When will animals be replaced in biomedical research?

Increased political pressure in Europe has raised expectations about the possible phasing out of animal use in scientific research, but what is the reality? In this EARA feature we look at the safety testing of drugs and chemicals, where the most progress with non-animal methods has been made, and consider what impact this will have on the overall number of animals used in biomedical studies.

There has been a long-standing commitment by the biomedical sector to using as few animals as possible in research. That commitment can be traced as far back as 1959, when the 3Rs (replacement, reduction, refinement) were established to address this issue.

For more than a decade it has also been enshrined in EU law (EU Directive 2010/63) that animals can only be used if there are no suitable alternative methods of study available. Yet while the trend is for a decreasing use of animals, there are still many millions being used each year across Europe and the rest of the world, leading to frustration among animal rights groups and keeping animal research as a hot issue for politicians.

One of the reasons for slow progress has been the sheer difficulty in finding scientific methods that can mimic the complexity of a whole organism. Different animals resemble humans in many different ways – for instance many species used in research, such as mice, zebrafish and fruit flies, share a majority of disease-causing genes that humans also have, making them valuable models. Meanwhile, there has also been some reticence by scientists to look beyond tried and tested methods and sometimes a lack of funding to develop alternative methods and get them adopted.

Are we seeing a quickening pace for animal replacement?

In recent years, helped along by advances in technology, new knowledge and better resources, the replacement or reduction in the use of animals in some areas has been quite noticeable. A particular area is in animal use for regulatory testing, which includes safety and toxicity testing of pharmaceuticals and chemicals (see graph below). Non-animal methods are also being used in the approval process for drugs and vaccines in humans, for example, and the testing of the safety of commonly used chemicals, such as pesticides, household products, or those used in industrial processes.

This decline in animal numbers has largely come about thanks to rapid developments in ways to assess the risk and safety of products and ingredients – techniques and technologies that have become known as new approach methodologies (NAMs), which are now legally permitted to take the place of animal studies in some parts of the world (see the EARA page on the 3Rs).

Nevertheless, it is still currently a legal requirement in some areas, such as drug licensing, that testing must also be conducted on animals (usually a mice or rat) as well as a non-rodent species (such as a rabbit or a dog) to identify any problems, or adverse effects – for instance the effect on the fetus.

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Why was regulatory testing in animals introduced?

For more than 80 years, animal testing for regulatory safety assessment has been the major route to the development, approval and use of drugs and vaccines in humans, and sometimes other chemicals.

One of the main reasons for animal testing was introduced was a major public health disaster in the USA in 1937. More than 100 people (many of them children) died from taking the antibiotic drug, elixir sulphanilamide, that had been deemed safe in laboratory tests to treat streptococcal infections. However, the failure to test it for toxicity and its effect on a living system meant it overlooked that the formulation in fact contained a deadly poison. In response, the 1938 Federal Food, Drug and Cosmetic (FD&C) Act was passed, requiring a proof of safety before drugs made it to market, and in turn requiring testing in animals as a safeguard that a similar tragedy would not happen again.

While some of these NAMs actively aim to replace the testing of animals, others are designed to work alongside animal studies to find better answers and solutions to scientific questions, which can also lead to a reduction in animal use.

Looking to the future, when it comes to protecting human health it is highly likely that a complementary approach, with different methods working alongside one another, including animal studies, will be used to investigate whether new drugs, therapies or particular chemical compounds will be harmful.

Political pressure to introduce NAMs

What has shifted in recent times is the exaggeration, by political groups and animal activists, about how widespread the use of NAMs is and the results that they can achieve. There have been broader claims that NAMs can replace the use of animals in regulatory testing right now and indeed that this is also possible for other areas of basic, or curiosity-driven research, most commonly conducted in academia. Some have claimed that animal research is obsolete and old-fashioned, rather than an essential method that continues to develop and innovate – even for cases where animals are known to have been involved in the recent development of life-saving treatments, for example for breast cancer.

