Reduction of use of animals in regulatory genotoxicity testing: Identification and implementation opportunities-Report from an ECVAM workshop.

In vivo genetic toxicology tests measure direct DNA damage or the formation of gene or chromosomal mutations, and are used to predict the mutagenic and carcinogenic potential of compounds for regulatory purposes and/or to follow-up positive results from in vitro testing. These tests are widely used and consume large numbers of animals, with a foreseeable marked increase as a result of the EU chemicals legislation (REACH), which may require follow-up of any positive outcome in the in vitro standard battery with appropriate in vivo tests, regardless of the tonnage level of the chemical. A 2-day workshop with genotoxicity experts from academia, regulatory agencies and industry was hosted by the European Centre for the Validation of Alternative Methods (ECVAM) in Ranco, Italy from 24 to 25 June 2008. The objectives of the workshop were to discuss how to reduce the number of animals in standard genotoxicity tests, whether the application of smarter test strategies can lead to lower animal numbers, and how the possibilities for reduction can be promoted and implemented. The workshop agreed that there are many reduction options available that are scientifically credible and therefore ready for use. Most of these are compliant with regulatory guidelines, i.e. the use of one sex only, one administration and two sampling times versus two or three administrations and one sampling time for micronucleus (MN), chromosomal aberration (CA) and Comet assays; and the integration of the MN endpoint into repeat-dose toxicity studies. The omission of a concurrent positive control in routine CA and MN tests has been proven to be scientifically acceptable, although the OECD guidelines still require this; also the combination of acute MN and Comet assay studies are compliant with guidelines, except for sampling times. Based on the data presented at the workshop, the participants concluded that these options have not been sufficiently utilized to date. Key factors for this seem to be the uncertainty regarding regulatory compliance/acceptance, lack of awareness, and an in many cases unjustified uncertainty regarding the scientific acceptance of reduction options. The workshop therefore encourages the use and promotion of these options as well as the dissemination of data related to reduction opportunities by the scientific community in order to boost the acceptance level of these approaches. Furthermore, experimental proof is needed and under way to demonstrate the credibility of additional options for reduction of the number of animals, such as the integration of the Comet assay into repeat-dose toxicity studies.

Follow-up testing of rodent carcinogens not positive in the standard genotoxicity testing battery: IWGT workgroup report.

At the Plymouth Third International Workshop on Genotoxicity Testing in June 2002, a new expert group started a working process to provide guidance on a common strategy for genotoxicity testing beyond the current standard battery. The group identified amongst others "Follow-up testing of tumorigenic agents not positive in the standard genotoxicity test battery" as one subject for further consideration [L. Müller, D. Blakey, K.L. Dearfield, S. Galloway, P. Guzzie, M. Hayashi, P. Kasper, D. Kirkland, J.T. MacGregor, J.M. Parry, L. Schechtman, A. Smith, N. Tanaka, D. Tweats, H. Yamasaki, Strategy for genotoxicity testing and stratification of genotoxicity test results-report on initial activities of the IWGT Expert Group, Mutat. Res. 540 (2003) 177-181]. A workgroup devoted to this topic was formed and met on September 9-10, 2005, in San Francisco. This workgroup was devoted to the discussion of when it would be appropriate to conduct additional genetic toxicology studies, as well as what type of studies, if the initial standard battery of tests was negative, but tumor formation was observed in the rodent carcinogenicity assessment. The important role of the standard genetic toxicology testing to determine the mode of action (MOA) for carcinogenesis (genotoxic versus non-genotoxic) was discussed, but the limitations of the standard testing were also reviewed. The workgroup also acknowledged that the entire toxicological profile (e.g. structure-activity relationships, the nature of the tumor finding and metabolic profiles) of a compound needed to be taken into consideration before the conduct of any additional testing. As part of the meeting, case studies were discussed to understand the practical application of additional testing as well as to form a decision tree. Finally, suitable additional genetic toxicology assays to help determine the carcinogenic MOA or establish a weight of evidence (WOE) argument were discussed and formulated into a decision tree.