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FRAME News Fund for the Replacement of Animals in Medical Experiments

Editor: Anne Jeffery

No. 65 October 2010

CHALLENGING Y EA R A H E A D A great deal of research is currently being carried out by FRAME and we are already committed to a programme of significant new activities for 2010–11. Among the projects underway is work on safer, cheaper biological medicines. FRAME’s office-based scientific staff have already carried out a review of biopharmaceutical testing methods that do not require the use of animals. Their work revealed that the predictive value of animal studies and traditional in vitro screens is questionable, with few existing methods able to predict immunotoxicity in a way that is useful for estimating risk for entire patient populations. Further desk-based studies will be carried out to assess the usefulness of


human cell-based tests and in silico prediction models. A new project in association with Kirkstall Limited, a Sheffield-based developer of in vitro testing systems, aims to develop artificial human livers. The new model, based on human cell cultures, can be used to study diseases or investigate variations in drug responses between patients. Unlike other cell culture models the new liver will enable repeat-dose testing, so it more closely mimics a patient’s experience of drug treatment. Inside this edition you can find more details of our work to promote the Three Rs among both scientists and the general public. That includes: our response to the latest Home Office statistics; plans to extend our successful experimental design training schools; distribution of a new CD for schools and colleges; and current research at the FRAME Alternatives Laboratory.

FRAME Chairman honoured


Award for FRAME trustee


Tadpole tissue repair


Home Office latest statistics


Education CD


I’m A Scientist Get Me Out of Here 6 Training School


News from the FAL


Dorothy Hegarty Award winners


Launch of CAAT-EU


Helping FRAME



on In Vitro Toxicology/13th Annual Congress of EUSAT/16th Linz Congress on Alternatives to Animal Testing. Read more on page 2.

Two FRAME Trustees have won international awards in recognition of their contributions to the development and validation of alternatives to animal experiments. FRAME Chairman Professor Michael Balls has received the Doerenkamp-Zbinden Award 2010 from the Swiss-based Doerenkamp-Zbinden Foundation and former Director of the FRAME Alternatives Laboratory (FAL) Dr

Dr Clothier was co-founder of the FAL and led its research programme for eight years, until 2005. During that time the laboratory developed a number of replacement methods in the field of toxicology. He also led successful collaborations with a number of industrial partners as well as working closely with the European Centre for the Validation of Alternative Methods (ECVAM). Read more on page 3.


Richard Clothier will be presented with the Björn Ekwall Memorial Award 2010 at this year’s FRAME Annual Lecture. Professor Balls became a Trustee of FRAME (Fund for the Replacement of Animals in Medical Experiments) in 1979, and has been Chairman of the Trustees since 1981. He was presented with his award at Linz, Austria, during the 16th ESTIV International Congress


AWARD 2010

FRAME Chairman Professor Michael Balls has been presented with an international award by The Doerenkamp-Zbinden Foundation for Alternatives in Biomedicine. The award recognises his contributions to the promotion of alternatives to the use of animals in medical and scientific research. He was presented with the award during an international conference in Linz, Austria. The citation that accompanies the award says: “The Doerenkamp-Zbinden Foundation for Alternatives in Biomedicine awards the DoerenkampZbinden Award 2010 to Professor Michael Balls (Fund for the Replacement of Animals in Medical Experiments, UK) in acknowledgement of his outstanding work in the promotion of alternatives to animal testing in biomedical research and education.” The DZ Foundation is based in Zurich, Switzerland. Its origins lie in the award of an annual prize for distinguished services to animal protection in science, which is designed not only to recognise significant work in the field, but also to

raise awareness of the need to search for alternatives to animal-based research. It can be awarded to teams and institutions as well as individuals and the decision on who should receive it is taken by the Foundation Board and its Scientific Advisory Board, based on nominations from key figures in the fields of replacement and reduction in animal experiments. Professor Balls’s nomination marks a long and distinguished career promoting the Three Rs. He gained a post as Senior Lecturer in the Department of

Human Morphology at the University of Nottingham Medical School in 1975 and soon afterwards became involved with FRAME. By 1979 he became a Trustee of the charity and has been Chairman since 1981. Since then he has acted as an adviser to the British Government during the drafting and passage through Parliament of the Animals (Scientific Procedures) Act 1986, and, from 1987–1995, was a member of the Animal Procedures Committee, which advises the Home Secretary on all matters related to animal experimentation. He was the first Head of the European Centre for the Validation of Alternative Methods (ECVAM) but retired from there in 2002. Throughout his career he has been recognised with many awards including the Russell & Burch Award of The Humane Society of the United States, the Marchig Animal Welfare Award of the World Society for the Protection of Animals, the SmithKline Beecham Laboratory Animal Welfare Prize, and the Michael Kay Award from the RSPCA.


