Category Archives: Michael Balls

The Factors Governing Progress

Progress toward implementation of the Three Rs is affected by a
complexity of personality and sociological factors, recognised in
The Principles in 1959, but no less effective in the 21st century

Michael Balls

One of the most challenging, but not totally discouraging, chapters in The Principles of Humane Experimental Technique is Russell and Burch’s Chapter 8: The Factors Governing Progress.1

Their main distinction is between individual personality factors and sociological factors, the first of which are dealt with somewhat summarily, with reference to “two pathological [sic] personality variables known to be important in determining attitudes to, and therefore treatment of, animals”. The first of these is the authoritarian factor, “now known to correlate significantly with hostile attitudes to animals, as well as stereotyped preferential treatment of particular species”. The second, “tentatively called the revolutionary factor”, finds its main expression in “a rigidly and fanatically anti-vivisectionist attitude”.

The positions of anti-vivisectionists and the organisation they manage or support are relatively transparent, but a discussion on the degree to which pro-vivisectionists and the organisations they manage or support are authoritarian in a pathological sense is a path down which I will not tread.

One of the great advantages of the Three Rs is the opportunity they provide for cooperation in the middle ground between the extremes and biases of the most trenchant anti- or pro-vivisection positions. This middle ground is very crowded these days, but only when we see what people and organisations actually do can we know whether their commitment to the Three Rs is genuine, or merely a convenient ploy to disguise their true authoritarian or revolutionary ambitions. One intriguing point made by Russell and Burch is that neither type of extremist would be likely to remain or become experimental biologists. Discussion of that point would make a good question in a university finals general examination.

The part of the chapter on the sociological factors begins with a discussion of the relationship between humanity and efficiency, which is where the undoubted power of the Three Rs becomes most effective. Efficiency is seen as “a balance between time, cost and efficacy”, where “refinement will inevitably increase efficacy, and may incidentally entail reduction as well”; reduction “must reduce costs”; and, while replacement may initially involve higher costs in terms of training and equipment, it may “represent a considerable gain in the speed of obtaining results”.

Russell and Burch emphasise that, “in terms of efficacy or yield of information, the advantages of humane experimental technique apply almost universally”, and warn against rationalisation, the mechanism of defence by which unacceptable actions are given acceptable reasons to justify them, while, at the same time, unwittingly hiding the true, but unconscious, motives for them. That reasoning represents a very serious challenge to the pro-vivisection apologists and organisations which operate to defend the use of animal experimentation in biomedical research, in the interests of overcoming the diseases which reduce the length and/or quality of human life.

At this point, Russell and Burch spell out what for me is the absolute highlight of The Principles, saying that it follows logically, that, “If we are to use a criterion for choosing experiments to perform, the criterion of humanity is the best we could possibly invent… The greatest scientific experiments have always been the most humane and the most aesthetically attractive, conveying that sense of beauty and elegance which is the essence of science at its most successful.”

The authors of The Principles then turn to flexibility and communication, beginning by lamenting “the long delay in the application of existing knowledge to the improvement of experimentation”, which they saw as “a sort of rigidity, or inertia — the maintenance of a habit long after information is available for its correction”. They considered this to be due to a lack of communication between research and applied science, and a failure of scientists to pay attention to work published in languages other than their own, but, above all, to “the great curse of modern science — division into specialisations”. The effects of this trend can be harmless, but only if “neutralised by skilled communication of their results by specialists and the provision of a class of avowed synthesisers”.

Russell and Burch say that, to some extent, “failure to make the necessary connections arises at the level of the individual”, but it is “intimately related to the sociological situation”, such as the “gradual growth of awe before experts”. However, “respect for expert specialist knowledge should never become uncritical”. They go on to say that “the problem of interspecialist communication merges into the general one of information retrieval”, and that “we now have far too much information as a species to digest as individuals”.

