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Alternative is the term traditionally used within the scientific community to describe the “3Rs”—methodologies to replace, reduce or refine animal use in an existing test or procedure. Since this section of our website addresses product safety and toxicity testing, the focus here will be on animal testing alternatives intended for toxicity testing applications that are being considered for acceptance by regulatory agencies.

Animal testing alternatives fall into three main categories. A replacement alternative is a test method that has been endorsed by an appropriate authority as being capable of fully replacing an existing regulatory test method with a method that does not use live animals: in vitro (primary cultures, cell lines, 3-D cell culture), ex-vivo (isolated animal tissues and organs) or in silico (mathematical models, computer simulations) methods. A reduction alternative is a method that reduces the number of animals used. And a refinement alternative is a method that reduces animal suffering.

There are a number of incentives for moving away from animal safety and toxicity testing and toward animal testing alternatives. One motivation is ethics, as these traditional safety and toxicity testing methods cause high levels of animal suffering. Scientific drivers are also providing motivation, as inherent differences in the biology of human and non-human animals limits the value of animals serving as human stand-ins. Also, there is the question of which humans are we expecting animals to model, as small genetic differences between humans leads to important variability not captured by lab animals. Other incentives for using animal testing alternatives include significant cost savings benefits from non-animal models and regulatory drivers forcing companies to rethink animal use in toxicity testing.

A new or alternative toxicity test method can be quite different from the existing animal test method, but it must provide the same or better prediction of human health effects for it to be accepted by regulatory agencies. To make this determination, representatives from government agencies, industry, and academia came together in the early 1990s to establish criteria and processes for evaluating new toxicity test methods. Test method validation criteria were established by three organizations in the 1990s: the Organization for Economic Cooperation and Development (OECD), the European Centre for the Validation of Alternative Methods (ECVAM) and the U.S.’s Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). Important terminology was defined as follows:

• Validation is “the process by which the reliability and relevance of a particular method or approach is established for a specific purpose.”

• Reliability is the measure of a test’s reproducibility, which is the degree of variation in test results obtained when the same method is performed in the same lab over time, or performed in different laboratories or by different technicians.

• Relevance is the assessment of a test’s ability to correctly predict the biological effect of interest.

A validation study is conducted to obtain the data needed to assess the reliability and relevance of a new or revised animal testing alternative to test toxicity. (It should be noted that the traditional Draize and LD50 tests were never subjected to a validation process). It is a large, expensive, and time-consuming process that takes place across several different laboratories. The data from the study are submitted to one of the validation centers, such as ECVAM or ICCVAM. The center, in turn, assesses the submitted data, and determines whether to move the method forward to peer review, where an assemblage of independent scientists evaluates the results and makes recommendations. ICCVAM or ECVAM’s advisory board then determines whether to recommend the test as scientifically valid for its intended purpose. If so, it is then called a validated method.

A validated method must still undergo further review before being accepted for use by a regulatory agency. Each agency makes its own determination of whether to accept data from the new method, and to what extent the new method can replace the existing regulatory test method (usually an animal test). This process is called regulatory acceptance.

What barriers stand in the way of widespread practice of non-animal testing approaches? Regulatory acceptance of animal testing alternatives is limited, as regulators may be reluctant to accept new approaches. They may prefer use of more traditional, less “risky” animal methods, in part, because they are more comfortable interpreting data from animal studies for which they have more experience. Data generated from methods which use animal testing alternatives is different and regulatory bodies may be unsure how to interpret it with respect to human risk assessment. Also, regulatory guidelines themselves may be misinterpreted, leading researchers and regulatory bodies to believe that animal data may be required when it isn’t. In addition, there is a great need to improve the global harmonization of data requirements.

Some scientific barriers must also be addressed, including the recognition that animal testing alternatives will not realistically replace traditional animal tests on a “like-for-like” basis, considering each new alternative method has limitations and could be recommended for use as a partial or full replacement method, or as a screening assay that can be used as part of a test battery (a group of tests used to make a determination) or a tiered (or integrated) testing strategy (a group of tests and possibly other data used together in a specific scheme or sequence to make a determination). Therefore, potential users of a new method must study the recommendations and limitations of the method, even when it is considered to be validated.

For a list of validated and accepted alternative methods, click here.

RESOURCES:

Burden, N., Sewell F., and Chapman, K. (2015).  Testing Chemical Safety: What Is Needed to Ensure the Widespread Application of Non-animal Approaches?  PLOS Biology.

Kandarova, H. and Letasiova, S. (2011).  Alternative methods in toxicology: pre-validated and validated methods. Interdiscip Toxicol.