BIOETHICS CONFERENCE FRAME Science Officer Michelle Hudson attended the London & Brighton Translational Ethics Centre (LABTEC) Conference on Bioethics & Experimental Neuroscience, which showcased a new five-year research programme being undertaken by LABTEC and CBAS, that is funded by a Wellcome Trust Biomedical Ethics Strategic Award. The research programme focuses on key developments in: O human embryonic stem cell research and neuroscience as critical instances of the translational interface between research and treatment, O the interface between science and medicine, including the use of inter-species embryos O boundaries and overlaps between experimental research and treatment O non-human primate research for neurological treatments The central ethical question is the acceptability of changing what it means to be human, but others include impact on


human and animal rights; the interests of future generations; interpretation of safety and risk; the relationship between personal beliefs and professional demands; the place of values in patient choice; and shifts in moral landscapes as research progresses. Michelle is currently working on a PhD project investigating attitudes toward the use of non-human primates in biomedical science. She said: “The work had many parallels with the kind of research I am conducting. It was a great opportunity to network and make new contacts with multidisciplinary researchers I would not normally meet. It also has a great deal of relevance to FRAME both in the context of exploring animal ethics and the impacts of animal experimentation and with public engagement. I hope to build on the contacts I have made and use this to enrich my research.”



MEMORIAL AWARD In recognition of his outstanding contributions in the field of in vitro toxicology, and in particular with respect to the development, implementation and validation of alternative toxicity test methods, and for his substantial contribution to the FRAME Research Programme, Dr Richard Clothier is the recipient of the Björn Ekwall Memorial Award 2010.

The Björn Ekwall Memorial Foundation (BEMF) was founded in 2001 to honour the memory of Dr Björn Ekwall (1940-2000), a Swedish scientist in the field of cell toxicology and the founder of the Scandinavian Society for Cell Toxicology (SSCT). It recognises achievements of outstanding scientists who have developed or used nonanimal models in toxicological investigations. University of Nottingham in 1985, to Reader in Cellular Toxicology in 1993, and became Associate Professor in 2005. Richard has been a FRAME Trustee since 1983.

Richard was a co-founder (in 1983) of the FRAME Alternatives Laboratory (FAL) at the University of Nottingham Medical School, and was director of the FAL from 1997 to 2005. He was promoted to Senior Lecturer at the

He has played a substantial role in the field of in vitro toxicology, by contributing to the development and application of alternative assays for replacing animal toxicity testing, e.g. the Neutral Red Uptake (NRU) and Neutral Red Release (NRR) tests for basal cytotoxicity, the Fluorescein Leakage assay, the Alamar Blue (Resazurin) metabolism test for toxicity, and many others. As an expert in national and international trials, he has contributed significantly to


the further advancement of the acceptance of alternative assays for toxicity through his commitment to a number of studies, including a FRAMEled study in 1983–1986, the EC/HO and COLIPA international validation study on alternatives to the Draize eye irritation test, the EU/COLIPA international validation study on the in vitro 3T3 NRU test for phototoxicity, and the ECVAM /ICCVAM assessment of the NRU assay for measuring basal toxicity. For many years, Richard collaborated with Björn Ekwall on the Multicenter Evaluation of In Vitro Cytotoxicity (MEIC) programme, aimed at the evaluation of the ability of in vitro basal cytotoxicity assays to predict human acute systemic toxicity. He is also one of the scientific leaders of the integrated EU-sponsored ACuteTox project, and is responsible for the generation of the basal cytotoxicity component of the in vitro database, contributing to the in vitro–in vivo evaluation of the data. Richard will formally receive the award and give the Björn Ekwall Memorial Lecture before this year’s FRAME Annual Lecture.

FRAME Office Research Among the subjects currently under study by FRAME scientific staff is the search for alternatives to adult animals for in vivo experiments. The Three Rs (Replacement, Reduction & Refinement) have been widely adopted but there is still a call for in vivo experiments. When such research is necessary, embryos and larvae of certain species can replace adult animals. Large numbers of individuals can be kept in simpler housing, making experiments cheaper and more manageable.

