Emerging Topics in Life Sciences (2019) 3 675–679

[Link]

Perspective

Animal experimentation: implementation and

application of the 3Rs
David I. Lewis
School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, U.K.
Correspondence: David I Lewis ([Link]@[Link])

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Despite the development of powerful molecular biological techniques and technologies,
studies involving research animals remain a key component of discovery biology, and in
the discovery and development of new medicines. In 1959, The Principles of Humane
Experimental Technique, the 3Rs (Replacement, Reduction and Refinement) were devel-
oped to provide a framework to ensure animal research was undertaken as humanely as
possible. Sixty years since their inception, the extent to which the 3Rs have been
adopted and implemented by the global scientific and medical research communities has
unfortunately been slow and patchy. However, this situation is changing rapidly as aware-
ness increases, not only of the 3Rs themselves, but of the impact of animal welfare on
the reproducibility, reliability and translatability of data from animal studies.

Introduction
The earliest records of the use of animals for scientific and medical research are from the fourth and
third centuries BC. Since then, the number of animals used for research worldwide has progressively
increased, particularly in the 18th and 19th centuries, a consequence of the dramatic increase in scien-
tific endeavours during this period. Despite the development of powerful molecular biological techni-
ques and technologies, rightly or wrongly, studies involving research animals, undertaken in parallel
with complimentary, non-animal experimental approaches, remain a key component of discovery
biology, and in the discovery and development of new medicines.
Animal experiments, by their very nature and purpose, will cause pain, suffering and distress, and
in many cases, the death, of the research animal. Morally and ethically, it is critical to minimise these
harms and to undertake these studies as humanely as possible. In short, to apply The Principles of
Humane Experimental Technique, the 3Rs, first articulated by Russell and Birch in 1959 [1]. Sixty
years on, this article does not seek to argue for or against the use of animals for research. Instead, to
reflect on the extent to which these Principles have been adopted, applied and developed by the global
research community.

What are the principles of humane experimental

technique, the 3Rs? [2]
• Replacement: Methods which replace the use of animals in research. These may be absolute i.e. no
use of animals or animal tissues at all (e.g. studies involving human participants, computer simula-
tions), or partial, i.e. the use of tissues or cells from humanely killed animals. In some jurisdictions,
for example the European Union, replacement could include replacement of protected species with
Received: 22 August 2019 non-protected species (e.g. immature vertebrates or invertebrates).
Revised: 2 October 2019 • Refinement: Methods which minimise the pain, suffering or distress of the research animal (e.g.
Accepted: 7 October 2019
administration of analgesics and anaesthetics, housing animals in social groups with appropriate
Version of Record published: enrichment of their environments to allow them to express natural behaviours, monitoring of
22 October 2019 welfare before, during and after experiments).

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 Emerging Topics in Life Sciences (2019) 3 675–679
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• Reduction: Minimising the number of animals required per study (e.g. making multiple measurements, and
at multiple time points, from the same animal), whilst maintaining robust experimental design to enable the
appropriate statistical analysis of the data. Alternatively, power calculations to calculate the optimal number
of animals required, although this does require pilot data or access to data from previous studies.
Since their introduction, additional Rs have emerged globally. The ‘Reuse’ of individual animals in further
studies where these studies cause no additional suffering for the animal, the ‘Rehabilitation’ or aftercare of the
research animal, a legal requirement in India, and in South Africa, the requirement to demonstrate ‘Respect’
for the animal. There is also Relevance, the need to use a specific species if, for example, you wish to explore
that particular species’ physiology, behaviour or neurobiology, or for the purpose of conservation biology,
where replacement with less sentient species, cells or computer modelling approaches would not allow you to
address these scientific questions [3].

