The Three Rs — Do We Need a New Principle?

A new generation of Russells and Burches are needed,
who will bring in a new era of biological science
that will truly benefit animals as well as humans

Christiaan Wittevrongel

Since the early sixties, much has been done in terms of Replacement, Reduction and Refinement. But are these Three Rs still the principles by which animal use in research can be avoided in the future? Not necessarily.

In 1959, the well-known concept of the Three Rs was published by William Russell and Rex Burch.1 Their approach to animal testing revolutionised the way in which laboratory animals were seen and used in scientific research. Consideration of the Three Rs changed the way experiments were carried out, with the development of non-animal methods (replacement) and the push toward experiments with fewer animals (reduction). This approach prompted changes in statistical methods as enabling factors to extract relevant information from the experimental data. Last, but not least, refinement led to progress in the relief of pain and suffering, which, in turn, has impacted on the quality of the experimental results.

The Three Rs have been in use for over 50 years. The question can be raised as to whether they still keep delivering. Let us look at the number of animals that are used in research in The Netherlands.2 The total number in 1991 was 1,117,234 and in 2011 it was 1,120,399. One may therefore be tempted to conclude that the efficacy of the Three Rs concept is running out of steam, because there is less well-documented agreement about a substantial reduction occurring during the preceding years. This levelling off can be partially explained by the increased interest in the use of genetically-modified animals, mainly rodents.

This brings us to the issue of surplus animals, i.e. animals that were to be used in experiments, but for some reason were not, and thus had to be killed. It so happens that, during the production of genetically-modified animals, some do not meet the requirements of the experiment. In many instances, only male animals are used, and this makes the females redundant. Last, but not least, poor planning may create animals that are not needed, or that cannot be used because they are at the wrong age. In 1991, the total number of surplus animals was 241,176, and in 2011 it was 530,446.2 It is obvious that no progress has been made in this area, whereas solutions seem to be relatively simple.
The conclusion seems to be that the Three Rs are still effective, but their effect is stabilising. The number of animal experiments for some purposes, has reduced considerably in the last few decades, due to replacement and reduction. The most spectacular success has been in diagnostics, where hardly any animals are now used, because of the arrival of new technology — for example, immunochemistry methods or the DNA-based polymerase chain reaction — that has made certain animal experiments obsolete and redundant.

It can be argued that additional approaches are needed, not just the Three Rs. The problem with the Three Rs approach is that it basically all starts from the consideration of the animal test. Replacement, if looked at in the traditional manner, has several disadvantages:

1. Laws on animal experiments mainly protect vertebrates. Therefore, legally, replacement is also achieved when invertebrates can be used. However, invertebrates are still animals, so this is not genuine replacement of an animal test. Unfortunately, this is not reflected in the total numbers of animals that are recorded. Since invertebrates are not protected by law (albeit with the exception of cephalopods following the introduction of the new European directive), they are not counted.3
2. If plans are being made to replace an animal experiment endpoint, then this might involve validation of the new endpoint in animal experiments that were meant to be replaced.

 Reduction and refinement are directly linked to the idea that a proper answer to a research question can be found by using animals. They have a very marginal effect on the change of general scientific thinking about the use of animals.

In the search for biological mechanisms of human disease and in the search for new drugs, the animal experiment is sometimes referred to as the gold standard. The validity of this statement has never been proven — and it seems that nobody has tried. It seems more likely that animal experiments are the only standard, because there is no alternative. It is well-known that the search for new drugs is very inefficient.4 Many drugs in development fail in clinical trials, due to lack of efficacy or unwanted safety profiles. These drugs have all been tested in animal experiments prior to the clinical trials. This means that something needs to change in the development of new drugs. It appears that animals and humans are very different after all. Not long ago, a discussion erupted on the use of animal models in inflammatory disease research.5 Researchers stated that animal models were not at all applicable for use in the development of drugs against, for example, type 1 diabetes.

