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SUMMARY

by xfabre last modified 27-03-2007 22:39
COMET ASSAY and CELL ARRAY for genotoxicity testing - non-animal alternative for REACH

PROJECT OVERVIEW


The methods to be developed will provide reliable in vitro assays for use in screening chemicals for potential genotoxic and cytotoxic effects, and will thus help to limit the amount of animal experimentation that needs to be carried out - notably, in connection with the new EC policy on the Registration, Evaluation and Authorisation of Chemicals (REACH). The methods will be subjected to rigorous testing in order to meet international validation standards and to be accepted by industrial users and regulatory authorities.



OBJECTIVES


  • To increase the throughput of the comet assay up to 20-fold, using multi-well format and ‘cell arrays’.
  • To develop further the cell array system as a parallel assay for cytotoxicity.
  • To seek optimal cell types for use in genotoxicity and cytotoxicity testing (e.g. metabolically active HepaRG cells).
  • To increase the speed of scoring of comets, by developing an alternative method based on differential fluorescence of DNA in heads and tails of comets, so that scoring is no longer a serious bottleneck.
  • To use lesion-specific enzymes and inhibitors to measure different kinds of DNA damage – oxidised and alkylated bases, UV-induced damage and bulky adducts.
  • To develop and compare methods for measuring DNA repair activity.
  • To develop an approach which combines fluorescent in situ hybridisation with the comet assay, allowing measurement of gene-specific DNA damage and repair.
  • To validate the comet assay in its various forms, assessing reproducibility and robustness, comparing results obtained with the same test system and the same chemical damaging agents in different laboratories.
  • To develop reference and internal standards for use in the comet assay.
  • To make the various innovative products available for use by companies and researchers investigating DNA damage and repair.


PROJECT ABSTRACT


A battery of reliable and validated in vitro assays is needed to test for genotoxic and cytotoxic effects of chemicals (as in the REACH programme) without resorting to animal experiments. The comet assay, a sensitive indicator of DNA damage, will be combined in this project with the Cell Array system, to establish and validate high capacity assays suitable for chemical testing. Up to 800 cell samples will be processed for comets on a single microscope slide. Arrays will use cells with different metabolic capabilities, and data on cytotoxicity will be obtained in parallel with DNA damage. A medium-throughput assay will also be developed. Comet analysis by differential staining of damaged/undamaged DNA using established and novel dyes combined with automated image analysis will be faster and more reliable than at present. A crucial aspect of the cellular response to DNA damage is DNA repair; variations between people can affect cancer risk. Genotoxic chemicals can act by interfering with repair. Two methods for measuring repair, one based on the comet assay, the other using a ‘Repair Chip’ approach, will be compared. In addition, fluorescent probes which lock onto the DNA after hybridization (‘padlock probes’) will be applied to study gene-specific DNA repair. The modified comet assay methods will be assessed for reproducibility and sensitivity in an inter-laboratory prevalidation trial, using a coded set of standard chemicals, to satisfy regulatory bodies as well as industrial users of the technology. The result will be robust, validated, high-throughput in vitro genotoxicity tests. This proposal brings together academic partners and SMEs, together with a large industrial concern, and the European Centre for the Validation of Alternative Methods (ECVAM). Dissemination through professional and trade publications, regulatory channels, and scientific conferences will lead to widespread adoption of the new methods.

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