AUTHOR: R. E. THORNTON
RET/LAP/46D
BIOLOGICAL MEETING HELD AT GR&DC,
19-20 MAY 1983
OBJECTIVES:
27 July, 1983
(1) To consider which are the most relevant
biological,
biochemical (or chemical) indices for the
development of
cigarettes of reduced biological activity.
(2) To review the state-of-the-art in the
development of
cigarettes of reduced biological activity.
(3) To make any necessary recommendations for
future work.
A MAIN CONCLUSIONS
1. A stepwise approach to Project RIO, the
development of
cigarettes of reduced biological activity was
recommended.
(a) Phase I would be the design of low activity
cigarettes
in the 5-10 mg range, using existing technology
and
tests.
(b) Phase II would be the further investigation of
such
cigarettes with additional tests, possibly after
their
introduction onto the market. Within the
aJditional
tests it is likely that there would -be a
requirement
for a long-term study; Dr. F. J. C. Roe
recoinmendad
that an inhalation study would be preferable,
providing
a viable test procedure was available.
(c) A further stage in the development of low
activity
products could be envisaged, involving more
speculative
procedures such as the adventitious addition of
materials
to cigarettes. However, the work associated with
the
develop-merit and evaluation of such cigarettes
would be
substantial and might pose problems similar to
those
faced by the pharmaceutical industry.
2 The collaborative programme involving the
laboratories at
Hamburg, Montreal -and Southampton is working well
and there-_
was support for the Fractionation programme
proposed for
Canada, and discussed at a sp~-cial meeting held
in April
(Appcndix A) .
3 While appropriate efforts should lie devoted to
th,~ on-goinj
progrn,.rm-c of. in-,~thod dcvelopment,
particularly Thos.--! showinq
Prcjr_,F-;c-, 7,uch as the P448 -_nzy!~e assay,
the of
lovi-acLivity cigarettes should ~),:: given th2
P--iority
in tl-':2 a1location cf- timr~ and r,-~zourcas.
C~o
_M:t.
CO
BATCo document for Province of BritiSh Columbia 3
November 1999


B SPECIFIC RESOURCES
1 . In the evaluation of Project RIO cigarettes,
the results
from tests related to cancer should be ranked
first.
2. The current status of Vitamin A as an
anti-cancer agent
should be reviewed in the context of the possible
addition
of Vitamin A (or some derivative) to tobacco.
3. The utility of certain short-term tests was
accepted,
notably the Ames test, related mutagenicity tests
and
possibly the P448 enzyme induction test.
Nevertheless the
improved Ames test (University of Saskatchewan,
Canada)
should be investigated. An easier way of scoring
the Ames
test was also required.
4. Irritation as a determinant of smoke inhalation
had important
implications, but needed further study in human
subjects to
confirm preliminary animal studies.
5. Some work was needed to determine how the
biological activity
of condensate varied with smoking parameters.
Model
pyrolysis studies might be useful in indicating
the range
of activity likely to be encountered in any
systematic
evaluation e.g. of human parameters.
6. Tests for promoting activity were not
sufficiently well
developed to justify investigation: indeed the
whole concept
of initiation/promotion was now in doubt (F. J. C.
Roe) .
7. Inhalation studies relating to cadmium exposure
and skin
painting studies relating to nicotine were
considered.
However, in both cases it was thought that, at
least for
the time being the results did not require changes
to be
made to the work programme.
8. The possibility of competitors producing a
biological
ranking of brands e.g. based on Ames test data is
real and
we should be in a position to respond to such a
situation.
co
CDC
BATCo document for Province of BritiSh Columbia 3
November 1999


C AGENDA ITEMS
Mutagenicity Tests
The development of the Test Battery now used at
Hamburg
and its relation to Group Activities was described
(Appendix 8)
and the results obtained are summarised in
Appendix C.
Inhalation Tests.
Inhalation toxicity studies have progressed along
two lines
in recent months.
1. Product orientated, where the effect of
additives (e.g.
BATIK) and changes in tobacco processing (use of
DIET expanded
tobacco) on the biologial activity of mainstream
smoke has
been evaluated.
The activity of cigarettes with reduced visible
sidestream
have also been compared, not only with each other,
but also
with a series of commercially available
cigarettes.
2. Fundamental studies, aimed at providing basic
information
on the comparative inhalation toxicity of low
delivery main-
stream products plus activity of sidestream and
ambient smoke.
Conditions necessary to produce changes with all
three smoke
types are now established.
The on-going programme advances the two themes
outlined
above. Studies examining the comparative activity
of different
tobacco types (including stem) and the influence,
if any, of
puff volume are partly completed. Ventilation and
filtration
effects will be studied together with parameters
such as
permeability and cigarette dimensions.
The use of sidestream/ambient with mainstream
using different
regimes is planned, allowing exposure of animals
to varying
ratios of particulate and vapour phase. This will
provide
fundamental information on the relative importance
of the two
phases of smoke, and perhaps more significantly
their inter-
action, and help explain the difference between
mainstream and
sidestream smoke at matched particulate phase
concentrations.
Test for Promoters
Dr. Roe said that the distinction between 'Pure'
initiators
and promoters, if it even existed, had become
blurred. There
was further confusion with the phrase
'co-carcinogen', referring
to a material acting synergistically with a
carcinogen (see
attached paper). Dr. Roe said that the most
meaningful way of
deciding on the biological activity of a
hypothetical smoke
component X would either be to add X to smoke, or
to totally
remove X.
Skin Painting: B15 Series E. B. Wilkes
Chemical Indices: Nicotine
Votes on the analyses of these experiments were
circulatr.-d,
and aill be formally reported. in suminary-the
results are givn
as A?pcndic-~s 0 and E.
CO
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BATCo document for Province of British Columbia 3
November 1999


