The Validation of Cell and Tissue Based Assays

The validation of cell- and tissue-based tests is the process by which the reliability and relevance of a test is established for a particular purpose [31]. Over the past 12 years, the European Centre for the Validation of Alternative Methods (ECVAM) has set up guidelines for validation, in cooperation with international experts. These guidelines have identified five stages in the evolution of new tests, namely development, prevalidation, validation, peer-review, and regulatory acceptance. These stages reflect the sequence of activities to be performed for a prospective validation exercise and are documented in detail in several publications [32-35]. This validation process has proved effective in establishing regulatory acceptance of several alternative tests [36]. Furthermore, these principles are generic and can be applied to any type of test, including in-vivo tests.

A typical validation programme lasts three years, and the subsequent regulatory implementation from two to seven years. At the time of writing, the cost for validation of a single test is approximately 300 000 Euro. The primary constraints in the validation process are the availability of reference substances and animal test data, the need for further optimization of test methods, the duration of financial/administrative procedures, and the ongoing consensus process of regulatory implementation.

Nine alternative methods for chemicals/cosmetics have been endorsed by ECVAM, including tests for skin corrosivity, skin sensitization, phototoxicity and embryotoxicity, as well as the percutaneous absorption of chemicals. In addition, seven methods reached scientific acceptance for the safety evaluation ofbiologicals such as vaccines. Currently, nine alternative methods for myelotoxicity, pyrogen testing and chronic toxicity in dogs are undergoing peer-review by the ECVAM Scientific Advisory Committee.

A number of the methods cited above have been accepted by both the European Commission (Annex V to Council Directive on dangerous substances 67/548/ EEC), as well as by the Test Guideline Programme ofthe Organisation ofEconomic Cooperation and Development (OECD, press release JRC April 2004), and three potency tests of biologicals have been accepted by the European Directorate for the Quality of Medicines (European Pharmacopoeia).

A number of organizations are engaged primarily in the validation of alternative methods to the use of animals. These include ECVAM, The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and specific actions by OECD. In the future, the recently established Japanese Centre for Evaluation ofAlternative Methods (JaCVAM) will hopefully also play a valuable role in this area. ECVAM operates as a co-coordinator of international validation studies, a focal point of information, a central database on alternative methods, a center of public dialogue, and a pre-normative research facility of the European Commission's Joint Research Centre. Participation in validation studies requires a running infrastructure, while active research maintains a practical and realistic view on science and technology. Furthermore, high-quality research ensures credibility in the scientific community.

An alternative, but complimentary, approach is used in establishing standards for the assay of biological medicines such as vaccines, insulin, growth factors, and monoclonal antibodies [37, 38]. In this model, batches of different control materials are prepared and distributed to a range of expert laboratories using a range of different assay methods. A nominal value for biological activity is assigned to the distributed materials, which are characterized in great detail through international collaborative studies in which different techniques and assays are used [39]. A number of organizations are engaged in standardization and the provision of reference materials (e.g., Institute for Reference Materials and Methods (Belgium), National Institute for Biological Standards and Control (UK), National Institute for Standards and Testing (USA), national pharmacopoeia institutes, WHO Expert Committee on Biological Standardisation). For more information on biological reference materials, see www.nibsc.ac.uk or http://www. who.int/bloodproducts/ref_materials/en/.

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