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IMPERIAL TOBACCO LIMITED
RESEARCH & ADVISORY SERVICES DIVISION
MONTREAL
PROGRESS REPORT
OCTOBER 1980 - DECEMBER 1991
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BATCo document for Province of British Columbia 19
April 1999


COPY NO . ......
IMPERIAL TOBACCO LIMITED
RESEARCH & ADVISORY SERVICES DIVISION
MONTREAL
RESTRICTED
PROGRESS REPORT
OCTOBER 1980 - DECEMBER 1981
Issued by: Mr. R.S. Wade Date Issued: March 18,
1982.
DISTRIBUTION:
Copy No.: 1. Library 18. Dr. A. Porter
2. Mr. R.M. Gibb 19. Dr. G.W. Boswall
3. Mr. R.S. Wade 20. Mr. G. DuPlessis
4. Mr. S.M. Candlish 21. Mr. L. Bouchard
5. Dr. T.A. Smith 22. Mr. J. Perry
6. Mr. E. Rittershaus 23. Mr. H. Roubicek
7. Dr. F. Seehofer 24. Miss R.R. Smith
8. Dr. C.J.P. De Siqueira 25. Mr. B.J. Stirlinq
9. Dr. L.C.F. Blackman---' 26. Mr. R.L. Rice
10. Mr. A.L. Heard 27. Mr. A.P. Schaffer
11. Mr. A.L. Heard 28. Mr. E.P. Gage
12. Dr. R.A. Sanford 29. Mr. P. Leblond
13. Dr. R.A. Sanford 30. Mr. W. Tennyson
14. Mr. R.G. Nicholls 31. Mr. W.J. Ross
15. Dr. M.H. Bilimoria 32. Mr. C.J. Brown
16. Dr. P.J. Dunn 33. Mr. C. Warren
17. Mr. M.P. Scherbak
-4
00
C7*1
BATCo document for Province of BritiSh Columbia 19
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INDEX
Paoe Code. Title Researcher(s)
a Summary of Activities
I T-0575 Delhi Research Station Samples for
Industry G.W.B., P.J.D.
Collaborative Evaluation & H.R.
8 T-0774 The Manufacture for Smoking Tests of
Cigars G.W.B. & H.R.
made of Tobacco from the L'Assomption
Experimental Farm
9 T-0821 Study of Methods to Produce Mature
G.w.B., P.J.D.
Flue-Cured Tobacco & H.R.
11 T-7278 Use of Oil/Water Partition to Compare
M.P.S., r,.W.B.,
Flue-Cured Grades & A.P.S.
13 T-7708 Microbial Mutagenicity Tests in Tobacco
M.H.B.
and Health Research
18 T-7710 The Effect of Tobacco Smoke on Drug
M.H.B. & D.J.E.
Metabolizing Enzymes in Mammalian Tissues
C0
BATCo document for Province of BritiSh Columbia 19
April 1999


a
SUMMARY OF ACTIVITIES
OCTOBER 1980 - DECEMBER 1981
AGRONOMY
When applied singly Ambush, an insecticide, and
Enide, a herbicide,
give excellent control of weeds and cutworms in
tobacco. Experiments
using one application of a tank mix of the two
chemicals to a narrow band
over the row have also given excellent results.
Therefore the application
of Ambush and Enide in a tank mix could be a
recommended treatment for
tobacco production if registration of these
chemicals can be altered to
include their use in this manner.
Blue mould was practically non-existent in Ontario
in 1981 perhaps
because Ridomil was used in all tobacco
qreenhouses and on much of the
tobacco in the field for at least one spray
application. Ridomil was granted
only a temporary registration for use on tobacco
in 1981 and as such it
automatically expired at the end of the year.
Ciba-Geigy is concerned that
strains of blue mould resistant to Ridomil may
develop if Ridomil only is
used as a control, and it is therefore promotinq
the idea that a mixture of
Mancozeb and Ridomil would be preferable for 1982.
This proposal has not
been favorably received by either the tobacco
producers or the tobacco
manufacturers.
Ethylene qas, although not registered for use in
Canada, has been used
as a curing aid by many farmers and is beinq
evaluated by the Delhi Research
Station and the tobacco manufacturers. In many
cases it improves the color
and can reduce the curing time, but in some
instances the Smokinn quality
of the treated tobacco is impaired. Testino
continues on samples from the
1981 crop.
Another herbicide. napropamide (Devrinol). if
registration is
granted may be recommended in addition to pebulate
in tobacco production.
When combined they have been shown to give
excellent control of both
grasses and broad leaf weeds without loss of
smoking quality in the treated
tobacco.
co
co
BATCo document for Province of British Columbia 19
April 1999


b
The licensing of new varieties is providing
farmers with a wider
choice of alternatives to qrowinq the old favorite
Virginia 115. Both
Delgold and Newdel which produce higher yields
than the previously
released Nordel are expected to be grown
extensively in 1982. Delgold
was estimated to have been grown on about 50% of
the 1981 Ontario acreage.
For the Maritime Provinces' tobacco production,
Islangold, a high yielding
variety with good quality leaf, and Windel, also
high yielding and very
tolerant to strong winds, have been licensed. The
two new varieties as
well as Delgold are expected to be grown
extensively in the Maritimes
in 1982.
Work continues on efforts to make use of oil/water
partition (D/WP)
data as a scientific aid to leaf blending. A new
tobacco blend generating
model which uses both OJWP data and discriminant
scores has been developed
at GR & DC. Blends to match individual grades will
be generated by this
new model, and cigarettes will be evaluated by a
smoking panel to determine
if the smoking characteristics of the blends are
similar to those of the
target grade.
GENETIC AND INHALATION TOXICOLOGY
Interest in short-term tests such as the Ames
bacterial mutagenicity
test. continues to grow. An interlaboratory study,
involving the Hamburg,
Montreal and Southampton laboratories is in
progress. in which four
cigarette types are being examined for
mutaqenicity in a blind study,
following closely the procedures used in Montreal.
The Hamburg and Montreal
laboratories have finished testing them, while
B.-A.T. Southampton are
setting up the procedure and will examine these
cigarettes in the near
future.
The mutagenicities of smoke condensates from
cigarettes made for the
Janus B-16 series have been determined and are
reported herein. The Janus
B-16 experiment was designed to study the effects
of a casing and of cocoa
and butter-fat on tobacco, on the tumorigenicity
of the smoke condensate.
The results obtained show that while addition of
cocoa has no influence
on the mutaqenicity of the condensate, both
butter-fat and casing.on tobacco
reduce mutagenicity.
L.n
00
BATCo document for Province of British Columbia 19
April 1999


