Sage Journals: Discover world-class research

Abstract

The Three Rs have become widely accepted and pursued, and are now the go-to framework that encourages the humane use of animals in science, where no other option is believed to exist. However, many people, including scientists, harbour varying degrees of concern about the value and impact of the Three Rs. This ranges from a continued adherence to the Three Rs principles in the belief that they have performed well, through a belief that there should be more emphasis (or indeed a sole focus) on replacement, to a view that the principles have hindered, rather than helped, a critical approach to animal research that should have resulted in replacement to a much greater extent. This critical review asks questions of the Three Rs and their implementation, and provides an overview of the current situation surrounding animal use in biomedical science (chiefly in research). It makes a case that it is time to move away from the Three Rs and that, while this happens, the principles need to be made more robust and enforced more efficiently. To expedite a shift from animal use in science, toward a much greater and quicker adoption of human-specific New Approach Methodologies (NAMs), some argue for a straightforward focus on the best available science.

Keywords

3Rs animal research humane NAMs new approach methodologies replacement reduction refinement Three Rs

An overview of the Three Rs and the case for change

In the UK, scientists are experimenting annually on approximately three times the number of animals that were being used for such experiments at the outbreak of the Second World War.¹ There has been a strong upward trajectory in animal use in the UK since the late 1990s, largely, and perhaps exclusively, due to the use of genetically modified (GM) animals — though this has turned into a downward trajectory since 2016.² Globally, an estimate of animal use for scientific purposes approached 200 million in 2015, including more than 12 million used in the EU.³ Statistics show that the total number of animals used in science in the EU decreased by 5% between 2015 and 2017 — however, the numbers had been largely static between 2005–2017 (decreasing by less than 10% over that time), with average annual reductions of only around 1%.^4,5

Breakdowns show that 60% of these animals were used in basic and applied research; three times the number used for regulatory purposes. This period also saw an increase of 21% in animals used in procedures classified as causing severe suffering, with 40% of all animals subjected to moderate or severe suffering, and an increase of 7% in non-human primate (NHP) use. All of this was despite myriad regulations being in place and other focused efforts, including promotion of the Three Rs principles. These various measures ostensibly aim to ensure that animal experiments are conducted only when absolutely necessary (for example, in efforts to understand and treat diseases that harm people, and when there is no scientific alternative to animal experiments), and when the harm that they cause to the animals involved is countered by a significant potential benefit for humans. There is, of course, some significant debate about the success of the Three Rs principles and the degree of positive impact they have had over time. This review critically addresses these questions, and looks at the evidence supporting the view held by some that the Three Rs principles need to change, in order to help realise what they set out to achieve.

The Three Rs principles — the Reduction, Refinement and Replacement of the use of animals in experiments — have been around since the 1950s. Conceived by zoologist and psychologist, William Russell, together with microbiologist, Rex Burch, the concept was considered ground-breaking at that time, providing guiding principles toward a more ethical use of animals in scientific research. Specifically, they defined the Three Rs, in their published book The Principles of Humane Experimental Technique⁶ as:

— Reduction: “reduction in the numbers of animals used to get information of a given amount and precision”. This is largely a matter of improvements in the design of experiments and analysis of the data they provide.

— Refinement: “any decrease in the incidence or severity of inhumane procedures applied to those animals that still have to be used”. This is after all possible steps have been taken to achieve reduction and replacement, and includes breeding, housing and husbandry.

— Replacement: “any scientific method employing non-sentient material, which may in the history of experimentation replace methods which use conscious living vertebrates”. This includes the strategic use of in vitro and in silico procedures and the ethical use of human volunteers.

The Three Rs concept was innovative when first proposed almost 70 years ago, but in practice it was largely ignored for many years. However, it received renewed interest during the 1980s and 1990s, when the UK and EU were developing new legal frameworks for animal research. Eventually, it came to be widely accepted and enshrined — at least to some degree — in law.⁷ We are therefore now entitled to ask a number of questions, such as:

1. What positive impacts have existing regulations, and the Three Rs principles, actually had on animal research and testing?

2. Given the rise in the numbers of animals used in science, as well as the prevalence of experiments that cause significant harm and suffering to animals, has it all failed to make significant positive impact?

3. Can an argument be made that the situation would be even worse in terms of animal use and welfare, if it weren’t for the regulations and the Three Rs principles?

4. Crucially: Can it be posited that the Three Rs principles have directed a great deal of effort away from the most important aspect — replacement — toward the other two Rs which, by their nature, tacitly accept the value and translational capacity of animal research to human benefit, and its necessity?

5. Finally, and perhaps most importantly of all: Despite the significant contribution to animal welfare that the Three Rs principles have made over time, do they still have a role to play, both right now and in the future of biomedical research and testing? Or is it time for something different — something more focused on replacement and on driving the adoption and acceleration of the use of human-focused methods, including high-tech New Approach Methodologies (NAMs), with all of their advantages?

Whatever opinions of the reader might have with regard to animal research, NAMs, the Three Rs principles, and the need for change in how research and testing are conducted, it is hoped that this article will provide some food for serious thought about how science can much more effectively ‘do its duty’ to many millions of animals in laboratories — in short, how greater and more rapid steps can be taken to remove animals from laboratories altogether. It is also hoped that this article will convey the urgent need for more focus on a change that many believe is sorely needed, from both animal-oriented and human-oriented ethical standpoints.

Though the Three Rs principles are well intentioned, some argue that they have had insufficient positive impact on the use of animals in science. A number of individuals with exceptional experience in, and knowledge of, the Three Rs, have proposed several key indicators to suggest that the application of the principles has failed to achieve its primary objective.