What strikes me about this first half of Chapter 8, is that, more than 50 years on, the difficulties identified by Russell and Burch in The Principles are still with us today — we are overwhelmed with information, much of which is not critically evaluated before it is dumped on the world, and we are surrounded by experts and specialists. What we need are more avowed synthesisers, capable of broad and lateral thinking.

Professor Michael Balls
c/o FRAME
Russell & Burch House
96–98 North Sherwood Street
Nottingham NG1 4EE UK
E-mail: michael.balls@btopenworld.com

Reference and Note
1 Russell, W.M.S. & Burch, R.L. (1959). The Principles of
Humane Experimental Technique
, xiv + 238pp. London,
UK: Methuen.
The Principles of Humane Experimental Technique is now out of print, but the full text can be found at http://altweb.jhsph.edu/pubs/books/humane_exp/het-toc. An abridged version, The Three Rs and the Humanity Criterion, by Michael Balls (2009), can be
obtained from FRAME.

Replacement

C elegans wormThe total replacement of animal procedures should be our common goal, not least because animal models cannot tell us what we need to know about human conditions and responses

Michael Balls

In The Principles of Humane Experimental Technique,1 Russell and Burch defined a Replacement technique as “any scientific method employing nonsentient material which may in the history of experimentation replace methods which use conscious living vertebrates”. They recognised that, while this definition can be applied readily to plants and microorganisms, “a more difficult question arises when we consider free-living metazoan invertebrates”.

They decided to exclude invertebrates “from consideration as objects of humanitarian concern”, describing their uses as comparative substitution and arguing that “to shed obsessional tears over the fate of these organisms would bring the whole concept of humanity into contempt”.

This may seem rather harsh in today’s animal welfare circles, but it is in line with many current practices involving vertebrates as well as invertebrates, including the use of pesticides, the trawling of fish from the sea, and many agricultural procedures.

Russell and Burch distinguished between absolute replacement, in which vertebrate animals are not required at all, and relative replacement, where animal are still required, but “are exposed, probably or certainly, to no distress at all”.

In the absolute replacement group, they included the use of metazoan endoparasites, higher plants, micro-organisms, and non-living physical and chemical systems. In view of the stance they had taken, they did not mention invertebrates, but nematodes (e.g. Caenorhabditis elegans) and insects (e.g. Drosophila melanogaster) would be regarded as absolute replacements today.

In the relative replacement group, they included “non-recovery experiments on living and intact, but completely anaesthetised animals”, and “experiments where animals are still required, but only to furnish preparations after being painlessly killed”. The last-named category included “work on the isolated cells, tissues, or organs of vertebrates”.

They saw tissue culture as a bridge between relative and absolute replacement, and considered mammalian tissue cultures to be “one of the most important replacement techniques, and indeed one of the most important developments in biology”. Most of us would have no hesitation in agreeing with that.

However, replacement can also be categorised in other ways. For example, a distinction can be made between partial replacement, where animals are subjected to regulated procedures as part of the programme of work, and total replacement, where animals are not subjected to regulated procedures at all. Partial replacement might involve the exposure of an animal to a toxic chemical, after which it is killed and its cells or tissues are subjected to further tests in vitro (i.e. ex vivo). By contrast, where all the experimental procedures are applied to cells or tissues in vitro, this would be classed as total replacement.

Nevertheless, in my opinion, one of the most important distinctions is between direct replacement and indirect replacement. In the former case, a replacement technique is used to give results which are directly comparable with those which would be obtained by the animal procedure that is being replaced.

A classical example of direct replacement is a test for irritancy, based on the application of chemicals to isolated rabbit eyes, instead of to the eyes of intact rabbits. The problem with this approach, where the aim is to identify chemicals likely to cause irritancy in human eyes, is that the uncertainty of the ex vivo/in vivo (i.e. isolated rabbit eye to in situ rabbit eye) equivalence must now be added to the uncertainty of the in vivo/in vivo (rabbit eye to human eye) equivalence. In addition, the isolated rabbit eye may be even more unlike the human eye than the in situ rabbit eye, so the data it provides will be even more difficult to interpret and apply.