This is because the immune system in animal models differs too much from that of humans. However, researchers are still obliged to show that drugs do work safely in animals, before clinical trials, which may lead to market access, can be started. Researchers acknowledge that there is a big problem in the development of drugs against conditions such as type 1 diabetes, and drugs have been discovered against type 1 diabetes, based on knowledge of the human biological system. The researchers know that these drugs will not work in animals, but they still have to show efficacy in animals before clinical trials can take place. Therefore these drugs might never be available on the market (Bart Roep, personal communication; Labyrinth Radio, Radio 1, 18 February 2013).

Comparison is sometimes possible in the field of toxicology, where some human data are available,specifically in studies that have been performed on the carcinogenicity of substances. However, if the comparison is conducted properly, it is usually found that results in animals and humans are not equal at all.6 In risk assessment, one needs to consider what must be prevented from happening. For example, in the case of carcinogenic substances, the development of a tumour is the event that needs to be prevented. Therefore, we need to take a closer look at the human biological systems behind this event, and then determine whether certain substances exert effects on these systems. Substance kinetics and DNA-damage need to be reinvestigated, specifically aimed at human biological systems, because these mechanisms form the basis of tumour development.

What do we need in addition to the Three Rs?

Generally speaking, animal experiments are carried out to either generate knowledge and understanding of human biology or to provide information as a basis for making decisions about either the safety of chemicals or the efficacy of medicines. This puts human biology at the centre stage. A true mind-shift needs to take place in the process of answering scientific questions about the human body and human diseases, their prevention and cure. What we really need to develop are methods to deliver just that, by making use of the achievements of modern medicine, but using these very same technologies in human models and systems, rather than in animals.7 This will also mean that we need a new generation of Russells and Burches, who will bring in a new era of biological science that will truly benefit animals as well as humans.

Dr Christiaan Wittevrongel
Senior Policy Officer
The Dutch Society for the Replacement of Animal
Testing (DsRAT)
Groot Hertoginnelaan 201
2517 ES The Hague, The Netherlands

References and Note
1 Russell, W.M.S. & Burch, R.L. (1959). The Principles of Humane Experimental Technique, 238pp. London, UK: Methuen. [Reprinted in 1992, by UFAW, South Mimms, Hertfordshire, UK.]
2 In this paper, we have used the number of animals as registered by The Netherlands’ authorities .
3 Anon. (2010). Directive 2010/63/EU of the European Parliament and of the Council on the protection of animals used for scientific purposes. Official Journal of the European Union L276, 20.10.2010, 33–79.
4 Paul, S.M., Mytelka, D.S., Dunwiddie, C.T., Persinger, C.C., Munos, B.H., Lindborg, S.R. & Schacht, A.L. (2010). How to improve R&D productivity: The pharmaceutical industry’s grand challenge. Nature Reviews Drug Discovery 9, 203–214.
5 Seok, J., Warren, H.S., Cuenca, A.G., Mindrinos, M.N., Baker, H.V., Xu, W., Richards, D.R., McDonald-Smith, G.P., Gao, H., Hennessy, L., Finnerty, C.C., López, C.M., Honari, S., Moore, E.E., Minei, J.P., Cuschieri, J., Bankey, P.E., Johnson, J.L., Sperry, J., Nathens, A.B., Billiar, T.R., West, M.A., Jeschke, M.G., Klein, M.B., Gamelli, R.L., Gibran, N.S., Brownstein, B.H., Miller-Graziano, C., Calvano, S.E., Mason, P.H., Cobb, J.P., Rahme, L.G., Lowry, S.F., Maier, R.V., Moldawer, L.L., Herndon, D.N., Davis, R.W., Xiao, W., Tompkins, R.G. & the Inflammation and Host Response to Injury Large Scale Collaborative Research Program (2013). Genomic responses in mouse models poorly mimic human inflammatory diseases. Proceedings of the National Academy of Sciences of the USA 110, 3507–3512.
6 Card, J.W., Fikree, H., Haighton, L.A., Lee-Brotherton, V., Wan, J. & Sangster, B. (2012). Lack of human tissue specific correlations for rodent pancreatic and colorectal carcinogens. Regulatory Toxicology & Pharmacology 64, 442–458.
7 Fentem, J., Chamberlain, M. & Sangster, B. (2004). The feasibility of replacing animal testing for assessing consumer safety: A suggested future direction. ATLA 32, 617–623.

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