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November 1999


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BATCo document for Province of British Columbia 5
November 1999


BRITISH-A14ERICAN TOBACCO COMPANY LTD.
LONG-TERM SKIN-PAINTING EXPFRDMqTS -
PROGRESS REPORT: JULY 19t)7
REPORT NO. B-8
DISTRIBUTION:
D.S.F. Hobson, Esq.
Sir Charles Ellis
Dr. S.J. Green
Dr. R.B. Griffith
L.C. Laporte, Esq.
W.W. Reid, Esq.
Dr. D.G. Felton
2.8.1967
AUIHOR: C.I. Ayres
Copy No. I
2
3, 4, 5
6
7
8
9, 10
COPY NO:
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BATCo document for Province of BritiSh Columbia 5
November 1999


LONG- MCIN-PAINTING FXPERDIaITS - PROGRESS REPORT:
JULY 1-1-67
SUMARY
In this Progress Report, an outline is given on
the results to
date and the mrocedures used in the series of
long-term skin-painting
experiments iemich are being carried out under
Project JANUS.
Although it is too early in the experiments to
draw firm
conclusions, attention is drawn to several trends
which are apparen':
in the resul:z. In particular, it should be noted
that in the first
experiment, Zxperiment BO, which has been in
operation for twelve
months, there appears to be a "high dose anomaly"
in that the hignest
dose of condmsate applied is giving rise to less
mice with tu;Tours
than is the agplication of the middle dose of
condensate.
BATCo document for Province of BritiSh Columbia 5
November 1999


INTRODUCTION
The major portion of the work under Project JANUS
is concerned
with a series of long-term skin-painting
experiments which are being
carried out by -die Battelle Institut, Frankfurt
am Main. In this
Progress Report, an outline is given of the
procedures involved in
these experiments and the results to date.
In brief, a solution of smoke condensate is
applied to the back
of a mouse at re-bular intervals throughout the
complete life-span of
the animal. By using large groups of animals and
recording the number
and time of appearance of tumours, the relative
tumorigenici ties of
different condensates can be compared.
DISCUSSION
In the operational plan, a new test is started
every three months.
The first experizent commenced in July 1966 and a
total of eleven
experiments is envisaged.
For each experiment, three groups of approximately
250 mice are
treated with different doses of smoke condensate.
At the time of
treatment, the solution of condensate is 24 hours
old and applications
("paintings") are made on TUesday, Thursday and
Saturday of each week.
The treatment is continued until a mouse dies or
is deliberately
sacrificed by the pathologist. Details are given
in the Appendices.
7 ne first ;ix of the experiments are either
already in hand or
have been planned. Details of the smoking
conditions, dose levels and
numbers of mice are given in Table-1.
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BATCO document for Province of BritiSh Columbia 6
November 1999


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TABLE 1
OUTLINE OF EXPERIMENTS
C1 gttj LW '1:11u1,ji1g
Dose of Number
Butt Puff Condensate of Mice
Code Dimensions Length " (mg) in Group
(mm) (mm) Volume Duration Frequency
(ml) (sec) (per min)
Experiment BO BO 70 x 25 2,3 35 2 1 25 252
50 252
Starting date: July 1966 75 252
Experiment Bl BO 70 x 25 23 10 2 1 50 252
25 2 1 50 252
Starting date: October 1966 50 2 1 50 252
Experiment B2 B2 85 x 25 28 35 2 1 25 252
50 252
Starting date: January 1967 75 252
Experiment B3 B3 70 x 25 23 35 2 1 25 252
50 252
Starting date: April 1967 75 252
Experiment B11 B4 70 x 25 23 35 2 1 25 252
50 2-32
Starting date: July 1967 75 252
ExperimwIL fill) B0 '10 X 2,5 3'~ P 1 25 25,-
50 252
Starting date: October 1967 75 252
35 2 3 50 252


