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3 Rs Of Research Papers

3Rs

Replacement, refinement and reduction

The MRC plays an active role in developing and disseminating the principles of the 3Rs (replacement, refinement and reduction):

Replacement

Replacement refers to methods that avoid or replace the use of animals defined as 'protected' under the Animals (Scientific Procedures) Act 1986 (ASPA) in an area where they would otherwise have been used. 'Protected' animals are all living vertebrates -except man and cephalopods, such as the octopus

Replacement methods can be absolute replacements - techniques which do not involve animals at any point, such as computer modelling, in vitro methodologies (e.g. tissue engineering), or human volunteers - or relative replacements, which avoid or replace the use of 'protected' animals with organisms not protected under ASPA, such as Drosophila (flies) or worms.

Growing 'mini-brains' from stem-cells
An example of Replacement in practice

Dr Madeline Lancaster and her team at MRC Laboratory of Molecular Biology employ cerebral organoids, also known as ‘mini brains’, grown from pluripotent stem cells to model human brain development in vitro. Using in vitro models has the potential to capture the intricacies of the human brain more accurately than animals, which do not have the anatomical and functional complexity of human brains. The challenge with using in vitro models has been to model the whole brain and its complex functions, rather than single cell types or processes individually. Using brain organoids is a way of overcoming this.

The process for growing these 3D tissues was first described in a paper by Dr Lancaster and colleagues published in Nature in September 2013. Her team has previously focussed on modelling neurodevelopmental disorders, such as microcephaly, a disorder characterized by a significantly reduced brain size (some cases may be associated with Zika virus infection). Her work is currently concentrated on other neurodevelopmental disorders such as autism and intellectual disability, by introducing mutations seen in these disorders into the cells used to make the organoids. This will give us a further understanding of the role these mutations play in the development of these conditions. The cerebral organoids are a major step towards reducing reliance on animals in studying neurological diseases and the development of new treatments, and have already been up taken by 16 other research laboratories. The paper authored by Dr Lancaster, who has recently given a TEDx talk about her research, won the 2015 NC3R’s 3Rs Prize for outstanding published research with 3Rs impact. 

Refinement

Refinement refers to improvements to scientific procedures and husbandry which minimise actual or potential pain, suffering, distress or lasting harm and/or improve animal welfare in situations where the use of animals is unavoidable. It applies to the lifetime experience of the animal. There is evidence that refinement not only benefits animals, but can also improve the quality of research findings.

Post-surgical care
An example of Refinement in practice

Dr Simon Milling’s lab at the University of Glasgow aim to better understand the adaptive immune responses against infections in the intestine, to help manage inflammatory bowel diseases and food allergies. It is thought that specialised dendritic cells, which travel from the intestine to the lymph nodes, are important in mediating these responses, but they can often only be obtained via surgical procedures. In addition to replacing the use of larger animals to surgically collect these cells, Dr Milling’s team have worked hard to refine the surgical cannulation technique in mice. Improvements in the surgical management of the mice have enabled larger volumes of lymph to be collected after surgery, and improved post-operative procedures and the use of a thoracic harness have enabled cannulated animals the full range of normal movement, within their normal cages. This also prevents the need for restraint in the post-surgical period. The three published papers using these refined techniques have been cited over 100 times in the last three years, and staff from laboratories in the UK, Sweden, and the USA have been trained to use the refined procedures.

Project reference G0900270

The Mouse House
An example of Refinement in practice

All mice like to create nests for sleeping in, giving birth and as somewhere to feel safe and secure from predators. However, animal technicians and scientists also need to check and observe the animals with the least amount of disturbance. Following work carried out by the National Institute for Medical Research (NIMR), the Medical Research Council (MRC) developed a red tinted plastic shelter as a means of providing an environmental enrichment strategy for mice.

Mice lack the ability to distinguish red from black, behaving the same in red light as they do in darkness (Spalding, J. (1969). Influence of the Visible Colour Spectrum on Activity in Mice. Laboratory Animal Care. Vol 19, No1, 50-54). After testing a range of red photographic filters, the final mouse house, launched in 2001, was made from red tinted plastic that has a wavelength of 593nm. This allows the mice to see some light through it and perceive shadows and movement. The suggestion made was that mice benefit from having a retreat that offers some ability to detect the presence of an external predator, a natural behaviour expressed even in the laboratory environment. The mouse house is used in many different countries improving the welfare of many thousands of laboratory mice.

