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

The Working Group (WG) dealt with the harmonisation of routine methodo logies of tests for unscheduled DNA synthesis (UDS) both in vitro and in vivo. In contrast to existing guidelines from OECD, EPA and EC on i n vitro UDS tests (there is no Japanese UDS guidline), the Working Gro up recommends that in general in vitro UDS tests should be performed w ith primary hepatocytes. For routine applications any other cell types would need special justification. Hepatocytes from male rats are pref eable, unless there are contra-indications on the basis of e.g. toxico kinetic data. According to the OECD, EPA and EC guidelines, UDS may be analysed by means of autoradiography (AR) or liquid scintillation cou nting (LSC). The WG recommends use of AR. LSC is less suitable due to the problem of differentiation between UDS activity and replicative DN A synthesis, and the disadvantage that cells cannot be analysed indivi dually. Since a specific cell type was recommended by the WG, methodol ogical aspects could be described in more detail than in the present g uidelines. For in vitro tests, it was agreed that the initial viabilit y of freshly isolated hepatocytes should be at least 70%. With regard to the need for confirmatory experiments in the event of a clear-cut n egative result, the majority view was that confirmation by a second (n ormally not identical) experiment is still needed; this is in line wit h the present OECD and EC guidelines. Evaluation of results from UDS t ests should be based primarily on net nuclear grain (NNG) values, alth ough 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 ca n be recommended for discrimination of positive and negative UDS resul ts. For in vitro assays, the criteria for positive findings go beyond those of the present guidelines and two alternative approaches are giv en which are based on (1) dose-dependent increases in NNG values and ( 2) reproducibility, dose-effect relationship and cytotoxicity. At pres ent there is no official guideline on the performance of in vivo UDS t ests. 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 recom mended. However, concerning the requirement to use one or two sexes, c onsistency with other in vive genotoxicity assays (e.g. the micronucle us 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 th ree animals per treatment group should be used. Evaluation of results should again be done on the basis of NNG values. The fundamental crite rion for a positive result is given by an increase of NNG values for a t least one experimental group. This NNG increase should be evaluated by consideration of (1) lab-specific historical controls or adequate s tatistics, or (2) interanimal variation, dose-effect relationship and cytotoxicity.