-4-
Biochemical Indices P. M. M. Godden
The research for biochemical tests which might
replace
animal tests, or model the human situation has
focussed on the
following:
Mucus Production - currently the effects of smoke
from
different cigarettes is being evaluated.
Short Term Tests for Carcinogenesis - encouraging
results
have beer. obtained in relation to the induction
of the P448
enzyme, where the level of induction may relate to
mouse skin
painting activity. This is a judgement made on
preliminary
results obtained at the University of Surrey
(Contract Research) .
other Areas - work had commenced on measuring
serum glyco-
proteins in animals, the levels of which are
elevated in smokers
and in various disease states. A contract was
being negotiated
for a study into the effects of smoking on
metabolism in humans.
Atherosclerosis - The complexities of developing a
model
had resulted in slow progress, but a new approach,
based on
measuring acute effects of smoke in humans, showed
promise.
Nitrogen Containing Compounds Dr. E. D. Massey
Dr. Massey's review has been incorporated into a
separate
document "Tobacco Smoke Mutagenicity: The
Influence of
Nitrogenous Compounds, Status Report. (Appendix
F).
Cadmium Dr. F. J. C. Roe
Recently, Takenaka and his colleagues at the
Frankhofer
institutc in Germany reported lung cancer
induction in rats
following their prolonged exposure to aerosols of
cadmium
chloride (Takenaka et al, 1983). If this finding
is confirmed
in further studies it will represent both a
break-through and
a challenge. Cadmium chloride is highly acidic and
is not
the form of cadmium to which humans are normally
exposed
industrially. Epidemiological studies have so far
revealed
no association between occupational exposure to
cadmium and
increased lung cancer risk. Thus, it is difficult
at present
to judge what, if anything, the findings of
Takenaka et al
mean for man.
Fractionation as a Means of
Identifying Chemical Indices Dr. E. D. Massey
The proposed programme of work is included as
appendix A.
Sidestream Smoke Dr. C. I. Ayres
The hypothesis that 'a whiff of nascent sidestream
smoke'
was as important as 'the inhalation of larger
quantities of
mainstream smoke was discussed. It was concluded,
on the
limited available evidence, that the
epideniological data
did not- support the hypothesis although it waz
not entirely
d i sin i ,-i s iv e .
CO
CO
C-14
BATCo document for Province of British Columbia 3
November 1999


-5-
Trends in Bioloqical Research Dr. F. J. C. Roe
Dr. Roe's views on the current status of testing
for
carcinogenicity are given in Appendix G.
Dr. Roe also stressed the growing importance of
peptide
hormones as regulatory agents in physiological
mechanisms, an
area that toxicological research had not yet
caught up with.
On the other hand, pharmaceutical companies were
very active in
this area. Dr. Roe also argued for a broader view
of the
processes leading to cancer: accidents to DNA may
not be terribly
important; more attention should be given to
immunotoxicity,
aging processes and cell regulatory mechanisms.
As an overview, Dr. Roe felt it unlikely that
fundamental
discoveries in cancer research would have a major
impact on the
tobacco industry in the immediate future.
0 C
cc
BATCo document for Province of British Columbia 3
November 1999


APPENDIX A
AUTHOR: E. D. MASSEY
EDM/LA-D/46D
28 April, 1983
MUIAGENICITY A1JDCIGARETTE SMOKE FRACTIOLNaTION
A Project for Dr. M. H. Bilimoria, Imperial
Tobacco Ltd, Canada.
The broad goal of this work is to determine the
overall chemicals
responsible for the mutagenic/tumourigenic
activity of cigarette smoke
condensate. Following the meeting at GR&DC
Southampton with the Company's
biological consultants, Dr. F. J. C. Roe,
Professor B. A. Bridges and
BAT scientists on 26 April, the following
guidelines are recormTended for
the fractionation/mutagenicity project.
(1) The Grimmer and Klimisch scheme of
fractionation should be used, this
involves partition between solvent systems and
thus minimises chemical
modification of the condensate during
fractionation. In addition,
the results can be compared with the mouse skin
painting carc.-nogen-
icity data available for this fractionation
procedure.
(2) The mutagenicity test system should be a
bacterial point mutation
assay using tty-- appropriate strains of S.
typhimurium and if time
permits E. coli. It is expected that the results
will generally
indicate w~~-ra the biological activity may b--
found following
fractionation. However, it should be remembered
that Professor Bcidges
did point out that compounds that showed strong
mutagenicity were not
necessarily that most potent carcinogens.
(3) Fractionation should be carried out on 3 types
of szmke condensate
w1 hich have different mutagenic activities by the
Ames test using S.
typhimurium strain TA 98.
(4) Accordingly, it is suggested that the
following plain cigarettes
manufactured by BAT Germany are investigated. I.
Gauloises (Flue),
II. Fenas (Oriental) and III. North State
(Virginia).
(5) Completion of the programme, up to and
including identification of
comr,*unds responsible for the bulk of
mutag(---iic activity, is likely
to involve a substantial amount of work, sorn of
which would have to
be carried out by GIR&DC Southampton, and may take
up to 5 years to
complete.
(6) The following should be instigated by ITL
Montreal:
I Negotiate with BAT Germany for the production of
cigarettes
II Condensate fractionation
III Perform Ames mutagenicity assays on the
fractions.
At Lhe end of this phase of the work, a further
rrt--eting will be held
to determine tha next stages which are envisaged
as subEractionat.-Lon
and chemical analyses with involvement of some of
the facilities at GR&DC
Southampton.
E. D. MPZ
co
co
BATCO document for Province of BritiSh Columbia 3
November `1999


0
0
CL
0
3
CD
0
0
CD
0
W
=r
0
0
E,
z
0
<
Cr
APPENDIX 5
SHORT TERIVI TEST BATTERY, RD HAMBURG
1. Repair test, E. col i pol A+/ pol A-
2, Ames test
2 a E.coli imutagenicity test E.c.K12, 343 113
3. CHO a) SCE
b) HGPRT
4. human Lym hocytes
a) S~E
b) Miki-onucleus test
c) Aberrations
DNA Repair Test (Unscheduled DNA Synthesis)
Ca Chromosomal aberrations in bone marrow
7 Lymphocytes in vivo
8. Sabaceous qlan'd test
RE, SEARCH & DEVELOPMENT AND PRODUCTION
K HH
M,HH,GRDC
M,HH
HH
OR/DG-
HH
HH
GR/DC
HH
GR/DCIHH
OR/DC/HH
HH
1983


APPENDIX C
OBJEKT- VARIATION
Burley
Tobacco Orient
Virgin
Sheet m 11/7
M 20
thin
Diameter normal
thick
Paper double
Additives K-acetate
(paper) No-acetate
Na- citrate
Na-cit rate - N a- acetate
Na-acetote + f 1 bre
Na-acetate+ f 1 bre
+succhrose
RESULT
0
0
0
+
Tobacco cut width
0
cc
-1 cc
D SVELCIPTMENT AND PnODUCTIOL 1,
BATCo document for Province of British columbia 3
November 1999


