COPY NO. .......
IMPERIAL TOBACCO LIMITED
RESEARCH & DEVELOPMENT DIVISION
MONTREAL 990UP k & D CENTRI
,-.N-tva- 29AUG1978
.....................
RESTRICTED
. ................
PROGRESS REPORT
RESEARCH DEPARTMENT
January - June 1978
ISSUED BY: Mr. R.S. Wade
DISTRIBUTION:
Copy No.: 1.
2.
3.
4.
S.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
Library
Mr. R.M. Gibb
Mr. R.S. Wade
Mr. S.M. Candlish
Dr. T.A. Smith
Dr. S.J. Green
Mr. E. Rittershaus
Dr. F. Seehofer
Dr. C.J.P. De Sequeira
Dr. D.G. Felton
Dr. D.G. Felton
Dr. C.I. AyresV
Dr. I.W. Hughes
Dr. R.A. Sanford
Dr. R.A. Sanford
Mr. R.G. Nicholls
Mr. R.G. Nicholls
Dr. M.H. Bilimoria
Dr. P.J. Dunn
DATE ISSUED: August 14, 1978.
20. Mr. M.P. Scherbak
21. Dr. G.W. Boswall
22. Mr. L. Bouchard
23. Mr. H. Roubicek
24. Miss R.R. Smith
25. Mr. B.J. Stirling
26. Mrs. J. Johnson
27. Mr. R.L. Rice
28. Mr. A. Schaffer
29. Mr. E.P. Gage
30. Mr. W.A. Gray
31. Mr. A.I. Kalhok
32. Mr. W.J. Ross
33. Mr. N.D. Maclennan
34. Mr. C.J. Brown
35. Mr. C. Warren
.tb-
(ZD
BATCo document for Province of British Columbia 23
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INDEX
Page Code Title Researcher(s)
a Summary of Research Department Activities.
1 T-0575 Delhi Research Station Samples for
Industry G.W.B., B.J.S.
Collaborative Evaluation. & H.R.
3 T-0770 Cigar Tobacco Production Committee's
G.W.B. h H.R.
Mechanization and Curing Experiments
Conducted at the L'Assomption Experimental
Farm.
6 T-0774 The Manufacture of Cigars for Smoking
Tests G.W.B. & H.R.
of Tobacco from the L'Assomption Experimental
Farm.
7 T-0820 Study of Methods to Promote Earlier
Maturing G.W.B., S.W.
of Flue-Cured Tobaccos. & B.J.S.
10 T-6420 Mechanization of Tobacco Production.
G.W.B., B.J.S.
& H.R.
12 T-8077 Studies in Human Smoking Behaviour
P.J.D.
T-7051
is T-7708 Microbial Mutagenicity Tests in Tobacco
and M.H.B.
Health Research.
17 T-7710 The Effect of Tobacco Smoke on Drug
Metabolizing M.H.B. & DA.E.
Enzymes in Mammalian Tissues.
1. Metabolic activation capability of
rodent tissue preparations.
2. Induction of AHH in Sprague-Dawley rat
tissues - Dose response curves using diluted
smoke produced in the B-A.T. Mason machine.
20 T-7709 Changes in Bronchial Epithelium and
Other J.J.. J.C.H.
Tissues Induced by Tobacco Smoke. J.B.R.
1. Bronchial Epithelia] Permeability.
CD
BATCO document for Province of BritiSh Columbia 23
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a
SU MMA RY OF RESEARCH DEPARTMENT ACTIVITIES
JANUARY - JUNE 1978
Agroloqy
Cooperative work with Agriculture Canada
Experimental Stations
continues in evaluating promising new varieties,
pesticides and tobacco
production practices. Favorable data from these
tests have resulted in
Metasystox-R, Vorlex CP and Terr-o-cide 15D being
added to the recommended
chemicals in the OMAF bulletin No. 298 "1978
Tobacco Production Recommenda-
tions". Ambush 50EC for the control of cutworms
has not been included
presumably because registration of the material
was not granted at the
time of publication.
The CTMC sponsored program which was carried out
at the Imperial
farm in previous years has been transferred to the
Delhi Research Station.
Under a cooperative agreement between Agriculture
Canada, Canadian Tobacco
Manufacturers' Council and Ontario Flue Cured
Tobacco Growers Marketing
Board, the Canadian Tobacco Research Group (not
Agri-systems Services Group
as mentioned in our previous report) has been
established to undertake
agricultural engineering and agricultural
economics studies of tobacco
production, thus filling a void in the Delhi
research program.
The Group has set the following project goals for
the engineering
research program.
A. By 1980 to provide sufficient instrumentation,
kiln modifications and
curing program information to improve cured leaf
quality and minimize
curing energy requirements.
S. By 1980 to develop material handling systems in
conjunction with a
mechanical harvester that minimize manpower
requirements and energy use.
c::)
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The evaluation in our laboratory of the
single-part smoking
duplicator designed and constructed for us by
Ontario Research Foundation
is nearly completed. Although certain faults were
found, they appear to
be readily correctable; hence a decision has been
taken to return the
duplicator to Ontario Research Foundation for
modification and to expand
it so that four cigarettes can be smoked
simultaneously.
Work employing the alveolar CO rebreathing
technique as a means of
assessing the degree of inhalation of smokers of
changed products
continues. Preliminary studies were conducted on
cigarettes differing in
degrees of ventilation. Significant changes in
alveolar CO levels from
those which might have been predicted from values
obtained by standard
machine smoking were observed. The changes found
were of sufficient
magnitude as to warrant a continuation of the
study with specially
prepared cigarettes in which the only design
change is the amount of
ventilation.
The study of cigarettes with a 30% increase in
nicotine delivery
and reported in the latter half of last year was
expanded from the
original four subjects to a total of 15. The
additional results confirmed
the original observations that the smokers, when
smoking the higher nicotine
cigarette, had significantly lower alveolar CO
levels than with the normal
nicotine delivery cigarette.
Of the recently developed in vitro short-term
tests for detecting
the potential mutagenic and carcinogenic activity
of chemicals, the one
most widely accepted as a good predictor of
carcinogenicity in man, is
the bacterial test developed by Professor B.N.
Ames. The wide acceptance
of this test may be attributed to the excellent
correlation that has
been obtained between mutagenicity in this test
and carcinogenicity in the
animal tests. Routine monitoring of tobacco
products, using these tests,
has begun with the testing of the Janus B-11
series. "Activation" of the
cigarette smoke condensates is being achieved
using a liver preparation
obtained from Arochlor-treated Sprague Dawley
rats.
C)
C)
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Biological research at McGill University is
progressing satisfactorily.
The recently acquired smoking machine has been
used extensively for the
program studying the effect of tobacco smoke on
drug metabolizing enzymes.
A dose response curve has been obtained with
respect to induction of aryl
hydrocarbon hydroxylase in rat kidney, and this is
being followed by studying
the effect of cigarette smoke on levels of this
enzyme in other tissues of
these species. This work will be extended to other
species with the hope
that when more information is available on these
enzymes in human tissues,
the most appropriate species can be selected for
bioassay work.
The permeability on the bronchial epithelium of
the guinea pig to
whole cigarette smoke has been shown to be
dependent upon the extent of
smoke exposure. Vapour phase alone, however, does
not result in increased
permeability. Results have been obtained, which
indicate that the site of
penetration of the tracer used to monitor
permeability is at the epithelial
tight junctions, thus confirming the work of Hogg
et al.
Studies to determine if smoke from different types
of tobaccos
causes differences in the permeability of the
bronchial epithelial tissues
of the guinea pig are underway. It is also planned
to investigate if the
permeability of the bronchial epithelium returns
to normal after exposure
to whole smoke has been terminated.
BATCo document for Province of British Columbia 23
April 1999