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Which R should take precedence?
Without question, the Replacement of animals with non-animal alternatives has to be the ultimate goal of all
those involved in animal experimentation. Any other position is morally indefensible. Whilst considerable
resources are being devoted to the development and validation of non-animal alternatives, unfortunately there
are still many scientific questions that can only be answered using research animals. If there is no alternative to
animal use, opinions on whether Refinement or Reduction takes precedence vary. In some countries, particu-
larly in the Emerging World, Reduction comes before Refinement, a consequence of limited resources, pressure
from ill-advised ethical review bodies [4,5, personal communications]. The result: studies that are under-
powered, with insufficient numbers of animals used to enable appropriate statistical analysis of the data
obtained to be undertaken. The data obtained is meaningless and the animals have suffered and/or died need-
lessly. We are the voice of the research animal; we should consider it from the perspective of an individual
animal, and their individual, specific suffering. For me there is no debate, Refinement has to take precedence
over Reduction. If suffering is unavoidable, it is preferable to cause limited suffering to a number of animals
rather than a large amount of suffering to an individual animal. Furthermore, multiple small refinements, for
example how animals are housed, handled, the presence of stimulatory environments/objects in their enclosure,
when provided together, can have a considerable, cumulative impact in reducing animal suffering and distress.

Adoption of the 3Rs globally

Sixty years on since they were first articulated, the extent to which the 3Rs have been adopted and implemented
by the global scientific and medical research communities has unfortunately been slow and, at best, patchy or
varied, both across continents and between research areas.
Globally, until the publication of the book ‘Alternatives to Animal Experiments’ in 1978 [6], there was very
limited awareness and therefore adoption of these Principles. With significant investment in the development
and validation of alternatives, particularly by global Pharma, the creation of national 3Rs Centres, and legisla-
tive changes, the situation is changing, particularly in Europe. Elsewhere in the world, progress is less forth-
coming. There is still a considerable amount of work to be undertaken, both in raising awareness in those
countries where knowledge of the 3Rs is currently limited, and in promoting their adoption and application by
the entire global research community.
Critical to this is the enshrinement within robust ethical review processes and law, and the establishment of
national 3Rs initiatives and centres. Many countries do not have research animal welfare legislation, animal
ethics committees (Institutional Animal Care and Use Committees) or indeed a legal requirement for the
ethical review of animal studies [7]. Research, including animal research, is a global endeavour. There needs to
be greater harmonisation, globally, of research animal welfare legislation and ethical review processes similar to
that that exists for human clinical trials. There also needs to be a mandatory, legally enforced focus on the
application of the 3Rs for all animal studies.

Adoption and application of each R

Replacement
There has been some notable recent successes in the development of non-animal alternatives, for example the
development of ‘Organ on a Chip’ technologies and the widespread use of databases for the toxicological evalu-
ation of potential new medicines. Indeed, the U.K. Governments strategy is to become the world leader in the

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Emerging Topics in Life Sciences (2019) 3 675–679
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development of alternatives over the next 25 years [8]. However, despite the considerable effort and resource
being devoted globally to the development of alternatives, there is considerable reluctance amongst the research
community to adopt them, the so called ‘3Rs valley of Death’- development but limited uptake or use. The
reasons for this lack of uptake could be many: a lack of awareness amongst the research community; an unwill-
ingness to let go of, or acknowledge, the limitations of animals preparations/techniques on which they have
published extensively; limited published historical or background data from these alternatives; minimal external
pressure to change. Driving forward replacement is going to take a concerted and more forceful effort from all
involved; Ethical review bodies, funders, regulatory authorities, journals, raising awareness but also refusing to
fund, approve or publish studies involving animals unless the researcher fully justifies why animals have to be
used.
Our goal is to replace animals but with what? Replacement of vertebrates with non-animal alternatives, for
example, computer simulations and ethically sourced human cell cultures, does not raise additional ethical

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issues, but what about with animals of lower sentience, those thought to be less able to perceive pain e.g. flies,
insects, worms? Most jurisdictions that place restrictions on species require the use of the species of lowest sen-
tience provided it is capable of addressing the scientific question under investigation [7]. However, it is becom-
ing increasingly apparent that invertebrates, particularly insects, exhibit aversive responses that do not meet our
current ‘criteria’ for a response to painful stimuli [9,10]. Is it morally justifiable to provide protection for one
species but not another? Should we treat all living creatures equally? In India, animal welfare legislation pro-
vides protection for all living beings, including insects, except man.

