Category Archives: OPINIONS

Laboratory Animal Sciences Training in Gulf Co- operation Council Countries: Think Globally, Act Locally

Steps need to be undertaken in the Arab countries of the Gulf Co-operation Council, toward improvement in experimental animal welfare through a formal laboratory animal science training course

 Syed Ilyas Shehnaz and Anoop Kumar Agarwal

Animal experiments are currently the mainstay in the determination of the biological activity of newly discovered compounds. The preliminary animal data serve as the basis for elaborate studies in the biological and medical sciences, such as the safety and efficacy  testing of drugs, research into genetic disorders, and the development of diagnostics. It is essential that the preclinical data generated should be reliable, accurate and precise, in order to optimise the subsequent clinical studies. Moreover, the handling of laboratory animals and the performance of animal experiments are an integral part of post-graduate  training in pharmacology, toxicology, biotechnology, physiology, molecular biology, microbiology and related courses. Many alternatives to animal experiments have been advocated globally. However, in view of the perceived limitations of these alternatives, animal experiments have not been totally replaced in most research institutions.

Biomedical research in Gulf Co-operation Council countries

The Arab countries of the Gulf region, i.e. Bahrain, Kuwait, Oman, Qatar, Saudi Arabia and United  Arab Emirates, together constitute the Gulf Co-operation Council (GCC). The number of biomedical research institutes in these countries has significantly increased in the past decade.1 This increase, taken together with the existence of 25 medical colleges2 and many other universities, means that research activities in the GCC countries have also concurrently increased. Nearly 7,000 MEDLINE®-listed research papers have been published from these countries over the past ten years.3 Kuwait and Saudi Arabia also featured among the top ten countries, with regard to biomedical research productivity in Asia.4 In spite of the active research profile of the GCC countries, to the best of our knowledge there is no formal training course that prepares the biomedical scientists in these countries to undertake animal research in line with the international standards on animal ethics, Good Laboratory Practice (GLP) or FELASA guidelines. Adherence to the principles of GLP is an essential requirement for all animal studies and must be indicated in the Investigational New Drug application, along with preclinical toxicity data, for clinical trials. Similarly, certification according to the FELASA guidelines is compulsory for biomedical scientists in most European countries.5

Laboratory animal sciences training

Several intrinsic and extrinsic factors have a bearing on animal experiments, and ignorance of these factors can often result in false positive or false negative data. The impact of these factors can be minimised through the formal training of the researchers involved in the planning, design and execution of animal studies. If undertaken, a structured training will help in troubleshooting, reduce errors and generate more-credible experimental data. Proper training will also help to reduce threats to the validity of, for example, neuro-behavioural studies involving whole animal responses, which are significantly affected by the environment and the experimental skills of the scientist.  A short training course in Laboratory Animal Sciences (LAS) was previously suggested,6 which can be adopted per se or modified as per the local needs and resources of the GCC countries. The characteristics of various animal species, their associated housing and handling practices, their suitability for different experiments, procedural skills, available alternatives and protocol design, were proposed as the main elements of the training course. Moreover, many ethical issues, such as pain and injury, rehabilitation, re-use, euthanasia, and the disposal of carcasses, have been adequately addressed.7  After this training, the researchers would be equipped to address the ethical issues with justification and  relevance, in line with the national regulatory requirements for animal experiments. The training could be offered by institutions and research centres, either  individually or at a centralised facility in each country, with the pooling of expertise and resources from  all the GCC countries. Owing to the charter  of the GCC countries, there is a likelihood that an initiative taken by one member country in this direction will promote and facilitate animal research training in other GCC countries. Apart from the aforesaid caveats, the training would assist the senior members of the Animal Ethics  Committee (AEC) to inspect and scrutinise proposals for animal research, in accordance with the principles of the Three Rs (Replacement, Reduction and Refinement). The training could also serve as a prerequisite for the induction of new members into the AEC. In most GCC institutions, the constitution of the AEC is based on the members’ professional experience and completion of the quorum which helps meet the requirements of international scientific journals.

The non-GCC countries of the Middle East, i.e. Turkey and Iran, have taken the initiative in  developing ethical regulatory systems for animal experiments by the setting up of AECs in almost all of their biomedical universities.8 An animal ethics course based on Islamic principles is being conducted in Iran as a mandatory requirement for personnel involved in animal research projects. The full training involves seven specific courses in addition to a general course.9 Attempts have also been made in North Africa and the Middle East to sensitise the scientific community to the advantages of alternatives to animal use, and guidelines have been outlined for the total replacement of animal experiments in education and research.10 The LAS course suggested for the GCC could assist in providing technical support for the effective execution of these guidelines.