APPENDIX D
SAMPLES USED IN JRNUS EXPERIMENT B.14
.. .................................. =
---------
I Sample ----------
I Nicotine ----------------
In condensate. --------------------------
I Nicotine in cigarette. I
I No. I
---------
---------------- I
------- I
----------------
I B14/1 1 1.47 mg nicotine per 1 0.59 mg nicotine
per
1 40 mg condensate. I cigarette.
1 B14/2 1 1.89 1 0
76
11 . 11
I B14/3 1 2.75 P 1.22
1
I B14/4 I 3.E8 1 1
70
--------- ---------- ---------------- .
------- -------------------
Blend: 'Ruslese'
36 cuts per Inch
Tobacco rod length 64 mm
circumference 25 mm
Tobacco weight per cigarette 0.877 9
Cigarette paper JANUS
Filter cellula:;e acetate ler.gth 20 rrm
circumference Z4.8 mm
Total cigarette weight 1.119 9
The nicotine figures for Bl-;/l are tht "natural"
del ]very figures for
the cigarette design ustd.The nicotine figures for
B1412 to 814/4 ere obta-.ed
by spraying the tobacco with nicotine citrate.
Dose levels of 28.3, 40.0, and
56.6 rig per application were used for each
condensate.
Each condaricate/dose used 120 animals. Thus 1Z ~
120 - 1440 animals were
used in this experiment.
C=)
cc
cc
Q.-4
BATCo document for Province of British Columbia 3
November 1999


ANALYSIS OF JANUS EXPERIMENT 8.14
.................................
Data used: All tumours size I mm or over.
1. Analysis of death rates.
In mouse skin painting experiments It has often
been observed that
nicotine exerts a toxic effect upon the animals.
The mortality experience of
the animals in this experinient was examined
regarding turr.our-bearir.g animals
as withdrawals. Assuming no difference in
mortality between condensates (but
assuming that differences in mortality between
doses will occur) the observed
and expected numbers of tumourless deaths were:
---------
1 1 ---- -----------
214/1 1 ---- -----------
B14/2 1 ---- -----------
314/3 1 ---- -----------
B14/4 I
I Dose I ---- ---------- I ---- ---------- I ----
---------- I ---- ---------- I
I level 1 0 1 E 1 0 1 E 1 0 1 E 1 0 1 E I
1 28.3 1
I 113 1 112.03 1 115 1 123.36 1 110 1 102.70 1 104
1 102.32 1
I
1 40,0 1
109 I I
1 95.86 1
109 I I
1 100.75 1
106 I I
1 119.29 1
96 I I
1 110.10 1
1 56.6 1 95 1 97.09 1 99 1 F34.72 1 98 1 106.05 1
83 1 76.355 1
lTotalt
1
-------- I D17
1
----- 1 304.98
1 1
---------- 1 316
----- 1 308.83
1 1
---------- 1 306
----- 1 327.a4
1
---------- 1 2133
1
----- 1 280.37 1
1 1
-----------
Chi -squared - 2. 121 w1 th 3 degreas of freedora.
Significant at the 45X level of confidence.
This -a lue of Chi -squared is not significant,
showing that the assumption
of no mortality difference between conden-.ate5
may be accepted. Thus it may be
assun-ed that nicotine toxicity had little 0~ no
effect on turnourlesi; deaths in
th I s exper I ment .
C=)
%10
0--
BATCo document for Province of British Columbia 3
November 1999


2. Analysis of tumour rates.
2.1 Parameter estimation.
Us I n9 the three -par &me ter We iUu I I d is tr
I but Ion to- mode I the
instantaneous tumour rates, common values of the
parameters k and w, and
separate values of the parameter b, were fitted to
the 12 groups of animals. The
(natural) logarithm of the parameter b is taken as
a measure of reTponse of the
animals to the 12 condensate/dose combinations.
The parameter values obtained
were!
--------
I
------- ---------
TABLE
--------- -------------
OF Ln(b).
------------- -----------
I
---------- I
I Dose level. I
I Samp I e I --------- ------------- ---------- I
I No. 1 28.3
--------- 1 40.0 1
- I---------- 56.6 1
---------- I
11314/1
1 1 -20.1904
. 1 -19.4334 1
I -18.7157 1
11314/21 1
-20.6043
I 1
1 -18.9835 1
I 1 1
-IB.7-~39 I
1
I
IS14/3 1 -19.7455 1 -19.5501 1
1 -18.5663 1
1
1
1914/4
------- 1
1 -19.2949
---------- 1
1
------------- -18.0728 1
-----------
k 3.9771
for all groups.
3.0503
C=)
\-0
cc
0 C
cc
BATCo document for Province of British Columbia 3
November 1999


2.2 Goodness - of - fit.
A goodness - of - fit test for these parameters
was carried out, the
results being:
---------------------------------
I OBSERVED & PREDICTED NUMBERS -------------
OF I
I TUMOUR -
----- BERRING RNINRLS
-----
--------- I
------------ I
I--------
I Weeks --
-
I Observ
------- -
-
ed I Predicted I
--- ----------- Chi-square I
------------
I 1- 50
1 1 28 1 33.4463 1
1 1 0.887 1
1
151- so 1
1 31 1 31.6190 1 0.021 1
161- 67 1 31 1 26.2638 1 0.265 1
168- 74 1 43 1 31.5673 1 4.141 1
175- 911 1 31 1 31.0671 1 0.000 1
182- 90 1 23 1 30.7980 1 1.974 1
131-lzl 1 31
------ 1 30.9485 1
--- ----------- 0.000 1
---------
I Totals
1
-------- -
I zie
1
---------
1 217.9100 1
1 1
---------------- ---
7.288 1
1
-------------
There is soi~e evidence to suggest a slight
over-prediction in the
early weeks of the experiment and a slight
under-predictior In the middle weeks.
The total Chi-squared of 7.288 is not significant
(confidence 1-tvel 71*/.)p
sho,,ing that the overall fit of the parameters to
the data Is quite good.
CC)
CC)
BATCo document for Province of British Columbia 3
November 1999


