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SID 5 Research Project Final Report

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NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects.

A SID 5A form must be completed where a project is paid on a monthly basis or against quarterly invoices. No SID 5A is required where payments are made at milestone points. When a SID 5A is required, no SID 5 form will be accepted without the accompanying SID 5A.

This form is in Word format and the boxes may be expanded or reduced, as appropriate.

ACCESS TO INFORMATIONThe information collected on this form will be stored electronically and may be sent to any part of Defra, or to individual researchers or organisations outside Defra for the purposes of reviewing the project. Defra may also disclose the information to any outside organisation acting as an agent authorised by Defra to process final research reports on its behalf. Defra intends to publish this form on its website, unless there are strong reasons not to, which fully comply with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.Defra may be required to release information, including personal data and commercial information, on request under the Environmental Information Regulations or the Freedom of Information Act 2000. However, Defra will not permit any unwarranted breach of confidentiality or act in contravention of its obligations under the Data Protection Act 1998. Defra or its appointed agents may use the name, address or other details on your form to contact you in connection with occasional customer research aimed at improving the processes through which Defra works with its contractors.

Project identification

1. Defra Project code VM0132

2. Project title

REVIEW OF THE DEVELOPMENT OF ALTERNATIVES AND REFINEMENTS TO USING ANIMALS IN THE TESTING OF VETERINARY PRODUCTS (I.E. IMMUNOLOGICAL AND PHARMACOLOGICAL

3. Contractororganisation(s)

Central Science LaboratorySand HuttonYorkYO41 1LZ                    

54. Total Defra project costs £ 30845

5. Project: start date................ 01/04/2006

end date................. 30 September 2007

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6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...................................................................................YES NO (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They

should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

(b) If you have answered NO, please explain why the Final report should not be released into public domain

Executive Summary7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the

intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.

Under EU Directive 2001/82/EC, it is necessary that an applicant for a Marketing Authorisation assesses the quality, safety (to target animals, humans and the environment) and efficacy of a veterinary medicinal product. In order to assess safety and efficacy and to set maximum residue limits, tests will typically be required on animal species. It is essential for ethical, scientific and legal reasons that welfare of animals used in drug testing is optimised. Hence the main aim of this study was to determine methods that would increase the welfare of animals in such studies and ensure that this information was readily accessible to relevant parties. The study therfroe, had three main objectives:

1. Complete a detailed survey encompassing both completed and ongoing research into the 3Rs (i.e Replacement, Reduction and Refinement) relevant to all veterinary medicine testing.

2. Identify and rank in order of priority areas for further consideration and/or research into the 3Rs with particular reference to veterinary medicine testing: Priorities were considered in the light of a) impact on animal testing, b) impact on the cost of testing, and c) scientific and technical feasibility.

3. To create a specific database for the implementation of the 3Rs to veterinary medicine Replacement : a wide range of methods such as in vitro methods based on cultured cells, tissues or sub-cellular fractions; the use of invertebrates, plants or micro-organisms; read-across approaches; and computer modelling (eg QSARs).

Reduction : relies heavily on the use of good experimental designs, particularly designs that avoid the unnecessary replication of control groups. For example the use of a "limit" test, where up to 10 animals are used to determine the minimum and maximum toxicity limits to establish the number of doses to be tested, has been shown to reduce the number of toxicity tests; such as a typical LD50 test that requires up to 200 animals.. A very basic method of reduction is simply the exchange of information between different laboratories to eliminate the need for undue re-testing of a drug. This is particularly apposite for veterinary medicines where around a third of medicines

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on the market have previously been sanctioned for human use. New monitoring techniques may improve precision such that fewer animals are required; for example, the use of MRI, MRS, and CAT scans can enhance detection of inflammation, tumours, and the deterioration of organs. The use of tailored testing that is driven by an exposure assessment of a substance may also provide a valuable approach to reducing the amount of animal testing.

Refinement : aim to minimise the suffering of experimental animals: for example, the provision of suitable analgesics and anaesthetic regimes, the administration of sedatives, and the setting of humane endpoints that allow the animal to be humanely killed before the onset of severe suffering without compromising the purpose of the experiment. In addition, there is the use of non-invasive techniques in place of more traditional measures (e.g. assessing hormone/chemical levels in urine or faeces as opposed to blood sampling).

