THE ROLE OF ECVAM IN PROMOTING THE REGULATORY ACCEPTANCE OF ALTERNATIVE METHODS Dr Andrew Worth European Centre for the Validation of Alternative Methods (ECVAM) Institute for Health & Consumer Protection European Commission Joint Research Centre Ispra, Italy Conference on Research into Alternatives to Animal Experimentation Brussels, 9-10 July 2002

OUTLINE 1) The evolution of regulatory tests - role of ECVAM - role of ESAC 2) The ECVAM validation process 3) Paths toward regulatory acceptance 4) Implications of the chemicals policy for the development and validation of alternative tests 5)The ECVAM report on alternative methods for chemicals testing, and its follow-up

THE ROLES OF ECVAM AND THE ESAC ECVAM was established by a Communication from the Commission to the Council and the Parliament in October 1991, which laid out the duties of ECVAM The main duty of ECVAM is to coordinate the validation of alternative tests at the EU level Validation is the process by which the reliability (reproducibility) and relevance (scientific basis and predictive capacity) of a method are established for a particular purpose ECVAM has a Scientific Advisory Committee The most important task of the ECVAM Scientific Advisory Committee (ESAC) to make formal recommendations on the scientific validity of alternative methods, by issuing statements on their applicability

EXAMPLE OF AN ESAC STATEMENT

THE EVOLUTION OF REGULATORY TESTS StageKey players 1Research and development Industry, academia National funding bodies & DG RTD 2Prevalidation 3Validation 4Independent assessmentScientific peer reviewers, ESAC, SCC-NFP Other expert groups 5Regulatory acceptanceRegulatory bodies e.g. EU Competent Authorities EDQM / European Pharmacopoeia ECVAM & national validation authorities

OVERVIEW OF THE ECVAM VALIDATION PROCESS Prevalidation Formal validation Catch-up validation Define structural and performance characteristics and criteria New test method Similar test method Scientifically validated test method Old test method Weight-of-evidence reviewRetrospective validation

Chemicals 1.For nephrotoxicity: Prevalidation of two renal cell lines (LLC-PK1 and MDCK), using trans-epithelial resistance and inulin as permeability endpoints 2.For acute thrombocytopenia: Prevalidation of Colony Forming Unit-Megakaryocyte assay 3.For acute lethal toxicity: Collaborative validation study with NICEATM Use of 3T3 fibroblasts and human keratinocytes to predict acute lethal toxicity in rodents and humans Biologicals 1.Validation of the Vero cell test for the safety testing of diphtheria vaccines 2.Validation of in vitro methods for pyrogenicity testing (DG RTD contract) PREVALIDATION AND VALIDATION STUDIES IN PROGRESS

PATHS TOWARD REGULATORY ACCEPTANCE Methods for Chemicals 1) ESAC endorsement of method Opinions of other Commission scientific committees 2a and 2b 2a) Regulatory acceptance in the EU EU National Coordinators for Test Methods EU Competent Authorities for Directive 67/548/EEC Adoption of Annex V Test Method 2b) Acceptance at OECD level National Coordinators of the OECD Test Guidelines Programme OECD Joint Meeting, EPOC and then OECD Council Adoption of OECD Test Guideline

REGULATORY ACCEPTANCE - Methods for chemicals On 4 February 2000, the EU Competent Authorities for Directive 67/548/EEC (on the Classification, Packaging and Labelling of Dangerous Substances) accepted three in vitro methods as replacement methods for the toxicity testing of chemical substances: 1.The rat skin transcutaneous electrical resistance (TER) method for skin corrosion 2.Human skin equivalents (that meet certain criteria) for skin corrosion 3.The 3T3 NRU method for phototoxic potential These methods have been incorporated into Annex V of Directive 67/548/EEC as Method B.40 (in vitro skin corrosion tests) and Method B.41 (in vitro 3T3 NRU phototoxicity test) On May 2002, the National Coordinators of the OECD Test Guidelines Programme approved Test Guidelines for these methods: TG 430: in vitro skin corrosion test (TER) TG 431: in vitro skin corrosion test (human skin model) TG 432: in vitro 3T3 NRU phototoxicity test

PATH TOWARD REGULATORY ACCEPTANCE Methods for biologicals 1) ESAC endorsement of method 2) Group of Experts of the European Pharmacopoeia: Group 15(human sera and vaccines) Group 15 V(veterinary sera and vaccines) Group 6(biological substances) Group 6B (human blood products) 3) Publication of draft / revision Monograph proposal 4) Commenting period 2 or 5 5) Regulatory acceptance by Council of Europe Countries European Pharmacopoeia Commission adopts Monograph Halder (2001). ALTEX 18, Supplement 1,

PROGRESS TOWARD REGULATORY ACCEPTANCE Methods for biologicals 1) Production of monoclonal antibodies: DG ENV has circulated the ESAC statement to the Competent Authorities for Directive 86/609/EEC 2) Batch potency testing of tetanus vaccines for human use Revised European Pharmacopoeia Monograph, including the ToBI and ELISA tests, has been published in Pharmeuropa for comment 3) Batch potency testing of erysipelas vaccines Revised European Pharmacopoeia Monograph, including the ELISA test, has been published in Pharmeuropa for comment

THE CHEMICALS POLICY AND ALTERNATIVE METHODS (1) 13 February 2001: European Commission adopts White Paper on a Future Chemicals Policy proposes REACH system for new and existing chemicals recommends that: Testing should generally be limited to in vitro methods for chemicals produced/imported in range of 1-10 tonnes Section 3.2 states that: … ECVAMs central role will be maintained, and it is expected that the development of alternative methods will be accelerated. Further research will be carried out both at Community and national level, in order to develop and validate novel testing strategies involving fewer or no animals, while enhancing the relevant information that can be obtained from testing without simultaneously increasing the number of animals involved.

