New Science 2010

ISSN: 2043-9881 (online)

Lord Dowding Fund for Humane Research

NEW SCIENCE ADVANCED TECHNIQUES: News from the world of research without animals

Progress in cancer study Implementation of alternatives Interview: Thomas Hartung Cataract lens replacement Animal testing directive ●

●

●

The Lord Dowding Fund for Humane Research

© T. Phillips / Lord Dowding Fund

EDITORIAL

The mission of the Lord Dowding Fund is to support, sponsor and fund advanced methods of scientific and medical research, which replace the use of animals; to fund non-animal medical research (fundamental and applied) in order to promote the adoption of nonanimal research methodology; to fund, promote and assist learning and training for the purpose of replacing animals in education and training.

Founded in 1973, the name of the Fund honours Britain’s Air Chief Marshal the Lord Dowding who, during his lifetime, was a President of our founding organisation, the National Anti-Vivisection Society.

The Fund awards grants totalling an average of £300,000 a year to researchers working in a wide range of fields.

Our projects have included cancer (breast, lung, metatastic); brain tumour, brain damage; brain toxicity; cataracts; ultrasound healing for cartilage; pain; vision; culture of tumours; infections including MODS and MRSA; computeraided drug design; biotechnology; computer-assisted science learning; microsurgery techniques; toxicity testing of dental fillings; safety testing; Parkinson’s; schizophrenia; cot death and kidney dialysis amongst others.

This ignores the reality of research – that if we are to get the best scientific endeavours, we need a raft of different techniques and we need to constantly develop new technologies – more accurate and more relevant to people. Animal research is the opposite of this; stagnant and scientifically unreliable. Tissue, organ and cell cultures, sophisticated imaging techniques, computer analysis, simulations and modelling such as QASARs, micro-dosing and accelerator mass spectrometry, as well as toxicogenomics and others are all moving forward, leaving animal research behind. It’s just that the regulators have not caught up, and in their anxiety about new methods, they allow the animal experimenters to keep them in the dark ages.

The Lord Dowding Fund is a department of the National Anti-Vivisection Society and Animal Defenders International.

The notion that experiments in a different species can deliver the information required for the human situation is a fallacy. THE single alternative is a myth too.

We must embrace a multi-disciplinary approach that delivers the right answers, and ensure that this is recognised and implemented by regulators.

New Science ISSN: 2042-9576 published by The Lord Dowding Fund for Humane Research

LDF UK: Millbank Tower, Millbank, LONDON, SW1P 4QP, UK Tel: +44 (0)20 7630 3340 Fax: +44 (0)20 7828 2179 e-mail: info@ldf.org.uk web: www.ldf.org.uk

Chief Executive, Jan Creamer, being interviewed at Aston Universityfor The OneShow.

Welcome to the latest edition of New Science, which brings you the latest news from the world of research without animals, as well as the project we support through the Lord Dowding Fund.

People often ask “what’s THE alternative to animal experiments?”, as if research is based on a single methodology. In reality, animal research is a backwater of the world of scientific and medical research; the majority of research is conducted without animals, using modern and superior technological and scientific methods.

Whilst animal experimentation is demonstrably limited, the options for non-animal replacements are almost limitless.

Animal experimenters like to perpetuate the myth that there needs to be just one alternative, with statements like “you can’t measure blood pressure in a computer”, when they know full well that it is the use of a range of techniques in combination which provides the replacement to animal use.

The regulators and politicians are comfortable with this simplistic one-modeldelivers-all notion. It fits easily into bureaucratic check-lists. As a result, the implementation of replacements is restricted and slow. The current revision of the European Directive on animal experiments has failed to properly address the issue of replacements, notably in fundamental (basic) research.

That’s our approach with New Science: To showcase the best of new scientific method and technology and to put this in the context of regulation and replacing animal experiments.

Should people really accept substandard science and animal suffering merely because regulators find implementation of the replacements too complex? We believe people, animals, and science deserve better.

LDF contact for USA: Animal Defenders International, 953 Mission Street, Suite 200, SAN FRANCISCO, CA 94103, USA Tel: +1 415 543 2344 Toll free: 800 978 ADII (2344) (inside US) Fax: +1 415 543 2343 e-mail: usa@ad-international.org web: www.ad-international.org

Jan Creamer Chief Executive

Help Finance an Evolution Will you leave a lasting legacy of compassion, and help scientific advancement?

South America: Apartado Postal 359888 BOGOTÁ, Colombia e-mail: info@ad-international.org web: www.ad-international.org Editors: Creamer/Phillips Design: Creamer/Phillips/Elson Contributors: Jan Creamer; Tim Phillips; Helder Constantino; Christina Dodkin; Jessamy Korotoga; Amanda Gent.

Cover image: LDF ©2010 LDF. All rights reserved. No part of this publication may be reproduced for commercial purposes by any means whatsoever without the written permission of LDF.

2

New Science

l

2010

© Lord Dowding Fund

LDF finances scientific and medical research without the use of animals. The projects featured in this publication have only been possible thanks to the generous donations and bequests of our supporters. Please consider a donation or legacy in your WILL to help our work. Your legacy could advance medical research and help to end unnecessary animal suffering. Help finance an evolution and leave a lasting legacy with a bequest or donation today, to the Lord Dowding Fund for Humane Research

Lord Dowding Fund for Humane Research

NEWS

© Lord Dowding Fund

LDF Project Report: Cartilage research showcased at international conference

NEWS IN BRIEF Cosmetic industry matches Commission in funding alternatives In July 2009, the European Commission called for proposals on the development of a strategy for alternatives to animal use in repeat dose systemic toxicity testing. The European cosmetics industry decided to match the research funds made available by the Commission, bringing the total available funds up to €50million.

The Commission will select up to seven projects to receive funding in areas including computational modelling, organ-simulating cellular devices and functional differentiation of human-based target cells in vitro. http://www.nc3rs.org.uk/news.asp?id=1127

© Lord Dowding Fund

AltTox – promoting alternative methods online

Dr. Vehid Salih (left) and Dr. Jamie Harle and their ultrasound exposure apparatus (inset).

In the last issue of New Science we highlighted the innovative research by Dr. Vehid Salih and Dr. Jamie Harle into a human, in vitro model of cartilage for testing the healing properties of ultrasound.

Currently, Drs Salih and Herle are working on modifications to the ultrasound exposure rig to expand the range of acoustic outputs available. The best scaffold composition will be used in the ‘exposure’ work for the next stage of the project. This will enable exposure of pulsed ultrasound signals, rather than the continuous wave form used in the work so far.