Seeking genuine indirect replacement procedures is much more intelligent, if it involves defining the information it was hoped to get from the animal procedure, then obtaining it from much more advanced experimental techniques. For example, the metabolism of drugs can be studied by using human hepatocytes in vitro, instead of administering the drugs to rats or dogs, which have different complements of drug metabolising enzymes. Direct replacement offers the possibility of direct relevance, where the object of interest can be studied, rather than an inadequate and imperfect model.

To return to the wisdom of Russell and Burch, it is interesting to note that it is in their chapter on Replacement that they discuss the difference between fidelity and discrimination, and the importance of the high-fidelity fallacy.2 The danger is in assuming that, since vertebrates — and especially the higher vertebrates, such as Old World monkeys — are generally similar to humans, data from experiments applied to them will be specifically relevant to humans. Russell and Burch believed that “progress in replacement has been restricted by [this] plausible, but untenable, assumption”. Common sense says that they were right, but the presumption of high-fidelity remains the justification for much research and testing with animal models today.2

Russell and I followed the same zoology degree course at Oxford, in what was then called the Department of Zoology and Comparative Anatomy. We studied every order of animals from the Protozoa (e.g. Amoeba) to the Hominoidea (great apes and humans), and the emphasis was on how animals had evolved from common ancestors via adaptive radiation, in ways appropriate to their particular environments and lifestyles, how originally-common features could evolve to be the solutions to different problems, and how the same kinds of problems could be solved in different ways. It would never have occurred to us that one type of animal could precisely and satisfactorily model another one.
While it may be true that our current thinking about replacement is somewhat different from that of Russell and Burch in 1959, to me, at least, their discussion on fidelity and discrimination is of timeless value, especially when coupled with the encouraging words of their view that “Replacement is always a satisfactory answer; but reduction and refinement should, wherever possible, be used in combination”.

Professor Michael Balls
c/o FRAME
Russell & Burch House
96–98 North Sherwood Street
Nottingham NG1 4EE
UK
E-mail: michael.balls@btopenworld.com

References
1 Russell, W.M.S. & Burch, R.L. (1959). The Principles of Humane Experimental Technique, xiv + 238pp. London,
UK: Methuen.
2 Balls, M. (2013). The Wisdom of Russell and Burch 3. Fidelity and discrimination. ATLA 41, P12–P13.

The Principles of Humane Experimental Technique is now out of print, but the full text can be found at http://
altweb.jhsph.edu/pubs/books/humane_exp/het-toc. An abridged version, The Three Rs and the Humanity Criterion,
by Michael Balls (2009), can be obtained from FRAME.

Russell and Burch on Refinement

The freedom of choice of the experimenter is often very much wider than it at first appears. The full use of this freedom is the mark alike of humane and successful experimentation. There is, perhaps, no limit in animal experimentation to the progress of refinement

Michael Balls

When introducing the Three Rs concept in The Principles of Humane Experi mental Technique, Russell and Burch defined Refinement as “any decrease in the incidence or severity of inhumane procedures applied to those animals that still have to be used” [after all possible steps have been taken to achieve Replacement and Reduction]. However, it was clear that its application would involve an extreme diversity of approaches, which made generalisation virtually impossible. They recognised two broad kinds of investigation, namely, stressful studies, which involved “the acquisition of knowledge about the mechanisms of pain and distress and/or their autonomic and endocrine consequences”, and neutral studies, “which did not have such objectives”. An important point was that “there seems at first sight to be an irreconcilable conflict between the claims of humanity and efficiency”

in stressful investigations, although Russell and Burch foresaw the possibility of being able to get “stress without distress”, by, for example, using decerebrate or anaesthetised animals. However, there would still be “a stubborn residue of inevitably inhumane study”, such as that involving fear, and a large part of the Refinement chapter in The Principles was devoted to “methods whereby fear itself can be studied with perfect humanity”.

