IMPERIAL TORAM) PRODUC-r!; DlVtSfON
OF IKASCO WILTED
RFSFARCII LAWRATORY
WNTREAL
PROnRESS REPORT
RMARCIT DFPARTI[r.rr
April June L971
Reserch &
20 DEC1971
. ....................
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by ....................
..................
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BATCo document for province of British Columbia U April 1999

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COPY NO... 17 ....
IMPERIAL T03ACCO PRODUCTS DIVISION
OF IMASCO LIMITED
RESEARCH LABORATORY Rik
MONTREAL Reyd 2 0 DECT971
A,',
...................
PROGRESS REPORT
RESEARCH DEPARTMENT
Fi.e ...... ..........I............
April - June 1971
Issued bv: Dr. T.A. Smith Date Issued-. October, 1971
DISTRIBUTION
Copy No. I Library 17 Dr. F. Hasiam
2 LibrAry 18 Dr. I.W. Hughes
3 Mr. R.M. Gibb 19 Dr. R.A. Sanford
4 Mr. R.S. Wade 20 Dr. R.A. Sanford
5 Dr. T.A. Smith 21 Xanalter. R. & D.D., Australia
6 Mr. S.M. Candlish 22 Manager, R. & D. D. , Australia
7 Mr. R.L. Rice 23 Dr. H. R. Bilimoria
8 Dr. M.A. Nisbet 24 Mr. E.R. Freiesleben
9 Dr. G.W. Boswell 25 Mrs. J. Johnson
10 Dr. S.J. Green 26 Mr. M.P. Scherbak
11 Dr. S.J. Green 27 Mr. W.A. Cray
12 Dr. S.J. Groan 28 Mr. A.F. El Nazgar
13 Dr. S.J. Green 29 Mrs. H.T. Schiff
14 Dr. S.J. Green 30 Miss R. R. Smith
15 Dr. D.G. Felton 31 Mr. B.J. Stirling
16 Dr. F. Haslam
rQ
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BATCo document for Province of British Columbia 12 April 1999

The Research Programme, issued for the first time in
July and henceforth being issued quarterly, records the objective
and current status of each project in the Research Department.
The present Progress Report given more details of work In some of
these areas, and relates to work done in April - June. 1971. In
future. such Progress Reports will be issued semi-annually.
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BATCo document for Province of British Columbia 12 April 1999

T-6900 Determination of Saccharin in and/or on Plastic Cizar Tips
A quantitative titrimetric method has been develooed and
applied for the determination of saccharin in and/or on
plastic cigar tips. The procedure involves the extraccion
of saccharin from a toluene solution with sodium carbonate
and conversion of sodium saccharin to mulphuric acid. The
famed sulphuric acid equivalent is titrated in an alcoholic
media with barium Perchlorate using thorin as indicator.
Hean recovery of known amounts of saccharin added to tip
material was 1002 with a standard deviation of +5.11.
Laboratory Report No. IOZL has been issued. (ERF)
T-6953 Determination of Sulphur Compounds in Tobacco and Tobacco Smoke
The indirect method for the determination of sulphate, based
on the spectrophoo ric measurement at 330 or 530 nm of the
chloroanilate ion formed, as applied in our laboratory for
the determination of sulphur on Wickbold combustion liquors
from smoke and tobacco products, has been replaced by a
direct titrimetric method.
The switch to the titrimetric method, in which sulphate is
titrated with barium Perchlorate in an 80% ethanol media at
a pH range between 2.5 - 4.0, using thorin 12(2-hvdroxv-3,
6-disulpho-l-naphtbylazo)-benzenearsonic acid] as indicator,
resulted from the finding that the indirect or calorimetric
method produced erroneous results often dependent on pH and
other anions.
The titrimetric method however, produces reliable results
within expected analytical tolerances. The averaite recovery
was 99.4% with a standard deviation of +6.8%. (ERF)
T-7084 Effect of Cuts Per Inch on the Nitromethane Fraction of Smoke
Condensate
We have examined the effect of cuts per inch (C.F.I.) on the
nitrovethane fraction (NMF) of smoke condensate. 74 mm
cigarettes, manufactured from flue-cured tobacco cut at 19.6,
32.2, 41.8, 54.3, 72.5 and 105.5 cuts per Inch, were smoked
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BATCo document for Province of British Columbia 12 April 1999