The database has been designed to operate in a similar manner to an internet search engine. Instead of typing in search terms the user selects categories in which they are interested under the headings of drug (e.g. antihelmintics), test (e.g. reproduction testing), taxon (e.g. caprine) and objective (i.e. type of replacement, reduction, refinement). The search can be made as general or specific as desired and more than one category can be selected under each heading.

The development of this database will allow veterinary medicine testing to employ searches in a more focused manner for specific alternative methods.

Project Report to Defra8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with

details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the scientific objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer).

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Date : 30/09/07

REVIEW OF THE DEVELOPMENT OF ALTERNATIVES AND REFINEMENTS TO USING ANIMALS IN THE TESTING OF VETERINARY PRODUCTS (I.E.

IMMUNOLOGICAL AND PHARMACOLOGICAL)

Ref No:VM0132

VMD Project Manager: Andrea Patterson

CSL Project Manager: Julie Lane

The aim of this study was to identify the areas of 3Rs research that are the most applicable and beneficial to the testing of veterinary products and to develop a 3Rs database specific to veterinary medicine testing.

Milestone 1: Establish current testing requirements for different classes of veterinary medicines

The current testing requirements for different classes of veterinary medicines were established from VICH guidelines and the testing requirements that are likely to have both an impact on animal welfare and potential opportunities for the implementation of the 3Rs have been identified (Appendix 1).

Milestone 2 Assess the impact of test procedures on animal welfare.

The numbers of animals used scientific procedures in the UK in 2006 was just under 3 million. Approximately 5% of these animals were used for applied veterinary studies (i.e 136,068). There is a downward trend on the use of animals with an 11% decrease in animals used in veterinary medicine compared to 2005.

Species usedThe majority of studies were carried out on birds (mainly poultry), fish and rodents (see below). Fish was the only species used in which there was an increase in numbers compared to the previous year. The use of fish as experimental animals is increasing worldwide. This increase is partly due to the use of fish as a replacement for mammals in biomedical, pharmacological and genetic research. Very few published studies concern fish welfare and the implementation of the 3Rs in this area seems very limited. Part of the reason for this seem to be due to a lack of public concern over the fate of these animals and the perception that they may not experience similar pain and suffering to

mammals and birds (although they do have the capacity to perceive noxious adverse states usually associated with pain in mammals)

Type of testToxicological tests are the most prominent both from the number of animals used and the potential severity. Single dose acute toxicity studies accounted for a large proportion of the animals used in toxicological studies in 2006. These studies are of concern as they are the only type where lethality is a defined endpoint. Safety testing also made up a very large proportion of the numbers used in toxicology testing. The problems of high injection volumes used in batch testing and the number of animals required for repeat testing are one of the main problems associated with this type of testing.

The species (fish) and test (safety toxicity testing) identified as high priorities were covered in the main topics of the workshop (please milestone 6)

Milestone 3 Completion of review of completed and ongoing research into relevant 3Rs and potential impact on vet medicine testing.

A review of replacement, reduction and refinement techniques applicable to these guidelines has been established and these have been ranked in order of priority and applicability. From these a range of search terms were generated and used to search 5 separate databases (Appendix 2)This searching was carried out using hosts such as Dialog-Datastar and STN, and commercial databases covering veterinary and clinical medicine, chemistry, the environment, and toxicology, agriculture, and the life sciences. As well as commercial hosts publications, extensive use was made of Web-based sources, including the Web of Science, the GrayLIT Network, the NTIS Electronic Catalog and the NASA Technical Reports Server for US Federal Agencies material. This involved reviewing published scientific papers and drawing on the current activities of and reviews carried out by such bodies as ECVAM, ICCVAM, ECETOC, OECD, FRAME, ECB, NC3Rs.

Milestone 4 Completion of priorities for research and possible assessment strategies after consultation with stakeholders.Initially the study plan stated that the review would be completed prior to the development of the database. However, on embarking on the review it was felt that it would be beneficial to develop the database at the same time. The reason for this was two fold:

i) it is more efficient as references can be added at the same time as they are reviewed.

ii) the set up of the database and how it is accessed and interrogated is dependent on the type and number of relevant references.