T-0575 Delhi Research Station Samples for Industry
Collaborative Evaluation.
Personnel: G.W. Boswall, B.J. Stirling, H.
Roubicek
Objective: To evaluate and compare cigarettes made
from varieties and
other flue-cured tobaccos produced under various
experimental
conditions by the Delhi Research Station (DRS).
Status: The Delhi Research Station produces
numerous tobacco samples
each year to evaluate the merits of different
varieties,
modified cultural and curing practices, and new
agricultural
chemicals such as pest control products for the
production of
flue-cured tobacco. A select number of samples
from the more
promising varieties and treatments are made
available to the
four Canadian cigarette manufacturers for their
evaluation of
smoking quality. As a result of testing samples
from the 1976
and previous crops, some new materials were to be
included in
the 1978 edition of the OMAF bulletin No. 298
Tobacco Production
Recommendations. However, Ambush 50EC for control
of cutworms
was not included, presumably because registration
had not been
granted at the time of publication. Therefore the
three new
additions to the bulletin are:
Metasystox-R for the control of aphids
Vorlex CP for the control of nematodes
Terr-o-cide 150 for the control of nematodes
Cigarette manufacturers are continuing to evaluate
cigarettes
from tobaccos produces at ORS in 1977 and the
following
samples have been allotted to the four cigarette
manufacturers
for cigarette making.
41-
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4 :h.
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-14
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April 1999


T-0575 (Cont'd)
5 Varieties (4 locations)
8 Varieties (DRS)
2 Insecticide samples
2 Fungicide samples
2 Herbicide samples
4 Nematicide samples
Imperial Tobacco
- Rothmans
- Macdonald Tobacco
- Macdonald Tobacco
- Macdonald Tobacco
- Benson & Hedges
The leaf samples have been delivered to the
tobacco manufacturers
and cigarettes have been received from Rothmans
and Macdonald
Tobacco. The four companies have agreed to use a
common
questionnaire when conducting the subjective
smoking tests
on these samples.
In addition to the above samples, Imperial has
agreed to
manufacture cigarettes containing blends of
regular tobacco
and PCL or PRT samples (made from DRS close
planted chopped
whole tobacco plants) for evaluation by the
subjective smoking
panels of the four companies.
CD
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CO
BATCo document for Province of British Columbia 23
April 1999