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to a 23 mm butt under standard restricted smoking
conditions of 35 al puffs of 2 seconds duration, taken
once a minute. The smoke condensate was collected by
electrostatic precipitation with sixty ciltarettes being
smoked per sample, and duplicate samples being.., used.
The per tent nitromethane fractions in the smoke conden-
sates were determined and are shown in table 1.
Table I
C.P.I. Wt. of Condensate Nitromethane Fraction Puff No. 1
9 9 z
105.5 1.4039 0.0936 6.67 9.4
72.5 1.8397 0.1242 6.75 9.6
54.3 1.8400 0.1293 7.02 9.9
41.8 2.0171 0.1428 7.08 10.3
32.2 2.0189 0.1450 7.19 10.8
19.6 1.8720 0.1399 7.47 11.4
The results clearly show a trend of decreasing per cent
nitromethane fraction in smoke condensate with increasing
number of cuts per inch, although the changes are on the
border line of significance. The puff number decreases
with increasing number of cuts per inch. The yields of
smoke condensate follow a similar trend to the puffs, but
the only significant change occured at 105.5 cuts per inch.
In table IE, three of these results are compared with
similar results obtained at Hamburg, and with long-term
mouse-skin painting, sebaccous gland and hyperplasia test
results from T.R.C., Battelle, Huntingdon and Hamburg.
M.:
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BATCo document for Province of British Columbia 12 April 1999

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Table 11 Biological Test Results
Caused by Changing Cuts Per Inch
indicates highest activity. although not always
numerically the highest).
FYPER-
C.P.I! LONG TERM SEBACEOUS GLAND PLASIA Z NMF
Battelle TRC
PSR (relative) Hamburg Huntingdon Battelle Hamburg Montreal
30 63* 1.07 (25)* 126* 36* 94 7.06* 7.19*(32)
60 60 1.00 (50) 117 67 97 6.70 7.02 (54)
120' 56 0.84 (100) 121 83 99* 6.54 6.67 (106)
t unless shown in brackets
It is clear that. with the exception of the hyperplasia
test results, all these results indicate that coarse cut
tobacco gives the highest activity, although in almost all
cases, the differences are very small. The trend for DCIP
reducing activity is the reverse of this, but is too small
to be significant (C. T-6256). (MP I AS)
T-7085 Effects of Tobacco Moisture an Some Aspects of Smoke Composition
Laboratory and consumer tests have compared the subjective
smoking properties of cigarettes at 13 and 152 moisture
levels. Clearly the lower level was perceived as stronger,
less mild, and harsher in the Consumer Test, although the
differences were not as marked in the laboratory trials as
had been expected. The effect of tobacco moisture content on
the deliveries of some smoke components has been determined
in a brief study.
The following table gives the percentage changes in deliveries
of HCN, total aliphatic aldehydes __ (TA AA)_ , acrolein, total
volatile acids, phanols and nicotine of the cigarettes on per
cigarette and pcr puff bases, to he expected from a decrease
in cigarette moisture content of V! from 13.9Z, as computed
from a study covering the range 8.3 to 15. 71.
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BATCo document for Province of British Columbia 12 April 1999