During the next phase of the work, researchers aim to investigate the biological responses of hMSC (human mesenchymal stem cells) for both diagnostic and therapeutic ultrasound exposure parameters. The exposure rig will be modified further allowing ultrasound signals to be amplified so that varying intensities of ultrasound can be delivered to the hMSC-alginate constructs.

The model will then be used to look for ultrasound-induced effects which are intensity-, exposure time-, or pulsing regime-dependent.

As the use of donor grafts to solve orthopaedic problems is not ideal, producing natural tissues artificially, with the use of such a scaffold to guide regeneration, is a good alternative. The regeneration of bone tissue can be greatly improved by regular treatment with therapeutic ultrasound. Therefore, the current research has great potential to develop these methods to greatly benefit patients with cartilage injuries.

The presentation was well received at the European Society for Biomaterials’ Annual Congress in September 2009, promoting the use of HA(hydroxyapatite)/alginate as an animal-free scaffold for an in vitro model of cartilage. The European Society for Biomaterials publishes a monthly journal and holds annual conferences to encourage progress in the field of biomaterials.

Lord Dowding Fund for Humane Research

AltTox (alttox.org) is a website which aims to advance non-animal methods of toxicity testing. The rationale for the site is to provide better protection for human health, animals and the environment. It also aims to reduce the number of animals being used in toxicity testing and the suffering that they undergo.

AltTox is composed of three different parts: Online forums – For posting news, information and for encouraging feedback. The forums are split into four categories: ‘Toxicity Endpoints & Tests’, ‘Emerging Technologies’, ‘Programs & policies’ and ‘Overarching challenges & opportunities’. Each forum’s moderator is an internationally recognised expert on the subject.

Information on toxicity testing – The TTRC (Toxicity Testing Resource Centre) is a comprehensive source of information on non-animal methods of toxicity testing. The topics are cross-referenced to the forums.

Invited commentaries – These are opinion pieces, written by experts, to accelerate progress in the development of non-animal methods.

New Science

l

2010

3

NEWS

The European Centre for the Validation of Alternative Methods’ (ECVAM) Scientific Advisory Committee (ESAC) is to be renewed. ESAC, which is known for publishing opinions on the validity of alternative test methods, will now gain support from the ECVAM Expert Pool (EPP), which is soon to be formed. An ECVAM Stakeholder Forum (ESTAF) will also be created in order to maintain an active dialogue with various stakeholders, including those with specific vested interests, including voicing societal concerns.

The new ESAC, comprising of senior, generalist scientists, will continue to issue opinions on ECVAM evaluated alternative test methods. The EPP will consist of a pool of highly specialised scientists who will cover a range of areas necessary for the validation of alternative tests, which would serve a wide range of applications.

ESAC working groups, dedicated to peer review submissions received or coordinated by ECVAM, will be chaired

by ESAC full members with the group containing mainly scientists from the EPP register. These working groups will prepare draft opinions for final adoption through ESAC.

© Tim Phillips / Lord Dowding Fund

NEWS IN BRIEF New ECVAM advisory structure

LDF Project Report: Neurotoxicity research goes from strength to strength As reported in the last issue of New Science, LDF is funding exciting work carried out by Professor Mike Coleman (pictured above) at Aston University. This research studies the reactions of brain cells to toxicity, using a human-based model to study neural function.

Most other cellular models use rapidly dividing cells, which does not occur in human brains where the cells are postmitotic (they have stopped growing.)

Professor Coleman’s model contains the most complete basic unit of the brain, the neurone/astrocyte combination. This is an improvement on many other models, which just look at neurones.

A new drug releasing coronary stent (tubular support placed inside a blood vessel) was investigated by scientists in a ‘first-in-human’ study. ‘MAHOROBA®’ was developed to treat coronary artery disease.

The study used 47 patients suffering stable and unstable angina and other cardiac problems. At six months, two patients had suffered heart attacks and 11 required repeated revascularisation because of ischemia (blood restriction) One of the features of the tested stent led to “positive results” in pigs, but this study “failed to establish the effectiveness of the MAHOROBA® stent”. A report on the study concludes that the “translation of results from animals to humans remains unclear” http://www.nature.com/nrcardio/journal/v6/n8/pdf/nrcardio.2009.117.pdf

4

New Science

l

2010

© Lord Dowding Fund

Coronary stent fails to live up to its promise

Ultimately, it should provide a better, more logical basis for re-creating the conditions of the neuronal/astrocyte partnership development. The model may also be used to understand neurodegeneration in the hope that this will lead to easier repair. As Professor Coleman states “we hope that our technique will provide scientists with a new and highly relevant human experimental model to help us understand the brain better and develop new drugs and treatments to tackle neurodegenerative disease.” This work recently received extensive media coverage including BBC and Sky News.

A neural network with astrocytes (shown in red).

Lord Dowding Fund for Humane Research

© Tim Phillips / Animal Defenders International

LDF PROJECT: ARTIFICIAL LYMPH NODE

Artificial Lymph Node: Testing human specific immune reactions It has been stated that the “development of therapeutical [sic] monoclonal antibodies has resulted in an increased need for primates in reproductive toxicology since the immune system in macaques is much more similar to humans compared with rodents.”1 However, as seen in previous disastrous human drug trials such as TGN1412, the immune systems of non-human primates are not sufficiently similar to those of humans to detect human immune reactions. The importance of testing human immune reactions to many modern biopharmaceuticals has emerged due to the very high degree of species specificity of drugs.

The lymphatic system is involved in human immunity2. Lymphatic fluid transports bacteria and other foreign agents along the lymph vessels to the lymph nodes. Lymphatic fluid is presented to antigens held in the nodes as it is filtered through them. As fewer vessels leave the node than enter, flow through the nodes is slow, allowing time for foreign agents to be destroyed by the antigens3. Lymphocytes, found in the lymph node, are antigens which are primarily responsible for specific immunological responses4.

As animals possess an often significantly different immune system, it is insufficient to carry out tests with any animal species to assess the efficacy and safety

of drugs in humans. The creation of a similar to that of the lymph node, it will fully human Artificial Lymph Node (ALN) be useful for looking at the effects of circumvents “the limitations associated drugs and cell therapies7. with the use of animal models”5.

Dr. Uwe Marx, one of the key scientists in the development of the ALN model, The ALN model uses a bioreactor which purports that in order to develop and is able to control the environment of the adopt such models across the EU, a tissues being grown. The model is also “human in vitro organ” program should reproducible which is important for its be designed, specifically “for predictive possible role in drug development and reliable substance testing” incorporating screening. The ALN mimics the disciplines such as tissue engineering, physiology of the lymph node in the bioreactors, and computational models8. human body by integrating mobile and immobile cell phases to ensure the cells interact effectively6.

