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Searching for Alternatives in Animal Research
The term "alternatives" in MEDLINE and other databases refers to documents discussing concepts relating to alternatives (Russell & Burch's 3 Rs, for example). Adding the term to other keywords relating to a specific study is not appropriate and will provide erroneous results (zero hits).

The Meaning of Alternatives
Searching for Alternatives
Understanding the Concepts
Conceptualizing the Search
Conducting the Search

The Animal Welfare Act, the implementing USDA regulations, and the U.S. Government Principles for the Utilization and Care of Vertebrate Animals used in Testing, Research, and Training, all require that consideration be given to alternative methodologies when planning research involving vertebrate animals. Researchers are always considering the use of emerging technologies to improve the quality of their research. Some of these new methodologies avoid the use of animals, reduce the number of animals required in a research study or lessen the impact on the animals used. Such techniques should be adopted as soon as they become available.

The search for "alternatives" is not optional for investigators conducting research involving vertebrate animals, but is an absolute requirement of all Federal agencies involved with funding for animal research. This search cannot be defined in a cookbook manner. Since it is intimately related to the research topic under consideration it must be as specialized as the research itself. Because no one understands the objectives of each technique to be used in a research study better than the investigator, no one is in a better position to evaluate the feasibility of the potential alternatives identified in a search. It is the purpose of this document to help clarify the goals of a search for alternatives and provide direction to some resources that may be helpful.

The Legal Basis

The PHS Policy
The PHS policy requires that investigators adhere to the US Government Principles. Principle III states, "Methods such as mathematical models, computer simulation, and in vitro biological systems should be considered". Principle IV states that "Proper use of animals, including the avoidance or minimization of discomfort, distress, and pain when consistent with sound scientific practices, is imperative" .While no guidance is provided regarding assurance of compliance is provided, the Policy declares the IACUC responsible for maintaining such compliance.

The Federal Animal Welfare Act (and USDA regulations)
The USDA regulations are the implementing rules for the Animal Welfare Act which gave the agency oversight authority. In Subpart C, section 2.31 the regulations state, “...the IACUC shall determine that the proposed activities or significant changes in ongoing activities meet the following requirements. .... (ii) The principal investigator has considered alternatives to procedures that 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”. Section 2.32(c)(5)(ii) also requires that the institution assure that personnel are trained in utilization of services providing information about alternatives.

The Meaning of Alternatives

Because the term "alternatives" was never completely defined in any Federal policy or regulation it led to considerable confusion among investigators and IACUCs. Often, research personnel interpreted the term to mean computer modeling or other purely non-animal models. Of course, it was quite clear to scientists that such "alternatives" were not applicable in a wide variety of situations. Recently, the Office of Technology Assessment clarified the issue by referring to the concept of alternatives as presented in Russell and Burch, The Principles of Humane Experimental Technique (1959). In their book, the authors present three encompassing categories which have been termed the "three Rs", replacement, reduction, and refinement. It is these three concepts that are now required by USDA policy to be considered in the search for alternative methodologies. The full text of the book is available on-line. Below is a description and some examples of applying the "three Rs" to animal research.

Replacement is one of the obvious alternatives. It is the use of non-animal techniques or the use of animals lower on the phylogenetic scale in the conduct of the research. Using Russell & Burch's concepts of "fidelity" and "discrimination" the replacement alternative emphasizes the use of low fidelity models (models that do not share a high proportion of characteristics with the human model) that have high discrimination (a model that shares ONE characteristic of the human model) for the process under investigation. Examples include use of cell culture or tissue culture, use of microbes for measurement of carcinogenic potential of compounds, use of invertebrates as has been done in the past (lobster and squid for nerve studies, limulus for visual electrophysiology). Some replacement alternatives may require the investigator to acquire new skills or invest in new materials and equipment. In many cases production of antibodies is performed in a live animal. While if properly done, this procedure causes little, if any, pain or distress in the animal subject, a model which produces a larger quantity of antibody is the fertilized chicken egg. Use of this procedure is more technically difficult and somewhat more expensive but should be considered when antibody production is required. Related to such a procedure is using mechanical biochemical systems for the production of hybridomas.