4
2 Changes in Delivery Caused by Changing
Cigt. Moisture Content from 13.9 to 12.9%
Per Cigarette
-Aper Puf.
Nicotine + 3.13 4.62
Phenols + 3.25 + 7.69
HCN - 1.42 + 1.17
TAA + 1.08 + 3.49
Acrolein + 0.58 + 2.41
Acids + 5.26 + 8.75
Since subjective properties are judged on a per puff basis,
it is to be expected from the results that a small drop in
cigarette moisture content would be perceived as an increase
in irritation. It is interesting that the relative increase
in acidic smoke components is greater than that of nicotine.
(TAS)
T-7086 Effect of Titanium Dioxide in Cigarette Paper on Smoke
Composition
Work at R & DE suggested that cigarettes wrapped in paper
which contained titanium dioxide gave an abnormally high
deliverv of carbon monoxide. We bad available samples of
paper which were identical except that one contained about
2.5% TiO2 and the other only traces of the oxide. Two ty es
.p
of cigarettes (flue-cured and blended) were manufactured.
each with the two types of paper, and the deliveries of
carbon monoxide and carbon dioxide were determined and
compared. All cigarettes were weight and pressure drop
selected, and were smoked on a Phipps and gird smoker to 9
puffs (taking 2 clearing puffs), the smoke vapour phase
being passed through Cambridge filters, and collected In
plastic bags. Twenty cigarettes were smoked per sample.
The CO and C02 contents of the smokes are shown in the table.
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BATCo document for Province of British Columbia 12 April 1999

r
Cigarette Weight Pressure Drop 7 TtO2 Ct C02
Type MR/cigt -@15 inches of 1120 0.1 in paper Z v/v V/v
flue-cured 1105 5.1 2.5 5. 7 10.2
T 5.9 10.7
5.8 10.2
5.9 10.4
flue-cured 1125 5.3 trace 5.4 9.5
5.4 10.1
5.5 lo.(
TZ -9. 9
olentied 1060 4.7 2.5 5.3 9.2
5.1 8.8
5.3 9.2
5.2 9.1
blended 1065 4.7 trace 5.5 9.0
5.5 8.9
5.6 9.6
9.2
The differences found are not significant, and hence it was
concluded that the incorporation of Tin, into cigarette paver
does not appreciably change the content of CO and C02 in the
smoke of Canadian flue-cured and blended cigarettes.
At the same time a comparison was made between gas chromatograms
of the vapour phase foreach type of cigarettes (flue-cured and
blended). The results showed no significant quantitative
differences, suggesting no influence of titanium dioxide on
the vapour phase composition of the smokes. (MPS)
T-6256 Studies an the Reducing Properties of Smnke
1. Effect of Paper Porosity and Salt @Zpltcat:@On
We are continuing our efforts at identtfyint smoke low in
biological activity as well as correlating the redox property
of smoke with other bioassays. ln this connection we have
looked at cigarettes made from isAver of different porosities
as well as the effect of noplication of salts to tobacco (to
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BATCo document for Province of British Columbia 12 April 1999

6 -
influence burn rate) at these porosities. From the results
of comparisons made with the OCIP redUCt ton tests and
summarized in Table 1. it will be seen that neither paper
porosity nor salt application influenced the DCIP-reducing
activitien of the condensates. Small differences seen were
not statistically significant.
TABLE I
rFFECT OF PAPER POROSITY AND SALT APPLICATION
ON DCIP REDUCTION BY CIGARETTE SMOKE C014DENSATE
SALT APPLICATION SPECIFIC DCIP-REDUCINC ACTIV ITY - "UNITS" PER mr. TPM
PAPER POROSITY - GREINER SECONDS
61 45 15
__L4
5% MRC12 6.53 7.34
7.54
SLOW BURNING
1% MRC12 6.60 6.8( 6.25
MEDILN BURNING
5% Y,;03 6.73 7.16 7.11
FAST BURNING
PLAYER'S CHECK 13 7.77 7.39 6.89
2. Effect of Tobacco Strand Width on I)CIP-reduSin& Activity
The effect of tobacoo strand width on biological activity has
been examined by several biological assays. While the long-
term and hyperplasia tests have not shown any differences due
to strand width, the sebaceous gland test has indicated that
condensate from finely cut tobacco has lower biological
activity. Consequently. we considered it interestint to find
out how this parameter would influence DC I ?-re du activity.
Condensates from cigarettes made usinr tobaccos cut at 20. 54
and 105 cuts per inch were examined for UCIP-reducinp activity
and from the results presented in Table 2. it will be seen
that no significant differences between the strand widths were
noted. (c.f. T-7081j. where it was found that the nitromethane
tended to decrease as the strand width decreased).
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BATCo document for Province of British Columbia 12 April 1999

