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Searching for Alternatives and the 3Rs

The concept of alternatives to the use of animals in research is not only espoused by animal welfare groups but required by federal law. The US Government Principles for the Care and Use of Animals Used in Testing, Research and Education state in part that “…Methods such as mathematical models, computer simulation, and in vitro biological systems should be considered.” The Public Health Service Policy endorses this principle and the Guide for the Care and Use of Laboratory Animals states that the ‘availability or appropriateness of the use of less-invasive procedures, other species, isolated organ preparation, cell or tissue culture, or computer simulation’ must be considered in the preparation and review of animal care and use protocols. The Animal Welfare Act states in section 2.31 “…(ii) The principal 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…”

What are Alternatives?
The use of animals remains necessary except where valid scientific alternative methods are available. The concept of alternatives was originally put forth in The Principles of Humane Experimental Technique by Russell and Burch (1959). In this book, the authors proposed three categories now commonly referred to as the “3 R’s”. These principles are:

Reduction – To minimize the number of animals used in a research protocol; to reduce the number of animals used to obtain information with statistically relevant results.

Refinement – Use of techniques that reduce the incidence or severity of pain and/or distress* in those animals that are used.

Replacement – Substitution of a lower species that may be less sensitive to pain and distress for a higher species or substitution of non-animal methods or material for animals.

The use or consideration of alternatives does not preclude the use of animals, but instead requires that all animal use be carefully considered and research protocols planned such that discomfort, distress or pain is eliminated or minimized wherever possible. As advances are made, the overall use of animals has decreased, however the use of live animals cannot yet be eliminated from research.

Distress – A state in which an animal cannot escape from or adapt to the external or internal stressors or conditions it experiences, resulting in negative effects upon its well-being.

Painful Procedure – as applied to any animal means any procedure that would reasonable be expected to cause more than slight or momentary pain or distress in a human being to which that procedure was applied, that is pain in excess of that caused by injections or other minor procedures.

An alternatives search must be performed for each painful or distressful procedure.  A search for alternatives to use of animals in research (reduction, refinement, replacement) is not the same as a search to show that the research is unique or not duplicative. It is also not a search to show that the intended animal use represents valid science. Keywords used for such searches would be unlikely to yield alternatives to the proposed animal use. Rather the search for alternatives focuses on the necessity of animals, species or techniques. The goal of an alternatives search is to locate, if possible, methods that would accomplish the goals of the research with less or no animal impact.

The search should include keywords relevant to the proposed research as well as appropriate alternatives terminology. Ideally there should be two phases to the search strategy. The first phase looks for reduction and refinement alternatives. This phase should focus on citations relevant to the proposed field of research. The second phase looks at replacement with non-animal alternatives (e.g. computer models, mathematical models, in vitro systems, epidemiologic studies) or alternative animal models (lower taxonomic vertebrates or non-vertebrates). Search results should be carefully examined to identify any techniques, procedures, parameters or methods that may lessen impact on animal use. The search for alternatives should ideally be performed prior to protocol formulation in an effort to save time on planning. and re-writing.

NOTE: With regard to the FSU Animal Use Protocol Form if one or more alternatives are identified during an alternatives search and will not be incorporated into the protocol, a written narrative must be supplied to justify why this alternative(s) was not used.

An alternatives search should build on the current literature pertinent to the research topic. Searches must be conducted in appropriate databases (see below) and should use appropriate keywords, subject headings and alternatives terminology. Combine these items logically using Boolean operators and use truncation symbols to get variant endings for search terms. Results of the search should be provided to the ACUC in a written format; search results should be quite clear and logical when reaching the final conclusion that no other methodology is available to replace, reduce or refine the use of animals in each protocol. The Committee will assess key words, databases used, search strategy and database returns to determine if the search has been thorough. Remember, there is no one perfect way to perform an alternatives search so there is not an exact formula for doing it. This is because most of the research protocols are unique, hence alternatives to specific procedures and techniques will need to be individualized.

There are a number of useful web sites that can help instruct faculty, staff and students in the art of performing an alternatives search. Some are listed below.

Search Terms and Strategies for Information on the Three Rs - FRAME - The most descriptive, one stop web site for alternatives searching, it discusses from start to finish why and how to perform a search for alternatives.

Tips for Searching for Alternatives to Animal Research and Testing – Animal Welfare Information Center, hosted by the USDA

Searching for Alternatives to Painful Procedures Used on Research Animals - NIH Library

Possible search terms for the 3Rs

*these are not all inclusive

General guidelines for first step - combine proposed animal testing procedure(s) with:
Alternative(s) +/- animal testing assay method model(s) or animal model(s) replace replacement surrogate system technique

Refinement - 'animal' combined with one or more of the following terms:      

analgesia analgesic
anesthesia anesthetic
anxiolytic cage
caging distress
enrichment enrichment environmental
enrichment behavioral euthanasia
handling housing
husbandry hypnotic
non-invasive pain
post-operative post surgery
restraint sedative
stress welfare

Reduction in the number of animals - with or without 'animal' or 'species'                      

experimental design model
reduce reduction
statistical combined with analysis or model

Replacement of animals with non-animal alternatives

artificial intelligence expert system
image imaging
interactive model
modeling mathematical model
theoretical model prediction
simulation software
structure activity relationship
teaching virtual instruction or teaching
aided or assisted instruction or learning