Xenopus — One suitable species is the African Clawed Frog (Xenopus). Xenopus is a useful model to study organ development in a vertebrate species. By manipulating gene expression is it possible to learn the role that specific genes play in organ development. These experiments can identify abnormalities that may lead to disease, such as some human heart problems that are thought to have their origin in embryonic developmental defects. This organism may be similarly valuable in toxicology, for the assessment of hazardous wastes or human toxicants. Larval frogs have the ability to regenerate their tails but, as larvae develop, they lose this capacity. Larvae thus provide a unique opportunity to investigate mammalian tissue repair. Frog eggs have provided an essential system in which to study signal transduction pathways (how cells convey external and internal signals) and understand basic cell biology.


FRAME Annual Lecture The Bill Annett Lecture This year’s FRAME Annual Lecture will be given by Dr Kelly BéruBé, Director of the Lung and Particle Research Group at Cardiff University. Her lecture is entitled "Alternative Experimental Models for Lung Research: between a rat and a hard place". The lecture will be held at the Kennel Club in London during November. Dr Clothier's title for the Björn Ekwall Memorial Lecture is “Experience in the Development and Evaluation of In Vitro Alternative Assays”.



medicine and dentistry and protection of man, animals and environment. This could be a reflection of the current economic climate and a shift by large pharmaceutical companies to reformulating existing drugs rather than researching and developing new ones. It is to be hoped that figures do not start to rise again as the economy improves.

For the first time in almost ten years the number of experimental procedures performed on animals decreased. Whilst 2008 saw the largest number of experimental procedures performed on animals since 1987 (Figure 1), the latest Home Office figures show that procedures started in 2009 declined by 1% to just over 3.6 million, with the actual number of animals decreasing similarly to 3.4 million. This reduction is very welcome and if the numbers of procedures for breeding purposes are removed there is in fact an 8% decrease in experimental procedures. However, this is a drop in the ocean when the accelerating increase in experimentation over the past decade is taken into account. The main reason the numbers appear to have fallen is because of a reduction in research into human

by Michelle Hudson

Following recent trends the majority of procedures were associated with breeding animals, followed by fundamental biological research and then procedures related to human medicine or dentistry (Figure 2).

Figure 1: Number of experimental procedures performed and animals used since 1987 in Great Britain 4.0

No. of Procedures 2009

Animal Type

Mouse 2,628,556 Rat 333,865 Other Rodent 25,748 Rabbit 16,562 Cat 275 Dog 5,923 Ferret 890 Other Carnivores 995 Pig 3,757 Sheep 38,003 Cattle 4,538 Other Ungulate 8,973 Primate 4,263 Other Mammal 1,315 Bird 126,781 Reptile/Amphibian 21,175 Fish 398,101 Total 3,619,540

Change from 2008 9% –6% –25% –3% –24% –3% –21% –21% –45% 6% 89% –10% –7% 34% 3% –35% –34% –1%

Mice (73%), fish (11%), rats (9%) and birds (3%) continue to be involved in the largest number of procedures. The use of mice, sheep, cattle, other mammals, and birds all increased while all other species decreased (Table 1). It is particularly gratifying to see a fall in the use of primates. FRAME is deeply concerned about the continued use of monkeys in UK research because they are highly intelligent and social animals that are likely to suffer more than others in laboratory situations. They can anticipate pain and it is possible that they can comprehend what is being done to them.


3.5 3.0 millions of procedures

Table 1: Percentage change in experimental procedures using animals in Great Britain

2.5 2.0 1.5 1.0 0.5 0

1987 1989 1991 1993 1995 1997 1999 2001 2003 2005 2007 2009 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 year

= procedures; = animals Number of animals used in procedures was not reported in the annual statistics until 1990. The number of animals used is less than the number of procedures as some animals may undergo more than one procedure e.g. having a compound applied and then having a blood sample taken.

Radio Ga Ga ‘People couldn’t give a flying fig about animal experiments done for medical purposes’ according to TalkSport radio presenter Duncan Barkes. However, on his Friday night discussion programme FRAME Scientific Officer Michelle Hudson, pointed out that people do indeed care, in particular, about the contentious issue of using primates in laboratories and that if everyone was given a choice of whether to have non-animal tested drugs, as is now the case with cosmetics, then interest would be even greater. She also provided an insight into what the current statistics mean in terms of the shift to genetically altered animals. Michelle took the opportunity to emphasise that the Three Rs must continue to be promoted and that more attention needs to be paid to ensuring scientists design and analyse their experiments correctly, as this is the most effective short term way of reducing the number of animals in laboratories now.