However, due to their current limitations, NAMs are unlikely to fully replace all aspects of animal use, even for regulatory testing, for some years to come. At present, around one in ten (13%) of the total number of animals used in the EU, in 2022, were in the field of regulatory testing, while almost three quarters (72%) were used in basic or translational/applied research, where non-animal methods still remain limited.

In the 2023 European Citizens’ Initiative (ECI) petition, Save Cruelty Free Cosmetics, there was also a call for limiting the use of animals in regulatory testing, beyond where science innovation currently stands. These calls can mislead the public and add confusion by implying that NAMs are a ready-made substitute for animals in all areas of testing and research. The response by the Commission was robust in defence of the continued use of animals (see quote below) when there are no NAMs available, but nevertheless emphasised that it would support ways to expand and accelerate NAMs wherever possible.

On Breast Cancer Awareness Day in 2023, the Eurogroup for Animals falsely claimed that cancer clinical trials rarely result in effective treatments due to a lack of relevance of animal studies. EARA, along with other advocacy groups, was quick to highlight that in fact, research in animals such as mice has led to the development of all major drugs for breast cancer, such as PARP inhibitors, which has led to the saving of numerous lives.

Meanwhile, politicians have also endorsed this misinformation, including Tilly Metz, a leading Green Party Member of European Parliament, who stated that “In certain areas like breast cancer, for example, we are stuck because we stick to animal testing.”
EARA added in another post: “Eurogroup suggests that non-animal methods can now replace animals in cancer research, which is far from the truth."

Considerable advances have been made in developing (non-animal) alternatives, but animal models remain unavoidable at the moment to understand some more complex biological or physiological processes involved in health, disease and biodiversity.

European Commission response to ECI petition, July 2023

Which non-animal methods are most frequently used?

Here are the main types of method used in regulatory testing, that can currently be used to assess chemicals and new drugs and that may either replace or reduce the number of animals used, with an outline of the advantages and limitations of each (see also Non-animal methods used in biomedical research at the end of the article).

Cultured cells and tissues (in vitro)

Whether human, animal or engineered, cells that are grown in the laboratory and maintained (described as cultured), outside a living organism, have been used within science for generations. The cells of interest can be extracted from living tissue to be examined more closely under carefully controlled conditions, allowing researchers to understand how tissue grows, test the effect of substances on cells, generate pharmaceuticals based on living cells and organisms, and much more.

When it comes to regulatory testing, lab-based (in vitro) methods are already well-established in assessing certain types of toxicity, such as for the skin, to test sensitivity and irritation. Here, different compounds that are found in common products, that may trigger an allergic reaction for instance, can be used to cause such a reaction in human tissue models, to understand the effect it will have.

Because these methods use human cells, they can be more accurate for predicting the effect in people compared to using animals like rodents, and are therefore also useful for identifying possible hazardous chemicals before they progress to any tests in animals.

An example of these models, for assessing skin irritation in the lab, is described in an article by EARA member, Charles River Laboratories. These models have certain properties akin to human skin, such as an outermost layer and underlying layer, that can give more accurate results than traditional skin irritation tests, such as the Draize test. This test, often conducted on rabbits (also to assess eye irritation) is now gradually being phased out.

There are some key limitations of using in vitro, the major one being tied to the very nature of these techniques, as cells in a dish are unlikely to behave in exactly the same way as in a living organism or their natural environment. Cells are also grown flat in a dish, whereas in the body they are surrounded by other cells in a 3D arrangement, with other complex inter-relationships – however a modern science of 3D cell and tissue cultures is now developing using organoids and organ-on-chips (see below).

Organoids (in vitro)

It has been calculated that preclinical animal tests can predict how a drug will behave in the living body in 86% of cases on average. Humans can nonetheless react differently to drugs compared to animals, and so additional complementary assessments in human-derived systems, such as organoids, can paint a more complete picture of possible toxicity in a variety of relevant organs.