While the figures for 2015–2017 (published in 2020) show that European use of animals for regulatory purposes is falling over time, the reduction in numbers is slight (around 7%). Although figures for specific tests appear to show encouraging decreases in animal use, such as those for skin and eye irritation, pyrogenicity and batch potency, there is evidence to show that validated, accepted non-animal methods are not being used sufficiently in accordance with the relevant EU Directive (e.g.^8,9). A confounding factor is that, while the latest data show, for example, that approximately 90% of studies conducted for skin and eye irritation between 2019–2022 were performed in vitro, the number of animals used specifically in the testing of chemicals under the EU REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals) regulation doubled between 2016 and 2019.^10,11 Grounds for serious concern therefore remain. While ethical concerns over animals used to test cosmetics drove the approval of a legislative EU ban on animal testing of cosmetics and cosmetic ingredients which was phased in between 2004–2013,¹² the implementation of REACH has resulted in an increase in new regulatory animal tests for chemicals.¹³ The nature and extent of this increase is difficult to ascertain, but overall numbers of animals used have been estimated at 4.2 million, just for reproductive, developmental and repeated-dose toxicity tests.¹⁴

Another seriously confounding factor in the attempt to assess the impact of the Three Rs principles is that, in the EU and globally, reporting has been weak at times, with relatively few countries reporting full statistics, and omitting details on regulatory use, severity, types of research, etc.^15,16 It therefore remains difficult to estimate areas of animal use in which any application of the Three Rs might have been successful to some degree, but some general statistics paint an overall picture of concern. There have been highly significant increases in animal use globally, both for procedural and non-procedural purposes, including the use of around 200,000 NHPs and 160,000 dogs.¹⁶ It is clear that animal use in science overall has increased over time, and that it remains an automatic choice in many areas. In fact, it is often a first, rather than a last, resort, with significant welfare issues persisting.¹⁷

The Three Rs may, in reality, have been used to justify and to perpetuate the continued use of animals, due to some researchers focusing more closely on refinement or reduction, instead of replacement. To some degree, this focus on refinement or reduction may be understandable from a solely practical level, at least for regulatory purposes. Conversations with stakeholders reveal a view that regulators will always require animal data if they are not provided, and so the animal data are prospectively obtained by those involved in drug and chemical development and testing. While some regulators argue that this is not the case (i.e. they do not require animal data, only appropriate data) they also admit that animal data are frequently provided and therefore they are obliged to consider them. Even if there is no direct legislative imperative to supply animal data to regulators, it feels to some that this imperative exists de facto.

In research, scientists may also still feel external pressure to use animals where there is no scientific rationale for doing so. To illustrate, a recent multi-stakeholder workshop concluded that there is widespread ‘animal methods bias’ in biomedical research, which results in the unnecessary use of animals in experiments and the continued reliance on animals in research — even where suitable non-animal methods exist.¹⁸ Recommendations have been made for journals, publishers, funders, governments and other policy makers, as well as the scientific community at large, to reduce the prevalence and impact of such ‘animal methods bias’, and these recommendations are in the process of publication at the time of writing;^19,20 however, the impact of these measures will not be immediate.

Unfortunately, these ‘drivers’ of animal use seem to have greater influence at the present time than other, perhaps less powerful, drivers that are associated with the use of non-animal alternatives — such as increased time and cost efficiency, less red tape/fewer regulations, better animal welfare/ethics, recognised scientific superiority and greater human relevance.

Ultimately, it is not a valid scientific approach to refine the procedures or to reduce the number of animals involved in any particular research project, if the animal experiments are of little or no human relevance. In such circumstances, replacement must be the only course of action. Key players, including the EU, agree that total replacement is their aim (for example, see the recent motion supported by the European Parliament).²¹ But the UK Government remains committed to all Three Rs, as shown by recent Government replies to parliamentary questions, which included statements such as the following:

“The use of animals in research is carefully regulated and remains important in ensuring new medicines and treatments are safe. At the same time, the Government believes that animals should only be used when there is no practicable alternative and it actively supports and funds the development and dissemination of techniques that replace, reduce and refine the use of animals in research (the Three Rs).”²²

The case for change

Such broad commitment to all of the Three Rs is out of line with the thinking of an increasing number of scientists, who acknowledge that the use of animals as models of human conditions is beset with translational issues at the very least, and could in fact be considered entirely ‘unscientific’ (i.e. not based on sound evidence). For decades, many researchers have laudably dedicated their working lives to the search for treatments and cures for all manner of human diseases — including cancer, dementia and diabetes. However, many of the results obtained with animal models fail to further our understanding of the causes and nature of human disease to any significant extent, and often do not lead to the successful realisation of safe and effective therapies and cures for many of them. For example, in my own work, I have authored and co-authored over 60 studies and reviews on this topic, all citing numerous supporting references to the work of scientists around the world. These include:

— two recent papers that examined the translatability of findings from diverse fields of animal research to humans and found it to be extremely poor;^23,24

— analyses of the contribution of chimpanzee research to the benefit of human health (including in fields such as HIV/AIDS and hepatitis C research), which helped to realise an end to federally funded chimpanzee research in the USA;^25–28

— critical reviews of NHP research in multiple areas and its translational failure, particularly in neuroscience;^29–32

— the need for a shift to human-focused research in the field of Parkinson’s disease research;³³

— how animal testing of potential new human drugs is insufficiently predictive of human safety and toxicity^34–37 (including developmental toxicology³⁸);

— why a shift away from animal use in science and to a complete focus on human biology is essential, due to inter-species genetic differences^39,40 compounded by the adverse and unavoidable physiological effects of stress for animals in laboratories.^41,42

There are also myriad works by others that augment the evidence base, including various published papers^43–52 and a number of substantial books.^53–55 In the field of regulatory toxicology and drug development specifically, the superior predictive nature of approaches based on human liver-on-a-chip for drug-induced liver injury (DILI) has recently been demonstrated. DILI is one of the most frequent reasons for the failure of new drugs in clinical trials, mainly as a result of the poor predictive nature of the animal methods used in its evaluation.^56,57 There are many more examples of this type of failing, all of which constitute substantial evidence for a paradigm shift in how we should approach research and testing.

