RECOMMENDATIONS FOR THE PERFORMANCE OF UDS TESTS IN-VITRO AND IN-VIVO

The Working Group (WG) dealt with the harmonisation of routine methodologies of tests for unscheduled DNA synthesis (UDS) both in vitro and in vivo. In contrast to existing guidelines from OECD, EPA and EC on in vitro UDS tests (there is no Japanese UDS guidline), the Working Group recommends that in general in vitro UDS tests should be performed with primary hepatocytes. For routine applications any other cell types would need special justification. Hepatocytes from male rats are prefeable, unless there are contra-indications on the basis of e.g. toxicokinetic data. According to the OECD, EPA and EC guidelines, UDS may be analysed by means of autoradiography (AR) or liquid scintillation counting (LSC). The WG recommends use of AR. LSC is less suitable due to the problem of differentiation between UDS activity and replicative DNA synthesis, and the disadvantage that cells cannot be analysed individually. Since a specific cell type was recommended by the WG, methodological aspects could be described in more detail than in the present guidelines. For in vitro tests, it was agreed that the initial viability of freshly isolated hepatocytes should be at least 70%. With regard to the need for confirmatory experiments in the event of a clear-cut negative result, the majority view was that confirmation by a second (normally not identical) experiment is still needed; this is in line with the present OECD and EC guidelines. Evaluation of results from UDS tests should be based primarily on net nuclear grain (NNG) values, although it is recognised that nuclear and cytoplasmic grains result from different biological processes. Since grain counts are influenced by a number of methodological parameters, no global threshold NNG value can be recommended for discrimination of positive and negative UDS results. For in vitro assays, the criteria for positive findings go beyond those of the present guidelines and two alternative approaches are given which are based on (1) dose-dependent increases in NNG values and (2) reproducibility, dose-effect relationship and cytotoxicity. At present there is no official guideline on the performance of in vivo UDS tests. Some fundamental recommendations given for in vitro methodology also apply to the in vivo assay. For routine testing with the in vivo UDS test, again the general use of hepatocytes from male rats is recommended. However, concerning the requirement to use one or two sexes, consistency with other in vive genotoxicity assays (e.g. the micronucleus assay) would be preferable. As for the in vitro methodology, AR is preferred rather than LSC. For in vivo UDS tests, a minimum viability of 50% is considered to be sufficient. Sampling of cells 12-16 h after treatment and, if this is negative, 2-4 h is recommended. At least three animals per treatment group should be used. Evaluation of results should again be done on the basis of NNG values. The fundamental criterion for a positive result is given by an increase of NNG values for at least one experimental group. This NNG increase should be evaluated by consideration of (1) lab-specific historical controls or adequate statistics, or (2) interanimal variation, dose-effect relationship and cytotoxicity.