Reducing and replacing animal tests

The three Rs

The concept of the three Rs was developed in the 1950s by William Russell and Rex Burch, and it is still regarded as the cornerstone of the most humane approach to using animals for research. The three Rs stand for reduction, replacement and refinement. We refer to the website of the British NC3Rs for information on this topic.

Refinement, referring to the means by which animal suffering is limited as much as possible, while paying maximum attention to animal welfare, is discussed in the context of laboratory animal welfare.


Whenever research can be conducted using fewer animals, without any drop in the quality of research, researchers at Ghent University are obliged to do so. The Ethical Committees oversee this, and can provide advice when necessary. The principle of reduction implies that as few laboratory animals are used as possible, without endangering the aims of the research.

The fewer animals, the better? Not necessarily: the scientific quality of research often depends on the statistical power, and this in turn depends on a sufficiently large number of samples, tests or other analyses being conducted. Thus it may be necessary to include a certain (sometimes large) number of laboratory animals in a study. Even then, it may be possible for the number of animals to be reduced, for example, if two tests can be conducted per animal. Of course, the latter cannot be done indefinitely: there are also strict guidelines on the reuse of laboratory animals.

As many as needed, as few as possible. This trade-off is part of the cost-benefit analysis which is conducted as part of research design. It is better to carry out a robust research project using 30 animals rather than an unreliable (and therefore useless) study with ten animals.

Whenever and wherever possible, animals are also used for multiple purposes. For example, tissue from an animal that was killed after use can be used for other research purposes if the initial examination cannot influence the results of the later research. Fortunately, there is often cooperation between institutes on this matter in order to thus limit the number of animals used.

Similarly, animals that are killed after testing (or animals that cannot be used due to their phenotype) are offered up for use in teaching: in this way, no additional animals are killed for this purpose.

Another example of reduction is when medical imaging techniques are used which make it possible to obtain information from the same animal over a longer period of time, for example, in relation to a progressive cancer, or a curative disease. This avoids the need to use multiple sick animals which would each have to be killed and examined during successive stages.

A final way of using fewer laboratory animals is to avoid unnecessary duplication of research. There is certainly room for improvement in this area, also internationally. This means avoiding research that has already been carried out but which has not been published in scientific journals because it did not produce publishable results. There is a growing awareness that studies with so-called negative results should at least be made public.

Fortunately, there are initiatives providing scientists the opportunity to consult databases to see which studies have been carried out in their field, regardless of whether the work was published. This good practice should definitely be implemented even more widely. Some examples from clinical research include and

Reduction, just like replacement and refinement, is extremely important when preparing applications for research funding. Any project can only start after receiving a positive evaluation from the responsible Ethics Committee. For each project the investigator has to explain what efforts will be made to reduce the number of animals used.


The principle of replacement in animal testing means that researchers try to use alternative, scientifically sound methods or other techniques that do not require the use of live animals.

There are many reasons why we should try to replace animal testing. The main one is ethical: any unnecessary suffering of animals must be avoided. Even keeping animals unnecessarily should be avoided if it could harm the animals. The 'necessity' and justification of animal research and testing, and thus of possible or probable animal suffering, is a complex ethical issue.

Other reasons for replacing animal research and testing are very practical. Using laboratory animals is often more complicated than other techniques, and above all very expensive. Just think of aspects such as the purchase or breeding of animals, keeping and caring for them in an adapted facility, training qualified personnel to conduct tests and provide care, internal checks by a veterinarian, and so on.

The best solution, of course, is to use a technique that no longer includes laboratory animals. Many people do not realise that in recent decades, animal research or testing is only been conducted whenever other methods are not available. The relatively high number of animal tests in the statistics rather reflects the scale of biomedical, veterinary and other research at Ghent University, and for example, in the whole of Flanders.

Even when laboratory animals are used in a research project, in vitro cell or tissue cultures are used also, and first, along with computer analyses, etc. The number and applicability of these and other techniques is constantly increasing.