c
Mainstream and sidestream smoke condensates,
collected from the
same flue-cured tobacco cigarettes, were also
examined for mutaqenic
activity, and it was found that both these
condensates were similar in
specific activity based on a unit of condensate.
The fungicides Ridomil, Maneb and Zineb were
tested for mutagenicity
in the Ames system and were found to be devoid of
activity. However, an
examination of smoke condensates showed that Maneb
treatment of tobacco did
increase mutagenic activity significantly while
Zineb treatment did not.
Application of Ridomil to tobacco also did not
increase the mutagenicity of
the condensate, although in one study, where the
supplier's instructions
were intentionally not followed and Ridomil was
applied well into the
harvesting season, a significant increase in
mutaqenicity of the condensate
was observed.
Cigarettes made for a study in which several sheet
materials were
being compared were also studied in the Ames test.
There was no difference
in mutagenic activity of any of the cigarettes in
which these sheets were
incorporated at the 10% level.
Inhalation studies involving tobacco smoke
exposure of small rodents
are continuing at McGill. Work on the induction of
rat pulmonary aryl
hydrocarbon hydroxylase (AHIH), an enzyme
important in the metabolism of
certain compounds to carcinogens, has been
extended to include a I mg.
cigarette. This study has been published
(Toxicology, 22, 205-10, 1981).
as well as reported as an ITL Research Laboratory
Report (No. 163, issued
on 1 Oct., 1981).
Milk and placental transfer studies in
smoke-exposed guinea pigs
have been completed and it has been shown that
smoke components were not
transmitted in breast milk, nor did they cross the
placental barrier in
quantities sufficient to induce A.HH in the pup or
foetal tissues of this
species. Work is now in progress to determine if
smoke components can
reach the young either transplacentally or via the
breast milk in another
c-n
BATCo document for Province of British Columbia 19
April 1999


d
species, the Spraque-Dawley rat. These studies are
relevant in that
there is increasing evidence that human foetuses
are susceptible to low
concentrations of a variety of chemicals, druqs
and environmental
pollutants resulting in cancer or other
abnormalities in later life.
BATCo document for Province of BritiSh Columbia 19
April 1999


T-0575 Delhi Research Station Samples for Industry
Collaborative Evaluation
Personnel: G.W. Boswall, P.J. Dunn, H. Houbicek
Objective: To evaluate and compare cigarettes made
from varieties and
other flue-cured tobaccos produced under various
experimental conditions by the Delhi Research
Station.
Status: This is an ongoing project which involves
the four major
cigarette manufacturing companies in Canada In
evaluating
tobacco samples from the more promising
experimental
varieties and cultural treatments under
investigation at
the Delhi Research Station.
Of the many promising samples from the 1980
experiments
submitted by Delhi to industry for smoking tests,
several
were found to have acceptable smoking
characteristics and a
number of these are commented on in the following
paragraphs.
Insecticides
It has been shown that an application of the
insecticide
Ambush as a tank mix with the herbicide Enide to a
10" band
over the row after planting the tobacco is very
effective
for controlling cutworms, grasses and broad leaf
weeds.
Because the chemicals are applied as a band no
materials
are wasted between the rows, the amount of
chemical applied
per acre is reduced and a substantial cost saving
results.
This treatment 13 being proposed by the station,
but tank
Six Use of Ambush and Enide is not Included in
their
registrations and this will be necessary before it
can be
recommended. (=D
1.0
BATCo document for Province of BritiSh Columbia 19
April 1999


2.
T-0575 (cont'd) Delhi Research Station Samples for
Industry
Collaborative Evaluation (cont'd)
Fungicides
Although blue mold occurred on only a few farms in
1980 and
1981, the threat of infection spreading from
Central
America and the United States remains high.
Consequently
the study of fungicides to control blue mold
continues and
several tobacco samples were produced in 1980
using the
following fungicide treatments.
Ridomil, 0.63 kg ai/ha (normal rate)
Ridomil, 1.25 kg ai/ha (2 x normal rate)
Bravo, 8.4 kg ai/ha
Bravo, 4.2 kg al/ha + Ridomil, 1.68 kg ai/ha
Prevacure, 1.68 kg ai/ha
Maneb + Ridomil (4:1) 1.68 kg ai/ha
SN78314, 1.68 kg ai/ha
Galben, 0.40 kg ai/ha in planting water
Ridomil EC incorporated in soil, 0.63 kg ai/ha
Ridomil EC sprayed on fumigation hill, 0.63 kg
ai/ha
Of the ten treatments inve3tigated, seven were
foliar
sprays and the remaining three Involved different
ways of
applying a fungicide to the 30il. Because of
faulty
experimental design and the re3Ulting differences
in smoke
chemical Composition (alkaloids level in
particular) of the
cigarettes compared by the subjective smoking test
panels,
the only conclusion that might be drawn is that
none of the
treatments had any negative effect on the taste
characteristics of the tobacco.
C>
N-i>
110
LM
BATCo document for Province of BritiSh Columbia 19
April 1999


3.
T-0575 (cont'd) Delhi Research Station Samples for
Industry
Collaborative Evaluation (cont'd)
In 1981 Mancozeb-Ridomil foliar sprays and Ridomil
incorporated into the soil were tested.
Mancozeb-Ridomil (8:1) 5.4 kg ai/ha (proposed
rate)
Mancozeb-Ridomil (8:1) 10.8 kg ai/ha (2 x proposed
rate)
Mancozeb-Ridomil (6.1) 4.2 kg ai/ha (proposed
rate)
Mancozeb-Ridomil (6.1) 8.4 kg ai/ha (2 x proposed
rate)
and one treatment with Ridomil incorporated into
the soil
i.e. Ridomil EC, 1 kg ai/ha in soil.
The chemical composition or the tobaccos from all
treatments including the untreated control were
very
similar, and the subjective smoking tests showed
no
significant differences between the treated
tobaccos and
the control.
Dithiocarbamate and Ridomil residues were
determined on
some samples from the 1981 experiments at the
Research
Station and these values are shown in Table I. The
residue
levels in these Canadian samples are not eXCe331ve
in view
of the German Cigarette Industry tolerance of 50
ppm CS 2
for tobacco
BATCo document for Province of BritiSh Columbia 19
April 1999


