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BATCo document for Province of British Columbia 14 April 1999

COPY No.
IMPERIAL TOBACCO LTD.
RESEARCH 6 OXVELOPMEMT DIVISION
MONTREAL
RESTRICTED
PROGRESS REPORT
OCTOBER 1985 - MAY 1986
DISTRIBUTION:
Or. P.J. Dunn Copy No. I
Dr. S.R. Massey 2
Mr. A.L. Heard 3
Dr. R. Binns 4, 5
Mr. E.E. Kohnhorst 6, 7
Mr. R.G. Nicholls 8, 9
Herr. E. Rittershaus 10
Dr. E. Kausch 11
Dr. C.J.P. de Siquelra 12
Library 13, 14. 15, 16, 17
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BATCo document for Province of British Columbia 14 April 1999

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WORK AREA 03
SMOKING BEHAVIOUR
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T-8079-1 Program for Upgrading the Recording/Duplicating System for Human
LMOA i nq
OblectIve:
To ensure that all aspects of human smoking are accurately and properly
monitored.
Status:
This is an ongoing activity to upgrade the recording and duplicating system
for monitoring human smoking behaviour.
The strategy is to replace the current ISC microprocessor used for duplication
with an Apple Ile/Isaac system. This. will result in a fully integrated
recording/duplicating system.
To date, phase I of the initial proposal has been achieved. This phase
consisted of writing and testing control procedures for the Isaac's data
acquisition and control of the puffing process..
Phase 2 is underway and will be finished by February 15, 1986. This phase
consists of using simulated puffs to test the software for control of the
stepping motors and switches. Tests an a prototype (with I piston) are also
underway.
The final phase is the validation procedure. This includes tests with real
data which should be completed by the beginning of March 1986. The new smoke
duplicator will then be extensively evaluated in terms of accuracy and
reproducibility.
Consideration is also being given to the feasibility of interfacing the Apple
Ile with the mainframe computer. This will allow us to transfer data directly
from the Apple to the mainframe so that SAS statistical packages may be more
readily accessed.
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T-8079-2 Alternative Methods for Duplication
Objecti ye:
To review the presently available methods. both within and external to the BAT
group, for measuring mouth level smoke constituents with the aim of assessing
the feasibility of adapting and/or developing a method to be used
alternatively or in conjunction with duplication.
Status:
Two methods were designated for assessment:
1) the BAT (UK & E) R & 0 computer program DELIVER.
2) the determination of puff by puff TPM by IR reflectance.
A preliminary investigation of the applicability of DELIVER to ITL has been
completed. The results of this investigation indicated that DELIVER was able
to predict total deliveries of tar and nicotine as determined by standard
smoking conditions to within 1.00% and 1.25% of the experimental values
respectively. however, this could only be achieved if some of the input
parameters were significantly altered from the original values. Furthermore
there appeared to be serious discrepancies between predicted (DELIVER) and
experimental values on a puff by puff basis. Differences ranged from 13-100%
higher for the first puffs and 4-16% lower for the last puffs. This
investigation is presented in an internal report (November 30, 1985, S.
Ke I eman) . The results have also been communicated to BAT (UK & E) R & 0 so
that further refinement of the model may continue. Based on current
information it is not recommended that DELIVER, in its present form. be
Incorporated Into the Human Smoking Behaviour methodology. However should the
model be sufficiently refined in the future, serious consideration should be
given to its inclusion as a Human Smoking Behaviour procedure.
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The feasibility of incorporating the determination of puff by puff TPM by IR
reflectance into the recording equipment for Human Smoking is presently being
assessed. Based on the method of Jenkins and Gale I an initial apparatus was
developed by BAT UK & E R & 0 during 1983-84. Before any significant
investigation is initiated, the work of BAT UK & E R & 0 in this area will be
reviewed. If these results seem promising, the apparatus could be developed
during 1966.
