Category Archives: Craig Redmond

‘One R’ is the new ‘Three Rs’

Craig Redmond

The Lush Prize, which rewards initiatives to end animal testing,

believes that more attention needs to be given to the ‘One R’

of absolute replacement, and that research methods that

exploit animals in any way (including tissues and cells)

should not be considered as ‘alternatives’

Introduction
Replacement of animal experiments is one of the Three R concepts (alongside Reduction and Refinement) first put forward by Russell and Burch in 1959.1 However, this can be either absolute replacement (i.e. methods that do not involve animals or animal tissues) or relative replacement (e.g. methods that use only cells or tissues of animals in vitro or ex vivo).

It has become accepted by many in the research community that some uses of animals can be classed as ‘alternatives’. In particular, the use of whole animals of species thought to either not experience pain or to have a lower level of sentience (e.g. fruit flies, nematodes and zebrafish), or of animal parts (including tissues, embryos, sera and cells). The use of these methods is reinforced by regulatory bodies, making it more difficult to reach a time when no animal use will occur in scientific research. André Ménache, of Antidote Europe, believes that in the region of 80% of ‘alternatives’ validated by ECVAM (the European Union Reference Laboratory for alternatives to animal testing) still use animals or animal tissues (personal communication, 04.09.13).

Founded in 2012, the Lush Prize rewards global initiatives to end animal testing, particularly in the area of toxicology. A total of £250,000 is shared annually between five prize categories covering science, training, young researchers, lobbying and public awareness. Lush Prize believes that more attention needs to be given to the ‘One R’ of absolute replacement, and that research methods that exploit animals in any way (including tissues and cells) should not be considered as ‘alternatives’.

Alternatives’ that still exploit animals
The United States Department of Agriculture refers to ‘alternatives’ as “a term that has different meanings to different people, and this difference largely depends on which side of the issue one is found”.2 So, for example, animal researchers might use relative replacement methods in addition to their use of animals (or look to refine existing animal tests), whereas abolitionists see ‘alternatives’ in terms of absolute replacement. Other examples of where the use of the term ‘replacement’ serves to reinforce the idea that relative replacement is routinely acceptable are:

— where Russell and Burch defined a replacement technique as “any scientific method employing non-sentient material which may in the history of animal experimentation replace methods which use conscious living vertebrates”.3 The words ‘non-sentient’, ‘conscious’ and ‘vertebrates’ ensure that the use of invertebrates and species considered as ‘lower organisms’ continues to be accepted.
— when, in its current “step-by-step approach to an alternatives search”, the Johns Hopkins University Center for Alternatives to Animal Testing (CAAT) suggests that, in addition to cell culture, tissue culture, models, simulations, etc., researchers “might look for a non-mammalian animal model
— fish or invertebrates, for example — that would still give you the data you need”. 4

Perhaps as a direct result of this widespread inherent acceptability of relative replacement alternatives, researchers at the University of British Columbia, looking into people’s acceptance of the use of particular species in laboratories, found that species such as fish and invertebrates “are typically rated below mammals, and, as such, are often considered an appropriate replacement for mammals in research”. 5

The philosopher Joel Marks notes that “developing alternatives to the use of animals can mean simply using a different animal” and considers that “the characterisation of the other animal […] as ‘lower’ on a phylogenetic ‘scale’ is arbitrary and disputed. The alternatives movement is therefore at risk of becoming a bait-and-switch con”.6 By this, Marks means that ‘alternatives’ are advertised as one thing (i.e. absolute replacement), but often turn out to be something completely different (i.e. simply the use of another species).

Some examples of relative replacement alternatives are:
Invertebrates: The horseshoe crab, Limulus polyphemus, is used in the Limulus amoebocyte lysate (LAL) assay. This method replaces the rabbit pyrogen test for the detection of endotoxin in, for example, hepatitis B vaccines. 7 The rabbit test involves injecting the test substance into a marginal vein of the ear of each of three rabbits.8 However, the LAL assay uses blood cells from the horseshoe crab, with up to 30% mortality resulting from the bleeding procedure.9

Fish: Zebrafish (Danio rerio) are widely used in research, including research on genetics, cancer and, increasingly, toxicology. The maintenance costs of zebrafish are less than one thousandth of the costs of maintaining mice,10 and they can produce 100–300 eggs per week, making their embryos useful for high-throughput screening.11 It is widely acknowledged that fish can feel pain, 12 with as much evidence for this as there is for birds and mammals.13 Other studies have shown that they have conscious awareness.14

Tissues: Hundreds of thousands of animals are bred and killed each year in Britain alone, solely to provide tissues for research.15 Human tissues to be preferred, due to species differences, yet animal tissue is often used on the grounds of cost and availability.16 Human tissue can be obtained from patients during diagnosis, removed as ‘waste’ during surgical operations, placentas or ‘afterbirth’, or tissues obtained after death.15

People can voluntarily donate blood or other tissue for transplantation or research, or their organs or bodies after death. Human tissue removed from the body in the course of disease diagnosis or treatment is the main source.17 However, although upwards of 600,000 residual surgical tissues are generated each year in England and Wales, only a tiny fraction of them are made available to researchers.18

The use of fetal calf serum
The move toward the use of in vitro cell culture to provide both human and animal cells for alternative methods is a step in the right direction. However, the use of fetal calf serum (FCS) as a cell culture media supplement is unacceptable, in the light of the availability of serum replacements and serum-free culture methods.19 collected for FCS production is obtained by cardiac puncture, performed by inserting a needle directly into the heart of the non-anaesthetised fetus.20 according to Jochems et al.20 it is very likely that the fetus is alive at the time of blood collection, and “will experience pain and/or suffering at the moment of heart puncture for blood collection and possibly for a period after that, until it actually dies”. The scientific validity of using FCS has been questioned. Risk of contamination is an issue,21 with the potential presence of viruses, bacteria, mycoplasma, yeast, fungi, immunoglobulins and endotoxins.20

Conclusions
These are just a few examples of animal use that some see as ‘alternatives’. The use of sentient animals, such as fish and horseshoe crabs, should not be accepted by those working in the field of alternatives to animal testing, despite the entrenched position within the research community and regulatory bodies. Neither should cruel processes such as the collection of FCS be condoned. In addition to greater humanity and greater acceptability, there are a multitude of clear scientific benefits to avoiding the use of animals or animal products.