This poor translation is underpinned by significant biological variation among the individuals of any species, so that no individuals or small groups of individuals can be used as models for all members of their own, let alone another, species. This also applies to humans,⁵⁸ and fortifies the argument for the need for change in how research and testing are approached generally. For example, genetic variability among regional populations of macaques means that they can differ from each other as much as they differ from another species entirely. Several rhesus macaque subspecies have been shown to display a variety of physiological and behavioural differences, and significant differences between distinct geographical populations affect macaque populations worldwide, influencing their susceptibility to a variety of diseases and how they metabolise drugs, for instance.^59,60 Yet, instead of responding to this type of considerable evidence, and resolving that future biomedical research and testing must maintain a focus on human biology from start to finish, either the evidence is largely overlooked, or there is a belief that these issues can be overcome with better experimental technique and/or analysis^61–64 and/or genetic modification.^16,65 This is in stark contrast to other scientific and engineering disciplines that try to rigorously challenge their models, and alter their approaches, based on solid data. But when it comes to biomedical science, despite many scientists trying to move away from using animals, the prevailing culture continues to view animal research as the ‘gold standard’, and takes the view that applying any of the Three Rs (i.e. refinement, reduction or replacement), to a greater or lesser degree, is sufficient.

This does not mean that no good has come from the Three Rs concept. However, this sense of doing enough, just because one is applying one or more of the Three Rs principles, has arguably resulted in the perpetuation of animal research and testing. One may feel that one is aiding animal welfare and the progress of biomedical science by practising and adhering to the Three Rs principles in any way, and indeed there may be some animal welfare benefits in practising reduction and refinement. However, the cost associated with that welfare benefit, in the form of a poor or absent focus on replacement, must be weighed against it. Arguably, a relative lack of effort on replacement may have stemmed from a defeatist attitude in some areas — i.e. it may be perceived as much easier to reduce or refine one’s habitual experiments in some areas, as compared to developing and implementing appropriate replacement methodologies.

The failure to focus on replacement involves the ongoing passive assumption that the animal methods being refined and reduced are scientifically valid, translatable to humans, and are the only way of mimicking a ‘living human’. The consequences of this assumption include: scientific failure (i.e. the lack of direct or indirect translation to clinical benefit from those experiments; see examples listed above); the ethical issues associated with animal suffering and death; and the significant impact on patients and their families, who are hoping for a better understanding of the diseases which affect them and that new, safe and effective therapies are available for their treatment.

Whilst animal experiments for research and testing purposes are still being carried out around the world (hopefully during the course of a wholesale transition away from them, and toward human-focused approaches), refinement and reduction perhaps still have a role to play. However, replacement should be overtly paramount and frameworks should be established to encourage and police this emphasis. Examples of such frameworks have been proposed. DeGrazia and Beauchamp detailed one approach in their book, Principles of Animal Research Ethics,⁶⁶ and this is also summarised in their paper.⁶⁷ In these publications, the authors convey their view that the Three Rs do not constitute an adequate “framework of principles displaying the core values of animal research ethics…to help in the guidance and proper oversight of animal research”. They also outline the evidence underpinning the need for a more effective framework, focusing on some of the ethical and scientific issues associated with animal research and testing, and propose some additional principles that are complementary to those of Russell and Burch. These additional principles are based on core values of societal benefit and animal welfare, which the authors argue are shared by researchers who use animals and those who advocate for animal protection. These ‘shared’ principles are thus considered more likely than other principles to generate agreement between interested parties. Overall, the case made is that: i) all ethically relevant aspects of an animal’s life must be considered, above and beyond those directly associated with the use of that animal in a scientific experiment; and ii) the value of any proposed social benefit and the likelihood of achieving that benefit should be considered fully, specifically with regard to how the social benefit might justify the costs and harms to the animals involved.

For further informed and detailed background and discussion on various similar arguments, the publications of Professor Michael Balls, former Head of the European Centre for the Validation of Alternative Methods (ECVAM), Life President of the UK’s Fund for the Replacement of Animals in Medical Experiments (FRAME), and expert in the Three Rs and the work of Russell and Burch, who has published extensively on the subject over many years, should be consulted (see, for example).^{7,17,24,68–72}

Arguably, much more replacement would have already been implemented, if there were sufficient desire for it. Perhaps replacement has become more important over time, yet reports from the UK’s National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) suggest that the proportion of funds allocated to it decreased from 85% in 2005 to 78% in 2017.⁷³ Either way, animal use in science is effectively plateauing, rather than decreasing. This is also reflected in the fact that millions of animals are still used in tests for which there are validated alternatives, as a result of the drawn-out approval and implementation of non-animal methods, as well as the poor enforcement of their use.^8,74 An optimistic attitude to the plateauing, rather than increasing, numbers of animals used in science holds the view that this is part of the process and nature of change, as established and habitual protocols and methodologies give way to new ones. Perhaps this reflects an increase in confidence of NAMs as a result of their greater use in biomedical science. This will result in the ongoing replacement of animals, as researchers, developers of new pharmaceuticals, funders and regulators more fully embrace NAMs in place of animal-based approaches.

Should we consider the replacement or refinement of the Three Rs themselves?

There have been arguments from many quarters that science needs to do something different with regard to the Three Rs as a concept. Perhaps, the Three Rs themselves now need replacing with something more effective; something that will result in the implementation of the most desirable of the Three Rs — replacement — and the consequent significant decline in animal experiments. Alternatively, some argue that it may be a case of augmenting and/or repositioning (i.e. refining) the Three Rs to make them more effective.

Proposals for something additional to the Three Rs — or something new altogether — have taken various forms. For example, various additional ‘Rs’ have been suggested, such as: Refusal, Registration, Rehabilitation, Relevance, Reporting, Reproducibility, Respect, Responsibility, Re-use, Rigour and Robustness (see, for example).^75,76 Occasionally, full substitution of the original set of Three Rs principles has been suggested, with some examples being: Dr Julia Fentem’s ‘Realism, Reassessment and Realignment’,⁷⁷ or the potential merits of ‘Relevance, Reliability and Responsibility^’ proposed by Animal Free Research UK. In the latter case, ‘Relevance’ would demand research that is directly relevant and specific to human beings, involving human-focused and human-derived methods, including NAMs (such as artificial intelligence), and the ethical use of human cells and tissues, organ-on-a-chip and stem cell technology. ‘Reliability’ might establish and encourage criteria for scientifically valid, standardised, high-quality methods that are more reproducible and human-relevant, reflect the diversity of the human population, are devoid of issues associated with interspecies extrapolation, and which would be optimisable over time.^78,79 ‘Responsibility’ could assure a strong commitment to animals currently used in laboratories, and also to human beings, relying on the best available science (in terms of translatability to human biology and clinical benefit), aligning with the UN’s Global Sustainable Development Goal 3, which aims to ensure healthy lives and well-being for all.⁸⁰