7 June 2001: Environment Council concludes (para. 23) that: Animal testing should be limited to the level necessary to deliver the objectives of the strategy ….... In addition to promoting this issue in ECVAM..., the Community should play a more active role in the OECD, to encourage wider adoption of validated, alternative, non-animal testing methods. 17 October 2001: Economic and Social Committee adopts opinion on the White Paper, in which it endorses the commitment to promote non-animal testing 15 November 2001: European Parliament adopts Schörling report, setting out rules to restrict the number of animal tests and foster the development of alternative methods THE CHEMICALS POLICY AND ALTERNATIVE METHODS (2)

THE ECVAM REPORT ON ALTERNATIVE METHODS FOR CHEMICALS TESTING The ECVAM report: 1)was written by ECVAM staff and members of the ECVAM Working Group on Chemicals, with input from the ESAC and other experts 2)describes the current status of alternative methods for human health endpoints 3)makes recommendations for research and development 4) provides a possible time-frame for validation Worth A.P. & Balls M. (2002). Alternative (non-animal) methods for chemicals testing: current status and future prospects. A report prepared by ECVAM and the ECVAM Working Group on Chemicals. ATLA 30, in press. Finalised draft available at:

OVERVIEW OF THE ECVAM REPORT Chapter 1Background: Chemicals Policy, alternative testing, role of ECVAM Chapter 2Principles and procedures of validation Chapter 3Scientific basis of chemical risk assessment Chapter 4Acute lethal toxicity Chapter 5Acute dermal and ocular toxicity Chapter 6Skin and respiratory sensitisation Chapter 7Biokinetics: barrier function, metabolism, mathematical modelling Chapter 8Target organ and system toxicity Chapter 9Genotoxicity and carcinogenicity Chapter 10Reproductive toxicity Chapter 11Endocrine disruption in humans Chapter 12 Summary of conclusions and recommendations Possible time-frame for validation

AVAILABILITY OF ALTERNATIVE METHODS (1) Alternative methods available immediately 1) Skin corrosion (in vitro – replacement) 2) Phototoxicity (in vitro – replacement) 3) Genotoxicity (in vitro – [partial] replacement) 4) Acute oral toxicity (in vivo – refinement and reduction) 5) Skin sensitisation (in vivo – refinement and reduction) 6) Developmental toxicity (in vitro – partial replacement) Short-term prospects for validation (by end 2003) - depending on availability of human and financial resources 1) Acute oral toxicity (in vitro – [partial] replacement) 2) Skin corrosion (in silico – partial replacement) 3) Eye irritation (in silico – partial replacement) 4) Skin sensitisation (in silico – partial replacement)

AVAILABILITY OF ALTERNATIVE METHODS (2) Medium-term prospects for validation (by end 2006) - depending on progress and availability of human and financial resources 1) Eye irritation (in vitro – [partial] replacement) 2) Skin irritation (in vitro – [partial] replacement; in silico – partial replacement) 3) Skin sensitisation (in vitro – [partial] replacement) 4) Acute oral toxicity (in silico – partial replacement) 5) Genotoxicity (in silico – partial replacement) Long-term (by end 2010; [partial] replacement ) - depending on progress and availability of human and financial resources 1) Respiratory sensitisation 2) Biokinetics 3) Target organ / system toxicity, including endocrine disruption 4) Chronic toxicity 5) Non-genotoxic carcinogencity 6) Reproductive toxicity

FOLLOW-UP TO THE ECVAM REPORT: IN SILICO METHODS A quantitative structure-activity relationship (QSAR) is a mathematical relationship between a quantitative measure of chemical structure, or a quantitative measure of a physicochemical property, and a biological activity ECVAM is currently planning an initiative on the Development, Validation and Dissemination of QSAR Models, in collaboration with the European Chemicals Bureau (also part of the IHCP, JRC Ispra) This will involve: a)the establishment of a network of experts in the EU Member States, Candidate Countries, the USA, Japan b)coordination with future OECD activities on QSARs Special session on QSARs at 34th Joint Meeting, 6 November 2002

FOLLOW-UP TO THE ECVAM REPORT: IN VITRO METHODS A coordinated action plan is needed within the European Research Area, to ensure the targeted development of new test methods and their subsequent validation The development challenge ECVAM will contribute by: 1)advising DG Research on priorities for the development of alternative tests relevant to the Chemicals policy 2)establishing a dialogue with industry and academic institutions involved in test development The validation challenge An enormous task - ECVAM and other validation authorities in the EU Member States will need to share the burden of validation work ECVAM is best placed to coordinate the overall validation process