Conclusions

Some Middle Eastern countries, such as Turkey and Iran, have passed regulations for establishing standards in animal research. Similar steps need to be undertaken in the Arab countries of the GCC, toward improvement in experimental animal welfare through a formal laboratory animal science training course.

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 Al-Maawali, A., Al Busadi, A. & Al-Adawi, S. (2012). Biomedical publications profile and trends in Gulf Cooperation Council countries. Sultan Qaboos University Medical Journal 12, 41–47.
2 Shehnaz, S.I. (2011). Privatization of medical education in Asia. South East Asian Journal of MedicalEducation 5, 18–25.
3 Deleu, D., Northway, M.G. & Hanssens, Y. (2001). Geographical distribution of biomedical publications from the Gulf Cooperation Council countries. Saudi Medical Journal 22, 10–12.
4 Rahman, M. & Fukui, T. (2000). Biomedical research productivity in Asian countries. Journal of Epidemiology 10, 290–291.
5 FELASA (1995). FELASA recommendations on the education and training of persons working with laboratory animals: Category A and Category C. Laboratory Animals 29, 121–131.
6 Shehnaz, S.I. & Agarwal, A.K. (2013). A structured course in laboratory animal science for postgraduates: Is it a necessity? Journal of Pharmacology & Pharmacotherapeutics 4, 67–68. 7 Shehnaz, S.I. & Agarwal, A.K. (2013). Animal ethics training for postgraduates in medical schools in India: Catch them young! ATLA 41, P2–P4.
8 Izmirli, S., Aldavood, S.J., Yasar, A. & Phillips, C.J. (2010). Introducing ethical evaluation of the use of animals in experiments in the Near East. ATLA 38, 331–336.
9 Aldavood, S.J., Fard, R.M. & Naderynezhad, F. (2013). Laboratory animal ethics course planning in Iran. ATLA 41, P40–P41.
10 Anon. (2010). North Africa and Middle East Seminar on Alternatives in Education and Training. ATLA 38, 201.

 

 

The Three Rs — Do We Need a New Principle?

A new generation of Russells and Burches are needed,
who will bring in a new era of biological science
that will truly benefit animals as well as humans

Christiaan Wittevrongel

Since the early sixties, much has been done in terms of Replacement, Reduction and Refinement. But are these Three Rs still the principles by which animal use in research can be avoided in the future? Not necessarily.

In 1959, the well-known concept of the Three Rs was published by William Russell and Rex Burch.1 Their approach to animal testing revolutionised the way in which laboratory animals were seen and used in scientific research. Consideration of the Three Rs changed the way experiments were carried out, with the development of non-animal methods (replacement) and the push toward experiments with fewer animals (reduction). This approach prompted changes in statistical methods as enabling factors to extract relevant information from the experimental data. Last, but not least, refinement led to progress in the relief of pain and suffering, which, in turn, has impacted on the quality of the experimental results.

The Three Rs have been in use for over 50 years. The question can be raised as to whether they still keep delivering. Let us look at the number of animals that are used in research in The Netherlands.2 The total number in 1991 was 1,117,234 and in 2011 it was 1,120,399. One may therefore be tempted to conclude that the efficacy of the Three Rs concept is running out of steam, because there is less well-documented agreement about a substantial reduction occurring during the preceding years. This levelling off can be partially explained by the increased interest in the use of genetically-modified animals, mainly rodents.

This brings us to the issue of surplus animals, i.e. animals that were to be used in experiments, but for some reason were not, and thus had to be killed. It so happens that, during the production of genetically-modified animals, some do not meet the requirements of the experiment. In many instances, only male animals are used, and this makes the females redundant. Last, but not least, poor planning may create animals that are not needed, or that cannot be used because they are at the wrong age. In 1991, the total number of surplus animals was 241,176, and in 2011 it was 530,446.2 It is obvious that no progress has been made in this area, whereas solutions seem to be relatively simple.
The conclusion seems to be that the Three Rs are still effective, but their effect is stabilising. The number of animal experiments for some purposes, has reduced considerably in the last few decades, due to replacement and reduction. The most spectacular success has been in diagnostics, where hardly any animals are now used, because of the arrival of new technology — for example, immunochemistry methods or the DNA-based polymerase chain reaction — that has made certain animal experiments obsolete and redundant.

