Rationalisation and Intellectualisation

Michael Balls

Russell and Burch saw failure to accept the correlation
between humanity and efficacy as an example of
rationalisation, a psychological defence mechanism

While wondering what I could discuss in this column I looked, as I often do, in the abridged version1 of The Principles of Humane Experimental Technique,2 at Russell and Burch’s introduction of what I call the humanity criterion. It is part of their discussion of the sociological factors which are among the Factors Governing Progress. This is how part of page 101 of the abridged version reads:
In fact, really informative experiments must be as humane as would be conceivable possible, for science and exploration are indissolubly linked to the social activity of cooperation, which will find its expression in relation to other animals, no less than to our fellow humans. Conscious good will and the social operational method are useless as safeguards against the mechanism of rationalisation (in the pathological sense of the term — i.e. the mechanism of defence by which unacceptable thoughts or actions are given acceptable reasons to justify them to oneself and to others, while, at the same tie, unwittingly hiding the true, but unconscious, motives for them).

The bold type indicates my explanation, and I have to admit that, six years after preparing the abridged version of The Principles, I now found it difficult to see what Russell and Burch had intended to convey. I therefore looked back at the original book, and found this paragraph on pages 156−157:
In efficacy, or yield of information, the advantages of humane technique apply almost universally. The correlation between humanity and efficacy has appeared so often in this book that we need not labour the point. There is, however, a more fundamental aspect of this correlation, specially important in research. Science means the operational method — telling somebody else how to see what you saw. This method is one of the greatest of all human evolutionary innovations. It has, however, one drawback. It prevents permanent acceptance of false information, but it does not prevent wastage of time and effort. The activity of science is the supreme expression of the human exploratory drive, and as such it is the subject to the same pathology. The scientist is liable, like all other individuals, to block his exploration on some front where his reactions to childhood social experiences are impinged upon. When this happens to the experimental biologist, we can predict the consequence with certainty. Instead of really exploring, he will, in his experiments, act out on his animals, in a more or less symbolic and exaggerated way, some kind of treatment which he once experienced in social intercourse with his parents. He can rationalise this as exploration, and hence fail to notice the block. But in fact such acting out invariably occurs precisely when real exploration is blocked, and must be relinquished before real exploration can begin again. Hence, such experiments will be utterly wasteful, misleading, and uninformative. The treatment of the animals, for one thing, will inevitably be such as to impair their use as satisfactory models. The interpretation of the results will be vitiated by projection. Really  informative experiments, must in fact be as humane as would be conceivably possible, for science and exploration are indissolubly linked to the social activity of cooperation, which will find its expression in relation to other animals no less than to our fellow humans. Conscious goodwill and the social operational method are useless as safeguards against the mechanism of rationalisation (in the pathological sense of the term).

Here, the underlining indicates what I omitted from the abridged version, and I now wonder why I did so. These words clearly reflect Russell’s interest in psychology — he later became a psychotherapist, and undoubtedly will have been influenced by discussions with his psychotherapist wife, Claire Russell. They could be seen as an explanation why some scientists did not appreciate the essential link between humanity and efficacy, and why Russell thought they needed what was offered by the Three Rs and the humanity criterion.

It is not clear what is meant by “the social operational method”, and consulting Google leads to only one hit — The Principles itself! “Conscious goodwill” is probably meant to contrast with unconscious rationalisation. Perhaps what Russell meant is that, however sincere the intention may appear to be, support for the Three Rs is useless, unless it leads to active and practical commitment to their development and application.

We are often confronted with rationalisation, the pseudo-rational justification of irrational acts,3 and its relative, intellectualisation, a different defence mechanism (or way of making excuses), “where reasoning is used to block confrontation with an unconscious conflict and its associated emotional stress, where thinking is used to avoid feeling. It involves removing one’s self, emotionally, from a stressful event. Intellectualisation is one of Freud’s original defence mechanisms. Freud believed that memories have both conscious and unconscious aspects, and that intellectualisation allows for the conscious analysis of an event in a way that does not provoke anxiety.”4

I am not a psychoanalyst, and I think it would be unwise, even dangerous, were I to seek to delve into the underlying reasons why some scientists are so keen to run to animal experimentation as the first resort and to do so little to make possible its replacement. Nevertheless, I can say, without fear of contradiction, that this is another great example of how Russell and Burch’s wonderful book continues to give us food for thought and calls for action.

Professor Michael Balls
E-mail: michael.balls@btopenworld.com

References
1 Balls, M. (2009). The Three Rs and the Humanity Criterion, 131pp. Nottingham, UK:  FRAME.
2 Russell, W.M.S. & Burch, R.L. (1959). The Principles of Humane Experimental Technique, xiv + 238pp. London, UK: Methuen.
3 Anon. (2015). Rationalization (psychology). San Francisco, CA, USA: Wikipedia Foundation, Inc. Available at: https://en.wikipedia.org/wiki/Rationalization_(psychology) (Accessed 26.08.15).
4 Anon. (2015). Intellectualization. San Francisco, CA, USA: Wikipedia Foundation, Inc. Available at: https://en.wikipedia.org/wiki/Intellectualization
(Accessed 26.08.15).
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. The abridged version, The Three Rs and the Humanity Criterion, can be obtained from
FRAME.

The choice of procedures

Michael Balls

The scientifically-justifiable choice of procedure
is a crucial issue in animal experimentation,
in the interests of both humanity and efficiency

In the opening of their chapter on Reduction and Strategy in Research in The Principles of  Humane Experimental Technique,1 Russell and Burch pointed out that “one general way in which great reduction can occur is by the right choice of strategies in the planning and performance of whole lines of research”.

They referred to the way Charles Hume had put it “in a searching essay”,2 that is, that “the central problem is that of choosing between trial and error on a grand scale and deductively inspired research”. The second type of choice can “take the form of testing deductions from well and consciously formulated hypotheses, or it may involve working from hunches — really the same thing, for where hunches are of any value, they are found to be based on equally precise hypotheses”. They said that the essence of the strategy “is that particular experiments are selected on some basis … from a larger set of experiments that  could have been performed”, and, as Hume had pointed out, “insighted” research “must be vastly less wasteful of animals”, where animals are to be used in the experiments.

I discussed this in an earlier comment on the Wisdom of Russell and Burch in relation to Reduction,3 with particular reference to experimental design and statistical analysis, but I now want to consider their discussion on The Choice of Procedures, in their chapter on Refinement.

Russell and Burch said that almost any research question “can always be answered in principle by a number of different procedures”, and the mark of distinction of “the great experimenter is the knack of choosing the most rapid, elegant and simple one”. But they then ask whether there are any simple rules in this context. One “general principle, important for both humanity and efficiency, is that of avoiding elaborate and roundabout methods”. Another rule is “the very careful formulation of questions”. One approach is to “first ask the question, then draw up, at least mentally, a list of procedures by which it could be answered”. If the list is long enough, consideration can be given to choosing the best procedure. If the list is too short, the question may need to be reformulated, to permit a wider range of procedural choice.

The greater the experimenter, in terms of ability and quality, the easier and better will be the choice of procedure. In addition, the humane experimenter will be careful to take account of Hume’s point about the wastage of animals, and Russell and Burch’s emphasis on the need for humanity and efficiency. This should be obvious, in terms of the use of resources, even if it were not a requirement of the laws under which animal experimentation is now permitted. It is a vital aspect of the education and training which must be regarded as essential for all those who are to undertake research, particularly if there is any risk of causing animal suffering, but also in the interests of the humans for whose benefit the research is being conducted.

It is encouraging that, in its response to the European Citizens’ Stop Vivisection Initiative,4,5 the European Commission has proposed four actions, one of which is to “analyse technologies, information sources and networks from all relevant sectors with potential impact on the advancement of the Three Rs”, in order to “present by end 2016, an assessment of options to enhance knowledge sharing among all relevant parties. The assessment will consider how  systematically to accelerate knowledge exchange through communication, dissemination, education and training.”

In order to aid researchers in their thought process when designing any project that could involve experimental animals, the FRAME Reduction Steering Committee designed a Strategic Planning Poster.6 The poster, which is available in several languages, guides the scientist through the decision points and steps needed when designing a whole programme of work, including the individual experiments within it. This resource, and the FRAME Training Schools in  Experimental Design and Statistical Analysis,7 encourage researchers to think about how to design their sequence of experiments, in order to minimise the number of animals that are exposed to the most severe procedures and to contemplate whether animals are needed at all.

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 Hume, C.W. (1957). The strategy and tactics of experimentation. The Lancet, 23 November, 1049–1052.
3 Balls, M. (2013). The Wisdom of Russell and Burch. 4. Reduction. ATLA 41, P24–P25.
4 Anon. (2015). Commission Response to the European Citizens’ Initiative “Stop Vivisection”. Brussels, Belgium: Intergroup on the Welfare and Conservation
of Animals. Available at: http://www.animalwelfare intergroup.eu/2015/06/05/european-commissiondoesnt- go-far-enough-to-meet-citizens-demands-toimprove-
animal-welfare/ (Accessed 08.07.15).
5 Balls, M. (2015). The European Citizens’ Stop Vivisection Initiative. ATLA 43, 147–150.
6 Gaines Das, R., Fry, D., Preziosi, R. & Hudson, M. (2009). Planning for reduction. ATLA 37, 27–32.
7 Fry, D., Gaines Das, R., Preziosi, R. & Hudson, M. (2010). Planning for refinement and reduction. ALTEX 27, Special Issue, 293–298.

 

In vitro models to mimic the endothelial barrier

Laurent Barbe, Mauro Alini, Sophie Verrier and Marietta Herrmann

Microfluidic technologies permit the replication in vitro
of geometrical features essential for the homeostasis of
all vascularised tissues in vivo, including the contribution
of pericytes to the endothelial barrier

Introduction

A functional microvasculature is critical for the homeostasis of all vascularised tissues.  accordingly, several diseases are associated with alterations in the microvasculature. For example, tumour angiogenesis is a major factor in determining the burden of the
disease. Furthermore, the formation of new vessels by angiogenesis and vasculogenesis is critical in the restoration of tissue function in ischaemic diseases. In tissue engineering, sufficient neovascularisation and early vessel anastomosis is thought to be a prerequisite
for the integration of the implant. These conditions have been extensively studied in animal models. However, in vivo studies have several limitations, including species differences and limited possibilities for imaging and tracking cells in the living animal. They also do not permit high-throughput and multiplexing applications. The development of microfluidic models of microvasculature and the endothelial barrier could help to overcome these problems and, most importantly, would replace a significant amount of animal experimentation. Nevertheless, microfluidic science is still an evolving research field, and many models do not address the endothelial barrier in its full complexity — for example, taking into account the contribution of pericytes.