The ‘humanity’ criterion

In their book on the Three Rs, published in 1959,⁶ Russell and Burch themselves suggested that applying the relatively simple criterion of ‘humanity’ when designing experiments could be the best possible approach:

“If we are to use a criterion for choosing experiments, that of humanity is the best we could possibly invent. The greatest scientific experiments have always been the most humane and attractive, conveying that sense of beauty and elegance which is the essence of science at its most successful.”⁶

The idea that we should “move away from the animal welfare focus of the Three Rs, in favour of a wider concept of humanity, which also embraces human welfare”⁷ has been discussed widely by Professor Balls.^7,17,68,71 Articles such as these should be consulted in their entirety to appreciate the argument fully, and save the need to repeat much of it here. In short, human welfare and animal welfare are both important, and ought to go hand in hand. However, a greater focus on human welfare will prioritise and accelerate replacement because it demands the best, most relevant, predictive scientific approaches — which are invariably human-focused. This human focus would consequently also benefit animal welfare to a considerable degree, largely through the increased use of more successful human-relevant replacement strategies leading to fewer animals being subject to experimentation.

Applying the humanity criterion to all biomedical research proposals would commit science to achieving the best possible human relevance, rather than replicating animal tests and data. It would ensure that inhumanity to animals (via all aspects of their breeding and experimentation) is properly considered. It would also guarantee that inhumanity to humans is fully taken into account. For example, the human consequences of poorly translational animal research and testing are fewer safe and effective drugs in the development pipeline and in clinical use. In addition, there are harmful consequences for personnel in laboratories in which animals are used in research and testing, which should be factored into any consideration of human harms.^81,82 Many such individuals have reported symptoms of traumatic stress, including compassion fatigue, which has been experienced by almost 90% of people involved in animal research.⁸³ The killing of animals in laboratories can represent a major psychological burden for laboratory staff. This, in turn, can adversely impact their ability to care for the animals, making it an ethical issue relevant to laboratory animals that is over and above the ethical issues associated with the actual experimental procedures being carried out.⁸⁴ Harmful consequences for personnel may be severe, including depression, substance abuse and suicide.^83,85 A full appreciation and use of the humanity criterion would therefore lead to optimal science that fulfils all the ethical criteria, aims, guidelines and legal imperatives, concerning both humans and animals.

Incorporating harm to humans into the overall consideration of harms and benefits seems entirely logical, and indeed could well be a crucial step on moving the Three Rs forward into a position of optimal efficacy and impact. The matter of how to do this may not be so simple, and would need considerable discussion to ensure this efficacy and impact are achieved.⁶⁸ But the need for incorporating this human harm is self-evident, because the harms that are currently considered are overwhelmingly focused on animal harm (however inadequate this focus might be). This additional and warranted human focus could, for example, all be incorporated into the application of the harm–benefit analysis, via the Bateson cube method, for example, which evaluates proposed research involving animals based on animal suffering, research quality and potential human benefit.⁸⁶ Factoring-in actual, rather than just potential, human benefit retrospectively is now done in some (but not all) cases. Although this is a positive step forward, consideration of human benefits alongside the consideration of human harms would enhance the evaluation process and make it more rigorous and selective. This process could be improved even further by ensuring that procedures are put in place to assess animal suffering more accurately and objectively. Indeed, the power of retrospective analyses, coupled with the need for more rigorous procedures, was comprehensively argued for by Pound et al.,⁴⁴ who concluded that so much was at fault with the system and application of animal-research evaluation as it stands, that only around 7% of the animal research studies they assessed might be permissible, if the criteria were more efficiently applied.

The way forward

It can therefore be argued that, whatever the Three Rs have delivered up to now, they have not delivered enough. This may be true particularly in more recent years, through a time of major technological advancements and replacement possibilities. Arguably, the Three Rs have, overall, been counter-productive, deflecting significant amounts of attention and effort away from replacement. This is evidenced by belated and relatively insufficient government investment in human-focused research and testing methods, and a continued defence of animal research and testing based on poor, or absent, evidence. There is, consequently, a formidable argument that significant change — in some format — must take place as a matter of urgency, with regard to how animal-based research and testing are funded, commissioned, reviewed, approved and reported.

All aspects and phases of animal research and testing should have much more critical, objective input — with a major, if not exclusive, focus on replacement. If done thoroughly and properly, this would quickly lead to only a small proportion of animal use in biomedical science being approved, and it would also highlight areas of focus for the complete replacement of animal use. It is generally accepted, with great reluctance in many quarters, that in reality this will take some time to happen — certainly, longer than evidence demands. While this change is taking place, with an overdue and welcome phasing in of NAMs and phasing out of animal experiments, some have asserted that it must remain an ethical duty for the ‘other’ two Rs of reduction and refinement to be applied to research involving animals. It would clearly be unacceptable for this not to be the case, and nobody would wish for a reduction- and refinement-free biomedical research and testing environment in the UK for any length of time. However, this must not be at the continued expense of replacement, as already noted.

The issue of funding and investment

It may be pleasing on one level to see that the UK’s NC3Rs has committed 63% of its research funding since 2004 toward replacement,⁷³ but there are two significant caveats. The first is that this means more than one-third of funding from the NC3Rs has been directed toward non-replacement efforts, and secondly, the amount that the NC3Rs has directed to replacement projects over almost two decades totals a relatively poor £46.7 million (equating to less than £2.5 million per year, on average). This compares to a total current UK Government R&D budget of £15–20 billion, while member organisations of the UK’s Association of Medical Research Charities (AMRC) spend around £1.5 billion on research annually. The average annual investment in replacement by the NC3Rs therefore amounts to around 0.01% of this total. At best, it has been estimated that “human-relevant NAMs funding” (not defined, but presumably encompassing development, validation and use in research) currently amounts to 0.2–0.6% of total biomedical research funding in the UK,⁸⁷ and less than 0.2% of the total budget for the UK Medical Research Council (MRC), Biotechnology and Biological Sciences Research Council (BBSRC) and Innovate UK combined (£1.144 billion).⁸⁷ In the EU, it has been estimated that just 0.036% of the research budget is dedicated specifically to the development and use of NAMs⁸⁸ (see also Marshall et al.⁵⁰).

