Very few, if any, scientists have argued that they would prefer to use animals even if they did not have to (Rowan, 1991, Silverman, 1993), and many of the companies using animals have contributed substantial time and money to the search for alternatives. This activity has been at least partly responsible for the dramatic decline in laboratory animal use over the past twenty years. Nonetheless, a significant segment of American scientists are uneasy about the term "alternative" (preferring to use adjectives like "adjunct" or "complementary") and are only slowly embracing the concept of alternatives.

Definitions

Although the word "alternatives" is used frequently, it does not always reflect identical intent by users. Some animal activists argue that all animal studies should be replaced immediately by "alternatives" although many experts on alternatives do not consider the total replacement of animals a possibility in the near future. Others who support the search for "alternatives" focus on decreasing animal use (rather than eliminating it) or on mitigating animal pain and distress.

Rowan (1984a) offers the following as the most widely accepted definition: "An alternative is any technique which could: (a) replace the use of animals altogether; (b) reduce the number of animals required; or (c) reduce the amount of stress suffered by the animal through suitable refinements in the techniques used." Replacement, reduction, and refinement are the "three Rs" as originally set out by Russell and Burch (1959). Rowan also stresses that any valid alternative system must provide data which leads to the same conclusion with at least the same degree of confidence as that obtained from the system being replaced. Many alternatives are improvements over traditional methods, which is the primary reason why scientists develop and apply them.

Replacement originally referred to the use of insentient material for conscious higher animals so that a fully anesthetized animal that did not recover could be regarded as a replacement to a conscious animal. Today, the idea of replacement is more restrictive and usually refers to the use of either tissue culture or some other experimental system that does not require either disturbing or killing an animal. Thus, the new pregnancy test kits and the Limulus Amoebocyte Lysate (LAL) test for pyrogenic [fever-producing] endotoxins are considered to be replacements of traditional tests which used rabbits. Even in these cases, however, there is some level of disturbance to animals. The antibodies used in the pregnancy kits are probably produced in living animals and horseshoe crabs (Limulus) are bled to provide a blood sample to manufacture the LAL reagent.

The question of what constitutes an animal in the alternatives framework is exemplified by attitudes toward Limulus, an invertebrate. Generally, invertebrates are perceived to be replacements. However, a few papers have discussed the sentience of insects and, recently, the British Home Office decided to add the octopus to the category of protected research animals. For the most part, however, invertebrates are considered to be acceptable as replacements, as are early-stage vertebrate embryos (e.g., chicken eggs).

Reduction refers to modifications in procedures that allow fewer animals to be used. In the assessment of acute toxicity, several reduction alternatives to the classical LD50 Test (which involves administering lethal doses of chemicals to from 20 to 50 animals) have been accepted by the Organization for Economic Cooperation and Development. These include the Fixed Dose Procedure, the Acute Toxic Class Method, and the Up and Down Procedure, all of which typically use fewer than 20 animals per test.

The distinction between replacements and reductions is not always clear (Russell, 1995). The introduction of non-animal methods (replacements) in the production of new batches of insulin and polio vaccine have led to dramatic reductions in animal numbers in the past twenty years (Hendriksen, 1988; Tretheway, 1989). Some observers would consider these cases to be examples of replacement techniques, while others would consider them to be examples of reduction in numbers. The former perspective focuses on the techniques employed (i.e., non-animal methods), whereas the latter focuses on outcomes (i.e., reduction in numbers).

Refinement refers to efforts to decrease the incidence or severity of painful or stressful procedures for animals which still are used in specific tests or research. For example, many research facilities have now instituted policies to restrict or eliminate the use of Complete Freund's Adjuvant (CFA) in immunization protocols. CFA causes an inflammatory reaction that can be very painful and, in the current climate, many institutional animal care and use committees (IACUCs) are focused on minimizing animal pain and distress.

Examples of Available Alternatives

An alternative is a new research technique that uses no or fewer animals or causes less animal distress. One of the features of biomedical and biological science is the increasing sophistication of research technology and the expanding ability to answer ever more complicated and detailed questions. Therefore, it is not particularly surprising that the on-going search for better research technology should be leading in many instances to a reduction in animal use and animal distress. Questioners often ask for more detail, however, and want to know "just what are the alternatives?" The following list provides some concrete examples of research technologies and approaches that have replaced, reduced, or refined animal use. However, readers should be aware that, given the broad definition of an alternative, most scientists are, or could be, engaged in the search for alternatives whether they realize it or not. It is part of the focus of The Humane Society of the United States to make scientists more aware of the issues and to encourage them to place the search for alternatives higher on their list of priorities.

Tissue culture (cell and organ cultures, organ slices, etc.) is widely used in biomedical research and testing. The use of tissue culture grew rapidly after the Second World War when antibiotics could be used to control the contamination of tissue cultures by micro-organisms. In the last ten to twenty years, the use of in vitro (literally, in glass) systems has grown dramatically across all disciplines, even in those like physiology and toxicology where whole-animal studies have been the mainstay. In monoclonal antibody production, for example, various in vitro approaches are replacing the use of the painful mouse ascites method.

Unfortunately, many cell cultures still require animal serum to grow properly and the cells themselves are sometimes obtained from animals killed for the purpose. Although these systems are not yet entirely "animal-free," the future potential of various tissue culture approaches to decrease reliance on animal use is considerable.