The contribution of pericytes to the endothelial barrier

Pericytes are vascular mural cells associated with microvessels. The most common definition of pericytes goes back to their localisation, embedded in the endothelial basement membrane.1 Besides sharing the basement membrane, close interactions between endothelial cells and pericytes have been described, such as peg–socket contacts representing tight-, gap- and adherence-junctions.2 The relationship between the two cell types, particularly the anatomy of the pericyte coverage, reflects the function of the individual tissues. In organs with high exchange rates of gas and metabolites, the  distribution of pericytes is such that diffusion is minimally hindered.1, 3 Pericytes play an important role in vessel stabilisation, and underlying molecular signalling pathways have been described. Here, signalling through angiopoietin and Tie2 is critical; mutation of either angiopoietin 1 or Tie2 leads to mid-gestational death by cardiovascular failure in mice.2 In angiogenesis, the recruitment of pericytes is required for the stability of newly-formed vessels. Various studies have identified platelet-derived growth factor B (PDGF-B) as a major chemoattractant for pericytes. 4  Capillary and arteriolar pericytes also play an important role in inflammatory processes by guiding extravasating leukocytes toward their target. 5  Pericytes are also involved in several disease states, including airway remodelling in chronic allergic asthma6 and stroke.7 The identification of mesenchymal
stem cells and progenitor cells at perivascular sites, and the subsequent isolation and characterisation of such cells, suggests that pericytes have a role as multipotent progenitors.8-10

Current animal models

The function of pericytes in physiologically and pathologically relevant situations has been studied in transgenic mouse models containing LacZ-expressing or fluorophore-expressing pericytes.5, 6, 11 The migration and stimulation of pericytes in different disease situations was subsequently studied by the administration of pro-inflammatory factors, or by  backcrossing mice to a transgenic disease mouse model.5, 12  Interactions between labelled leukocytes and pericytes have been studied by lethal irradiation and the subsequent injection of bone-marrow cells from GFP mice.5 In addition, specific knockout lines have been used to study specific components of the cell junction and signalling complexes involved in pericyte– endothelial cell crosstalk, e.g. Sparc-deficient and Ccn2-deficient mice.13,14 Various animal models have also been developed, for studying the basic mechanisms of angiogenesis and vessel sprouting, including the cornea model, the chick chorioallantoic membrane (CAM) model, matrigel plug assays, and the dorsal skin fold chamber.15

Current microvascular models

Many factors determine the phenotypes of cells in vivo, including cell–cell interactions, interaction with the surrounding extracellular matrix, and the influence of various paracrine factors. Current cell culture vessels (dishes, flasks) cannot recapitulate these
aforementioned complex interactions. However, in recent years, microfluidic technologies have shown the potential to more-closely mimic the cellular microenvironment, at both the spatial and temporal levels.16 Typical microfluidic systems have geometrical features ranging in size from tens to hundreds of microns, and can host single cells or millions of cells arranged in a 2-D or a 3-D fashion. Microfluidic devices can accommodate flow control and therefore induce shear stress, which is known to have significant effects on the endothelial layer.17 This shear stress is absent in classical culture dishes. Furthermore, microfluidic technologies enable the generation of gradients over long periods of time, and also permit the control of paracrine factors in complex co-culture systems.18

In the past, various strategies were pursued to generate perfused microvessels in vitro.19 Different  levels of complexity were achieved, ranging from straight or branched channels within a material such as polydimethylsiloxane (PDMS), toward more complex microvascular networks within extracellular matrix hydrogels. In order to study cell–cell and cell– matrix interactions, microfluidic chips were designed that comprised two or more parallel channels, allowing the seeding of different cell types or hydrogels next to each other.20-22 Chen et al. reported on an alternative approach to studying the interactions between endothelial cells and other cell types.23  Here, each cell type was seeded in a different layer of the microfluidic device, separated by porous membranes; perfusion was applied to the endothelial layer of the chip.23 The embedding of microvessels in a 3-D matrix, potentially mimicking the perivascular tissue, represents another level of complexity. Several groups have addressed this challenge by generating endothelial cell-aligned microchannels within a collagen hydrogel.24–28 Such a set-up also allows the incorporation of pericytes in the hydrogel, which might eventually reassemble at the perivascular site of the endothelial cell layer.24, 26, 28, 29

Conclusions

Microfluidic technologies permit the replication of geometrical features found in vivo (i.e. small channels mimicking capillaries). In addition, due to the small dimensions involved, screenings are much less costly and time-consuming, as compared to similar in vivo studies. This is particularly interesting for applications such as testing the delivery of drugs. For the in vitro models to be successful, it is critical to consider all parameters of the endothelial barrier, including cell–cell and cell–matrix interactions, and the surrounding perivascular tissue.

Acknowledgements

The research of the authors is supported by the AO Foundation and the 3R Research Foundation Switz erland (Reduction, Refinement and Replacement of animal experimentation).

Dr Laurent Barbe
CSEM
Bahnhofstrasse 1
7302 Landquart
Switzerland

Dr Mauro Alini
AO Research Institute
Clavadelerstrasse 8
7270 Davos Platz
Switzerland

Dr Marietta Herrmann
AO Research Institute
Clavadelerstrasse 8
7270 Davos Platz
Switzerland

Author for correspondence:
Dr Sophie Verrier
AO Research Institute
Clavadelerstrasse 8
7270 Davos Platz
Switzerland
E-mail: sophie.verrier@aofoundation.org

References
1 Armulik, A., Genové, G. & Betsholtz, C. (2011). Pericytes: Developmental, physiological,  and pathological perspectives, problems, and promises. Developmental Cell 21, 193–215.
2 Armulik, A., Abramsson, A. & Betsholtz, C. (2005). Endothelial/pericyte interactions. Circulation Res earch 97, 512–523.
3 Allt, G. & Lawrenson, J.G. (2001). Pericytes: Cell biology and pathology. Cells, Tissues, Organs 169, 1–11. P36
4 Aguilera, K.Y. & Brekken, R.A. (2014). Recruitment and retention: Factors that affect pericyte migration. Cellular & Molecular Life Sciences 71, 299–309.
5 Stark, K., Eckart, A., Haidari, S., Tirniceriu, A., Lorenz, M., von Brühl, M.L., Gärtner, F., Khandoga, A.G., Legate, K.R., Pless, R., Hepper, I., Lauber, K., Walzog, B. & Massberg, S. (2013). Capillary and arteriolar pericytes attract innate leukocytes exiting through venules
and ‘instruct’ them with pattern-recognition and motility programs. Nature Immunology 14, 41–51.
6 Johnson, J.R., Folestad, E., Rowley, J.E., Noll, E.M., Walker, S.A., Lloyd, C.M., Rankin, S.M., Pietras, K., Erik sson, U. & Fuxe, J. (2015). Pericytes contribute to airway remodeling in a mouse model of chronic allergic asthma. American Journal of Physiology. Lung Cellular
& Molecular Physiology 308, L658–L671.
7 Bai, Y., Zhu, X., Chao, J., Zhang, Y., Qian, C., Li, P., Liu,  D., Han, B., Zhao, L., Zhang, J., Buch, S., Teng, G., Hu, G. & Yao, H. (2015). Pericytes contribute to the disruption of the cerebral endothelial barrier via increasing VEGF expression: Implications for stroke. PLoS One 10, e0124362.
8 da Silva, M.L., Caplan, A.I. & Nardi, N.B. (2008). In search of the in vivo identity of mesenchymal stem cells. Stem Cells 26, 2287–2299.
9 Crisan, M., Yap, S., Casteilla, L., Chen, C.W., Corselli, M., Park, T.S., Andriolo, G., Sun, B., Zheng, B., Zhang, L., Norotte, C., Teng, P.N., Traas, J., Schugar, R., Deasy, B.M., Badylak, S., Buhring, H.J., Giacobino, J.P., Lazzari, L., Huard, J. & Péault, B. (2008). A perivascular
origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell 3, 301–313.
10 Chen, W.C., Park, T.S., Murray, I.R., Zimmerlin, L., Lazz ari, L., Huard, J. & Péault, B. (2013). Cellular kinetics of perivascular MSC precursors. Stem Cells International
2013, 983059.
11 Tidhar, A., Reichenstein, M., Cohen, D., Faerman, A., Copeland, N.G., Gilbert, D.J., Jenkins, N.A. & Shani, M. (2001). A novel transgenic marker for migrating limb muscle precursors and for vascular smooth muscle cells. Developmental Dynamics 220, 60–73.
12 Pfister, F., Feng, Y., vom Hagen, F., Hoffmann, S., Molema, G., Hillebrands, J.L., Shani, M., Deutsch, U. & Hammes, H.P. (2008). Pericyte migration: A novel mechanism of pericyte loss in experimental diabetic retinopathy. Diabetes 57, 2495–2502.
13 Rivera, L.B. & Brekken, R.A. (2011). SPARC promotes pericyte recruitment via inhibition of endoglin-dependent TGF-beta1 activity. Journal of Cell Biology 193, 1305–1319.
14 Hall-Glenn, F., De Young, R.A., Huang, B.L., van Handel, B., Hofmann, J.J., Chen, T.T., Choi, A., Ong, J.R., Benya, P.D., Mikkola, H., Iruela-Arispe, M.L. & Lyons, K.M. (2012). CCN2/connective tissue growth factor is essential for pericyte adhesion and endothelial basement membrane formation during angiogenesis. PLoS One 7, e30562.
15 Staton, C.A., Reed, M.W. & Brown, N.J. (2009). A critical analysis of current in vitro and in vivo angiogenesis assays. International Journal of Experimental Patholology 90, 195–221.
16 Bersini, S. & Moretti, M. (2015). 3D functional and perfusable microvascular networks for organotypic microfluidic models. Journal of Materials Science. Materials in Medicine 26, 5520.
17 Song, J.W. & Munn, L.L. (2011). Fluid forces control endothelial sprouting. Proceedings  of the National Academy of Sciences of the USA 108, 15,342–15,347. 18 Bae, H., Puranik, A.S., Gauvin, R., Edalat, F., Carrillo-Conde, B., Peppas, N.A. & Khademhosseini, A. (2012).
Building vascular networks. Science Translational Medicine 4,160ps123.
19 Zervantonakis, I.K., Kothapalli, C.R., Chung, S., Sudo, R. & Kamm, R.D. (2011). Microfluidic devices for studying heterotypic cell–cell interactions and tissue specimen cultures under controlled microenvironments. Biomicrofluidics 5, 13,406.
20 Bischel, L.L., Young, E.W., Mader, B.R. & Beebe, D.J. (2013). Tubeless microfluidic angiogenesis assay with three-dimensional endothelial-lined microvessels.
Biomaterials 34, 1471–1477.
21 Jeon, J.S., Zervantonakis, I.K., Chung, S., Kamm, R.D. & Charest, J.L. (2013). In vitro model of tumor cell extravasation. PLoS One 8, e56910.
22 Zervantonakis, I.K., Hughes-Alford, S.K., Charest, J.L., Condeelis, J.S., Gertler, F.B. & Kamm, R.D. (2012). Three-dimensional microfluidic model for tumor cell
intravasation and endothelial barrier function. Proceedings of the National Academy of Sciences of the USA 109, 13,515–13,520.
23 Chen, M.B., Srigunapalan, S., Wheeler, A.R. & Simmons, C.A. (2013). A 3D microfluidic platform incorporating methacrylated gelatin hydrogels to study physiological
cardiovascular cell–cell  interactions. Lab on a Chip 13, 2591–2598.
24Chrobak, K.M., Potter, D.R. & Tien, J. (2006). Formation of perfused, functional microvascular tubes in vitro. Microvascular Research 71, 185–196.
25 Price, G.M., Wong, K.H., Truslow, J.G., Leung, A.D., Acharya, C. & Tien, J. (2010). Effect of mechanical factors on the function of engineered human blood microvessels in microfluidic collagen gels. Biomaterials 31, 6182–6189.
26 van der Meer, A.D., Orlova, V.V., ten Dijke, P., van den Berg, A. & Mummery, C.L. (2013). Three-dimensional co-cultures of human endothelial cells and embryonic stem cell-derived pericytes inside a microfluidic device. Lab on a Chip 13, 3562–3568.
27 Morgan, J.P., Delnero, P.F., Zheng, Y., Verbridge, S.S., Chen, J., Craven, M., Choi, N.W., Diaz-Santana, A., Kermani, P., Hempstead, B., López, J.A., Corso, T.N., Fischbach, C. & Stroock, A.D. (2013). Formation of microvascular networks in vitro. Nature Protocols 8,
1820–1836.
28 Zheng, Y., Chen, J., Craven, M., Choi, N.W., Totorica, S., Diaz-Santana, A., Kermani, P., Hempstead, B., Fischbach- Teschl, C., López, J.A. & Stroock, A.D. (2012). In vitro
microvessels for the study  of angiogenesis and thrombosis. Proceedings of the National Academy of Sciences of the USA 109, 9342–9347.
29 Bichsel, C.A., Hall, S.R., Schmid, R.A., Guenat, O. & Geiser, T. (2015). Primary human lung pericytes support and stabilize in vitro perfusable microvessels. Tissue Engineering. Part A [E-pub ahead of print.]