4.
T-0575 Ccont'd) Delhi Research Station Samples for
Industry
Collaborative Evaluation (cont'd)
Table I
RESIDUES OF DITHIOCARBAMATE (EBDC) AND RIDOMIL
(METALAXYL)
IN TOBACCO OF TEST CIGARETTES
ZINEB EQUIVALENT METALAXYL
TREATMENT EBDC ppm ppm
MANCOZEB-RIDOMIL (8:1) 5.4 kg/ha 12 0.45
MANCOZEB-RIDOMIL (8:1) 10.8 kg/ha 37 0.95
MANCOZEB-RIDOKIL (6:1) 4.2 kg/ha 17 0.58
MANCOZEB-RIDOKIL (6:1) 8.4 kg/ha 34 1.20
RIDOMIL INCORPORATED IN SOIL 1.0 kg/ha 3.3 0.14
UNTREATED CONTROL 1.8 o.o&
To date Ridomil is the only fungicide for which
Agriculture
Canada has granted even a temporary registration
(expires
at the end of the calendar year it was granted)
for
controlling blue mold. Ciba-Geigy, the producer of
Ridomil, is concerned that resistant strains of
'Peronospera tabacinal are likely to develop if
Ridomil 13
used alone and it is promoting the idea that a
mixture of
fungicides, such as a dithiocarbonate with Ridomil
should
be used as a foliar spray. In fact Ridomil may be
withdrawn by Ciba-Geigy in favor of a
Mancozeb-Ridomil
mixture. Both the tobacco growers (OFCTGKB) and
the
tobacco manufacturers (CTMC) have gone on record
as
preferring the foliar application of only Ridomil.
Full
registration of Ridomil is very difficult to
obtain in
Canada because of Ottawa's insistence on extensive
residue
& metabolite data which have not been provided to
date in
sufficient detail.
BATCO document for Province of BritiSh Columbia 19
April 1999


5.
Herbicides
A herbicide napropamide (Devrinol) at an
application rate
of 1 kg/ha could become a recommended chemical in
tobacco
production to control annual grasses if
registration is
granted to the Stauffer Chemical Company, the
supplier. It
has been tested alone and with pebulate and in the
latter
case excellent control of both grasses and broad
leaf weeds
is achieved.
Chemical Curing Aids
Although ethylene gas is not registered for use on
tobacco
in Canada, it is estimated that it has been Used
by perhaps
20% of Ontario growers who believe that it
improves the
color of the tobacco and decreases the time and
cost of
curing. Samples obtained by the Delhi Research
Station
have been evaluated by the tobacco companies for
smoking
quality, but the results to date are rather
inconsistent.
Where effects were noticeable the smoking quality
of the
ethylene treated tobacco was considered inferior.
Therefore the companies cannot at this time
endorse the use
of ethylene 633. Further testing will be carried
out on
samples from the 1981 crop.
Varieties
Throughout the 1970'3 Virginia 115 was the popular
variety
with Ontario flue cured tobacco growers and it was
grown on
more than 50% of the total acreage rising in 1976
to a high
of 90%. In spite Of Some shortcomings such &3
'difficult
to top early' and the production of grey spongy or
muddy
faced tobacco, no other variety could overcome the
popularity of Virginia 115 under Ontario's growing
INC
conditions.
Lt
N-C
C71
BATCo document for Province of BritiSh Columbia 19
April 1999


6.
T-0575 (cont'd) Delhi Research Station Samples for
Industry
Collaborative Evaluation (cont'd)
In recent years the Delhi Research Station as a
result Of
an intensive breeding and selection program,
developed
several new strains which have performed extremely
well in
comparative variety tests. As licensed varieties,
some
should gain favour with growers and tobacco
processors
alike because of superior leaf quality and higher
yields.
The first to be released was Nordel (strain N3)
whose
background includes N. RUStica, Delhi 34 and
Virginia 115.
Although it produces a superior quality of leaf,
its modest
yield (about 100 lb per acre le33 than Virginia
115) will
probably make it unattractive to a lot of growers.
Recently 8 sister line MB) to be known as Newdel
was
licensed. Its leaf is Of similar quality to Nordel
but its
yield exceeds that of Virginia 115 by about 100 lb
per acre.
Another new variety that has been licensed is
Delgold,
previously known a3'sirain N2-E. Delgold has a
background
of N. Rustica, Hicks Broadleaf and Virginia 115,
outyield3
Virginia 115 by about 300 1b per acre and 3hOW3
great*r
tolerance to black root rot and weatherfleck. The
grade
composition Of its leaf is Considered superior to
that of
Virginia 115 by both dOMe3tic and export buyer3,
and it has
about 0.5% higher total alkaloid3. It has been
estimated
that Delgold made up at least 50% of the tobacco
acreage In
Ontario In 1981.
C=)
BATCo document for Province of BritiSh Columbia 19
April 1999


7.
T-0575 (cont'd) Delhi Research Station Samples for
Industry
Collaborative Evaluation (cont'd)
In the Maritime Provinces a lot of low quality
tobacco is
produced and again there is a recognized need for
a variety
to replace Virginia 115. Delgold may fill the need
to 30MO
extent, but two other new varieties, Islangold and
Windel,
have been licensed especially for that region.
I31angold
and Windel, previously known as strain 77C15
(Delhi 34 x
Speight G7) and strain 77C11 (Delhi 34 x Virginia
115)
respectively, stand up well to the rigours of the
Maritime
climate and the yields Of Usable tobacco surpass
that of
Virginia 115. Although the alkaloid level or
Islangold is
lower than most varieties by about 0.5% it
produces a high
yield of good quality usable leaf. Windel has
shown
superior tolerance to high winds and prodUC03 a
high yield
of quality tobacco whose sugar and alkaloid levels
are
comparable to Virginia 115.
C:
%-4
11 C
CX
BATCo document for Province of BritiSh Columbia 19
April 1999