A perfusion of cells Artificial Lymph Node and media flow from media exchange cycled cell suspension an outer culture space into a central culture space, which is a fixed porous membrane PCS matrix containing HFM immobile cells. This CCS gas, media, allows cells flowing substrates through the system to be presented to the fixed cells. This immobile matrix cell-network emulates the actions which occur in the mobile cells lymph node when the lymphatic fluid flows into the node, passing Concept of mobile and immobile cell phases interacting in the artificial lymph by the lymphocytes node bioreactor. The suspended lymphocytes pass the CCS and come into close contact with the immobile dendritic cell network in the embedding which carry out an matrix of the CCS. (HFM = hollow-fibre membrane. PCS = peripheral immune response. As culture space. CCS = central culture space.) this system creates an environment which is

1. http://ec.europa.eu/health/ph_risk/committees/04_scher/docs/scher_o_110.pdf 2. http://uhaweb.hartford.edu/BUGL/immune.htm#top 3. Marieb, E.N (1992) Human Anatomy and Physiology 2nd Edition, The Benjamin/Cummings Publishing Company, Inc. California 4. Miller, J.F.A.P, Lymphocytes, In: Dulbecco, R (Ed) (1991) Encyclopedia of Human Biology, Vol. V, Go-Me, Academic Press, Inc USA 5. http://www.probiogen.net/english/news/view_newsfile.php?id=134 6. Pörtner, R and Giese, C, An Overview on Bioreactor Design, Prototyping and Process Control for Reproducible Three-Dimensional Tissue Culture. In Marx, U and Sandig, S (2007) Drug Testing In Vitro: Breakthroughs and Trends in Cell Culture Technology, WILEY-VCH, Germany 7. Hitchcock, T and Niklason, L (2008) Lymphatic Tissue Engineering Progress and Prospects, Annals of the New York Academy of Science Vol. 1131, pp: 44-49 8. Pers. Comm. Dr Uwe Marx 14/04/08

Lord Dowding Fund for Humane Research

New Science

l

2010

5

Above: Wild, free macaque.

EXPERT OPINION

Dr. Thomas Hartung is a leader in the field of toxicology who was, in 2009, appointed Director of the Johns Hopkins Centre for Alternatives to Animal Testing (CAAT). As an advocate for alternatives to animal testing he has assisted in the development of several test strategies, revolutionising the way in which chemical safety assessments are carried out in Europe. In this exclusive interview, Dr. Hartung reveals his motivations and hopes for the future of advanced methods to replace animal testing.

New Science (NS): What does your new role at CAAT involve? How does this differ from your previous role at ECVAM (European Centre for the Validation of alternative Methods)?

I would characterise it as two sides of the same coin: both institutions have been promoting alternative methods for many years. ECVAM is giving scientific policy support, especially to the European Commission, and is responsible for the formal validation of methods; CAAT has, for 28 years, been a key information hub sponsored by private foundations and industry. CAAT is not working primarily on validation. As part of Johns Hopkins, one of the leading universities worldwide, it is primarily bound to sound science and more broadly to humane science approaches and the paradigm shift in toxicology. I aim to further develop CAAT toward becoming a think tank of toxicology. Last but not least, education of students is a key role of an academic institution and we try to promote humane science in various curricula.

NS: What motivates you to be involved in the development of alternatives to animal testing?

I want to move the science of risk assessment forward and make it more humane, so it is about safety of patients and consumers AND animal welfare. I believe that alternative methods are the forerunner for the revolution of risk assessment sciences ahead of us.

Alternatives are the pilots for a new toxicology and for quality assurance in the life sciences in general. There is no other field, where the same effort is spent to ascertain the predictive value of models and to quality control them.

That’s where science has to move – we are publishing far too much, which is not reproducible, is irrelevant or simply artefact.

6

New Science

l

2010

NS: What barriers, do you think, prevent researchers in academia and working in pharmaceutical companies from using alternatives as opposed to animals?

There are a few barriers: traditions, regulatory requirements, lack of information on opportunities. However, on a positive note, there is also pressure for change such as the biotech and informatics revolution, which provided new tools, companies commercialising these, legislative pressures such as the European animal welfare legislation, REACH and the 7th amendment of the Cosmetics Directive [total ban on animal testing regardless of available alternatives]. Furthermore, needs for higher testing throughput and increasing awareness of the shortcomings of current tests help a lot.

NS: What are the advantages of using non-animal methods in toxicology?

Simply, to overcome the limitations of animal testing. These are not only ethical ones: animal tests are costly, have low throughput and do often give wrong results. Some were developed 40-80 years ago, especially in regulatory toxicology. In the meantime a body of knowledge has been built on mechanisms, molecular targets and modes of action. These must be fully exploited before targeted, hypothesisdriven testing in animals starts, if still necessary.

NS: Do you think there will be a time when animal testing will not be a legal requirement in the regulatory testing of drugs?

Yes – we already see that we are harming the drug development process with wrong decisions. Killing valuable substances under development also kills people. Furthermore, more than half of the new drugs are human proteins or antibodies to human proteins making

© Dr. Thomas Hartung

Dr. Thomas Hartung, Director of the Johns Hopkins Centre for Alternatives to Animal Testing and former Head of ECVAM, in conversation with New Science

classical approaches useless. If we can show that we can handle these properly without animal testing, why shouldn’t we do so for small molecules?

NS: Do you believe that the use of primates in experiments can be replaced and, if yes, how?

In the end by moving to human testing, therefore bypassing non-human primates, e.g. microdosing. This certainly requires another level of certainty in the non-animal methods and non-primate models. The trend to use human proteins is certainly prompting a lot of primate use. However, the tragedy of the Tegenero antibody [TGN1412], which had no effect at 500 times higher doses in primates and almost killed instantly the first six volunteers treated, has shown that also this testing has questionable results.

NS: Do you regard the new Directive on animal testing, as drafted by the European Commission, as a good text to regulate animal testing and promote alternatives?

Europe has, since 1986, had the most progressive legislation for animal welfare worldwide. The new draft is moving this even further – exactly how far will be determined by the political decision process. It will certainly raise standards further but also leave room for improvement for the next revision…

NS: Would you agree with the statement that the use of animals in experiments is a scientific dogma that must be challenged?

I do not consider it a dogma – something believed to be true without question. I consider it a tool with increasingly known limitations. The more we make these limitations obvious and develop other tools, the less useful, and less used, the animal tools will become.