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Single housing of primates in US laboratories: a growing problem with shrinking transparency

Alka Chandna, Michael Niebo, Stacy Lopresti-Goodman and Justin Goodman

Thirty years ago, the United States took steps to enhance
the psychological well-being of primates in laboratories,
including the introduction of social housing requirements.
Now, in an apparent response to questions about
the effectiveness of these measures, federal authorities
are completely shutting down public access to
information on the implementation of social housing

Historical context

In 1985, in response to high-profile cases documenting the mistreatment of non-human primates used in experiments, 1 the US Congress amended the federal Animal Welfare Act (AWA) to mandate that institutions take steps to promote the psychological wellbeing of primates in laboratories.2 As a result, the US Department of Agriculture (USDA) created regulations pertaining to environment enhancement for primates, including provisions aimed at addressing their social needs.3 In federal regulations and guidelines, social housing of primates is now deemed to be the ‘default’, with exemptions permitted only for veterinary or experimental reasons with appropriate documentation and approval.4 In cases in which an experiment-related exemption was granted, institutions have been required to submit these written and approved justifications to the USDA with their annual reports on animal use. These documents — which report the number of primates singly housed for experimental reasons, and why it was deemed necessary — were then made publicly available.

Animal welfare science

The USDA’s primate social housing requirements are evidence-based, as social housing is universally acknowledged to be a key factor in the welfare of primates in laboratories. Moreover, it is well documented that housing primates alone, or single housing, is detrimental to their development, physical health, and psychological well-being. In rhesus macaques, physical contact with conspecifics is essential to normal development, and the amount of time spent caged alone is a significant predictor of stereotypic and self-injurious behaviour — including repeated pacing, circling, hyper-aggression, depression, hair plucking, or self-biting.5, 6  Psychological distress stemming from being caged alone has also been documented in cynomolgus monkeys,7  pigtailed macaques,8 chimpanzees,9 – 11 and baboons.12, 13 In one modified preference test involving capuchin monkeys, the value of social companionship was so high that the primates chose it in lieu of food.14 Singly housed primates have also been documented as having suffered from physiological abnormalities, including depressed immune function and higher incidence of coronary atherosclerosis.15, 16

Surveys on housing of non-human primates

Despite this evidence of the harms caused by single housing, as well as federal regulations and guidelines promoting social housing, the data indicate that many primates in US laboratories continue to be housed alone. A 2000–2001 USDA survey found that 34.7% of primates in US laboratories were housed individually — although the USDA admitted that this was likely a low estimate, as primates who had been housed only temporarily with other primates for breeding purposes were classified as being socially housed.17 A 2003 survey found that 54% of primates at 22 laboratories were singly housed — although this was also likely to have been a low estimate, as the study ill-advisedly included in its definition of “social housing” instances of “grooming-contact” housing, in which singly housed primates have some limited tactile contact with one another through barred or mesh barriers.18 A survey of primate housing at the Washington National Primate Research Center from 2004 to 2006 found that at least 63% of the monkeys were singly caged.19

An ongoing concern is that laboratories sometimes permit single housing of primates for the sake of convenience rather than necessity. For example, many laboratories singly house primates who have had surgical implants, such as head posts or other equipment, even though it is possible to successfully house them socially.20

Given the government mandate that social housing of primates in laboratories should be the default position, the aforementioned figures are cause for concern, particularly because they indicate that rates of single housing may be increasing.

Preliminary analysis of primate single-housing data

In the interest of conducting a current and more comprehensive evaluation of trends in primate single housing and the justifications provided, we attempted to undertake a new analysis of all single housing exceptions submitted in annual reports by laboratories to the USDA from 2010 to 2013 — the years for which data are currently available online. While the total number of facilities that confined primates (191 in 2010, 188 in 2011, 192 in 2012, and 184 in 2013) stayed relatively flat over the four years, there was a steady increase in the number of facilities that reported single-housing exceptions, from 30 (16%) in 2010 to 53 (29%) in 2013. However, when we attempted to look more closely at these exceptions, in order to determine trends in the numbers of singly housed primates and the  explanations given, we found glaring inadequacies in the data available on the USDA site. This inadequacy stemmed mainly from the failure of laboratories in meeting reporting requirements. From 2010 to 2013, the percentage of laboratories reporting singly
housed primates that failed to specify the number of primates singly housed for experimental reasons and the scientific justification for it — both required by law — increased from 36% to 47%. Worryingly, it also appeared that some, or all, of the required information was improperly redacted from many facilities’ reports. We also observed that several facilities that had provided very detailed exception letters in 2010 produced only very vague information in 2013 or, as noted above, redacted all the information, showing
a growing trend toward secrecy.

Government response

In December 2014, we informed the USDA of the problematic reports and requested the agency’s assistance  in securing the missing data. In February 2015, we received correspondence from the USDA (a personal communication), which stated, “We have had
discussions and are finalising our position.” We heard nothing further, but in March, the USDA sent an email to all its stakeholders announcing changes to its Inspection Guide — used by its inspectors to identify violations of the AWA at the facilities they inspect.
The announcement noted that the revisions included a “more consistent procedure for reporting exceptions and exemptions on the Annual Report.”21 Upon closer examination, it became clear that the revised inspection guide now specified that exemption of a primate from “some or all of the environmental enhancement plan” — which would include social housing — “should not be reported on the Annual Report” [emphasis in the original].22 Prior to the recent revision to the inspection guide, it was standard operating procedure for laboratories to report single housing of primates.

Rather than ensuring that laboratories were properly reporting single housing of primates, the USDA instead — we suspect in consultation with the laboratory community — took the backward step of simply exempting facilities from having to submit this information at all.

Discussion

A 2011 study seeking to assess the effectiveness of the 1985 AWA primate psychological health amendments determined that “the current system of laboratory animal care and record keeping is inadequate to properly assess AWA impacts on primate psychological wellbeing and that more is required to ensure the psychological well-being of primates.”17 It was already difficult to ascertain this information, and now the USDA has made this task virtually impossible. Even with the reporting requirement in place, laboratories were often failing to approve, document, and report the single housing of primates. For instance, a 2011 USDA inspection report for a US contract laboratory cited the company for singly housing 83% of the more than 6,000 primates at the facility  without securing the necessary justifications or reporting the matter to the USDA.23,24 This number amounts to more than 4% of all primates housed in US laboratories.

Any previously reported figures are also underestimations, because institutions have only been required to report the numbers of primates who are singly housed for ‘experimental’, but not veterinary, reasons; this could account for a third more singly housed primates.25

Conclusions

On the 30th anniversary of the amendments to promote the psychological well-being of primates, the evidence that singly housed primates suffer is overwhelming, as is the proof that the US government and laboratories are failing to confront this rapidly growing problem effectively. The USDA’s recent revisions to reporting requirements will limit the availability of data, and consequently will stifle informed debate on the suffering of primates in laboratories and failures of the existing regulatory system. To address this issue meaningfully, we need more transparency and accountability, not less.

Author for correspondence:
Dr Alka Chandna
People for the Ethical Treatment of Animals
501 Front Street
Norfolk, VA 23510
USA
E-mail: AlkaC@peta.org

Michael Niebo
People for the Ethical Treatment of Animals
501 Front Street
Norfolk, VA 23510
USA

Dr Stacy Lopresti-Goodman
Department of Psychology
Marymount University
2807 N. Glebe Road
Arlington, VA 22207
USA

Justin Goodman
People for the Ethical Treatment of Animals
501 Front Street
Norfolk, VA 23510
USA
and
Department of Sociology
Marymount University
2807 N. Glebe Road
Arlington, VA 22207
USA