Details of the cigarettes used in the experiments
are given below.
Cigarette BO
Tobacco
c.p.i.:
Paper:
Average weight:
Average draw resistance
CN102 lamina, i.e., does not contain CRS.
55
014, Verge -46-41 mm.
1-C9 g.
9.7 cm W.G.
Cigarette B2
This cigarette is a typical blended American
cigarette with casing
and flavouring. As the cigarette is an 85 mm
filter-tipped cigarette,
a butt len6th of filter + 8 mm tobacco is used in
the experiment.
Cigarette B3
Tobacco IC,--;-. PCL.
The PCL was made by the Imperial Tobacco Co. of
Canada, Ltd. from Blend
BO lamina and Canadian stem (VS). Me flour was
prepared from 2,150 lbs
of BO lamina and 715 lbs VS. The Binder was
prepared from 2,000 lbs
VS; the flour:binder ratio was .3:1. '-%e
cigarettes were made at R. & D.E.,
Southampton. The dimensions, paper and c.p.i. of
the tobacco were the
same as Cigarette BO.
Average weight: 1.15 9-
Average draw resistance: 9.7 cm W.G.
Cigarette B4
Tobacco: A =ixture of equal portion of CN102
lamina
(55 c.p.i.) and CRS (180 c.p.i.).
The CRS was made from Canadian stem from the same
batch as that used
in the manufacture of PCL. The paper and
dimensions of the cigarette are
the same as Cigarette BO.
BATCo document for Province of British Columbia 5
November 1999


Avtrage weight: 1.05 9
Av2rage draw resistance: 9.2 cm W.G.
RESULTS
The information obtained from the experiments will
be:
(a'. An indication of the possible importance of
smoking parameters
on the :=,origenicity of the condensate
(Experiments BO, Bl and B5). it
is know-- (1) that the chemistry of cigarette
smoke changes as the volume
of the ;uff used to smoke the cigarettes is
altered. Farther it has
recent!T been shown (2) that the concentration of
benzo(a)pyrene in
smoke .4Z lower when cigarettes are smoked at
three puffs a minute than
when ci~-arettes are smoked at one puff a minute.
Thus by changing the
smoking -:arameters it is possible to obtain large
changes in the
composi-:fon of the smoke condensate without
changing the tobacco.
(b' A comparison of the condensate from a U.S.
blended cigarette
and a c--'-arette made from flue-cured lamina
(Experiment*B2 v. Fxperiment BO).
(c, The effect of PCL (Experiment B3) and of CRS
(Experiment B4). As
the PCL ~_:nd the CRS/lamina cigarettes were made
from approximately equal
weight cf starting tobaccos, an indication of the
importance of the
processfag involved in PCL manufacture will also
be obtained.
The results to date are summarised in Tables 2-6.
A certain number of
"replacenent mice" are available to substitute for
any mice which die
during zEie first 8 weeks of an experiment.
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BATCo document for Province of BritiSh Columbia 5
November 1999


T A' =-E 2
CONT.FCL GROLTIPS
Replacement mice: Untreated = 2
Solvent treated - 2
No Treatment Solvent TreF-----d
(0-3 ml acetone :wa-zer (9:1))
No. of Mice Total No. of Mice Total
Weeks A live Number A live N-=b e r
of Mice TO of %K, ce T.6?
Without With EN Without W --* th EN
Total Tumours Tumours H
Total Tumou rs T=ou rs
NH EAP NH Z3~?
0 126 126 - - 126 126
4 126 126 - - 126 126
8 123 123 - - 125 125
12 123 1 2_3 - - 123 123
16 123 123 - - 122 122
20 122 122 - - 122 122
24 122 122 - - 122 122
28 122 122 - - 121 121
32 122 122 - - 121 121
36 122 122 - - 121 121
40 119 119 - - 119 119
4-4 117 117 - - 118 118
48 115 115 - - 118 118
52 114 114 - - 117 117
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BATCo document for Province of British Columbia 5
November 1999