For a full version of this interview, please visit www.ldf.org.uk Lord Dowding Fund for Humane Research

VALIDATION AND IMPLEMENTATION

Guide to validation and implementation of alternatives © Tim Phillips / Lord Dowding Fund

strategies can help to overcome some of these obstacles.

Networking between stakeholders and involving regulators in the validation process and research programs have also been suggested as ways of alleviating some of these problems3.

Another ECVAM workshop pointed out the difficulties of using traditional test methods, such as in vivo, as reference against which to compare new alternatives. It is pointed out that assumptions about the toxicity of test compounds are based on these ‘traditional methods’ which may or may not accurately reflect human data. The report of the workshop points out that “the majority of these traditional tests themselves were never validated” and that, although traditional test methods are broadly accepted, they do not necessarily qualify as reference test methods4.

Some useful methods are being used by the European Commission to monitor/track the progress of alternative tests, such as the Tracking System for Alternative Test Methods Review (TSAR). This system tracks methods from the point when they are purely scientific protocols submitted for pre-validation, to their active use in a regulatory context5.

Many scientists are researching methods which are able, or have the potential, to provide alternatives.

However, the reality is that their implementation depends on more than just development.

1. http://ecvam.jrc.it/page_pdf.cfm?voce=m&idvoce=1 Acessed 29/09/09 2. Balls, M et al (1995) Practical Aspects of the Validation of Toxicity Test Procedures: The Report and Recommendations of ECVAM Workshop 5, IATLA 23, 129-147 3. Bottini, A. A et al (2008) Optimisation of the Post-validation Process: The Report and Recommendations of the ECVAM Workshop 67ª, ATLA, 36, 353 - 366 4. Hoffman, S et al (2008) Points of Reference in the Validation Process: The Report and Recommendations of ECVAM Workshop 66ª, ATLA 36, 343 - 352 5. http://tsar.jrc.ec.europa.eu/ Accessed 12/10/09

Lord Dowding Fund for Humane Research

Above and below: Non animal testing methods in use for the neurotoxicity research project at Aston University, as described on p4.

© Tim Phillips / Lord Dowding Fund

animal welfare communities. Among other aims, the panel should consider whether the purpose and objectives of the validation study have been met.

5. Progression toward regulatory acceptance – The validated methods need to receive widespread international recognition. For example through the Mutual Acceptance of Data Agreement, the OECD (Organisation for Economic CoOperation and Development) agreement sets out that member countries agree to accept data from tests which are performed to OECD test guidelines. These guidelines are used in Europe, Japan and North America.

Validation and acceptance problems Recent publications have focused on the need to ensure that once methods have passed the validation process they are accepted and implemented by regulatory authorities. An ECVAM workshop on the optimisation of the post-validation process highlighted some obstacles to regulatory acceptance of validated alternative methods: ● Reluctance to share data by some companies as it may harm their competitive position. Data sharing would, however, avoid duplication. ● Insufficient communication, for example, between scientists and regulators. ● The tendency of regulators to favour refinement and reduction over replacement, due to a familiarity with animal procedures and the ease of modifying these instead of replacing methods altogether with alternatives. ● Scientists lack knowledge about the regulations and the opportunities which could influence those regulations.

There are ways to overcome these obstacles. By taking advantage of joint initiatives such as the European Partnership for Alternative Approaches to Animal Testing (EPAA) the mapping of existing research and development of

© Lord Dowding Fund

Many scientists, such as those funded by the LDF, are working to develop methods which could and should be used in place of tests on animals. However, the actual regulatory acceptance, implementation and dissemination of these methods is a long and sometimes difficult process.

The validation process The European Centre for the Validation of Alternative Methods (ECVAM) was established in 1991 as part of the European Commission’s Joint Research Unit. The duties of ECVAM include: the validation of alternatives; being a focal point for the exchange of information on development of alternatives; to promote dialogue between legislators, industry, scientists, consumer organisations and animal welfare groups on the issue of alternative methods1.

The ECVAM validation process involves establishing the reliability and relevance of an alternative procedure for a specific purpose in five stages2: 1. Test development – The test must be described and its scientific purpose defined. A protocol must be produced which is comprehensive enough that the test may be carried out by another laboratory (interlaboratory transferability).

2. Prevalidation – This stage optimises and standardises the protocol. It is important for ensuring that methods entering a formal validation study have a greater likelihood of fulfilling expectations of scientists and regulatory bodies.

3. Validation – An interlaboratory blind trial is carried out at this stage, with ‘preliminary’ and ‘definitive’ stages where coded chemicals are tested.

Finally, data is analysed and the outcome of the studies evaluated.

4. Independent assessment – The published results of the validation studies are considered by one or more independent assessment panels.

These panels need to be independent of the validation study in question and representative of the scientific, toxicological, industrial, regulatory and

New Science

l

2010

7

Below: Accelerator Mass Spectrometry (AMS) equipment in use at Glasgow University. AMS offers precise analysis of samples from volunteers who have received micro-doses of test compounds.

LDF PROJECT: BLOOD BRAIN BARRIER

© Lord Dowding Fund

Progress in brain tumour project Above: A slide showing stained endothelial cells, which line the blood vessels.