Funding, therefore, is perhaps the major issue, and consequently one that is receiving a great deal of attention from groups seeking to realise change in how biomedical science is done. Whatever the exact percentages and whatever the details, it can be argued that the numbers presented above mean that the situation cannot be dressed up as anything other than disastrous for laboratory animals, and also for the humans relying on the best and most appropriate science to understand and find treatments for their diseases.

This is not just a case for a greater proportion of funds to go toward replacement, and the development and more widespread use of NAMs. It is a demand for the considerable redirection of funds that support science involving the use of animals, which demonstrably needs to be phased out as a matter of urgency, as it is simply not delivering the benefits that are demanded of it (which are claimed with little or no convincing evidence). The monetary figures may sound high, but the point is that research funding is never sufficient, and so the funding is relatively scarce and hard to come by. The fact that much of it is not dedicated to the best, most human-focused research most likely to translate to clinical benefit is a salient point that needs urgent attention.

Cause for optimism

While multi-pronged and determined efforts are still ongoing to elicit changes in how research and testing are best conducted to benefit both animals and humans, there is much cause for optimism and there are signs that change is happening. For instance, the UK NC3Rs has set up its NAMs methodologies hub, including a ‘NAMs Network’ community of “...researchers, developers and industry and regulatory end-users working together to accelerate the use of new approach methodologies” (see https://www.nc3rs.org.uk/3rs-resources/new-approach-methodologies-hub), and the US National Institutes of Health (NIH) announced, in January 2024, its ‘Complement Animal Research in Experimentation' (Complement-ARIE) programme, to catalyse the development and use of NAMs, and to help the replacement of animals in research (https://commonfund.nih.gov/complementarie).

Human-focused methods and NAMs are also being used widely in many different areas of research and testing — for example, in DILI studies^56,57 (mentioned already) and botox testing,⁸ as well as in research into Parkinson’s disease, Alzheimer’s disease, rheumatoid arthritis, respiratory diseases and HIV/AIDS.^23,33 Those who are seeking to expedite these changes for the better should be able to make the following simple statement — “Of course NAMs are being used much more widely now!” Indeed, this wider use should indeed be the obvious response to our increasing knowledge of factors such as:

— the existence of intractable and (crucially) insurmountable species differences, and how they underpin the poor translation of animal data to humans;

— the myriad differences in gene complement and gene expression between species;

— the significant human variation that must be, and can be, factored into research and testing (but which is impossible when using animals);

— the misleading differences in pathologies in humans with diseases and in animals used to model those diseases (including many different types of tumours);

— how the physiological relevance of research can be substantially greater when using advanced, more sophisticated 3-D human tissue culture models, with their potential to incorporate dynamic and shear stress aspects, serum specificity, etc.;

— how these human-focused methods can be used in concert with each other, and with computational methods and clinical research data, to give an optimal picture of the question at hand in so many areas; and

— how such approaches are being used for the repurposing of existing drugs, mitigating losses in drug development due to the poor predictive nature of animal tests.

All of the above points have been summarised in my own published work over the years (see https://www.researchgate.net/profile/Jarrod-Bailey/research), and corroborated by the published work of many others.

A recent (2023) report commissioned by the UK’s NC3Rs is further cause for optimism.⁸⁹ After reviewing the oversight of animal research and testing in the UK, and how this oversight impacts the Three Rs and the mission of the NC3Rs, some critical conclusions were drawn that should be fully considered and appreciated by those involved in such activities. Some of these are listed below, followed by what I believe are logical progressions (given in parentheses):

— replacement is not well covered by review processes (those processes must therefore be revised and improved);

— inspectors rarely suggest replacements, at least partly because they cannot have sufficiently detailed knowledge of the many areas they need to cover (inspectors need access to broad and diverse expertise);

— Institutional Animal Welfare Ethical Review Bodies (AWERBs) assume that researchers whose applications they receive have properly considered replacement (protocols should be put in place and adhered to, critical input should be provided to ensure that replacements have been fully and properly considered, and assumptions should cease);

— attitudes of many journals lead researchers to assume that they will be required to support publications on NAMs with animal data (journals should move away from this attitude);

— researchers need greater and easier access to information about replacement technologies and methods, including via resources provided by funders and institutions, etc. (this information should be provided);

— funders have a role to play to facilitate replacement, via critical and constructive input, as well as dedicated funding (steps should be taken to encourage funders to do this);

— proposed animal use should be substantially challenged early in the process (critical input on research proposals should be demanded in all cases, and be substantial; for instance, there could be pre-registration and pre-submission processes);

— there is a need to shift from ‘box ticking’ to a genuine commitment toward replacement (processes should be put in place to seek such commitments).

This is all very honest, open, welcome and warranted. The points listed above must shock anybody who possesses a genuine passion for the replacement of animals in research. But this passion, this shock, and these points, must lead to significant action — action that has arguably been hugely inadequate for a long time. The first step is a full appreciation of the situation and need for change. Encouragingly, it does appear that change is afoot to improve the situation, and to give replacement the status it should have had all along.

As the use of, and confidence in, methods that replace the use of animals increases, investments in their further development and improvement might become more self-sustaining and perpetuating. We are already seeing trends that point toward the greater use of NAMs, as well as an increasing level of NAMs-related publications and investment, while animal use is plateauing. This is surely a sign of change and an impending tipping point.^90,91 This could (and should) all be supported and expedited via a concerted and sustained focus on replacement strategies of the type outlined in this article, and also on the application of the humanity criterion.