GENETICALLY ALTERED ANIMALS Table 2: Procedures involving genetically altered animals by species in Great Britain, 2009

The 2009 figures show that, for the first time, the number of genetically modified animals and animals with harmful genetic defects (collectively called genetically altered) exceeded ‘normal’ animals. However, of these, 67% were conducted for breeding purposes, including generating founder animals and maintaining breeding colonies. The mouse most frequently undergoes genetic alteration but other species are also used (Table 2). After breeding procedures the majority of genetically altered mice are used in fundamental research. While the total number of procedures involving fish decreased, the percentage of the animals that were genetically altered has risen from 31% in 2008 to 33% last year, so perhaps continuing the trend for increasing

Species of animal

No. of procedures involving genetically altered animals

Mouse Rat Pig Sheep Domestic Fowl Amphibian Fish

1,737,123 20,697 178 43 490 7,309

66% 6% 5% <1% <1% 35%



alteration of this species for a variety of experimental purposes. The unabated rise in experiments involving genetically altered animals is of particular concern because these animals have unique welfare requirements and the methods used to

HOME OFFICE CONSULTATION ON THE PUBLICATION OF THE ANIMAL STATISTICS The Home Office has also published the results of a consultation exercise about the way the figures are published and what they contain. FRAME was one of the organisations consulted during the process.

Percentage of all procedures involving that species

recommendations will be taken on board and look forward to further improvements in the future.

produce them are currently inefficient, leading to a great deal of animal wastage. Whilst the main reason for creating these models is the hope that they will better mimic the human conditions they are altered to suffer from, they often turn out to be less useful than anticipated. FRAME would like to see further information in the animal statistics on the types of procedures used in the course of breeding programmes to evaluate how the persistent rise can be halted using Three Rs principles. It is also important that a thorough critical assessment of the usefulness of the currently available strains is done before this large scale production continues unchecked.

Figure 2: Primary purpose of experimental procedures on animals in Great Britain, 2009 0.1% education

FRAME is pleased to see that this yearâ&#x20AC;&#x2122;s figures have been published in more than one format, making them easier to interpret and compare. Data table design and format has also been improved so that the figures are clearer and easier to read. We are pleased that the Home Office has taken these recommendations into account. The changes have increased clarity and transparency and are a significant step in engaging more closely with interested parties. We hope that more of the





2% other 3%

protection of man, animals or the environment

18% 0.1% training

32% 1%

= fundamental biological research; = human medicine or dentistry.

= veterinary medicine;


direct diagnosis

= breeding;



Schools throughout the UK have received a new CD ROM that includes electronic versions of the Focus on FRAME series of educational leaflets.

aimed at sixth-formers it is also accessible at GCSE level and has been used successfully in bioethics sessions by first-year undergraduates.

The leaflets provide an introduction to the topic of animal experiments, what kinds of tests are used and why they are carried out. They also look at what valid alternative procedures are available to scientists that do not involve the use of animals, or reduce the number used, and minimise distress where there is no alternative.

Professor Michael Reiss of the Institute of Education at the University of London said: “These are an excellent set of materials from FRAME to help students address important questions. I wish I had had them when I was teaching.”

The CD provides ideal source materials for science lessons, projects and discussions. Although the material is

Senior Lecturer at the University of Derby Dr Heidi Sowter said: “Animal experimentation is an area subject to misunderstanding, and often students are confused as to the difference

between procedures used in medical and cosmetic research. This CD cuts through to the heart of the matter, and explains UK legislation, types of experiments and risk in an interesting and clear way.” The CD will be useful in science lessons but could also encourage discussion in other subjects such as English, Religious Education and Citizenship. It has been produced and distributed by FRAME with financial assistance from The Marjorie Coote Animal Charity Trust.



TEST METHODS FRAME Scientific Officer Michelle Hudson has been taking part in an online contest designed to help schoolchildren learn about what life is really like for working scientists in the UK. The scheme, called I’m a Scientist Get Me Out of Here, ran for two weeks, during which time Michelle answered written questions and took part in on-line chat sessions with students aged 14–19. There were no limits on topics and students could ask anything they wanted, science-based or not. In the second week there were daily votes to evict the least popular scientist until only one remained. Michelle survived until round three, defeating opponents who were working on cell-based and computer-modelled drug development. At the end of the experience she said: “I have really enjoyed taking part in the chats, although the questions could be very challenging. It has helped me to understand much more about communicating with young people about science. “I realised as the days went on that my answers were getting shorter and clearer because the students were quick to tell me if they could not understand my first reply.” The winning scientist was awarded £500 to fund a sciencebased project of their choice