With 25% of cancers found in the body and endothelial cells to either side of a the BBB a highly effective biological spreading to the brain and worsening membrane filter. This membrane is barrier so these results indicate that patient prognosis, it is vital to understand coated with extracellular matrices (ECM) astrocytes improve the TJ formation. the mechanisms by which cancerous and the whole model is grown in human Currently, the addition of pericytes to cells metastasise (move) into the brain, serum supplement. create a tri-culture has not improved tight thereby spreading the cancer. In vitro The initial stages of the study were junction formation. However, this is constructions generally use animal performed to analyse the components of possibly due to the pericytes blocking the tissues, such as rat and mice cells, to the BBB model, to aid the development endothelial cells, preventing them from model the blood-brain barrier (BBB), the of the Transwell® model. In these first producing a monolayer. The team intend layer which covers and protects the stages, different cancer cells were added to repeat the experiment, with the brain. However, these animal models do to various cultures of brain cells. Results pericytes at the bottom of the Transwell® not reflect the situation in humans. found lung cancer cells, which were so that they are not in direct contact with Those models which do use human cells observed over 48 hours, to move the endothelial cells. are often grown in media which include through an endothelial cell mono-culture, It is hoped the model will identify the foetal calf serum, a product which is demonstrating the cancer’s high pathways malignant cells take to cross ethically unsound. metastatic potential. the BBB and, once fully developed, could Exciting new progress has been made Aspects of the cells were measured and be used to study metastases. In however, in the development of an allobserved using state of the art addition, because of the importance of human tissue model of the BBB at techniques such as ECIS™, which uses the BBB in conditions of the Central Portsmouth University, where Professor electrical currents to measure the Nervous System, such as stroke and Geoffrey Pilkington and his team have resistance of cells and cell behaviour, brain trauma, this model has the been testing various permutations of cell treatment with immunoflorescence and potential to improve the treatment of cultures in the Transwell® model. The time lapse microscopy. these conditions. Excitingly the model researchers are using astrocytes could also be used as a tool by drug Recent preliminary results have revealed (supporting cells) from two different companies to discover if, and how, that co-cultures of astrocytes and areas of the brain; endothelial cells, therapeutic agents pass through the endothelial cells have shown better which line the blood vessels, and BBB. formation of ‘tight junctions’ (TJs) than pericytes. Pericytes are a less well endothelial cell mono-cultures. TJs make known component of the BBB and are thought to regulate Professor Pilkington’s Blood-Brain Barrier proliferation and differentiation of endothelial cells. Many other researchers do not include pericytes in in vitro models due insert to the difficulties involved by Endothelial cell growth medium introducing them into the BBB model. However, the team at Portsmouth has persevered with Endothelial cells this element as they believe their Membrane filter (white) ECM components (blue) inclusion is needed to produce a true reflection of the in vivo Astrocytes situation.

Astrocyte growth medium The Transwell® model is set up by adding the human astrocytes

8

New Science

l

2010

Lord Dowding Fund for Humane Research

LDF PROJECT: CATARACTS

Cataract lens replacement tested in fully human system tissue culture at varying concentrations of serum.

Once this was established, the first IOL was implanted into an eye capsule. An eye bag without an IOL was used as a control. The observations, to monitor any sign of PCO indicated by cell re-growth, were made using phase-microscopy. The IOL did slow the development of PCO, but did not prevent it completely; cells grew in sufficient number to cover the posterior of the capsule. Wrinkling of the

1. http://www.cks.nhs.uk/patient_information_leaflet/cataracts_age_related

Lord Dowding Fund for Humane Research

capsule’s posterior also occurred, but less severely. In the human patient this would have resulted in impairment of the patient’s sight.

The initial IOL had round edges and it is known that IOL designs with a square edge are better at preventing PCO. Therefore, the next lens to be tested will be square-edged IOL which, if there is seen to be further retardation of cell growth, will provide further evidence for the use of the current model.

© Lord Dowding Fund

Cataracts are cloudy patches in the lens inside the eye and are the main cause of impaired vision in the world, with an estimated 30% of over 65 year olds having cataracts in one or both eyes.

There are various risk factors for the condition, which include a family history of cataracts, smoking, poor diet and exposure to sunlight1.

According to an article in the British Medical Journal, “cataract surgery is possibly the world’s oldest surgical procedure”, being brought to Europe from India by the armies of Alexander the Great2.

The only cure for cataracts is to remove the lens from the eye, which is done by either surgically removing the nucleus of the lens as a whole or by ultrasonically fragmenting the nucleus, which is then sucked out of the eye. The latter procedure requires a smaller incision, causes less inflammation postoperatively and rehabilitation takes 1-3 weeks, rather than 2-4 months for the former process.

Once the affected lens has been removed, it is replaced with an intra ocular lens (IOL). However, some lens’ epithelial cells still remain after the surgery. In approximately 25% of patients this leads to the development of Posterior Capsule Opacification (PCO), a condition where the epithelial cells spread across the visual axis of the eye.

The design of IOLs can help prevent PCO, but current research into IOL designs is largely conducted on rabbits.

LDF sponsored research by Dr. Michael Wormstone at the University of East Anglia is using ‘eye bags’, from human donors, to carry out such tests. Dr. Wormstone recently established the optimum level of human serum in which to maximize cell growth on the lens. This was done by counting cell growth in

2. Spalton, D.S & Koch, D (2000) “The Constant evolution of cataract surgery”, BMJ ,vol.321. pp. 1304

New Science

l

2010

9

Below: A piece of lens cultured in human serum. Cell growth can be seen in purple.

LDF PROJECT: EDUCATION

EU survey of animal use LDF and Profess in education Left: Dewhurst computer, 1988.

implementation of alternatives will be investigated. The results of the research, which is being overseen

by Professor David Dewhurst at the University of Edinburgh, will be a unique tool for targeted replacement of animals in education. In addition, it will demonstrate the feasibility of gathering quantitative data in order to assess possibilities for the implementation of advanced techniques in other areas of research where animals are currently used.

Questionnaires will be sent to universities and professional bodies in the target member states, to gather information on the use of alternatives to animals and specific data regarding animals, such as where they are used and why. The researcher conducting the survey will follow up the questionnaires with phone calls and emails to maximise the number of responses, thereby giving LDF a more detailed picture of the situation.

At the end of the survey it is hoped that a comprehensive picture of the use of animals across the EU will have been gained, along with information on how and where LDF can push alternatives in order to reduce the number of animals used from 200,000 to ZERO. We will report on the progress of the survey, and its outcomes in future editions of New Science and in our sister magazine Animal Defender.

Below: Early computer simulation, 1993.

© David Dewhurst

© David Dewhurst

Below: Prof. Dewhurst receiving the Queen’s Anniversary Prize for Higher and Further Education, 2005.

Right: Workshop in India, 2006.

Below: Prof. Dewhurst receives the Doerenkamp-Zbinden Foundation International Prize for Animal Free Research, 2006

Right: The physiology and pharmacology simulation, having been subjected to the RECAL process, 2009.

10

New Science

l

2010

Lord Dowding Fund for Humane Research

© Lord Dowding Fund

© Christina Dodkin / Animal Defenders International

© David Dewhurst

The latest figures from the 25 EU Member States, reporting animal use in 2005 (2004 for France), state that 198,994 animals were used for “education and training”. This is staggering in its enormity and also the diversity of species used in this area.

The report gives scant information on the exact use of these animals, which include 86,597 mice, 50,048 rats, 3,856 rabbits, 5,854 pigs, 1,391 cattle, 15,666 amphibians and 23,796 fish.

The figure fell since the preceding reporting period, 2002 (2001 for France) which reported that 341,967 animals were used. The latest report states that the “decrease of animals used for education and training can be attributed to both an uptake of alternative techniques and the re-use of animals.”

However, we believe that many more animals are being used in dissections or other procedures where statistics are not published.