Another way to reduce animal suffering through replacement is the right choice of animal species. The higher the level of consciousness in a species, the higher potential it has for psychological suffering. Invertebrates usually have a (far) lower degree of consciousness, and less potential for psychological suffering. Therefore they are often chosen. Amongst others, fruit flies or the roundworm C. elegans are used. Because of this reason, in European legislation invertebrates (with the exception of cephalopods) are not included in the definition of laboratory animals.

Proper justification must therefore be given if a species with a higher level of consciousness is selected. If a study can be done equally well with zebrafish or with mice, one must opt for the zebrafish.

For every application for animal research or testing submitted to their Ethics Committee, the researcher should explain what efforts are being made to replace animal testing with other practices. Examples of replacement are described below.

Alternative methods

Alternative techniques are, simply put, techniques that do not involve the use of animal research or testing. Not every alternative method that is ‘free of laboratory animals’ is completely ‘animal-free’. For example, in vitro and some other techniques still require animals to be killed to obtain cells or antibodies. Completely ‘animal-free’ alternatives are methods which do not require the need of animals at all.

The alternatives can be subdivided according to two criteria:

  • on the basis of period of original development:
    • long-standing alternatives – which we now often no longer regard as an alternative, for example, in vitro trials in general
    • recently developed alternatives – for example many animal models for educational purposes, but also recent and innovative in vitro techniques
    • alternatives that have already been developed but have not yet been effectively rolled out (for example, because they have not yet been sufficiently tested, or received validation)
  • on the basis of intentionality:
    • techniques that have not been developed with the aim of replacing animal testing, but nonetheless do so. For example, some new medical imaging techniques making certain animal tests redundant, or the ELISA-technique (Enzyme-Linked Immuno Sorbent Assay)
    • alternative techniques developed with the specific aim of replacing animal use, e.g. organoids, educational animal models, and bioreactors

There are various initiatives which seek to encourage the use of alternatives and maximise the scope for the application of these. There exist databases of alternative techniques, including, for example, RE-Place in Flanders and NORINA in Norway.

Some promising alternative methods:

  • organoids: in vitro growing (human) cells that form a realistic 3D structure, and whose interactions mimic a real organ
  • organs-on-a-chip: human cell cultures that grow on membranes in channels in a plastic device, effectively mimicking organs in terms of  physiology and mechanics, among others
  • in silico models: computer models (i.e. without living tissue) of, for example, physiological processes, which have already proved their worth in, amongst others, pharmacokinetics (e.g. CADD or Computer-Aided Drug Discovery and SARs or Structure Activity Relationship programmes)

The complexity of human physiology, with countless interactions between countless often as yet unknown and insufficiently understood molecules, cell types, etc., means that many investigations can unfortunately not (yet) be carried out using these kinds of alternative techniques. For example, certain processes, such as ageing, cannot be practically mimicked in an organ-on-a-chip.

There is also a lot of potential in the continuous advancement of medical techniques. For example, ever smaller doses of drugs can be tested on human subjects, which could partly replace intermediate steps using animals.

The search for alternatives

Innovative research that seeks to develop or improve new alternative methods is challenging and, almost by definition extremely difficult: the ‘easiest’ options have, after all, already been discovered. For example, most product tests on animals have been replaced (many have also since long been banned in the EU).

An additional obstacle is the potentially difficult comparability of research results based on alternative methods with those of other studies. On top of that, an alternative method is often only interesting if it is recognised, i.e. validated, by authorities. An important player in this field is the European Centre for the Validation of Alternative Methods (ECVAM).

Ghent University is an active player in the national and Flemish consultations between the scientific institutions, the government and other partners. The voice of the scientists is very crucial here, because apart from validation, funding is also a tricky issue, not sufficiently supporting this type of research: the combination of the highly innovative character and the lack of guarantee of success or quick applicability deters financiers such as the government.

Read more about international initiatives on alternatives here: