Abstract for TestSmart--A Humane and Efficient Approach to Screening Information Data Sets (SIDS) Data
Manfred Liebsch
ZEBET
This talk is given on behalf of Professor Michael Balls, Head of ECVAM
Basis for European Centers
DIRECTIVE 86/609/EEC
- Article 23:
- The Commission and Member States should encourage research into the development and validation of alternative techniques which could provide the same level of information as that obtained in experiments using animals...
...and shall take such other steps as they consider appropriate to encourage research in this field. The commission and Member States shall monitor trends in experimental methods.
- Article 7.2:
- 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.
European Centers for Alternatives to Animal Testing

Stakeholders in ECVAM's Woi

ECVAM's Mission
To coordinate the independent evaluation of the relevance and reliability of tests for specific purposes, and in particular through prevalidation and validation studies, so that chemicals and products of various kinds, including medicines, vaccines, other biologicals, medical devices, cosmetics, household products and agricultural products, can be manufactured, transported and used more economically and more safely, whilst the current reliance on animal test procedures is progressively reduced.
The Duties of ECVAM
- To coordinate the validation of alternative test methods at the European Union level.
- To act as a focal point for the exchange of information on the development of alternative test methods.
- To set up, maintain and manage a data base on alternative procedures.
- To promote dialogue between legislators, industries, biomedical scientists, consumer organisations and animal welfare groups, with a view to the development, validation and international recognition of alternative test methods.
(Commission Communication to Council and the Parliament, 29 October 1991)
ECVAM Scientific Advisory Committee (ESCA)
Member state representatives
- Prof. J Castell (ES)
- Dr. D. Clark (UK)
- Prof. A. Guillouzo (F)
- Dr. C. Hendriksen (NL)
- Prof. R. Lorenzini (I)
- Prof. G. Papadopoulos (G)
- Prof. C. Regan (IR)
- Prof. V. Rougiers (B)
- Prof. AMVM SOARES (P)
- Prof. H. Spielmann (D)
- Prof. O. Svendsen (DK)
- Dr. M. Viluksela (SF)
- Prof. E. Walum (S)
Organisation representatives
- Dr. B. Blaauboer (ERGATT)
- Dr. P. Botham (ECETOC)
- Dr. B. Garthoff (EFIPA)
- Dr. B. Rusche (Eurogroup f. Animal Welfare)
- Dr. O. de Silva (COLIPA)
EC/JRC representatives
- Mr. H. Allgeier (JRC&IHPC)
- Prof. M. Balls (ECVAM)
- Mr. G. Corcelle (DG XI)
- Dr. F. Drion (DG XXIV)
- Dr. M. Halder (ECVAM)
- Ms. S. Louhimies (DG XI)
- Dr. E. Sabbioni (ECVAM)
- Dr. G. Vollmer (ECB)
- Mr. GP Tartaglia (IHPC)
Definition of "Alternative Method" According to: the Three Rs of Russell & Burch
Reduction: any decrease in the numbers of animals used to obtain infor-mation of a given amount and precision.
Refinement: any decrease in the incidence or severity of procedures applied to animals necessarily used.
Replacement: the substitution of conscious living higher animals by non-sentient material.
Validation of Alternative Methods--AMDEN I
"The process by which the reliability and relevance of a procedure are established for a particular purpose"
This definition stems from the 1990 Amden CAAT/ERGATT workshop and Frazier's 1990 report to the OECD.
What is Meant by Relevance, Reliability, and Purpose?
Relevance: scientific value and practical usefulness in relation to a clearly defined and specific purpose.
Reliability: reproducibility of results within and between laboratories and over time.
Purpose: relevance and reliability in relation to a specific aspect of in vivo toxicity (according to type of toxicity, target species, degree of toxicity, and spectrum of test materials and products).