To reproduce the complex conditions that exist within a living organ, researchers have over the last 30 years developed organoids, opening up new dimensions in both regulatory testing and wider research. Organoids are small groups of cells that grow and arrange themselves into a 3D system and can mimic many aspects of a specific function of a human organ – making them suitable to study the effect of new drugs and other substances.

Skin model (left) with blood vessels (right).
(Credit: MPI for Medical Research)

The cells of organoids can be derived from stem cells – either from animals or humans – that have the ability to develop into any kind of cell in the body and turn into multiple cell types found in a specific organ. Using specialised cell-culturing techniques, scientists can now grow organoids of almost any organ in the body, including the brain, heart, liver, kidney, and lungs.

Human brain organoid (red) grows on microelectrode.
(Credit: MPI for Molecular Biomedicine)

There is an unmet need to improve laboratory-based test systems to use in drug development. We need tests that are reliable, accurate and reflect what will really happen when patients take a new drug in clinical practice.

Robert Fontana, University of Michigan, USA

A study at the University of Michigan, USA, used liver organoids, grown from human stem cells, to assess the toxicity of drugs to the liver, finding that these 3D models had a greater diversity of liver cell types that could effectively recreate injury to the human liver caused by toxicity.

In work at EARA member Wageningen University & Research, Netherlands, researchers developed organoids, featuring multiple cell types and with a blood-like fluid, for testing new medicines. By including more cell types and structures the team increased the resemblance to the human body, therefore providing a good simulation of tissue function and biological reactions to chemicals.

However, organoids are not actually mini organs. While they consist of multiple cell types, they lack immune cells and cells lining the blood vessels, meaning that they cannot grow very much without dying. It is also very challenging to mimic the microenvironment where these cells are found.

Organoids are really simplified models that help us study specific aspects of how an organ works, but they cannot replace the complete functionality of real organs. There is limited capacity to join organoids with other organoids, and connect them to other types of cell or tissue, which limits their usefulness for studying the full repertoire of interactions within the body – a major drawback compared to research using animals, where the systemic response in the whole body and all organs can be studied.

Organ-on-chips (in vitro)

These miniaturised devices consist of cells and/or tissue grown in microfluidic chips, which are composed of a clear flexible polymer – the chips are designed to closely mimic the human physiology of a specific organ. Also known as microphysiological systems (MPS), organ-on-chips can be developed using material derived from a range of organs, including the heart, kidneys, liver and lungs, and have broad applications in drug development and toxicology.

Specific versions of MPS allow for a rapid screening of different compounds as either toxic, or promising for use in drug development. They have also been developed to study the liver in a way that provides a more cost-effective alternative (drug development is often lengthy and expensive).

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A recent development, by US-based company Tara Biosystems and EARA member GSK, engineered a ‘heart-on-a-chip’ system that was able to replicate how humans would responds to drugs that are already known to be toxic, to eventually help to predict how human hearts could react to different drugs.

Multi-organ chip developed by TU Berlin and TissUse (Credit: TissUse GmbH)

The platform enables researchers to gather human-relevant functional data in the lab, providing a surrogate measure for how effectively the human heart pumps blood in the presence of potentially toxic drugs.

Misti Ushio, Tara Biosystems

Nonetheless, organ-on-chips are still a very new technology and their widespread rollout faces practical issues, such as in their manufacture to scale, which has yet to be standardised with universal materials and practices. And although organ-on-chips do mirror aspects of both healthy and diseased tissue and organ function, there remains the challenge of replicating the physical characteristics of organs, the interactions between different organs and how they respond to stimuli.

There have also been advances to better understand the different interactions between the organs of the human body. Organ-on-chips or organoids can be combined to create Multi-Organ-Chips (MOCs) to better study these interconnections and the exchange of materials – such as substances that break down drugs (metabolites). The number of organs connected in MOCs is constantly increasing, but is still far from a reflection of a complete organism.

