Research Laboratory Report No. 164
Copy No.
IMPERIAL TOBACCO LIMITED
RESEARCH LABORATORY
MONTREAL
SUMMARY OF AMES TESTS
FOR MUTAGENICITY OF SMOKE CONDENSATES
CONDUCTED BY ITL, MONTREAL
Project No. T-7708
Authors:
M.H. Bilimoria
R.S. Wade
Issued by: R.S. Wade Date Issued: July 2, 1981.
Distribution:
Copy No. I. Library 18. Dr. P.J. Dunn
2. Mr. R.M. Gibb 19. Mr. M.P. Scherbak
3. Mr. R.S. Wade 20. Dr. G.W. Boswall
4. Mr. S.M. Candlish -21. Dr. A
Porter
5. Dr. T.A. Smith 22. Mr. .
L. Bouchard
6. Mr. E. Rittershaus 23. Mr. H. Roubicek
7. Dr. F. Seehofer 24. Miss R.R. Smith
8. Dr. C.J.P. De Siquejra 25. Mr. B.J. Stirling
9. Dr. L.C.F. Blackmw 26. Mr. R.L. Rice
10, Mr. A.L. Heard 27. Mr. A. Schaffer
11. Mr. A.L. Heard 28. Mr. E.P. Gage
12. Dr. I.W. Hughes 29. Mr. P. Leblond
13. Dr. R.A. Sanford 30. Mr. W. Tennyson
14. Dr. R.A. Sanford 31. Mr. W.J. Ross
15. Mr. R.G. Nicholls 32. Mr. C.J. Brown
16. Mr. R.G. Nicholls 33. Mr. C. Warren
17. Dr. M.H. Bilimoria
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SUMMARY OF AMES TESTS FOR MUTASEMICITY OF SMCKE
CONDENSATES CONDUCTED BY ITL, MONTREAL
Mutagenicity of Janus experimental cigarettes in
the Ames test and
comparison with carcinogenic activity.
Introduction
Traditional methods employing small rodents for
predicting carcinogen-
icity of chemicals and complex mixtures such as
tobacco smoke are both expensive
and time consuming, and neither resources nor
manpower are available to test
all the compounds that man is introducing into his
environment. For these
reasons considerable interest has existed in
finding short-term tests that
could reliably predict the carcinogenicity of
chemicals. Of the several
short-term tests that have recentTy been proposed
for detecting the mutagenic/
carcinogenic potential of chemicals, the one
developed by Professor Bruce
N. Ames appears to be the most reliable, and
consequently has gained world-
-aide acceptance. On account of the excellent
Correlation obtained between
Atnes bacterial mutaqenicity and rodent
carcinogenicity of pure compounds,
considerable interest has also developed in
studying the correlation between
mutagenicity and carcinogenicity of complex
mixtures s*uch as tobacco smoke
condensates.
Thus, we developed the capability of conducting
the Aines test on smoke
condensates in a quantitative manner, and decided
to measure the mutagenicities
of condensates from several series of experimental
Ciqarettes (Janus series)
produced by B.-A.T., Southampton. These cigarettes
were designed to study
several parameters thought to be important in
determining the carcinogenic
activity of cigarette smoke. Since B.-A.T. has
undertaken carcinogenic
studies on most of these cigarettes, they were
ideal candidates for a
comparative study of mutaaenicity and
carcinogenicity of cigarette smoke.
GR & DC have followed our studies closely and are
plannina a meeting in
july '81 to review these data as well as those
obtained on some of the same samples
oy B.-A.T. Hamburg using the Ames test and another
predictive short-term
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test utilising mammalian cell cultures. The
followinq is a summary of
data obtained at ITL, Montreal.
~ljmmary of Results
Janus B9
This series was designed to study the effect of
extracting tobacco with
ethanol as well as incorporating into PCL the
additives employed in the
Gerlach process of making reconstituted tobacco.
Gerlach reconstituted
tobacco is made commercially in Germany and has
been demonstrated to have
an uniquely low biological activity. B.-A.T., some
years ago carried out
these studies to try to determine the reasons for
the low biological
activity. The followinq conclusions were drawn
from the mutagenicity and
carcinogenicity studies:
(a) Both tests show that ethanol extraction
reduces activity
significantly, and returning the extract restores
original
activity.
(b) Both tests show that the sheet materials are
much lower than
tobacco, and Gerlach is significantly lower than
PCL made
using Gerlach additives.
(c) The carcinogenicity test shows that Gerlach
made from ethanol-
extracted tobacco is significantly lower than
Gerlach made using
unextracted tobacco. In contrast, the mutagenicity
test shows
the former to be slightly higher in activity.
janus Bll
Janus Bll cigarettes were produced to compare
sheet materials made by
the PCL and Schweitzer paper process. The
following conclusions were drawn:
(a) Both mutagenicity and carcinogenicity tests
showed that the
sheet materials were significantly lower in
activity than
flue-cured tobacco.
/3....
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(b) The mutagenicity showed the two sheets to
possess equal
activity, while the carcinogenicity test showed
that the
Schweitzer sheet had significantly lower activity
than PCL.
Janus B14
This Janus series was designed to study the effect
of added nicotine
to tobacco on the long-term skin painting activity
on account of reports
indicating that nicotine may be a co-carcinogen.
Conclusions drawn from
the experiments are as follows:
(a) A dose-related increase in Ames mutagenicity
with increasing
total nicotine alkaloid content has been observed.
A similar
result is being obtained in the long-term
carcinogenicity test
by B.-A.T., Southampton (Binns, personal
communication).
(b) Both mutagenicity and carcinogenicity tests
show that oniy at
the highest concentration of nicotine is there
statistical
significance in difference from control.