It can be argued that additional approaches are needed, not just the Three Rs. The problem with the Three Rs approach is that it basically all starts from the consideration of the animal test. Replacement, if looked at in the traditional manner, has several disadvantages:

1. Laws on animal experiments mainly protect vertebrates. Therefore, legally, replacement is also achieved when invertebrates can be used. However, invertebrates are still animals, so this is not genuine replacement of an animal test. Unfortunately, this is not reflected in the total numbers of animals that are recorded. Since invertebrates are not protected by law (albeit with the exception of cephalopods following the introduction of the new European directive), they are not counted.3
2. If plans are being made to replace an animal experiment endpoint, then this might involve validation of the new endpoint in animal experiments that were meant to be replaced.

 Reduction and refinement are directly linked to the idea that a proper answer to a research question can be found by using animals. They have a very marginal effect on the change of general scientific thinking about the use of animals.

In the search for biological mechanisms of human disease and in the search for new drugs, the animal experiment is sometimes referred to as the gold standard. The validity of this statement has never been proven — and it seems that nobody has tried. It seems more likely that animal experiments are the only standard, because there is no alternative. It is well-known that the search for new drugs is very inefficient.4 Many drugs in development fail in clinical trials, due to lack of efficacy or unwanted safety profiles. These drugs have all been tested in animal experiments prior to the clinical trials. This means that something needs to change in the development of new drugs. It appears that animals and humans are very different after all. Not long ago, a discussion erupted on the use of animal models in inflammatory disease research.5 Researchers stated that animal models were not at all applicable for use in the development of drugs against, for example, type 1 diabetes.

This is because the immune system in animal models differs too much from that of humans. However, researchers are still obliged to show that drugs do work safely in animals, before clinical trials, which may lead to market access, can be started. Researchers acknowledge that there is a big problem in the development of drugs against conditions such as type 1 diabetes, and drugs have been discovered against type 1 diabetes, based on knowledge of the human biological system. The researchers know that these drugs will not work in animals, but they still have to show efficacy in animals before clinical trials can take place. Therefore these drugs might never be available on the market (Bart Roep, personal communication; Labyrinth Radio, Radio 1, 18 February 2013).

Comparison is sometimes possible in the field of toxicology, where some human data are available,specifically in studies that have been performed on the carcinogenicity of substances. However, if the comparison is conducted properly, it is usually found that results in animals and humans are not equal at all.6 In risk assessment, one needs to consider what must be prevented from happening. For example, in the case of carcinogenic substances, the development of a tumour is the event that needs to be prevented. Therefore, we need to take a closer look at the human biological systems behind this event, and then determine whether certain substances exert effects on these systems. Substance kinetics and DNA-damage need to be reinvestigated, specifically aimed at human biological systems, because these mechanisms form the basis of tumour development.

What do we need in addition to the Three Rs?

Generally speaking, animal experiments are carried out to either generate knowledge and understanding of human biology or to provide information as a basis for making decisions about either the safety of chemicals or the efficacy of medicines. This puts human biology at the centre stage. A true mind-shift needs to take place in the process of answering scientific questions about the human body and human diseases, their prevention and cure. What we really need to develop are methods to deliver just that, by making use of the achievements of modern medicine, but using these very same technologies in human models and systems, rather than in animals.7 This will also mean that we need a new generation of Russells and Burches, who will bring in a new era of biological science that will truly benefit animals as well as humans.

Dr Christiaan Wittevrongel
Senior Policy Officer
The Dutch Society for the Replacement of Animal
Testing (DsRAT)
Groot Hertoginnelaan 201
2517 ES The Hague, The Netherlands
E-mail: Wittevrongel@proefdiervrij.nl

References and Note
1 Russell, W.M.S. & Burch, R.L. (1959). The Principles of Humane Experimental Technique, 238pp. London, UK: Methuen. [Reprinted in 1992, by UFAW, South Mimms, Hertfordshire, UK.]
2 In this paper, we have used the number of animals as registered by The Netherlands’ authorities .
3 Anon. (2010). Directive 2010/63/EU of the European Parliament and of the Council on the protection of animals used for scientific purposes. Official Journal of the European Union L276, 20.10.2010, 33–79.
4 Paul, S.M., Mytelka, D.S., Dunwiddie, C.T., Persinger, C.C., Munos, B.H., Lindborg, S.R. & Schacht, A.L. (2010). How to improve R&D productivity: The pharmaceutical industry’s grand challenge. Nature Reviews Drug Discovery 9, 203–214.
5 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. & the 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.
6 Card, J.W., Fikree, H., Haighton, L.A., Lee-Brotherton, V., Wan, J. & Sangster, B. (2012). Lack of human tissue specific correlations for rodent pancreatic and colorectal carcinogens. Regulatory Toxicology & Pharmacology 64, 442–458.
7 Fentem, J., Chamberlain, M. & Sangster, B. (2004). The feasibility of replacing animal testing for assessing consumer safety: A suggested future direction. ATLA 32, 617–623.