References

1 Carbone, L. (2004). What Animals Want: Expertise and Advocacy in Laboratory Animal Welfare Policy, 304pp. Oxford, UK: Oxford University Press.
2 US Congress (1985). Public Law 99-198, Food Security Act of 1985, Subtitle F — Animal Welfare. Title XVII. Available at: https://awic.nal.usda.gov/public-law-99-198-food-security-act-1985-subtitle-f-animalwelfare (Accessed 31.05.15).
3 US Department of Agriculture (1991). Final Rules: Animal Welfare; Title 9, CFR (Code of Federal Register) Part 3. Standards. Federal Register 55 (No. 32), 6426–6505. Available at: https://awic.nal.usda. gov/final-rules-animal-welfare-9-cfr-part-3 (Accessed 31.05.15).
4 National Research Council (2011). Guide for the Care and Use of Laboratory Animals: Eighth Edition, 248pp. Washington, DC, USA: The National Academies Press.
5 Baker, K.C., Bloomsmith, M.A., Oettinger, B., Neu, K., Griffis, C., Schoof, V. & Maloney, M. (2012). Benefits of pair housing are consistent across a diverse population of rhesus macaques. Applied Animal Behaviour Science 137, 148–156.
6 Bayne, K. (2005). Macaques. From the booklet series Enrichment for Nonhuman Primates. Washington, DC, USA: Department of Health and Human Services. Available at: http://grants.nih.gov/grants/olaw/ Enrichment_for_Nonhuman_Primates.pdf (Accessed 05.04.15).
7 Shively, C.A., Clarkson, T.B. & Kaplan, J.R. (1989). Social deprivation and coronary artery atherosclerosis in female cynomolgus monkeys. Atherosclerosis 77, 69–76.
8 Bellanca, R.U. & Crockett, C.M. (2002). Factors predicting increased incidence of abnormal behavior in male pigtailed macaques. American Journal of Primatology 58, 57–69.
9 Brent, L., Lee, D.R. & Eichberg, J.W. (1989). The effects of single caging on chimpanzee behavior. Laboratory Animal Science 39, 345–346.
10 Kalcher, E., Franz, C., Crailsheim, K. & Preuschoft, S. (2008). Differential onset of infantile deprivation produces distinctive long‐term effects in adult ex‐laboratory chimpanzees (Pan troglodytes). Developmental Psychobiology 50, 777–788.
11 Nash, L.T., Fritz, J., Alford, P.A. & Brent, L. (1999). Variables influencing the origins of diverse abnormal behaviors in a large sample of captive chimpanzees (Pan troglodytes). American Journal of Primatology 48, 15–29.
12 Coelho, A.M., Carey, K.D., & Shade, R.E. (1991). Assessing the effects of social environment on blood pressure and heart rates of baboons. American Journal of Primatology 23, 257–267.
13 Kessel, A. & Brent, L. (2001). The rehabilitation of captive baboons. Journal of Medical Primatology 30, 71–80.
14 Dettmer, E. & Fragaszy, D. (2000). Determining the value of social companionship to captive tufted capuchin monkeys (Cebus apella). Journal of Applied Animal Welfare Science 3, 293–304.
15 Lilly, A.A., Mehlman, P.T. & Higley, J.D. (1999). Traitlike immunological and hematological measures in female rhesus across varied environmental conditions. American Journal of Primatology 48, 197–223.
16 Doyle, L.A., Baker, K.C. & Cox, L.D. (2008). Physiological and behavioral effects of social introduction on adult male rhesus macaques. American Journal of Primatology 70, 542–550.
17 Balcombe, J., Ferdowsian, H. & Durham, D. (2011). Self-harm in laboratory-housed primates: Where is the evidence that the Animal Welfare Act amendment has worked? Journal of Applied Animal Welfare Science 14, 361–370.
18 Baker, K.C., Weed, J.L., Crockett, C.M. & Bloomsmith, M.A. (2007). Survey of environmental enhancement programs for laboratory primates. American Journal of Primatology 69, 377–394.
19 Thom, J.P. & Crockett, C.M. (2008). Managing environmental enhancement plans for individual research projects at a national primate research center. Journal of the American Association for Laboratory Animal Science 47, 51.
20 DiVincenti, L., Jr & Wyatt, J.D. (2011). Pair housing of macaques in research facilities: A science-based review of benefits and risks. Journal of the American Association for Laboratory Animal Science 50, 856.
21 USDA (2015). USDA Revises Its Inspection Guide to Improve Oversight of Research Facilities. Washington, DC, USA: United States Department of Agriculture. Available at: http://content.govdelivery.com/ accounts/USDAAPHIS/bulletins/f4e94e (Accessed 31. 05.15).
22 USDA (2015). Animal Welfare Inspection Guide, 424pp. Washington, DC, USA: United States Department of Agriculture. Available at: http://www.aphis. uda.gov/animal_welfare/downloads/Animal%20Care %20Inspection%20Guide.pdf (Accessed 31.05.15).
23 USDA (2011). Inspection Report for SNBL USA, Ltd. Washington, DC, USA: United States Department of Agriculture. Available at: http://www.mediapeta. com/peta/PDF/July132011-78percent singly housed.pdf (Accessed 25.06.15).
24 USDA (2011). Annual Report for SNBL USA, Ltd. Washington, DC, USA: United States Department of Agriculture. Available at: http://www.mediapeta. com/peta/PDF/SNBL-AnnualReportfor2011.pdf
(Accessed 25.06.15).
25 Lee, D.R. (2013). Social housing strategies for nonhuman primates [PowerPoint slides]. Available at: http://www.aclam.org/content/files/files/forum2013/aclam_forum_2013_lee_social.pdf (Accessed 05.04. 15).

Download a pdf of the article here: Primate single housing.

Renewed concern about the welfare of laboratory primates

PiLAS staff writer

One of the key points about the Three Rs is that, as Russell and Burch emphasised in The Principles of Humane Experimental Technique,1 “the humanest treatment of animals, far from being an obstacle, is actually a prerequisite for successful animal experiments”. Indeed, they said, the “wages of inhumanity” are “paid in ambiguous or otherwise unsatisfactory experimental results”. The PiLAS article by Chandna et al. on the commonplace single housing of primates in US laboratories,2 is therefore a matter of great concern, for both scientific and humanitarian reasons. It even appears that, far from trying to solve the problem, the US Government may be trying to cover it up.

In the UK, the NC3Rs is putting a great deal of effort into improving the welfare of the non-human primates used for research,3 but one has to wonder what fundamental and significant changes have taken place since the Home Secretary of the time, Douglas Hurd, accepted all but one of 17 proposals put to him by FRAME and CRAE (Committee for the Reform of Animal Experimentation) on the day that the Animals Scientific Procedures Act 1986 came into force.4,5 One of the points highlighted by FRAME and CRAE6 was that: “The very nature of a primate is such that you cannot institutionalise it in the laboratory and have a healthy animal. A primate is such that isolating in itself is deleterious.”

The current initiatives of the NC3Rs deserve to be applauded, but why do non-human primates continue to be used in research and testing at all, and how relevant and reliable are the data obtained to the understanding and treatment of diseases? Commenting on a recent report that the cynomolgus macaque is resistant to doses of paracetamol that would be fatal in humans,7 FRAME’s Scientific Director, Gerry Kenna, said in FRAME News that “This new research raises significant concern about the scientific validity to humans of drug safety studies undertaken in primates. The use of non-human primates in non-clinical safety testing is ethically undesirable and, in view of the substantial cost of such studies, can be expected to increase, markedly, the cost of drug development. Such studies should be considered only if they can be shown to be scientifically justifiable and there are no valid alternatives.”

The FRAME News item concluded by saying that “More time and money should be invested in cell-based and computer models that would be more reliable.”

1 Russell, W.M.S. & Burch, R.L. (1959). The Principles of Humane Experimental Technique, 238pp. London, UK: Methuen.
2 Chandna, A., Niebo, M., Lopresti-Goodman, S. & Goodman, J. (2015). Single housing of primates in US laboratories: A growing problem with shrinking transparency. ATLA 43, P30–P33.
3 Anon. (2015). The Welfare of Non-human Primates, 16pp. London, UK: NC3Rs. Available at: https://www.nc3rs.org.uk/welfare-non-human-primates (Accessed 16.07.15).
4 Anon. (1987). The Use of Non-Human Primates as Laboratory Animals in Great Britain, 16pp. Nottingham, UK, and Edinburgh, UK: FRAME and CRAE.
5 Anon. (1987/88). Response of the Home Secretary to the FRAME/CRAE primates report. FRAME News 17, 6−9.
6 Chivers, D. (1984). Comment in discussion session on Laboratory Primates. In Standards in Laboratory Animal Management, pp. 272. Potters Bar, UK: UFAW.
7 FRAME. (2015). Non-human primates and drug testing. FRAME News 74, 3.

Download a pdf of this article here.

Turning Apples into Oranges? The Harm–Benefit Analysis and How to Take Ethical Considerations into Account

Herwig Grimm

How can expected study benefits and animal harms be weighed
against each other? What is the unit and common currency
that allows this weighing to be performed?

Suppose you are a scientist, working in the field of oncology and using live animals in your studies. Furthermore, suppose you have an excellent track record, you are well respected in the research community, and you regularly publish in high-ranking journals. One day, a person that you have not met before, wants to see you and talk about your work. Despite the fact that your time is extremely scarce, you invite this very person to your office and postpone your work on a follow-up research proposal. It turns out that the person who wants to talk to you is a member of a major animal protection group. She asks you the following question: “I came across a project summary, published according to Article 43 of Directive 2010/63/EU. Knowing your research, I think it is your project. Can you ethically justify the use of animals in your work? What I mean is: Do the benefits really outweigh the harms? And which ethical considerations do you take into account?” The animal protectionist is actually asking something for which you should, in fact, be well prepared. Directive 2010/63/EU1 was transposed into the national laws of the EU Member States. In Article 38(2) of the Directive, it is emphasised that a harm–benefit analysis of any project involving the use of animals must be carried out, in order to assess whether the harms related to the project are outweighed by the expected benefits. Furthermore, the relevant passage stipulates that ethical considerations have to be taken into account in this assessment (emphasis added by the current author):

“The project evaluation shall consist in particular of the following: d) a harm–benefit analysis of the project, to assess whether the harm to the animals in terms of suffering, pain and distress, is justified by the expected outcome taking into account ethical considerations, and may ultimately benefit human beings, animals or the environment.”

Consequently, the question arises as to how the harm–benefit analysis can be carried out, and how the term “taking into account ethical considerations” might be understood in this context. The meaning of this term is of major importance, since it provides a legally binding basis for the approval or rejection of projects. In other words, everyone who aims to secure the authorisation of a project in the EU has to make sure that the expected benefits outweigh the harms, and the justification must take into account ethical considerations, whatever that in fact means.All this has to be done on legal grounds — it is not just some fancy idea of animal protectionists.
The harm–benefit analysis:
A challenge or mission impossible?

At present, and to my knowledge, it is not at all clear how to prove, in a transparent and objective manner, that the expected benefits of an experimental study outweigh the expected harm to the animals to be used. Moreover, the actual meaning of the term “ethical considerations” remains vague, to say the least. How can expected study benefits and animal harms be weighed against each other? What is the unit and common currency that allows this weighing to be performed? And can ‘ethics’ help to turn apples into oranges, so that only comparable weights are on the scales? Since the Directive does not provide any specifications on standards for the harm–benefit analysis and how to take ethical considerations into account, the passage invites the reader to speculate.

The working document on Project Evaluation and Retrospective Assessment (WD 2013),2 from September 2013, is only of limited help. It provides important criteria and ideas, but it leaves the reader without help when it comes to a methodology for transparent decision-making. It refers to the Bateson Cube, which indicates what should be taken into account, but whether its dimensions (i.e. benefit, likelihood of benefit, harm to animals) are ethical in nature, and whether these dimensions are sufficient for the harm–benefit analysis, remains open to question. Furthermore, no measure is provided to allow the various dimensions to be made comparable. Take, for example, ‘benefit’. Here, a set of analytic questions is given (WD 2013, p. 21):
What will be the benefits of the work?
Who will benefit from the work?
How will they benefit/impact?
When (where possible) will the benefits be achieved?

But, even if we had the answers to all these questions, how can they be integrated in the harm–benefit analysis? Does this mean that there should be no research on orphan diseases, because only few people can benefit? Does it mean that it matters who benefits in terms of age? Does this mean that a new cold remedy is more important than a new cancer treatment, since many more people will use it and it therefore has a greater impact? Is research more important, if it will bring about practical benefits sooner, and should this influence the harm–benefit analysis?

Similar questions arise on the harm side. Is the severity classification enough? Shall we add all harms done to individual animals, and if so, how do we deal with harms that are related to the project indirectly, such as harm to animals that were necessary to establish the particular mouse strain used? Is the absolute number of animals used in an experiment something that should be taken into account — or is it acceptable to adhere to the Three Rs criterion of reduction, and to use the minimum number of animals? And if the absolute number should count, what is a ‘high’ number (100 dogs, or 12,000 mice?) and does ‘high’ vary, depending on the research field in question? And if we knew all that and more, how could we bring all these criteria into one methodology, in order to carry out a transparent harm–benefit analysis? At the moment, this seems to be a mission impossible, rather than a challenge to be dealt with.