Russell and Burch emphasised that, by contrast, there should be “no insuperable obstacles to the refinement of neutral studies”. It was also concluded that “there are many refinement procedures, applicable in many different kinds of experiments, which can be added to or superimposed upon the particular procedure chosen for an experiment”. They considered this R to be “one of the most obviously, immediately, and universally advantageous in terms of efficiency” of all the modes of progress. It is, they suggested, “desirable in any procedure, however humane, which employs large numbers of animals in one laboratory”, and achievable “by the right choice of strategies in the planning and performance of whole lines of research”. The “most generally important of all is anaesthesia, the supreme refinement procedure”, which has brought about “the greatest single advance in humane technique, and has at the same time been virtually indispensable for the advance of experimental biology”. Russell and Burch saw euthanasia as possibly being even more important than anaesthesia, not least in relation to relative replacement, where animals are still required, though not exposed to any distress in experiments. For example, if cells or tissues are needed for in vitro procedures, it is important to avoid the biochemical and physiological disturbances induced by a stressful death. This is another situation where humanity and efficiency go hand-in-hand. Two more-specific aspects of refinement are considered — namely, choice of procedure and choice of species.

Fundamental research involves new challenges in every experiment, in contrast with testing, where standard protocols tend to be followed. As Russell and Burch pointed out, “almost any research questions can be answered in principle by a number of different procedures. The mark of distinction of the great experimenter is a knack for choosing the most rapid, elegant, and simple one”. This is also important for maximising humanity, as well as efficiency. Choice of species is no less important, and, as a fellow zoologist, I share Russell’s concern that this should involve “a wide knowledge of the special advantages of particular species for particular purposes”. The aim should be to “match procedure to species, and species to objectives”. Failure to do this can lead to inhumanity, if, as often happens, an attempt is made “to force the wrong species to conform to the requirements of the investigation”. Bearing this in mind, Russell and Burch noted that the overwhelming bulk of the animals used in laboratories were mice, rats, guinea-pigs and rabbits. They saw this as “yet another expression of the high fidelity fallacy”.

The overall conclusion to the Refinement chapter is very challenging: “The freedom of choice of the experimenter is often very much wider than it at first appears. The full use of this freedom is the mark alike of humane and successful experimentation. There is, perhaps, no limit in animal experimentation to the progress of refinement.” It is linked, of course, to the essence of Russell and Burch’s Three Rs, as stated in the Humanity Criterion: “If we are to use a criterion for choosing experiments to perform, 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.”

The Principles of Humane Experimental Technique is now out of print, but the full text can be found at http://altweb.jhsph.edu/pubs/books/humane_exp/het-toc. An abridged version, The Three Rs and the Humanity Criterion, by Michael Balls (2009), can be obtained from FRAME.

Reduction

Russell & Burch book cover

Michael Balls

Reduction should be the easiest of the Three Rs to achieve,
but it will not happen without a revolution in education
and training on the design and analysis of animal experiments

I have read The Principles of Humane Experimental Technique1 many times, but I have to confess that I have always had difficulty in understanding much of Russell and Burch’s chapter on Reduction. They saw this R as “one of the most obviously, immediately, and universally advantageous in terms of efficiency” of all the modes of progress. It is, they suggested, “desirable in any procedure, however humane, which employs large numbers of animals in one laboratory”, and is achievable “by the right choice of strategies in the planning and performance of whole lines of research”.

The main issue, as discussed “in a searching essay” by Charles Hume,2 founder of UFAW and initiator of the Three Rs project, is “choosing between trial and error on a grand scale and deductively inspired research”. The first approach has been used to test “a constant and huge stream of chemical substances” randomly directed at major medical targets, such as cancer. The second “may take the form of testing deductions from well and consciously formulated hypotheses, or it may involve working from hunches”.
Russell and Burch suggested two main conclusions: that, “wherever it is possible directly to compare guided and random research, the former is seen to be the most efficient”; and that, where trial and error methods are used, “it is desirable in terms of humanity, cost, and effort, for the trial and error to be applied to replacement methods or subjects”.