8.
T-0774 The Manufacture for Smoking Tests of Cigars
made of Tobacco
from the L'Usomption 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: The L'Assomption Experimental Farm
operates a subjective
smoking test panel, composed of members recruited
throughout the Agriculture Canada organization, to
evaluate
the cigar tobaccos produced in field experiments.
The
tobacco samples are subjected to essentially the
processes as commercial cigar tobacco by being
routed
through the growers cooperative at St. Jacques,
our cigar
processing plant at Joliette, and finally
manufactured into
cigars by normal production equipment in Montreal.
During 1981 Imperial made cigars from short filler
samples
of new breeding strains and varieties produced in
1979 for
evaluation by the Agriculture Canada smoking
panel. In
recent years the Vk3somption Experimental Farm has
not
identified any new strain or variety worthy of
licensing.
The Imperial cigar smoking panel evaluated the
smoking
quality of cigar tobaccos which had received
foliar sprays
of Ridomil. The normal rate of application had no
effect
on the smoking quality of the tobacco, but double
the
normal rate produced a smoke which tended to have
better
aroma, more taste and more irritation, but not
sufficiently
so to be significant.
Ly
BATCo document for Province of BritiSh Columbia 19
April 1999


9.
T-0821 Study of Methods to Produce Mature
Flue-Cured Tobacco
Personnel: G.W. Bo3wall, P.J. Dunn, H. Roubicek
Objective: To evaluate some cultural practices
which have been
suggested as factors contributing to the
production of more
mature tobacco with better rlavour
characteristics.
Status: This experiment is being conducted at the
Delhi Re3each
Station as a cooperative project with Imperial
Tobacco
Ltd. There are four treatments which are
replicated four
times. All Plots are harvested at the same time as
the
control which is harvested at a time determined by
its
state of maturity as judged by the Research
Station staff.
The project was begun in 1980 and is likely to be
carried
on for at least three years.
Treatment Spacing Topping
A 42" x 241 15-16 leaves (early bud stage)
B (Control) 42" x 24" 17-18 leaves (late bud
stage)
C 42" x 28" 15-16 leaves (early bud stage)
D 42" x 28" 17-18 leaves (late bud stage)
The trends noted from the first year's data (1980
crop) may be
summarized as follows:
1. Spacing on the row (24" v3 28") was not a
significant factor
for Most measured attributes.
2. Earlier and lower topping reduced yield by
about 250 lb. per
acre and reduced monetary value by $300 per acre.
3. Earlier and lower topping produced a somewhat
more mature
type of leaf Whose nicotine content Was about 0.5%
higher.
4. Smoking tests of the tobaccos from each
treatment indicated
only that the lower and earlier topped treatment
resulted In
a marginally less acceptable taste quality with a
slight 110
strength Increase especially with respect to
impact.
f%0
C=>
C=)
BATCo document for Province of BritiSh Columbia 19
April 1999


10.
T-0821 (cont'd) Study of Hethod3 to Produce Mature
Flue-Cured Tobacco
(cont,d)
MATURITY EXPERIMENT
1980 DATA
YIELD VALUE NICOTINE R. SUGAR TAR NICOTINE CO
TREATMENT lb3/Ac. $/Ac. % % mg/cigt. MR/cigt.
mg/cigt.
A 2192 3123 2.76 24.0 26.6 2.84 22.b
B 2538 3555 2.33 24.9 26.7 2.43 21.8
C 2242 3189 2.74 22.0 26.1 2.88 22.5
D 2393 3364 2.19 23.9 26.4 2.36 21.7
%.C
Lr
BATCo document for Province of BritiSh Columbia 19
April 1999


11.
T-T2T8 Use of Oil/Water Partition to Compare
Flue-cured Grades
Personnel: M.P. Scherbak, G.W. Boswall, A.P.
Schaffer
Objective: To study the smoke differences among
cigarettes of
representative single grades or flue-cured
tobaccos from
Canada together with some grades from the United
States so
as to obtain a better understanding of the scope
for
achieving product development goals through lear
blending.
Status: This project is being conducted to
determine If smoke
characteristics Of individual grades can be used
to
generate a blend of grades whose smoking
characteristics
match those of a target grade. The chemical
composition of
smoke from rlue-cured tobacco grades, as reflected
by
chromatographic analysis of tobacco smoke (Using
the O/WP
technique) is being investigated as an 'objective'
measure
for the characterization of tobacco grades.
Traditionally tobacco is sorted into relatively
homogeneous
types (grades) and at Imperial Tobacco these are
arranged
into groups in a leaf classification grid whose
aXe3 are
stalk Position and maturity-bleMi3h. Computer
operated
di3criminant analysis has shown that O/WP data
provide
sufficient objective information, using specific
O/WP
peaks, to generate a mathematical classification
grid.
The power of the model is such that
reclassification Of the
grades by the model places them in the same grid
positions
as did the leaf graders. The specific O/WP peaks
selected
by di3criminant analysis for classification
purposes have
been Used in a linear programing system (MPS) to
derive a
blend or grades to match a target grade. With
hand-made
cigarettes we have had inconsistent results when
smoking
characteristics of the blends were compare to
those of the
target grade.
Lr
BATCo document for Province of BritiSh Columbia 19
April 1999


12.
T-7278 (cont'd) Use of Oil/Water Partition to
Compare Flue-cured Grades
(cont'd)
Dr. George Few, Southampton, Visited Us in
December 1981
and introduced a new blend generating model,
recently
developed at GR & DC, which utilizes both O/WP
data and
d13criminant scores. Since both the chemistry of
the smoke
(O/WP data) and d13criminant scores (a reflection
of the
leaf blenders expertise) are combined in the
model, more
precise matching may result. Fxperiment3 will be
undertaken to determine if it is superior to the
old model.
Lil
C:>
BATCo document for Province of BritiSh Columbia 19
April 1999


13.
T-7708 Microbial Mutagenicity Tests in Tobacco and
Health Research
Personnel: M.H. Bilimoria
Objective: To use microbial mutaqenicity tests for
the detection of
potential mutagens and carcinogens in smoke, and
for
comparing tobacco products, both commercial and
experimental.
Status: 1. Janus B-16 ciqarettes
This series, consisting of 14 samples. was
desiqned to study
the bioloqical effects of the application of a
casing
(formulation available at B.-A.T. Southampton) and
of cocoa
and butter-fat on tobacco. As the Hunter Committee
did not
permit the use of cocoa as a tobacco additive in
the U.K.,
B.-A.T. Southampton decided not to undertake skin
carcinogenicity
and inhalation tests on this group of cigarettes.
However, since
cocoa is used to a large extent in the U.S.A. and
to a lesser
extent in Canada, it was considered useful to
examine this
Janus series for their Ames mutaqenicity.
Combinations of cocoa
having butter-fat contents of 11 and 21% were used
to introduce
different levels of butter-fat in tobacco. The
mutagenicities
were expressed as the number of His* revertants
per Ug of
condensate at dose levels of 50. 100, 150, 200 and
300 pq per
plate. Means and standard deviations were
calculated for each
cigarette from a total of 20 data points. An ANOVA
was carried
out to determine whether the differences between
the means
%.C
BATCo document for Province of BritiSh Columbia 19
April 1999