Reduction

Reduction refers to methods which minimise animal use and enable researchers to obtain comparable levels of information from fewer animals or to obtain more information from the same number of animals, thereby reducing the future use of animals.

Single cell modifications
An example of Reduction in practice

Dr Gavin Kelsey and his team at the Babraham Institute use genetically altered strains of mice to investigate how environmental factors, such as diet and early life experiences, alter epigenetic programming and contribute to disease development later in life. A novel method, developed and published by Dr Kelsey’s team in 2014, allowed profiling of these epigenetic modifications in single cells (Smallwood et al. Nat. Methods 2014). Therefore, sufficient cells can be obtained from a small number of mice, meaning that larger groups of animals are no longer required for profiling experiments, thereby reducing the numbers of mice used in the study very substantially.

Publication of this this new profiling method has gleaned significant interest from the community and Dr Kelsey’s team plan to work to implement this method with national and international collaborators.

Project reference: MR/K011332/1

The National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs)

The MRC is a major funder of The National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), a scientific organisation which leads the discovery, development and promotion of new ways to replace, reduce and refine the use of animals in research and testing (the 3Rs). The NC3Rs is the UK’s major sponsor of 3Rs research.  The NC3Rs have issued various guidance documents, and provide an extensive library of 3Rs resources. These can be found here.

3Rs impact in MRC funded research

Currently, about one third of MRC–funded research programmes involve the use of animals under the Animals (Scientific Procedures) Act. Each year, the MRC collects information from its researchers on how they have implemented 3Rs in their work via the evaluation tool, Researchfish. Some examples of how researchers implement the 3Rs in practice include:

  • Replaced some animal use with alternative technique
  • Reduced number of animals required (e.g. improved experimental design or statistical analysis)
  • Refinement or development of experimental techniques or procedures to improve animal welfare
  • Improved housing, including environmental enrichment
  • Substitution by a species of lower neurophysiological sensitivity
  • Shared use of tissues, organs or other material.
  • Changes resulting in downgrading of severity limits for procedures/protocols
  • Avoidance of specific procedures or adverse effects
  • Other

Most awards (59%) implement more than one of these measures. The most frequently reported implementation measure is ‘Reduced number of animals required’ (34%) followed by ‘Replaced some animal use with alternative technique’ (21%) and ‘Avoidance of specific procedures or adverse effects’ (15%).
 
Around 17% of MRC-funded researchers proposing to use animals in their research reported making additional changes to further reduce, refine or replace animal use during the course of their project. Furthermore, around 12% were able to refine or develop methodology with 3Rs impact that could be shared/adopted by others.

Joanne Zurlo, Deborah Rudacille, and Alan M. Goldberg
Article reprinted from "Environmental Health Perspectives,"
 August 1996, vol. 104, no. 8


Laws that mandate replacement alternatives, reduction alternatives, and refinement alternatives (the Three Rs) in scientific research have been passed in the United Kingdom, Germany, the Netherlands, the United States, and the European Union over the past decade. Full implementation of this newly developed legislation depends upon scientists' ability to understand animal welfare issues and to accept the legitimacy of the public's interest in the conduct of science.

The European Centre for the Validation of Alternative Methods (ECVAM) established by the European Commission in 1991 to promote the scientific and regulatory acceptance of alternative methods, recently sponsored a workshop to discuss the current status of the Three Rs and to make recommendations aimed at achieving greater acceptance of the concept of humane experimental technique.

Twenty-one scientists professionally committed to the Three Rs were invited to attend the conference, which was chaired by Michael Balls, head of ECVAM, and Alan M. Goldberg, director of the Johns Hopkins Center for Alternatives to Animal Testing (CAAT). The conference was held in Sheringham, Norfolk, UK on May 30, 1995-June 3, 1995. A report based on the conference was published by ECVAM in December 2, 1995.