Computer modelling (in silico)

As computers have developed they have become a significant way to reduce reliance on animals in research in certain areas. Data on toxicity can be obtained using different computational models – for example, models that can work out the potential toxicity for one species based on what is already known about another. Increasingly artificial intelligence (AI) and machine learning are also helping to drive improvements in how researchers design their experiments, analyse data and predict outcomes.

Researchers at Chalmers University of Technology, Sweden, developed an AI method that improves the identification of toxic chemicals, based on their chemical structure, at an early stage, which holds the potential to reduce the reliance on animal tests.

However, because AI’s predictive power relies solely on existing data and predefined algorithms determined by humans, it can miss unforeseen effects that would otherwise be identified in animal tests, as it cannot account for the biological variability that takes place in organisms. For AI to accurately predict the effect of a drug, science would have had to already arrive at a place where all human processes are fully described and detailed, and we are still very far from that. AI can therefore supplement the approval of new drugs and medicines, but does not meet all the requirements for safety and ethical approval that would bypass the need for animals. Many regulatory bodies will therefore still require that these compounds be tested in animals.

AI can also integrate data from different studies to help identify which procedures or experiments, that would otherwise involve animals, are unnecessary and can be skipped. Training AI for the best results can be done using both animal data, that incorporates findings from previous research, and human data, from results in the clinic or clinical trials.

Where have NAMs already replaced animals in testing?

The desire of agencies and bodies to reduce animal testing, wherever possible, has spurred on the development of NAMs. Potentially, the successful introduction of NAMs could mean not just better data that is more predictive for effects on humans than animal studies, but also cheaper and faster results. In general, NAMs are developed in areas of regulatory testing where animal tests have not accurately predicted the potential effects of substances on humans, or where there is potential for technology to make further scientific advances.

The rabbit pyrogen test (RPT), which is used to detect particles derived from microbes or chemicals (pyrogens) that can cause a fever (an inflammatory immune response), is currently being phased out. This is largely because alternatives to the RPT, such as the cell-based monocyte activation test (MAT), have demonstrated that they can be used successfully instead of rabbits.

However, there are only a few instances where NAMs exist as a direct replacement for animals in regulatory testing. These specifically are in areas that assess fast-acting and short-term effects only, such as eye irritation or skin sensitisation (see earlier section on cultured cells and tissues). Here, NAMs are also beginning to show their value in predicting how toxic a chemical will be, based on the chemical’s structure itself – certain chemical structures are known to cause negative reactions in people – by using AI to assess thousands of chemicals at a time. However, these methods are still far from being routine and it is noticeable that NAMs have been adopted more often for external aspects of the body, such as eyes and skin, rather than for methods that assess the internal organs.

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MAT scientist (Credit: MAT Research)

There are also concerns that NAMs cannot yet reliably assess complex health effects in humans. A 2022 article in Chemical & Engineering News highlighted the differing views of scientists about the question of using animals versus NAMs in chemical testing. Among the concerns raised about a phase-out of animals in this field, was that NAMs can sometimes capture unintended effects that are not the target, and cannot yet fully address the ‘endpoints’ that are used to assess the effects that can arise from daily, repeated doses of a particular substance, or evaluate the long-term exposure and its accumulation in the body (known as dose toxicity).

For this, animals such as rats remain the standard for understanding, for instance, the effect on organs as the body ages. The animals are regularly monitored for any unexpected changes, while tissue, blood, urine and other samples are taken and analysed in the lab. Since the current way to test for toxicity is already effective, there are still many scientists that argue that it will take a lot more evidence to make the case that NAMs could eventually take the place of routine procedures involving animals.

I am sceptical of using only NAMs to prove safety.

I don’t think we are there yet. But NAMs could help us understand which chemicals are probably problematic.

Anna Lennquist, toxicologist, International Chemical Secretariat (ChemSec), which advocates for substitution of toxic chemicals to safer alternatives

Ultimately, the goal is to improve the overall efficiency of safety testing – which can come about in some cases by reducing the dependency on animals – but might not always lead directly to the replacement of animal tests with a particular NAM. The purpose of NAMs in these contexts is to provide more relevant information on the chemical in question, but not to be a repetition of the animal tests carried out for the same chemical. Nonetheless, there are groups that believe that NAMs will replace animal testing in the short-term, provided that the necessary developments take place (see next section, What prevents NAMs being introduced right now?)