The development of physical and chemical techniques over the past fifty years has been nothing short of spectacular. The new genetic engineering technologies in particular allow laboratories to do in a day what took a year or more only a few decades ago. In addition, the new imaging technology (e.g. ultrasound, nuclear magnetic resonance) allows research scientists to do more and more investigation without invading body cavities or killing animals (refinement). The much greater sensitivity of new laboratory technology, coupled with non-invasive imaging, has played a major role in reducing the demand for laboratory animals over the past twenty years. For example, diagnostic kits have replaced rabbits in human pregnancy testing, high-performance liquid chromatography (HPLC) has replaced mice in insulin potency testing, and various physical-chemical techniques, including HPLC, have replaced rats and other animals in assays for several vitamins (see Stephens, 1990).

Not surprisingly, human studies can provide considerable information on human biology and disease. Although 40% of the NIH budget already goes to support human studies, more could be done using human clinical studies, epidemiological approaches, and autopsy material. However, human research is more expensive than animal studies, and the Helsinki Declaration on the use of human subjects in research specifically states that humans should not be used in research unless appropriate studies have already been conducted in animals. Clinical and post-mortem studies of Alzheimer's patients have aided our understanding of this form of senile dementia. Epidemiological studies, mostly occupational research, have identified most of the substances known to cause cancer in humans. The same imaging techniques that can make animal studies more humane (see above) can also expand the capabilities of ethical human studies.

Depending on one's perspective, organisms that are sometimes considered to be less-sentient such as invertebrates, early-stage vertebrate embryos, and micro-organisms can be viewed as either replacements or refinements when used in lieu of vertebrates. The CAM Test, or Hen's Egg Test, is used in lieu of rabbits as a screen for eye irritancy. Bacterial cultures have replaced animals in several vitamin assays. The Ames Test using Salmonella bacteria is an alternative for detecting chemicals that cause mutations.

State-of-the-art approaches to biomedical research and testing increasingly incorporate mathematical or computer modeling into their descriptions of living systems. Toxicologists use mathematical models known as Quantitative Structure Activity Relationships (QSARs) to predict biological activity (in this case, toxicity) from chemical structure. Pharmacologists use sophisticated computer models to develop new drugs in a process known as rational drug design. Although the potential scope of mathematical and computer modeling have been somewhat oversold by some advocates as alternatives to animal research, these methods have been influential in decreasing animal use.

Audiovisual guides and aids have been promoted as alternatives to animals in the field of education, and the past decade has seen a marked growth in the diversity and availability of alternatives to dissection and other classroom exercises that traditionally use animals. These materials, including CD-ROMs, offer the advantages of repeated and play-back viewing as well as animations and views unobservable in real specimens, and they often allow the viewer to study procedures on humans instead of animals. More than a dozen published studies indicate that these materials are competitive with animal-based counterparts as effective, time-saving learning tools. In the long run, most also cost less than having to re-purchase animals.

Not surprisingly then, there is some evidence that animal use has declined in teaching at all levels. In addition to the greater diversity and availability of alternatives, other indicators of this trend include increased numbers of students who request alternatives to dissection, and the enactment of laws supporting a student's right to choose alternatives.

Nevertheless, the adoption of alternatives in education is slower than might be expected. Dissection has a long tradition in biology education, and it is passed on from one generation of teachers to the next. Also, alternatives are not without their limitations, a notable example being the teaching of practical skills, such as performing veterinary medical procedures, learning laboratory techniques, and developing animal handling skills. For these, much can be accomplished by using animals in a clinical setting, such as veterinary students performing spay/neuter surgery on cats and dogs who are then returned to an animal shelter for adoption.

Refinement approaches tend to be rather ad hoc and hence do not fall into a few discrete categories as do the replacement and reduction alternatives mentioned in the foregoing paragraphs. A few examples will serve to illustrate the diversity of refinement approaches.

Some companies have substituted the Limit Test for the LD50 Test in assessing acute toxicity. In the latter, some animals are deliberately given chemical doses high enough to cause death. In the former, dosing stops at a fixed level, so that the animals are typically spared a painful death. Housing and husbandry refinements include housing social primates in pairs or groups rather than solitarily, providing dogs with daily play time rather than keeping them alone in their cages all the time, and providing mice with paper toweling with which to build nests. Some of the model systems for detecting anti-anxiety drugs are clearly more distressing than others. In one, mice are first deprived of water, then given access to drinking apparatus that delivers electric shocks on a random schedule. Another method by contrast, simply gives mice the opportunity to enter a brightly lit area. The latter model is both more benign and gives the animals control over the situation (they decide when to venture into the open field).

Interest in Alternatives

In the face of lingering controversy over use of the term "alternatives" and arguments over the potential utility of alternatives, it may be useful to review some of the highlights of the growth of interest in alternatives and the growing public and corporate acceptance of the approach.

Since 1985, so much has happened in the "alternatives" arena that a chronological listing of important happenings becomes too overwhelming. For example, many corporations have become active developers and promoters of alternatives (e.g., Colgate-Palmolive, Exxon, Gillette, Hoffman-La Roche, L'Oreal, Procter and Gamble, Unilever and Zeneca have been major players). In 1993, the first director for the European Centre for the Validation of Alternative Methods (a new European Union unit) was hired; the first World Congress on Alternatives was held in Baltimore; and a U.S.-government-sponsored international meeting to examine potential replacement methods for rabbit eye irritancy testing was organized. The National Institute for Environmental Health Sciences now has an Interagency Coordinating Center for the Validation of Alternative Methods (ICCVAM) staffed by an Interagency Center for Evaluation of Alternatives Toxicological Methods, which oversees related program activities.