T-0770 Cigar Tobacco Production Committee's
Mechanization and
Curing Experiments Conducted at the L'Assomption
Experimental Farm
Personnel: G.W. Boswall, H. Roubicek.
Objective: To evaluate the subjective smoking
properties of cigar filler
samples harvested and cured by various methods.
Status: Cigar filler production in Quebec has been
decreasing
dramatically in recent years. Consequently a Cigar
Tobacco
Production Working Committee composed of
representatives of
the cigar manufacturers, the Socidtd Cooperative
de Montcalm,
and the Canada and Quebec Departments of
Agriculture. was
formed in 1973 to study the situation. It became
obvious
that cigar tobacco lacks both the modern
production techniques
(mechanization) and the dollar returns~ that
farmers experience
from other crops. The committee instituted an
experimental
program to modernize the harvesting and curing of
cigar filler
which, if successful, would hopefully encourage
farmers to
resume cigar tobacco production. Cigar samples
have been
made from tobaccos produced during the first three
years of
the experiment. but only samples from the first
two years
have been evaluated by the subjective smoking
panel. It has
been found that bulk curing of primed cigar leaf
gave a product
having a different appearance to that produced by
the traditional
air curing of stalk cut tobacco, but it was rated
similar in
smoking quality. However, because of the increased
cost of
fuel used in bulk curing, emphasis in 1978 will
return to air
curing stalk cut tobacco to find practical ways of
mechanizing
the operations and reducing labour requirements.
Trials with different prototype machines for stalk
cutting cigar
C:)
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C:)
BATCo document for Province of BritiSh Columbia 23
April 1999


T-0770 (Cont'd)
tobacco will be continued. A CTMC designed machine
cuts
the plants and conveys them up to the back of the
machine
where the operator spears the stalks on laths.
Laths with
plants are either stuck in the ground for plant
wilting
or they can be loaded directly on a wagon for
transportation
to the curing barn. Balthes Farm Equipment Co.
plans to
manufacture two such machines (with some
modifications) for
the 1978 trials. Other machines are under
development by
the Engineering and Statistical Research
Institute, Ottawa,
for stalk cutting air cured tobacco (burley and
cigar) and
another developed by a farmer in Quebec for cigar
tobacco.
Some two-row versions of some of these machines
will be
available for trials in 1978.
Other phases of investigation of harvesting and
curing
procedures for air cured tobaccos are to be
undertaken by
different people at various locations as Indicated
in the
following summary of work proposals, most of which
will
likely be actively pursued this summer.
Agr. Canada, L'Assomption Agr. Canada, Ottawa Aqr.
Canada, Harrow
P.P. Lukosivicius G. Hergert T. Welacky
G. Laporte and 1. Ogilvy
CIGAR CIGAR BURLEY
Bulk:
1) Use variety L.A. 201
2) Harvest in 3 stages
3) Use mechanical aids
for harvesting
4) Re-evaluate heat
exchanger
1) Modify existing
barns for racks
2) New loading methods
for racks
3) Further development
of harvester
4) Staples to replace
spearing
5) Stripping Aid
1) Continue machine
harvesting evaluation
2) Studies of rack curing
in modified barn
3) Study stripping aids
4 b.
C__
BATCo document for Province of BritiSh Columbia 23
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Agr. Canada, L'Assomption Quebec Dept. of Aqr.
Balthes Farm Equipment Co.
P.P. Lukosivicius J. Allard and E. Stampfer
G. Laporte and 1. Ogilvy L. Bernard
CIGAR CIGAR BURLEY and CIGAR
Stalk-Cut Curing:
1) Modify present plastic 1) Evaluate Beauregarde
1) Construction of two modified
covered barn into 2-tier Tobacco Plant versions of
C.T.M.C. plant
barn. Harvester cutter. One available for
2) OR construct 2-tier 2) Wilted tobacco cigar and
the other for
rigid plastic roof transported to barn burley
barn 3) Spearing tobacco
3) OR look at feasibility at barn
of constructing low-
profile (2-tier)
permanent type barn
Curing Chambers
Improve ventilation and
humidity control
_L__
BATCO document for Province of BritiSh Columbia 23
April 1999