That is all very sound and reasonable, but it is the following 26 pages of the book chapter which are somewhat overwhelming. This is where Russell focused intensely on his particular interest in the problem of variance and how it could be tackled, stating that “Relative accuracy depends on the size of the sample, the extent to which individuals of the species vary [e.g. in response to a drug] and the efficiency and design of [the] experiment”, and that the key to reducing the effects of this variance is “the ingenious design of experiments”.

Statistical advice is important at the outset, since “every time any particle of statistical method is properly used, fewer animals are employed than would otherwise have been necessary”. However, the nature and quality of such advice are variable, leading to controversies, some of which are still unresolved today. One example is toxicity testing, where Hume said that “the fallacy consists in supposing that, in order to obtain a broad inductive basis, a heterogeneous stock [of animals] should be used”.

This, he said, is like estimating the value of a pocketful of coins “by counting the coins as coins, without sorting them according to their different values”. The proper procedure is “to use several different homogeneous samples”, and “to allow for the variance between samples”. This particular controversy continues today, as Michael Festing3 pointed out in an issue of ATLA on Reduction, in a special volume to mark FRAME’s 40th anniversary.

It is particularly disappointing that, while the numbers of procedures applied to laboratory animals fell steadily during the 1980s and 1990s, they have increased year on year since then. This was almost exclusively attributed to the breeding and use of genetically-modified animals and animals with harmful genetic defects, but in the UK in 2011, the larger increase was on procedures on normal animals, and especially for fundamental research on cancer, immunology and physiology.4

Another cause of the failure to achieve reduction is that scientists are not sufficiently well trained in how to perform experiments and how to analyse the data they obtain from them. Of course, this problem is not confined to experiments on animals, but applies to other kinds of experiment as well. Russell and Burch were aware of this problem, when, referring to the choice of strategies in planning and performing experiments, they said that “most of us settle early in our research career on some strategy that appears to suit our temperament, and are liable never to raise the question again”. That is nothing short of shocking, as it takes into account neither the scientific demands of the purpose of the experiment, nor, in the case of animal experimentation, the costs to the animals involved. I remember being deeply disturbed, many years ago, on learning that rhesus monkeys were kept in primate chairs with head restraint for up to six hours a day, five days a week, with electrodes implanted into their brains. They would have been there for longer, but the scientists involved didn’t like working in the evenings or at weekends.

The lack of adequate education and training in the design and analysis of experiments has long been a matter of concern to a small number of people and organisations. FRAME has been successfully running courses for some time,5 but the numbers of participants involved can only be small in relation to the total number of animal users.

Michael Festing, who I have always regarded as the natural successor to William Russell on these issues, has contributed an excellent and challenging comment to this issue of PiLAS.6 He emphasises that animal experiments should not only be well designed, but also repeatable, if the use of the animals is to be justifiable and the results are to lead to reliable improvements in human health. Unfortunately, this is often not possible, as illustrated by three reviews he cites which show the magnitude of the problem in research on oncology, amyotrophic lateral sclerosis and drug development.

The solution to the problem lies partly with those who fund research and the editors of the journals that publish its outcomes, but it is primarily the responsibility of the research scientists themselves and those in their institutions who evaluate their progress as professionals. The laws and regulations which apply to the use of animals for scientific purposes also have an important role to play, as has been recognised in various countries in the past, including The Netherlands and the UK. In the EU, it can be expected that Directive 2010/63/EU,7 which came into force at the beginning of 2013, will have a significant impact in the 27 Member States, as it specifically refers to education and training in Article 23, Competence of personnel:

2. The staff shall be adequately educated and trained before they perform any of the following functions: (a) carrying out procedures on animals; (b) designing procedures and projects; (c) taking care of animals; or (d) killing animals.

