Documento 1 del GTEMA

OVERALL REVIEW, SUMMARY AND CONCLUSIONS OF THE MEETING OF THE ICLAS/CSIC WORKING GROUP ON COMPLEMENTARY METHODS

Talavera de la Reina (Toledo, Spain). 28-29 April 1995.

PREFACE

In September 1994, the International Council for Laboratory Animal Science(ICLAS), a non-governmental organization for international cooperation inlaboratory animal science, created a working group to promote complementaryand alternative methods to reduce, refine and replace the use of laboratory animals.

A meeting of the ICLAS/CSIC Working Group on complementary methods was heldin Centro Regional de Salud in Talavera de la Reina, Toledo Spain on April 28-29, 1995 under the chairmanship of Dr. Eduardo de la Peña, ICLAS Scientific Member for Spain.

The objective of this meeting was to obtain a list of recommendations to be presented to the ICLAS Governing Board and General Assembly that was held in Helsinki, Finland, on July 1-7, 1995. Professor Garcia Partida, President of the ICLAS Spanish committee, expressed his gratitude to all those who participated and stressed the significance of this report of the meeting.

INTRODUCTION

Dr. Eduardo de la Peña, coordinator of the ICLAS/CSIC working group on complementary methods, explained the format of the meeting at the start of the first session. The meeting was divided into six sessions. In each session (except for session six) a fifteen minute presentation by a speaker with expertise on a specific topic was followed by a one hour discussion.

The session rapporteur, moderator and secretary of the working group were responsible for preparing a document summarizing the major issues and conclusions of each session.

During Session Six, the rapporteurs of each previous session read their respective documents. Participants then discussed these reports in order to obtain a final report on the entire meeting.

GENERAL SUMMARY

The scientific rationale for alternatives development is based upon the 3Rs concept of Russell and Burch--replacement, reduction and refinement. In general, sessions focused on the harmonization of approaches to the development and validation of alternative methods. Validation was defined as the process whereby the relevance and reliability of a procedure are established for a particular purpose.

Several issues related to validation were discussed including criteria, approaches, and problems associated with previous studies. Several recommendations focusing on the role ICLAS can take in fostering the development and implementation of alternatives were also formulated.

SESSION I: INTERNATIONAL HARMONIZATION OF TEST METHODS FOR HAZARD CHARACTERIZATION TAKING INTO ACCOUNT ANIMAL WELFARE ISSUES

SPEAKER: Herman B.W.M. Koeter

MODERATOR: Dr. Pilar Goya

RAPPORTEURS: Drs. Carmen Pueyo, Elina Valcarce, Bartolome Ribas, and Ana Guadaño

Discussion focused on the structure and role of the OECD, with special mention of the following aspects:

* the OECD serves as forum for discussion for its 25 member countries

* the OECD works to harmonize different assay methods, with goals of reducing animal use and mutual acceptance of data (MAD).

* OECD functions to develop and update guidelines that are binding to all members and used as a reference in non-member countries.

* until today, no formal proposal for the adoption of any alternative test has been received by the OECD. The absence of harmonized validation criteria may have discouraged scientists from participating in formal validation studies.

Harmonization of OECD guidelines for acute toxicity testing has resulted in a reduction of animal numbers for safety testing of chemicals since results are now accepted internationally. Such harmonization and acceptance could also be achieved for pharmaceuticals where considerable numbers of animals are currently being used for acute toxicity testing. Since 1990, the international conference on harmonization (ICH) is making an effort to harmonize test methods for pharmaceuticals between the U.S., Japan and the E.U.

SESSION II: THE VALIDATION OF IN VITRO TOXICITY TEST PROCEDURES IN EUROPE

SPEAKER: Prof. Horst Spielmann

MODERATOR: Prof. Paulino Garcia Partida

RAPPORTEURS: Drs. Guillermo Repetto, Isabel Rodriguez and Kai Pelkonen

Relevance and reliability of a test or an assay should be established during the development of a new test, from the beginning through the process.

We have learned that some in vivo tests are not relevant and reliable for human toxicity assessment. This is not a major problem with respect to legislation, because such assessments are viewed as a "package" and there is a sufficient safety margin. However, the in vivo methods should be updated in the future.

SESSION III: IN VITRO ASSAYS IN THE DEVELOPMENT OF DRUGS AND CHEMICALS

SPEAKER: Dr. Mª Jose Gomez Lechon

MODERATOR: Dr. Jose V. Tarazona

RAPPORTEURS: Drs. Francisco O. Gonzalez Mencio, Angustias Herrera and Herman Koeter

In vitro assays have been steadily refined and are being used for toxicological testing of a number of substances. Assays employing human cells are to be encouraged. When designing new in vitro assays, systems which use genetically engineered cells as models of human metabolism look promising. Immortalization of cells is another promising field of activity because of its potential for standardizing tests.

Standard cell lines are already available and could be used even more extensively than at present. Primary cultures are also relevant for certain uses. The main goal for the future is the development of reasonably standardized human cells with known metabolism. Knowledge of metabolism is also crucial in selecting appropriate animal species for toxicity testing.