The recent EU approval of the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) regulation is just one result of the increased need to assess the effects of a huge number of chemicals. A team led by Emilio Benfenati of the Istituto di Ricerche Farmacologiche ‘Mario Negri’ in Milan, Italy, has compared in vivo, in vitro and computer-based (in silico) methods to estimate their relative strengths, weaknesses, opportunities and threats (SWOT analysis). In a paper published in FRAME's scientific journal ATLA they conclude: “The current scheme for evaluation of chemicals needs to be reshaped, in the face of the much larger numbers of chemicals which need to be examined and the availability of a diversified set of tools.” They identified several serious obstacles to the integration of different methods such as in vivo, in vitro and in silico. Among them was a lack of common language between the various specialities. Others were related to techniques and the overall framework of risk assessment. The paper calls for a number of considerations to be made in validating test methods, including verifying that language used is clearly defined, ensuring any uncertainties in various methods are known and understood, using measurable parameters, and avoiding subjective evaluation. Full details can be found in: Emilio Benfenati, Giuseppina Gini, Sebastian Hoffmann and Robert Luttik. Comparing In Vivo, In Vitro and In Silico Methods and Integrated Strategies for Chemical Assessment: Problems and Prospects. ATLA 38, 153–166, 2010



STATISTICS TRAINING SCHOOL FRAME has staged another highly successful training course in Experimental Design and Statistical Analysis of Biomedical Experiments. This year’s course was held at the University of Nottingham and more than 30 people attended. Numbers were slightly down because the Icelandic volcano interrupted travel, so some overseas delegates were unable to reach the UK. The courses were first organised by the FRAME Reduction Steering Committee (FRSC) three years ago and each one has been welcomed by the delegates who attended. The University of Manchester has incorporated a version of the course into its postgraduate syllabus since hosting one on behalf of FRAME. Feedback from delegates on the latest course was overwhelmingly positive. They were asked to complete a questionnaire at the end of the three day event and their comments showed a high level of satisfaction with both the conduct and the results of the course. Most said the aims and objectives of the course were clear and that it met with their expectations. They welcomed the opportunity to work on practical examples on computer and to compare experiences with others in similar fields. They felt they had learned new knowledge and practices that would help them in their work. The majority would recommend the course to colleagues. Course organiser, FRAME Scientific

Feedback from Delegates The computer session was by far the most useful and interesting, it puts the rest of the course into a practical context. Group exercises are enjoyable and were written well.

Officer Michelle Hudson, said: “It was very pleasing to find that several of the delegates would be interested in taking a second course that would help them pass on their new skills to others. We hope that these courses are a foundation for spreading Reduction skills among researchers across Europe.” FRSC is now planning further stage-one courses in other areas of the UK and hopes to extend into Europe next year. The committee is also looking into ways to set up a second-stage course to train past delegates to run courses at their own universities and laboratories.

Good planning and design can have a major impact on reducing animal usage in research programmes and on the welfare of the animals used. Experimental design text books usually offer no guidance on how to design individual experiments to reduce the number of animals needed or to minimise the severity of the procedures involved.


Lectures in progress

Networking over dinner

Group exercises were good for interacting with other participants on the course and were useful to apply lectures to examples. Very good and helpful course. The course was useful for initiating collaborations with colleagues from different areas. It was interesting to spend time with people from different areas of scientific research. Great help to improve my teaching to research students and my own research. Excellent course, I learned a lot. This course has reinforced my existing methods, whilst reminding me of many I had forgotten. The content of the course was extremely relevant and useful to me and I will definitely recommend the course to others. I found the course helpful and it made me think about lots of different factors.


THE FRAME ALTERNATIVES LABORATORY (FAL) PRIMARY CELLS AND IMMORTAL CELL LINES By Rita Seabra Primary cells are cultured directly from the living organism, from tissue biopsies or blood samples. A number of advantages can be gained by using them rather than cell lines, for research and testing. Primary cells have not been subjected to genetic manipulation (for example to immortalise them) and are not generally subjected to repeated growth cycles or freeze-thawing, so they are more likely to retain key functions of cells within the living organ or tissue. The biggest problem facing researchers who wish to use human liver cells is acquiring hepatocytes that retain key metabolic functions. The quality of the liver tissues from which these cells are isolated is crucial and requires that researchers are on call to prepare cells from freshly biopsied tissues. A second problem is keeping the cells in suitable conditions during growth and transport. In fact, hepatoctyes can only go through a set number of replications. Once they reach this point, morphological and biochemical changes take place and the cells no longer display functions as in life. This means that the researcher has a very small window in which to conduct studies