Conclusions

This focus on replacement and application of the humanity criterion, would also help address a number of important issues associated with the use of animals in science that were originally acknowledged and warned against by Russell and Burch in their 1959 book.⁶ These include:

— the lack of knowledge about what is being modelled (i.e. human disease), and about the models themselves (i.e. animal models);

— the considerable impact of species differences and the ‘high fidelity fallacy’ of assuming that animal data will reliably translate to human biology due to relatively superficial anatomical, physiological and genetic similarities; and

— the need to take full account of human variation and diversity, as well as the many factors that exacerbate their biological consequences (see also¹⁷).

A greater knowledge of human biology will be acquired through clinical research, supported and complemented by advanced human cell and tissue cultures; this knowledge will aid our increased understanding of species differences and their adverse effects on extrapolation to humans and translation to clinical benefits. Only through human-focused research will we have the ability to factor in, and take full account of, human diversity, and thus fully embrace and expedite the necessary shift toward personalised medicine.⁴⁹ A comprehensive shift to human-oriented research is of paramount importance, essential and urgent — and making replacement the greatest priority is a crucial aspect of achieving this goal.

Footnotes

Acknowledgments

The input of Michael Balls, Geoff Pilkington and Mike Philpott is greatly appreciated.

Declaration of Conflicting Interests

The author declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was funded by Animal Free Research UK, London, UK.

References

Rowan

. A brief history of the animal research debate and the place of alternatives. J Oral Tissue Eng 2007; 5: 113–121.

UK Home Office . Statistics of scientific procedures on living animals, Great Britain: 2022, https://www.gov.uk/government/statistics/statistics-of-scientific-procedures-on-living-animals-great-britain-2022 (2023, accessed 23 January 2024).

Taylor

Gordon

Langley

, et al. Estimates for worldwide laboratory animal use in 2005. Altern Lab Anim 2008; 36: 327–342.

European Commission . Report from the Commission to the European Parliament and the Council 2019 report on the statistics on the use of animals for scientific purposes in the Member States of the European Union in 2015–2017, https://eur-lex.europa.eu/legal-content/EN/TXT/?qid=1581689520921&uri=CELEX:52020DC0016 (2020, accessed 23 January 2024).

Cruelty Free Europe . Briefing on number of animals used in experiments in the EU, https://www.crueltyfreeeurope.org/sites/default/files/GeneralEUstatsbriefing.pdf (accessed 23 January 2024).

Russell

WMS

Burch

. The principles of humane experimental technique. London: Methuen 1959, 238 pp.

Balls

. It’s time to reconsider the principles of humane experimental technique. Altern Lab Anim 2020; 48: 40–46.

Taylor

Gericke

Alvarez

. Botulinum toxin testing on animals is still a Europe-wide issue. ALTEX 2019; 36: 81–90.

Taylor

Ten years of REACH — An animal protection perspective. Altern Lab Anim 2018; 46: 347–373.

10.

ECHA . The use of alternatives to testing on animals for the REACH Regulation, Fourth report under Article 117(3) of the REACH Regulation , https://echa.europa.eu/documents/10162/0/alternatives_test_animals_2020_en.pdf (2020, accessed 23 January 2024).

11.

ECHA . The use of alternatives to testing on animals for the REACH Regulation, Fifth report under Article 117(3) of the REACH Regulation , https://echa.europa.eu/documents/10162/23919267/230530_117_3_alternatives_test_animals_2023_en.pdf (2023, accessed 23 January 2024).

12.

Hartung

. Food for thought… on evidence-based toxicology. ALTEX 2009; 26: 75–82.

13.

Knight

Rovida

Kreiling

, et al. Continuing animal tests on cosmetic ingredients for REACH in the EU. ALTEX 2021; 38: 653–668.

14.

Knight

Hartung

Rovida

. 4.2 million and counting… The animal toll for REACH systemic toxicity studies. ALTEX 2023; 40: 389–407.

15.

Taylor

Rego

. EU statistics on animal experiments for 2014. [Letter.] ALTEX 2016; 33: 465–468.

16.

Taylor

Alvarez

. An estimate of the number of animals used for scientific purposes worldwide in 2015. Altern Lab Anim 2019; 47: 196–213.

17.

Balls

It’s time to reconsider fundamental issues involved in using laboratory animals as models for humans in biomedical research and testing. In Presentation at the Third Meeting of the APPG on Human Relevant Science, London, UK, 12 May 2021, https://www.humanrelevantscience.org/all-party-parliamentary-group/ (2021, accessed 23 January 2024).

18.

Krebs

Camp

Constantino

, et al. Proceedings of a workshop to address animal methods bias in scientific publishing. ALTEX 2023; 40: 677–688.

19.

Krebs

Camp

Constantino

, et al. Author guide for addressing animal methods bias in publishing. Adv Sci 2023; 10: e2303226.

20.

Physicians Committee for Responsible Medicine . Addressing animal methods bias through building evidence and mitigation strategies, https://www.pcrm.org/ethical-science/animalmethodsbias (2023, accessed 23 January 2024).

21.

European Parliament . Motion for a European Parliament resolution on plans and actions to accelerate the transition to innovation without the use of animals in research, regulatory testing and education, B9-0425/2021, https://www.europarl.europa.eu/doceo/document/B-9-2021-0425_EN.html (2021, accessed 23 January 2024).

22.

Solloway

. Department for Business, Energy and Industrial Strategy. Animal experiments, Question for Department for Business, Energy and Industrial Strategy UIN 34616, tabled on 16 July 2021, https://questions-statements.parliament.uk/written-questions/detail/2021-07-16/34616 (2021, accessed 23 January 2024).

23.

Marshall

Bailey

Cassotta

, et al. Poor translatability of biomedical research using animals — A narrative review. Altern Lab Anim 2023; 51: 102–135.

24.

Bailey

Balls

. Clinical impact of high-profile animal-based research reported in the UK national press. BMJ Open Sci 2020; 4: e100039.

25.

Bailey

An assessment of the use of chimpanzees in hepatitis C research past, present and future: 2. Alternative replacement methods. Altern Lab Anim 2010; 38: 471–494.

26.

Bailey

An assessment of the use of chimpanzees in hepatitis C research past, present and future: 1. Validity of the chimpanzee model. Altern Lab Anim 2010; 38: 387–418.