T-0774 The Manufacture of Cigars for Smoking Tests
of Tobacco
from the L'Assomption Experimental Farm.
Personnel: G.W. Boswall, H. Roubicek
Objective: To evaluate the subjective smoking
properties of cigar tobaccos
from various varieties and various plant breeding
and cultural
experiments.
Status: Most of the 1976 L'Assomption variety
samples (Preliminary Test,
H-series and the Final Test, V-series) have been
manufactured
into cigars and are being delivered to
L'Assomption for smoking
tests by the L'Assomption operated panel composed
of members
recruited throughout the Agriculture Canada
organization. The
remaining variety samples from the 1976 crop will
be manufac-
tured into cigars as soon as possible.
C=)
LM
r"i
BATCO document for Province of BritiSh Columbia 23
April 1999


T-0820 Study of Methods to Promote Earlier
Maturinq of
Flue-Cured Tobaccos.
Personnel: G.W. Boswall, S. Webster. B.J. Stirling
Objective: To evaluate and compare the cultural
practices required to
achieve early maturity and the completion of
harvest by
students before they return to classes.
Status: A report entitled "A Report on CTMC
Sponsored Activities
Conducted on the Imperial Leaf Tobacco Company
Farm in 1976"
was issued in April. This experiment was
summarized as
follows:
"In an effort to promote maturity and complete
harvest early
in September, different combinations of cultural
treatments
were applied to five plots of tobacco, one being a
control
using normal tobacco culture. All plots were
harvested on
schedule before students returned to school. The
cured
leaf was evaluated by a leaf grader and samples
were sub-
jected to chemical analysis for reducing sugars
and total
alkaloids.
All four experimental treatments differed from one
another,
and in addition all differed from the control in
one common
respect, namely that topping was done at the late
bud stage
as opposed to the usual three flower stage. Cured
tobacco
from these four plots was Judged to be of similar
value,
but somewhat better than the control tobacco.
The advantage of establishing the crop in the
field early
was clearly demonstrated because a delay of one
week in
transplanting seedlings to the field reduced the
total
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C:)
BATCo document for Province of BritiSh Columbia 23
April 1999


T-0820 (Cont'd)
alkaloids, increased the reducing sugars (both an
indication
of immaturity) and generally resulted in a lower
amount of
mature unblemished tobacco. Measurements taken at
different
times throughout the growing season showed that
the size
of plants and leaves was significantly reduced by
the delay
in transplanting.
Seedling hardening procedures which were applied
in the
greenhouse seemed to produce more vigorous plants,
but these
differences tended to disappear toward the end of
July as
harvest time approached."
The cultural practices applied to each plot are
summarized
on the next page.
A formal report covering the 1977 activities in
this project
will be prepared as soon as the Delhi Research
Station completes
the chemical analysis of the leaf from small
replicated plats.
All CTMC sponsored activities in tobacco
production have
been transferred to the Canadian Tobacco Research
Group at
the Delhi Research Station, and we will not be
undertaking
any further field work in this project in 1978.
NJ
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BATCO document for Province of BritiSh COluMbia 23
April 1999


0
0
CL
0
0
(D
SU mmARY OF CULTURAL PRACTICES
1+
-h
PFamuff MST B
Smomm TEST A
SEOMEA" TEST 9
0 OMBIM PPM9JOr 12Sr A SM m
0.5 0.5
4 4 9a. ft.
I oz. seed per 3000 Sam as
Green-hou.. I oz. seed per 2200 9q. ft-
d6 nirP of alants bv
Sx-e as plants
t
Extrav hardm-o 0
to
hmxk-ng Of Plants
N.KrAl Har
-trq
forkinq 6ut not to bem 10 davs rrtor
:- -3--,
W."=~ Sam as -7--"
(D by fmkLrw and waterug lor -7 to 4 days .
=.
t and oret-me .-.d
0 .~Oo thereafter to outs:Lde ermi-rent.
h
Field fertilizer and
NIGUM pesticide traat~ts Enor
to t .. planting - the
field
4
VAY 29
mey 29
te
i may 77 ard May 20 May 27 and Hey 28 June
0 Punt
ng De '
0 ' 48" x 20' 48' x 20' 48* x 20
E, Flnt Spacing 46' x 20' 48' x 20
3 Swe as By hand at late-bud stm* by hand at late-
5, 1-8
C7 TcMing By hand at the three flower By hand at
late-bW
(twloe) to IT leaves to 14 leaves h4 tAqe t
leaves
Stage to Is leaves st-a
W
t=* Of
Delete applied at
Sam as 'Vm"
Sam as "PTA"
Sme as *M*
> Sucker Control Delete appLied prior to
to
pim ,
toppuq or soon a-ter e
~
p w
O
h SWO as Sm" as 'PM' e.-Pt na M
SWIff AS
Barvest-ing In S pass" begun Amp-=t e
exceat t
C
S~ &9
h W be tips to hrve--LW becauSe
9 d -.tLn..d qVmurately St
fter the 4th
d of low topping
weekly a
har-st
SS160VZOP


