of knowledge, or the good of society (PHS Policy: U.S. Interagency Research Animal Committee Principle II). The relative value of the study is a particularly important consideration in potentially painful category D and E experiments where there is an ethical imperative that the benefits of the research clearly outweigh any pain, discomfort and distress experienced by the animals. The investigator is requested to carefully consider the potential value of the project in relation to the ethical costs of the research, i.e., animal pain, morbidity and mortality.

  2. Selection of an Animal Model. Selection of an appropriate animal model is an important consideration, particularly at a time when alternative models for animal research are being emphasized. It is the investigator's responsibility, therefore, to select the optimal species for a particular project with consideration given to any uncontrollable financial and housing restraints.

    When a proposal requires the use of a particular species, the investigator should carefully consider why accomplishment of research objectives requires utilization of that species. Justification of a species should be based upon relevant anatomical, physiological or behavioral characteristics required for the research as well as any need to use established, comparative data. Monetary cost should not be a significant factor in choosing a particular species and is generally considered inadequate justification.

  3. Alternatives to Animal Use. It is recognized that virtually every major advance in health care stems in whole or in part from research involving animals and, in many research protocols, there is simply no alternative to the use of live animals. Despite this social imperative for animal experimentation, all investigators have an ethical obligation to explore ways in which animals can be partially or totally replaced (PHS Policy: U.S. Interagency Research Animal Committee Principle III). When a research question can be meaningfully pursued using reasonably available alternatives to the use of live animals, the investigator should choose these alternatives.

    The term "alternative" is interpreted liberally and includes four broad categories: 1) Modification of existing use of animals; 2) Use of animal-derived material in place of whole animals; 3) Replacement of living systems with non-living ones; 4) Use of mathematical or computer models. Each of these categories is discussed in more detail below. It should be noted that many of these alternatives are not new. Indeed, the entire history of the use of animals in research can be viewed as an ongoing process of the refinement of use of animal models with many factors (ethical, humane, scientific and economic) driving the refinement process.

    1. Modification of Existing Animal Use. It is sometimes possible to substitute one species of animal for another. For example, lower vertebrates or invertebrates may be substituted for "companion animals" such as dogs or cats. Such substitutions are usually advocated on the grounds that species differ in their capacity to suffer pain or distress, and it is generally assumed that invertebrates or lower vertebrates will suffer less than higher vertebrates, and laboratory species less than companion species. The truth of this assumption is still untested.

      If the research is to yield useful results, the animal species selected should be the one which will best fulfill the requirements of the model or most closely mimic the condition being studied. Substitutions, although they can and in some cases have yielded fruitful results, may raise as many difficulties as they resolve. In addition, selection of a particular species for a research project is constrained by many considerations, and there may be few or no other species that satisfy all the requirements of the model. In biomedical research, where the models are often of human diseases, invertebrates or lower vertebrates may share so few relevant characteristics with humans such that substitution is impossible. In basic biological research, the problem being studied may be specific to a particular species or group of species and may not even occur in other groups.

      Plants and microorganisms have also been suggested as substitutes for animals. For example, Salmonella is used in mechanistic studies in genetics, and the active steroid hormones found in yeasts are used in some endocrinological and immunological studies. Use of plants and microorganisms as substitutes, however, is limited by their evolutionary distance from humans and other higher vertebrates and by their own unique characteristics.

    2. Use of Animal-Derived Material. Although critics of animal research see alternatives as a way to eliminate animal use in research, many suggested "replacements" consist of animal-derived material. Examples of use of animal-derived material include cell, tissue and organ cultures. Working with culture specimens avoids potentially painful manipulations of live animals, although these materials must originate in a living animal. Use of such models may reduce the number of animals needed for research, as when several researchers needing small tissue samples of different organs are able to share a single animal. In some cases, cell culture studies can utilize cell lines derived from propagation of relatively few cells. Tissue or cells can also be obtained from national banks of animal or human cell lines, human donors, cadavers or placentas. Ultimately, however, hypotheses and data derived from these must be checked in the whole organism.
    3. Chemical, Physical and Mechanical Models. In some instances, it is possible to use physical or chemical models to study living systems. The study of many biochemical mechanisms, for example, make use of materials isolated from organs or tissues. Some physical and mechanical models have been developed mainly for educational uses, like Resusci-Dog, a canine cardiopulmonary resuscitation training mannequin used for veterinary training.
    4. Mathematical and Computer Models. Whenever a function or a relationship within a living system can be described mathematically, the possibility exists for developing a mathematical model. Scientists have long employed such models in biological and medical research because they provide the opportunity to vary the parameters involved and to predict what effects different parameters will have on the system. A complete inventory of information and parameters that should go into a model is not available until after extensive experimental work has been done on a living system, however, the final stage of this process must be to go back to the organism to check the accuracy of the model's predictions.

      It has been suggested that computer technology is now so advanced that computer-based models can completely substitute for the use of animals in research. Computers analyze data; they do not generate it. To make use of a computer model, a researcher or instructor must supply the computer with whatever information is needed for the model. If a living system is being modeled, the only source of this information is the living system itself. The more detailed the information supplied to the model, the better the model is likely to be.

      Based on information derived from animal studies, computer models have been developed to analyze relationships within and between living systems. Computer models have been particularly useful in modeling feedback systems. In animal behavior, for example, game theory has been used to construct computer models which would predict how an animal might behave during aggressive encounters. Biomedical applications of computer models include aspects of kidney, cardiac and lung functions, sensory physiology, neurophysiology and developmental biology. Biochemical applications of computer models include recognition programs to identify toxic substances. Similar attempts to identify carcinogens by computer have, so far at least, been less successful.

  4. Minimization of Animal Usage. The number of animals utilized in a protocol should be minimized consistent with sound scientific and statistical principles (PHS Policy: U.S. Interagency Research Animal Committee Principle III). Careful thought should be given to experimental design, detailed planning, and consultation with a biostatistician. Experiments should be designed that derive the maximum amount of information from the minimum number of animals. Statistical evaluation designed to provide the correct number of animals to be used may not always reduce the numbers in a given experiment, but by assigning the correct numbers to the original protocol one prevents the wastage involved in repeating a project due to inadequate numbers of animals in the initial experiment or involved in wrongly concluding that interventions are without effect. Good animal management and healthy stock selection can also reduce numbers by preventing losses from disease and injury.

    Investigators should also consider whether or not it is possible to share individual experimental animals or animal tissues between compatible research groups with IACUC-approved protocols. As long as sequential experiments are not deemed inhumane or scientifically conflicting, animal sharing results in animal conservation. Investigators are, however, reminded that non-related multiple major surgeries are prohibited.

  5. Alternatives to Potentially Painful Procedures*.  It is a moral imperative that pain be either eliminated or maximally reduced whenever possible. Therefore, it is required that investigators search for alternatives and justify the use of any potentially painful procedures. 

    * A "potentially painful" procedure is one that would result in animal pain if anesthetic or analgesic drugs were withheld. Note that a procedure can be potentially painful and the animal can experience no pain.

  6. Refinement of the Protocol to Reduce Potential Pain. Investigators are reminded that protocols should be carefully refined in order to minimize pain and distress (PHS Policy: U.S. Interagency Research Animal Committee Principle IV). For example, in toxicologic and carcinogenic research, animals should be euthanized whenever possible before they become moribund or develop large, painful tumors. If an investigator feels that death is a required end-point in order to preserve experimental validity, this must be strongly justified. In general, the IACUC does not approve such studies. With respect to the LD50, the FDA and many other agencies no longer require data obtained by the classical and statistically precise LD50. Investigators should make every attempt to alter protocols toward modified LD50 tests. These tests can provide most of the information expected from conventional LD50 type studies with fewer animals required and a reduction in animal pain and suffering.

  7. Restraints. Physical restraint procedures should be used on awake animals only after alternative procedures have been considered and found to be inadequate. If a restraint must be utilized, the animal should be trained or conditioned to the restraining device, using positive reinforcement, prior to the beginning of the experiment. The restraining device should provide the minimum restraint consistent with the maximum security and comfort of the animal. In addition, the restraining device should provide the animal with the greatest possible opportunity to assume its normal postural adjustments.

    Awake animals should not be subjected to prolonged physical restraint. Physical restraint should not be used on any animal that will not or cannot adjust to the restraining device and displays behavioral or biochemical/physiological signs indicative of stress.

  8. Pain Control During Acute Procedure(s). If a procedure will cause more than momentary slight pain or distress to the animals, the pain must be minimized to the greatest extent possible both in intensity and duration through the administration of appropriate anesthetics, analgesics and tranquilizers consistent with acceptable standards of veterinary medicine (PHS Policy: U.S. Interagency Research Animal Committee Principle V). It should be emphasized that the requirement for the alleviation or reduction of pain applies not only at the time the procedure is being conducted but also following the procedure until such time that the pain is either alleviated or reduced to an acceptable tolerance level. Where there is a question concerning whether or not an animal is or will be in pain that animal should be treated for pain prophylactically.

    Mechanisms of action of the major analgesics, tranquilizers, and anesthetics are fairly well elucidated. However, considerable differences in the action of these drugs in different species and in individual animals are known. A potential danger of inadequate treatment for pain exists when pharmacologic data are extrapolated from one species to another. Investigators should be familiar with the pharmacologic actions of the various anesthetic and analgesic drugs and must use the most effective drug available.

    The IACUC recognizes that in certain research protocols (category E), the administration of appropriate anesthetic or analgesic agents or both will compromise the scientific validity of the experiment. Such experiments should be carefully justified and described in terms of scientific design and potential value in the IACUC application. The withholding of these drugs must be based on referenceable scientific fact or experimental data and not intuition. In addition, pain, discomfort and distress levels must be carefully monitored. There is a limitation on the pain to which an experimental animal may be exposed. An animal that is observed to be in a state of severe pain which cannot be alleviated should be euthanized immediately. Protocols which produce severe, unrelieved pain in animals are not approvable.