AMDEN II
Practical Aspects of the Validation of Toxicty Test Procedures: The Report and Recommendations of ECVAM Workshop 5
Michael Balls, Bas J. Blaauboer, Julia H. Fentem, Leon Bruner, Robert D. Combes, Björn Ekwall, Robin J. Fielder, Andre Guillouzo, Richard W. Lewis, David P. Lovell, Christoph A. Reinhardt, Guillermo Repetto, Dariusz Sladowski, Horst Spielmann and Flavia Zucco
ATLA 23: 129-147, 1995
AMDEN II: Validation Study Management

ECVAM Guideline for Prevalidation
The Role of Prevalidation in the Development, Validation and Acceptance of Alternative Methods
Rodger D. Curren, Jacqeline A. Southee, Horst Spielmann, Manfred Liebsch, Julia H. Fentem and Michael Balls
ATLA 23: 211-217, 1995
- protocol refinement (1 laboratory)
- protocol transfer (2 laboratories)
- protocol performance (blind trial in 3 laboratories)
Harmonization of Validation and Acceptance Criteria
ECVAM
- 1995 Amden II and 1998 Amden III (with ERGATT)
- statement with ECB in 1995
ICCVAM
- workshop in 1995
- report in 1997
- formal establishment in 1997
OECD
- workshop and report in 1996 (Solna)
- guidance to be published in 1999
Key points:
- regular exchange of views and personnel
- broad agreement on criteria
- agreement on interpretation and application still to be reached
Up Front a Prevalidation Study Info Must Be Provided
- Description of the basis of the method
- Definition of scientific purpose and practical application
- Test protocol / Standard Operating Procedures
- specification of endpoint
- derivation and expression of results
- their interpretation & application, via Pred. Model
- appropriate controls & historical data
- Limitations of the method
- Data supporting
- intralaboratory reproducubility
- if available, interlab. transferability
ECVAM Procedures

ECVAM's Criteria for Acceptibility of a Validation Study
- Clarity of defined goals
- Quality of overall design
- Independence of management
- Independence of test chemical selection
- Independence of test chemical coding and distribution
- Number and properties of test chemicals studied
- Quality of interpretation of results
- Performance of method(s) in relation to goals of the study
- Reporting of outcome in the peer review literature
- Availability of raw data
- Independence of assessment of outcome
Example 1: Validation of the 3T3NRU-PT in vitro phototoxicity test (1992-1998)
EU-COLIPA Project
Goals
- Validate one new in vitro phototoxicity test: 3T3 NRU-PT
- Evaluate "in house tests" used in industry
- photohaemolysis in red blood cells (RBC)
- primary human keratinocytes
- skin models (LSE & Skin2)
- histidine oxidation
- photobinding to proteins
- photoactivation of complement
Participants
ZEBET-BgVV, Beiersdorf AG, Unilever, ESL, Novartis, Hoffmann-La Roche, Henkel KGaA, Procter & Gamble, FRAME, University of Warsaw
Time Frame
- 1992 - 94: Prevalidation Study
- 1994 - 96: formal Validation Study
- 1997 - 98: Special UV filter Study
- 1998: Draft OECD TG
Sponsors
EU DG XI, ECVAM, ZEBET-BgVV, COLIPA
Design of 3T3NRU-PT
3T3 monolayer => 24 hours => chemical => 1 hour => UVA-vis/dark => 24 hour => viability test (NR Uptake)
Prediction model: comparison of "dose-responses" obtained in concurrent (+UVA) and (-UVA) exp.