In addition, after consultation with colleagues in the veterinary medicine field, it was felt that the original idea of consulting stakeholders (in the form of a workshop) before production of the database would be unlikely to achieve the aims of the exercise. The most important objective within this study programme is the production of a user-friendly database that contains relevant information. In order to achieve this we need to ensure both that the information contained is all encompassing and relevant and that the accessibility to this information is user-friendly. Hence it was agreed that the best approach to this was to provide a prototype database that can be demonstrated and trialled to relevant stakeholders. From this it is hoped that we can elucidate any changes that may increase relevance or amount of information available from searching of the system and also ensure that the database is easily accessible.

Milestone 8 (moved forward) - Development of 3Rs web-based database.

A web-based database system has been created to allow scientists and other users to quickly and easily find references to literature on the three Rs of relevance to their application.

ArchitectureThe system has been created using PHP (http://www.php.net/), a web scripting language, with MySQL (http://www.mysql.com/) providing the database back-end. These software systems are freely available and open-source. The combination is widely used in the industry and has good support from hosting providers. An attempt to maintain compatibility with a wide range of versions has been made to maximise the portability of the system to different hosting solutions. Industry standard techniques such as a modular architecture, separation of presentation from data handling, in-code documentation with developer documentation generated using phpDocumentor (http://phpdoc.org/), version control (with subversion, http://subversion.tigris.org/) and suits of regression tests have been employed to create a high quality, maintainable and extensible system.

User InterfaceThe system operates in a similar manner to an internet search engine. Instead of typing in search terms the user selects categories in which they are interested under the headings of drug (e.g. antihelmintics), test (e.g. reproduction testing), taxon (e.g. caprine) and objective (i.e. type of replacement, reduction, refinement). The search can be made as general or specific as desired and more than one category can be selected under each heading. Below the search boxes a glossary is provided where each of the categories is defined. A paged list of references and/or relevant websites is returned from a search in a format very similar to results from a search engine. Basic details are displayed in the list and full details, including the abstract, can be viewed for each one individually. Work is in progress to add a facility to allow search results to be downloaded as a spreadsheet or in RIS format.

Admin Interface Limited management interface, requiring login, has been created. This has the facility to import new references from a RIS file and allow the categories and glossary entries to be edited.

Milestone 6 Host one- day workshop to present and discuss findings with stakeholders.The workshop was hosted by CSL in July and attracted 35 delegates. These delegates were from industry, academia and government bodies. The institutes represented were: FRAME, VPC, VMD, RSPCA, Covance, AstraZeneca, Pfizer, VPC, Brantom RA, Intervet, University of Leeds and CSL.

The main aims of the workshop were to:

i) To give an overview of the 3Rs and their relevance to veterinary medicine testing

ii) To demonstrate the database and assess (with potential users) the relevance and accessibility of the information that it provides.

iii) To discuss areas or techniques that could be employed in the testing of veterinary medicines.

The programme was designed to deliver information on all the 3Rs and to raise awareness of issues particularly associated with veterinary medicine testing. This included information on fish welfare and safety testing (by Jane Cooper RSPCA), as those areas were highlighted as priorities in the review. I

Invited speakers and their presentations were :

Introduction to the 3Rs Bob Combes (FRAME)

Replacement –

Use of QSARs in regulatory testing Qasim Chaudhry (CSL)

Reduction Use of statistics in regulatory testing Tony Prickett (CSL)

Refinement Human/animal interactions- effects in the laboratory Katja Van Driel (CSL)Refining husbandry and procedures Penny Hawkins (RSPCA)3Rs in batch testing of veterinary vaccines Jane Cooper (RSPCA)

The afternoon session was specifically aimed at introducing the database to the stakeholders and gaining feedback on the relevance and accessibility of the information. The attendees were asked to use the database and then complete a questionnaire (appendix 3).

Milestone 7 : Report based on findings of workshop.