Persons carrying out the functions referred to in point (b) shall have received instruction in a scientific discipline relevant to the work beingundertaken and shall have species-specific knowledge. Staff carrying out functions referred to in points (a), (c) or (d) shall be supervised in the performance of their tasks until they have demonstrated the requisite competence. Member States shall ensure, through authorisation or by other means, that the requirements laid down in this paragraph are fulfilled.

3. Member States shall publish [on the basis of the elements set out in Annex V] minimum requirements with regard to education and training and the requirements for obtaining, maintaining and demonstrating requisite competence for the functions set out in paragraph 2.

The Directive also requires that Member States shall ensure that the number of animals used in projects is reduced to a minimum without compromising the objectives of the project. If this requirement is strictly enforced and competently and sincerely adhered to, the steady upward trend in the numbers of animals used should be reversed and the number of procedures conducted progressively reduced, at least in the EU, in line with Russell and Burch’s central Three Rs thesis that “the humanest possible treatment of laboratory animals, far from being an obstacle, is actually a prerequisite for successful animal experiments”.

References and Note
1 Russell, W.M.S. & Burch, R.L. (1959). The Principles of Humane Experimental Technique, xiv + 238pp. London, UK: Methuen.
2 Hume, C.W. (1957). The strategy and tactics of experimentation. The Lancet, 23 November, 1049–1052.
3 Festing, M. (2009). Fifty years after Russell and Burch, toxicologists continue to ignore variation in their test animals. ATLA 37, 1–5.
4 Hudson-Shore, M. (2012). Statistics of Scientific Procedures on Living Animals 2011: Another increase in animal experimentation, but is there a shift in emphasis? ATLA 40, 211–219.
5 Hudson, M. & Howard, B. (2009). The FRAME Reduction Steering Committee: Reflections on a decade devoted to reducing animal use in biomedical science. ATLA 37, 23–26.
6 Festing, M. (2013). We are not born knowing how to design and analyse scientific experiments. ATLA 41, P19–P21.
7 Anon. (2010). Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes. Official Journal of the European Union L276, 20.10.2010, 33–79.

The Principles of Humane Experimental Technique is now out of print, but the full text can be found at http://altweb.jhsph.edu/pubs/books/humane_exp/het -toc. An abridged version, The Three Rs and the Humanity Criterion, by Michael Balls (2009), can be obtained from FRAME.

Fidelity and Discrimination

Progress in replacement has been restricted by certain plausible, but untenable, assumptions, summed up as the high fidelity fallacy.

One of the most important and challenging sections in The Principles of Humane Experimental Technique is Russell and Burch’s discussion of models and their proper use. Research on human health and disease, and tests on the effects of substances and pathogenic organisms, frequently involve establishing a model (a laboratory animal) of the system to be investigated (the human organism), then studying the model in ways which would not be possible with the system being modelled (for practical or ethical reasons).
By their very nature, models must differ from what is being modelled, and the importance and consequences of this difference depend on two major factors, fidelity and discrimination.

For a pdf of the full article click here.

New Directive Comes into Force in the EU

Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes, came into force on 9 November 2010, to replace Directive 86/609/EEC. The 27 EU Member States were required to transpose its requirements into their own national legislation, so that they could be implemented by 1 January 2013.

To download a copy of this article as a pdf CLICK HERE.

The Concept, Sources and Incidence of Inhumanity and its Diminution or Removal Through Implementation of the Three Rs

Michael Balls

The concepts expounded by W.M.S. Russell and R.L. Burch in the 1950s in their outstanding book, The Principles of Humane Experimental Technique, are now the basis of many national and international laws and regulations on the proper use of laboratory animals.

Bill Russell and Rex Burch
Bill Russell and Rex Burch

For a pdf version of the full article CLICK HERE.