In vitro (literally “in glass”) experiments often include animal or human cells. These can be either primary cells or cell lines. The FAL is working to lower the barriers hindering the use of human cells, which are more suitable than animal cells when investigating human diseases. This is not only of interest to scientists carrying out basic research but also for companies carrying out toxicity testing. By increasing the longevity of primary liver cells and creating relevant human immortal cell lines, the FAL is contributing to replacing and reducing the number of animals used for research and drug development. on these cells, and is working against the clock to distribute cells to other laboratories. Given these concerns, the FAL is collaborating with a Welsh company (Abcellute) to optimise a method for preserving primary liver cells. So far, research at the FAL has demonstrated that it is possible to coldstore hepatocytes for up to a week with minimal loss of viability and cellular function. In addition, specialised media and protocols to enable transport of hepatocytes have been developed. Unfortunately, primary cells are not always an option. Many in vitro experiments use immortal cell lines because these cells are able to undergo unlimited divisions without losing their characteristics. In order to achieve immortalisation, cells are infected with viral genes or part of a telomerase enzyme (hTERT) that maintains telomere length. The FAL has exploited both approaches to derive cell lines from human primary cell lineages


that include hepatocytes, skeletal muscle myoblasts, and aortic endothelial cells. The resultant cell lines are characterised by using omics approaches and functional assays over many growth cycles to confirm functional and phenotypic stability.

Impact of the FAL The FAL was recently featured in the University of Nottingham students’ magazine Impact. The article, called “Lab Rats–the Nottingham Connection”, gave a very positive impression of the work carried out at the FAL. The journalist visited the laboratory and spoke to director Dr Andrew Bennett. The article explained FRAME’s position and outlined the Three Rs, as well as discussing work carried out at the FAL. It described the FAL as “an absolutely essential facility”.


2009 DOROTHY HEGARTY AWARD PRIZE GOES TO ANGLO–GERMAN TEAM The award is named after Mrs Dorothy Hegarty who founded FRAME in 1969. She began fundraising after learning about replacement from biologist Dr Charles Foister. The south London housewife was convinced that the only way to reduce the number of animals used in experiments, and ultimately replace them, was to make a determined effort to find alternative methods. In its earliest days FRAME was run from a spare room in Mrs Hegarty’s home.

British team l-r: Emma Bishop, Marianna Gaça, Fiona Cunningham, Oluwatobiloba Oke, Natalia Cockcroft and Ian Fearon

German team member Roman Zantl.

The Dorothy Hegarty Award is presented annually by FRAME to the authors of a paper published during the previous year in Alternatives to Laboratory Animals (ATLA), FRAME’s scientific journal. It is presented to the paper that is judged by members of the Editorial Board to be likely to make the most significant contribution to the Three Rs, reduction, refinement and replacement, in animal experimentation.

a large variety reach the shortlist each year. The 2009 Award goes to a joint UK/German team from British American Tobacco and ibidi GmbH. The abstract for the paper can be found below.

“With this information, next year we will begin an internal validation in which we will examine the reproducibility of data obtained using the assay.

Lead author Natalia Cockcroft said: “Since we published the development of the perfusion system in ATLA, we have been further refining the monocyte adhesion assay in particular, and testing the responsiveness of this assay to both particulate and vapour phase cigarette smoke extracts.

“Following on from this we will potentially use the assay as part of our process for testing reduced toxicant prototypes and we are very excited about integrating such a physiologically-relevant system into our product assessment framework.”

Each member of the ATLA Editorial Board can nominate up to five papers so

ABSTRACT: An In Vitro Perfusion System to Examine the Responses of Endothelial Cells to Simulated Flow and Inflammatory Stimulation Natalia Y. Cockcroft, Oluwatobiloba Oke, Fiona Cunningham, Emma Bishop, Ian M. Fearon, Roman Zantl and Marianna D. Gaça British American Tobacco, Southampton, UK; ibidi GmbH, Martinsried, Germany Atherosclerosis is a disease process which develops at the arterial branches and curvatures of medium to large arteries. Local haemodynamic flow patterns in these vessels play an essential role in the formation of atherosclerotic lesions. To simulate pro-atherogenic blood flow patterns, we have developed a perfusion system with the ability to simulate in vivo patterns of blood flow in vitro. In this system, human umbilical vein endothelial cells were seeded in y-shaped microslides, in which they were exposed