2.3 Discussion of results.
The (natural) logarithms of the I-Jelbull
parameter b are graphed in
Plots 1. and Z. An analysis of variance of these
data shows that the addition of
nicotine tin the form of nicotine citrate) to the
tobacco leaf has had a highly
significant effect upon the tumorigentelty of the
condensate. (S I cin If icance
level > 99/). If a regression-type model Is fitted
to the data then the
Incidence of tumour-bearing animals can be
adequately described using only
three terms: one for the overall mean response,
the second to take account of
the effect of the nicotine dose, and the third to
account for the effect of the
ni cot I me -free dose. The par arneterz of the
regress ion show that the turnor igen I c
effect of nicotine appears to be about 70% to SOY
of that of the nicotine-free
condensate. The regression results are shouin in
Plot 3.
This r-zsult must be treated with caution, for two
reasons.
I. Plot 1 . shows that the signi f 1cince of the
result Is heart 1y lmfluenc,d
by the response for S14/4. It c5n be -;houjn that
314/4 has a significantly
higher response than the other three condensatec,
ard that these three
I.e. 014/1, B1,2, and 814/3, do not differ
significantly amongst
themselves. If the B14/4 orcups of animals are
dropped from the analysis
then the effect of nicotine becomes
non-significant. Thus the outco,r,e of
the experiment Is dominated by the rezponse from
E114/4.
1 I The B14/1 condensate does not form a proper
control for this e.periment.
As mentioned earlier, the nicotine content of the
tobacco was adjusted
by spraying the cut tobarco with a -;oluticn of
nicotine cttrate. This
solution contained ethanol. For EiI4~1 to have
been an adequate control
it too should have been sprayed wi th a s Imi )ar
solvent to that used to
apply the m Icat ine. Rr it is, the observed resu
Its may be part ial 1y
explained as sol%jent ef;ectz;.
cc
cc
c:::)
BATCo document for Province of British Columbia 3
November 1999


Plot 1 .
B.14 All Tumours Size 1 mm or over.
Observed Data.
Predic ted D-ata.
110- 1 a Ln b -I O.e7771L-21 +
0 . 72 1 IiB4 1 . 9'~ 6 i L , I D,
CXD
cc
BATCo document for Province of British Columbia 3
November 1999


Plot 2.
B-14 All Turriours Size 1 rrim or over.
Observed Data.
14 ico tin. concertration in SQ/40 dos-
_18.,, Ln(Wwibj11 par-tor b)
Hi'j.conc.
. ..........
-13.5 ................ ...............
..................................................
......... ..... ;-,, ....................
89 ..........
-19.8 . .............
...............................
........ .......... .....
...... ...............................
Hic
.. ......... ......
-19.s . ............. ........... .............
..... ..... ................................
...................
-28.0 . ....... .... ............
............. .............. . .............
.............. ....
.................
-20.S . ............
:.................. ...............
...............
. ............. . ...... . ...........
-21.21
0.0 0.2 0.4 8.6 0.1 1.0 .2
Lr.(nj-tt- d- .9)
Predicted DaataL.
4,
. . .. ........ ..................
................ ....
......... ... .. ..... ...
. ....... ....
-19.0 . ...........
............................... .... .. .. .
. ......... . .... .... . .... ... ...
. .......... ......
... .........
-19.s . ............. ...........
..............
........................ I ..... ...............
..........
.............
. .. ....... ;............... ...
............... . ........
................. ............. . ...... .. ..
..... ... ..... .......
69
-20.9 . ............
.................
.......... ...............
............... .... ......... ........... ... . :
... ...........
0.0 0.2 0.4 0.6 0.9 1.0 1 .2
Mouel r Lrt(b)- -2,S.,SS.13 0-0777'?1"21
00-
D 1. C-d--q, cc
BATCo document for Province of British Columbia 3
November 1999


Plot 3.
B.14 All Tumours Size I mm or over.
P red i c ted Data .
I c a t i no concentration in mg/40 mg dos-
Ln(W*ibull parameter b
18.
HIC --
47
19.S .
............................................
................. . . .. .............. . .
........... .. .. .......... ......
C-1 ------
. ............. ............................ . ...
.......... .. . ........ ...........
Hic
-
-
.
7
j.
' ....................... ..........
............... ........................
Hic
C."r . .............. .......... . ...............
........... .. .................
.......................
i
6 a
-20.S . ........ ....
................................ ........ ...
..... ............................ .
............... ............... .
3.2 3.3 3.4 3.S 3.6 3.7 3.8 .9 4 .0
L~ Olic -Itt- f, d- g)
P red i c ted D~.-it A-
L We I b. I I b)
7
-13.S . ....... ..
........... ........ .............. ....... . ..
... .. .......... ....... ......
H i,: conc
. ............. .................................
......... .... .... ... ....
.............................. .....
C7 S'- -L9.S . ............. .............. .
........
. . ......... ................. ... .......... .
nc -20.0 . ........ ..... .... ..... . ......
.. .... . ... .... ..........
.... . . ....
3 6a
-213 S
............. ......... ..... .. ..... . . . .
...... ....... .............. ...............
.................... .....
........ ...
0.0 0.2 0.4 0.11 0.8 1.3 1 .2 1 .4 1.6
[To-Je.: L-L- -:'~.S-~ 4
CDO
cc
BATCo document for Province of British Columbia 3
November 1999