Origins of the Three R's

The Three R's originated in a proposal made in 1954 by Charles Hume, founder of the Universities Federation for Animal Welfare (UFAW) that the UFAW should undertake a scientific study of humane technique in laboratory animal experiments. The project was managed by a committee under the chairmanship of Sir Peter Medawar, the Nobel prize-winning immunologist, with William Lane-Petter, Secretary of the Research Defense Society of Great Britain, among its members. Christine Stevens, founder of the Animal Welfare Institute (AWI) in the U.S., provided financial support for the project. W.M.S. Russell, a zoologist, and R.L. Burch, a microbiologist, were appointed to carry out the work, which led to the publication of the book The Principles of Humane Experimental Technique in 1959.

At the time of the book's publication, Charles Hume said:

  • "This deserves to become a classic for all time and we have great hope that it will inaugurate a new field of systematic study. We hope that others will follow up the lead it has given, and that a generalized study of humane technique, as a systematic component of the methodology of research, will come to be considered essential to the training of a biologist."

Hume's predictions regarding the book's impact have been realized as the concepts of replacement alternatives, reduction alternatives, and refinement alternatives have become established in law. However, at the present time, a thorough working knowledge and acceptance of the principles of humane experimental technique among scientists in general remains at best elusive and at worse ignored.

Scientific and Ethical Justification

Current legislation in Europe and the United States decrees that all proposed use of laboratory animals should be subject to review to determine whether such use appears to be scientifically and ethically justifiable. Individually and collectively, such laws not only recognize Russell and Burch's concept but place legal and moral obligations on all concerned to replace, reduce and refine laboratory animal experimentation wherever possible.

The degree to which proposed animal use is reviewed varies from country to country. For example, in the United Kingdom, a working party of the Institute of Medical Ethics concluded that a project using animal subjects should only be done when the review committee ascertains that the aim of the project is worthwhile, that they use the minimum number of animals necessary, and that the investigators document that they have adequately considered alternatives to any procedure that causes more than momentary pain or distress (either with or without the use of anesthetics). Guidelines for searching for alternative procedures have been prepared to assist investigators and IACUC members in these considerations.

Reduction Alternatives

The term reduction alternatives describes methods for obtaining comparable levels of information from the use of fewer animals in scientific procedures or for obtaining more information from a given number of animals so that, in the long run, fewer animals are needed to complete a given research project or test. The greater the number of animals used, the greater will be the overall costs in terms of animal suffering. Therefore, the number of animals used should be the minimum that is consistent with the aims of the experiment.

There is evidence that poor experimental design and inappropriate statistical analysis of experimental results leads to inefficient use of animals and scientific resources in toxicological research (5,6). Previous studies of statistical methods used in other areas of biomedical research reveal similar findings (7). In some cases, the level of statistical expertise appears to be so low that investigators are either unaware of the potential value of obtaining statistical advice, or they are unable to obtain appropriate statistical advice because there are so few biometricians with experience in their field of interest.

A basic understanding of experimental design and statistics is necessary for all scientists. For investigators with no previous training in statistics, this level of expertise can probably be obtained from an introductory course. There are many texts on statistical methods, which can be used for both learning purposes and as reference books. Biomedical research workers should have more detailed training in biometrics and statistics so that they can act as consultants to other investigators in their own institutes.

Refinement Alternatives

Refinement alternatives encompass those methods that alleviate or minimize potential pain and distress and enhance animal well-being. Distress is an aversive state in which an animal is unable to adapt completely to stressors and the resulting stress and, therefore, shows maladaptive behavior. The stressors may induce physiological, psychological, or environmental stress. Pain results from potential or actual tissue damage, such as that caused by injury, surgery, or disease, and can lead to distress (8-10).

Much potential pain and distress can be avoided or alleviated with the proper use of anesthetics, analgesics, and tranquilizers. This critical component of any comprehensive program of veterinary care provides for frequent observation of the animals by trained veterinary staff to detect and relieve pain and distress. However, a substantial number of animals used in research and testing experience unrelieved pain and distress.

At present, we do not have a convenient and standardized way of objectively assessing animal pain and distress. Rather, the assessment is generally based on subjective clinical signs of abnormal behavior and appearance. Although the implementation of refinement alternatives depends largely on the ability of scientists to observe and understand the behavior and needs of laboratory animals, many experimenters are as lacking in ethological knowledge as they are in statistical training. The best approach to pain and distress is to assume that a procedure that inflicts pain and distress in humans will inflict at least as much pain and distress in animals unless there is evidence to the contrary.