T-8079-3 Assessment of the Feasibility gf Measuring Inhalation PatteEns and
Human Retention of Cigarette Smoke
objective:
To investigate the usefulness and feasibility of measuring human inhalation
patterns and retention of smoke for possible inclusion into human smoking
behaviour studies.
Status:
The project was initiated in October 1985. To date a report concerning this
subject has been completed.
This report Is a review of methods developed both within and external to the
BAT group for the measurement of human Inhalation and retention of cigarette
smoke.
I Behaviour Research Methods.' Instr. & Computers, 1984, 16(3),
263-267. KAn Instrumental Cigarette Smoke Monitor Designed for the
Direct Measurement of Smoke Particulate Matter Generated in Human
Smoking Studies".
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It is made up of three parts:
a) Smoking patterns (Inhalation/Exhalation) and puff parameters.
b) Human retention of cigarette smoke (nicotine and tar).
O Human retention of carbon monoxide from cigarette smoking.
The conclusions and recommendations are summarized as follows:
of all the methods reviewed In this project, only the chest respiratory
inductive plethysmagraph combined with a modified cigarette holder or a cheek
plethysmograph appears to be feasible for use in Human Smoking studies. This
method would allow the measurement of both inhalation and puffing parameters.
The results obtained appear to be relatively accurate, reliable and
non-obtrusive. Although quite expensive. this method permits measurements in
a 'natural' environment. However. the decision to include such a method in
Human Smoking Research will also be based on the relative value of the new
Information which would be added by the method.
Many methods have been developed for measuring smoke retention. Experimental
procedures are similar throughout all research groups and implementation
appears to be relatively simple. The same provisos mentioned above for
inhalation also apply to the adoption of a method for retention.
T-8077-17 Investigation of the Effects of Nicotine Enhancement an Human
Smoking Behaviour
Obiective:
To assess the changes, if any, occurring in Human Smoking Behaviour as a
result of cigarettes with enhanced levels of nicotine.
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20
Status:
As a result of the pilot study conducted in April 198S, the appropriate levels
of nicotine enhancement were determined to be 15 and 30% for mid to high
delivery products and 30 and 45% for low delivery products. These levels are
sufficiently high that changes in smoking behaviour and human delivery can be
attributed to increases in available nicotine and not to variability in the
apparatus.
Du Maur I er K.S. and matinde E.R. K.S. (15 and 4 mg tar delivery respectively)
were selected as the brands for study. Cigarettes were injected with the
appropriate quantities of nicotine citrate solution to achieve increases in
smoke nicotine content of 15 and 30% for du Maurier K.S. and 30 and 45% for
Matinie E.M. K.S. The du Maurl er K.S. samples had to be rejected since a
distinct off-taste was noticed during subjective evaluation. Panelists had
difficulty in differentiating between the control Ratin6e E.M. K.S. and the
nicotine enhanced samples. The leaf and smoke analyses were conducted
concurrently with the subjective evaluation. Leaf analysis indicated the
appropriate increases in blend nicotine. However, smoke analysis showed a
poor transfer of injected nicotine from tobacco to smoke. This may have been
due to an uneven distribution of nicotine along the tobacco rod when the
samples were injected with nicotine citrate. It may have also contributed to
the off-taste found for du Maurier K.S.
In light of the above, small quantities of Matinte E.N. K.S., du Maur I er K.S.
and du Maurier Light K.S. tobaccos were sprayed with sufficient nicotine
citrate to increase the smoke level of nicotine by 15, 30 and 45%. Smoke
analysis results indicated that the smoke nicotine of these experimental
cigarettes was appropriately increased.
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BATCo document for Province of British Columbia 14 April 1999

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Subjective evaluation of the Mat I n@e E.M. samples Indicated a small but
definite difference between the control and enhanced nicotine cigarettes. The
nicotine enhanced cigarettes were perceived to have more impact and to be
slightly more irritating than the controls, but no off-taste was detected. du
Maurior K.S. cigarettes with 30% more nicotine were also tested subjectively.
