Proponents of animal testing often criticise animal-free research methods for not being validated. This is quite simply a falsehood. Many such methods have already successfully been validated. What has never been demonstrably validated, however, are the animal experiments. If the animal-free methods were as bad at predicting results in humans and as poorly reproducible as animal experiments, they would not survive the validation process. Fortunately, they are superior and the list of validated cruelty-free tests is getting longer all the time.

What Does Validation Mean?

Validating animal-free test procedures is a major issue when it comes to establishing these methods as “alternatives” to animal testing. But what exactly does this term mean? In general, validation is understood to mean the verification of previously defined usage criteria of a product/test procedure. Ultimately, the decisive factor here are the users or usage goals, which can be defined individually. A successful validation is intended to show the user that the product/test is reliable and meets crucial requirements. In the case of animal-free methods, many manufacturers in Europe want to achieve validation accepted by the European Commission and the Organisation for Economic Cooperation and Development (OECD), an association of the most important industrialised nations, so that the product or test is officially declared an “alternative” to animal testing. The road to a test procedure’s regulatory acceptance is long and rocky, involving many defined steps and examination by a number of committees, who decide based on various criteria whether the respective method is suitable (1). Validating the test is only the first step, followed by further stages it must go through before finally being accepted by regulators. Validation itself is a rigorous process that must meet the exact criteria set out by ECVAM and the OECD (2).

Our NAT database provides the possibility of filtering results according to validated methods. These make up only a fraction of all database entries, which reflects the situation in real life. Most animal-free methods developed worldwide are in fact not validated. They are often used for academic or basic research, in which the focus is usually not on the validation of a research method, but on new scientific insights. In this context, research models and methods are simply means for researching scientific facts. There is also little or no state funding for the validation of methods. State funding is supposed to promote the development of new knowledge. In most cases, it is companies that invest large sums of money in validation when they are interested in a particular method.

Validating Animal-Free Testing Methods at the European level

The European Centre for the Validation of Alternative Methods (ECVAM) (3), is responsible for validating alternative methods to animal experiments in Europe. Its task is to accompany and coordinate the path of animal-free procedures towards regulatory acceptance, so that they can be used in safety assessment instead of previously mandated animal tests. Analogous institutions in countries outside Europe cooperate closely with ECVAM, and there is also an ongoing exchange with the OECD. This is very important in light of our globalised economy, so that ideally authorities worldwide will accept a test method as an alternative to animal testing, for instance when it a chemical substance is registered or a new product is developed. ECVAM is part of the JRC (Joint Research Centre) and reports to the European Commission (4).  

When an animal-free test method is registered with ECVAM, several committees and groups of stakeholders are consulted before the validation process begins. These assess the relevance of the method. Only then does the extremely complex validation process begin, akin to a quality assurance procedure. The ECVAM specifies specific experimental criteria the test must fulfil. These are strict requirements that demonstrate the reliability of the test procedure and ensure that the test always produces consistent results, regardless of by whom or where it is conducted. This means that the results must display a certain degree of reproducibility or be the same when delivered by different laboratories.

The validation studies are usually carried out in ECVAM’s own laboratories or in the so-called NETVAL laboratories (Network of Laboratories for the Validation of Alternative Methods), about forty accredited laboratories at various locations in Europe, including Germany, that cooperate with ECVAM (5). After validation, the results are reviewed by the ECVAM Scientific Advisory Committee (ESAC). Finally, ECVAM, in cooperation with other bodies of stakeholders and experts, prepares a recommendation for a method. This recommendation is then published and submitted to the European Commission. It should be noted that this recommendation is not binding and does not entail a statutory requirement, but should be taken into account and adapted by decision-makers and authorities in the Member States. The last such recommendation was made in May 2020 regarding the use of animal-free antibodies, with the document clearly stating the distinct advantages they offer compared with animal antibodies (6).

The final step is the official regulatory acceptance of a procedure as an “alternative to animal testing”. This method can then be used for certain regulatory tests instead of testing on animals, for instance when investigating whether a certain chemical irritates the skin. The problem here is that although the competent European authorities usually accept these tests, animal-free methods that are accepted instead of animal experiments do not exist for all the numerous test criteria to be examined. Further preparatory work is therefore needed to completely ban testing on animals.

The entire validation process leading up to official recognition and inclusion in legislation takes several years. Unfortunately, even validated and recognised non-animal methods are often conducted parallel to animal experiments, for instance the pyrogen test. This has nothing to do with ECVAM, however.

Failure of the 3Rs

Many animal-free in vitro tests were validated and accepted by regulators when the EU issued a ban on animal testing in the field of cosmetics (7). This ban came into force in 2013 and the situation is a good example of how important clear legal bans on animal testing are for promoting non-animal research and its regulatory acceptance. By contrast, 10 years after the EU Directive on the protection of animals used for scientific purposes came into force, a reduction in animal experiments is nowhere to be seen, because it does not impose any clear bans and leaves too much leeway for conducting animal experiments (8).