The replacement alternative for some procedures must often wait for the development or validation of new technologies. The Johns Hopkins Center for Alternatives to Animal Testing is actively pursuing the development and validation of such alternatives.

Reduction requires limiting animals used in the study to the absolute minimum required for scientific validity. This minimum may be based on a statistical analysis (power analysis), or an estimate of the minimum quantity of antibody or tissue required by a study. Another statistical method currently being employed to reduce the numbers of animals is sequential analysis. For those studies where this method is appropriate a notable decrease in animal numbers can be realized. Changes in experimental design can also reduce the number of animals required. For example, use of an unbalanced design (fewer control animals than experimental animals) may be possible for some studies. Other studies may permit use of the animals as their own control thus eliminating the need for a separate control group. In some cases a control group may not be necessary at all. For example, in a study of a new immunosuppressive drug an investigator may want to include a control for the vehicle in which the drug was dissolved. However, previous studies of immunosuppressive drugs already had used this vehicle as a control. It was not necessary to repeat the control. The use of animals to determine standard baseline values is also not necessary in many cases since many such values may be obtainable from the literature.

Less obvious methods include the use of genetically homogeneous animals from known stocks to reduce the variability due to the organism itself. Controlled environments and standardized husbandry practices also contribute significantly to the reduction of organismic variability and thus to the reduction of the number of animals required for a study. Animal sharing, if done appropriately, can reduce the number of animals used in research. The key is appropriate use. Animal sharing does not mean the use of an animal in multiple painful or stressful procedures. A clear example is the use of an animal under anesthesia in a feasibility study just before it is to be euthanized for tissue harvest by use of anesthesia overdose in a second study.

Refinement is perhaps the most elusive concept when considering alternatives to experimental procedures. Refinement requires the reduction of animal pain and distress to the minimum level consonant with the study under consideration. The most obvious example is the use of anesthetics, analgesics, and tranquilizers to reduce pain and distress chemically. However, the number of methods that can be employed to improve animal subjects' well-being and comfort within the confines of an experimental design is not limited to the chemical methods noted above.

Refinement methods begin with the housing and environment in which the animal spends most of its time. Adequate cage size and nutritious food are clearly mandated. However, the use of enrichment, exercise programs, and social groupings can all help to decrease stress and improve well-being among animal subjects. Preconditioning animals to test environments (mazes, restraint apparatus, etc.) both decreases stress and improves experimental results. Positive reinforcement is preferable to noxious stimuli but when painful or distressing stimuli must be used animals should be able to escape. Early euthanasia (euthanasia of an animal prior to the normal end of the study) must be performed if the animal stops eating, drinking, grooming, or ambulating as a result of illness or disability due to the experimental procedures. Death as an endpoint must be avoided unless there is a compelling scientific rationale since it is assumed that there is significant pain or distress when an animal is in extremis.

Even though antibody and hybridoma production seems relatively painless and of relatively low stress, inappropriate use of adjuvants or use of the incorrect adjuvant can cause lesions and other problems among experimental animals. Investigators should choose techniques and adjuvants which exhibit the least irritating effects on the animals used. An information source for adjuvants and antibody production is available for consideration by investigators.

Searching for Alternatives

Understanding the concepts
The search for alternatives is not simply a literature search that strings a number of keywords together to demonstrate that use of a living animal in a particular study is required and that the study is unique. It is also not a search to establish the scientific validity of the study. While searches for scientific validity and non-duplication are required, these searches are inadequate for determining the possibilities for alternatives. The USDA web site on alternatives provides some examples of a successful search.

Once scientific validity has been established, the search for alternatives must focus on the techniques and procedures of the study. It is these that are the sources of potentially painful and stressful events that must be minimized or eliminated. The search for alternatives is conducted in an effort to find methods that will accomplish the goals of the research with less or no impact on animals. Medline includes the descriptor "Alternatives to Animal Testing" which covers the topic of alternatives (not specific alternatives) and is useful to find papers that may assist the investigator in understanding the approaches and concepts involved in alternative searching.