14.
T-7708 Microbial Mutagenicity Tests in Tobacco and
Health Research
(Cont'd.) (cont'd.)
were significant or not, and the
Student-Newnan-Keul test to
determine which of the means (mutagenicities) were
significantly different from each other. A
description.of
the cigarettes and results obtained are presented
in Table 1.
as well as described below:-
(a) Cocoa as an additive: When mutagenicity is
plotted
aoainst percent butter-fat added to tobacco as
shown in
Figure I it is evident that the addition of
butter-fat
appears to reduce the mutagenicity of the
condensate.
If the data in Figure I are analysed by a linear
regression.
a regression line Y - 3.67 - 2.98X is obtained
with
correlation coefficient r --0.47 (p<0.09). If the
first
four points are omitted from the analysis
(reduction in
mutagenicity manifests only at concentrations of
butter-fat
higher than 0.5%), the negative association
between
mutagenicity and butter-fat is Considerably
increased. The
regression line now obtained is Y = 4.13 - 6.14X
with the
correlation coefficient r - -0.66 (p<0.03). From
the fioure
it will also be seen that the addition of cocoa
itself has
no influence on the mutaqenicity of the condensate
(r =-0.12;
P>0.25).
(b) Casing of tobacco: From Table I it will be
seen that
in every instance. the mutagenicity of the
condensate was
reduced, by the application of casing to the
tobacco. The
drop in mutagenicity is also evident from a plot
of mutaqenicity
against butter-fat content of tobacco as shown in
Figure 1.
where every condensate from a cigarette made with
cased
tobacco was lower in mutagenicity than the
corresponding
cigarette made with uncased tobacco. A paired
't'-test
showed a significant difference between the
uncased and cased
tobaccos. However, each of these individual
differences was c:;)
small and not significantly different by the
Student-Newman- %-D
Keul test.
Qn
BATCo document for Province of BritiSh Columbia 19
April 1999


15.
T-7708 Microbial Mutaqenicity Tests in Tobacco and
Health Research
(cont'd.) (cont'd.)
2. Mainstream and sidestream smoke condensates
Recent reports on the effects of passive smoking
on
respiratory function and lung cancer have led to a
renewed
interest in sidestream and ambient smoke. Since
B.-A.T.
Southampton had available an inhalation system for
exposing
small rodents to sidestream smoke, a visit there
in July 1980
resulted in our obtaining four lots of mainstream
and side-
stream smoke condensates collected from the same
flue-cured
tobacco cigarettes. These condensates, collected
on Cambridge
filter pads were extracted with 95% ethanol and
tested for
mutagenicity by the standard procedure followed in
this
laboratory. Condensates from both mainstream and
sidestream
smoke were found to be similar in terms of
specific mutagenic
activity. The results have already been reported
in a
Research Laboratory Report (No. 165 issued 13 May,
1981).
3. Fungicide-treated tobacco cigarettes
The blue mould epidemic in 1979 which resulted in
the
destruction of a considerable portion of the
Ontario flue-cured
tobacco crop, has stimulated interest in fungicide
application
to tobacco and the possible biological effects in
humans of
such treatment. In order to prevent a repetition
of the 1979
disaster, Ridomil was widely used on the 1980
Ontario crop
with no loss of tobacco to the fungus. Other
fungicides, which
have been considered for the control of tobacco
blue mould in
the field in Canada. are Maneb and Zineb, which
are dithio-
carbamates containing manganese and zinc,
respectively.
The fungicides Ridomil, Maneb and Zineb were not
mutagenic in
the Ames system either with or without the
addition of a rat
liver activation system. However, the
dithiocarbamates were
found to be quite toxic in the Ames system, and it
is quite
possible that this toxicity was responsible for
the above
negative Ames test. C=)
Un
rl.-)
C=)
BATCo document for Province of British Columbia 19
April 1999


16.
T-7708 Microbial Muta enicity Tests in Tobacco and
Health Research
(cont'd.) (cont'd.?
An examination of smoke condensates from
Ridomil-treated
tobacco ciqarettes, suggests that this fungicide.
if applied
ccording to the manufacturer's instructions, does
not
:
ignificantly increase the mutaqenic activity of
the smoke
condensate, althoUqh siqnificant increases in
mutagenic activity
were obtained when Pidemil was aoplied into the
harvesting
period. (see ITL Research Laboratory Report No.
162 issued
May 13, 1981). An examination of condensates from
Maneb and
Zineb-treated tobacco cigarettes yield interestinq
results
in that Zineb treatment did not result in anv
increase in Ames
mutagenic activity, while Maneb treatment did
increase
mutagenic activity very significantly. These
results are
presented in Table 2 and Figure 2. Since the only
difference
in Maneb and Zineb resides in the inorganic
portions of the
molecules it is obvious that the manqanese ion is
associated
with increased mutagenicity. These results suggest
that
Maneb itself be re-examined in a modified Ames,
should these
dithiocarbamates be needed for any future use in
blue mould
control.
4. Project T-5568: The comparison of AMF sheet
with regular
PCL and with two experiment4l PCL's.
This project was designed to compare the physical
and chemical
properties and smoking qualities of the current
ITL sheet
(PCL) with a new sheet (MCF) made by AMF Inc.. for
trial by
ITL, as well as with two experimental ITL sheets.
The new
sheets were produced as part of an ongoing effort
to improve
PCL made by ITL.
Since it was considered important to know whether
incorporation
of these new sheet materials into cigarettes would
alter the
biological properties of the smoke, a request was
made by
Mr. R.M. Gibb to examine these cigarettes for
their Ames
mutagenicity.
N-
BATCo document for Province of BritiSh Columbia 19
April 1999