SESSION IV: DEVELOPMENT OF ALTERNATIVE METHODS AND THE MODULAR APPROACH TO VALIDATION

SPEAKER: Prof. Alan M. Goldberg

MODERATOR: Dr. Elina Valcarce

RAPPORTEURS: Drs. Jose V. Tarazona, Covadonga Caballo and Michael Balls

This presentation focused on issues of mechanism toxicity and the

usefulness of alternatives in risk assessment. Questions raised during the presentation included the need for mechanistically-based tests, incorporation of in vitro assays as a first approach in evaluating the safety of chemical, therapeutic and other commercial product; the use of human cells and cataloging regulatory requirements to encourage in vitro assays.

The presentation also provided an approach to validation that is based on fundamental principles and modeled after actual practice in other areas of methodology acceptance. It was recognized that the validation process is but one aspect of incorporation of in vitro approaches into safety evaluation and risk assessment.

SESSION V: THE DEVELOPMENT, VALIDATION AND ACCEPTANCE OF ALTERNATIVE METHODS

SPEAKER: Prof. Michael Balls

MODERATOR: Dr. Carmen Barrueco

RAPPORTEURS: Drs. M. Jose Gomez Lechon, Jorge Zapatero, and Alan Goldberg

This session dealt with the development, validation and acceptance of alternative methods in the context of directive 86/609/EEC, which requires that experiments must "use the minimum number of animals" and "must cause the least pain, suffering, distress or lasting harm", which is consistent with providing satisfactory results, and that an experiment on animals "shall not be peformed, if another scientifically satisfactory method of obtaining the result sought, not entailing the use of an animal, is reasonably and practically available."

Where the replacement of animal tests required by regulatory guidelines is sought, formal validation is necessary. ECVAM (The European Centre for Validation of Alternative Methods) has been set up by the European Commission, with the principal task of coordinating the validation of alternative methods at the European Union level. By the year 2000, The EU aims to reduce the number of vertebrates used for experiments and other scientific purposes by 50% .

ECVAM has established criteria for the Centre's priority areas. The criteria are based on numbers of animals, amount of possible suffering, and the likelihood that valid and acceptable replacement alternative methods can be found.

CONCLUSIONS

Sequential approaches hold great promise for reducing animal numbers in toxicity testing. The use of in vitro assays should be encouraged, although in some cases they may be more time-consuming and expensive than in vivo assays.

The acceptance of in vitro tests may be facilitated by conducting both in vitro and in vivo studies in parallel, until sufficient confidence is built with the in vitro tests to delete/replace the in vivo counterpart.

Although there will be many different approaches to validation methodology evaluation, several validation studies have proven that no more than 4-5 experienced laboratories are required to validate a single assay.

In many cases the animal tests do not correspond to the human response. Therefore, the aim of alternative methods should not be to simply reduce the numbers of animals used, nor to replace the in vivo tests, but to develop new technologies and new strategies which may result in different and more useful information.

There is a need for development of genetically modified cell lines which could result in metabolic mechanisms similar to those of human hepatocytes.

Cell models expressing specific functions are also necessary. For example, the use of human cells of known metabolism can facilitate the choice of more appropriate species for toxicity testing of particular substances.

Insufficient toxicological data exists for a considerable number of

chemicals. To enable initial hazard /risk assessment of these chemicals within a reasonable time frame, relatively simple, fast test methods are needed. For this purpose, non-animal (in vitro) methods should be considered as part of the initial safety assessment process.

In vitro studies should be incorporated into the development of new

products as early as possible.

Batteries of non-animal tests will be required. As they become available, mechanistically-based tests will be required.

The optimization of assays is an essential component prior to formal validation.

Replacement of single animal tests, although useful, is not necessary. The use of alternative methods cannot be reduced to simply reducing numbers of animals, but must focus on the development of new technologies resulting in new information.

There is an urgent need to establish courses for scientists and students both on non-whole animal alternatives and on ethics of experimentation in the biomedical sciences.

Acceptance of new animal methods should be subjected to the same criteria applied to alternative methods. To establish an order of validation areas, criteria must be developed to permit the most efficient use of resources and to produce validated methodology. In the area of vaccines and medicines, adequate biological understanding of mechanisms exists to provide a reasonable assurance that work in this area will lead to validated alternatives.

RECOMMENDATIONS

ICLAS should take an active role in educating its members about

developments in ethics, statistics, alternatives (3Rs), and their validation.

The ICLAS/CSIC workshop suggested that more fundamental research is essential to develop new non-animal methods. A reduction in animal use can be achieved if procedures involving animals were better designed and analyzed, and if stronger scientific justification were required before animal experiments were permitted.

The ICLAS/CSIC workshop recommends that new animal tests should be required to pass the same criteria as non-whole animal methods to achieve regulatory acceptance.

Although there are countries with minimal legislation on human and animal experimentation, changing the site of animal testing to those countries in order to avoid more stringent regulations in other nations is immoral and unethical. More stringent practices should be endorsed by all member countries.