L -T-
TAR[ F
UPERIMENT BO
Repl acement mice- 25 mg group 5
50 mg group 25
75 09 group 24
25 mg Does 50 ag D03e 75 g Doze
No. of Mice "Abt8l No. of Mice Total No. o f Mice
Total
Weeks Alive Number rap Alive Nmber w Al ive Number
YLP
of Mice
ENH of MIce
UT.-
H
c.
of MI -
LNH
Witbout W1 th
x 10 Itho with I
u
x 10-)
W
e
ithout Wi th
10
Total Tumours Tumour. Total TU.-
Tu-,a
r Total Tuiaoura Tumoure
% rAp NH L&P ;i N, w
0 252 2-52 252 252 252 252
4 252 2ci2 252 252 251 251
8 249 20-
243 243
206
206
12 247 247 - 240 240 Igo 198
16 247 2-7 - 2~9 239 192 192
20 2166 2-6 238 238 181 181
24 245 245 2,36 235 1 7 179 17-Q
28 244 2&A 2,35 232 3 18 176 176
32 243 2&3 2,35 226 9 47 175- I7-, 4 26
36 240 236 4 18 235 , 215. 20 94 171 16A 8 47
40 219 234 5 20 235 210 2*3 107 166 156 9 48
44 2'58 2,13 6 22 230 199 32 126 161 1%9 14 1 69
48 M 2,V 8 2a 226 185 44 162 156 136 22 102
52 234 221 14 45 218 170 54 187 152 122 33 144
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BATCo document for Province of British Columbia 5
November 1999


TAMX 4
E(PERIKENT 31
Replacement micei 10 ml g~p - 12
25 -1 &-p - 3
... 50 -1 g-p - 6
10 al Puff Volume 25 .1 Puff Volun. 50 ml P uff
Volume
No. o f Mice Total No. of Kies No. o f Mee Total
Weeks Al ive Number Ea Alive Number YAP Al i-
umoer
N
of Mice -I
ENH of Kies
DIN r Kies
o
D;H
Without With _.
10
(x Without With
(x 10~) Without W
ith ( x 10-
Tht.1 Tumoure Smours Tumours, Tumoure To
tal Tumours, T-oure
NH MP N H FAP N, Ea
0 252 252 252 252 - - 25Lf 252
4 213 213 230 230 - - 238 238
8 lq~, Ig-, 229 229 - 238 238
12 18B 186 228 226 - 235 235
16 180 180 227 227 - 23i 231
20 177 177 226 226 - 225 225
24 175 173 2 18 22.3 223 - 222 221 1 7
28 170 168 2 15 221 219 2 13 216 214 2 12
32 168 16o a 55 219 212 7 .39 215 214 2 11
36 164 153 12 75 215 205 10 50 211 208 3 15
40 161 143 19 109 213 197 16 72 2og 195 14 63
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BATCo document for Province of British Columbia 5
November 1999


-9-
TABLE 5
EXPERDUNT B2
Replacement mice: 25 mg group - 15
50 mg group - 14
75 -g group - 5
25 ~g Do.. 50 mg Dose 75 mg Dose
No. of Mice Total No. of Mice Total No. o f Mice
Total
Alive N-b.r Alive Number
~Lp
RE Al ive Number rZY
Weeks of Mice of Mice of Mi.. -
Without With FNH Without With ENH
10-)
( Without Wi th DH
-
Total Tumours Tumours x
Thtal Tumoure Tumour. Total Tumour. Tumou, (x 10
% rAp N H NH ra
0 252 252 - - 252 250 252 252
4 252 252 - - 252 252 252 252
8 250 250 - - 249 249 245 245
12 250 250 - - 245 245 - 238 238
16 249 249 - - 245 245 - 235 235
21) 247 24T - 244 244 235 235
245 245 - 243 240 3 20 2,33 23-3
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BATCo document for Province of British Columbia 5
November 1999