TABLE 2
EFFECT OF T011ACCO STRAND WIDTH ON DCIP-REULCENG ACTIVITY
OF CIGARETTE SMOKE CONDENSATE
STRAND WLUTH WT. OF DRY SPECUIC ACTIVITY
OF TOBACCO- TP.%I-
CUTS PER INCH mg PER urrrs 'P R MuM L)CIP
CICARETTE ml. PER mg RLDUCED PLR
TPM ml PF.R Mr,
TPM
105 16.9 7.86 J 424.4
54 21.8 7.05 380.5
20 2L.4 6.32 341.1
PLAYER'S 20.1, 7.04 38(.4
CAMK 1.3
45
J. L)CIP-reducinr Activities of Condenmates from Water
and Ethanol Extracted Fjue-cumJ Tob@a co
Work carried out in R & DE hnq indicated that ethanol extrac-
tion of flue-cured tobacco mav result in lower biological
activity. Consequently, flue-curcd tobacco extracted in
M water and (II) 7OZ ethanol in water. has been dried.
powdered and -moked In specialty made cigarette tubes.
The DUP-reducing activities of TP.11's obtained from the
smoking of these extracted tobaccor have been compared with
unextracted flue-cured tobacco condensate obtained under
identical Rmokinx conditions.
From the results presented in Table 3, It will be seen that
specific activttv of the TPA obtained from the water extracted.
tobacco is much lower than the specific activities of the TPM's
from both the ethnnol extracted and cnntrol flue-cured tobacco.
Stntistical analyses have shown that the lower value obtained
in the case of water extracted tobacco is indeed significant
BATCo document for Province of British Columbia 12 April 1999

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An analysis of variance has shown that the Specific activities
are different at the 51 level of significance and Tukev's test
W. Snedecor, Statistical Methods. p. 251-2, The Iowa State
College Press, Ames, Iowa. 5th ed. . 1956) was employed to
locate the differences. This test showed that while there was
noLl'iffef'ence 'IL . the ethanol extracted and control flue-
cured tobaccos, the water extracted flue-cured tobacco was
distinctly lower titan both the above mentioned types.
TABLE 3
UCIP-REDUCING ACTIVITIES OF WATER AND 70% ETHANOL EXTRACTED TOBACCOS
CIGARETTES FROM WT. OF DRY NO. OF PUFFS DCIP-REDUCING ACTIVITY
TP:I-i4G PER PER SPECIFIC ACTIVITY T?M/V P.
5 CNARETTES CIGARETTE "UNITS" PER MG. RATIO'
TPM
WATER EXTRACTED 129.5 20 5.81 28
FLUE
70Z ETHANOL 103.7 15 7.29 29
EXTRACTED FLUE
CONTROL FLUE 88.4 12 7.64 27
4. The Influence of the Smoking Vehicle on DCIP-reducing
Activity
Air-cured tobaccos. when smoked in the form of cigars and in
stainless steel pipes, give TPH's that are much lower with
respect to DCIP-reducinst activity than the TPM obtained by
smoking Canadian flue-cured cigarettes. However. this
difference between air- and flue-cured tohaccos is not as
marked when they are smoked in the form of cigarettes using
specially made cigarette tubes. Also, some earlier preliminary
studies had suggested that cigar filler tobacco, when crushed
with the hand and smoked in a stainless steel pipe. Rave
condensate with such a low DCIP-reducinn activity as to be
bareiv detectable under the conditions of our assav.
BATCo document for Province of British Columbia 12 April 1999