Refinement: Lessons from the 2012 Olympics

Until the day comes when animals are no longer needed for research, refinement needs to be a priority for the scientific community

Elliot Lilley and Maggy Jennings

British road and track cycling has gone though a major renaissance recently, with enormous success at the London Olympics and with Sir Bradley Wiggins winning the Tour de France. One of the reasons that British cycling is leading the world at the moment is the leadership of the head of UK cycling, Dave Brailsford, and in particular, his approach of ‘aggregation of marginal gains’.

What does ‘aggregation of marginal gains’ mean in relation to professional cycle racing? If you wanted to surpass your opponents in terms of team performance, then you could invest your time in looking for the next big paradigm shift in technology (e.g. the Boardman bike), in order to overtake the rest of the field in a single step. Alternatively, you could break down all of the component parts that contribute to a successful cycling performance, and make every single one just a little bit better in a systematic, iterative way; e.g. slight changes to front fork, helmet, and suit design, and improvements to dietary, physiotherapy and psychological support to riders. The combination of a number of small improvements like these can lead to a significant improvement in performance for the elite, with the added benefit of raised standards for all of the team. But what has this to do with refinement in relation to animal experimentation? In our view, ‘aggregation of marginal gains’ is a great principle that can also be applied to refinement.

The effective implementation of refinement requires the systematic breakdown of the lifetime experience of the animals involved into its component steps, in order to identify the potential for physical and psychological suffering and to put in place measures to eliminate or ameliorate this suffering. This process should begin during the planning stage of the experiment, and should then evolve as the project unfolds. Refinement has been represented as a ‘loop’,1 and this is a good way to think about it. There should be an ongoing process of assessment of suffering, identification of refinement opportunities, implementation of refinement, then back to assessment of suffering and so on. Welfare science is a continually developing field, and thus a regular appraisal of the literature is essential to stay abreast of the current state of play with regard to optimising laboratory animal welfare.

Refinement practices, whilst apparently widespread (based on our interactions with the scientific community), are largely absent from the methodological sections of scientific research publications. One reason for this is that individual refinements are often small iterative changes that alone do not appear to be overly significant. However, refinements are not generally implemented in isolation, and the aggregated benefit of several small refinements can have a significant impact on the reduction of suffering and improvement of the welfare of animals used in research.

Efforts to reduce suffering are not simply a moral obligation or an exercise in sympathetic anthropomorphism. Refinement and improved welfare standards are broadly acknowledged to be essential for good quality science. This was well-articulated by Russell and Burch over 50 years ago: “It is widely recognised that the humanest possible treatment of experimental animals, far from being an obstacle, is actually a prerequisite for successful animal experiments”,2 and it has been repeated by many others since then.1,3 Poor animal welfare can often be a confounding factor in an experiment, and will therefore increase variability and error,4 yet some people still fail to understand this principle and fail to acknowledge the many factors that can affect welfare. Therefore, it is critical that all the sources of suffering that can occur in an experiment are identified and their impact is minimised.

Under the current UK legislation (the Animals [Scientific Procedures] Act 1986, as amended in December 2012 in compliance with EU Directive 2010/63/EU5), there are three categories of suffering that animals can experience, namely, Mild, Moderate and Severe. To date, there are no clear data on the proportion of the 3.7 million animals used last year (or indeed any year before that) that underwent procedures that caused severe suffering. Thankfully, this is going to change, as, from January of this year, establishments are required to assess the actual level of suffering experienced by animals undergoing regulated procedures. This is good news, for three reasons. Firstly, it will give the general public a clear indication of the levels of suffering experienced by laboratory animals. Secondly, it will give establishments an opportunity to evaluate how successful their refinement programmes are in reducing suffering and to highlight areas where more work is needed. Finally, this new information will allow welfare organisations to focus their resources on areas of research where suffering is high and refinement is lacking, and to track the progress of Three Rs-related programmes targeted at ending this suffering.

All suffering is of concern, but procedures and models that have the potential to cause severe suffering are of particular concern to animal welfare organisations and to the general public. Annual opinion polls (conducted by Ipsos MORI) report that about 85% of the public are ‘conditional acceptors’ of animal research, i.e. that they are willing to accept animal research, as long as there is a clear medical benefit and as long as suffering is minimised, thus indicating that suffering is an important issue.