Week.
0
4
8
12
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TPJ= 6
EOPOUMENT B3
Replaostfamt aloes 25 mg group - 3
50 -g group - 3
75 -a group - 2
25 mg Dosse 50 ~g Wee 75 mg Dome
No. o f Mioe Total No. o f Mi.. Total No. f Mi..
Total
All~e Nuaber Ali,. Nub.r Al i- N-b.r
of Kloe TAP
of Mi..
f KI.. Eap
W
ithout WI th
NH
(x 10-) thou t
wi Wl th INK
(x 10-1) Without WI th ENH_.
(x 10
Total Tumouts TU..u
r. Total Tunsoure Tumoure Total Twoura Tumoure
N
H rAP N
H rAP N
H rAP
252 252 252 252 - 252 252
2-_;2 252 252 252 - - 252 252
248 248 251 251 - - 249 249
247 247 249. 249 - - 249 249
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BATCo document for Province of British Columbia 5
November 1999


The initial guide to the progress of an experiment
is given by the
column headed FLP, i.e., the total number of mice
which have tumours. A
simple comparison of the T treatments can, on
.4 F values for the various
some occasions, be somewhat misleading. For
example, if a condensate
leads to a high mortality of the mice, then there
are less mice available
to get tumours. Consequently, at the present time,
use is being made
of the ratio ZA-P which, it is hoped, will provide
a better guide to the
ENH
relative tumorigenicity of the condensate. The
denominator of this ratio
is obtained by summating the number of mice alive
which do not have
tumours (N ) at the end of each four week period.
The reasons for the
H I
choice of this ratio are given in Appendix IV.
At the present time it is too early in the
experiments to draw
major conclusions. Several comments, however, can
be made.
Control Groups
A very low death-rate is being experienced in the
control groups.
At the present time, the death-rate is lower than
that observed in the
"First Main F-xperiment" at the TRC Laboratories,
Harrogate (-3).
Experiment BO
The major point to notice is that the highest dose
level (75 mg) is
giving rise to less tumours than is the middle
dose level (50 mg). The
reason for this is not known although several
possible explanations can
be advanced. These are associated with:
(i) The high dose level killed more animals in the
early weeks
than did the 50 mg dose level. Thus it is possible
that a
greater proportion of the mice which are
susceptible to
tumour formation have been killed in the 75 mg
group.
00
BATCo document for Province of BritiSh Columbia 5
November 1999


(ii" The dose-response relationships of the irice
to tumorigenic
materials and to "anti-tumorigenic" materials are
different
and are such that the nett experimental re&ult
emerges.
(iii) The observation is the result of an artefact
(not yet defined)
arising from the way the experiment is perforned.
For
example, the different dose levels are applied in
a constant
volume (0.3 ml) of solution, i.e., there is
approxima'-ely a
factor of three in the concentrations of the
solutions being
applied. It is feasible that the response of t-he
mice is
sensitive to.factors such as the spreading of the
condensate
on the back of the mouse or the penetration of the
skin by
smoke constituents.
Experf--ent BI
There fs an indication from the early results that
condensate pro-
duced at a low puff volume (10 ml) might give rise
to more tumours than
dose the ccne-ensate produced at a high puff
volume (50 Ml)-
An unexpected observation was the difference in
the number of mice
killed in '2:e first 8 weeks of the experiment:
10 ml group 73 mice died
25 ml group 28 mice died
50 ml group 20 mice died.
The majority of the mice which died had the
symptoms of "nicotine
poisoning". The nicotine content of the painting
solutions is, however,
approximatel7 independent of the puff volume used
to smoke the cigarettes.
Eliminating from consideration the replacement
mice, the number of animals
which died from "nicotine poisoning" are given in
Table 7.
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BATCo document for Province of BritiSh Columbia 5
November 1999


