The conditions under which noscapine causes high levels of polyploidy in vitro in human lymphocytes were investigated to try to determine its mode of action and to assess whether it was likely to be a genotoxic hazard when used as an antitussive agent. Irrespective of duration of treatment or type of medium, there seemed to be a threshold for polyploidy induction between 15.0 and 30.0 micrograms/ml and a maximum between 100.0 and 150.0 micrograms/ml noscapine. High levels (10.0-20.0%) of noscapine-induced polyploidy were never found with 4 h treatments or with RPMI 1640 medium plus 15% (v/v) foetal calf serum; the use of Iscove's modified Dulbecco's medium and 24 h treatments were needed. The reasons for these observations seemed to be the faster cell division and greater sensitivity of cells grown in Iscove's medium. There was conflicting evidence about the mechanism of polyploidy induction by noscapine; either spindle damage or cell fusion remain as possibilities. The need for prolonged exposure and the precise nutritional requirements suggest that a short exposure, albeit at high concentration, in the upper gastro-intestinal tract is unlikely to be a hazard for humans. Furthermore, evidence of a threshold at approximately 20 micrograms/ml plus the virtual elimination of noscapine-induced polyploidy by microsomal metabolism (S9 mix) together with published metabolic data imply that the low-level systemic exposure after absorption may well not be hazardous. We conclude that the use of noscapine in cough mixtures does not pose a significant potential hazard for humans.

The importance of polyploidy as a genotoxic lesion is uncertain and there have been few publications and no reviews which have included data on spontaneous or induced polyploidy in routine genotoxicity screening. We have attempted to clarify some of the issues by reviewing the published literature and by reference to our historical data base for metaphase analysis of cultured human lymphocytes. In our studies on pharmaceutical compounds polyploidy was the lesion most often found, being induced by approximately 40% of the compounds tested. The mean spontaneous frequency was between 0.1 and 0.3%, and values for polyploidy induction were 5-fold to > 100-fold the spontaneous value. Spontaneous polyploids tended to be near-exact multiples of the haploid chromosome number whereas induced 'polyploids' were, in fact, very heteroploid with a wide range of chromosome numbers. Polyploidy induction often occurred at non-toxic concentrations, usually there were well defined no-effect (threshold) levels and it was unrelated to other genetic effects. Such observations would be expected for inducers of polyploidy because the target molecules are not DNA and for these non-DNA targets there is usually a degree of redundancy. Therefore, inducers of polyploidy are only likely to be a hazard for humans if they are positive at or below therapeutic concentrations. We conclude that polyploidy/near-polyploidy (shown as 'polyploidy' throughout) should be scored as accessory data which becomes important only when induction occurs at therapeutic levels.

The definition of a negative result is a problem in genetic toxicology. Here we suggest that a result may be considered biologically unimportant (negative) if it falls within the limits of variation usually found in the negative controls of the particular test. To determine 'usual' variation, we have set 95% confidence limits on three indices of variation, calculated from historical values for duplicate negative control data from several genotoxicity tests. These tests showed four characteristic types of response and the appropriate index of variability was determined for each. Where there was little test-to-test variation in true mean (micronucleus test and metaphase analysis), confidence limits set on the overall distribution of negative controls were the best index of variability. In other assays (Ames, yeast and mouse lymphoma), there was considerable test-to-test variation so that differences between, or ratios of, the members of control duplicates were the preferred measure of variability. This approach can define what is biologically unimportant in terms of the test system. However, no inference can be drawn as to potential importance. Thus the main use is the removal of the positive 'label' from statistically significant results which fall within the usual range of spontaneous variation for the assay under consideration.

The optimum concentrations of Aroclor-induced rat liver S9 microsomal fraction for the mutagenic activity of the four standard mutagens 2-aminofluorene (2-AF), acriflavine (ACR), benzo[a]pyrene (BP) and cyclophosphamide (CP) were determined in four mutation assays. The four assays were the Ames test using Salmonella typhimurium strain TA100, cycloheximide resistance in the yeast Saccharomyces cerevisiae, the mouse lymphoma TK assay and the human peripheral lymphocyte cytogenetic assay. BP was the only mutagen to be most active at comparable S9 concentrations, of approximately 1%, for all four assays. The optimum S9 concentrations for each of the remaining three mutagens varied substantially between the four assays. ACR was a potent direct-acting mutagen in both mammalian cell assays. The mouse lymphoma TK assay results showed similar optimal values of 1.5% S9 or below for each of the four test agents. The assay with the largest variation of optimal S9 values for the four mutagens was the Ames test in strain TA100, although it also had the widest peaks of activity over the range of S9 concentrations tested. It is likely that the diversity of findings is due to a variety of metabolites affecting the different genetic endpoints that are measured in these assays. Thus from these results it is not possible for bacterial optimization data to be related to other routine in vitro systems. The use of more than one concentration of S9 would contribute useful information.

Micronucleus tests are generally analysed statistically for differences between the means of treated and control groups. 'Outliers' may either be rejected or grouped together with data from less responsive animals. In either case, a valuable indicator of a small, more sensitive (responder) population sub-group may then be missed. To alleviate this problem, we have developed an additional strategy, based on historic data, for the detection of any single animal with a significant increase in micronucleated polychromatic erythrocytes in an otherwise insignificant treatment group. Forty-one sets of negative control data (of five male and five female CD-1 mice each) have been analysed. Within each set there were no significant male to female differences and data were consistent with a Poisson distribution. Pooled data from all 41 sets showed slightly hyper-Poisson variation and were adequately described by the negative binomial distribution. The negative binomial probability generating function was used to show that six or more micronuclei per 1000 polychromatic cells from one treated animal would be significant for our laboratory, methodology and strain of mouse, provided that concurrent negative control data conformed with historic values. Changes in methodology desirable for this type of analysis include increasing the number of mice in each test group and possible compensation by a reduction in the number of test groups.