It is not a matter of if but of when. With further scientific and technology development, policy change and smarter validation, NAMs will gradually replace animal testing.

Susy Brescia, regulatory toxicologist, UK Health and Safety Executive

What prevents NAMs being introduceed right now?

Despite the growing prevalence of NAMs, especially in the areas of toxicology and safety testing, there remain significant hurdles to getting these technologies to a place where they are widely established and accepted, from both a scientific and regulatory standpoint.

These include:

Data: For risk assessment – where all possible toxic effects of a particular chemical are identified and evaluated for their risk on people, animals and the environment – there needs to be more data to validate NAMs, as well as the establishment of common procedures to collect and share this data. In addition, NAMs are generally limited by the fact that they cannot be predictive for or applied to all classes of chemicals, and cannot fully represent biological complexity such as organ-organ interactions.

Standardisation: On a global scale this is needed to define how NAMs are used practically in order to ensure that exactly the same testing is carried out everywhere. This includes how different NAMs with different capabilities and applications may be used together to improve results.

Training and education: Not surprisingly, researchers and regulators need to have the skills to handle and implement NAMs, as well as to develop how they are used.

Consequently there is still a lack of broad acceptance of these technologies by regulators, governing bodies and some parts of the scientific community – and in turn their broader use. The hope is that when these different criteria are addressed and met, NAMs should in principle contribute to enriching the ‘toolbox’ of methods at our disposal for carrying out regulatory science and managing harms and risks. A wide range of strategies is certainly ideal, but jumping to conclusions prematurely, without comprehensive scientific consensus, carries significant risk, most importantly for human health further down the line.

A recent case about antibodies is a prime example of the dangers of going too fast too soon. Many therapeutic antibodies that are used for a range of purposes, from detecting infections and tumours, to being vital components of analytical techniques, are derived from animals like rabbits, chickens and camels. Yet a recommendation by the EU Reference Laboratory for alternatives to animal testing (EURL ECVAM) in 2020 claimed that animals were no longer needed in antibody research.

The report was widely criticised by the biomedical community as well as EARA, with a European National Committees sub-working group recently concluding that non-animal-derived antibodies were not able to take the place of animal-derived ones at present.

... an uncritical and full application of the recommendation during the approval process of animal experiments in Europe could create a serious hindrance to the future development of antibodies as diagnostics, in research, for purification of compounds and as therapeutics.

European National Committees

sub-working group

A complete phasing out of animal testing was previously suggested in the Netherlands, when the Dutch government announced its ambition in 2016 to end all regulatory testing by 2025, and significantly reduce numbers in basic and translational research as well. However, once confronted by the reality of the situation, the Netherlands government has since backtracked on this claim and has said instead that it wants to position itself as a ‘forerunner’ in animal-free innovation with no set deadline dates. Similarly, the US Environmental Protection Agency (EPA) also recently ditched a controversial plan to phase out all use of mammals to test the safety of chemicals by 2035.

For the foreseeable future, it is unlikely that any legislation in Europe will lead to an outright ban on animals across all areas of testing, as this may be a risk that politicians would be unwilling to take. In addition, animals will always be required to develop veterinary medicine and so animal tests will always exist in order to understand the effect of drugs on animals specifically.

In a study involving EARA member Maastricht University, Netherlands, a cancer vaccine was found to work for both lab mice and pet dogs with cancer – insights which could also be useful in developing a vaccine for human cancer. This highlights the complementary nature of research, and that the translatability from animals to humans can sometimes start from an animal health perspective, not a human one.

And while certain NAMs may be able to replace and reduce the number of animals used in testing, there will also be cases where some findings obtained using NAMs will still need to be verified in animals. For example, if there is limited information available on the mechanism by which a particular NAM works to give the intended effect, comparisons will need to be drawn from a model where this data is available – often animals.