17.
T-7708 Microbial Mutagenicity Tests in Tobacco and
Health Research
(cont'd.) (cont'd.)
From Table 3 it will be seen that there was no
difference in
mutagenic activity of any of the cigarettes in
which these
sheets were incorporated, all of them being
similar to the
Players Check cigarettes used as control. This is
not
surprising when it is considered that the amount
of tar
contributed by the sheets at 101, incorporation
will be
very small compared to that produced from the
tobacco.
It would have been interestino to examine
cigarettes made
from 100% sheet material, but these were not
available.
A Laboratory report has been prepared and will be
issued
shortly.
5. B.-A.T. collaborative Aines testing of
cigarette smoke
condensates.
At the July 1981 Biological Research Conference it
was decided
that the B.-A.T. laboratories at Hamburg. Montreal
and
Southampton undertake a collaborative study of
four cigarette
brands employing the Ames test as developed in
Montreal.
It was agreed that B.-A.T. Hamburg would select
the cigarettes
and distribute them blind to the three
laboratories involved.
Hamburg and Montreal were to beqin testing
immediately, while
Southampton was to begin as soon as they had set
up the Aines
assay procedures.
Hamburg and Montreal have now completed the
testing of these
cigarettes. Complete details of this study will be
published
as a joint B-A.T. report as soon as Southampton
has finished
the testing of these same cigarettes.
C.Pb
1.30
N)
C:Z>
CO
BATCo document for Province of BritiSh Columbia 19
April 1999


TABLE I
Mutaqeniclty of smoke condensates from cinarettes
belo-inq to the Janus B-16 series
Specific mutau4nicity-
cod, Casing Cocoa Butter-f.t of His+ revertants
%
,
-98 per ug
condensate.*
616-1 Nil Nil N 652120.79
"
9164 8.7 Nil Nil :128ho.s)
816-1 Nil 0.41 O.OS ).S10810.69
616-4 8.7 0.37 0.05 1.4818.t0.69
616-5 Nil 0.82 0.10 3.9584*0.7S
6-6
1 0.6 0.75 0.09 3.6722-0.77
.4
:
Nil
0.41
O.OR
3.9636,0.SS
1-8 OJ 0.37 0.07 LMA-.0.88
16.9 Nil 0.02 0.16 3.:0010.11S
16-11)
: 8.6 0.74 0.14
60110.93
2.
1
WI Nil 0.41 1
0. 1
3. 37820.94
3
.6-12
: 8.7 0.37 0.;0 10-99
2-910
816-13 Nil 0.81 0. 2 3.103 !0 .78
2
8.6 0.74 O.zo 2.s945,0.67
SIqnIf1cance of the difference In means by the
Student-N-q-#'.-~e.l test
Code No.
1116-1111116-2 $16-3 516-4 816-5 816-6 616-71616-8
016-91916-10 616-11 B16-12 616-1: 81W4
1 its pis PIS PIS NS PIS HS "S S "S S Ns Ss
is PIS :S :S S Ns IIS NS PIS HS Ns 4S
S S
S
S
S
hs
Pis
NS
PIS
NS
"S
NS Ss
:S Ns NS NS NS N5 HS IIS
"
S PIS "s NS S
S S us "S NS NS HS "S IS PIS Ss S Ss
"S NS PIS "S NS NS PIS Ils 5 NS 5 MS ss
14 S 5 "S NS NS NS NS NS SS NS S5 S SS
RS NS
I'S
"S NS S
: NS NS S PIS S NS Ss
NS MS
I
:S
:5 PIS 5 NS NS NS NS ?is PIS S
S 1,
S
S
S
S
S S
SS
S
N
s "s NS
S
N
S PIS Ns NS PIS NS NS 5 Ns 11 N
S
S 'is "S NS SS S ss S
: NS NS PIS NS PIS
PIS .45 "S NS S NS S S NS NS Ns Hs 4S
SS PIS SS S SS SS ss ss S Ns NS
*7 he value% given are Mean IS.D.. calculated from
4 replications at S concentrations of condensate,
each concentration In trfplicdte.
NS x not significantly different; S siqnlficantly
different at pcO.05; SS siqnificantly different at
P 40.01,
Wq/6601


FIG. BUTTER-FAT CONTENT OF TOBACCO AND AMES
MUTAGENICITY
.$do C"ING
No COCOA
3-5-
g:bCASING
V) NOCOr-OA
3-0,
w 2-S
2
2-0 O~is
0 0.0s 0
pER CENT BUTTER-FAT
0-20
-j
t_n
NJ
BATCo document for Province of British Columbia 19
April 1999


0
0
CL
0
M
0
0
(D
0
_01
0
0
.JL
V
TARLE
NU TACtNIC ACTIVITIES- BY TNE AMES TEST OF
SMOKE CONDENSATES FROM
CICARETTES
MADF IJST14C MANrB AND Z1Nt -T1EATFD TOBACCOS,
Cod.
ungicide applied
to t-b.cc.
og.ritheic
.... I.ti..
Coefficient
ransfor
lope R-g,.6.1 ...
ati.n of data
nt*rC~Pt
(MUTACENICIT free
ntr... (.,
orrtlation
Ir coefficient
ad data
I.P.
NUTAGENICIT) MUTACENICITY
NO. of H10'
":`;It
T ::t,
c OR
. 0
M-tS.D. Significance of the diff .......
Ift muts9-nicitle. by the
--t-11 .......... test
Cod. no.
239 463 611 Check 11
239 - control 0.9699 1.09)3 0.6S07 0.9619 2.0741
"S 38
463 - Zin.b 0.9629 1.2600 0.6650 0.9410 2.4705
2.1737to.35 HS SB "S
611 - 14 ... b 0.9775 1.2900 0.9571 0.973? 3.6333
3.146420,59 53 Ss SS
check 23 0.9112 1.2563 0.6424 0.9632 2.4156
2.10t310.35 us No
*Calculated two- four replications at five
concentrations of condensate, each concentration
to triplicate.
tcalculeted separately at each concentration of
snake condensate.
Is a not significantly difterm.tj SS Z
signittContly different at p~0.01.
IIZSL6601


R92: Ant es Mutagenicity of Smoke Condensates from
Maneb
and Zineb-Treated Tobacco Cigarettes
1500
CL
1-11
CD
E
1000-
E
0
.500-
0
6
z
1
DMoneb
0 Zineb
0 Check 23
0 Control
100 200 300 400
Cigarette Smoke Condensate -mg/Plate
BATCo document for Province of British Columbia 19
April 1999