APPENDIX E
DFSCRJPT',OI4 OF ERMPLEE, USED 114 JANUS
[XPFRII1EIJT S.-
-
.................................................
:
---------
IS?, rr,p I e I ----------
------------------------
Paper. I-Filte,l -----------
Type c-
1 I----------- --------------- I I
I No. I
I I- Type.
---------- I Porosity I
---- I---------- I p.d. I
------- tipplr--.
-------
I I
1
3 E5C I I
1 95 1
I
Lo. I
I
st-01-
I
I I
I M 100C
1
I H, gh I
I st.-14-
4 1
I
I
DM95C
1 7 00 1
Lo~- I
St.nd*-
1 6 1
I I
7 1
1
I
H, oh I
I
St&rck,
I
6 1 1 1 11 100-'
9 1 W.ttq,ns 200 1 1600 1
I I High I
I St-di-
I
I I I I ,I Icir,
95 P 1 7 00 1 H, 91, 1 Si i, r.dz -
1
--------- 565C ep
---------
----------------- L,,. 1
-------- 516-1-
----------
iY, p-os it,,, datb bre in WTU (norreii--0 vblue -
suppl le-
ri 1000 it b pei-f-ted tipping - fltilaucer- ute7d
L-,th t Iy pcirc- PIL1q--"Bj'
Frou. %I-is desi9r, sanirle-, 1 to 8 al low thi,
ric-rot: ity, f, Iter
pre::~-ljre drc-p, ;=nc' tir %,enl i Itticn, to be
tested. 4, '1 ~!' 9, arid 1".
-.te,j -,),.e rar- o-' the dblt. Thi, eti~:ct of
ci:.-,- f r.,per addit-
car. be ti,stecl rtmpie= ", a- Th,!- effect of IKA
(c,trate
cher.ge froii. I-ahly po,c.,.;s r-tr-tr xv Ierf,
of ;:, IC,;- 1:-C-,Cl:",.
paiper cat. be 't-ted using sz&~.rjl- 5 and
C=)
11 C
CC
BATCo document for Province of BritiSh Columbia 3
November 1999


REVIE14 Or RESULTS. JRHUS EXPERINE11T 2.15
.........................................
rjat& Lis ed: R I I tumour s & i ze I ff- or owe r
.
-----------------------------------------
I Exterided dose/r espor-e curve.
5 arnples used: B15/01
The e~tendeci dose/rerpc-r-e- cur%,e. used for
E!4.1,,'C,l -, I-ear. The
variarece. sh-rs that only -the log 1-tar -,errr.
of the fitted r--.UI~
r,9-f-r-t. Pl~-t 1 th~ 1--a, ty of tht dal,, h,,d
th- -;~It s~--
that the dc--~- le.,els used 4r,, tht re,.a-der of
'the L-perin-rit a- u,e-ll ujithir,
V- I-r--ar portic- of the dons-'re-tpo-ze. curv-
CD
11 C
cc
cc
BATCo document for Province of British Columbia 3
November 1999


A14RLYSIS OF JR14US EXPERIMENT B-15
.................................
Ihis experin-rit. may be considered in 5 parts.
I Extended dose/resportse curve.
Samples used: B15/01. No. --alt 496
Tests
for efiect of porctity, 4ilter pre-s-sure, drop,
~-d -r-
S
narf,ples used: E15/01 to B115/06. No.
I;ote: Only three of the five dot-e --ed for
E-15/01 are i-lvde-c.
Extended range of porosity.
Samples used: E15104, E15,07 to B15110. No.
4. Effect of e~lectrottatic pe7rio-tior-
Sample, used: B15/05 arid F,4.5/12. No. '5E,
S. Effect of change of p&pe-r addit,-.
Sa".ples used: B15/07 ar,d P15/11. 14c. 756
Cc
BATCo document for Province of British Columbia 3
November 1999


Plot 1 .
D.15 Exte-rided Dcse/Respon-Se Curve.
t. I - -:'l Ni2 ILr. I j-. I)
CO
CO
BATCo document for Province of British Columbia 3
November 1999


4.
4. Effect of & lectrOstbtlc per forat sort.
Sa.ples used: 13,11505 arid 815/12.
The chance, from a h.ighl%,, porous paper to an
electrostatically perforated
paper (of low porosity) had an effect which wall
significant at the: 94% level
of confidence. Rithough this - just below thL,
usually accepted level of 95"
for significance, this result must be tater, tr
casting a dzubt ower the.
continued use of electrostatically per4orhted
cigarette paper u-Fiere a paper o-'
equivalent "natural" porosity is availible.
Certainly if there are cor..pellir-
co-rierc-I reasons for the continued u- c-f
electr-tatic perforatior, then th--
r--ult r--riti further invest igaticn.
The Utta bre oraphed in plo.t 2.
CO
-0
BATCo document for Province of British Columbia 3
November 1999


P I o t '2 .
6 H -a t u r a 1 v E 1 ec t r o S t a t i c Pap e
r
Observed Data.
T.,g.t t~ "OL UTU.
L~(U.ib.ll b)
. ... .........
..............
.. ................ .........
.........
I I at . .............................. ..
................ ...... ..
-IS.0 . .............................. ..
............
-IF.E . ............. .... ...
I S 0 . ...................... .... . ... ........
..... .... . ..
3.7 E 3.9
P r d i t e d If t
T-q,t t ;:,- ty 70C LITLI
L~ I L4. tu I I t.
--ral
...... ........ ..... . .. ... .... .... ......
....... ........ .............. .............. .
..
. ........ . .... . ........ . .
r. . ............... ........ ........
............ ... . . .... 7 ...... ........ .
. ......... ....... ..
. ... ..... ..
....... .. ...... ... .... .. ...
.3 3.4 4 C'
1 1 L 7j C C,
E C C C. C
C CO
cc
BATCo document for Province of British Columbia 3
November 1999


S. Effect of change of paper additive.
Sanples used: B15/07 and B15/11.
The change from citrate to phosphate as a paper
additive hac an effect
which is significant at the 64% level of
cor.;idt-cL. Thir is a "half-way housL"
sort c4' result. The effect is neither larve
erough to be conclurive roor S~.all
er-oug'- to be ignored. The conc)ucion would
teerr. to be that phospl-te. p?pers
zhc-ld r.-! Le used urless the!re are oood e~~sc-s
for doir- ~c- 1-1 i.--
c.nt-.~d use of photphate p.perr 1,ke.ly -~-- thiz
result shc.uld bL- ---.-ttrj
,.Nore rioc.routly. It rriust be- remeriebered
that 7SE ar,irf.wl: u,er&
-'s ccrz.~a-zor.; a larger t~periment may .-ell
.,,eld k -h,oh1v
Fio, 3. illu!:trat&s the data.
C-1
00
CC
_Z~-
U-11
Q-71
C-:)
BATCo document for Province of British Columbia 3
November 1999


P I o t
D 1 c" C i t r a t e v . Ph c, s h a t e A ci d i
t i v e
Observed Data.
Lr.(W#!buI1 b I
. ................................:.............
.. .
. ............... ........................ ......
.. ...... ....
.............. ... . . . .....
4 .0
P e d i t. d D a t s.
LIS C 11EIII
. .. .. ..... .... . . . . .. . . . ... ...
..........
.3 3.4
CO
cc
4 M.
BATCo document for Province of British Columbia 3
November 1999