The search must concentrate on those procedures which have the potential to cause pain or distress. Since animals cannot verbally describe the sensations engendered by a procedure we must rely on a rule of thumb. Any procedure which would induce pain or distress in a human must be considered potentially painful or distressful in animals. In practice, there are many signs of pain that can be observed in animals by one who is familiar with their behavior. However, many animals will not show signs of pain until it is extreme - an adaptive behavior to prevent attack by predators when an animal is sick or injured. Some investigators believe that use of analgesics or anesthetics for a potentially painful procedure renders it "nonpainful" and thus relieves them of the responsibility for searching for alternatives. The USDA standards would define the procedure as "painful" (since analgesia or anesthesia was required) and therefore require that alternatives be considered. Under this standard, euthanasia of an animal to harvest tissue or fluids is considered a painful procedure and an alternative search must be conducted.

Conceptualizing the search
There is no simple method to describe how the search is to be approached. A study may contain many techniques and procedures and each of them capable of inducing pain or distress would have to be considered in the alternatives search. A study that involves implanting a jugular catheter to measure blood oxygenation and inducing a specific pulmonary infection in a rabbit would require two different approaches to the search. To string together the keywords rabbit, catheter, and pulmonary infection would not lead to useful results, if indeed any results are obtained at all. The first step would be to examine the nature of the measurement and to determine if a transcutaneous method is possible. In the second instance, it would be more useful to review the literature on lung infection without reference to "rabbit". To limit to a specific species is to ignore the fact that the infection may not be specific to a particular species and alternatives may be adapted from studies of other animals. While in some cases the techniques are unique to the field of the research and the search should proceed in the scientific literature of that discipline, in many cases the methods are sufficiently general to permit searching across a number of areas of interest.

Conducting the search
There are a number of sources that can be consulted in conducting the search. Since the USDA requires that a search for alternatives must include searching a literature database, that would seem to be a logical place to start. It is important to remember that USDA policy requires that the database searched, the dates covered by the literature search along with the date of the search and the keyword(s) used must be provided to the IACUC as substantiation of the alternative search. Not only must the content of the database to be searched be considered but the coverage of the database is especially important. For example, Medline does not cover books but is limited to journal articles. If an investigator was interested in anesthetics or analgesics, several recent books might provide the necessary information. A database would have to be selected that includes book coverage. Dialog is an information provider that encompasses over 350 databases. Each database is described in a Blue Sheet that is available on the internet. Each Blue Sheet describes the contents of the database, the types of publications it covers, the years of coverage, and the searchable fields. The importance of choosing an appropriate database cannot be overemphasized

Much information on useful alternatives can be found in databases and sources which appear quite distinct from those where the goals of the experiment would be found. Some useful sources include the National Library of Medicine's bibliographies, information and valuable links to other resources in Alternatives to the Use of Live Animals in Biomedical Research and Testing  while the European Center for the Validation of Alternative Methods supports a website of alternatives databases. As noted above, the Johns Hopkins Center for Alternatives also publishes bibliographies and articles on alternative methods. Finally, the Animal Welfare Information Center of the USDA manages a website that contains a great deal of information relating to alternatives including the development of searches and provides an example of a search. In addition the information center will assist investigators in developing a search strategy for alternatives and will, for a small fee, conduct the search. It is to the advantage of any scientist to consult the library information specialist for assistance in conducting the alternatives search.

This site has been established to provide access to all Federal State, and Institutional animal welfare laws, regulations, policy and guidelines concerning vertebrate animals used in research and teaching. In addition, the latest versions of IACUC record keeping forms and protocol review forms are available for downloading. Finally, information on alternatives searching and links to other animal welfare-related informational sites have been provided. For comments or questions regarding this site contact the Webmaster
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