These results showed du Maurler K.S. with added nicotine to have an equal or
slightly greater taste level, more impact and to be slightly more irritating
than the control. Subjective evaluation of the du Maur Light K.S. clearly
showed an increase in taste, impact and irritation of the product with an
increase in nicotine level. Of the three brands tested du Maurier Light K.S.
appeared to be the most acceptable and will therefore be the product used for
further study. The human smoking study is expected to start in April 1986.
T-8082-1 Application of Delta for Product Screening and Evaluation
a) Project Omega
Object I Ye:
To determine the relative positions of Mat I n@e K.S. and Regular in the market.
Status:
MatiOe , in both king size and regular lengths. was evaluated by the Delta
panel relative to the major competitors. For both lengths, subjective
evaluation Indicated that Matinie was milder than its competitors, having loss
irritation. taste and after-effects. For the king size brands, Craven A
(Rothmans) appeared to be most similar to Matin6e, while for the regular
length brands, Belvedere E.H. (B & H) was the closest match to Matin6e.
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b) Project Swirl
Objective:
To further evaluate the subjective characteristics of two novel filters
designed to Improve the smoke quality of low delivery cigarettes by changing
the smoke pattern.
Status:
Due to differences in filtration efficiencies and ventilation. direct
comparison of swirl filters and regular cellulose acetate filters on the
subjective smoke qualities of Medallion was not possible. For this reason the
contribution of swirl to subjective improvement was assessed by comparing
swirl filters both with and without a plastic sleeve (which disrupts swirl).
In the previous progress report (May 1985) it was noted that the sleeve Itself
caused a subjective change. The sleeve has now been redesigned and using the
new sleeve it has been shown that swirl does Improve the subjective response
to Medallion. which delivers nominally I mg of tar. The next phase will
consist of measuring the deliveries of these products under both standard and
human smoking conditions.
T-8082-3 investigation of the Effect of Brand Imagery on Subiective Evaluation
ObJective:
To study the Influence of brand identification and pack imagery on subjective
evaluation.
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Status:
The initial study was reported in the previous progress report and details are
given in an internal report (June 1985, C. Boisjoly) . A second study was
designed to:
1) control for product familiarity by using foreign products.
2) Isolate the pack effect from the brand name effect by evaluating empty
packages and subsequently masked products In different packages.
Panelists were asked to evaluate:
1) empty packages in terms of product expectations.
2) masked products alone.
3) masked products presented in their own packages.
4) masked products In different packages.
5) unmasked products alone.
At this point the first four stages have been completed. Each evaluation was
repeated twice with an Inter-evaluation interval of two weeks. In any of the
evaluations 85-90% of the variation has been explained and the inter-trial
correlation has exceeded 0.90.
The fifth and final phase is presently underway and will be completed by the
end of May.
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T-8082-4 Investigation of Product Quality Attributls
Objective:
To determine the threshold level at which subjects start to perceive a
difference in the firmness of cigarettes.
Status:
Since the initial study investigating cigarettes differing in weight by 50
mg. a new study was initiated Investigating cigarettes with weight differences
of + 25 mg. Results indicate that, on average, subjects were still able to
differentiate the firmness between the cigarettes, despite the fact that
approximately 25% of the respondents claimed their answers were guesses and
all found the test very difficult. It appears, therefore, although the group
results indicate that subjects can perceive tactile differences, a smoker
would probably not spontaneously recognize this difference as he picks a
cigarette out of pack. Therefore it is suggested to further study the impact
of these firmness changes on the sensory evaluation of the product.
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WORK AREA 04
BIOLOGICAL RESEARCH
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T-7709: Mutagenicity Studies
aLer
To determine those parameters which affect mutagenicity of smoke condensate,
and. by monitoring market brands for condensate mutagenicity on a periodic
basis, to help ensure that ITL brands rank favourably.
Status:
1. Mutagenicity of Commercial Cigarettes
Since the last status report, we have extended our studies to include more
N. American and European brands. In an examination of the mutagenicity of
a group of low delivery, ventilated Canadian brands. it was found that a
particular opposition brand was significantly lower than 3 other brands,
produced by ITL and Rothmans. This result is interesting since our
earlier studies showed that a brand from the same competitor had the
lowest mutagenicity among a group of 26 N. American brands.