The RSPCA is committed to ending severe suffering in scientific research that uses animals, and has initiated a project comprising three parallel strands, in order to achieve that goal. The first of these is to identify refinements that have already been implemented to reduce suffering in severe models, and to encourage researchers and animal-care staff to promote their refinement approaches to the wider scientific community, in order to benefit as many animals as possible. The second strand is the establishment of working groups made up of researchers, animal-care staff, and veterinarians, who have expert knowledge of specific models and procedures that have the potential to cause severe suffering. These working groups will be tasked with establishing ‘state-of-the-art’ refinement principles for specific models or research areas, and with drawing up a set of recommendations for publication.

We have already established two such groups, focusing on: a) procedures and models that involve seizures;6 and b) experimental autoimmune encephalomyelitis (EAE).7 Two further groups will be initiated in 2013, to look into refinements for models of rheumatoid arthritis and of sepsis. The third strand of our approach is called the ‘stretch objective’ — this is human resources jargon for a programme of work which, despite being challenging, is both achievable and worthwhile to strive for. The idea is to get establishments to think critically about their research programmes and to challenge the necessity and justification for severe procedures. We strongly believe that ending severe suffering is a desirable and achievable goal, and that refinement must play a key part in delivering it. This brings us back to the ‘aggregation of marginal gains’. An end to severe suffering could be achieved through a single large paradigm shift, i.e. by implementing an immediate UK ban on all severe procedures. This is unlikely to be achieved in the current scientific and political climate, and could result in under-reporting of actual severity and/or higher numbers of severe procedures being performed overseas. Alternatively, the many factors related to suffering could be systematically identified and addressed. This could take the form of pushes to achieve better welfare assessment, better housing and care, the implementation of humane endpoints, rigorous ethical review, better experimental design, greater sharing of refinement information, and a greater proportion of life science research funding going to research into alternatives. This could not only result in ending severe suffering, but it would also have the added effect of reducing the suffering of all animals used in research. This is because refinement principles can be applied to reduce the suffering of all animals used in research, and not only those who suffer most.

References
1 Lloyd, M.H., Foden, B.W. & Wolfensohn, S.E. (2008). Refinement: Promoting the Three Rs in practice. Laboratory Animals 42, 284–293.
2 Russell, W.M.S. & Burch, R.L. (1959). The Principles of Humane Experimental Technique, xiv + 238pp. London, UK: Methuen.
3 Baumans, V. (2005). Science-based assessment of animal welfare: Laboratory animals. Revue Scientifique et Technique (International Office of Epizootics) 25(2), 503–514.
4 Poole, P. (1997). Happy animals make good science. Laboratory Animals 31, 116–124.
5 Anon. (2010). Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes. Official Journal of the European Union L276, 20.10.2010, 33–79.
6 Wolfensohn, S., Hawkins, P., Lilley, E., Anthony, D., Chambers, C., Lane, S., Lawton, M., Robinson, S., Voipio, M-H. & Woodhall, G. (2013). Reducing suffering in animal models and procedures involving seizures, convulsions and epilepsy. Journal of Pharmacological & Toxicological Methods 67, 9–15.
7 Wolfensohn, S., Hawkins, P., Lilley, E., Anthony, D., Chambers, C., Lane, S., Lawton, M., Voipio, M-H. & Woodhall, G. (2013). Reducing suffering in experimental autoimmune encephalomyelitis (EAE). Journal of Pharmacological & Toxicological Methods 67, 169–176. P29

Learning to be Humane

Coenraad Hendriksen and Iris Boumans

More effort should be put into reducing
severe pain and distress in animal experiments
by applying more-humane endpoints

These are exciting times for the Three Rs community. Innovative technologies that might have a broad window of application in biomedical research (e.g. organ-on-a-chip and tissue engineering) are on the radar for Three R spin-offs. And what about new testing
strategies, such as Tox21 and ITS, that have the potential to improve the quality of science while reducing the numbers of animals required? At the same time, however, we have to acknowledge the fact that not all we do in biomedical research will benefit from these Three Rs technologies and strategies, so we will continue to have to use laboratory animals. This also includes performing in vivo studies
that are known to induce significant (> moderate) pain and distress. According to the Dutch statistics on animal experimentation, these accounted for 12.5% of total animal use in 2011.1 The models we have for studying rheumatic arthritis or pandemic influenza infections are some of the clear examples of severe suffering. For these scientific purposes, replacement is not yet feasible, and reduction strategies provide only a few possibilities. In these cases, one of the last resorts to make science more humane (or ‘less inhumane’, as Michael Balls, former head of the European Centre for the Validation of Alternative Methods [ECVAM], encouraged us to say), is to limit the period of severe pain and distress the animals have to go through.