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1hese results suppested an important role for the vehicle
in which tubacco is smoked In Peneratinz DUP-reducitir
activit-o. Hence a systematic examination of three tobacco
types was undertaken to find out whcther the condensates
have low activity when they are smoked in stainless
pipes. Table 4 records the typical rcgults for there.
It will be seen that as the time of smoktng in pipes increases,
the specL[ic UCIP-raducinit activity falls quite markedIv. If
wet weiznts of TPM are used to calculate specific activitv.
then even lower specific activities are obtained. At ttie nresent
tine we are not sure whether thexe low snecific activities
indicate a change in the nature of tire MI is biornLnr in the pipe
proceeds or thev nre mere1v artifacts of smoking due to trapnin2
of reducing substances hv writer accumii1atLin! in the pipe. Tile
results in the table also show the accumulation of a larue -amount
of water on the Cambridge pads which increases as smakinc tire
increases. This water may have some role in the low activities
observed. OM)
T-7703 Effect of Tobacco Smoke Condensate an the Microsomal Electron
Transport Svstem
In our studies on the interraction of tobacco smoke condensate
with c".Ponents of microsom 1 electron transoort we have found
that ciznrette smoke TM can oxidi%e the microsomal pigment
cvtochrome h5. This electron transfer cnmponent which I,; isolated
in the oxidised fnrm was reduced using the reduced form of nico-
tinamide adenine dinucleatttle, and a crude preparation of the
microsormal enzyme. NADII-cytochrome b5 reductase. Addition of TPM
to this en;,vmattcally reduced cvtochrnre hS resulted in raold
reoxidation of the cytochrome as Illustrated in fleure 1. This
reaction is interesting. particularly in view of some recent work
that hns implicated the m1crosonial electron transport system
involving NAVII-cytochrome b5 reduct-nse and cytochrome b5 in
preventing oxidation of ascorbate. (Ml I a)
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BATCo document for Province of British Columbia 12 April 1999

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TABLE .4
1) 1@ ACTIVITIES OF AIR- AND FLUE-CURED
TOBACCOS SMOKED IN STAINLESS STEEL PIPES
S: 'rl'!'
v , .- IWT. OF WT. OF WT. F PERLENT LOSS DCIP-REDUCING
IN HINS. TOBACCO WET TPM DRY TPM IN WT. ON ACTIVITY-
BURNED- IN GM IN GX DRYLK SPECIFIC
I N rl ACTIVITY
"UNITS" PER
RG TPM
FLUE-CURED
5 0.21 0.0062 0.0058 6.5 1 6.94
1( 0.55 O.OL75 0.0148 15.4 3.41
20 0. 8f) 0.0437 0.02on S4. 2 1.85
40 1.55 0.1832 0.0367 M. 0 2.17
64) 2.19 G. J582 0.0465 87. @0
AIR-CURED CICAR
5 0.40 0.0054 0.0049 9.3 2.91
lo O.hl 0.0068 0.0060 11.8 1.42
20 0.94 0.0415 0.0100 75.9 1.85
40 2.00 0.1920 0.0456 if). 3 n.23
hu 2.60 0.4756 0.0743 94.4 0.20
BURLEY
to 0.25 0.0191 0.0083 56.5 1.54
20 0.72 0.0473 0.009( 91.0 0.53
140 1.75 0.1777 0.0342 80.8 0.10
hO 2.35 0.3794 0.0549 .45.5 0.38
Ln
BATCo document for Province of British Columbia 12 April 1999