In a recent examination of a French brand we speculated that the version
of this brand available In Canada appeared to be lower in mutagenicity
than similar cigarettes available here a few years ago due to the former
being manufactured with significantly lower amounts of dark fermented
tobacco. Consequently, we tested two French brands available in Canada
with those available in France, and found that for one brand there was no
difference in activity, while for the other brand there was a significant
difference. This suggests that the Canadian version of the latter brand
contains significant amounts of a lower activity tobacco (possibly
flue-cured).
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All our studies have shown that French cigarettes containing dark
fermented tobaccos are much higher in activity than blended U.S. brands.
Other European countries such as Spain and Portugal appear to use similar
tobacco in their cigarettes and therefore, we obtained and examined one
brand from each of these countries. This study showed that the Spanish
cigarette was much higher in activity than the Portuguese cigarette which
was similar in activity to a U.S. blended cigarette. Both brands had
significantly higher activities than Canadian brands containing only
flue-cured tobacco.
2. Mutagenicity of Condensate from Player's Cigarettes Modified by Electro
Medica Office
mutagenicity testing of these cigarettes along with market samples of the
same brand showed that the two were similar in activity, and that the
Electra Medica modification does not affect the Ames biological activity.
3. A Comparison of Rat and Guinea Pig Metabolic Activation Systems
The standard Ames test employs Aroclor-treated rat liver post-
mitachondrial supernatant (S-9) as the activation system. Earlier studies
in this laboratory suggested that a similar preparation from the guinea
pig was more active in converting cigarette smoke condensate to mutagenic
intermediates in the Ames test. Preliminary results obtained suggest that
the initial observation is true and that the concentration of S-9 might
play a role in the level of activation obtained.
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4. Selenium-and Cicarette Smoke Condensate (CSC) Mutagenicity
Selenium as sodium selenite has been shown to be an effective inhibitor of
the mutagen1c activity of several chemicals such as 2-acetylaminofluorene,
2-hydroxyacetylamino fluorene, and 7,12-dimethylbenzanthracene, which
require metabolic activation before they manifest their activity. It is
also known to reduce the mutagenic activity of chemicals such as
N-methyl-NI-nitro-N-nitrosoguanidine, malonaidehyde and propiolactone
which are direct acting mutagens. Hence, we thought it useful to
determine if selenium could reduce the mutagenicity of CSC.
Preliminary studies undertaken using Ames strain TA-98 suggest that sodium
selenite can reduce the mutagenicity of CSC, while sodium selenate can
increase the activity of CSC. The increase in activity appears to be due
to the positive mutagenicity of sodium selenate Itself with strains that
detect frameshtft mutagens. The reduction In mutagenicIty of CSC by
selenite is an interesting observation and more work is planned to confirm
it.
T-6115 Microbiological Examination of Tobacco Products
Oblective:
To survey the microflora in our own and opposition tobacco products.
Status:
1. Cigarettes
Most of the 7 brands selected were examined by B.A.T. Southampton in
1982-83. In that study the total bacterial counts for the Canadian
cigarettes were found to be an order of magnitude higher than the counts
obtained for cigarettes of U.K. manufacture. The present Montreal study
of Canadian cigarettes found similar levels of bacteria to those found by
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Southampton two years earlier, Indicating a consistency in the handling of
the tobacco during cigarette manufacture.
Of interest is the fact that the two brands with the highest bacterial
counts also showed the lowest specific mutagenic activity in the Ames
test. Hence one may speculate that similar grades of tobaccos are used
for these brands. Whether the microorganisms themselves have any role In
lowering mutagenicity is unknown.
The main difference between this ITL study and the Southampton examination
of 2 years ago was the lower numbers of thermophilic actlnomycetes
recorded in the former study. This would suggest that more care has been
taken in the handling of tobacco or In cigarette manufacturing In the last
2 years. There was nothing remarkable about the col If orm bacteria. which
were not detected, nor in the mould content of the cigarettes.