Janus B15
This series, consisting of 12 samples, was
desiqned to study the effect
of ventilation on biological activity. Increasing
use of ventilation in
commercial ciqarettes has rendered such a study of
areat importance to the
tobacco 'Industry. Ventilation was achieved by
using papers of different
porosities and by usinq a ventilated tip. A study
of pressure drop was aiso
built into the design of the experiment. Final
carcinogenic data are not as
yet available, and the following conclusions have
been drawn from mutagenic
data only:
(a) Higher pressure drop appears to be associated
with higher
mutagenicity, while hiqher paper porosities
resulted in
lowering mutagenic activities. The differences
were
statistically significant.
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(b) There was no difference between cigarettes
made using non-
ventilated and ventilated tipping (30 percent
ventilation).
As a result of these findings it has been decided
to conduct mutaqenesis
tests on commercial Canadian low-tar cigarettes in
the near future.
Janus B16
The B16 series was designed to study the effects
of application of
casing" to tobacco as well as the effects of other
additives, viz., cocoa
and the butterfat therefrom. These additives were
studied because research
at the National Cancer Institute, U.S.A.,
indicated that increased rodent
carcinogenicity was associated with their use on
tobacco.
Due to a policy decision to stop long-term mouse
skin painting tests,
B.-A.T., Southampton will not be doing the above
long-term test on this
series, and a comparison with mutaqenicity will
not be possible. However,
on the basis of the Ames test, the following
conclusions may be drawn:
(a) Application of casing appears to lower
mutagenicity, but the
differences obtained, although significant, are
small indeed.
(b) The addition of cocoa to tobacco has no effect
on mutagenicity,
but the amount of cocoa butterfat added is
significantly
associated with lowering of mutaqenicity.
11. Mutaoenicity tests on other exoerimental and
commerciaT cigarettes.
Introduction
Gradual acceptance of the Ames test as a good
predictor of the
carcinogenicity of pure chemicals and complex
mixtures, such as tobacco siroke,
was considered encouraging enough to test for
mutagenic activity, a variety
of condensates from experimental as well as
commercial cigarettes, and the
results have been summarized below.
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Summary of Results
Agriculture Canada Phase I Cigarettes
This series of 26 samples produced by the Delhi
Research Station
represents a carefully planned experiment to
evaluate the chemistry of
flue-cured and Burley tobaccos, as well as the
effect of plant position,
inclusion of stalk, conversion into sheet of the
tobacco materials, etc.,
on the biological activity of smoke condensates.
We selected 15 of these
samples for mutaqenicity testing and the following
conclusions have been
drawn:
(a) Lamina from the lowest plant position showed
the highest
mutagenic activity, which was significantly
different from
the activities obtained for the middle plant
position and
whole plant laminas.
(b) Sheet materials made from Burley and
flue-cured laminas were
significantly lower in activity than the original
materials.
(c) Burley lamina was significantly hiqher than
flue-cured.
(d) Mutagenicity is significantly correlated with
the total and
protein nitroqen contents of tobacco, confirming
the results
obtained in an earlier Japanese study.
French cioarettes
Evidence for the higher carcinogenicity of
cigarettes made from dark
highly fermented tobaccos, led us to evaluate
three French cigarette
brands, viz., Gauloise, Gallia and Celtique. The
following conclusions
were drawn:
(a) All three brands produced condensates which
were
significantly hiqher in mutaqenic activity than
condensates
from Players plain cigarettes.
(b) Gallia, which is sold as a low biological
activity cigarette,
was significantly lower than the other two brands,
which
were similar in mutaqenicity.
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Blue Mould infected tobacco
A considerable amount of blue mould damaged
tobacco was purchased
for cigarette making in 1979. Since concern was
expressed about the use
of this damaged tobacco, Ames tests were conducted
on condensates from
cigarettes made from this tobacco. Severely
damaged "light" and "dark"
tobaccos of both lug and cutter grades were
employed in this study.
This study led to the following conclusions:
(a) Blue mould damage did not affect mutagenic
activity of
condensate.
(b) Condensates from both blue mould infected and
uninfected
dark tobaccos were significantly higher than
infected and
uninfected light flue-cured tobaccos.
Ridomil and Ridomil-treated flue-cured tobacco
Severe damage by blue mould to a considerable
portion of the Ontario
tobacco crop in 1979 led to the use of Ridomil on
almost the entire Ontario
tobacco crop in 1980 when it was found to be very
effective. In both these
years the Delhi Research Station produced Ridomil
treated tobacco on an
experimental basis to study the effectiveness of
the fungicide under Ontario
growing conditions. Since nothing was known about
the mutagenicity of
Ridomil or the effect of Ridomil treatment of
tobacco on the biological
activity of cigarette smoke, it was decided to
examine both Ridomil as
well as experimental cigarettes made with the 1979
and 1980 Ridomil treated
tobaccos in the Ames mutagenicity system. The
results obtained are
summarized below:
(a) Ridomil was not mutagenic in the Ames system
either with or
without the addition of a rat liver activation
system.
W In 1979 when Ridomi7 was applied in late August
and early
September (too late in the growing season), a
small but
progressive increase in mutagenicity with
increasing rate
of application was observed for the 3 levels of
treatment. 7i_1
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(c) In 1980 two rates of Ridomil were applied at
the proper
times in June and July, and no differences from
the controls
were found.
Conclusions
1. The Ames mutagenicity test is relatively
inexpensive, easy to perform
and permits quantitative comparisons of smoke
condensates to be made.
2. Although correlations with the long-term
carcinogenicity test are not
perfect, any substance damaging the human genetic
material should be of
concern to man. Thus, tobacco smoke mutagenicity,
should be studied
for its own sake and not because it happens to
correlate with its
carcinogenicity.
BATCo document for Province of British Columbia 8
November 1999