10.
T-6420 Mechanization of Tobacco Production
Personnel: G.W. Boswall, B.J. Stirling, H.
Roubicek.
Objective: To investigate the various forms of
mechanization that might
be adapted to tobacco production and reduce its
manual labor
requi rement.
Status: A report entilted "A Report on C7MC
Sponsored Activities
Conducted on the Imperial Leaf Tobacco Company
Farm in 1976"
was issued in April. This project was summarized
as follows:
"Cross-Flow Modular Tobacco Curing System:
The Cross-Flow Modular Tobacco Curing System was
constructed,
and operated at only 25% capacity using six
modules to evaluate
the prototype design. Six curing cycles were
completed during
the season with rather poor results in the earlier
cures but
better results were obtained in later cures,
largely as a result
of modifications to curing modules to improve the
air distri-
bution through the tobacco. Uneven drying and the
presence
of fat stems were obvious in the first two cures,
but in
subsequent cures tobacco from modified modules was
of acceptable
quality. Other weaknesses of the curing system.
such as
restriction of the flow of outside air to the kiln
due to a
faulty heat exchanger, and the fixation of green
in cured
tobacco adjacent to the positive pressure plenum
were identified.
It will be necessary to introduce and evaluate
further modi-
fications in order to achieve what is believed to
be the full
potential of the system.
Curing Air Management:
The survey of curing air management procedures as
practiced by
a small sample of tobacco farmers showed a large
variation in
NJ
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BATCo document for Province of BritiSh Columbia 23
April 1999


T-6420 (Cont'd)
curing efficiency in terns of thermal energy
required to cure
tobacco.
The energy required to produce a pound of cured
tobacco
ranged from 12,786 B.T.U. to 19,414 B.T.U. for the
older
forced air kilns and from 8,900 B.T.U. to 10,640
B.T.U. for
bulk kilns. These variations are attributed mainly
to kiln
construction (air leakage versus tight kilns) and
to the
degree of air exchange permitted by the cureman
during the
high temperature drying phase of the curing
cycle."
This project has been transferred to the Canadian
Tobacco
Research Group (previously referred to as the
Agri-systems
Services Group) at the Delhi Research Station
where these
mechanization and curing studies are being
continued. When
the 1978 studies have been completed the Canadian
Tobacco
Research Group will issue a report covering both
the 1977
and 1978 activities in this project.
The 1978 program has been outlined as follows:
A. 1. to evaluate the effectiveness of various
kiln
insulating materials.
2. to develop and analyse kiln heat exchangers for
commercial application.
3. to improve environmental control for curing and
conditioning through instrumentation and through
kiln
modifications.
4. to define optimum environmental conditions for
curing.
B. 1. to investigate leaf orientation techniques
that are
adaptable to existing tobacco combines.
2. to develop and analyse containerized curing
systems.
C~
N;
Ur.
BATCo document for Province of British Columbia 23
April 1999


T-8077, Studies in Human Smoking Behaviour
T-7051
Personnel: P.J. Dunn
Objective: To study the real effects on smokers of
modifying products
in order to reduce the intake of harmful
substances.
Status: The single-port smoking duplicator, being
constructed by
Ontario Research Foundation is in the final stages
of evaluation
in our laboratory. Signals generated from the
present Sanborn
differential transducer in conjunction with the
modified
Southampton mouthpiece of 0.75 cm P.D. are at the
lower signal
to noise ratio limit. Although flow measurements
can be
successfully measured with this transducer, more
sensitive
transducers, within the range of measurement with
this mouth-
piece will be required. The present transducers
will be used
for P.D. or effort measurements.
The microprocessor is programed to standardize the
stepping
motor-valve apparatus over a series of 5 different
flows up to
maximum velocity of 3000 cc/min. This is repeated
4 times
:
Uch that a correction feedback signal is placed in
the memory
in order to correctly duplicate the required human
flows.
Presently there is a lag time of approximately 0.3
sec. for the
reproduced flows to reach maximum response with
the master
signal. This will be corrected by adjustment of a
set-screw of
the piston as well as software correction for flow
duplication.
Despite this problem as well as small inconsistent
leaks within
the temporary artificial mouth apparatus, true
duplicated volumes
(as measured with bubble tube) are within 7% of
the actual taped
volumes (measured in the same manner) of a variety
of human puffs.
Nonetheless the microprocessor read-out of
reproduced volume
responds to within 1% of the input volume signal.
There is a designed delay in the feedback
correction of 40
milliseconds between original and corrected flows,
with a
200 millisecond total delay time between actual
and reproduced
smoking signals. The 40 millisecond delay time is
chosen such
M;
00
BATCo document for Province of BritiSh Columbia 23
April 1999