This approach, also known as the use of humane endpoints, has been given increasing attention, not least because it is specifically mentioned in the new EU legislation, Directive 2010/63/EU.2 Article 13.3 of the directive requires that “Death as the end-point of a procedure shall be avoided as far as possible and replaced by early and humane end-points. Where death as the end-point is unavoidable, the procedure shall be designed so as to: (a) result in the deaths of as few animals as possible; and (b) reduce the duration and intensity of suffering to the animal to the minimum possible and, as far as possible, ensure a painless death”.

Apart from legal aspects, there are also moral and scientific reasons to consider the use of humane endpoints. Certain levels of pain and distress simply can’t be accepted, whatever justification for them
may be given. And what about the investigators who claim that applying humane endpoints will interfere with the scientific output, while they don’t mind about pain and distress being a bias in their studies?
Several approaches for implementing humane endpoints have been published, such as for vaccine potency testing,3,4 for shock research,5 and for experiments on fish,6 while some standardisation bodies such as the Organisation for Economic Co-operation and Development (OECD)7 and the European Pharmacopoeia8 explicitly refer to the use of these endpoints in their monographs.

The question is whether this has resulted in an overall application of humane endpoints in day-to-day animal experimentation. We don’t think that this is the case. For instance, a search on the internet will show that mice are still used for production of monoclonal antibody production by injection of hybridoma cells into the abdominal cavity. This is a procedure of high severity, due to extensive growth of abdominal tumours. Ascites production in itself is shameful, as a range of in vitro techniques exists nowadays, that enable the production monoclonal antibodies at almost every magnitude of volume that is needed. But even if ascites production might be the only option, there are recommendations to limit the suffering of the animals by restricting the number of taps, and only allowing multiple taps if the animal does not exhibit signs of distress.9

Nevertheless, some websites of commercial companies offer ascites production to a maximum of up to 6000 animals (!), while they indicate that the animals can be used for multiple taps. Also, in vaccine potency testing that is based on challenging immunised animals with a lethal dose of virulent microorganisms, some companies continue to use death as an endpoint, even when earlier predictive clinical endpoints have been described.

It is claimed that relatively simple refinement strategies, such as the use of humane endpoints, are difficult to implement. Why is this? One reason might be blunt ignorance or a lack of awareness of what is going on in the animal facility. Quite often, severe pain and distress is part of routine procedures, and, since innovations in these procedures are not very catchy, there is little incentive to change. Another reason might be the fact that implementing humane endpoints requires that animals are monitored more frequently. Ultimately, this will increase the costs of the animal procedure. A more scientifically valid argument against applying humane endpoints is that death as an endpoint is 100% objective, while clinical signs involve an element of subjectivity. This could increase test variability or result in incorrect predictions, for instance,
if survival-time after challenge with the virulent microorganism
is one of the test parameters.10

Several suggestions have been made to encourage a positive attitude. These range from improving institutional policies to increase awareness in the animal facility, to a focus on refinement strategies in project approval by ethics committees. Regulatory bodies could interact with company policies, in case humane endpoints have not been addressed in product licensing dossiers or batch release dossiers. We feel that, in particular, the training of laboratory facility staff and investigators is extremely important in encouraging the improved implementation of humane endpoints. Awareness with regard to possible humane endpoints requires both a fine-tuning of attitude and improving clinical monitoring skills.

With the financial support of the Dutch funding organisation, ZonMw, and the Foundation Alternatives to Animal Experimentation, we recently established a website on humane endpoints, www.humaneendpoints.
info, with a focus on rats and mice. The website is currently available in the English language, while both Dutch and French versions are under construction. The website has a publicly accessible part that provides general information on humane endpoints, on the relevant literature, and on regulations. The starting point is that, in order to recognise clinical signs, the characteristics of healthy animals must be known. Thus, chapters are included on normal behaviour, including physiological reference values, and on how to monitor the behaviour of animals. More in-depth information is given in a secure part of the website that is only available after registration. This part includes the core of the website on signs of pain and distress and opportunities for local or general analgesics, on general and specific clinical signs, and on the implementation of humane endpoints. The information is accompanied by photographs and videos. Annexes to the website are an extensive glossary and links to relevant organisations. In addition, a training section is included, where visitors are invited to recognise clinical signs, to decide on the application of humane endpoints in specific situations, and to work on case studies.