Steps to tackle the problem: Criteria, methodologies and committees

One could of course go on and on with this list of open questions. In order to answer at least some of them, researchers from various fields — and in particular, ethicists — try to take on the challenge. For example, the Messerli Research Institute (Vienna, Austria) hosted an international symposium in March 2013, in order to discuss possible steps toward overcoming the aforementioned problems. A conference on the harm–benefit analysis was also held in Bergen in 2014, and we debated the issues at the World Congress on Alternatives and Animal Use in the Life Sciences, held in Prague in late 2014. Many well-known experts in the field of ethical evaluation of animal experiments took part. At all these meetings, the aim was to bring together state-of-the-art knowledge with regard to the issues. For example, in Vienna, 22 speakers from eight European countries and the USA discussed their experiences and the current situation surrounding these issues in their respective countries. The lively discussions went to show that many challenges remain, but some issues can be solved.

In the course of the Vienna symposium, not only the criteria and aspects that should go into the harm–benefit analyses were debated. Importantly, different methodologies such as checklists, scoring systems or comparative methodologies, were also introduced. Most of the experts emphasised the importance of independent and well-balanced committees, and the integration of lay people (i.e. non-specialists) and representatives of animal welfare organisations into these committees. Taking into account the lay people’s perspectives and current public opinion would contribute to an up-to-date ethical evaluation of animal experiments. But, by taking all of these factors into account, are we any way nearer solving the problem of how to transparently weigh apples against oranges successfully?

Although no ‘super-theory’ to resolve all of the issues was identified, the challenges became much clearer. Furthermore, things to be avoided came to the table: A particular and major threat that has to be avoided when developing methodologies for the harm–benefit analysis became very clear, and that is over-bureaucratisation. Any methodology for the harm–benefit analysis has to be a user-friendly tool that leads to deeper reflection on individual animal experiments. The different forms of methodologies were summarised in the following three groups:

— comparative methodologies that use positive lists (white-lists) and negative lists (black-lists) of animal experiments, in order to evaluate the project in question;
— scoring strategies that quantify the extent to which relevant criteria are met, and that provide an algorithm for calculating the harms and benefits of projects; and
— check lists that provide binary (yes/no) evaluation methods, e.g. in the form of decision trees.

Whether these methods are used within or without the committee structure makes a big difference, and both scenarios are indeed possible. Ideally, applicants should follow a structured procedure and provide the relevant information according to a set of clear standards and criteria that have to be met. Interdisciplinary committees would then be able to evaluate the projects according to the same standards and criteria. These evaluations could inform the competent authority’s decisions.

Many things could be said, and indeed have been said in the past, about methodologies, and a great deal has also been written on the subject. Needless to say, we did not come to any final conclusions at the symposium in Vienna, nor in Prague, nor in Bergen, on this complex but vital matter.

How to proceed from here?

In order to reach a clearer vision of how the harm–benefit analysis can be brought into a feasible methodology, any ideas are welcome. Exchanging ideas and arguments might inspire and boost the debate. This short article serves as an open invitation to all interested experts in the field to start such a debate. Since this should happen in a focused way, the following topics might be useful to guide the discussion:

Committees and their limitations and advantages: A great number of EU Member States have established local and national committees to support the authorities in decision-making on submitted proposals. Certainly, such committees have the advantage of bringing skilled experts from the sciences, statisticians, representatives of animal protection groups and lay people, to work together in order to formulate a statement on harms and expected benefits. However, these committees often work without explicit methodology or criteria. So the question arises as to how they can safeguard transparent and non-arbitrary decision-making when they carry out harm–benefit analyses. I am sure that many of the readers are experienced members of such committees, and it would be very useful, if they would contribute with their experience, knowledge and ideas.
Methodologies: It would be of great interest to share knowledge on the advantages and disadvantages of methods used. If committees and the national authorities apply consistent methods and explicit criteria, it would be of utmost importance to get into an exchange of views and experience on whether and how such methods can support and improve the decision-making process.
Ethics and Law: A third question relates to the terminology used in the Directive. If ethical considerations should be taken into account, should these considerations exceed existing law or is “ethics” to be understood within legal limits (and is not allowed to exceed existing law)? Here, ethics runs the risk of contradicting the principle of legality in constitutional states. In other words: How is the term “taking ethical considerations into account” interpreted in different countries. It would be great to get some insight into this.
Experience from the past: Generally, since many countries have carried out harm–benefit analyses in the past, knowledge of their experiences could contribute to future developments.
Ideas for the future: Finally, a possible thought experiment is to think about where we are going to be in 20 years’ time. How will the debate look in 2035? Will we still be trying to weigh apples against oranges?

These questions and statements aim to initiate a debate that is relevant to all EU Member States and everybody involved in animal research. It would be very useful, if experts in this forum were willing to find some time to contribute to a lively and future oriented discussion, in order to solve at least some of the open questions mentioned above. The idea is to continue to build up knowledge on the process of harm–benefit analysis in animal research, and maybe improve the situation for both animals and researchers. Perhaps this forum could bring us closer to the point where researchers were able to respond to the question as to whether, and indeed which, projects involving live animals are justifiable, and which are not. Being able to respond to the question as to whether a project is worth carrying out or not, could demonstrate that scientists are able to take on this responsibility in a knowledge-based society and thus can contribute to ethical welfare.

Prof. Dr Herwig Grimm
Messerli Research Institute
Veterinary University of Vienna,
Medical University Vienna,
and University of Vienna
Veterinärplatz 1
1210 Vienna
Austria
E-mail: herwig.grimm@vetmeduni.ac.at

References
1 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, 22.10.2010, 33–79.
2 Anon. (2013). National Competent Authorities for the Implementation of Directive 2010/63/EU on the Protection of Animals Used for Scientific Purposes. Working Document on Project Evaluation and Retrospective Assessment, 42pp. Brussels, Belgium: European Commission.

Download a pdf of the article here: Discussion Grimm.

The Three Rs: The Way Forward

Michael Balls

It is now 20 years since Russell and Burch
last met to discuss, with others,
the way forward for the Three Rs concept

 

Sitting at my computer, faced with the challenge of deciding what I could say in Wisdom 16, I suddenly realised that it was almost 20 years, to the day, since Bill Russell and Rex Burch met in Sheringham, a small seaside town in Norfolk, UK, for the first scientific meeting they had attended together since the publication of The Principles in 1959 (Figure 1). Sadly, it was also to be the last such meeting, as Rex died a few months later.

Rex Burch and Bill Russell 31 May 1995 and Ecvam Workshop report

 

The meeting took place on 31 May to 3 June 1995, in the form of an e, which I chaired in partnership with Alan Goldberg of CAAT. Our reason for being in Sheringham was that Rex was too ill to go more than a few miles from home, so Alan and I decided that we would invite some of those committed to the Three Rs, to travel to meet him. The other participants included Claire Russell, and some of our colleagues from Germany, Italy, The Netherlands, the UK and the USA (Figure 2).

Workshop participants May 1995

 

An opening ceremony was held in the council chamber of Sheringham Town Hall, where Rex had rented space for his microbiology laboratory since the early 1970s. All the participants made a brief statement, and Bill sang a song, as he always did on special occasions. These opening proceedings were recorded on videotape. The rest of the workshop took place at the Links Country House Hotel in West Runton, about a mile from where I now live.

The principal aims of the workshop were 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, and, in the interests of both scientific excellence and the highest standards of animal welfare, the more active implementation of reduction alternatives, refinement alternatives and replacement alternatives.

The report of the workshop was published in the November/December 1995 issue of ATLA.1 It reviewed the origins and evolution of the Three Rs concept as originally outlined in The Principles,2 the selection of appropriate animal species, reduction alternatives, refinement alternatives and replacement alternatives, education and training, and certain special considerations (vaccines and immunobiologicals, transgenic animals, special protection for selected animals, benefit and suffering, and the setting of targets). It concluded with 58 conclusions and recommendations, which were preceded by the following remarks: The workshop participants unanimously reaffirmed the principles put forward by Russell & Burch, that humane science is good science and that this is best achieved by vigorous application of the Three Rs: reduction alternatives, refinement alternatives and replacement alternatives.

Thus, the only acceptable animal experiment is one which uses the smallest possible number of animals and causes the least possible pain or distress which is consistent with the achievement of a justifiable scientific purpose, and which is necessary because there is no other way of achieving that purpose. Any proposed experiments on animals should be subjected to prior and effective expert review by an ethics committee or an equivalent body. The Three Rs should be seen as a challenge and as an opportunity for reaping benefits of every kind — scientific, economic and humanitarian — not as a threat.

Many of the conclusions and recommendations are as relevant today as they were in 1995. The workshop was a memorable occasion in many other ways. It was run according to the ECVAM tradition — five days of hard work, interspersed with good food and good wine, with a determination to have the words of detailed conclusions and recommendations down on paper by the end. One of my lasting memories will be witnessing the pleasure shown by Bill and Rex, as they took the opportunity to sit and talk quietly together after a gap of more than 30 years. All I have to show of that is one out-of-focus photograph, taken during the final reception and dinner at Blickling Hall, on 2 June 1995 (Figure 3).

Bill Russell and Rex Burch 2 June 1995

Many developments of many kinds have taken place since 1995, but, as Roman Kolar spells out in the latest issue of ATLA,3 there is still much to be achieved, if the aims of the workshop and of The Three Rs Declaration of Bologna,4 to which it led, are to be achieved, resulting in the revolution in biomedical research and its application which was proposed in The Principles. For my own part, I am concerned that stating allegiance to the Three Rs concept has become a convenient smokescreen, to which lip service can be paid, whilst little is actually permitted to change. I have therefore proposed that the focus should now be more squarely on humane science, which avoids the problem of seeming conflicts between human benefit and animal welfare,5 and I am rather confident that Bill and Rex would approve.

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 Balls, M., Goldberg, A.M., Fentem, J.H., Broadhead, C.L., Burch, R.L., Festing, M.F.W., Frazier, J.M., Hendriksen, C.F.M., Jennings, M., van der Kamp, M.D.O., Morton, D.B., Rowan, A.N., Russell, C., Russell, W.M.S., Spielmann, H., Stephens, M.L., Stokes, W.S., Straughan, D.W., Yager, J.D., Zurlo, J. & van Zutphen, B.F.M. (1995). The Three Rs: The way forward. The report and recommendations of ECVAM Workshop 11. ATLA 23, 838-866.
2 Russell, W.M.S. & Burch, R.L. (1959). The Principles of Humane Experimental Technique, xiv + 238pp. London, UK: Methuen.
3 Kolar, R. (2015). How long must they suffer? Success and failure of our efforts to end the animal tragedy in laboratories. ATLA 43, 129-143.
4 Anon. (2000). The Three Rs Declaration of Bologna. ATLA 28, 1-5.
5 Balls, M. (2014). Animal experimentation and alternatives: Time to say goodbye to the Three Rs and hello to humanity? ATLA 42, 27-333.

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.