13.
T-8077
T-7051 (cont'd.)
that for each 10 millisecond period, feedback
correction can
be directed to a 4-port multismoker. The total
delay time
between actual and duplicated signals is corrected
on the
microprocessor screen such that simultaneous
overlap can be
observed; different coloured data points designate
where the
duplicated profile velocities are in error
relative to the
original smoking profile.
The actual smoking profiles can be transferred
from the tape
recorder during smoking duplication to floppy disc
tapes for
storage. A printer in conjunction with the
microprocessor is
planned for data retention. The equipment is being
returned
to O.R.F. in August for completion of the
multi-port phase.
An expanded study covering in total 15 smokers
with a
cigarette of 30% increase in nicotine delivery is
completed,
and will be reported as an addendum to R.L. Report
#156. This
larger study verified the results of the
preliminary report
wherein alveolar CO levels were significantly
lower with this
cigarette than with the normal brand, despite both
cigarettes
having similar CO and tar yields as measured by
standard
machine smoking.
A group of 9 Matinde K.S. smokers (Tar: 13 mg.,
Nic: 0.65 mg.)
has been monitored with its normal brand as well
as with a
cigarette of 30% less tar and nicotine. (The
cigarettes varied
in design and tobacco blend.) An increase in mouth
nicotine
with a significantly larger increase in alveolar
CO levels was
noted for all smokers with this changed product,
relative to
those deliveries which may have been anticipated
from standard
smoke delivery data.
A group of 4 Medallion smokers (Tar: 1 mg.. Nic:
0.1 mg.) when
given cigarettes of lesser ventilation (with
variation in tobacco
blend) obtained correspondingly larger mouth
nicotine levels
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4 ~--
CD
(XI
NO
BATCo document for Province of BritiSh Columbia 23
April 1999


14.
T-8077 (cont'd.)
T-7051
and alveolar CO levels. These smokers found the
cigarettes
delivering 0.2 - 0.3 mg. more satisfying but also
slightly more
irritating than their normal cigarette. Both of
the above-
mentioned studies will be repeated with specially
prepared
blend-matched cigarettes such that variations in
ventilation
will be the only product design change. As well,
it is hoped
that the new mouthpiece-transducer apparatus will
be available
so that duplication of this smoking study can be
carried out
when construction of the multi-port puff
duplicator has been
completed. This will provide true mouth nicotine
and CC delivery
levels, which will be useful in better
understanding those factors
actually affecting alveolar CC levels.
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BATCo document for Province of BritiSh Columbia 23
April 1999


1 1) .
T- 7708 Microbial Mutagenicity Tests in Tobacco
and Health Research.
Personnel: M.H. Bilimoria.
Objective: To use microbial mutagenicity tests for
the detection of
potential mutagens and carcinogens in smoke, and
for comparing
tobacco products, both commercial and
experimental.
Status: Prior to utilizing the Ames bacterial
mutation test for
evaluating the B-11 (Janus) series and a series of
samples
provided by Dr. B. Zilkey of the Delhi Research
Station. it
was decided to examine certain steps in the test
procedure.
Most laboratories throughout the world are using
liver
preparations from Arochlor-treated rats in
performing the Ames
test. Since there were no reports in the
literature on the
use of liver preparations from guinea pigs, and
because
metabolic differences between guinea pig and rat
tissue
preparations were noted in one laboratory, we
considered it
worthwhile to compare liver preparations from
these two species
for their aryl hydrocarbon hydroxylase (AHH)
content, and their
ability to activate cigarette smoke condensate
(CSC) to
intermediate(s) mutagenic towards S. typhimurium
TA-98. To
induce the microsomal enzymes, which are
responsible for the
metabolic activation of CSC and other carcinogens,
we employed
either Arochlor 1254, which is a polychlorinated
biphenyl mixture
or 3.4-benzo(-)pyrene. At appropriate intervals
after treatment
with these chemicals, the animals were sacrificed,
the livers
homogenized in Tris buffer, and the homogenates
centrifuged at
9,000g for 15 minutes. The supernatants were
assayed for their
AHH content, and used to determine their abilities
to convert
CSC to mutagenic compound(s).
Arochlor and 3.4-benzo(-)pyrene were equally
effective as
inducers of AHH in the rat liver and always
induced higher levels
of AHH in rat than in guinea pig liver. The guinea
pig liver
preparation had a further disadvantage in that in
the Ames test
it gave high control mutation rates at the higher
CSC concentrations.
Since the liver preparation from the
Arochlor-treated rat appeared
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BATCo document for Province of BritiSh Columbia 23
April 1999


16.
T-7708 (cont'd.)
to give a more linear dose response curve than the
liver
preparation from the benzo(-)pyrene-treated rat,
it was decided
to use this chemical for induction of the
microsomal enzymes.
and the rat liver preparation for all our studies
involving
comparison of condensates from different smoking
products.
We have finished examining cigarettes belonging to
the B-11
series (Janus), and are in the process of
evaluating the data
statistically. Work has commenced on the series of
cigarettes
provided by Dr. B. Zilkey. A report covering these
two series
will be issued in due course.
C=)
BATCO document for Province of British Columbia 23
April 1999