At a workshop held at the 8th World Congress on Alternatives and Animal Use in the Life Sciences, which took place in Montreal, Canada in 2011, Hendriksen and Griffin addressed the opportunities for ending severe pain and distress in animal experiments by the year 2025. There was consensus about the fact that eliminating severe pain and distress is in the interest of animal welfare and the quality of science. However, it was also felt that a ban on experiments with severe pain and distress will not be an option, but that there should be an increased emphasis on making those involved in animal experiments
more sensitive to the consequences of pain and distress. We believe that the use of the website on humane endpoints could be one of the instruments to support such an approach, for instance in training
programmes for young scientists.

Professor Coenraad Hendriksen
Faculty of Veterinary Medicine
Utrecht University
PO Box 80166
3508 TD Utrecht
The Netherlands
E-mail: C.F.M.Hendriksen1@uu.nl
Iris Boumans
Animal Production Systems Group
Wageningen University
PO Box 338
6708 WD Wageningen
The Netherlands
E-mail: Iris.Boumans@wur.nl

References
1 NVWA (2012). Zo doende 2011. [Annual report of the Netherlands Food and Consumer Product Safety Authority (NVWA) about animal experimentation and laboratory animals in 2011], 52pp. Utrecht, The Netherlands: Neder landse Voedsel- en Warenautoriteit.
Available at: www.rijksoverheid.nl/documenten-enpublicaties/
rapporten/2012/10/01/zo-doende-2011.
html (Acc essed 28.04.13).
2 Anon. (2010). Directive 2010/63/EU of the European
Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes. Official Journal of the European Union L276, 20.10.2010, 33–79.
3 Hendriksen, C.F.M., Steen, B., Visser, J., Cussler, K., Morton, D. & Streijger, F. (1999). The evaluation of humane endpoints in pertussis vaccine potency testing. In Humane Endpoints in Animal Experiments for Biomedical Research (ed. C.F.M. Hendriksen & D.B.
Morton), pp. 106–113. London, UK: Royal Society Medicine Press.
4 Cussler, K., Morton, D.B. & Hendriksen, C.F.M. (1999).
Humane endpoints in vaccine research and quality control. In Humane Endpoints in Animal Experiments for Biomedical Research (ed. C.F.M. Hendriksen & D.B. Morton), pp. 95–101. London, UK: Royal Society
Medicine Press.
5 Nemzek, J.A., Xiao, H.Y., Minard, A.E., Bolgos, G.L. & Remick, F.G. (2004). Humane endpoints in shock research. Shock 21, 17–25.
6 Sneddon, L.U. (2009). Pain perception in fish: Indicators and endpoints. ILAR Journal 50, 338–342.
7 OECD (2000). Guidance Document on the Recognition, Assessment, and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation, 39pp. Paris, France: Organisation for Economic Co-operation and Development. Available at:
http://www.oecd-ilibrary.org/environment/guidancedocument-
on-the-recognition-assessment-and-use-ofclinical-signs-as-human-endpoints-for-experimentalanimals-used-in-safety-evaluation_9789264078376-en (Accessed 28.04.13).
8 Castle, P. (1999). The European Pharmacopoeia and humane endpoints. In Humane Endpoints in Animal Experiments for Biomedical Research (ed. C.F.M.Hendriksen & D.B. Morton), pp. 15–19. London, UK: Royal Society Medicine Press.
9 Committee on Methods of Producing Monoclonal Antibodies; Institute for Laboratory Animal Research; National Research Council (1999). Monoclonal Antibody Production, 74pp. Washington, DC, USA: The
National Academies Press.
10 Hendriksen, C., Cussler, K. & Morton, D. (2010). Use of humane endpoints to minimise suffering. In The COST Manual of Laboratory Animal Care and Use: Refinement, Reduction, and Research (ed. B.
Howard, T. Nevalainen & G. Perretta), pp.333–353.
Boca Raton, FL, USA: CRC Press.

Responsibility for Animal Experiments: Where the Buck Stops

bunny and arm

Sadly, just after the Animals (Scientific Procedures) Act 1986 had been amended1 to bring into force in the UK the new European Directive 2010/63/EU on the protection of animals used for scientific purposes,2 allegations were made about the abuse of laboratory animals at one of the country’s leading academic research establishments.3 Nothing more must be said about that, pending the outcome of various investigations. However, it raises the question of who is responsible for ensuring that animals are used properly, and who is culpable, when things go wrong.