Very little research funding is available to support efforts to investigate and refine experimental techniques and scientific procedures. Furthermore, there is no readily available up-to-date knowledge base on refinement. Techniques that are developed to refine a procedure are frequently not reported in the scientific literature or are established simply as standard operating procedures (SOPs) within an institution.

To establish best practice and to advance the implementation of refinement alternatives, it is important to share such experience, data, and SOPs. Sharing of data and theories is normally accomplished via the scientific literature, but there has been a marked lack of opportunity to discuss and provide information on refinement alternatives in the main biological journals. Consequently, scientists are not sufficiently aware of the concept of refinement alternatives and in general do not recognize the importance of refinement in their research.

The concept of recognizing, minimizing, and eliminating pain and distress in laboratory animals should be included in training programs for all persons involved in the care and use of laboratory animals. Details of refinement and animal welfare considerations should routinely be included in scientific papers and publications.

Replacement Alternatives

Replacement alternatives encompass those methods that permit a given purpose to be achieved without conducting experiments or other scientific procedures on animals. Russell and Burch distinguished between relative replacement e.g. the humane killing of a vertebrate animal to provide cells, tissues, or organs for in vitro studies and absolute replacement in which animals would not need to be used at all, e.g. the culture of human invertebrate cells and tissues.

The range of replacement alternative methods and approaches includes the improved storage, exchange, and use of information about previous animal experiments to avoid unnecessary repetition of animal procedures; use of physical and chemical techniques and predictions based upon the physical and chemical properties in molecules; use of mathematical and computer models; use of organisms with limited sentience such as invertebrates, plants and microorganisms; use of in vitro methods including subcellular fractions, tissue slices, cell suspensions, and perfused organs; and human studies including use of human volunteers, postmarketing surveillance, and epidemiology.

In many areas of the biomedical sciences, in vitro methods are increasingly used as the methods of choice in place of animal studies, not because they provide precisely the same information, but because they offer the best scientific approach.

Russell and Burch (2) discussed the relative merits of fidelity and discrimination models, noting that high-fidelity models, as exemplified by the use of rodents and other laboratory mammals in toxicity testing, are used because, in their general physiological and pharmacological properties, they are similar to humans. High discrimination models, on the other hand, "reproduce one particular property of the original, in which we happen to be interested." (2)

Russell and Burch warned of the high-fidelity fallacy and of the danger of expecting discrimination in particular circumstances from models that show high fidelity in other, more general terms--a prediction illustrated by other more recent analyses of the differing molecular responses to certain chemicals by the rat, the mouse, and the human. Russell and Burch pointed out that the fidelity of mammals as models for man is greatly overestimated; however, replacement alternatives methods must be based on good science, and extravagant claims that cannot be substantiated must be avoided.

The development and acceptance of replacement alternatives for both research and testing must be based on a sufficient understanding of the molecular and cellular mechanistic basis of what is being studied or measured i.e. on sound science.

Education and Training

The successful implementation of the Three Rs depends upon the education and training of those involved in research and testing. Education is defined as the didactic presentation of the information and theories of animal use that will contribute to the development of proper attitudes toward the use of animals in scientific procedures. Training is defined as the acquisition of practical knowledge and skill directly associated with animal handling and procedures.

The objective of the education and training is to provide sufficient information to allow scientists to conduct animal procedures to high standards of both science and animal welfare, following proper evaluation of the scientific and ethical considerations that should govern the use of laboratory animals.

Course-work should contribute to a scientist's ability to design experiments properly and to plan research strategies, to become competent in animal handling and the performance of scientific procedures, to make decisions with regard to the ethics of using animals in experiments, and to determine whether alternatives are available.