All attendees were fully cognisant with the 3Rs and were keen to implement these in their studies and work programmes. However, all agreed that implementation of the 3Rs was not a fundamental part of all veterinary medicine testing and that the 3Rs could be much more widely used in this field. Some attendees stated that they had colleagues were unaware of what the 3Rs were and the impact that their implementation could have on the welfare of the animals and scientific quality of the studies. Particular issues raised were:

i) Regulatory testing – it was agreed that the regulator needs to be more pro-active with respect to implementation of the 3Rs and allow more flexibility with respect to the data requirements. In particular with regards to accepting studies carried out with less animals (that are statistically viable) and with replacement techniques. It as felt that using alternative for regulator studies was very difficult to achieve with all the onus being on the individual investigators to provide all the data necessary to accept non-animals models for all or some of the required data. In addition no incentives were given to scientists to reduce or refine their experiments and hence it results in all studies being performed to the exact requirements of the regulators. It was felt that in order to fully implement the 3Rs in vet med testing that the regulators should be approached and a better system designed in which alternatives and changes to the requirements could be implemented without fear of losing regulatory approval. It was understood that health and safety and scientific robustness was a priority for these authorities but that the possibility of alerting the experimental protocols whilst still maintaining the integrity of the results should be investigated.

ii) Priorities for 3Rs research – it was agreed that although replacement should be the ultimate goal more emphasis should be placed on refinement in the interim. It was felt that housing, and refinement of procedures (eg use of analgesics and maximum injection volumes) were issues that were not fully addressed . In particular, fish welfare was often overlooked with environmental enrichment and humane end points being areas for future study.

All delegates were asked to fill in a questionnaire (appendix 3) mainly relating to the database – a summary of the outcome of these is detailed below:

1) Database is a very useful tool which would have a positive impact on veterinary medicine testing

2) Useful to have abstracts and links to full papers: unfortunately due to copyright laws in many cases this is not possible. The only potential way of addressing this would be to write summaries of each paper which would be very time consuming and expensive

3) 3Rs categories should be more specific to allow for more detailed searches: this has now been addressed and he 3Rs categories changed to include descriptors (e.g. refinement – housing, refinement – analgesics)

4) Newsfeed – this was generally accepted as being helpful

5) More data included –data from 5 search engines have now been included to extend the database whilst still ensuring it is small enough to remain relevant and specific

6) Chat exchange – it was felt that a chat room feature/forum would be useful were users could register and ask questions or great ethical discussions. Although it was felt this would be useful it would require high levels of security and maintenance which were beyond the reach of the current project

7) Vet medicine/ 3Rs forum – it was generally felt that a forum that met annually to discuss relevant new development would be a worthwhile venture and most attendees felt that this could be financed within their company.

A report of this workshop and summaries of the talks are to be published in LASA’s newsletter in Jan 08.

Milestone 9 Linkage of database to relevant systems

This study has demonstrated that a 3Rs database specific to veterinary medicine is a useful tool which would be highly utilised. The database developed is a prototype to demonstrate the most effective method of searching and displaying the results. It is now available from an external source on:

http://vmd3rs.csl.gov.uk/

This database, would require a high level of input and maintenance to keep the information in the searches relevant and current.

APPENDIX 1 -VICH GUIDELINES USED

1. Studies to evaluate the safety of residues of veterinary drugs in human food:

a) Reproduction testing

b) Genotoxicity testing

c) Carcinogencicity testing

d) Repeate dose toxicity testing

e) Developmental toxicity testing

2. Efficacy of antihlemintics:

a) general requiremts

b) specific recommendations for bovines

c) specific recommendations for ovine

d) specific recommendations for caprines

e) specific recommendations for equine

f) specific recommendations for swine

g) specific recommendations for canine

h) specific recommendations for feline

i) specific recommendations for poultry

3. Target animal safety – examination of live veterinary vaccines in target animals for absence of reversion to virulence

APPENDIX 2 – REVIEW DATA

1. Literature searches – summary

The following search logic was used in the OVID BIOSIS Previews database, <1985 to 2006 Week 49>

Search Strategy:

1 (reduction and refinement and replacement).mp (176)

2 ((animal$ adj5 (experiment$ or laborator$ or test$ or evaluat$ or develop$)) and (3Rs or 3 Rs or three Rs or reduction or refinement or replacement or alternativ$)).mp (11433)

3 atla alternatives to laboratory animals.jn. (412)

4 (veterinary and (pharma$ or medicin$ or drug$ or compound$ or product$ or vaccin$ or antihelmint$ or analgesi$ or antiparasit$ or antibiotic$ or treatment$)).mp (15780)

5 1 and 4 (10)6 2 and 4 (167)7 3 and 4 (7)8 5 or 6 or 7 (170)9 from 8 keep 1-170 (170)

This retrieved 170 items. Trial uploads have been made to a Reference Manager 11 database. Of the 170 items circa 150 have been identified as useful to the project and categorised as relevant to Replacement, Reduction, Refinement.