Tests for effect of porozss~y, filter pressure
crop, tip -ritilatior-
Samples used: B15/01 to B15/08.
This is the main part of the -periff.ent. B15,101
to B15zOE are used In a
2 >. 2 design to test for the effect of a charice
it-, paper porosity, a
char,ge- fr. the preriure cirop of the filter,
arid the u--L of tip ~ertolbt,on.
!nt-actions 1-tween these mair, ef4ec-s may &)so
te e-r.-ned.
None c-f these main e-ifec-t-c reatched -.
1grtifica-cet at '.he ~-5:.' level of
confider-ce. Paper porosity was -significant at
the 84:: le,,el, filter pres~Ure
drop at the 70% I-el, and the iritroduction of tip
-er.tilat-,
t h e S 5;' 1 ~- .,e I . Hone of the i r, t. er bc
t i or,z tier e s e 9r. f., c a r, t . TI-t - a r
i orc
9 ~ i n I e f t i r- the "ha 1 f - w& %, hour e" -
i t -_ t i ort . These i9r.if litarice level- -e
r.r-t
1 c.L.- enough to be ignored, nor yet high er,ough
to bc cc-clut-c-
seeir.- tome however thtt thecori:istertcio4 the
--g-ficnact- te
ltrics support to the conclu:ion that thefe
rerult= ar e ir, fact 17er-rL- :%q- ficar.1
tl-.,,n the Ttat~st,cal c&lculttiont tugg-st.
Cor,tic1tr the. fc I 1--r.9 title:
--------------------------
I Levels of S,q-f,c-.e I
I------------------------ I
I I I
1 55 WTU 1 700 PITU I
---------------------------------- ----------
-------------
I
Tip ~_-fect,oiver, Filterz.,Doses 1 86;: 1 16X 1
1 Filter effect, giver, Tipt,Doset. 1 11 % I E: 4
- - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - -
If u)e lool, at the retult- for tht ti~o le-tlt r
p ~; r t~ r rcrot I:.- tep-- -,A 1~
t 1. r. t h t r t a L. I e , h o u, s 't h e c r,
n f i d I I c L e c, i I h I c I c. , i c c e . f
ei;ect. c.; ch~,rglrq the tip from tC v-t
-,j tj- :f4eCt 4
-cre~;ring the prensure circ-P of the filttr.
The lur.-c-rige-city -' -h~ de;-.deFt upori, a'--2
~th=.
of zolbuztic-, whic!, tLirr. dti,tr.r! r.1 upc-
cc
cc
BATCo document for Province of British Columbia 3
November 1999


t e c ond er. ; a -, e . As t t-., F. c. , c, , t
5, c. 4 % V. e p - p i_- r, , s e s a . ... e f f
t c 1 0 4 t , p
,.,."t 16% I.r. d ii-nishes as mor e and more di
lut. son 6 ir comes in through the papee
Find aq~-6 in, in the extreme case of zero paper
poros ity changes in the pressure
drop of the filter wil I ha- little or no
influence --nce all of the- air drawn
clu- ing the puff must flow over the coisl. Rs
paper porosity rises ch&naes in the
pre-Ure drop of the fi Ite, ht.e a gres,%er
opportunity to alter the airflow
t hr ough t he pa per a rid a r ound thi c. ob I .
. Following this line of trgurrent, this table
reflect; the pi2ti&rr. of
out cor-es thal one would ewpect tc zee: tip
vitnti I&I-r. -portant A'-, us.,
lou, F-rjrosit,,, paperv and filter pretsure drop
of irr.po-C&nce whtn using high
poros ity
papers . The fact that. thi -. pi,ttern o; e--ntt'
I,a: err.L-r qed fr orr, the 6~ t
:.eems to me to ind icate that a I though the tr
ad it ic-I I levels of ; ignif icjr.ce.
have root beer. &ttained the re-sult,- are
ric-c-the.-Iess I" . This cor-clus-r. ':
;OtO ~LIJDJI- ted by the ftct that if ije
cori..p?,re 81S/01 utith Pic,/08 I c-. a te:,t E.;
the cc-b-ed -iiect of a s chanc;e - &I I three
rr~ain effect=
highly sq-i car.t result I-- obtained (S7;..
Plott 4. -,o S. i I lu.-tr&te t~.e, dita, for
these result:.
cc
cc
Z:-
BATCo document for Province of British Columbia 3
November 1999


Plot 4 .
P.1S Effect of Paper Porc.-Sity.
0 bS e r e d D a
Lm Ww t bu I I b
IV
9 S UTV
. .... .. ....... ....... ........... ..........
.. ...... .. ..... .. ... ...
-1 S.0 . .............. ......... ..... ..
........ .......... .
-1 S.S . .. ... .. ...
. ....... ...... ... ... . .....
. ... ...... ... .. . ... ........ ........ ....
.... ......... . . ..........
Z F z 4.0
P r d i d D z-:L t a
D.
-1-.0 L-L'.,bull b)
. . . ........ .
,C
. ..........
3.E
C C ft Oc
Oc
BATCo document for Province of British Columbia 3
November 1999


F1 o t
E' . 15 E f f ec IL of Tip Venti lation
Observed Data. Pa p r Porosi t-,) 91, biTli .
L~W-b.ll b)
. ....... .. ......... . .......... ... ........ .
.... ..
T
. ... ........................... ............. ..
. .
. . . . ......
. .......... ..
.. .. .. ...... ..
.................................. .
............... ............ . . ...... .. ..
...... ... ..
5 .7
F redic ted 11 a t -a Pa, Pi r F',:. r c. -S i t 9
S 1.0 T 1-1
L I tle , t, I I I, I
. .... ... ..... .............. .. ........... ...
. . ..... . ........ .. ...
C. . .. . ............ ... .. ... . .... .... .
.... ... ...
.. ...... . .
.......... .. ..... ..... ... ..
cc-C 'C'
BATCo document for Province of British Columbia 3
November 1999