to a variety of flow patterns. Besides being able to reproduce the disturbed flow involved in the development of proatherogenic events within the arterial wall, the system also permitted the assessment of the pre-conditioning/priming effect of oscillatory flow on endothelial cells. The system was further capable of integrating multi-endpoint assays relevant to cardiovascular disease. We show that oscillatory flow primed endothelial cells, making them more sensitive to subsequent treatments. The treatment of oscillatory flow-primed cells with TNFα

resulted in the detection of enhanced levels of pro-inflammatory and chemoattractant factors such as IL-8 and MCP-1. These measurements were facilitated by the small volumes of medium circulating within the perfusion system. Oscillatory flow also altered the characteristics of monocyte adhesion to the endothelial layer. In summary, this system allows the monitoring of multiple endpoints and biomarkers, and provides an alternative to the use of in vivo and ex vivo models of cardiovascular disease.



experience are inevitably involved in what is under consideration. The solution is fairness, honesty and transparency, together with the need to explain publicly all major decisions.


Animal Welfare versus Animal Experimentation

Conflicts result from the actual or perceived opposition of values, needs, interests, actions and/or directions. Those of us who seek a revolution in tests and testing need to recognise the many real or potential conflicts which face us, then determine whether they are irreconcilable, or whether they can be exploited to the benefit of all that seems to be in conflict. I think that we have to try to avoid the absoluteness of the love/hate, totally for/totally against ways of thinking, whilst being wary of compromise as an easy way out. One of our biggest and most common problems is conflicts of interest. It is unavoidable, because those with the necessary specific

As CAAT Comes to Europe, Let’s:

is irreconcilable as far as laboratory animals are concerned. Applying the Three Rs and conducting rigorous benefit–suffering analyses is the best we can do.

Hazard versus Risk Risk = Hazard x Exposure There are attempts to make decisions about risk on the basis of knowing only something about hazard. Hazard is inherent in what is under consideration. Risk can be managed by avoiding, limiting or controlling exposure to the hazard concerned.

Risk versus Safety Safety is the state of being protected from something that is undesirable, of being free from risk. It is not possible to test whether or not something is “safe” in a general sense — tests can only tell us about conditions under which it is “unsafe”. The misuse of “safety”, and the false promise of providing it, are matters of disgrace. One great conflict remains:

O Make ourselves fully aware of the conflicts

Humans and Animals are Different

O Avoid their pitfalls O Benefit from their challenges O Compete in putting forward new ideas O Collaborate in putting the best of them into practice O Join together to lead the much-needed revolution O Improve the scientific basis of hazard prediction and risk assessment by developing, validating and employing advanced methods and replacing the use of flawed animal tests


The problem of species differences is insuperable. Despite many attempts at rationalisation, we rarely, if ever, know, with sufficient certainty, whether animal data are

O the degree of animal suffering (lowest) O the quality of the research (highest) O the potential medical benefit (highest) That which doesn’t satisfy these criteria should not be permitted. Replacement is the only truly satisfactory way forward.



Low Low Low

Certainty of medical benefits

su Ani ffe m rin al g

I welcome the formation of CAAT-EU. There is much to be done, and the key to success will be the dedicated commitment and positive contributions of those with the necessary expertise and experience.

Bateson’s cube evaluates proposed research through three criteria:

Quality of research

FRAME chairman Prof Michael Balls spoke at the official opening of the new European Center for Alternatives to Animal Testing (CAAT-EU) at the University of Konstanz. Here are extracts from his speech entitled “Alternative Methods: Servants of Two Masters”, which focused on some of the conflicts involved in the search for alternatives to animal experiments. The full text can be found on the FRAME website ATLA pages under Free Access Editorials in ATLA 38.3.


sufficiently relevant and reliable for use in human hazard prediction and risk assessment. This, together with ethical concerns for human welfare and animal welfare, is the principal force which drives the immediate and pressing need for a revolution in toxicology and toxicity testing, based on mechanistically relevant and human oriented non-animal procedures.

Competition versus Collaboration Competition can stimulate originality, but it can also be destructive and be used to serve vested interests. Collaboration can be mutually beneficial, and lead to added value, but it can also lead to centralisation, narrow thinking and the concentration of power. It is essential to find a balance between the two, so that their combined advantages can be exploited and their disadvantages can be avoided.