The 3R principle enshrined in the Directive has also failed to achieve its objective, because if the two Rs Reduce and Replace had worked, there would have been a significant decline in the number of animal experiments in the EU after 10 years. This is, however, not the case (9). There are many reasons why the 3Rs failed. For instance, unfortunately many researchers hide behind the third R, Refine, which entails “improving” animal experiments or the conditions under which animals are held. Enlarging the cages of “laboratory animals” by a few square centimetres or providing them with additional activity material does not even begin to solve the problem, yet such measures are nonetheless recognised as a contribution towards animal welfare.

Examples of Validated Test Methods

Many tests were validated as part of the EU ban on animal testing for cosmetic products, especially tests using human 3D cell culture models or tissue models. These include three-dimensional models of human skin (10), the eye (11) and the respiratory tract (12). In these tests, the complex cell models are sited on a cell culture plate and the substance to be tested just needs to added. In most cases, a photometer detects a colour reaction, the intensity of which reflects the extent to which the test substance damages the cells. For all these models there are now also approved regulatory tests that can be used to prove required test criteria such as eye irritation, skin irritation, irritation of the respiratory tract, etc., without having to use animal experiments.

A detailed overview of all test methods that have already been accepted or are currently in the validation process can be found in the TSAR (Tracking System for Alternative Methods towards Regulatory Acceptance) database (13). However, caution is also advisable here, because not all of these methods are actually free of animal tissues or components, but rather are simply guaranteed not to be tested on living animals. 

Especially in the area of safety testing for chemicals, the computer-based (in silico) methods must be emphasised (14). They have established themselves as validated and accepted regulatory “alternative” methods to animal experiments and are now frequently used, for example for approving chemicals or assessing whether the substance could have harmful effects on the human organism.

We Need to Rethink

Despite the increasing number of validated methods that facilitate animal-free testing, validation is unfortunately subject to double standards. Animal testing is still the measure of all things in the regulatory field, the so-called gold standard, and to make things worse, human-based animal-free methods must be measured against it. This is even though it is well-known and has been scientifically proven time and again that the predictive power of animal experiments is poor – even in safety tests and toxicological investigations. Why should an innovative test procedure based on human cells, which thus logically better reflects the situation in humans, have to prove its suitability in animal experiments? This is absurd and also a dilemma in strategic considerations for the abolition of regulatory animal tests. This is why in this article we don’t speak of “alternatives” and why we put the term in quotation marks. An “alternative” implies that something equivalent is being replaced. However, human-based methods are not equivalent, but better.

There needs to be a general rethinking, a departure from the classic “replacement” of prescribed animal experiments, because that’s exactly what won’t work. According to the European Chemicals Agency (ECHA), certain animal experiments can now be “replaced” by alternative methods quite well, even completely, but this is not yet possible for more complex studies (15). One example cited is the difficulty simulating in vitro a 90-day toxicity test on a rat. This is precisely the wrong approach. The goal must not be the in vitro simulation of a currently used demonstrably unreliable rat test, but to keep in mind that we wants to investigate the toxic long-term potential of a substance in humans. Who says that the classic 90-day test, in which the rat is administered a potentially toxic substance orally via a throat tube every day(!), is the method of choice? Also, this test has never been validated, but simply gained a foothold and has now been carried out as a standard for decades. If this method were to be validated correctly, it would most likely fail, because the susceptibility of the test to errors and its lack of transferability to humans is undeniable.

Conclusion

Unfortunately, there is still no solution to these complex problems. However, it is good and pleasing that there is open and critical discussion with all stakeholders, including NGOs, European authorities and decision-makers. Together with our European umbrella organisations ECEAE (European Coalition to End Animal Experiments) and Eurogroup for Animals, we are also committed to ensuring that problems are not hushed up and that concrete ideas and strategies are developed so that decision-makers at national and European levels focus clearly on abolishing animal experiments. This includes two things in particular: giving the innovative non-animal methods the trust they deserve; and not looking away from the fact that animal experiments with their lack of meaningful results must not be allowed to be the research model for the future.

13 July 2020
Dr. rer. NAT. Tamara Zietek

Sources

1. EU Science Hub: Validation and submission process
2. OECD: ENV/JM/MONO(2005)14
3. EU Science Hub: EU reference laboratory for alternatives to animal testing
4. EU Science Hub: Joint Research Centre (JRC)
5. EU Science Hub: EU-NETVAL
6. Barroso V et al. EURL EVCAM Recommendation on non-animal derived antibodies. 2020
7. End of cosmetics animal testing! Doctors Against Animal Experiments, 12.03.2013
8. EU Animal Experimentation Directive – Background information. Doctors Against Animal Experiments, 27.01.2014
9. New statistics: Numbers of animal experiments in the EU did not fall in 2017. Doctors Against Animal Testing, 02.02.2020
10. SkinEthic™ RHE reconstructed human epidermis. Episkin
11. EpiOcular™. MatTek Life Sciences
12. MucilAir™. Epithelix
13. EURL ECVAM Tracking system for alternative methods towards regulatory acceptance (TSAR)
14. EU Science Hub: Computational Methods
15. ECHA: The use of alternatives to testing on animals for the REACH Regulation 2020; DOI:10.2823/509114