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Difficult Issues in Protocol Review

Potential for Pain and Distress

The National Research Council book entitled Recognition and Alleviation of Pain and Distress in Laboratory Animals (1992), states “that in most experimental and husbandry situations, laboratory animals need not experience substantial pain and stress and that prevention and alleviation of pain or stress in laboratory animals is an ethical imperative.” Likewise, Article IV of the U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research and Training states that, “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.” Minimizing pain and distress, consistent with meeting experimental objectives, therefore, is a key feature of IACUC/institutional responsibility.

The relief of pain and distress in research animals is not only ethically sound, humane and necessary for regulatory compliance, it also promotes good science. Minimizing animal pain and distress can minimize physiologic and psychological changes that can skew experimental data. The IACUC needs to know what the potential is for pain or distress associated with experimental procedures, including clinical and behavioral abnormalities associated with spontaneous or induced animal models (e.g., mutant and transgenic mouse strains). By regulation, veterinary staff must be consulted in the planning of studies involving potential pain or distress. Planning may involve both the preparation of the protocol for IACUC submission and training in the experimental methods. Veterinary consultation is usually valuable for understanding:

• regimens for use of anesthetics, analgesics, and tranquilizers, including monitoring;
• how to recognize species-specific signs of pain and distress;
• surgical approaches and aseptic technique; and
• post-procedural monitoring and intensive care.

One of the most difficult issues for both research staff and the IACUC is discussion and evaluation of methods to minimize pain and distress. This inevitably involves a balancing act between experimental objectives and animal welfare. When there is potential for pain or distress, researchers are expected to carefully monitor animals and relieve the pain or distress whenever possible. One method to accomplish this is by administration of anesthetics or analgesics. Pain and distress may also be reduced by euthanizing an animal. Defining so-called humane endpoints (e.g., limits on the size of tumors in cancer studies) is an important aspect of protocol review. If unexpected pain or distress occurs and is more than an isolated incident, the PI must submit a protocol amendment describing the problem and stating the proposed resolution (e.g., administration of analgesics, lowering the dose of the drug administered, etc.).  Sometimes, the PI may justify the need for unrelieved pain or distress in the amendment, or in the case of regulated species, as a “Column E justification.” [See USDA Pain Categories in previous lesson.]

Recognizing pain in an animal can be subjective. Animals should be monitored for pain and distress by trained individuals as appropriate for a particular species, condition and procedure. Pain should also be considered as an individual experience, since each animal may respond differently to a painful stressor. Animal care technicians may be the first to pick up on subtle changes in appearance or behavior because they work with the animals closely every day. The obligation to reduce pain or distress, therefore, does not end with the IACUC review of an animal use protocol. It is the responsibility of animal care staff, investigators and research staff, and veterinarians, to continue to monitor animals for pain, distress, illness, morbidity, or mortality during the course of a research study.

The AWRs require that research facilities file an annual report that accounts for numbers of animals used, by species and category of pain or distress. [See USDA Pain Categories in previous lesson.] Some IACUCs require that this information be provided in the protocol form, and the IACUC may provide additional guidelines for determining the appropriate pain category. This determination is not always straightforward.

Alternatives

Federal regulations require that investigators provide a written narrative of the sources consulted to determine whether or not alternatives exist to procedures that may cause pain or distress in animals. The IACUC, through the protocol review process, must determine that this has been done appropriately.

The concept of minimizing animal exposure to pain and distress by limiting numbers and considering non-animal alternatives was popularized in 1959 by Drs. Russell and Burch in their book The Principles of Humane Experimental Technique. This concept is now widely known as the “Three R’s” or replacement, reduction and refinement. Alternatives are taken to mean not just replacement of animals with non-animal methods, such as computer simulations, but also reduction of animal numbers to the minimum necessary to satisfy experimental objectives, and refinement of methods to minimize pain and distress. Current regulatory expectations are that, in most cases, the PI perform a literature search for alternatives and the results must be recorded on the animal use protocol form. A number of databases are available for alternative searches, including:

• Agricola
• ALTWEB, the Johns Hopkins Center for Alternatives to Animal Testing (CAAT)
• Animal Welfare Information Center (AWIC) (an information service of the National Agricultural Library)
• Biological Abstracts
• Current Contents
• Current Research Information Service (CRIS)
• NORINA
• PubMed
• University of California Center for Animal Alternatives

An excellent compilation of alternatives search resources in table form can be found at the University of Miami School of Medicine’s Office of the Animal Care and Use Committee website. The narrative describing the alternative search should be detailed enough that the IACUC can readily assess whether the search topics were appropriate and whether the search was sufficiently thorough. The minimal information required by the IACUC includes: the database(s) or sources searched, the date of the search, the years covered by the search, and the keywords used by the investigator.

Justification for Using Animals, Species, Numbers Used

US Government Principle II states that research using 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.” Animal use protocol forms typically have a question that asks about the purpose and importance of the work, and PIs should use this as an opportunity to provide basic background information so IACUC members can understand why the animal use activity is necessary.

Regulations require that investigators provide a justification for using animals, and for the specific species and numbers of animals to be used. These are sometimes difficult issues to address, but involve basic questions that should be considered in the planning of any experiment using animals. Implied in these questions is the idea that animals should not be wasted. The prevailing view is that animals should not be used in research, teaching or testing unless there is no other way to accomplish the objectives of the study. The justification should include the rationale for selection of the specific species chosen, a brief description of the experimental design, and the basis upon which the total number of animals requested was determined.

There is a common misconception that animal numbers in a study must always be minimized. In fact, the requirement to justify animal numbers means that there has been due consideration of the experimental design of an experiment, including determination of the optimal number of animals. Use of too few animals in an experiment may fail to yield interpretable results and can also be wasteful. Proper experimental design includes statistical considerations, and some IACUCs may have expectations that investigators consult with a statistician prior to completing an animal use protocol. Some institutions even have statisticians as IACUC members, in order to provide advice in this area. In the final analysis, however, IACUCs are still expected to determine that the numbers of animals requested are appropriate for the intended purpose.

Survival Surgical Procedures / Multiple Survival Surgeries

Federal regulations are specific regarding surgical procedures on research animals. The regulations require:

• that survival surgery be performed using aseptic technique (a set of specific practices and procedures performed under carefully controlled conditions – e.g., the use of sterile surgical gloves and instruments — in order to minimize contamination by pathogens);
• that personnel be adequately trained to perform the surgical procedure;
• that adequate anesthesia and postoperative care (including analgesia) are addressed in the animal use protocol; and
• that appropriate records are maintained.

Appropriate surgical records should include a brief description of the procedure, a record of the drugs administered, a description of any complications that develop, a description of postoperative care, and a record of who performed the surgery. In addition, if controlled drugs such as ketamine or pentobarbital are used, a separate controlled drug log must be maintained.

Studies proposing multiple survival surgeries must be scientifically justified in the animal use protocol, and approved by the IACUC, or required as routine veterinary care to protect the health or well-being of an animal as determined by the attending veterinarian.

Transgenic Animals

The term “transgenic animal” refers to an animal in which there has been a deliberate modification of the genome — the material responsible for inherited characteristics. Since 1981, when the term was first introduced, genetically-engineered animals have become increasingly important as research subjects. Transgenic animals are used, among other things, in the basic biological study of gene regulation; in biomedical research to identify the functions of specific factors in complex systems as models of human disease; in toxicology as responsive test animals; and in biotechnology as producers of specific proteins. Transgenic animals are an extremely powerful tool for development of disease models, and in some cases can be considered a replacement alternative. For example, the use of transgenic mouse models which more closely mimic the human disease can replace the need to use more sentient animals as models. The better specificity of transgenic models may in time also lead to a reduction in the number of animals used.

Along with the development of transgenic technology, ethical concerns have arisen about the use of this technology. These concerns are wide ranging and include animal welfare, human health and environmental issues. They include animal suffering caused by how the transgenes manifest themselves, sometimes inducing tumors or neurodegenerative diseases, for example, the possible escape of transgenic animals into the environment; and the possibility for the modification of the human genome. Some considerations that IACUCs must address in reviewing transgenic animal protocols include:

• known traits that will affect breeding and lifespan;
• abnormalities that are expected or known to exist in the animals, and whether they may cause pain or distress;
• methods for monitoring, recording and alleviating potential pain or distress;
• criteria for experimental endpoints; and
• possible biological containment and security procedures.