2. Fine Cut Tobaccos
Eight samples were selected for this study which included I of the 3
brands examined by Southampton In 1982-83. The bacterial levels recorded
in this ITL study of 24 samples from 8 brands were about three times
higher than those recorded in 1982-83. However, a direct comparison
cannot be made an account of the different samples examined as well as the
small number tested earlier. As in the case of the cigarettes, the
Montreal study showed fewer actinomycetes in the fine cuts than the
Southampton study of 2 years ago, indicating a possible general
improvement In the handling and processing of the tobacco. Both the
Southampton and Montreal studies failed to detect co I A form bacteria or
fungi In these products.
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3. Pipe Tobacco
An examination of 4 brands was undertaken by ITL. Old Port showed the
highest number of bacteria (9 x 106/gram) followed by Mac5aren (3 x
106). The other two brands, Skandinavik and Borkum Riff showed numbers
of bacteria 2-3 orders of magnitude lower.
A considerable number of thermaphilic actinomycetes were also found in Old
Port (6.5 x 102). However. this number is considerably lower than the
value of 1.9 x 104 recorded for this product 3 years earlier.
Skandinavik and Borkum Riff did not show the presence of thermophilic
actinomycetes. No coliforms or Fungi were detected in these products.
4. Cigars
In the 1985 ITL study 12 brands of cigar and cigars were examined for
their microbial content. Ten of these had been examined by Southampton in
1982-83 hence a good comparison can be made. O'Sherry Filter Tip (RJR)
examined earlier was not available in 1985, and it is not known whether El
Producto (RJR) examined by Southampton were cigars or cigarillos.
Bacterial counts for El Producto cigars and cigarillos were very similar
in both the ITL and Southampton studies. once again Southampton recorded
much higher counts of thermophilic actinomycetes than did ITL.
While this is most probably due to a general improvement in the handling
and processing of tobacco. it is also possible that differences in
microbiological procedures such as sampl ing, as well as accuracy of the
incubation temperature ( so-C) , account for the difference.
Neither study detected co I if orm bacteria in any sample, and there was
nothing remarkable about the numbers of fungi in the ITL study.
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T-7710 Biochemical Effects of-Tobacco Smoke Exposure in ExRerimental Animals
@@t I VI I
To study the effect on protective mechanisms in experimental animals of
exposure to mainstream and sidestream cigarette smoke.
Status:
Cigarette smoke is known to contain oxidants which could affect levels of
endoqencus protective mechanisms in the lung. Studies have shown that when
animals are exposed to cigarette smoke there is an Increase in levels of
glutathione (GSH), an antioxidant present in significant quantities in
tissues. It has also been shown that when experimental animals are treated
with SSH-depleting chemicals such as diethylmaleate. the rapid depletion of
GSH is followed by a re-synthesis of this antioxidant. Such a re-synthesis of
GS14 was not considered in the above mentioned studies involving cigarette
smoke. Hence it was considered Important to determine whether exposure to
cigarette smoke would lead to an Immediate loss of GSH followed by a recovery
of normal levels such as occurring after treatment with diethylmaleate.
Adult male, Sprague-Dawley rats were exposed to dilute cigarette smoke using
the 8 A. T. -Mas on Inhalation System. Animals exposed to 40, 120 and 240 puffs
were sacrificed immediately after smoke exposure. The rats were euthanlzed
and tissues (lung, liver and kidney) were removed and used for determination
of their glutathione (GSH) content employing El Iman Is reagent (515-dithiobts
(2-nitrobenzoic acid)).
Exposure to 40 puffs of a 1:5 diluted smoke (3.33 minutes) resulted in a
slight lowering of GSH in the lung, but did not affect levels of this
antioxidant in the liver and kidney. Increasing smoke exposure to 120 puffs
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BATCo document for Province of British Columbia 14 April 1999