0-7 ADDITION
OF TPIA
0 @10 Cr
0-4-
4.
O-3--
0-2.-
G
0 2
TIME, IN MINUTES
F@I G. IOXIDATION OF MICROSOMAL CYTOCHROME b5
BY CIGARETTE SMOKE CONDENSATE.
The sclub' isacL aration o; c
11 I jtormi@rowoc b.5 w"
anzjrnabca4y recLice7uncter aerobic conditions using
NAJ)H (10-""[;) anct 0. cru arabon of
er (O I
NADH- cy tcc6ome bs Zftaf;'e -33 un't) anot
6M). TPM soluti,
C-Ytachrome b,5 (G-2x to- on was
a'ade.cL to bet6 blank a"at samf/c cuvettes as
dlustr-ateoL in the f!Y Ure.
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BATCo document for Province of British Columbia 12 April 1999

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'r- 76 3 Z RioLogical Effects of SmokeLlusidation of the Hechnnism
ActLun of Cigarette Smoke on Hemoglobin
In the continued effort to determine the mechanism of action
of cigarette smoke on hemoglobin, the behaviour of the water-
soluble fraction of cigarette smoke condenSAte on solutions of
oxyhemoglobin and methemoglobin was compared to that of known
hemolytic agents (Sodium nitrite. phenv1hydrazine, sodium
dithionite. potassium ferricyanide and quinone). Fnr this
study, .2 Biological (xviten monitor, filled with a Clarke Type
electrode. wRs used to measure the oxygen uptake or evolution
during the reactions.
3.0 ml samples of oxyhemoglobin or methemov.1ohtn solutions were
equilibrated at 30% in the incubation chamber of the monitor,
and aliquots of known concentrations of smoke solution or test
compound were introduced into the chamber through a vent in the
electrode, using a microlitre syrinpe. Subsequent evolution or
uptake of 02 caused during the reaction was immediately recorded.
The reactions of the comnounds known to oxidise oxyhemoelobin to
.methemoglobin. indicated that the test compounds could be classed
into 2 groups, on the basis of the initial response- one group
causing uotake (reducina agents) and thc- otbar causinc liberation
(oxidisinq agents). (See Table I and Flp. 1). It can be seen
that smoke caused a slow uptake of oxvpen and can therefore be
classified as a reducing agent.
The same comocunds when reacted with methemoalobin again fall into
2 groups. Potassium ferricvanide, quinone and sodium nitrite (tire
oxidizing agents) caused no uptake or liberation of oxyzen while
T)henvlhydrazine and dithlonite (reducing agents) produced oxypen
uptake (See Fig. 11). Smoke solution. al-to caused 02 uptake. thus
confirming its behaviour as a reducing apent.
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inh.
BATCo document for Province of BritiSh Columbia 12 April 1999

- 13
The exnerimental nbservations can be rationnIlsed hv the cvclic
scheme shown below:
lib(021 -- ) 4ETHFIMLORIN ---+SULFIIEM0rLOBIN
(0xvhemojtIobin)
@O2 Hb
(hemoglobin)
The reaction mechanism for reducing agents Ls typified by phenyl-
hydrnzine (1), oxidisinst agents are represented bv potassium
ferricyanide (2).
Hb+02- Hh' + H02-
2HO2- + $N11 - ';H-) 2H2n2 4- @Nfl - NH
(phenvldiimide)
Hb+02- + Fe(CN)3- 4-
6 ---4 Hb+ + 02 + Fe(Csi)6 (2)
TABLE I
Compound Reaction with HbI02J Reaction with lietHb Cla3S
Uptake 02 Liberation I
Uptake OZ
Pheny1hydrazine + + + . . . Red.
01thionite + + + . . . Red.
Potassium + + + Ox.
ferricyanide
Sodium nitrite + + ox.
Quinone + + + ox.
Smoke + + Red.
Red. - Reducing agent Metill) - I'lethemoRlobin
Ox. - OxIdising agent + + + - Knpid
Hb[021 - OxyhemoglobLn + - Slow Wj)
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BATCo document for Province of British Columbia 12 April 1999

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