-13-
TABLE 7
DEATHS FROM "NICOTINE POISONING"
Puff Volume (ml)
10 25
1 35
(from Expt. BO)
50
Delivery per cigarette
Condensate (mg) 11-5 27.9 32.6 38.7
Nicotine (mg) 1.04 2.42 2.70 3.19
Dose
Condensate 50 50 50 50
Nicotine (mg) 4-52 4-34 4.15 4.12
Deaths due to
"nicotine poi soning"
I week - - -
2 weeks 16 8 4 2
3 weeks 15 8
9
5
i 4 weeks 11 3 2 3
5 weeks 14 1 1 -
6 weeks 3 1
7 weeks 1 2
8 weeks -
TOTAL 6o 20 20 10
Experiments B2 and B3
The numbers of mice which died in the early weeks
were small-.and, as yet,
there are insufficient animals with tumours to
make any initial conclusions
worthwhile.
Finally, it is of interest to examine the numbers
of animals which have died
in the various rooms (Table 8).
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BATCo document for Province of BritiSh Columbia 5
November 1999


TABLE 8
CC'vTARISON OF ROCM3 : DEAD MICE
Rocm A! Room B Room C
October 1966
Mice entering room 353 352 359
Dead mice 45 ~11 59
Januar-j 1967
Mice entering room 614 611 616
Dead mice 99 81 108
April 1967
Mice entering room 876 870 885
Dead mice 143 103 145
June 19b7
Mice entering room 1130 1125 1140
Dead mice 178 131 177
From these early results, i t would appear that
there is a tendency
for less mice to die in Room B than in Rooms A or
C. There is, however,
a partial explanation for the observed result. In
Room A, there was a
minor accident in which an air-h ose blew apart.
Although it was corrected
within seconds, the sudden noise caused 15 mice to
die from shock. In
Room B, 25 mice escaped through the bars of the
cages and were sacrificed.
The mice which have tumours are largely lo cated
in Experiment BO.
Combining the various dose levels, the number o f
tumour-bearing animals
is given in Table 9.
BATCo document for Province of British Columbia 5
November 1999


-15-
A =- L r-
T
=ERI,-'La-'!T EO: M-TARISON OF R00`*,S
Room A Room B Room C
Week 7LIP 'r-,
ap FNH i fa IH ap H
(X (X (X 10-1)
4
8
12 -
16 -
20 -
24 - - 1 8
28 1 6 1 6 6
32 4 22 5 206 4 23
36 11 56 13 62 9
40 12 55 15 65 12
44 15 63 24 95 1.5 55
48 22 86 28 103 24 95
52 32 118 33 114 36 134
From these results, there was an indication after
44 weeks that in
Room B, which has the lowest mortality rate, there
is a trend for a
higher proportion of the mice to develop tumours.
During the last
eight weeks, however, this trend has been
eliminated.
Finally, however, it must be emphasised that at
present it is only
possible to indicate trends in the results and it
is too early in the
experiments to draw firm conclusions.
BATCo document for Province of BritiSh Columbia 5
November 1999