LDF is funding an exciting new project to clarify exactly what these animals are being used for, to gain valuable information regarding their use and also to target the implementation of alternatives to certain subject or geographical ‘hot-spots’.

This is the first LDF funded survey, and it will be on a massive scale! The aim of the survey is to find out exactly where animals are being used in higher education, for which subject and in what numbers. In addition, the availability and

LDF PROJECT: EDUCATION

sor David Dewhurst – 24 years of Progress 1986

LDF’s funding of Dr. Dewhurst began in order to strengthen the NAVS Violence Free Science campaign. Nerve physiology simulation for BBC microcomputer released

© David Dewhurst

physiology, Frog Heart, Neuromuscular Pharmacology and ‘Rat stack’ (rat 1987-9 Muscle dissection) simulations released

© David Dewhurst

© David Dewhurst

Below: Prof. Dewhurst at Balkan University, 2006.

1989

New projects to develop computer-based alternatives in pharmacology and rat dissection (interactive video-disk) commenced. Conversion of BBC microcomputer versions to IBM (DOS) versions

1991

Replacement of all frog, rat, rabbit and guinea pig practicals with computer simulations at Liverpool and Leeds Polytechnics. Several new simulations released including Guinea Pig Ileum, Cat Nictitating Membrane, Frog Skin, Intestinal Absorption in Rat

1993

Evaluations of educational effectiveness of the simulations carried out in small number of UK universities – all supported the view that students’ learning using simulations was at least comparable to that in animal laboratory classes

1996

One of the simulations, Rat Blood Pressure wins the 1996 Multimedia Festival held at the 2nd World Congress on Alternatives and Animal use in the Life Sciences, Utrecht, The Netherlands

1997

Megan Quentin-Baxter, who developed the interactive video-disk of a rat dissection, was awarded her PhD which was largely based around this work

1998

Work commenced on rewriting simulations for the MS Windows environment alongside the development of new simulations

1999

New project supported by LDF to continue redevelopment of simulations for MS Windows began at the University of Edinburgh

2001

The European Resource Centre for Alternatives in Higher Education (EURCA) is set up by David Dewhurst (University of Edinburgh) and Jan van der Valk (University of Utrecht) with the help of LDF and the Doerenkamp Zbinden Foundation

2003

The funding of RECAL began, which should eliminate the need for rewriting computer programs to keep pace with technological advances and enable teachers to assemble personalised teaching and learning materials for replacing animals in higher education

2004

A lecture tour of Brazilian Universities was carried out by Professor Dewhurst to promote computer-based teaching packages to replace animals

2005

Professor Dewhurst’s team awarded the Queen’s Anniversary Prize for Higher and Further Education, for their work in using e-learning to support medical and veterinary education in Edinburgh

2006

Workshops held in China and India to promote computer alternatives to higher education professionals. A workshop was also held for teachers of physiology and pharmacology at Balkan Universities. Professor Dewhurst awarded the 2006 Doerenkamp-Zbinden Foundation International Prize for Animal Free Research at a ceremony in Linz

2007

With seventeen computer programmes having been successfully taken apart and subjected to the RECAL process, further funding was granted for the project’s continuation

2010

LDF continues to fund Professor Dewhurst’s projects at the University of Edinburgh

Lord Dowding Fund for Humane Research

New Science

l

2010

11

EU DIRECTIVE ON ANIMAL EXPERIMENTS

New dawn or bureaucratic impasse?

Opposite, from main picture, clockwise: Jan Creamer addresses the Progress Without Pain conference at the European Parliament.

Alongside (from left) are Commissioner Dimas, Neil Parish MEP, and LDF grant holder Professor Paul Furlong.

The European Commission.

Amongst ADI’s supporters in the European Parliament were Jens Holm MEP, Caroline Lucas MEP, Mojca Drcar Murko MEP. ADI Campaigns Director Tim Phillips and Head of Parliamentary Affairs Helder Constantino leave Parliament after giving oral evidence to the House of Lords Committee reviewing the new EU rules for lab animals.

© Lord Dowding Fund

Below: Alternative research methods in progress at Aston University; ADI produced many scientific and legal Technical Briefings in up to 12 languages.

The new Directive’s regulations will represent a significant step forward for animal welfare compared to those of 86/609/EEC, defined nearly a quarter of a century ago. However, whether the Directive will have a positive impact on those who are promoting, developing and using nonanimal research methods remains to be seen.

The European Commission’s proposal on the use of animal testing, issued in November 2008, has entered the final stage of the EU legislative process. The Council of Ministers, chaired by the Swedish Presidency, and the Rapporteur of the European Parliament Elizabeth Jeggle MEP (Germany, EPP) reached a compromise in December 2009.

However, the political groups of the European Parliament have yet to approve the Council’s amended text. In fact, the Greens/EFA Group has announced that it will reject the current text, with one of the major issues at stake being the weakening of the requirement to use alternative methods.

Yet, their promotion was originally one of the main aims of the new Directive.

In Recital 11 of the version presented by the European Commission in 2008, the Directive states that “the principles of replacement, reduction and refinement should be implemented through a strict

12

New Science

l

2010

hierarchy of the requirement to use alternative methods.” Thus, the text starts with a very positive signal that replacement is the highest goal of the Directive.

The ‘3Rs’ principle is implemented in Article 4: “where a method of testing not involving the use of animals exists and may be used in place of a procedure, Member States shall ensure that the alternative method is used.”

In paragraph 2, the principle of reduction is laid out, followed by refinement in paragraph 3; both apply as soon as animals are used, including in breeding, accommodation and care. However, the replacement principle in the new Directive is not new, it was included in Article 7 of the 86/609/EEC Directive, in equally strong terms: “an experiment shall not be performed if another scientifically satisfactory method of obtaining the result sought, not entailing the use of an animal, is reasonably and practicably available.”

The real innovation of the Commission lies elsewhere. In order to accelerate the development and validation of alternatives, the Commission created National Reference Laboratories (NRLs) in Article 46: “each Member State shall (…) designate a national reference laboratory for the validation of alternative methods replacing, reducing and refining the use of animals.” The article details specific requirements and objectives to NRLs. It states they must be staffed with personnel qualified in alternatives and have appropriate infrastructures and

equipments. In addition their function is to participate, in coordination with the Commission, to: validate alternative methods; provide scientific assistance and information to authorities; and provide training in alternative methods.

LDF welcomed this proactive approach which could multiply the number of validated alternatives and contribute to alleviate the bottleneck at the European Centre for the Validation of Alternatives (ECVAM). ECVAM was created by the Commission to support EU objectives contained in Directive 86/609/EEC. Due to lack of funding ECVAM has only validated 27 methods since its creation in 1991, since it could only review a small proportion of the alternative methods submitted. This bottleneck considerably slows down ethical and scientific progress in Europe.