0
0
CL
0
r+
0
o
=r
0
0
>
V
code a.. -4
Met typ.
Play.,. Check 23
*45 - FCL
143 - Aff P,-...
$20 - ITL Modified
.1
F ..1.
TAILE 3
Mutamenic activitlee* by the A- t..t Of a,o0ke
--d.n..t, ..Je --t-t rgur
different sheet materials at t he lot level
(Froijet T-5~6g).
Itegf.a.ions IT" MTAGE14IC17Y- SIg.IfIc.... of the
diff ... n-
mi. + 1. mut.g.nkitl.o by the Swd..t-
Logarithmic tra,mefocimation of date
got.... ad date
'07
onto
Me - .-K..1 teat
C.'r.l.tion I.t.-Pt lCorrelstion U.P. code
III.P. -d-.t. f
coefficient Caefficl-t (Hutagentelty) Ik.n I S.D.
Check 23 605 14) $53
52.0
0.97" 1.2606 0.3520 0.9634 3.05?9 2.471610.46 - PS
"s "s ws
0.9771 1.2640 0.5540 0.9621 ).1131 2.4788t0.49 us
- "S p$ HS
0.9164 1.2191 0.3620 0.9714 2.8053 2.5255t0.43 H3
"S - PS Pis
0.9741 1.2734 0.5488 0.9519 3.0554 2.4639to.so "s
FS "S - HS
0.991) 1.3127 0.3452 0.9723 3.1190 2.4455!0.s0 14S
VS NS "S
0
:.4
.
6 1.59
.73, 0
:
:
Calcolot ed fro. f"r replications at live
conceatrat ion. of conden-ate, each coscentretion
In triplicate.
Calculat e4 9*lpsrac*ly at each com-tration of
sooke cond-ate.
us-"t 816.1fic.mly diff-eat


18.
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) - Supported in part by C.T.M.C.
Objective: The main objective of this study is to
explore the
biochemical mechanisms whereby tobacco smoke
exerts its
toxic effects in animal and human tissues.
Status: 1. Sprague-Dawley rat pulmonary AHH-Dose
response relationships:
Since considerable induction of rat pulmonary AHH
enzyme
was obtained at all smoke dilutions of Players
Check and Matin6e
Extra Mild cigarettes. it was considered necessary
to include
an even lower tar delivery cigarette in this dose
response study.
Medallion Ultra Mild cigarettes, which deliver I
mg. tar per
cigarette, were selected. From the results
presented in Figure 1.
it will be seen that an increase in AHH activity
is still
observed at 200 and 300-second exposure of the 1:5
dilution of
smoke from.these 1 mg. cigarettes. Thus, rat lung
AHH appears
to be an extremely responsive enzyme to very low
levels of
inhaled cigarette smoke and has potential for
development into
a short-term inhalation bioassay for the
assessment of modern
low-tar delivery cigarettes. However, this extreme
sensitivity
is also a drawback in that higher tar products
(>15 mg. tar)
will be difficult to assay unless the B.-A.T.
machine is
modified to obtain dilutions higher than the 1:40
that is
NJ
BATCo document for Province of BritiSh Columbia 19
April 1999


19.
T-7710 The Effect of Tobacco Smoke on Drug
Metabolizing Enzymes
(cont'd.) in Mammalian Tissues (cont'd.)
presently possible.
The data obtained in the above study were
sufficient to
investigate the lower end of the dose response
curve, but
were inadequate to make a quantitative comparison
between
the cigarettes. In order to compare cigarettes in
this
inhalation test, more attention would have to be
paid to
monitoring tar in the exposure chamber as well as
to the
amount of smoke the rodents actually inhaled.
Initial studies
would involve comparison of ciqarettes made from
vastly
different tobaccos, but delivering the same amount
of tar.
achieved either by cigarette dilution or by
dilution of
smoke in the inhalation machine. Finally, it would
also be
important,to employ the same batch of animals in
each study,
which was not done in the previous experiments.
2. Effects of smoke exposure on perinatal animals:
Epidemiological studies suggest that babies born
to smoking
mothers generally are lower in weight at birth
than are those
born to non-smokers. It is also known that smoke
constituents
such as nicotine, carbon monoxide and cyanide will
cross the
placental barrier. Thus it was considered of
interest to
determine whether exposure of rodents to cigarette
smoke would
result in other potentially hazardous smoke
components reaching
the pups either transplacentally or via the breast
milk.
Tissue responses of the perinatal animals to smoke
components
inhaled by the dams was assessed by the analysis
of AHH. an
enzyme important in the production of "ultimate"
carcinogenic
intermediates from the polycyclic hydrocarbons.
(a) The guinea pia
The results of experiments undertaken to study
possible
transfer via breast milk in the guinea pig are
presented in
Table 1. From this table it will be seen that
smoke
components did not appear to be present in
sufficient U1
BATCo document for Province of British Columbia 19
April 1999


20.
T-7710 The Effect of Tobacco Smoke on Druq
Metabolizing Enzymes
(cont'd.) in Mamma lian Tissues (cont'd.)
quantities in the breast milk to siqnificantly
influence
the levels of AHH in pup tissues. Statistical
significance
was attained in the dam lung and kidney as well as
in pup
liver. However, of these, only the tissue response
in dam
kidney was of biological sionificance. a
conclusion reached
based on our previously published results. The
statistical
significance attained in the dam lung and pup
liver may be
artefactually due to possible seasonal variations
in enzyme
levels. Difficulties in obtaining sufficient
numbers of
pregnant guinea pigs resulted in our studying
control
pregnant guinea pigs at a different period in time
from the
smoke-treated animals.
With the negative results obtained in the milk
transfer
experiments, it was considered necessary to
determine
whether young guinea pigs would respond to direct
exposure
to cigarette smoke. From Table 2 it will be seen
that ANH
is induced only in the kidney of 2-day old guinea
pigs
exposed directly to cigarette smoke and in this
respect is
similar to the responses of adult guinea pigs.
In experiments designed to study the placental
transfer of
smoke components, pregnant guinea pigs were
exposed to
cigarette smoke till near term. at which staqe the
animals
were sacrificed and the tissues assayed for AHH.
The results
presented in Table 3 show that smoke components
did not
appear to cross the placental barrier of the
guinea pig in
concentrations sufficient to induce AHH in foetal
tissues.
Finally. it may be mentioned here that these same
tissues
which have been examined here for AHH activity,
will also
be used to test whether or not their metabolic
activation
capability, in an Ames system, has been altered.
Smoke
BATCo document for Province of British Columbia 19
April 1999