T-7710 The Effect of Tobacco Smoke on Drug
Metabolizing Enzymes in
Mammalian Tissues
Personnel: M.H. Bilimoria and D.J. Ecobichon
(Professor of Pharmacology,
McGill University)
Objective: With Dr. Hogg's and Dr. Witschi's
departures from Montreal in
August last year, a re-evaluation of the project
formerly
Entitled "Biochemical Effects of Tobacco Smoke
Exposure in
Experimental Animals" was made. It was decided
that the
project will continue under the new title shown
above and will
concentrate an studying the induction by tobacco
smoke of tissue
enzymes known to be involved in the metabolism of
drugs as well
as the conversion of procarcinogens into
"ultimate" carcinogens
in adult and very young animals.
The project has received support from the Canadian
Tobacco
Manufacturers Council since the beginning of May
this year
through a grant to McGill University and guidance
is being
given by Dr. D.J. Ecobichon, Professor of
Pharmacology in the
Department of Pharmacology and Therapeutics. This
work continues
to be carried out at the university.
Status: 1. Metabolic activation capability of
rodent tissue preparations.
We have been studying the effect of cigarette
smoke inhalation on
levels of aryl hydrocarbon hydroxylase (AHH) in
rodent tissues.
We have also been monitoring these tissues from
smoke-exposed
animals for their abilities to transform
carcinogenic agents,
such as cigarette smoke condensate (CSC),
benzo(-)pyrene and
2-aminoanthracene, to intermediate(s) mutagenic to
Salmonella
typhimurium TA-98. Such studies would give us a
clue as to what
happens when inert carcinogens inhaled from
polluted air or from
cigarette smoke remain lodged in the lung or other
tissues until
they are metabolized and/or cleared. While we can
activate CSC,
B-naphthylamine and benzo(-)pyrene to
intermediate(s) mutagenic
towards S. typhimurium TA-98, when we use rodent
liver preparations,
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BATCo document for Province of BritiSh Columbia 23
April 1999


18.
T-7710 (Cont'd.)
we have, thus far, failed to activate the above
chemical agents
when we employ lung and kidney preparations from
normal or
smoke-exposed animals. These same lung and kidney
preparations
can activate compounds such as 2-aminoanthracene
and 2-acetylamino-
fluorene, which are not known to occur in tobacco
smoke to any
significant extent. Since our main interest is
CSC, we would
like to be able to activate this agent as well as
other chemicals
known to occur in it, to mutagenic compounds
employing lung tissue
preparations. The lung is the prime target organ
of an agent like
tobacco smoke which is inhaled.
To find out whether or not this inability is due
to the low levels
of microsomal enzymes that are present in these
tissues in contrast
to the liver, we have been examining, for their
metabolic
activation capability, lung and kidney
preparations obtained from
rodents treated with more potent inducers of
microsomal enzymes,
such as benzo(-)pyrene and Arochlor 1254. In the
last semi-annual
report we had reported that we had been successful
in converting
benzo(.)pyrene to intermediates mutagen1c towards
S. typhimurium
TA-98, when we employed a lung preparation from
benzo(-)pyrene-
treated guinea pigs. We now wish to report that a
kidney preparation,
obtained from Arochlor-treated guinea pigs, is
capable of converting
CSC as well as benzo(-)pyrene to intemediate(s)
mutagenic towards
S. typhimurium TA-98. Surprisingly, the lung
preparation from
these same Arochlor-treated guinea pigs did not
possess this
metabolic capability. Further, both lung and
kidney preparations
from Arochlor-treated Sprague-Dawley rats failed
to activate CSC
as well as benzo(-)pyrene to mutagenic compounds,
even though the
AHH content of the rat kidney preparation was
equal to that of the
kidney preparation from the guinea pig. These
results once again
show that ability to metabolize certain
carcinogens, is a function
of the species and its enzymatic make-up. Since
such studies are
of interest to scientists working in the field of
tobacco and
health, further investigation is merited.
BATCo document for Province of BritiSh Columbia 23
April 1999


