MAMMALIAN-CELL GENE MUTATION ASSAYS WORKING GROUP-REPORT

As part of the International Workshop on Standardization of Genotoxici ty Test Procedures, in Melbourne, 27-28 February 1993, various interna tional guidelines were examined with respect to protocol issues in the area of mammalian cell gene mutation assays. The working group on mam malian cell gene mutation assays discussed a wide range of protocol is sues related to study design; in most cases the recommendations are re asonably consistent with existing guidelines. Agreement was reached on several issues as follows. The upper limit of concentration for testi ng non-toxic substances should be 10 mM or 5 mg/ml, whichever is lower . For testing toxic substances the criteria of an acceptable upper lim it of concentration should yield 10-20% survival. Any of several estab lished mammalian cell mutation assays (L5178Y TK+/-, CHO/HPRT, AS52/XP RT, V79/HPRT) can be used to evaluate mutagenesis in mammalian cells; the ouabain (Na/K-ATPase) system is not an acceptable mutation assay f or routine evaluation of mutagenesis in mammalian cells. Ability to re cover small colonies must be convincingly demonstrated when using the L5178Y TK+/- mouse lymphoma assay. In the mouse lymphoma assay (L5278Y TK+/-), colonies in positive controls and at least two (if available) representative positive doses of the test compound should be sized if a positive response is seen; in the event of a negative response due to the test compound, colony sizing of the positive control is necessa ry to validate the conduct of the assay. Testing both in the presence and absence of S9 metabolic activation is necessary. It was not possib le to come to a firm conclusion about the length of treatment. There w as a general agreement that extended treatment times (>2 cell cycles) often bear more disadvantages than advantages and should only be used with adequate justification. It is not necessary to repeat clear posit ive or clear negative tests when the assay has been adequately perform ed; this recommendation differs significantly from the UK guidelines. If treatment groups are not replicated, the numbers of doses tested sh ould be increased; this recommendation differs significantly from the UK guidelines. Each laboratory should establish a historical database for the performance of a given assay in that laboratory.