Caroline Lucas MEP (UK, Green Party) criticised it in October 2008 in a parliamentary question which said that “the Commission has repeatedly stated that it would not allow validation to become a ‘bottleneck’ in the process of bringing new non-animal test methods into use, and yet it appears that this is exactly what has happened.”

NRLs could be seen as the Commission’s response to criticism: a step to diversify centres for validation. It was, however, an incomplete response as the first casualty of NRLs was ECVAM itself. ECVAM has a fragile legal existence: it is based on a communication from the European Commission. The Directive was an excellent opportunity to strengthen it, but the Commission failed to mention it in its draft. Moreover, the funding envisaged for NRLs in the Commission’s Impact Assessment of the Directive was extremely low: approximately €100,000 per Member State. How can such a tiny budget overcome ECVAM’s shortcomings when the latter, already very backlogged, receives about €2million per year? When the draft Directive went Lord Dowding Fund for Humane Research

Lord Dowding Fund for Humane Research

New Science

l

2010

© Tim Phillips / Animal Defenders International © Tim Phillips / Animal Defenders International

Council’s version, to be launched whenever the Commission wishes, instead of biannually.

These amendments combined may put the development of alternatives in Europe in a bureaucratic impasse and will allow some industries and universities to continue using animals where advanced methods are available, simply because there will be insufficient means to validate them.

This situation is due to the lack of vision from some key Member States, who are unwilling to fund the effort into alternatives, despite potentially huge benefits for science and research in the future.

The Directive is described in Recital 8 as “an important step towards achieving the final goal of full replacement of procedures on live animals”. However, if the situation is not improved in the last stage of the negotiations, these will remain empty words.

There is still room for negotiations and there will be opportunities to table amendments in the European Parliament. LDF will not fail to continue to lobby, for the sake of science and animals.

© Tim Phillips / Animal Defenders International

vivisection industry, to undermine the requirements to use alternatives.

Firstly, the very cornerstone of the duty to use available alternatives was damaged. A new Article 13 was added to interpret the principle of replacement as the duty to ensure “that a procedure is not carried out if another method or testing strategy for obtaining the result sought, not entailing the use of a live animal, is recognised by Community legislation.” Originally, the principle was to use any existing alternatives. This drastic narrowing of the scope of the duty to use alternatives is a huge step back, not only from the Commission’s draft, but from the old Directive 86/609/EEC; both provided a much broader obligation.

The consequence is that only the handful of methods validated by ECVAM concerning cosmetics and toxicity testing will be strictly compulsory.

ECVAM disappeared and was replaced by a ‘Community Reference Laboratory’, responsible for “coordinating the validation of alternatives” and “exchange information”. Although it can still validate methods, it lost its remit with regards to conducting and commissioning research into new alternatives, as well as implementing strategies to put the 3Rs into action.

The NRLs also disappeared, and were replaced by a general duty for Member States to contribute to the validation of alternatives and “assist the Commission in identifying and nominating suitable specialised and qualified laboratories to carry out those validation studies.” These therefore have a very similar profile to NRLs, but again with fewer commitments. For instance, NRLs were created to validate but also provide training and technical assistance: this crucial aspect has been removed following the Council’s amendments. The thematic reviews survived but are, in the

© Animal Defenders International

to the European Parliament for amendments, LDF successfully lobbied to improve the Commission’s text.

ECVAM was firmly anchored to the new Directive with a new Article 45a, aimed at coordinating NRLs, but also continuing to validate alternatives and research into their development etc. In that framework, NRLs would not be created at the expense of ECVAM but would work together.

An amendment was voted to strengthen the duty for Member States to contribute financially to alternatives. This mechanism was completed by two amendments creating regular thematic reviews. This key proposal from Animal Defenders International, on behalf of LDF, aims at keeping replacement of animal testing on the political agenda, instead of waiting another 25 years for new legislation.

A new Article 53a was created, which gives the Commission the duty to “conduct a thematic review of the use of animals in procedures every two years commencing two years after the entry into force of this Directive. The review shall examine the impact of developments in technological, scientific and animal welfare knowledge, and set targets for the implementation of validated replacement methods.” The concept of targets to replace animal testing was therefore introduced in legislation for the first time, which was a groundbreaking achievement. A very similar amendment to Article 8 on primates was passed to have reviews and targets specific to these species.

Unfortunately, this progress is now in jeopardy in the Council of Ministers, who can amend the Directive, following the vote in first reading in the European Parliament (which took place in May 2009). The Swedish Presidency came under strong pressure from key Member States, themselves lobbied by the pro-

© Animal Defenders International

© Tim Phillips / Animal Defenders International

EU DIRECTIVE ON ANIMAL EXPERIMENTS

EU DIRECTIVE ON ANIMAL EXPERIMENTS

Above: ADI’s Technical Briefing on the EU Severity Classification System.

© Louise Wallis / Animal Defenders International

1. http://ec.europa.eu/environment/chemicals/lab_animals/pdf/ staff_work_doc_sec1455.pdf 2. http://www.biosecurity.govt.nz/files/regs/animalwelfare/pubs/naeac/animal-use-statistics.pdf

14

New Science

l

2010

The proposed categories of severity are: mild, consequential, significant, severe (transient), severe and prolonged or non-transient (which will not be authorised). We believe that these are more reflective of the situation than mild, moderate and severe.

Classification is based on the duration and intensity of pain, suffering, distress and enduring harm. It also depends upon the frequency and number of interventions, the shortfall in the animal’s behavioural needs and whether or not anaesthesia and analgesia are used.

The severity categories represent the maximum impact / maximum severity that will be suffered by a single animal throughout the procedure. Repeated interventions or those in combination can have the effect of increasing the project’s severity. Actual suffering rather than intended suffering should be identified, reported and made public.

The components of suffering are the aspects of an animal’s life which must be satisfied in order to maximise its wellbeing; any diversion from the norm in these areas can affect the animal, its wellbeing and the data gathered from it.

The components of suffering are: ● Mental state and dignity – animals should not suffer fear, stress or undignified treatment. Mental suffering should be avoided ● Food and water – animals should be free from thirst and hunger with access to fresh water and a diet enabling the maintenance of full health ● Environmental challenges – the animal’s environment should not cause it discomfort ● Injury / Functional impairment / Disease – these should be kept to a minimum by ensuring rapid diagnosis and treatment. The degree of induced disease should be kept to a minimum.

