Abstracts of the 43rd Annual Meeting of the European Environmental Mutagen Society Lancaster University UK 6–10 July 2014

Mutagenesis vol. 29 no. 6 pp. 497–559 doi:10.1093/mutage/geu053 Abstracts of the 43rd Annual Meeting of the European Environmental Mutagen Society Lancaster University UK 6–10 July 2014 Sunday CEP, Comet Assay: Talk 1/5 Sunday CEP, Comet Assay: Talk 3/5 How to perform a high quality in vivo comet assay that is acceptable to the regulators? Ulla Plappert-Helbig Novartis Institutes for BioMedical Research, Basel, Swizerland Combination and Integration of the Comet assay in vivo: Experiences with repeated dosing of methyl methanesulfonate, temozolomide and dibenzopyrene Melanie Guerard, Juergen Funk, Andreas Zeller Pharma Research and Early Development, Basel, Switzerland The revised ICH S2 ‘Guideline for genotoxicity testing and data interpretation for medicines intended for human use’ recommends the in vivo comet assay as a second in vivo genotoxicity endpoint. Furthermore the guideline includes also the new option in the genetic toxicology testing strategy ‘battery without in vitro mammalian cell assay but two in vivo endpoints.’ The combined micronucleus/comet assay test fits perfectly into that new strategy. Therefore, the in vivo rodent alkaline Comet assay has increasingly been used for regulatory genotoxicity testing in recent years. The assay has now been validated formally with a standardized study protocol in the international validation study led by the Japanese Center for the Validation of Alternative Methods (JaCVAM). The result of the validation study was submitted to the OECD for the establishment of the OECD test guideline. The experience from the international JaCVAM validation study as well as further analysis and discussions in the meetings of the IWGT (International Workshop on Genotoxicity Testing) revealed that a crucial part is the proper experimental performance of the assay. Examples will be shown how experimental conditions can influence data variability and assay sensitivity. Recommendation will be given how positive and vehicle control data as well as historical control data should be used to show the validity of the assay and the proficiency of the laboratory. Furthermore the influence of experimental design, cytotoxicity and selection of organs on data interpretation and the study conclusion will be discussed. Sunday CEP, Comet Assay: Talk 2/5 Interpretation of comet data - what does it all mean? Carol Beevers Covance Laboratories Ltd, UK The alkaline comet assay is frequently described as a simple and quick technique for examining genotoxicity effects in a wide range of animal tissues. However, interpretation of the results may not always be so straight forward and a simple and quick approach to data evaluation could lead to an incorrect conclusion. DNA strand breaks are the endpoint measurement in the assay but are not formed solely by direct chemical interaction with the DNA. A variety of other mechanisms such as indirect secondary effects following target tissue toxicity, oxidative stress, pharmacological activity of the test chemical and mechanical shearing during tissue processing can produce comets identical in appearance to those formed following genotoxic damage. Interpretation of all comet data should include an assessment of other supportive information and may trigger further experimentation in order to reach a final conclusion on the genotoxicity of the test compound. The comet assay is frequently used as a second in vivo endpoint to determine the genotoxic potential of new drug candidates. Due to a testing design comparable with the micronucleus test (MNT) in vivo, both assays can be combined within one experiment. While the MNT is often integrated in repeat dose toxicology studies, a survey among pharmaceutical companies demonstrated that this is not regularly done for the comet assay. The main reasons for not combining or integrating theassay are; the complexity of tissue sampling during necropsy, the risk of cytotoxicity impacting the comet read-out after repeated dosing and the potential impact of an additional administration required before necropsy on other toxicological endpoints. By using different genotoxic test compounds, i.e. methyl methanesulfonate, temozolomide and dibenzopyrene, we assessed whether an integration of the comet assay in a repeat dose study is feasible. We used a Pig-a assay with a dosing period of 5 to 28 days and a treatment free period of 28 days. In addition, MNT in bone marrow and yH2AX staining as well as histopathological examinations in liver were performed. In order to assess DNA damage by the comet assay, animals were also treated following the recovery period over 2 or 3 days, just as in an acute stand-alone comet experiment. The data obtained demonstrated that the comet assay can be combined with a variety of genotoxicity endpoints and even in a repeat-dosing regimen, without compromising the comet read-out. The results obtained therefore are in favor for combining the comet assay with additional genotoxicity endpoints and further its integration into repeat dose experiments. Further data is needed as during our exploratory studies, a considerably lower amount of tissue samples was obtained compared to a regulatory repeat dose toxicology study. Sunday CEP, Comet Assay: Talk 4/5 Results of the EFPIA survey on the in vivo comet assay Bas-jan van der Leede Janssen Research & Development, Belgium In the 2012 annual meeting of the EFPIA Preclinical Development Committee (PDC) and the EMA Safety Working Party regulators, one of the European regulatory agencies mentioned the perception that a number of pharmaceutical companies had been encountering difficulties with the interpretation of some findings in the in vivo comet assay. On request of the EMA Safety Working Party, the PDC organized a survey to investigate among the European representatives of pharmaceutical companies their experience with the in vivo comet assay focused on issues/problems © The Author 2014. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: . 497 Abstracts with interpretation of study results and regulatory acceptance of 2 negative in vivo (comet and micronucleus assay) to mitigate a positive in vitro mammalian cell assay following the current ICH S2 guidance. A group of comet assay experts was identified by the PCD to collect and analyze the survey responses, to review the studies with less interpretable findings in more detail, and to report the survey results. The survey reports on the incidence of studies with positive, negative, equivocal or inconclusive results, the rationale for conducting the studies, further exploration of the equivocal studies (i.e. not clearly interpretable as positive or negative or leading to debate with the regulatory authorities) and the studies showing positive results, and the regulatory acceptance of the assay. More than 140 studies have been conducted (in-house or outsourced) by the (...truncated)