21.
T-7710 The effect of Tobacco Smoke on Drug
Metabolizing Enzymes
(cont'd.) in Mammalian Tissues (contd.)
exposure of dam and subsequent transfer of smoke
components
through the milk or placenta could well alter the
metabolic
activation capability of pup tissue without
changes in AHH
activity.
(b) The Sprague-Dawley rat
On account of the negative results obtained in the
guinea pig,
it was important that we study at least one other
animal
species to confi rm whether smoke components can
reach the
young either transplacentally or via the breast
milk.
Because of the sensitivity of rat tissues to
induction by
low concentrations of components in smoke.studies
were
initiated using timed-pregnant Sprague-Dawley rats
with
measurement of control AHH levels in tissues of
the dams
and foetuses at 12. 15. 18 and 22 days of
pregnancy as is
shown in Tables 4 and 5. While the assays for AHH
activity
in tissues of these control animals were
completed, these
tissues were lost when the -800C freezer in which
they were
stored broke down and we could not use them for
metabolic
activation studies in the Ames type of
mutagenicity test.
Tissues from dams and 7-day old rat pups were also
lost in
this accident, as were tissues from smoke-exposed
dams and
pups that were allowed to nurse on these exposed
mothers for
20 days. These rat experiments are now being
repeated. It
will take about 4-6 months to finish these rat
transplacental
and milk transfer studies to a stage where we can
conclude
whether the negative results obtained in the
guinea pig are
also applicable to the Spraque-Dawley rat.
NJ
BATCo document for Province of BritiSh Columbia 19
April 1999


cc
0
0
CL
0
0
CD
0
CD
0
=r
0
0
Cr
A C014MERCIAL NON-FILTER CIGARETTE B
(20m9 TAR) 1.5-
C
o 1:10
C 0
1 ;5
.n 1.0
x
0
0
. 0) V.. - 1:20
6 E
W 1-40
5
0 of
.
0 1~0 2~ 3~
1.0
EXTRA MILD CIGARETTE C
k4.0mg TAR) 1.5-
1.0
1 :5
0.5- ........ 4
1 :20
1 :40
_W.
0 100 2~ 300
Duration of Exposure in Seconds
0.5-
-5
1 :40
0 100 200 300
FIGURE 1. The dose-r4sponse relationships between
rat pulmonary aryl hydrocarbon hydroxylase
activity
and the duration of exposure to different
dilutions of smoke generated from commercial non-
filter (A), extra mild (B) and ultra wild (C)
cigarettes, each of which produce a different
concentration of tar as Is shown. The points
marked with an asterisk are not significantly
different from control values (p>0.05). Each point
represents the mean value of a minimum
of four animals.
I ZSL'660 I
ULTRA MILD CIGARETTE
(I-0mg TAR)


TABLE 1.
Hilk transfer experiment
ARH levels In tissues from smoke-exposed &ulna&
9IR
dams and pups allowed to nurse for 7 days*
ARR activity - ng 3-0H benzo[alpyrane/=ln/mg of
protein
Guinea pit
Lung Liver Kidney
Dam
Smoke-expomod 4.23il.33(3)t 15.17 2.53(3)
0.30tO.04(3)t
Control 1.7610.51(5) 23.00 14.14(5) 0.1110.05(3)
PUP
Smoke-exposed 4.13.tl.97(9) 19.4814.14(10)t
0.06tO.02(10)
Control 2.90i1.63(20) 31-6918.84(20) O.OStO.03(20)
*The dams were exposed to 40 puffs of a ItS
dilution of a cigarette
smoke twice a day, for 7 days, and the newborn
pups allowed to aurais
ad libitum for this time period.
tSignificaatly different from control values
(ptO.05).
C=1
1.0
BATCo document for Province of BritiSh Columbia 19
April 1999


TABLE 3.
Placen tal transfer experiment
Snake exposure of preRnant aulnen pigs and levels
of
AHM In tissues from dams and pups*
ARK activity ng 3-0H benzolo)pyrene/sin/ug of
protein
Guinea pig
Lung Liver Kidney Placenta.
Dam Suoke-ezposed
Smoke-*zposed 3.94t1.76(14) 14.990-58(14)
0.70to.29(10)t 6.020.8204)
Control 2.58i1.83(6) 13.8717.07(8) 0.0610.03(s)
Control
Fetuses
Suoke-exposed 1.5211.46(55) 0.22tO.27(49) 0.14
0.06(57)
control 1.86 1,77(27) 0.26tO.22(29) 0.13to.07(27)
*Pregnant guinea pigs at 50-55 days and 60-65 days
of &,estation
Vero exposed to 40 puffs of a 1:5 dilution of
cigarette smokop
twice daily until term. The pups Vera removed by
Casaarlam
ctlon and tissues from dam, and pup rsmov,sd for
ANN assays.
t:i"SuLficantly different from control (p40.05).
BATCo document for Province of British Columbia 19
April 1999
Pj


0
0
CL
0
n
M
0
<
S'
n
CD
0
M
0
0
F
3
Cr
S,
V
to
CO
Age of foetus
-days
12
15
is
Term
Pup 7-day old
WS16601
TABLE S.
ARH levels in the perinatal SpraRue-Dawley rat
AHH activity - ng 3-OH benzola)pyrene/min/ag of
protein
Placenta Foetus Lung Liver
0.2010.06(6)
0. 14 to. 07 (a)
0.3710.16(8)
0.1710.08(6)
0.03tO.O2(6)
0.2ltO.03(3)
0. 08t0 . 05 (5)
2. 84t1. 080)
Pregnant rate were sacrificed at different stages
of pregnancy and tissues removed for AHH assays.
Whole foetuses vera homogenized and 9.000 a super-
natant@ prepared as done for the tissues of the
dam.
The numbers given are the MeantS.D., vith the
number of animals In parenthesis.
Kidney
0.3610.10(6)
0-39:0.050)


C. q
J
-4 ~ F,
rjr-7-p
j
4.7
EZ
A-Z
ZZZSL'6601
BATCo document for Province of British Columbia 19
April 1999