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Wisdom 16

In Vitro Methodologies in Ecotoxicological Hazard Assessment: The Case of Bioaccumulation Testing for Fish

Helmut Segner

The concerted research efforts undertaken in recent years have
highlighted the potential of in vitro approaches, as part of an
integrated testing strategy, to replace or reduce in vivo bioaccumulation testing in fish

Worldwide programmes for the regulation of chemicals require an assessment of the risks of chemicals to human and environmental health based on three categories of concern: Persistence, Bioaccumulation and Toxicity (PBT). Among these three categories, bioaccumulation refers to the enrichment of environmental chemicals in organisms. It encompasses the absorption, distribution, metabolism and excretion (ADME) of a chemical inside an organism, and ultimately determines the internal toxic dose. For the aquatic environment, the most widely used parameter to estimate the bioaccumulation potential of a chemical is the so-called Bioconcentration Factor (BCF). The BCF represents the ratio of the steadystate chemical concentration in the organism and the chemical concentration in the respiratory medium, i.e. water. For the experimental determination of the BCF, the test procedure as described in OECD Test Guideline 3051 represents the current ‘gold standard’. In this test, fish are exposed to a chemical for 28 days, to reach an equilibrium of chemical concentration between fish and water, followed by a 28-day depuration period to measure the elimination rate. This test, in addition to being lengthy and costly, requires a high number of animals (> 100 fish per test).

Regulatory programmes require bioaccumulation information for chemicals which are lipophilic (for example, those with a log Kow > 3), and which are produced at a certain tonnage (for example, the European Community REACH Regulation requires BCF information for lipophilic chemicals that are produced at > 100 tonnes per year). Experi mentally determined BCF data are not available for the vast majority of existing compounds. For instance,  in a Canadian investigation of 23,000 existing chemicals, it was found that bioaccumulation data existed for less than 4% of them (cf. Nichols et al.2). If the missing BCF data had to be generated by means of the OECD 305 test, this would entail a drastic increase in animal use.3,4 Therefore, there is an urgent need to develop alternative methods to reduce the number of fish used for in vivo bioaccumulation testing. The bioconcentration of chemicals in fish results from the competing rates of chemical uptake via the gills and skin (k1) and chemical elimination via respiratory exchange (k2), faecal egestion (ke) and metabolic biotransformation (km).5 In addition, dilution as a result of growth (kd) can influence bioconcentration.

With the involvement of these different processes, it is clear that non-animal approaches to bioconcentration assessment cannot be based on one single method, but have to rely on an array of methodologies.2,4,6 An initial non-animal based approximation of the bioconcentration potential of an organic chemical in aquatic organisms can be obtained from an in silico hydrophobicity model, which considers bioconcentration as a passive partitioning process resulting from the competing uptake and elimination processes. In this model, bioconcentration can be predicted from the lipophilicity of a chemical, as estimated from its octanol–water partition coefficient, Kow.5 Also, it can actually be measured by using artificial membranes which simulate the passive diffusion processes across the respiratory epithelia.7

The development of in vitro methods

Diffusion-based methodologies have proven instrumental in the prediction of the BCF values of lipophilic chemicals that undergo no endogenous metabolism in the organism. However, as they are not able to take into account chemical loss due to biotransformation (km), they overestimate the BCF values of metabolisable xenobiotics. To correct for the influence of biotransformation on fish BCF values, a possible approach is the use of metabolically competent in vitro assays that show which chemicals are biotransformed, and at what rates. In mammalian toxicology, in vitro assays for the analysis of xenobiotic metabolism largely rely on liver preparations such as subcellular liver fractions (S9, microsomes) and isolated hepatocytes, as the liver is the organ with the highest metabolic activity. Corresponding technologies are also available for fish, and it has been demonstrated that they are suitable for determining biotransformation parameters (see Segner & Cravedi8 and Fitzimmons et al.9). However, their reproducibility and their capability of predicting in vivo BCF values remain to be demonstrated.

In recent years, intensive efforts have been undertaken — largely coordinated by the Health and Environmental Sciences Institute (HESI) — to advance the development of piscine in vitro assays for regulatory purposes. After an initial phase of reviewing the available knowledge and technologies,2,6 in the next step, standardised protocols for liver S9 preparations and isolated hepatocytes from rainbow trout were established.10,11 A major drawback experienced in these studies, particularly with freshly-isolated hepatocyte suspensions, was the between-isolate variability of metabolic capabilities, which is related to factors such as seasonal oscillation, and the nutritional status, gender or genetic background of the donor fishes. Here, a major step forward was the introduction of a cryopreservation method for fish hepatocytes,12 enabling the year-round provision of uniform batches of metabolically characterised hepatocytes to laboratories worldwide. By using a standardised assay protocol, Fay et al.13 recently performed an international ring study with cryopreserved rainbow trout hepatocytes, and were able to demonstrate the good interlaboratory and intra-laboratory reproducibility of the metabolic rate values obtained with the in vitro hepatocyte assay.

To be able to extrapolate from the metabolic rate values measured in the isolated fish hepatocytes to the metabolic rate value (km) in the intact fish, physiologically-based prediction models were developed. 14,15 These models initially scale from the clearance rate of the isolated liver cells to that of the whole liver, and from there to the metabolic transformation rate of the whole fish. The predicted km values are then used to calculate the in vivo BCF value of the test chemical. Currently, the availability of data on BCF values  predicted from in vitro assays is still limited, and it is still too early to come up with a conclusive statement on the predictability of the in vitro approach, partly also because of the variable quality of the in vivo BCF data; however, the existing results look promising.

Looking to the future

There are lessons to be learned from the recent development of in vitro assays as components of alternative integrated testing strategies for the assessment of bioaccumulation in fish. Although a broad spectrum of in vitro assays and methods have been available in ecotoxicology for a while,16 they have never made their way to regulatory implementation. Partly, this is due to the fact that they were considered to be technically not ready nor sufficiently standardised. In the case of the piscine in vitro metabolism assays, this obstacle has been overcome through targeted and internationally concerted research efforts on the standardisation and harmonisation of the assay protocols. Another constraint to the regulatory acceptance of in vitro assays is that they were considered not to be appropriate for the protection goals of ecotoxicology, which are ecological entities such as populations and communities. However, ecotoxicological hazard assessment largely relies on classical toxicity tests for measuring organism-level endpoints such as lethality (cf. Segner17), and these endpoints may well be predictable by in vitro assays, provided that: a) the in vitro assays are rationally selected to represent the critical toxicological processes; b) the assays are standardised; and c) valid extrapolation models are available. These requirements are fulfilled in the case of bioaccumulation assessment — i.e. the in vitro assays measure biotransformation as the critical toxicokinetic process, they are standardised, and there exist physiologically-based models for the scaling of the in vitro metabolic rate values to the in vivo metabolic rates. As ecotoxicology deals with a huge diversity of species, the interspecies scaling of metabolic rates is another critical issue, but this question is also currently under investigation. In conclusion, the concerted research efforts undertaken in recent years have substantially moved the field ahead, and the results obtained highlight the potential of in vitro approaches, as part of an integrated testing strategy,4 to replace or reduce in vivo bioaccumulation testing in fish.

Acknowledgements

The financial support of Stiftung Forschung 3R, ünsingen (Switzerland) and the Health and Environmental Sciences Institute (HESI) is gratefully acknowledged.

Prof. Dr Helmut Segner
Centre for Fish and Wildlife Health
Department of Infectious Diseases and Pathobiology
Vetsuisse Faculty
University of Bern
PO Box 8466
CH 3012 Bern
Switzerland
E-mail: Helmut.segner@vetsuisse.unibe.ch

References

1 OECD (2011). OECD Guideline for Testing of Chemicals No. 305. Bioaccumulation in Fish: Aqueous and Dietary Exposure, 72pp. Paris, France: Organisation for Economic Co-operation & Development.
2 Nichols, J.S., Erhardt, S., Dyer, M.J., Moore, M., Plotzke, K., Segner, H., Schultz, I., Thomas, K., Vasiluk, J. & Weisbrod, A. (2007). Use of in vitro Absorption, Distribution, Metabolism, and Excretion (ADME) data in bioaccumulation assessments for fish. Human & Ecological Risk Assessment 13, 1164–1191.
3 de Wolf, W., Comber, M., Douben, P., Gimeno, S., Holt, M., Léonard, M., Lillicrap, A., Sijm, D., van Egmond, R., Weisbrod, A., & Whale, G. (2007). Animal use replacement, reduction,  and refinement: Development of an integrated testing strategy for bioconcentration of chemicals in fish. Integrated Environmental Assessment & Management 3, 3–17.
4 Lombardo, A., Roncaglioni, A., Benfenati, E., Nendza, M., Segner, H., Fernández, A., Kühne, R., Franco, A., Pauné, E. & Schüürmann, G. (2014). Integrated testing strategy (ITS) for bioaccumulation assessment under REACH. Environment International 69, 40–50.
5 Arnot, J.A. & Gobas, F. (2006). A review of bioconcentration factor (BCF) and bioaccumulation factor (BAF) assessments for organic chemicals in aquatic organisms. Environmental Reviews 14, 257–330.
6 Weisbrod, A.V., Sahi, J., Segner, H., James, M.O., Nichols, J., Schultz, I., Erhardt, S.,  Cowan-Ellsberry, C., Bonnell, M. & Hoeger, B. (2009). The state of   science for use in bioaccumulation assessments for fish. Environmental Toxicology & Chemistry 28, 86–96.
7 Kwon, J.H. & Escher, B.I. (2008). A modified parallel artificial membrane permeability assay for evaluating bioconcentration of highly hydrophobic chemicals in fish. Environmental Science & Technology 42, 1787–1793.
8 Segner, H. & Cravedi, J.P. (2001). Metabolic activity in primary cultures of fish hepatocytes. ATLA 29, 251–257.
9 Fitzsimmons, P.N., Lien, G.J. & Nichols, J.W. (2007). A compilation of in vitro rate and affinity values for xenobiotic biotransformation in fish, measured under physiological conditions. Comparative Biochemistry & Physiology 145C, 485–506.
10 Han, X., Nabb, D., Mingoia, R. & Yang, C. (2007). Determination of xenobiotic intrinsic clearance in freshly isolated hepatocytes from rainbow trout (Oncorhynchus mykiss) and rat and its application in bioaccumulation assessment. Environmental Science & Technology 41, 3269–3276.
11 Johanning, K., Hancock, G., Escher, B., Adekola, A., Bernhard, M.J., Cowan-Ellsberry, C., Domodoradzki, J., Dyer, S., Eickhoff, C., Embry, M., Erhardt, S., Fitzsimmons, P., Halder, M., Hill, J., Holden, D., Johnson, R., Rutishauser, S., Segner, H., Schultz, I. & Nichols, J. (2012). Assessment of metabolic stability using the rainbow trout (Oncorhynchus mykiss) liver S9 fraction. Current Protocols in Toxicology 53, 14.10.1–14.10.28.
12 Mingoia, R.T., Glover, K.P., Nabb, D.L., Yang, C.H., Snajdr, S.I. & Han, X. (2010). Cryopreserved hepatocytes from rainbow trout (Oncorhynchus mykiss): A validation study to support their application in bioaccumulation assessment. Environmental Science & Technology 44, 3052–3058.
13 Fay, K.A., Mingoia, R.T., Goeritz, I., Nabb, D.L., Hoffman, A.D., Ferell, B.D., Peterson, H.M., Nichols, J.W., Segner, H. & Han, X. (2014). Intra- and inter-laboratory reliability of a cryopreserved trout hepatocyte assay for the prediction of chemical bioaccumulation potential. Environmental Science & Technology 48, 8170–8178.
14 Nichols, J.W., Schultz, R.I. & Fitzsimmons, P.N. (2006). In vitro–in vivo extrapolation of quantitative hepatic biotransformation data for fish. I. A review of methods, and strategies for incorporating intrinsic clearance estimates into chemical kinetic methods. Aquatic Toxicology 78, 74–90.
15 Cowan-Ellsberry, C.S., Dyer, S., Erhardt, S., Bernhard, M.J., Roe, A., Dowty, M. & Weisbrod, A. (2008). Approach for extrapolating in vitro metabolism data to refine bioconcentration factor estimates. Chemosphere 70, 1804–1817.
16 Castano, A., Bols, N.C., Braunbeck, T., Dierickx, P., Halder, M., Isomaa, B., Kawahara, K., Lee, L.E.J., Mothersill, C., Pärt, P., Repetto, G., Sintes, J.R., Rufli, H., Smith, R., Wood, C. & Segner, H. (2003). The use of fish cells in ecotoxicology. The report and recommendations of ECVAM workshop 47. ATLA 31, 317–351.
17 Segner, H. (2011). Moving beyond a descriptive aquatic toxicology: The value of biological process and trait information. Aquatic Toxicology 105, 50–55.

