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The validation of alternative methods - ecvam

Objective

Specific Objectives

General
- publication of a comprehensive review on the use of alternative test methods and testing strategies in relation to the Future EU Chemicals Policy;
- organisation of a conference to review the current status of alternative methods and future prospects for their development, validation, acceptance and application;
- publication of a report on Good Cell Culture Practice;
- continuation of the ECVAM PECO project on the Three Rs and advanced testing methods and their validation;
- participation in the 4th World Congress on Alternatives and Animal Use in the Life Sciences, to be held in New Orleans, USA, in August 2002
- further establishment and integration of expertise in analytical chemistry and in molecular biology;
- completion of a number of major external study contracts.

Task 1: Validation
- publication of an ESAC statement on the scientific validity of the GM-CFU assay for acute neutropenia;
- publication of an ESAC statement on in vitro tests for percutaneous absorption;
- publication of an ESAC statement on acute systemic toxicity testing;
- publication of an ESAC statement on the target animal safety test (TEST);
- publication of an ESAC statement on physicochemical methods for the potency testing of recombinant follicle stimulating hormone ( rFSH );
- follow-up of the pre-validation study on skin irritation, initially with a preliminary blind trial with refined protocols, followed by a validation study;
- initiation of a pre/validation study on the neutral red release assay;
- initiation of a validation study on computer models for predicting pharmacotoxicological endpoints;
- initiation of a pre/validation study on biocompatibility testing of dental filling materials.

Task 2: Acute and Systemic Toxicity
- setting-up of a validation study with ICCVAM on the use of cytotoxicity tests to predict lethal toxic doses/concentrations in rodents and humans;
- establishment of a database on the in vitro cytotoxicity of chemicals;
- setting-up of a collaborative study on the ability of cytotoxicity tests to predict maximum tolerated doses of anti-cancer drugs in rodents and humans.

Task 3: Barriers, Nephrotoxicity and Chronic Testing
- further evaluation of cellular and molecular mechanisms involved in damage to the renal barrier;
- follow-up of a pre-validation study on the blood-brain barrier;
- initiation of studies on human intestinal cell models;
- evaluation of a cell culture medium supplement to replace the use of animal sera;
- refinement of miniaturised perfusion cell culture systems for long-term studies.

Task 4: Reproductive Toxicity and Cardiotoxicity
- establishment of genetically engineered embryonic stem cell clones for detecting damage to the neuronal and skeletal systems;
- establishment at ECVAM of commercially-available human embryonic stem cell lines;
- establishment of a cardiotoxicity assay based on cardiomyocytes derived from embryonic stem cell lines;
- development of tests based on differentiating mesenchymal stem cells and osteoblasts.

Task 5: Haematotoxicity and Anti-cancer Drugs
- development of a test based on erythroblastic progenitors;
- prevalidation of miniaturised clonogenic assays for studying delayed myelotoxicity;
- study of gene expression in cord blood -erived haematopoetic stem cells.

Task 6: Metabolism and Neurotoxicity
- initiation of prevalidation phase 3 of the SCA project on novel pyrogen tests based on the human fever reaction;
- production of a tier testing strategy for identifying metabolism-mediated toxicity;
- contributions to the development of a tier testing strategy for identifying neurotoxic compounds.

Task 7: Metal Toxicity and Carcinogenesis
- completion of a screening study on concurrent cytotoxicity and morphological transformation induced by 58 metal compounds in the BALB/3T3 cell line;
- standardisation of the SHE cell assay for the carcinogenic potential of selected metal compounds, and comparison of the two cell transformation assays;
- completion of screening of the cytotoxicity of 50 metal compounds in the HaCaT, LLC-PK1, MDCK, PC-12 (tet-off, tet-on), V79, Caco-2, Hep G2 and Hep G2 (+ hsp promoter) cell lines
- completion of the development of a screening test for metal-induced effects on the innate and adaptive immune capacities of human lymphocytes.

Task 8: Biologicals
- publication of a report on the Three Rs contributions of ECVAM in relation to biologicals;
- critical review of existing laboratory animal procedures in Ph. Eur. monographs;
- comments on new/revised Ph. Eur. monographs;
- publication of outcome of a study of the relevance of the target animal safety test for the quality control of veterinary immunobiological medicinal products;
- publication of outcome of a study on physicochemical methods for the potency testing of recombinant follicle stimulating hormone (rFSH)

Task 9: Human Studies, and Topical Toxicity
- publication of a report on identification of new endpoints for predicting skin irritation in vitro;
- participation in a project on skin irritation testing in human volunteers with non-invasive technology;
- follow-up of recommendations of a study on the use of reference standard compounds in the establishment of the relevance and reliability of in vitro tests for eye irritation;
- production of a review on the status of alternative methods for testing surfactants and surfactant-based products.

Task 10: Computer Modelling and Integrated Testing
- proposal of an integrated testing strategy for existing and new chemicals;
- development of user-friendly software for predicting target organ toxicity;
- further development of structure-activity relationships for toxicological endpoints.