27.

Bailey

An assessment of the role of chimpanzees in AIDS vaccine research. Altern Lab Anim 2008; 36: 381–428.

28.

Bailey

Balcombe

Capaldo

. Chimpanzee research: An examination of its contribution to biomedical knowledge and efficacy in combating human diseases, https://releasechimps.org/resources/publication/chimpanzee-research (2007, accessed 23 January 2024).

29.

Bailey

Arguments against using nonhuman primates in research. In: Robinson

Weiss

, (eds) Nonhuman primate welfare: From history, science, and ethics to practice. London: Springer Nature, 2023, pp. 569–588.

30.

Bailey

Taylor

. Non-human primates in neuroscience research: The case against its scientific necessity. Altern Lab Anim 2016; 44: 43–69.

31.

Bailey

Taylor

. The SCHER report on non-human primate research — biased and deeply flawed. Altern Lab Anim 2009; 37: 427–435.

32.

Bailey

Capaldo

Conlee

, et al. Experimental use of nonhuman primates is not a simple problem. Nat Med 2008; 14: 1011–1012; discussion 1012.

33.

Cassotta

Geerts

Harbom

, et al. The future of Parkinson’s disease research: A new paradigm of human-specific investigation is necessary… and possible. ALTEX 2022; 39: 694.

34.

Bailey

Balls

. Recent efforts to elucidate the scientific validity of animal-based drug tests by the pharmaceutical industry, pro-testing lobby groups, and animal welfare organisations. BMC Med Ethics 2019; 20: 16.

35.

Bailey

Thew

Balls

. Predicting human drug toxicity and safety via animal tests: Can any one species predict drug toxicity in any other, and do monkeys help? Altern Lab Anim 2015; 43: 393–403.

36.

Bailey

Thew

Balls

. An analysis of the use of animal models in predicting human toxicology and drug safety. Altern Lab Anim 2014; 42: 181–199.

37.

Bailey

Thew

Balls

. An analysis of the use of dogs in predicting human toxicology and drug safety. Altern Lab Anim 2013; 41: 335–350.

38.

Bailey

Developmental toxicity testing: Protecting future generations?

Altern Lab Anim 2008; 36: 718–721.

39.

Bailey

Monkey-based research on human disease: The implications of genetic differences. Altern Lab Anim 2014; 42: 287–317.

40.

Bailey

Lessons from chimpanzee-based research on human disease: The implications of genetic differences. Altern Lab Anim 2011; 39: 527–540.

41.

Bailey

Does the stress of laboratory life and experimentation on animals adversely affect research data? A critical review. Altern Lab Anim 2018; 46: 291–305.

42.

Bailey

Stress in animals in laboratories is inherent, unavoidable, causes psychological and physiological harm, and significantly confounds experimental results. Altern Lab Anim 2017; 45: 299–301.

43.

Pound

Ritskes-Hoitinga

. Is it possible to overcome issues of external validity in preclinical animal research? Why most animal models are bound to fail. J Transl Med 2018; 16: 304.

44.

Pound

Nicol

. Retrospective harm benefit analysis of pre-clinical animal research for six treatment interventions. PLoS One 2018; 13: e0193758.

45.

Ritskes-Hoitinga

Pound

. The role of systematic reviews in identifying the limitations of preclinical animal research, 2000–2022: Part 1. J R Soc Med 2022; 115: 186–192.

46.

Ritskes-Hoitinga

Pound

. The role of systematic reviews in identifying the limitations of preclinical animal research, 2000-2022: part 2. J R Soc Med 2022; 115: 231–235.

47.

Cavanaugh

Pippin

Barnard

. Animal models of Alzheimer disease: Historical pitfalls and a path forward. ALTEX 2014; 31: 279–302.

48.

Pippin

Cavanaugh

Pistollato

. Animal research for Alzheimer disease: Failures of science and ethics. In: Herrmann

Jayne

(eds) Animal experimentation: Working towards a paradigm change. Boston, MA: Brill, 2019, pp. 480–516.

49.

Ingber

. Human organs-on-chips for disease modelling, drug development and personalized medicine. Nat Rev Genet 2022; 23: 467–491.

50.

Marshall

Triunfol

Seidle

. Patient-derived xenograft vs. organoids: A preliminary analysis of cancer research output, funding and human health impact in 2014–2019. Animals (Basel) 2020; 10: 1923.

51.

Pistollato

Cavanaugh

Chandrasekera

. A human-based integrated framework for Alzheimer’s disease research. J Alzheimers Dis 2015; 47: 857–868.

52.

Pistollato

Ohayon

Lam

, et al. Alzheimer disease research in the 21st century: Past and current failures, new perspectives and funding priorities. Oncotarget 2016; 7: 38999–39016.

53.

Pound

. Rat trap: The capture of medicine by animal research — and how to break free. Market Harborough: Troubador Publishing Ltd, 2023, 230 pp.

54.

Herrmann

Kimberley

(eds). Animal experimentation: Working towards a paradigm change. Boston, MA: Brill, 2019, https://www.jstor.org/stable/10.1163/j.ctvjhzq0f (2019, accessed 23 January 2024).

55.

Balls

Combes

Worth

(eds). The history of alternative test methods in toxicology (History of toxicology and environmental health). Cambridge, MA: Academic Press, 2018, 350 pp.

56.

Ewart

Apostolou

Briggs

, et al. Performance assessment and economic analysis of a human liver-chip for predictive toxicology. Commun Med (Lond) 2022; 2: 154.

57.

Zhang

Meyer

O’Meara

, et al. A human liver organoid screening platform for DILI risk prediction. J Hepatol 2023; 78: 998–1006.

58.

Das

Tatarinova

Galieva

, et al. Editorial: Association between individuals’ genomic ancestry and variation in disease susceptibility. Front Genet 2022; 13.

59.

Kanthaswamy

Satkoski Trask

, et al. The genetic composition of populations of cynomolgus macaques (Macaca fascicularis) used in biomedical research. J Med Primatol 2013; 42: 120–131.

60.