It is still not possible to replace animal testing for chemical safety assessments for all (eco-)toxicological endpoints. For some endpoints, further research is necessary. For other endpoints, non-animal testing is currently not satisfying fully the regulatory needs, e.g. as regards the quantitative assessments of hazards and risks.

European Commission response to the European Citizens’ Initiative Save Cruelty Free Cosmetics, July 2023

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NAMs in cosmetic testing

A full EU ban on animal-tested cosmetic products came into effect in 2013, as part of EU Regulation 1223/2009. Before this ban was implemented, safety assessments involved the use of animals, mainly rodents and rabbits, to determine if cosmetic products and ingredients were safe for humans (toxicology endpoints). Due to the development of non-animal techniques, it became apparent that these animal studies were no longer required, hence why it was possible to introduce the ban on animal tested cosmetics and their ingredients.

There are also new techniques that are being developed to improve toxicity assessments of, for example, the skin, including engineered skin tissue, developed from human skin cells, that can accurately describe toxic effects of cosmetics, rather than just predict how they will behave in the body in people (traditionally done using animals). NAMs are also being tested on new or non-standard cosmetics ingredients, with some of the largest beauty companies exploring how these methods can be increasingly used in future testing.

However, as well as being subject to the EU Cosmetic Regulation, chemicals used in cosmetics and their ingredients are also subject to the REACH Regulation, which requires companies who make and supply chemicals to provide information about the properties of these substances, to control the risks they may pose to humans (primarily industrial workers) and environmental health. This means that, under REACH, certain cosmetics ingredients still need to be tested on animals to assess the hazards for both the production workers, who are exposed to larger quantities of a substance than consumers, and the potential effects on the natural environment.

The future of NAMs

Measures that aim to usher in NAMs are a welcome move in the transition towards scientific innovation, or away from animal studies, in instances where there may be improved alternatives now or in the near future. However, the anticipation of bringing NAMs to the fore should never surpass the science of what they can actually achieve, and here lies another important challenge – combatting information that is overblown or false (this tends to occur in efforts to slow or stop animal research), and ensuring the public and non-specialist groups are clearer about the progress of NAMs and the specific areas in which they can play a role.

NAMs and other alternatives to animal research pose both exciting prospects and stimulating challenges in biomedical research, drug and safety testing, and beyond. What they can potentially contribute to scientific discovery, regulatory confidence and computing efficiency has huge implications for how research and testing might be carried out in the future. However, while the use of NAMs is expected to grow, some also predict that the use of animals will rise as well.

With legislative developments already indicating a move away from animal tests in some areas (such as the recent phase-out of the rabbit pyrogen test in Europe); the existence of co-ordinated bodies like the European Partnership for Alternative Approaches to Animal Testing (EPAA); and the continuous dedication of research groups, 3Rs initiatives and bodies to the development of animal research alternatives, it is evident that the momentum is there.

The European Commission has now begun a process to build a roadmap towards the eventual phase out of animal testing in favour of NAMs, but only in the area of chemical safety assessments, accounting for less than 2.5% of all animal use for scientific purposes in the EU and UK.

EARA has responded to the Commission’s requests for comments and highlighted that despite significant progress, the transition to NAMs still faces many challenges and advised that time should be allowed ‘to gain experience and confidence with NAMs and technologies to maintain the high level of protection that EU citizens are used to’.

Overall the strategy should recognise that the best method available should be selected to ensure the safety of humans, animals and the environment, including animal methods, and that a complementary approach should be considered where a variety of methods need to be employed.

EARA response to Commission consultation on roadmap for NAMs

EARA has also published a policy statement, on behalf of its members, about NAMs and animal research. In the statement, EARA highlights its view that a balanced and science-based dialogue on the use of animals and NAMs is needed. By being practical about what NAMs can achieve at present and realistic about where and how far the transition can be made society can move, with confidence, towards an ever-more capable and diverse set of alternative non-animal methods.