  9. Estimation of Potential Post-Operative or Post-Intervention Pain. Whereas it is recognized that humans have no direct knowledge of the quality of pain an animal may or may not feel, there is no reason to believe that an animal's perception of pain is significantly different from a human's subjective interpretation of a painful event unless behavioral or clinical signs prove otherwise (PHS Policy: U.S. Interagency Research Animal Committee Principle IV). The American Physiological Society has defined stimuli as painful to animals if those stimuli are: 1) detected as pain in humans; 2) approach or exceed tissue damaging proportions; and 3) produce escape behavior in animals. In addition, animal pain, like human pain, is influenced by psychological and physiological variables. It should be noted that virtually any use of a laboratory animal may potentially result in some pain, discomfort or stress.

    If a procedure (e.g., thoracotomy, amputation, implantation of an artificial joint, etc.) or condition (e.g., tumor, radiation sickness, toxicity, etc.) has associated pain, discomfort or distress, it is imperative that the investigator estimate in advance the probable occurrence, magnitude and duration of the pain associated with that procedure/condition. Estimation of pain in advance is necessary in order to develop plans to prevent, monitor and relieve as much pain as possible during the post-procedure period.

  10. Post-Procedure and Chronic Care. It is the responsibility of the investigator to ensure that adequate post-surgical and post-procedure care is provided to all animals as often as necessary and for as long as necessary including nights and weekends. This care must meet acceptable standards in veterinary medicine. All category D and E protocols require some form of post-procedure care. Most category D1 and a few category C experiments require monitoring of the animals.

    The IACUC is particularly concerned about the possibility that an animal may experience pain, discomfort or distress as a result of a procedure or induced condition. The investigator must take all necessary steps to assess the presence of pain, discomfort and distress. In assessing pain, the investigator should use behavioral signs based upon the normal behavior pattern of the species under study. (Species-specific signs of pain are listed in the SOP on Recognizing the Potential Causes, Intensity and Clinical Signs of Pain.) In some circumstances, it may be desirable to use physiological parameters (e.g., plasma cortisol, catecholamine, white blood cell counts and cardiovascular parameters). Where there is a question concerning whether or not an animal is or will be in pain, the animal must be given the benefit of the doubt and the existence of pain assumed unless proven otherwise. That animal should, therefore, be treated prophylactically for pain.

  11. Euthanasia/Disposition of Animals. Euthanasia is the act of inducing painless death. The selected method of euthanasia must be consistent with the recommendations of the current AVMA Guidelines on Euthanasia. The selection of the method of euthanasia should be according to the following criteria: 1) its ability to produce death without causing pain; 2) the time required to produce loss of consciousness; 3) the time required to produce death; 4) its reliability; 5) any hazard to personnel; 6) its potential for minimizing psychologic distress; 7) its compatibility with the requirements and purpose of the research; 8) its emotional effect upon observers or operators; 9) its economic feasibility; 10) its compatibility with histopathologic evaluation; and 11) drug availability and abuse potential. Some methods of euthanasia require an additional physical method of euthanasia be performed to ensure death.

    The most desirable euthanizing techniques are those that produce a rapidly occurring unconsciousness followed by cardiac or respiratory arrest. Selection of the most appropriate method of euthanasia is dependent upon the species and weight of the animal involved, available means of animal control, skill of personnel, numbers of animals, objectives of the protocol and other considerations.

    Physical methods such as decapitation and cervical dislocation are the least desirable euthanasia methods but can be humane techniques when justified and carefully performed. The use of ether is discouraged because of its ability to produce stress in rats and its flammability, and halothane is not recommended because of its potential for liver toxicity. Use of an injectable agent such as an overdose of pentobarbital is considered the most desirable method. Carbon dioxide is a permissible inhalant agent.

    Whenever possible, animals should not observe euthanasia of others, especially their own species. Distress vocalizations, fearful behavior, and release of certain odors or pheromones by a frightened animal may cause anxiety and apprehension in other animals. However, there is at least one study that shows that there may be no effect of nearby euthanasia on animals.

    Distress may occur among personnel directly involved in performing repetitive euthanasia. The investigator, therefore, should be sensitive to this possibility.

  12. Investigator(s) Qualifications, Training and Experience. Procedures involving the use of animals must be performed by or under the immediate supervision of a qualified individual (PHS Policy: U.S. Interagency Research Animal Committee Principle VIII). If an investigator requires training, the IACUC and the Director of Comparative Medicine will provide or assist the investigator in obtaining the necessary training. The application must list for all persons involved 1) their role in the protocol, 2) their training related to the study and species to be used and 3) their relevant training and experience. All investigators and other personnel with animal contact must have completed required training before protocols, continuing review of protocols or requests for changes in protocols will be approved.

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