Groves

. Primate taxonomy (Smithsonian series in comparative evolutionary biology). Washington, DC: Smithsonian Books, 2001, 350 pp.

61.

Bailoo

Reichlin

Würbel

. Refinement of experimental design and conduct in laboratory animal research. ILAR J 2014; 55: 383–391.

62.

Smith

. Guidelines for planning and conducting high-quality research and testing on animals. Lab Anim Res 2020; 36: 21.

63.

Hubrecht

Carter

. The 3Rs and humane experimental technique: Implementing change. Animals (Basel) 2019; 9: 754.

64.

von Kortzfleisch

Karp

Palme

, et al. Improving reproducibility in animal research by splitting the study population into several ‘mini-experiments’. Sci Rep 2020; 10: 16579.

65.

UK Home Office . Statistics of Scientific Procedures on Living Animals, Great Britain 2014, https://www.gov.uk/government/statistics/statistics-of-scientific-procedures-on-living-animals-great-britain-2014 (2015, accessed 24 January 2024).

66.

Beauchamp

DeGrazia

. Principles of animal research ethics. Oxford: Oxford University Press, 2020, 168 pp.

67.

DeGrazia

Beauchamp

. Beyond the 3Rs to a more comprehensive framework of principles for animal research ethics. ILAR J 2019; 60: 308–317.

68.

Balls

. It’s time to include harm to humans in harm–benefit analysis — but how to do it, that is the question. Altern Lab Anim 2021; 49: 182–196.

69.

Balls

. Why are validated alternatives not being used to replace animal tests? Altern Lab Anim 2018; 46: 1–3.

70.

Balls

Combes

. Questionable progress in the application of the Three Rs to improve science, human well-being and animal welfare. Altern Lab Anim 2018; 46: 55.

71.

Balls

. Animal experimentation and alternatives: Time to say goodbye to the Three Rs and hello to humanity. Altern Lab Anim 2014; 42: 327–333.

72.

Balls

Combes

. Promoting the Three Rs: Actions speak louder than words. Altern Lab Anim 2000; 28: 741–742.

73.

NC3Rs . Research review 2019, https://nc3rs.org.uk/sites/default/files/2021-09/NC3RsResearchReview2019.pdf (2021, accessed 24 January 2024).

74.

Taylor

Modi

Bailey

. Recent trends in the use of New Approach Methodologies (NAMs). In submission.

75.

Lee

Kang

. The ‘R’ principles in laboratory animal experiments. Lab Anim Res 2020; 36: 45.

76.

Ogden

. Principles of animal research: Replacement, reduction, refinement, and responsibility. Animal L 1996; 2: 167.

77.

Fentem

. Conference report: COLIPA symposium on alternatives to animal testing. Altern Lab Anim 1996; 24: 119–121.

78.

Hartung

De Vries

Hoffmann

, et al. Toward good in vitro reporting standards. ALTEX 2019; 36: 3–17.

79.

Bracher

Pilkington

Hanemann

, et al. A Systematic Approach to Review of in vitro Methods in Brain Tumour Research (SAToRI-BTR): Development of a preliminary checklist for evaluating quality and human relevance. Front Bioeng Biotechnol 2020; 8: 936.

80.

United Nations Department of Economic and Social Affairs . Sustainable Development Goal 3: “Ensure healthy lives and promote well-being for all at all ages”, https://sdgs.un.org/goals/goal3 (accessed 17 March 2023).

81.

Rohlf

Bennett

. Perpetration-induced traumatic stress in persons who euthanize nonhuman animals in surgeries, animal shelters, and laboratories. Soc Anim 2005; 13: 201–219.

82.

LaFollette

Riley

Cloutier

, et al. Laboratory animal welfare meets human welfare: A cross-sectional study of professional quality of life, including compassion fatigue in laboratory animal personnel. Front Vet Sci 2020; 7: 114.

83.

Thurston

Chan

Burlingame

, et al. Compassion fatigue in laboratory animal personnel during the COVID-19 pandemic. J Am Assoc Lab Anim Sci 2021; 60: 646–654.

84.

Mike

Hazem

. Animal researchers shoulder a psychological burden that animal ethics committees ought to address. J Med Ethics 2022; 48: 299.

85.

Grimm

Suffering in silence. Science 2023; 379: 974–977.

86.

Bateson

When to experiment on animals. New Sci 1986; 109: 30–32.

87.

Alliance for Human Relevant Science . Bringing back the human: Transitioning from animal research to human relevant science in the UK, https://www.humanrelevantscience.org/wp-content/uploads/APPG-report-March-2022.pdf (2022, accessed 24 January 2024).

88.

Alliance for Human Relevant Science . Accelerating the growth of human relevant sciences in the UK, https://www.humanrelevantscience.org/wp-content/uploads/Accelerating-the-Growth-of-Human-Relevant-Sciences-in-the-UK_2020-final.pdf (2020, accessed 24 January 2024).

89.

Rawle

. The role of review and regulatory approvals processes for animal research in supporting implementation of the 3Rs. A report by Dr Frances Rawle, commissioned by the NC3Rs , https://nc3rs.org.uk/role-review-and-regulatory-approvals-processes-animal-research-supporting-implementation-3rs-2023 (2023, accessed 24 January 2024).

90.

Hutchinson

Owen

Bailey

. Modernizing medical research to benefit people and animals. Animals (Basel) 2022; 12: 1173.

91.

Centre for Economics and Business Research (Cebr) . The economic impact of the UK’s New Approach Methodologies sector. A Cebr report for Animal Free Research UK, https://www.animalfreeresearchuk.org/wp-content/uploads/2021/09/Animal-Free-Research-UK_Economic-Report-2.pdf (2021, accessed 24 January 2024).

It’s Time to Review the Three Rs,to Make them More Fit for Purpose in the 21st Century

Abstract

Keywords

An overview of the Three Rs and the case for change

The case for change

Should we consider the replacement or refinement of the Three Rs themselves?

The ‘humanity’ criterion

The way forward

The issue of funding and investment

Cause for optimism

Conclusions

Footnotes

Acknowledgments

Declaration of Conflicting Interests

Funding

References