Chemicals Tested
- Prevalidation Study: 11 PT, 9 NPT, not blind
- Validation Study: 25 PT, 5 NPT, blind trial
- UV Filter Study: 10 PT, 10 PT blind trial
Outcome
- sensitivity: 84%
specificity: 93%
- sensitivity: 100%
specificity: 98%
Independent Experts Involved
- Chemical selection for prevalidation: COLIPA
- Assessment of outcome of prevalidation: ECVAM Workshop 2 on Phototoxicity Testing
- Chemical selection for Validation study: ECVAM Workshop 2 on Phototoxicity Testing
- Assessment of outcome of formal Validation: Scientific Committee of Cosmetology (SCC) and later ESAC
- Request for additional UV Filter Study: SCC
- Selection of chemicals for UV Filter Study: SCCNFP and COLIPA expert group
- Assessment of outcome of UV Filter Study:.ESAC and later SCCNFP
Example 2: ECVAM Skin Corrosivity Validation Study (1994-1998)
Tests
- TER Assay
- EPISKIN
- CORROSITEX™
- Skin™ ZK 1350 Assay
Participants
- TER
- Unilever ESL, UK $
- ZENECA CTL, UK
- Corning Hazleton, UK
- EPISKIN
- Rhone-Poulenc Agro, F $
- Agence du Medicament, F
- INRS, F
- CORROSITEX
- MA Inc, USA $
- MA Ltd, UK
- Sanofi Recherche, F
- $ = lead laboratory
Chemicals
60 selected by independent CSSC Outcome
- TER - sensitivity: 88%, specificity: 72%, acceptable
- EPISKIN - sensitivity: 83%, specificity: 80%, acceptable
- CORROSITEX - sensitivity: 71%, specificity: 76%, only 60% of chemicals reacted with CDS, sensitivity too low
- Skin2 ZK 1350 - sensitivity: 53%, specificity: 100%, unacceptable underprediction (10 sec application = too insensitive)
Independent Experts Involved
- Assessment of outcome of prevalidation: ECVAM Workshop 6
- Chemical selection for formal Validation Study: CSSC (it took one year!)
- Selection of appropriate statistics to be.applied in formal Validation Study: Humboldt University and ECVAM
- Assessment of outcome of formal Validation Study: MT of ECVAM SCVS & Biostatisticians later: ESAC
- Decision to conduct Special Study on SAR, pH and acid- alcaline reserve ECVAM
ECVAM is Validating Test Strategies
An Evaluation of the Proposed OECD Testing Strategy for Skin Corrosion
Andrew P. Worth, Julia H. Fentem, Michael Balls, Philip A. Botham, Rodger D. Curren, Lesley K. Earl David J. Esdaile and Manfred Liebsch
ATLA 26: 709-720, 1998
- SAR
- pH < 2 and > 11.5
- acid reserve, alcaline reserve
- EPISKIN
- TER
- rabbit
Example 3: Planning of the ECVAM Skin Irritation Prevalidation Study (1998-1999)
Alternative Methods for Skin Irritation Testing: the Current Status
ECVAM Skin Irritation Task Force Report 1
Philip A. Botham, Lesley K. Earl, Julia Fentem, Roland Roguet and Johannes J.M. van de Sandt
ATLA 26: 195-211, 1998
Recommendations
- consider human and animal skin organ cultures for prevalidation
- reconstituted human skin / epidermis models are relevant: support prevalidation
- ECVAM Task Force shall act as preliminary interim Management Team of a prevalidation study
Preparation of a New Study
- In the first report of ECVAM Task Force "Skin Irritation" a list of 10 chemicals was issued, for which human and rabbit data of high quality existed. Test developers were asked to submit their test protocols and data obtained with this set of 10 refernce chemicals (April - June '98)
- In July 98 the TF selected four promising tests:
- EpiDerm Skin Iritation Test
- EPISKIN Skin Irritation Test
- PrediSkin™ (human skin ex vivo)
- non-perfused pig ear model
- A call for tenders for an ECVAM-funded prevalidation study on these four in vitro skin irritation tests was issued in July 1998.
- Under management of Unilever SEAC, twelve laboratories are presently being contracted to conduct the prevalidation study in 1999/2000.
Conclusions
So far, ECVAM has sucessfully promoted three tests to regulatory acceptance by sponsoring and managing Prevalidation Studies, followed by formal Validation Studies and additional Special Studies.
The duration of the processes was four years in case of Skin Corrosivity Tests, and seven years in case of the 3T3NRU Phototoxicity Test.
Apart from the peer review of the publication mauscripts, during these processes, both, the methods and results obtained had undergone scientific reviews by various expert groups, finalizing in the respective ESAC statements.
In contrast, the ESAC statement "on the scientific acceptability and practical availability of in vitro methods for the production of monoclonal antibodies" (31 March 1998) was based on conclusions of an ECVAM Workshop a peer review report produced for ESAC.