P I t E.
E.16 Effect of Tip Ventilation.
Ob-Served Data. Paper Pc-r-------=itV 700 IATU.
Jj4t& r-t%c-I*d owor filt-r.
..............
S .7 .6 4.0
4.1
Predicte-d Data. Pape'r Forozit--.; C-1 T
--q- It., r .
F .. .... .......
.. . ... ..................
.... . .......
-:E.( . . ...... ..............
.............. ....... . .. .. ... .....
............ .. . ....... ...... ........ .....
. ........ .............
. ... ........ ............. ....... . ..
cc
BATCo document for Province of British Columbia 3
November 1999


P I o t 7
B.15 Effect of Filter Pressure Drop.
Observed Data. Paper Porosi t,,) WTU .
TI. t . p... d
. .................... ........... . ... ...... .
..... ..
.......... ....... . ... ...
.
... .
.
.
.
.
.
........ .
.
...
..
...... .. .
... . ........... ..
. ..... . ................. ...... ...
............ ... . .... ...... .. .. .. .... .
......... .. ... . ........ ..
2.7
P r e d i t 11 -a t -a F -a p r F o r o t E 6.1 T
1-1
L,. W, i hu I I
.... ........
...... ..... .... ... . ....... ..
..... . . .... . ............ ......... .
. . .............. ....... .. .... . ..
-I i E . ..............
......... ..
-17.0
3.4
r: fz..
CC
-C~-
L-11
BATCo document for Province of British Columbia 3
November 1999


Pl 0t 8.
B.16 Effect of Filter Fres-sure Drop..
Observed Data. Pap e r- P c, r c. s i t 700 WTLI .
L, (W. i b. I I b 1
..... .. ...... ........ .. . . ...... .. ......
M ...........
. . .. ....... .......
...... ..... . ... ...
...... ........ ..... . ....... . . ... .
......... .. .. ...... .
. ........................ ... . .
....................
. .. .............................
. ...... ...... .... . ... ........ .......
........ .............. . .......
Predict4---d D-=--ta. Paper '7 C1 ~-i I.J T 1-1
Lr. I L't, I tO
,06'
..... .. ..... .... ... . ... .......... ......
.............. .. .......... ....
. .................................. . ...........
. .. . ..... .. ....... ... .. . .....
. ............... ........... ..... ...
-17
-2 3.-
C=)
CO
cc
rz,
BATCo document for Province of British Columbia 3
November 1999


Flot 9
1 T. C o at b i rt ed Effect of Parameter-.
Observed Data.
................ ............ .. ... .... . . .. .
. .............. . ...... ...
. ............... .................. .........
..... .. . ... . ...... --- . . ....
I E
. ............... ....... .........
................ .. ...... . .... .. . . . ...
....
. .. .. .. ..... . .. .. ...... . ............. .
. ... ...
......... ... ...... ....... . .... ..... .. .. .
..
1.4 2A 4 .
-1( S
tic 8 L Z E 1
cc
(DO
411
BATCo document for Province of British Columbia 3
November 1999


3. E.tended range Oi Porosity.
Sarnples used: B15/0D, B15,'04~ B15~07 to 915,10.
I n th i s sect I on of the exper v rrent sanip I
es B1 5/03, El 5/04 . &red saffip I es
-R15,07 to B15,10 e,e used to provide an e,t&,,ded
r&r,-oe of pape, p-osity data.
Frorr, the analysis of this data it- is clear that
incre-irig paper porosity a
significant effect on the turrorigenicit, of thL-
cordericate. If the porosity s,-
L-r,LtSed ar 100`WTU (I.e. a reciproi:7,1
relatic-:hip) 'then the porosity e44ec-.
is I near .
The d&ta i-re 111--trated in p1r,% 1r- Fron.
ti-,e--L- plots it car. t- seen t;-
e a r-.c,r o~ i ty o; about 40C, WTU i -. reached
the:r. fur ther trocr ta, t~ i r, po, c,
P, .,e- I,,, t I e or ro tf fec the t -or i c:er,
i c it y of t he c or.der, ~kte . I he ef f L c-,
- the r ar-ge ztr r, -r.- 20C, 141-L1 is quite
rrar ~ td. I I rr--t he rioted hc-u,ver that
t j -, --- 1-ax icn :.; .ne curve: , houir, i r. I
h t- ) rjwf-r f igure of p I r- 10 ar f based or.
c r, e c: z, tp o r, t s at E~S, 700 a-d IE00
14TU.
CZ)
C=)
BATCo document for Province of British Columbia 3
November 1999


plot 10.
p 16 Plots of Response v. Porosity.
R e S p o n -S e v . 1 C'0zLJTU
L~(W.ib.ll b)
D- 1-1
.. ........ .... .. . ... . .. ... ...... .... ...
. ... ....... ... . . . . .....
. . .......
- - - - - - - - - - - - - - - - - -
I C, E' .............. ........... ... .... ...
...... ..... .. .. ...... 777'= - - - - - - - - -
- --
Q Z 1.6 Z
11 TU,
R e *S p k T L
F.*rpL,r,e i Ir tul I c r.+ ter t
t-I
- - - - - - - - - - - - - _ - - - - - - - - - - -
- - - - I
....... ..... . . . . . . . .. ..... . . ..
. ......... .. ......... .. ............. ..
_1 ~ A,
-If E
20 C
(Z)
cc
0-
xz.
ON
BATCo document for Province of British Columbia 3
November 1999