All measures to minimise and ameliorate pain and suffering should be implemented ● Behaviour – the animal’s behaviour should reflect its natural behavioural repertoire as closely as possible.

Lord Dowding Fund for Humane Research

© Animal Defenders International

Right: These rats are suffering excruciating pain; their legs are swollen and inflamed with artificially-induced arthritis. It is vital that the European Severity Classification System introduces an upper limit on levels of pain which can be inflicted.

On the basis of the European Parliament’s amendments, the European Commission convened an expert working group to address this issue (ADI did not participate). The group met for two days in July 2009 and came to a consensus on the classification of various procedures and also a minimum level of suffering which needed to be categorised, but did not stipulate a maximum suffering level for animals. The principle of a maximum level of suffering is used in New Zealand. It states in the Animal Use Statistics that “it may be decided that the impact of a procedure on the animal is so great that it should not proceed, no matter what the potential benefit is.”2 Whilst we oppose all animal experimentation as unnecessary, unreliable and unscientific, until animal experiments are a thing of the past the reality of life in laboratories should be clear for all to see. A transparent and truly reflective system, which realistically portrays the suffering of laboratory animals, is one step towards ensuring this.

ADI, unconvinced of the robustness of the classification system which was formulated, wanted to provide policy makers and other interested parties with a comprehensive alternative which accurately reflects the situation for millions of laboratory animals. So we published the ‘EU Severity Classification System’, an outline of which is given here.

The ADI classification system has two elements: severity classes and components of suffering, which are derived from systems currently in use in New Zealand, Switzerland, Canada and the UK. The titles of the severity categories have been chosen to be reflective of the actual situation the animal is experiencing without being emotive.

It is vital that any system of categorisation is easily understood and provides an accurate portrayal of suffering, even to the general public.

© Terry Hill / Animal Defenders International

According to the latest EU statistics, over 12 million animals were used in experiments in one year1. It is vital, for the clarity of research and public accountability, for every EU citizen to be able to see, preferably at a glance, exactly what level of pain, suffering and stress these animals endure.

Disappointingly, the proposed revision of the EU Directive on animal experiments and the amendments by MEPs lack any solid, scientifically based categorisation of the pain and suffering that animals experience each year in the EU Member States.

© Adam Thomas / Animal Defenders International

ADI publishes EU Severity Classification System Briefing

PAIN RELIEF IN EXPERIMENTS

The paper highlighted worrying laxity in reporting procedures, which included: ● “dose was often given in mL/animal or mg/animal rather than mg/kg” ● “In both time periods, duration of systemic analgesia administration was not specified in 46% of papers that reported the use of a systemic analgesic” ● “When the administration of a systemic analgesic was specified, the frequency of systemic analgesic administration was not specified in the majority of cases (68% of papers in both time periods)” ● “There was a wide reported dose range of analgesics for many species” – the authors explain how, in rabbits, with one compound, the dose “varied by 30-fold”, which “suggests uncertainty about dose” ● Duration of analgesic administration did not always increase with the severity of the procedure. The authors concluded that “duration of analgesic administration may often be inappropriate” ● Eight papers stated analgesia was given “as necessary”, but none described how pain was assessed or the signs that were used as indicators of pain.

© Jan Creamer / Animal Defenders International

A paper in the journal Laboratory Animals recently cast a shadow over the use of analgesia in laboratory situations and its appropriate use in experimental animals.

The research assessed how frequently analgesia was used, which agents were used and determined if this use was related to the severity of the procedure. The papers included in the review covered thoracotomies, orthopaedic procedures, skin incisions, craniotomy, laparotomy and burn studies. In total, 149 papers were reviewed, 74 from 2000-2001 and 75 from 2005-2006. The articles came from 61 different journals.

© Jan Creamer / Animal Defenders International

Questions over pain relief in experiments

In the discussion, the authors state that “given the sensitivity surrounding the use of animals in experiments, particularly when they involve non-human primates and companion species such as dogs, it is surprising that some peer-reviewed papers still do not report the administration of any form of analgesic.”

The paper states how “overall, rabbits, pigs, sheep, dogs and non-human primates were more likely to receive analgesics following potentially painful experimental procedures than has been reported in laboratory rodents but analgesic administration to ‘large’ laboratory species is still not optimal.”

Coulter, C.A. et al (2009) “Reported analgesic administration to rabbits, pigs, sheep, dogs and non-human primates undergoing experimental surgical procedures”, Laboratory Animals, vol.43, pp.232-238

Lord Dowding Fund for Humane Research

New Science

l

2010

15

Our

research includes cancer

cancer

© Rob Hill / Animal Defenders International

computer assisted learning

hearing Alzheimer’s

pain

toxicity testing

healing The

vision

difference is ... ... we NEVER fund animal experiments

Investing in the future with Good Science, Saving Animals and a Clear Conscience

❑ Yes! I would like to support Lord Dowding Fund for Humane Research (LDF), ✓ and support medical and scientific research that helps people AND animals.

Please complete this form in BLOCK CAPITALS, using a ball point pen, and return by post to Lord Dowding Fund for Humane Research, Millbank Tower, Millbank, LONDON, SW1P 4QP, UK.

Please accept my donation of ■ £100 ■ £75 ■ £50 ■ £25 ■ £15 ■ £10 ■ £5 £ ........................................ other (minimum £4)

■

Please accept my Cheque/Postal Order, made payable to LDF, or please debit my: Card number

■■■■■■■■■■■■■■■■ Please send me leaflets to promote your work ■ 5

■■■■ ■■ ■■

■ Visa ■ Mastercard ■ Switch/Maestro, Issue Number Valid from Date

■ 10 ■ 25 ■ 50 ■ 100

■■ ■■

Expiry Date

................ (other).

Name: .................................................................................................................... Address: ......................................................................................................................... ......................................................................................................................................................................................................................................................................... ...................................................................................................................................................................................... Postcode: ................................................................. email address ..................................................................................................................................................................................................................................................

We are sometimes asked by similar organisations if they may write to our supporters. We would allow this only if the organisation is reputable. This allows us to raise funds for our work. If you DO NOT wish your name to be included, please tick here ■

Lord Dowding Fund for Humane Research www.ldf.org.uk UK: Millbank Tower, Millbank, LONDON, SW1P 4QP, UK. US: 953 Mission St., Suite 200, SAN FRANCISCO, CA 94103, USA. South America: Apartado Postal 359888 BOGOTÁ, Colombia. e-mail: info@ldf.org.uk

Tel: +44 (0)20 7630 3340 Tel: +1 415 543-2344