In the UK, we have long experience of the clearly-defined responsibilities of the Home Office ministers and officials, including the Animals in Science Regulation Unit (ASRU) Inspectorate, and various named persons in the breeding or user establishment, including the certificate holder, the day-today care person, the veterinary surgeon, the project licensees and the personal licensees. The former EU Directive, Directive 86/609/EEC, said relatively little about specific responsibilities, but the new Directive says far more in several of its articles, as follows:

Article 23 requires Member States to ensure the competence of all staff, by seeing that all staff are adequately educated and trained before they perform any of their functions.

Article 24 spells out the specific requirements for personnel to be responsible for overseeing the welfare and care of the animals in the establishment, ensuring that the staff dealing with animals have access to information specific to the species housed in the establishment, and ensuring that the staff are adequately educated, competent and continuously trained, and supervised until they have demonstrated the requisite competence, whilst also ensuring that projects are carried out in accordance with the project authorisation.

Article 25 requires Member States to ensure that each establishment has a designated veterinarian with expertise in laboratory animal medicine, charged with advisory duties in relation to the well-being and treatment of the animals.

Article 26 lays down the requirement for an animal welfare body, while Article 27 outlines its tasks in relation to the welfare of the animals, the application of the Three Rs, the internal establishment and review of operating procedures with regard to monitoring, reporting and follow-up, including the development and outcomes of projects and their further contributions to the Three Rs.
Article 30 requires the Member States to ensure that all establishments keep records of the number and species of animals involved, their origins, their use and their death or re-homing.

Article 34 specifies that the competent authorities of the Member States must carry out regular inspections, to verify compliance with the requirements of the Directive, taking into account the number and species of animals involved, the record of the establishment in complying with the requirements of this Directive, the number and types of projects carried out, and any information that might indicate noncompliance.

Article 35 states that, when there is due reason for concern, the Commission shall undertake controls of the infrastructure and operation of national inspections in Member States, and the competent authority of the Member State concerned shall undertake measures to take account of the results of the control.

There are, of course, other individuals and organisations with responsibilities. These include the research funding bodies and the peer reviewers who assess research grant proposals, as discussed in a useful booklet, Responsibility in the use of animals in bioscience research,4 published by the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs). Also of importance are the editors and editorial boards of the journals which publish articles which involve the use of laboratory animals, as was considered in a thoughtful article by Helen Galley.5

Given the clarity of the legislation and the specified responsibilities of so many different kinds of people, how can things go so wrong, as happens, as far as we know, on relatively rare occasions? First of all, human beings are involved, and whether those involved are doctors, lawyers, airline pilots, other professionals or even parents, we are a fallible
species and sometimes do wrong things, accidentally or deliberately. We need rules within which to work, and people in place to try to ensure that we do so, but they can’t watch all of us all of the time. In the end, it all depends on personal integrity, and, in the case of performing potentially-harmful experimental procedures on living vertebrates, the ultimate and inescapable responsibility must rest with those who actually perform the procedures. That is where the buck stops.

References
1 Anon. (2012). The Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012. Statutory Instrument No. 3039, 57pp. London, UK: The National Archive. Available at: http://www.legislation.gov.uk/uksi/2012/3039/contents/made (Accessed 23.05.13).
2 Anon. (2010). Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes. Official Journal of the European Union L276, 20.10.2010, 33–79.
3 Woolf, M. & Jacobson, S. (2013). Inquiry into ‘abuse’ of lab rats at college. The Sunday Times, 14 April 2013, p. 8.
4 Anon. (2013). Responsibility in the use of animals in bioscience research, 24pp. London, UK: NC3Rs. Available at: http://www.nc3rs.org.uk/downloaddoc.asp? id =719 (Accessed 23.05.13).
5Galley, H.F. (2010). Mice, men and medicine. British Journal of Anaesthesia 105, 396–400.

Animal Ethics Training for Postgraduates in Medical Schools in India: Catch Them Young!

Adequate training in ethics for young scientists will serve as an important means of improving animal-based research in India.

In India, students specialise in pharmacology after their graduation from medical or other health-related courses. Animal experiments form an integral part of the postgraduate curriculum in pharmacology, and equip the aspirants with the technical know-how and skills for future research. Therefore, these young researchers have an obligation to fulfil the course requirements before their final assessments.
The conduct of these experiments demands a considerable knowledge of animal ethics with respect to humane treatment, gentle handling and proper utilisation of laboratory animals, based on the Three Rs concept, in order to provide high-quality information.

For a pdf copy of the full article click here.