THE MUTAGENICITY BY THE BACTERIAL TEST OF SMOKE CONDENSATES FRO,
AGRICULTURE CANADI.- PrLkSE I CICAU-TTES
M. H. BILIMORIA
BACKGROUND
The mutagenicity testing by the Ames procedure of fifteen samples
of cigarettes made for Agriculture Canada Tobacco and Health Study
(Experiment No. 40), has now been completed. These cigarettes were of
interest to us not only because they have been examined by several'short-
term tests, but also because they include a wide variety of tobacco
materials produced under controlled conditions, for which detailed
chemical analyses are available. Included in this report are the NMF
determinations carried out at Imperial Tobacco Ltd. , Montreal, the
mutagenesis tests carried out by Dr. (','II P.1". Easrur at Guelph
University and the sebaceous &land tests done at Hamburg, Germany.
The mutagenesis assay was carried out according' to the procedures
described by Ames et al. (Mutation Res., 31, 347, 1973). The cigarettes
were tested for mutagenicity in groups of five, which always included
Player's Check 20 for intercomparison of samples, and each group was
tested six times. Dose levels of 50, 100, 150, 200 and 400 Ug condensate
per plate were tested in triplicate. Regression lines were computed for
the relationship between the number of revertant colonies and amount of
smoke condensate (dose response). Correlation coefficients and slopes
were calculated from these regression lines and the slopes were used to
express mugatenicities of the condensates.
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RESULTS
A brief description of the cigarettes employed in this study is pre-
sented in Table 1. As will be seen from the table, the cigarettes were
made from a variety of tobacco materials which were produccd under con-
trolled conditions. The mutagenicities of these cigarettes, expressed
in terms of these regression lines, are presented in Table 2. From the
correlation coefficients presented in Table 2, it will be seen that the
dose responses are linear. Thus, slopes of these dose response curves
can be used to calculate the mutagenicities of the condensates. Logarithmic
transformations of the data were also carried out, but the slopes of these
regression lines were not parallel as will be. seen from the results of the
analysis of variance (ANOVA) presented in Table 3 (p<0.001). Because tile
slopes of these regression lines were not parallel, the intercepts could
not be used to express the mutagenicities of the condensates.
Since these sixteen cigarettes were tested in four grcups, it was
necessary to find out if the data from these groups could be pooled for
statistical evaluation. Consequently, an ANOVA test was performed to
determine whether there were any differences in the mutagenicities of the
Player's Check cigarettes tested with each of the four groups. The results
of this ANOVA test, presented in Table 4, show that there was no significant
difference between these Player's cigarettes tested at four different
periods in time (p<9. 5).
Having shown that the results of the Player's cigarettes were similar
the data-from these-four groups of experiments were pooled, and an ANOVA
test was performed to find out if there were significant differences
between the mutagenicities of these sixteen cigarettes. From Table 5 it
will be seen that the mutagenicities (slopes) were indeed. significantly
different (P<0.001). A Student-hTewman-Keul test was performed to detect
differences between cigarettes, and these results are su=arized in Table
6. From this table it can readily be determined whether the difference in
mutagenic-ity between any two pairs of cigarettes is significant or not.
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From Table 7 it will be seen that homogenization of tobacco to make
PCL leads to a decrease in mutagenic activit7, which, with the exception
of PCL made from flue-cured whole plant leaf blend, is significant at the
5% level. The latter FCL, however, is significantly lower only at the
102 level. In this table are also given the differences between the flue-
cured tobaccos and burley.
In Table 8 a comparison is drawn between the mutagenicities as
determined at Imperial Tobacco and Guelph University (Dr. Basrur),
Sebaceous gland as done at B.A.T. Hamburg and M-IF, as done at I.T.L.
Montreal. The data in each test have been ranked and the rankings are shown
in brackets in the table. Correlation coefficients have been obtained for
pairs of data from regression lines as well as by the Spearman rank test.
These results are presented in Table 9. That the highest correlation was
obtained between the mutagenicity tests as done at 1. T. L. and Guelph, should
not be surprising. The correlation is, however, rather low probably because
different procedures were employed in the mutagenicity testing in these two
laboratories. Dr. Basrur in her study erploycd whole smoke, whereas at
Imperial, condensate was used, and whole smoke is likely to be more cyto-
toxic because of the presence of vapour phase components. The mutagenicity
test correlated significantly with the NMF and not at all with the Sebaceous
gland.
Finally, an attempt was also made to find out whether mutagenicity in
the Ames test is in any way related to the chemical composition of the
tobacco or the smoke condensate obtained therefrom. This statistical
treatment of the data is presented in Table 10. From this table, it will be
seen that a negative correlation appears to exist between mutagenicity and
the tar-nicotine ratio of smoke as well as the reducing sugar content of
tobacco. However, these correlations are significant only in the non-para-
metric Spearman test, and a larger sa=ple will have to be studied before con-
cluding that such correlations exist. On the other hand, mutagenicity is
significantly correlated with total and protein nitrogen.contetits of tobacco.
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Such a correlation with the nitrogenous content of tobacco has also been
observed in a Japanese study. Information such as this may be of value
to the tobacco industry.
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BATCo document for Province of British Columbia 15 April 1999