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Current Dilemmas Segner

Coffee in Class: An Alternative to Animal Experiments in Pharmacology?

Anoop Kumar Agarwal, Syed Ilyas Shehnaz, Razia Khanam and Mohamed Arifulla

The stimulant effect of coffee on psychomotor performance was
introduced as a potential alternative clinical pharmacology
experiment for medical and pharmacy students

Animal experiments have been designed and standardised to demonstrate the effects of certain drugs on body organs, as part of undergraduate health professional education. However, the logistics of animal availability, the expenses incurred, increasing awareness of concerns about animal welfare1–3 and the ‘Three Rs’ concept (i.e. Replacement, Refinement and Reduction),4 have often either reduced these experiments to tutor demonstrations or have resulted in their complete withdrawal from the undergraduate curriculum.5

As an alternative to satisfy the ethical concerns of animal rights activists, Computer Assisted Learning (CAL) was introduced.6–8 Although CAL is an effective means of fulfilling the educational objectives of laboratory sessions, the lack of hands-on experience with living tissues, the lack of practical experience to facilitate the future application of the procedures in research, as well as the absence of biological variation, are the major limitations of CAL.9

In view of the current scenario, we considered it necessary to investigate alternative exercises which would expose the students to experimental methodology with scientific explanation. Clinical pharmacology experiments, such as dosage calculations, rational drug selection, evaluation of drug information, and the analgesic effect of NSAIDs, have been used to supplement CAL.10 In an effort to identify alternatives to animal experiments and CAL at the undergraduate level, the Department of Pharmacology, Gulf Medical University, Ajman, United Arab Emirates, introduced a new experiment in the teaching curriculum of Bachelor of Medicine/Bachelor of Surgery (MBBS) and Pharm D (Doctor of Pharmacy) programmes. The aim of the experiment was to demonstrate the stimulant effect of coffee on psychomotor performance in students, by using simple paper and pencil tests, namely, the Six-Letter Cancellation Test (SLCT) and the Digit/Letter Substitution Test (DLST). These tests objectively assess the psychomotor functions of an individual, and integrate different mental functions, such as perception, recognition, integration and reaction, in the assigned task. These tests are not meant for assessing memory or intelligence. Since they are speed tests, performance is influenced by mental alertness, concentration and coordination abilities. Both tests consist of three sections: instructions, the key (target) letters, and the working-out part. In the SLCT, the subject identifies the key letters in the working-out part, whereas in the DLST, the numbers in the working-out part have to be substituted by the corresponding letters given in the key. The duration of each test is 90 seconds. In both the tests, the extent of the working-out part exceeds the potential for completion in the stipulated time. The maximum and minimum scores for these tests vary in different subgroups. Parallel worksheets (with a different key) are used on each occasion (Figure 1 and Figure 2), to nullify the effect of memory.11

Minimal materials are required

The equipment required for the experiment is readily available at low cost. It comprises three sets of parallel worksheets for the SLCT, three sets of parallel worksheets for the DLST, a stop watch, an office bell, and standard hot coffee (2g instant coffee/200ml).

Figure 1

The protocol

The experimental protocol is divided into five individual stages:

1. The practice session: The tutors familiarised themselves with the tests and planned the experiment to ensure smooth implementation. Ethical approval was granted from the Institutional Ethics Committee prior to the conduct of the experiment. The tests were administered during the pharmacology laboratory sessions to seven batches of 25–30 medical and pharmacy students. The students were told about the importance and relevance of the tests, and were issued with the instructions necessary for performing the tests. Written consent was obtained from those who volunteered to participate in the experiment. One practice session was organised, in order to familiarise the participants with both the tests.

2. The pre-coffee session: The worksheet for the SLCT were distributed, and the students were asked to write their names on the back of the sheet. This was fllowed by the first bell, indicating the beginning of the ‘working-out time’, which ended with a second bell after 90 seconds. The students were asked to start and stop immediately when the bell rang, and strict monitoring of time was ensured. The sheets were randomly exchanged among the students for score calculations. The second test, the DLST, was administered in a similar manner, after an interval of 5 minutes.

3. The post-coffee session: A 20ml cup of standard coffee was served to each student. After 20 minutes, the two tests were re-administered as before, with parallel worksheets, i.e. a new key was used in each session, to nullify the effect of memory.

4. Interpretation of scores: The students were asked to record the scores of the two tests in the three sessions (practice, pre-coffee and post-coffee) in their record books, and to draw conclusions based on a pharmacological explanation. The mean scores of both the tests for the practice, precoffee and post-coffee sessions were expressed as the mean ± standard deviation (SD). Comparison of scores was done by using the Wilcoxon signed-rank test. The significance level was set at 0.05.

5. Student feedback: Feedback on the experiment was obtained by using a structured, content-validated and pre-tested questionnaire on a five-point Likert-like scale (strongly agree to strongly disagree). The statements enquired about the ease of understanding and performing of the tests, the appropriateness of the time allowed and the methodology, the understanding of the concepts and the generation of links between theory and actual effects of CNS stimulants, the interest levels generated, and the willingness to perform similar experiments in the future. In addition, open-ended responses about the students’ experiences were also encouraged. Voluntary participation was emphasised, and full confidentiality of the data was ensured to all the
participants.

Agarwal table 1Figure 2

Results

Of a total of 180 medical and pharmacy 162 participated in the experiment (response rate 90%). The mean scores (± SD) of the SLCT for the practice, pre-coffee and post-coffee sessions are given in Table 1. Although there were no significant differences between the practice and pre-coffee session scores for both the tests, a statistically significant difference (p < 0.05) was observed between the pre-coffee and the post-coffee scores. Furthermore, a small number of students obtained a low score (5%), or had no change in their scores after coffee intake (6%).

The students gave a very good feedback on the experiment, as reflected in the questionnaire (Table 2). However, in the free responses, a few students (12%) reported that they lost interest by the third session, due to the simplicity and repetitive nature of the test, and that, as a result, they felt that they did not perform at their best.

Table 2

Discussion

We have endeavoured to introduce a clinical pharmacology experiment as an alternative to CAL and animal experiments, for use in undergraduate health professional education. This experiment has very few requirements, and can be easily performed within laboratory session time-frames. The experiment also reinforces the concept of the mild stimulant effect of coffee. However, test performance is affected by various factors, including motivation, understanding, interest, mood, environment, quality of the worksheet, and personality type, so scores obtained may vary for different groups and sub-groups. Moreover, a clear understanding of the principle of the paper and pencil test among the faculty is important, in order to plan the experiment in an organised manner. Although these tests had been used earlier as a teaching tool among medical students, the perceptions of the students had not been obtained.11 The experiment was reintroduced in our setting with certain modifications, and student feedback was obtained with regard to its acceptability and relevance. This experiment had been introduced for the first time in the Pharm D curriculum to demonstrate the effects of the drug.

The test scores of both the tests (SLCT and DLST) showed a significant increase in psychomotor performance after coffee intake, suggesting a stimulant effect. However, a number of sources of variation were identified: differences in quantity of coffee consumed, loss of interest due to repetitive nature of the test, and anxiety after coffee intake. The latter is a known side-effect of coffee in certain individuals. Students who did not show an increase in score stated that they frequently consumed coffee during the day.

The student feedback revealed that the majority found the experiment interesting and informative. This could probably motivate them to learn more about the drugs and their effects. However, in the open-ended responses, a few students felt that the tests were too simple and that a higher degree of complexity was necessary to keep up their interest.

Author for correspondence:
Dr Syed Ilyas Shehnaz
Department of Pharmacology
Gulf Medical University
PO Box 4184
Ajman
United Arab Emirates
E-mail : shehnazilyas@yahoo.com

References

1 Gitanjali, B. (2001). Animal experimentation in teaching: Time to sing a swan song. Indian Journal of Pharmacology 33, 71.
2 Solanki, D. (2010). Unnecessary and cruel use of animals for medical undergraduate training in India. Journal of Pharmacology & Pharmacotherapeutics 1,59.
3 Desai, M. (2009). Changing face of pharmacology practicals for medical undergraduates. Indian Journal of Pharmacology 41, 151–152.
4 Guhad, F. (2005). Introduction to the 3Rs (refinement, reduction and replacement). Contemporary Topics in Laboratory Animal Science 4, 58-59.
5 Setalvad, A.R.N. (2009). Medical Council of India, New Delhi, Amendment Notification of 8 July 2009 to the Minimal Standard Requirements for Medical Colleges with 150 Admissions Annually, Regulations 1999. Available at: http://www.mciindia.org/helpdesk/how_to_start/STANDARD%20FOR%20150.pdf (Accessed
01.08.14).
6 Dewhurst, D. (2004). Computer-based alternatives to using animals in teaching physiology and pharmacology to undergraduate students. ATLA 32, 517–520.
7 Badyal, D.K., Modgill, V. & Kaur, J. (2009). Computer simulation models are implementable as replacements for animal experiments. ATLA 37, 191–195.
8 Wang, L. (2001). Computer-simulated pharmacology experiments for undergraduate pharmacy students: Experience from an Australian University. Indian Journal of Pharmacology 33, 280–282.
9 Kuruvilla, A., Ramalingam, S., Bose, A.C., Shastri, G.V., Bhuvaneswari, K. & Amudha, G. (2001). Use of computer assisted learning as an adjuvant to practical pharmacology teaching: Advantages and limitations. Indian Journal of Pharmacology 33, 272–275.
10 Gitanjali, B. & Shashindran, C.H. (2006). Curriculum in clinical pharmacology for medical undergraduates of India. Indian Journal of Pharmacology 38, Suppl., 108–114.
11 Natu, M.V. & Agarwal, A.K. (1997). Testing of stimulant effects of coffee on the psychomotor performance: An exercise in clinical pharmacology. Indian Journal of Pharmacology 29, 11–14.