Keywords from these records will now be employed in searching a wider range of biomedical databases.

In September / October 2007 a second round of searching was carried out.

The Dialog commercial host was used, the databases chosen for searching being:

File 5:Biosis Previews(R) 1926-2007

File 50:CAB Abstracts 1972-2007

File 155:MEDLINE(R) 1950-2007

File 34:SciSearch(R) Cited Ref Sci 1990-2007

File 144:Pascal 1973-2007

File 156:ToxFile 1965-2007

File 71:ELSEVIER BIOBASE 1994-2007

File 185:Zoological Record Online(R) 1864-2007

The search logic employed being:

S1 REDUCTION(S)REFINEMENT(S)REPLACEMENT

S2 JN=ATLA, ALTERNATIVES TO LABORATORY ANIMALS

S3 (ANIMAL?(N3)(EXPERIMENT? OR LABORATOR? OR TEST? OR EVALUAT? OR DEVELOP?)) AND (3RS OR 3()RS OR THREE()RS OR REDUCTION OR REFINEMENT OR REPLACEMENT OR ALTERNATIV?)

S4 VETERINARY AND (PHARMA? OR MEDICIN? OR DRUG? OR COMPOUND? OR PRODUCT? OR VACCIN? OR ANTIHELMINT? OR ANALGESI? OR ANTIPARASIT? OR ANTIBIOTIC? OR TREATMENT?)

S5 S4 AND (S1 OR S2 OR S3)

S6 RD S5 (removes duplicates – giving unique items)

Items already retrieved by earlier searching were excluded. Titles were printed and from these 101 additional records were selected for downloading and addition to the database.

Total of 281 records

2. Search Terms Replacement

modelsurrogatein vitroartificial intelligencecomputer prediction/simulation expert systemmathematical model (modelling)QSARSARstructure-activity relationship theoretical model (modelling)virtualartificial physicochemicalcell culturetissues culturetissue engineeringabattoir autopsy biopsy cadaver cellcell linecellularculturecytosolic fractiondermal equivalentembryo

explantex-vivofragment humanisolatedmethodmicrosomalmodelorganorganelle slicesubcellular fractiontechniquetissuetissue equivalentalgaebacteria fungushydrainsectinvertebratemicro-organismplantprotozoanyeast

Reductionexperimental designphased/sequential designstatistical design

Refinementcagecaginghousinghusbandrywelfarebehavioural/environmental enrichment euthanasiahandlingrestraintnon-invasive stress anaesthesiaanaestheticanalgesiaanalgesicanxiolytichypnoticsedativeNSAID

3RS AND VETERINARY MEDICINE WORKSHOP

CSL, Sand Hutton, YorkTuesday 17 July 2007

Questionnaire

Please could you fill in the questions below either as part of your group or individually. Thank you!

1. Do you think that a specialised database will assist in the implementation of the 3Rs to the testing of veterinary medicines? Do you think there will be any disadvantages to suing a specialised system?

2. Do you think that the format of this database is easy to use? If not, what changes would you recommend?

3. Do you think any more or less categories should be used?

4. Do you think the newsfeed is helpful/useful

5. How do you think that this database could be improved?

6. How and where do you the database be advertised/promoted (eg NC3Rs, NOAH )?

7. Do you think there would be any value in establishing a veterinary medicine/3Rs Forum that meets annually?

8. What do you think should be the priorities for 3Rs research and veterinary medicine (eg humane endpoints)?

APPENDIX 3 – WORKSHOP QUESTIONNAIRE

References to published material9. This section should be used to record links (hypertext links where possible) or references to other

published material generated by, or relating to this project.