Monoclonal Antibody Production

Monoclonal antibodies (MAbs) are exceptionally pure components of the immune system that are able to recognize and bind to a specific antigen. Following the development of this technology in 1975, MAbs have become an important resource for medical research, diagnosis, therapy, and basic science. The first step in the production of MAbs is to immunize a mouse with an antigen. When the mouse begins to produce antibodies to the antigen, its spleen is removed. Antibody-producing cells from the spleen are then fused with a myeloma (a type of immune cell tumor arising in the bone marrow) cell line, one which does not produce antibodies and has been maintained in culture. The new fused cell line, which does produce antibodies, is grown briefly in culture and then re-injected into another mouse’s peritoneum. Finally, the ascites fluid which contains monoclonal antibodies is harvested from the mouse.

In an attempt to avoid pain, discomfort, or distress in animals from the production of monoclonal antibodies using the mouse ascites method, both NIH and the USDA encourage the use of alternative methods to produce monoclonal antibodies in vitro without compromising the aims of the study. IACUCs are now expected to critically evaluate all protocols which propose using the mouse ascites method for monoclonal antibody production, and only allow in vivo production on the basis of strong scientific justification.

Prolonged Physical Restraint

Physical restraint may be a requirement of the research. However, such restraint must be made as painless as possible for the animals, should be of as short a duration as possible, and should utilize the most animal-friendly restraint system possible. The need to restrain animals should be fully described and justified in the protocol. Justification is particularly important if the restraint is of a long duration or if the restraint mechanism is non-standard. Similarly, acclimation to a restraining device or procedure may be required prior to its use on study.

Food or Fluid Restrictions

Animal care and use standards require that animals receive food and drink consistent with their species and its natural needs. Deviation from the standard diet of food or constant availability of water must be scientifically justified in the protocol and approved by the IACUC.

Humane Experimental Endpoints

Experimental studies may involve procedures that cause clinical symptoms or morbidity (death) in animals. The IACUC must consider the selection of the most appropriate experimental endpoint(s). This requires careful consideration of the scientific requirements of the study; the expected and possible adverse effects the research animals may experience (pain, distress, illness, etc.); the most likely time course and progression of those adverse effects; and the earliest most predictive indicators of present or impending adverse effects.

The effective use of endpoints requires that properly qualified individuals perform both general and study-specific observations of the research animals at appropriate time points. Under optimal conditions, studies are terminated when animals begin to exhibit clinical signs of disease if this endpoint is compatible with meeting the research objectives. Such endpoints are preferable to death or moribundity (nearness to death) since they minimize pain and distress. The term “death as an endpoint” refers to a situation in which the unassisted death of an animal – not euthanasia of the animal – is needed as a time point in a study. For instance, it may be necessary in the study of a particular disease to learn how long a treatment prolongs life. In that situation, a case could be made by an investigator that intervening to euthanize an animal prior to its “natural” death would be detrimental to the study and would preclude collection of necessary data.

In general, death as an endpoint must be extremely well justified before an IACUC will approve it in a study. The Johns Hopkins Center for Alternatives to Animal Testing maintains a humane endpoints database that can be helpful under some circumstances. Another helpful document is the OECD Guidance Document on the Recognition, Assessment, and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation (2000).

Method of Euthanasia

The term euthanasia is derived from the Greek terms eu meaning “good” and thanatos meaning “death.” A “good death” would be one that occurs without pain and distress. In the context of this course, euthanasia is the act of inducing humane death in an experimental animal. According to the AVMA Guidelines for the Euthanasia of Animals (2013), euthanasia techniques should result in rapid unconsciousness, followed by cardiac or respiratory arrest and ultimate loss of brain function. In addition, the technique should minimize any stress and anxiety experienced by the animal prior to unconsciousness. Stress may be minimized by technical proficiency and humane handling of the animals to be euthanized.