- it,:) -
AFP2-D1K I
rE-:E ANre.*;.L HOUSE
It wan necessary for Battelle to build a Aninal
House for the
long-ter-m tests and a two storey building has
been constructed. Th e
experi.mental work is carried out on the upper
storey as this houses the
animal rooms, smoking facilities and
pat~nologistls laborator-J. Th e
lower storey, which is a semi-basemenL, houses
several necessary features
such as the air-conditioning plant, cage-washing
facilities, cold store
and conditioning chambers for cigarettes, fodder
storage eLc.
The Animal House is divided into "clean" and
"dirty" areas.
Stringent precautions are taken in the operation
of the "clear." area in
which the mice are housed. All operators have to
strip, snower and dress
in sterile clothes prior to entering the "clear.
area". Personnel a-so
have regular medical checks to limit as far as
possible the risk of
infection entering the Animal Rooms.
The air is conditioned to a temperature of 22 +
0.50C ard humidity
of 60 + 2f, The air is washed and filtered
(particles greater t-lian 1 k
are removed) and 50% of the air is re-cycled. The
Animal Rooms operate
under positive air-Pressure and a mat soaked in
11,55 Incidine is placed at
each door. Incidine is a product marketed by
Altromin G.m.b.H. and
consists of tri-n-butyltin benzoate and formalin
in isopropanol/ethanol.
Several points in the operating procedure are
outlined below.
Mice
Female, S.P.F. mice of I.C.I. type are supplied by
Animal Suppliers
(London) Ltd. The mice, which are 4 weeks + 2 days
old or, 'arrival, are
housed in a quarantine room for four weeks prior
to entering the Animal
Roo.ms.
BATCo document for Province of BritiSh Columbia 5
November 1999


Distribution of th--~ in nr
For each experizmer.:~, 756 mice are divided
evenly into three Animal
Rooms (Rooms A, B and C). In each room, the mice
are divided into three
groups which receive the different doses of
condensate, i.e., the 25 :.-.g
group, the 50 mG Croup and the 75 mg -,rouP. Thus,
in each experiment,
the mice are distributed throughout the total
%rorking area. The alter-
native systez; of EAperlment I in Room A,
Experiment 2 in Room 3 etc. was
rejected as possible "room effects" would remain
undetected. Although
all attempts are made to standardise the
techniques of the operators and
the conditions in the r3oms, the possibility
cannot be excluded that
minor differences in techniques or conditions
could lead to apparent
differences in condensaiz;e tumorigenicizy
emerging.
To avoid any Itime of painting' effect, the order
of painting the
groups of mice with the different doses of
condensate is sequentially
changed.
The mice are shaved over an area approximately
from the nape of the
neck to the tail as and -when necessary, using
electric clippers.
Cages
The mice are housed in Macrolen poly-ester cages.
Three mice are
housed per cage: the in-dividual. mice are
distinguished by ear-clippin.-.
At the end of each month, vacancies in the cages
are filled and the total
number of cages reduced. This enables a worthwhile
saving to be made in
cage requirements and space.
The cage bedding is wood powder (free of terpenes
and dust) which
has been autoclaved. The cages are held in racks
(84 per rack) and tte
position of --'-e rack in --he Animal Room is
changed periodically.
The cages are washed and autoclaved every three or
four days.
BATCo document for Province of British Columbia 5
November 1999


Fccd
The mice receive tao-water and oterilise,-: food
a-I T!-,e
food is sterilised by the supplier (Altr~Zge).
Record Keeping
Each mouse is assiGned a number and a detailed
rccord is maintained
in triplicate of all treatments the mouse
receives, weirhl~ gain, illness,
tu;-nour appearance etc. The patholorist is the
sole judge of when a
papilloma is diagnosed, when the treatmert of a
mouse should be suspended
or when Ue mouse should be sacrificed. It u-az
considered essential to
apply unified criteria in all Animal Rooms and for
all experiments. A
post-mortem examination of all mice is undertaken
and, az far as possible,
the cause of death ascertained.
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BATCO doCUMent for Province of British COIUMbia 5
NoVernber 1999