The Lush Prize promotes the ‘One R’ of Replacement over all of the ‘Three Rs’, believing that the  true absolute replacement of animals is essential for ethical and scientific progress.

Craig Redmond
Lush Prize
Unit 21
41 Old Birley Street
Manchester M15 5RF
UK
E-mail: craig@lushprize.org
References
1 Russell, W.M.S. & Burch, R.L. (1959). The Principles of Humane Experimental Technique, 238pp. London, UK: Methuen.
2 Taylor Bennett, B. (1996). Alternative methodologies. In Essentials for Animal Research. A Primer for Research Personnel, pp. 9–17. Darby, PA, USA: Diane Publishing.
3 Balls, M. (1994). Replacement of animal procedures: Alternatives in research, education and testing. Laboratory Animals 28, 193–211.
4 Altweb Project Team (undated). Search for Alternatives. Baltimore, MD, USA: CAAT, Johns Hopkins
University. Available at: http://altweb.jhsph.edu/resources/searchalt/index.html (Accessed 22.10.14).
5 Ormandy, E.H., Schuppli, C.A. & Weary, D.M. (2012). Factors affecting people’s acceptance of the use of zebrafish and mice in research. ATLA 40, 321–333.
6 Marks, J. (2012). Accept no substitutes: The ethics of alternatives. The Hastings Center Report 42, Nov–Dec (Special Report), S16–S18.
7 Park, C.Y., Jung, S.H., Bak, J.P., Lee, S.S. & Rhee, D.K. (2005). Comparison of the rabbit pyrogen test and Limulus amoebocyte lysate (LAL) assay for endotoxin in hepatitis B vaccines and the effect of aluminum hydroxide. Biologicals 33, 145–151.
8 WHO (2013). The International Pharmacopoeia. Preface: 3rd Supplement. Geneva, Switzerland: World Health Organisation. Available at: http://apps.who.int/phint/en/p/docf/ (Accessed 07.09.13).
9 Leschen, A.S. & Correia, S.J. (2010). Mortality in female horseshoe crabs (Limulus polyphemus) from biomedical bleeding and handling: Implications for fisheries management. Boston, MA, USA: Massachusetts Division of Marine Fisheries. Available at: http://www.mass.gov/eea/docs/dfg/dmf/publications/mortality-in-female-horseshoe-crabsabstract.
pdf (Accessed 25.10.14).
10 Reed, B. & Jennings, M. (2011). Guidance on the housing and care of zebrafish Danio rerio, 64pp. Horsham, West Sussex, UK: Research Animals Department, Science Group, RSPCA.
11 van Vliet, E. (2011). Current standing and future prospects for the technologies proposed to transform toxicity testing in the 21st century. ALTEX 28, 17–44.
12 FAWC (1996). FAWC report on the welfare of farmed fish, 43pp. London, UK: Farm Animal Welfare Council.
13 Braithwaite, V. (2010). Do Fish Feel Pain?, 208pp. Oxford, UK: Oxford University Press.
14 Cottee, S.Y. (2012). Are fish the victims of ‘speciesism’? A discussion about fear, pain and animal consciousness. Fish Physiology & Biochemistry 38, 5–15.
15 Focus on Alternatives (undated). Focus on Human Tissue in Research, 4pp. Available at: http://www.frame.org.uk/dynamic_files/foa_humantissue.pdf (Accessed 03.09.13).
16 EMA (2012). Committee for Advanced Therapies (CAT) Scientific Workshop: Reducing the number of laboratory animals used in tissue engineering research — 11th October 2012 — European Medicines Agency, London [EMA/CAT/708346/2012], 4pp. London, UK: European Medicines Agency. Available at: http://www.ema.europa.eu/docs/en_GB/document_library/Report/2012/12/WC500136419.pdf (Accessed 25.10.14).
17 Nuffield Council on Bioethics (1995). Human Tissue: Ethical and Legal Issues, 182pp. London, UK: Nuffield Council on Bioethics. Available at: http://nuffieldbioethics.org/project/human-tissue/ (Accessed 25.10.14).
18 Bunton, D. (2011). The use of functional human tissues in drug development. Cell & Tissue Banking 12, Issue 1, 31–32.
19 Newman, C. (2003). Serum-free cell culture — the ethical, scientific and economic choice. The Biomedical Scientist, September 2003, 941–942.
20 Jochems, C.E., van der Valk, J.B., Stafleu, F.R. & Baumans, V. (2002). The use of fetal bovine serum: Ethical or scientific problem? ATLA 30, 219–227.
21 Focus on Alternatives (2009). Serum-free Media for Cell Culture, 52pp. http://www.frame.org.uk/dynamic_files/foa_fcs_free_table_may09.pdf
(Accessed 03.09.13).