Dr. de la Peña closed the session by recommending that ICLAS use the terms "alternative" and "complementary" as synonyms. He also remarked on the need to make the scientific community aware of the development and use of complementary alternative methods and also of the diffusion of those methods. He also recognized the contributions of speakers, moderators, rapporteurs, translators, secretary members and all members of the ICLAS/CSIC Working Group on Complentary Methods to the creation of an excellent meeting. Special thanks were extended to ICLAS scientific member for Cuba, Dr. Gonzalez Mencio and ICLAS national member for Spain, Prof. Paulino Garcia Partida and Dr. Juan Atenza, director of the Centro Regional de Salud Publica, Talavera de la Reina, for their kindness and the facilities of their Center.

ANNEX 1: TERMINOLOGY

Alternative methods include any procedure that reduce the number of animals used, that refines a method in order to reduce the amount of stress and suffering endured by the animal, and a procedure that replaces the use of animals. In vivo and in vitro methods are complementary.
Prevalidation, see interlaboratory assessment.
Validation is the process by which the reliability and relevance of a procedure are established for a specific purpose.
Reliability of a procedure describes whether it can be performed reproducibly within and among laboratories and over time.
Relevance of a procedure describes whether it is meaningful and useful for a particular purpose.
Test development is the process by which the components of a protocol (for example, experimental system, exposure conditions, endpoint, endpoint measurement and data analysis procedures) are defined for a specific purpose, and is normally carried out in the laboratory of origin.
Interlaboratory assessment establishes whether or not a test can be successfully transferred from one laboratory to another. It should include two phases: prevalidation, which results in fine-tunning of the study design and final optimisation and agreement of the standard protocol to be used, and formal validation, in which a set of test chemicals is evaluated by the standard protocol.
Procedure refers to a test, test battery or testing strategy.
Test refers to the combination of the experimental system used, exposure conditions, endpoint, endpoint measurement and data analysis method.
Endpoint refers to the processes, responses or effects assessed.
Endpoint measurement refers to the techniques used to assess endpoints.
Protocol refers to the precise step-by-step description of a test.

ANNEX 2

LIST OF PARTICIPANTS

E. de la Peña 1; J. Atenza 2; M. Balls 3; C. Barrueco 4; C. Caballo 5; P. Garcia Partida 6; M. Garcia Vedia 1; A. Goldberg 7; M.J. Gomez Lechón 8; A. Gonzalez Coloma 1; F. Gonzalez Menció 9; P. Goya 10; A. Guadaño 1;A. Herrera 5; H. Köeter 11; A. Lopez de Ceraín 12; K. Pelkonen 13; C.Pueyo 14; G. Repetto 15; I. Rodriguez 16; H. Spielmann 17; J.V.Tarazona 18; E. Valcarce 1; and J. Zapatero 19.

CENTRES:

Centro de Ciencias Medioambientales, Serrano 115 dpdo 28006 Madrid, Spain
Centro Regional de Salud Pública. Talavera de la Reina, Toledo, Spain
ECVAM, JRC Environmental Institute, 21020 Ispra (Va) Italy
Centro Nacional de Alimentación, Instituto de Salud Carlos III, 28200 Majadahonda, Madrid, Spain
Subdirección General de Sanidad Ambiental, Ministerio de Sanidad y Consumo. Spain
Facultad de Verinaria. Universidad Complutense, Madrid, Spain
CAAT Center for Alternative Animal Testing. Johns Hopkins School of Public Health. 11 Market place, Suite 840. Baltimore, Maryland 21202-6709.USA
Centro de Investigación. Hospital Universitario La Fé. Avda. Campanar 21. E-46009 Valencia. Spain
Centro de Toxicología, CENPALAB Centro Nacional de Producción de Animales de Laboratorio, Bejucal, Habana, Cuba
CSIC. Relaciones Internacionales c/ Serrano 113, Madrid, Spain
OECD Environmental Health and Safety Division. 2 rue Andre-Pascal, 75775 Paris, Cedex 16. France
CIFA, Universidad de Navarra, Pamplona, Spain
Dpt. Physiology. University of Kuopio, Finland
Dpt. of Biochemistry and Molecular Biology, Universidad de Cordoba, Cordoba, Spain
Instituto Nacional de Toxicología, Apdo. 863. 41080 Sevilla, Spain
GAIKER, Zamudio (Bilbao) Spain
ZEBET at the Bgvv. Diedersdorfer Weg 1. D-12277 Berlin, Germany
CISA/INIA Valdeolmos (Madrid) Spain
CIDA. (Barcelona) Spain.

INSTITUTIONS

CAAT = Center for Alternatives to Animal Testing
CICyT = Comisión Interministerial de Ciencia y Tecnología
CSIC = Consejo Superior de Investigaciones Científicas (Spanish Research Council)
ECVAM = European Center of the Validation of Alternative Methods
ICLAS = International Council on Laboratory Animal Sciences
MSyC = Ministerio de Sanidad y Consumo
ZEBET = National German Center for Documentation and Evaluation of Alternative Methods to Animal Experiment