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Discussion Agarwal

Likelihood Ratios in Assessing the Safety of New Medicines

Robert A. Coleman

Until we know the true predictive value of animal-based methods
for predicting clinical safety issues, it is impossible to assess
the advantage or otherwise of non-animal based approaches

The use of animals in the discovery and development of new medicines has generated debate for decades. For much of this time, contrasting views have been primarily polarised on the basis of practicality versus ethics, with proponents arguing that for the development of new medicines to treat human disease, the end justifies the means, while the opponents’
chief objection has been the associated animal suffering. Commercial and public health pressures over the years have ensured that those in the ‘practicality camp’ have held sway.

More recently, however, the issue has become increasingly complex, with growing concerns that, irrespective of ethical considerations, data generated in animal (i.e. non-human) models are not necessarily or sufficiently relevant to human patients.1–3 There is now general consensus that inter-species variability is a real issue, and that animal models are far from perfect for the purpose of ensuring either the efficacy or the safety of potential new medicines intended for human subjects. Supporters of the continued use of animals argue that, while they do not provide an absolute indication of either efficacy or safety, in the absence of any other approach, one that is somewhat unreliable is better than none at all.

Such an argument has some merit, if indeed it is valid. However, in this field, all may not be entirely as it seems. Firstly, human-based in vitro and in silico alternatives are becoming ever more sophisticated,4 thus overcoming many of the criticisms originally directed toward them. For example, it has long been held that it would be impossible to model the complexity of the intact patient through a study of isolated cells and tissues, and while this problem may never be wholly overcome, the gap gets ever smaller. Secondly, it is important to understand that we really don’t know how good existing animal-based methods are. In the field of efficacy, there is a wealth of evidence that results obtained by using experimental animals can be hugely misleading.5–11 Here, we have the advantage that drugs that promise efficacy in patients on the basis of animal data can advance into clinical testing, and their utility can be directly assessed. For the majority of these drugs, the clinical outcome has been disappointing. With safety, the issue is different, as drugs with identified safety issues in animals will seldom, if ever, advance to clinical testing, thus the relevance of the animal data to safety in humans may never be determined. However, what we do know is that many drugs identified as safe in pre-clinical profiling eventually prove to cause serious and use-limiting side effects in human subjects. 12 The key question is, “Could such failures have been avoided, had we relied on human-based test methods?” Until we know how frequently non-animal methods could have identified safety issues that were missed by animal tests, it is impossible to assess the advantage or otherwise of those methods. It is a fact that, despite the continued use of animals as human surrogates in pharmaceutical research, there has never been a solid, published, peer-reviewed study demonstrating fitness for purpose, whereas reviews identifying the shortcomings are abundant.

Assessing the value of animal studies

It is for this reason that any information that sheds light on the actual value of animal-based testing for its intended purpose is of inestimable worth. Until recently, much evidence, while valuable, has been indirect. For example, a recent telling study demonstrated that pre-clinical fast-tracking (i.e. abbreviated safety testing) of potential new medicines resulted in no increase in the proportion of candidates that subsequently proved toxic in human subjects.
13 In another study, the ability of animal studies to detect serious post-marketing adverse events was demonstrably poor.14 While such reports add to the volume of data providing witness to the shortcomings of animal-based approaches to ensuring clinical safety, they do not provide a robustly measurable metric of predictive efficiency. In view of the colossal amount of data generated over the years in pre-clinical safety studies on thousands of new potential medicines, many of which have progressed to clinical testing and even to market, it is amazing that, until recently, no comprehensive analysis of such data has been applied in order to explore the value of the current approach to safety testing.

Likelihood ratios

In the light of such a background, it is of considerable significance that serious attempts are now being made to extract intelligence from the wealth of information available in publicly accessible sources, in order to shed more light on the actual predictive power of animal-based safety testing. A particular example is the utilisation of the Safety Intelligence Programme (SIP),15 which overcomes semantic issues to extract valid information from all available data sources. SIP has been used, for example, to explore the predictive power of animal models for the detection of liver toxicity associated with a wide range of human medicines,16 highlighting the highly variable efficiency of different models in combination with different drug toxicities. More recently, SIP has been used to particular effect in two studies that have explored directly the value of dogs, mice, rats and rabbits in predicting safety issues in human subjects.17,18 While most previous studies have relied on determining ‘concordance’ between animal and human data, that tells only a part of the story, and is too simplistic a measure to be of much real value. Its
problem is that it only deals with positive correlation, i.e. the frequency that toxicity in experimental animals and in human subjects coincide, ignoring the issue of true prediction. What is needed is a determination of likelihood ratios (LRs),19 both positive (PLRs) and negative (NLRs), to gain a more complete picture. What emerged when LRs were determined was that, although there was indeed some measure of concordance between positive toxicity data between animals and humans, in terms of LRs, none of the species proved to offer any useful level of real predictive power. Although the studies and their conclusions did not escape criticism from some quarters,20 the suggested limitations, real or perceived, are arguably irrelevant to its overall validity.21

What did emerge from the application of this approach were absolute values for both PLRs and NLRs for a wide range of specific drugs. The importance of this is that, for the first time, such measures can provide a robust yardstick against which to evaluate the relative merits of alternative approaches to toxicity testing.

Conclusions

To summarise, the use of more-rigorous approaches to the evaluation of animal models as predictors of the likelihood that any chemical will be similarly toxic or non-toxic in human subjects provides not only a realistic measure of their actual fitness for purpose, but crucially, also a basis by which the efficiency of other, ideally human-based, approaches can be evaluated through their exposure to the same range of drugs. The use of the same drugs ensures that any criticisms related to potential bias, or other potentially confounding factors, are negated. Such a prospective study would be of inestimable value. Dare we hope that the government and pharmaceutical companies will take up the challenge and fund
such a study?

Dr Robert A. Coleman
Independent Consultant
UK
E-mail: robt.coleman@btinternet.co
m

References

1 Wall, R.J. & Shani, M. (2008). Are animal models as good as we think? Theriogenology 69, 2–9.
2 Hartung, T. (2013). Food for Thought… Look back in anger — What clinical studies tell us about preclinical work. ALTEX 30, 275–291.
3 Pippin, J.J. & Sullivan, K. (undated). Dangerous medicine:
Examples of animal-based “safety” tests gone wrong. Washington, DC, USA: The Physicians Committee for Responsible Medicine. Available at: http://www.pcrm.org/research/animaltestalt/animal testing/dangerous-medicine-examples-of-animalbased-tests (Accessed 03.11.14).
4 Ashton, R., De Wever, B., Fuchs, H.W., Gaça, M., Hill, E., Krul, C., Poth, A. & Roggen, E.L. (2014). State of the art on alternative methods to animal testing from an industrial point of view: Ready for regulation? ALTEX 31, 357–363.
5 Pound, P., Ebrahim, S., Sandercock, P., Bracken, M.B. & Roberts, I. (2004). Where is the evidence that animal research benefits humans? British Medical Journal 328, 514-517.
6 Kaste, M. (2005). Use of animal models has not contributed
to development of acute stroke therapies: Pro. Stroke 36, 2323–2324.
7 Pippin, J.J. (2005). The Need for Revision of Pre-Market Testing: The Failure of Animal Tests of COX-2 Inhibitors, 23pp. Washington, DC, USA: The Physicians Committee for Responsible Medicine. Available at: pcrm.org/pdfs/research/testing/exp/COX2Report.pdf
(Accessed 04.11.14).
8 Hackam, D.G. & Redelmeier, D.A. (2006). Translation of research evidence from animals to humans. Journal of the American Medical Association 296, 1731–1732.
9 Knight, A. (2008). Systematic reviews of animal experiments
demonstrate poor contributions towards human healthcare. Reviews on Recent Clinical Trials 3, 89–96.
10 Matthews, R.A.J. (2008). Medical progress depends on animal models — doesn’t it? Journal of the Royal Society of Medicine 101, 95–98.
11 Seok, J., Warren, H.S., Cuenca, A.G., Mindrinos, M.N., Baker, H.V., Xu, W., Richards, D.R., McDonald-Smith, G.P., Gao, H., Hennessy, L., Finnerty, C.C., López, C.M., Honari, S., Moore, E.E., Minei, J.P., Cuschieri, J., Bankey, P.E., Johnson, J.L., Sperry, J., Nathens, A.B., Billiar, T.R., West, M.A., Jeschke, M.G., Klein, M.B., Gamelli, R.L., Gibran, N.S., Brownstein, B.H., Miller-Graziano, C., Calvano, S.E., Mason, P.H., Cobb, J.P., Rahme, L.G., Lowry, S.F., Maier, R.V., Moldawer, L.L., Herndon, D.N., Davis, R.W., Xiao, W., Tompkins, R.G. & Inflammation and Host Response to Injury, Large Scale Collaborative Research Program (2013). Genomic responses in mouse models poorly mimic human inflammatory diseases. Proceedings of the National Academy of Sciences of the USA 110, 3507–3512.
12 Li, A.P. (2004). Accurate prediction of human drug toxicity: A major challenge in drug development. Chemico-biological Interactions 150, 3–7. 13 Arnardottir, A.H., Haaijer-Ruskamp, F.M., Straus, S.M., Eichler, H.G., de Graeff, P.A. & Mol, P.G. (2011). Additional safety risk to exceptionally approved drugs in Europe? British Journal of Clinical Pharmacology 72, 490–499.
14 van Meer, P.J., Kooijman, M., Gispen-de Wied, C.C., Moors, E.H. & Schellekens, H. (2012). The ability of animal studies to detect serious post marketing adverse events is limited. Regulatory Toxicology & Pharmacology 64, 345–349.
15 Berry, S. (2012). Safety Intelligence Program Provides Insight into Drug-Induced Cardiac Effects. Stone, UK: Instem plc. Available at: http://www.biowisdom.com/content/safety-intelligence-program (Accessed 04.11.14).
16 Fourches, D., Barnes, J.C., Day, N.C., Bradley, P., Reed, J.Z. & Tropsha, A. (2010). Cheminformatics analysis of assertions mined from literature that describe drug induced liver injury in different species. Chemical Research in Toxicology 23, 171–183.
17 Bailey, J., Thew, M. & Balls, M. (2013). An analysis of the use of dogs in predicting human toxicology and drug safety. ATLA 41, 335–350.
18 Bailey, J., Thew, M. & Balls, M. (2014). An analysis of
the use of animal models in predicting human toxicology and drug safety. ATLA 42, 181–199.
19 Altman, D.G. & Bland, J.M. (1994). Diagnostic tests 2: Predictive values. British Medical Journal 309, 102.
20 Brooker, P. (2014). The use of second species in toxicology
testing. ATLA 42, 147–149.
21 Bailey, J. (2014). A response to the ABPI’s Letter to
the Editor on the use of dogs in predicting drug toxicity
in humans. ATLA 42, 149–153.

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