IWGT comet final2

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Published on October 30, 2007

Author: Nivedi

Source: authorstream.com

The Comet Assay Working Group:  The Comet Assay Working Group 4th International Workshop on Genotoxicity Testing San Francisco, CA September 10, 2005 Slide2:  Purpose: To enhance the ability of Comet assay test results to be used for regulatory decision making Primary Focus: The rodent alkaline (pH>13) Comet assay as a regulatory replacement/alternative for the in vivo unscheduled DNA Synthesis (UDS) assay Protocol Issues Validation Issues Establishing minimum reporting standards for Comet assay test results 4th IWGT Comet Assay Working Group Slide3:  In situations where evident toxicity is not present, can the limit dose only be tested in studies to evaluate the in vivo genotoxicity of a test substance? No! Downturns in response exist (bell-shaped dose-response curve) that are, for example, possibly due to altered bioavailability at higher dose levels Also, positive responses at multiple dose levels reinforce the biological relevance of the result. Rodent Alkaline (pH>13) Comet Assay: Multiple Dose Levels vs Limit Dose Slide4:  Does the method used to process tissues make a difference in the accuracy of the assay? Do we have enough data to decide? No! But we consider the issue unlikely to be a problem However, any international validation study would consider both processing methods for different tissues using reference chemicals with diverse mechanisms of action and covering a range of potencies Rodent Alkaline (pH>13) Comet Assay: Tissue Processing - Whole Cells vs Isolated Nuclei Rodent Alkaline (pH>13) Comet Assay: Do We Need to Include Measures of Cytotoxicity?:  Rodent Alkaline (pH>13) Comet Assay: Do We Need to Include Measures of Cytotoxicity? Possible methods include: Dye exclusion tests for membrane integrity (e.g., trypan blue) Dual dye tests for membrane integrity and metabolic competency Determining the frequency of comets that represent “dead“ cells Determining the frequency of cells with low molecular weight DNA Using histopathology in the event of positive findings Rodent Alkaline (pH>13) Comet Assay: Do We Need to Include Measures of Cytotoxicity?:  Rodent Alkaline (pH>13) Comet Assay: Do We Need to Include Measures of Cytotoxicity? Yes, measures of cytotoxicity need to be included! Studies should include histopathology to evaluate for necrosis and apoptosis when results are positive need to standardize ways to present histopathological findings The neutral diffusion assay can be used to identify the frequency of cells with low molecular weight DNA (i.e., dead cells) need to better define diffusion Can include membrane integrity dyes (e.g., trypan blue) if appropriate for the procedure used to isolate cells Slide7:  Image analysis (IA) or manual scoring IA is preferred but not required Scoring methodology (should all comets be scored?) Hedgehogs should be excluded from IA data collection; determining their frequency might be useful for data interpretation Which measure(s) of DNA migration can be used? For IA-based studies, tail moment, tail length, or % tail DNA can be used to evaluate for genotoxicity % tail DNA appears to be the most linearly related to dose and the easiest to compare across studies If tail moment is used, need to present tail length and % tail DNA data as well Data on the distribution of migration among cells should be presented Rodent Alkaline (pH>13) Comet Assay: Scoring - Measures of DNA Migration Slide8:  Need to develop and include historical negative/ positive control data The minimal number of studies needed is not defined but need enough studies to demonstrate stability of the extent of migration for negative/positive controls Criteria for determining the acceptability of new studies, based on historical control data, should be developed for each tissue by each lab The negative control should exhibit measurable DNA migration; a mean extent of migration corresponding to 10-20% tail DNA is useful if DNA crosslinking agents are to be detected, or the presence of crosslinks must be demonstrated using a second genotoxic agent Quality Control Issues Minimal Reporting Standards:  Minimal Reporting Standards To ensure that all studies can be independently evaluated, minimum reporting standards for regulatory submissions and publications will be developed that consistent with OECD in vivo genetic toxicology test method guidelines Comet Assay Validation (1):  Comet Assay Validation (1) Validation discussed briefly; the need is to: Establish an international “Management Team” Obtain funding, at least for chemical purchase and distribution Review current status of the rodent alkaline Comet assay (need to obtain raw data) Identify most appropriate protocol(s) Identify chemicals to test coded in order to compare Comet assay performance against UDS, MN, & carcinogenicity test results Identify participating labs (preferably GLP-compliant) Develop optimal statistical methods for evaluating validation data Comet Assay Validation (2):  Comet Assay Validation (2) Conduct phased/modular approach Phase 1 - generate historical negative/positive control data Phase 2 - test 3 coded substances to demonstrate cross lab performance (some labs may be excluded after this phase) Phase 3 - test x coded substances to demonstrate reproducibility within and across labs Phase 4 - test additional coded substances to demonstrate accuracy Data analyzed at each phase by the Management Team for lab performance and for assay relevance (accuracy) and reliability

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