19.
T-7710 (cont'd.)
2. Induction of AHH in Spraque-Dawley rat tissues
- Dose
response curves usinq diluted smoke produced in
the B-A.T. -
Mason machine.
In earlier dose response studies employing
random-bred male
guinea pigs, we used the Hogg smoking machine in
which the
animals were exposed to undiluted smoke. A linear
increase in
renal AHH was obtained upto about 12 puffs of
undiluted smoke.
In this machine there was no way to dilute the
smoke in order
to increase the precision of the dose response
study, or for
that matter, simulate more realistic smoking
conditions. In
the recently acquired B.-A.T.-Mason smoking
machine it is possible
to use dilutions of smoke ranging from 1:5 to
1:40.
Using this new smoking machine we have already
carried out a
dose response study with respect to induction of
renal AHH in
the rat, at smoke concentrations of 5, 10, 20, 40
& 80 puffs,
and at 1:5, 1 :10 and 1:20 dilutions of cigarette
smoke. An
increase in AHH induction with increase in puff
number has
been obtained, while increasing the dilution of
smoke has
resulted in a decline in AHH induction. This study
will be
followed by one in which the effect of diluted
smoke exposure
on aryl hydrocarbon hydroxylase levels in other
tissues, such
as lung and liver in the male and female of this
species, will
be studied. This will be followed by dose and
time-response
studies using both sexes of other species such as
guinea pigs,
hamsters and gerbils. Eventually, when comparisons
to human
tissues are possible, this information will be
useful in
selecting the most appropriate species for
bioassay purposes.
-L-.
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BATCO document for Province of BritiSh Colurnbia
23 April 1999


20.
T-7709 Chanqes in Bronchial Eyjth~jiuni and Other
Tissues Induced
by Tobacco Smoke.
Personnel: J. Johnson, J.C. Hogg (University of
British Columbia),
J.B. Richardson (McGill)
Objective: To establish biochemical parameters of
tobacco-smoke induced
lesions in the bronchial tree and pulmonary
parenchyma. It
is hoped that one or several parameters can be
defined which
could at some time be developed into a short term
screening
test for the evaluation of product improvement. To
maximize
the relevance of such a test to the effects of
smoking on
humans, priority was given to studies of the
tracheobronchial
tree, over those involving lung tissues.
Status: 1. Bronchial Epithelial Permeability
We have previously reported that exposure of
cigarette
smoke to tracheostomized guinea pigs affects
bronchial
mucosal permeability as reflected in increased
rates of
accumulation of the cytochemical tracer,
horseradish
peroxidase (HRP) in plasma of the systemic
circulation.
We have explored this phenomenon and have
established dose
responses to increasing numbers of puffs of whole
smoke as
well as of vapour phase. We have shown that
exposure to
5 puffs whole smoke has little effect on the mean
rate of
HRP accumulation compared to the control while
exposure to
20 puffs causes a moderate increase. 100 puffs,
however,
results in an approximate 3-fold increase in the
rate of
accumulation of HRP in plasma over the control.
The exposure
regimen of 100 puffs was therefore chosen for
comparative
studies in subsequent experiments. In contrast to
the effect
seen with 100 puffs whole smoke, exposure to
vapour phase does
not cause increased rates of plasma HRP
accumulation over the
control. It is planned to investigate if the
permeability of
the tracheal bronchial epithelium returns to
normal after
exposure to whole smoke has been terminated.
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BATCO document for Province of British Columbia 23
April 1999


T-7709 (cont'd.)
Dosimetry studies varying the dilution and the
number of
puffs are underway with the recently acquired
B.A.T.
animal smoke exposure machine. Comparisons of
cigarettes
differing widely in composition have also been
started
in order to determine if there are different
effects on
the permeability of the bronchial tissue. The
factors
being investigated are the effect of
reconstitution by
the PCL process, stem and elevated nicotine level.
We have also attempted to define the site or sites
of
action of cigarette smoke on mucosal permeability.
Normally
HRP (a glycoprotein of 40,000 daltons) is
restricted from
penetrating the epithelium. This restriction is in
part
afforded by the tight junctions of the epithelial
cells.
The observed effects of increased rates of HRP
accumulation
in plasma following smoke exposure is suggestive
of a
loosening up or opening of these tight junctions.
(Other
studies have shown that even subtle modifications
in the
configuration of these junctions could account for
various
physiopathological phenomena by influencing the
overall
permeability of substances across the epithelium).
Hogg
and Inoue, collaborators in this project, used
freeze-
fracture techniques to morphologically examine the
epithelial
junctions of tracheal epithelium of control and
smoke-exposed
guinea pigs. In the normal epithelium the tight
junction is
seen to be composed of distinct fibrils that join
together to
form discreet compartments - a configuration
associated with
relatively impermeant epithelia. In contrast,
there is a
"beading" of the fibrils resulting in multiple
free fibrillar
endings causing a loss of compartmentalization, in
smoke-
exposed epithelium. Similar changes to these have
been
associated with changes in permeability in in
vitro situations
and it seems likely that these changes relate in
our case to
the increased HRP fluxes.
In summary:
1. Exposure to whole cigarette smoke is associated
with
increased tracheal permeability to HRP.
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22.
T-7709 (Cont'd.)
2. Freeze fracture techniques indicate that the
site
of action may be at the level of the epithelia]
tight junctions.
3. The principal active agents promoting this
lesion
do not appear to reside in the vapour phase, or
at least do not appear in the absence of the
particulate phase.
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April 1999


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BATCo document for Province of British Columbia 23
April 1999