APPENDIX F
LAI~2 vmxp
T
AUTHOR: E. D. MASSEY & G. A. FEW
EDM/GAF/LAP/46D 20 July, 1983
TOBACCO SMOKE MUTAGENICITY:
THE I14FLUENCE OF NITROGENOUS COMPOUNDS
STATUS REPORT
The influence of nitrogenous compounds, especially
nitrates
and proteins/amino acids, on the mutagenic
activity of cigarette
smoke condensate (CSC) was reviewed in 1982 (1).
In essence,
pyrolysis products of proteins/amino acids are
powerful mutagens.
Furthermore, tobacco nitrate varies between being
heavily impli-
cated in CSC mutagenicity and not being implicated
(1) . This
confusion requires clarification and can be
accommodated within
the proposed work. Previous experiments in this
area, mainlv
by the Japanese Tobacco and Salt Public
Corporation (JTS) , ha~
utilised natural tobaccos and hence were studying
the effect
on CSC mutagenicity of multi-variables (1) .
Similar experiments
were carried out at GR&DC to confirm the JTS
results (1) and
suggested that the area may be fruitful in
designing cigarettes
with a lower biological (mutagenic) activity. To
concentrate
on one class of chemicals, which the Industry may
be able to
selectively remove from tobacco, the emphasis was
put on
proteins/amino acids. The most simple way to probe
this area,
to examine if a fuller project is worthwhile, was
to add these
compounds to tobacco, manufacture cigarettes and
assess the
mutagenicity of the CSC, it was this approach that
will be
reported below.
MATERIALS AND METHODS
A flue-cured tobacco, cut blend 42 as used for
manufacturing
GR&DC reference cigarettes P161, was analysed for
its free amino
acid content (2). Subsequently, an aqueous mixture
of those
amino acids was prepared and sprayed on the
tobacco, being
intended to give a 10X increase in free amino
acids. The
tobacco was then reconditioned and the free amino
acid content
re-analysed and plain cigarettes manufactured.
Control tobacco
was sprayed with distilled water only. Cigarettes
were then
smoked, condensate collected and assayed for
mutagenicity using
S. typhimurium TA 98 as described previously (5).
RESULTS
The analyses of the amino acid enhanced tobacco
showed the
free amino acid content of the tobacco had
increased approxi-
mately 8 times (3) . Furthermore, the CSC from the
amino acid
enhanced cigarettes was considerably more
mutagenic than that
from control cigarettes, fiaure 1.
DISCUSSION
With this small series of experiments a two phase
investi-
gation has now been completed in this area'
............ Cont'd.
cc
C)c
-r--.
L.14
c7 N
IN.)
BATCo document for Province of BritiSh Columbia 3
November 1999


-2-
I. A series of experiments to confirm the work of
JTS (1)
The cigarettes with highest protein levels were
found to produce
the most mutagenic CSC. However, confounding
variables such as
variation in tobacco nitrate and sugar content,
which are thought
to influence CSC mutagenicity, were present, as in
the JTS
experiments. Thus, absolute interpretation of the
results
regarding tobacco protein/amino acids content in
determining
CSC mutagenicity is difficult.
II. The second experimental series was able to
determine
the effect on CSC mutagenicity of a single
variable, i.e. the
amino acid content of the tobacco. This
establishes that the
tobacco content of amino acids profoundly
influences CSC muta-
genicity.
These results therefore suggest that removal of
proteins/
amino acids from tobacco may diminish the
mutagenic activity
of CSC and would be an useful addition to
approaches to project
RIO.
PROPOSALS FOR FUTURE WORK
Processed Tobacco
e.g.
Protein Cylproc
removed
by enzymes
k""-z
Combinations of tobaccos
and tobacco extracts
Natural Tobacco
Project Mini Brochure
Liner Programming
Change 1 tobacco component
independent of others
cigarettes
Mutag :y Assay
The developments in this field should be centred
upon natural
and processed tobacco as detailed above.
Furthermore the systems
are designed not only around an experimental
approach but also
around one that is industrially feasible.
I. PROCESSED TOBACCO
C:)
1--C
These experiments involve removal of protein
material from LY--
either partially cured yellow tobacco (Cyloroc) or
from cured cc
tobacco using enzymes that digest proteins. The
liquor extract
along with treated or control tcbacco can then be
used in a
series of 'a66ition' ex::,2riments to investigate
the effect of
............ Cont'd.
BATCo document for Province of British Columbia 3
November 1999


-3-
these processes on CSC mutagenicity. In add.~tion,
the liquor
extracts may be fractlonated such that the single
class of
compound of interest may be added or subtracted
from the tobacco
of interest. This latter concept is perhaps an
important area,
for general tobacco extracts may be deleterious to
smoke character-
istics, e.g. proteins are flavour negative whilst
sugars and
free amino acids have flavour enhancing
characteristics (4) .
Thus, in such experiments to provide the potential
customer
with an acceptable product with low biological
activity, a
balance between removal bf proteins, but with free
amino acids
and sugars remaining must be maintained.
II. NATORAL TOBACCOS
This approach to the study will be carried out
with the aid
of linear programming, using the project Mini
Brochure and the
wealth of data concerning tobacco characteristics
that is being
collected. Hence, for the first time, it will be
possible using
natural tobacco to maintain fixed parameters
whilst altering
fewer variables than previously possible.
Using the two experimental designs outlined above,
the data
should indicate the mutagenic activity of
cigarettes with reduced
proteins/amino acids. The systems will probe the
scientific
principles to further establish the rational of
deproteinised
tobacco as a basis to a product of reduced
biological (mutagenic)
activity.
Additionally, within this project not only will
proteins/
amino acids be investigated but also the role of
tobacco nitrate
and mutagenicity.
I *"~T_
E. D. MASSEY
Biological Sciences
Smoking & Health
A -
G. A. FEW
Leaf & Biotechnology
DISTRIBUTIO14:
Mr. A. L. Heard.
cc
Dr. C. I. Ayres,
Mr. D. E. Conway,
Dr. R. E. T~ocnton,
Mr- T. G. Ifitchell.
cc
CDC
C_
BATCo document for Province of British Columbia 3
November 1999


-4-
REFERENCES
(1) MASSEY, E. D.
Reduction of Tobacco Smoke Mutagenicity: The
Influence of
Nitrogenous Compounds.
GR&DC Reference EDM/JP/46D, 5 July, 1982.
(2) BURDEN, A.J.
Amino Acid Analysis - Sample P161.
Technical Memorandum No. TM 82-07-013, July 1982.
(3) BURDEN, A.J.
Amino Acid Analysis of Treated-P164 Blends.
Technical Memorandum No. TM 83-04-008, April 1983.
(4) CRELLIN, R.A.
Personal Communication.
(5) BAT Reoort No. RD 1874-C Restricted.
cz:0
cc
cc
crl~
-Y1
BATCo document for Province of British Columbia 3
November 1999


FIGURE 1
The Effect of Enhancing Amino Acids on
Smoke Condensate Mutagenicity
Cf)
M
0
z
cn CO
I-- C)
z
LU
LLI
0
z 0
z 0
< Ln
LLI
0
X
z
0
0
0
Cl)
-0
-0
Cl)
0 LLJ
0
C14
CL
z
LU
M
z
0
0
0
C:D
11 C
cc
cc
BATCo document for Province of BritiSh Columbia 3
November 1999