The Way Forward

The use of the term alternatives to encompass all of the Three Rs is now widely accepted in many countries, enshrined in legislation, and incorporated into the names of various centers throughout the world. However, some scientists see its use as being driven by political and social forces rather than by scientific issues. This is partly due to a lack of appreciation of the basis of the Three Rs concept as proposed by Russell and Burch (i.e. that scientific excellence and the greatest humanity in the use of laboratory animals are inextricably linked). It also stems from a defensive attitude among some scientists, perhaps resulting from the campaigns of some anti-vivisection organizations and from insufficient dialogue among the scientific and animal protection communities.

In the mid-1990's, the question we face is whether there will be a revolution in thinking and practice, which is what is needed if the principles of humane experimental technique are to be brought fully and effectively into operation. Much has been achieved, but there is still considerable room for progress and improvement.

The Sheringham workshop participants propose several general recommendations:

  • Existing laboratory animal protection laws should be fully implemented.
  • All countries should have a legal framework that actively incorporates the Three Rs into all animal-based research, testing, and education.
  • There should be formal and informal mechanisms for the education and training of academic, industrial and government scientists and officials in the Three Rs to ensure compliance with the spirit and letter of laboratory animal protection legislation and regulations.
  • There should be international discussion and agreement on what levels of animal suffering should not be permitted in any circumstances, regardless of any likely or potential benefits.
  • It is unacceptable to export scientific work involving laboratory animals to avoid scientifically realistic, but more stringent, animal welfare codes.

The participants of the Sheringham workshop unanimously reaffirmed the principles put forth by Russell and Burch that humane science is good science and that this is best achieved by vigorous application of the Three Rs. The only acceptable animal experiment is one that uses the smallest number of animals and causes the least possible pain or distress, is consistent with the achievement of a justifiable scientific purpose, and is necessary because there is no other way of achieving that purpose.

Any proposed experiment on animals should be subjected to prior and effective expert review by an ethics committee. Scientists should be better informed about the Three Rs concept and should be encouraged to see it as an opportunity for reaping benefits of every kind--scientific, economic, and humanitarian. Only in this way can the aspirations of all those who have worked for the good of both human and animal welfare be achieved at last.

References

  1. Balls M, Goldberg AM, Fentem JH, Broadhead CL, Burch RL, Resting MFW, Frazier JM, Hendriksen CFM, Jennings M, van der Kamp MDO (1995). The Three Rs: the way forward. ATLA23:838-866.
  2. Russell WMS, Burch RL. The principles of humane experimental technique. London: Metheun, 1959.
  3. Howard-Jones N (1985). A CIOMS ethical code for animal experimentation. WHO Chron29(2):51-56.
  4. Stokes WS, Jensen DJB (1995). Guidelines for institutional animal care and use committees: consideration of alternatives. Contemp Top Lab Anima Sci34(3):51-60.
  5. Festing MFW (1992). The scope for improving the design of laboratory animal experiments. Lab Anim26:256-267.
  6. Festing MFW (1994). Reduction of animal use: experimental design and quality of experimetns. Lab Animals28:212-221.
  7. Altman DG (1982). Statistics in medical journals. Stat Med1:59-71.
  8. Institute of Laboratory Animal Resources. Recognition and alleviation of pain and distress in laboratory animals. Washington D.C.: National Academy Press, 1992.
  9. Flecknell PA (1994). Refinement of animal use--assessment and alleviation of pain and distress. Lab Anim28:222-231.
  10. Morton DB. Recognition and assessment of adverse effects in animals. In: Animals in science conference: perspectives on their use, care and welfare; Proceedings, April 1995. Research Ethics Unit (Johnston NE, ed). Clayton, Vic., Australia: The Unit, 1995; 157-167.
  11. van Zutphen LFM, Baumans V, Beynen AC, eds. Principles of laboratory animal science. A contribution to the humane use and care of animals and to the quality of experimental results. Amsterdam: Elsevier, 1993.
  12. Wilson MS, Berge B, Maess J, Mahony G, Natoff I, Nevalainen T, Van Zutphen LFM, Zaninelli P (1995). FELASA recommendations on the education and training of persons working with laboratory animals: categories A and C: reports of the Federation of Laboratory Animal Science Associations Working Group on Education accepted by the FELASA Board of Management. Lab Animal29:121-131.
  13. National Research Council. Education and training in the care and use of laboratory animals: a guide for developing institutional programs. Washington, D.C: National Academy Press, 1991.