Physicochemical Systems                                                    

artificial chemical physical
any of the above possibly combined with assay, membrane, method, model, system or technique

In Vitro Systems - appropriate combinations of the following:                                                  

abbatoir assay autopsy
biopsy necropsy cadaver
cell cell line cellular
culture cytosolic fraction dermal equivalent
embryo explant ex-vivo
fragment human isolated
method microsomal model
organ organelle slice
tissue tissue equivalent technique
vitro (Note: "in" is often a stop word which will be stripped from the search)

Non-mammalian organisms        

algae amphibian bacteria
fish fungi or fungus hydra
insect(s) invertebrate(s) microorganism
plant protozoa yeast


There are many databases that can be searched for alternatives. Some are specific to research needs (e.g. ToxNet) while most are fairly broad in nature. Below is a partial list of databases that may be of use while performing searches for alternatives. Most of the subscription databases are available to FSU employees and students via the Florida State University Libraries Database service. Teaching and training protocols should perform searches in NORINA. An excellent resource for those performing a search for teaching and training protocols is the UC Davis Center for Animal Alternatives – Education.

AGRICOLA – National Agricultural Library's database containing citations for books, articles, conference proceedings, and technical reports in agricultural and veterinary sciences and animal welfare. Coverage: 1970-present

ALTBIB (National Library of Medicine Bibliography on Alternatives to the Use of Live Vertebrates in Biomedical Research and Testing) - The intent of the bibliography is to assist in identifying methods and procedures helpful in supporting the development, testing, application, and validation of alternatives to the use of vertebrates in biomedical research and toxicology testing.

ALTWEB – Johns Hopkins Alternatives to Animal Testing on the Web; A great site for news, information, discussion, and resources from the field of alternatives to animal testing. A joint effort of Johns Hopkins’ Center for Alternatives to Animal Testing (CAAT): and a number of other public and private organizations, it is the most comprehensive resource on animal alternatives for scientists, educators, veterinarians and individuals throughout the world. Altweb includes links to other sources of information on alternatives, and has access to Internet search engines that can be used to search many databases.

Antibody Resource Page – A guide to antibody research and suppliers.

BIOSIS Previews - Comprehensive reference database for life science research, covering original research reports and reviews in biological and biomedical areas; includes conference proceedings, meeting abstracts, technical reports, and patents. Coverage: 1985-present

Cell Line Databases – searchable databases of cell lines, including hybridomas. This includes American Type Culture Collection (ATCC), HyperCLDB and the European Collection of Cell Cultures (ECCC).

Center for Animal Alternatives at the University of California - This site provides investigators who use animals with information on the most current methods for improving all aspects of animal care during their work. It also provides an extensive section on searching for alternatives (including an alternatives search service) as well as search templates; information resources guides, a readings and resources section, electronic versions of their newsletter (UC Alert), and links to other sites of interest.

CORDIS – information on research, development and innovation activities in the European Union

CRIS - information on research projects supported by the United States Department of Agriculture

CRISP - information on research projects supported by the United States Public Health Service

DoD Biomedical Research Database – information on research projects supported by the United States Department of Defense

DoD Defense Technical Information Center - A resource to prevent unnecessary or redundant research from being performed at taxpayer expense. DoD-funded researchers are required to search DTIC's collections of technical reports and summaries of ongoing research to ensure that unnecessary research is not undertaken.

European Centre for the Validation of Alternative Methods (ECVAM) Scientific Information Service - The ECVAM is an international reference center for the development, scientific and regulatory acceptance of alternative testing methods aimed at replacing, reducing or refining the use of laboratory animals. The Scientific Information Service (SIS) is a database of the European Commission Joint Research Centre. SIS provides factual and evaluated information on advanced non-animal test development and validation for toxicology assessments coming from a wide range of international information sources.

EMBASE - A major biomedical and pharmaceutical database that also covers topics on health policy and environmental health. Coverage: 1980-present

Environmental Enrichment for Primates - annotated bibliographical database

The Interagency Coordinating Committee on the Validation of Animal Models (ICCVAM) - The site provides information on models that have been validated, those under development, and how the process of validation works.

Invitroderm - information on alternatives to in vivo skin irritation testing.

MEDLINE – National Library of Medicine's premier database covering biomedical literature, related life sciences and research. Coverage: 1966-present.

Model Organisms of Biomedical Research – NIH web site with information about national and international activities and major resources that are being developed to facilitate biomedical research using various animal models of great diversity.

Mouse and Rat Strains - information on the characteristics of inbred strains from Jackson Laboratories

NCA Database – recently started database on research projects related to the development of alternatives to animal use in The Netherlands

NIH Office of Animal Care and Use (OACU).

NORINA - database on alternatives to animals in education

PsycINFO - Contains citations and summaries of journal articles, book chapters, books, dissertations, and technical reports from the professional and academic literature in psychology and related disciplines. Coverage: 1887-present

TOXNET - A cluster of databases on toxicology, hazardous chemicals, and related areas.

Web of Science - A collection of multidisciplinary citation databases, including Science Citation Index; the information includes the article's cited reference list and allows cited reference searching. Coverage: 1975-present

Zoological Record - A subset database of BIOSIS. Zoological and animal science literature covering all research from biochemistry to veterinary medicine; references are from international serial publications, books, meetings, reviews and other non serial literature. Coverage: 1978-present