Task 11: ECVAM Scientific Information Service (SIS)
- completion of evaluations on four topics before their entry into the SIS databases;
- creation of a new data sector on basal cytotoxicity testing (with Task 2);
- beginning of the development of SIS as a combined and interconnected system with complete online Internet availability;
- updating of the validation study database;
- following the second circulation of the draft Thesaurus on Advanced Alternative Methods (TAAM), to be concluded in December 2001, adaptation of the electronic version of the TAAM;
- development of an Internet version of the TAAM and its presentation for public comment and use;
- extension of the TAAM to topics other than non-animal toxicity testing, on which it is focused at present.
Planned Deliverables

It is vital that a strong support is got by JRC services as well as a strengthened collaboration is set up with the DGs with which ECVAM works.

Summary of deliverables made by: 31/12/2001
General
- publication of ECVAM workshop report 44, on the establishment of human research tissue banking (ATLA 29, 125-134, 2001)
- acceptance by the Group of Experts 15V of the European Pharmacopoeia of the ToBI and ELISA for the batch potency testing of veterinary tetanus vaccines;
- publication of a review on the importance of the prediction model in validation (ATLA 29, 135-143, 2001);
- publication of a review on the role of ECVAM in promoting the regulatory acceptance of alternative methods in the European Union (ATLA 29, 2001, in press).

Task 1: Validation
- publication of ESAC statements on the scientific validity of three in vitro tests for embryotoxicity.

Task 2: Acute and Systemic Toxicity
- The Task Leader did not take up her appointment until July 2001.

Task 3: Barriers, Nephrotoxicity and Chronic Testing
- completion of study on in vitro models of the blood-brain barrier;
- publication of ECVAM workshop report 45, on long-term testing in vitro (ATLA 29, 393-426, 2001);
- publication of ECVAM workshop report on in vitro models of the intestinal barrier (ATLA 29, 2001, in press).

Task 4: Reproductive Toxicity and Cardiotoxicity
- the Task Leader for this task took up her appointment in May 2001;
- establishment of a reporter gene assay for developmental cardiac toxicity.

Task 5: Haematotoxicity and Anti-cancer Drugs
- optimisation of protocols for human BFU-E and CFU-E assays;
- establishment of a new cell line (LA) from CD40L transgenic mice;
- development of cytofluorometric assays for the induction of apoptosis and the modification of telomerase activity in human bone-marrow and leukaemic cells;
- characterisation of various cell lines for protein expression in relation to key haematopoietic stem cell genes.

Task 6: Metabolism and Neurotoxicity
- development of a test system employing polymorphic forms of human cytochrome P450 enzymes in genetically engineered cell lines, for studying the effects of human polymorphism on metabolism-mediated toxicity;
- publication of ECVAM workshop report 43, on novel pyrogen tests based on the human fever reaction (ATLA 29, 99-123, 2001).

Task 7: Metal Toxicity and Carcinogenesis
- completion of the protocol for the BALB/3T3 mouse fibroblast assay, and its further development for predicting the cytotoxicity and carcinogenic potential of a wide range of metal compounds
- development of HaCaT, LLC-PK1, MDCK, PC12 (tet-off, tet-on), V79 and CaCo-2 Hep G2 and Hep G2-containing hsp 70 promoter cell lines for screening studies on the basal cytotoxicity of metal compounds.

Task 8: Biologicals
- the Task Leader for this task took up her appointment in May 2001
- completion of evaluation of the relevance of the target animal safety test (TAST)
- comment to European Pharmacopoeia on the draft revision of the monograph on veterinary vaccines.

Task 9: Human Studies and Topical Toxicity
- publication of a review on the neutral red release method (ATLA 29, 2001, in press).

Task 10: Computer Modelling and Integrated Testing
- the Task Leader for this task took up his appointment in May 2001; because of his previous work at ECVAM, he was one of three winners of the JRC Young Scientist Award for 2001;
- development of proposals on the use of bootstrap resampling.

Task 11: ECVAM Scientific Information Service (SIS)
- continuation of production of SIS database entries on hepatotoxicity, metabolism-mediated toxicity, percutaneous absorption and neurotoxicity;
- completion of the initial Internet version of SIS, containing selected databases;
- completion of the draft version of the Thesaurus on Advanced Alternative Methods (TAAM) in consultation with the Head of the Thesaurus Section of the US National Library of Medicine, and its circulation to experts for comment.

Output Indicators and Impact
% Actual impact will depend on DGs application and will be discussed with them in the coming future.
Summary of the project

ECVAM will play a leading role at the European level in the independent evaluation of the relevance and reliability of alternative (non-animal) toxicity and safety tests and testing strategies for specific purposes, through research on advanced methods, new test development and validation, and the operation of specialised databases.
See also the related EU enlargement process pre-accession (pa) project.

Rationale

Improvement of the regulatory process is necessary, so that chemicals and products of various kinds, including medicines, vaccines, 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.

Funding Scheme

JRC - Joint Research Centre research

Coordinator

Institute for Health and Consumer Protection
Address

Ispra
Italy