Refinement
There is an increasing realisation that animal welfare, the minimising of pain suffering and distress, can have a
substantial impact on the reproducibility and reliability of the data obtained [11]. The data from the majority
of pre-clinical animal studies is not reproducible in humans [12]. Morally, should we continue with such
studies if the data obtained cannot be trusted?
Until recently, it was believed that standardisation of everything was the answer. However, it is becoming
increasingly apparent that this is not the case; housing, environment, handling, diet, gut microbiome and many
other factors can have a substantial impact on the data obtained, and its reproducibility [13,14]. Animals, like
humans, are a heterogeneous population, living beings rather than research tools; we should view them as such
if we want to increase the validity of animal studies. We also need to adopt many of the practices involved in
clinical research: randomisation of studies, double blinding, multicentre studies, minimisation of bias and
robust experimental design [15]. Without these interventions, data is only reproducible in the laboratory in
which it was gathered at that particular moment in time.
There is a need to raise awareness in the research community of all the known factors that could influence
the reproducibility and reliability of their data, for research into the impact of each intervention on experimen-
tal outcomes, and greater communication and collaboration between researchers and animal welfare experts,
and the sharing and promotion of each other’s 3Rs resources. Increased use of PREPARE guidelines [16] in
designing studies, and a requirement to fully detail (as per the ARRIVE [17] or similar guidelines) all welfare
interventions in publications should be in place. Researchers should treat with caution and question the validity
of any data where the 3Rs have not been applied or fully described.
Current initiatives, led by animal welfare organisations, to abolish studies that cause severe suffering to
animals [18] or the use of procedures, such as the forced swim test [19,20], which have questionable validity or
translatability to human disorders, should be encouraged and implemented. Similarly, there should be more
non-regulatory expert working groups working collaboratively to develop recommendations to refine existing
animal models or procedures in specific areas of research, for example animal models of sepsis and septic
shock [21], in order to reduce animal use and suffering.

Reduction
The number of animals used for scientific and medical research worldwide is unknown; most countries do not
keep records. Animal activist organisations estimate that over 100 million animals may be used worldwide
every year. Because of the lack of records, nobody knows whether this figure is increasing or decreasing, and
therefore whether researchers are implementing the principle of reduction.
In the U.K., one of the few countries to collate and publish the numbers of research animals used annually
within its jurisdiction from the late 1930s to the present day, the numbers have cycled between 1 million and

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 Emerging Topics in Life Sciences (2019) 3 675–679
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5.5 million [22]. Whilst the number of wild-type or non-genetically altered animals is falling, which is to be
encouraged, the total number of procedures on animals in the U.K. has increased over the last 15 years, plat-
eauing at the current level of ∼4 million per year [22]. This increase in numbers is due to the creation, breed-
ing, but not necessarily use, of genetically altered animals or animals with a harmful spontaneous genetic
mutation, whereas the number of wild-type animals used is falling [22]. Genetically altered animals, which are
bred but not used, are included as procedures in both U.K. and EU statistical reports. In addition to counteract-
ing the principle of reduction, this increased creation, breeding and use of genetically altered animals raises
other significant ethical issues: the excessive wastage of animals, the increased likelihood of them experiencing
suffering including severe suffering, whether they are accurate models of human disease to name but a few.

The way forward

Whilst the 3Rs are increasingly being implemented by the global research community, for the sake of the

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research animals and the validity of the ensuing data, there needs to be far greater momentum and impetus.
There has to be far greater raising of awareness, globally, of the 3Rs and their impact, mandatory education,
training and continuing professional development, and increased collaboration between all involved students,
early career and established researchers, vets, animal caretakers and technologists, animal welfare experts and
animal ethics committee members. There needs to be more funding for 3Rs interventions, greater sharing and
promotion of 3Rs resources including those developed by other agencies or bodies, the global harmonisation of
animal welfare legislation and the strengthening of ethical review processes. Collectively, we should all be striv-
ing to go beyond what is legally required, developing and fostering a ‘Culture of Care’ [23,24], a commitment
to improving animal welfare, scientific quality, care of staff and transparency for stakeholders, within our orga-
nisations and practices when animals have to be used.

Competing Interests
The Author declares that there are no competing interests associated with this manuscript.

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678 © 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society and the Royal Society of Biology