FRAME REDUCTION STEERING COMMITTEE UPDATE To help scientists to reduce the number of animals they use in their research, the FRAME Reduction Steering Committee has developed a Strategic Planning flowchart for planning a programme of experiments. It outlines steps to follow during research involving animals, taking account of ethical considerations. This double sided poster has been designed to be displayed in laboratories and staff meeting areas. The FRAME Reduction Committee was established in 1998 and has now developed into the FRAME Reduction Steering Committee overseeing a number of working parties. Members of the committee are representatives from industry, the Home Office, and academia, with expertise in statistics, experimental design, animal welfare and alternatives research. The Committee’s main aim is: ‘To reduce the number of animals used in research, education and testing without compromising the quality of research or hindering scientific progress’


FRAME relies heavily on its corporate supporters. Without regular donations from companies in various industries it would be very difficult to maintain The FRAME Research Programme and promotional work. FRAME receives no financial support from local or central government so regular gifts from commercial supporters are vital. Annual support from companies is particularly important because FRAME needs a reliable income in order to


The flow chart has been distributed widely via the journals ATLA and Laboratory Animals as well as at various scientific meetings and the FRAME Training School (see page 2). Information on both the Training School and the Strategic Planning flowchart were presented in a poster to delegates at the LASA Winter Meeting last year. The committee now plans to translate the flowchart into several other languages to further increase access to this very useful resource. Feedback received so far has been extremely positive, with scientists saying the chart is extremely helpful and provides a useful training aid. Committee members have written two scientific articles which expand on how to use the poster effectively and why it is important to consider every step in the experimental process. These were published in ATLA and ALTEX. The flowchart is available to download from the FRAME website (http://www. For hard copies please contact Michelle Hudson at

plan and move forward with its high quality research. Corporate sponsorship can also help with events like last year's series of scientific conferences or with educational projects such as the Focus on FRAME leaflets designed for undergraduate science students.

The FRAME rabbit roundel logo may be used on products and marketing materials by companies who can prove that they have considered or implemented ethical standards of animal welfare, and who actively support FRAME.

Companies can become FRAME associates for as little as £500, but those who give more can benefit from a huge range of services that we offer in return. That can include listing on the FRAME website and in the annual report, personal invitations to our events, access to FRAME’s scientific expertise or use of the FRAME logo on products.

Use of the logo does not imply that products have not been tested on animals – only that the company supports FRAME and has acceptable policies. Not all companies who support FRAME have chosen to use the logo but it has been shown to raise the profile of those who do.

If your company would like to support FRAME, email for more details


O There is no worldwide agreement that protects the welfare of animals either in the wild or in laboratories.

HOW CAN YOU HELP FRAME? FRAME relies entirely on grants and donations to carry out its vital work promoting the development of new and valid methods that will replace the need for laboratory animals in medical and scientific research, education, and testing.

O UK law says that animal-free research and testing methods should be used if they are available, but there is no specific legal requirement to look for alternatives. O FRAME is helping to find those alternatives. Without the backing of commercial sponsors and individual supporters the work would be impossible.

It receives no financial support from local or central government so any gifts from supporters, either individuals or companies, are always gladly received. As an independent charity, FRAME welcomes any donation, however small. You can send donations directly to our office (address on the left) as a cheque made out to FRAME. Log on to and click on the button marked “Donate Now”. (Please give reference FRAME News) Getting married soon? Did you know that couples who ask for charity donations instead of wedding gifts raise an average of £918?

Hold an event

O FRAME’s ultimate aim is the elimination of the need to use laboratory animals in any kind of medical or scientific procedure.

Why not hold an event locally to raise money for FRAME? Email with the details and we can supply you with leaflets and other items to give away to your guests. If you are thinking of holding an event for FRAME why not use our JustGiving site to boost the funds even further. It’s easy to register on our site ( then tell all your sponsors to make their donations through it. The site handles all the paperwork and will even collect Gift Aid from taxpayers. That can make your donation worth up to 28 per cent more to FRAME.

Published by: FRAME Russell and Burch House, 96 – 98 North Sherwood Street, Nottingham NG1 4EE Phone: 0115 958 4740 E-mail: Registered Charity No. 259464

Email to be placed on our electronic mailing list. This saves on print and postage costs and helps us to direct more of our funds to the vital research work being undertaken. It can cost a lot of money to have a scrap car taken away and you might not recoup the cost in value. A new salvage service called Giveacar can collect your vehicle, free of charge, and then either re-sell it or recycle it on FRAME's behalf. Log on today and help us AND the environment.

Corporate sponsorship Ask your boss if your company can become a corporate sponsor of FRAME. There are many advantages. Email for details of the scheme.

FRAME News October 2010  

The latest news from FRAME - the Fund for the Replacement of Animals in Medical Experiments

FRAME News October 2010  

The latest news from FRAME - the Fund for the Replacement of Animals in Medical Experiments