A-IFENDIX I!
PRODUCTION OF COI\'D---!SATE
Smoking Machines
Smoking machines supplied by R.W. Mason Ltd. are
used. In principle,
a disc which holds 24 cigarettes is rotated so
that each cigarette in turn
is connected to a suction port. The smoke is
collected in a glass trap
(essentially that of Elmenhorst (4)), cooled in
acetone/solid carbon
dioxide. The smoking machine is a "free smoker".
In Experiment B5, a puffing frequency of 3 times
per minute will be
obtained by modifying the. machine so that three
suction ports are spaced
a-~ equal intervals around the disc, e.g.,
cigarettes 1, 9 and 17 are puffed
simultaneously.
Painting Solutions
The doses of condensate which are applied to the
mice are based upon
"rotary evaporated condensate", or, as it is known
in Battelle, "Dry
Condensate", i.e., the material which remains when
a solution of smoke
condensate in acetone is evaporated to constant
weight in a rotary
evaporator at 400C and water pump vacuum. The
solvent for the painting
solution is acetone:water (9:1).
The initial procedure used to prepare the painting
solutions was:
The condensate from three or four Mason smoking
machines was
dissolved into a single acetone solution. An
aliquot, which corresponded
to approximately 200 cigarettes, was taken for
evaporation and hence the
weight of Dry Condensate in the acetone solution
was determined.
The concentration of the solution was adjusted to
75 mg Dry Conden-
sate in 0.3 ml either by a small amount of
evaporation or dilution with
acetone. The water content of smoke is such that
the solvent approximates
BATCO document for Province of BritiSh Columbia 5
November 1999


-20-
to acetone:water (9:1) and this solution was used
to paint the 75 mg
group of mice.
7 he painting solutions for the lower doses were
obtained by suitable
dilution with acetone:water (9:1).
The evaporation stage of this procedure is
time-consuming and
caused a bottle-neck as the number of experiments
in hand was increased.
Consequently in April 1967, the procedure was
modified:
Battelle had recorded the delivery of Crude
Condensate (RC) which
is obtained from the weight of the cold trap
before and after smoking.
It was found that the ratio of Dry Condensate to
Crude Condensate (DC:RC)
was constant (Table 10).
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BATCO document for Province of BritiSh Columbia 5
November 1999


BATCo document for Province of British Columbia 5
November 1999
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Mqwriment BO Experiment BI Experiment B1
Experiment B1 Experiment B2
(35 -1) (10 .1) (25 -1) (50 -1)
A,Z) (Av ) (,) M, *) ) A.- A, v
mg -9 % (mg (.9 (%
RATIO
-21-
TABLE 10
OF DRY CONDENSATE : CMJDE CONDENSATE


Consequently the new procedure which was adopted
is:-
Stage 1
In the first three months of a new experiment the
original procedure
is followed, i.e., aliquots are taken for
evaporation etc.
Stage 2
The information obtained in this period is used to
establish a value
for the ratio of DC:RC.
Stage 3
After the first three months, the evaporation of
an aliquot is sus-
pended. 'Ihe weight of crude condensate is
measured and the weight of dry
condensate calculated from the ratio obtained in
Stage 2.
Considering Stage 2 in slightly more detail:
During the first month, the operators are not
completely familiar
with the cigarette and condensate. Consequently
the ratio of DC:RC is
established from the data obtained in the second
and thi2~d month. In
Table 11, the validity of this procedure is
demonstrated.
0
BATCo document for Province of British Columbia 5
November 1999


-23-
TABLE 11
COMPARISON OF DC:RC RATIOS
Crude Condensate Dry Condensate Ratio DC:RC
AV.
(mg)
Experiment BO Months 1-8 50.0
Months 2 and 3 47.1
Experiment B1 Months 1-5 19.3
(10 ml) Months 2 and 3 19.3
Experiment Bl Months 1-5 41.3
(25 ml) Months 2 and 3 41.1
Experiment Bl Months 1-5 60.2
(50 ml) Months 2 and _3 62.7
Experiment B2 Months 1 and 2 37.1
Month 2 3b.6
Cr
(mg)
3.4
1.8
3.4
2.2
3.2
3.0
5.2
4.7
1.6
1-3
V
W
6.9
2.9
17.5
11.4
7.8
7.2
8.5
7.5
4.3
3.5
AV. a
(mg) (mg)
34.1 2.5
31.1 1.3
13.5 2.6
13.6 1.7
28.6 2.5
28.4 1.9
39.4 3.1
40.7 2.4
21.3 1.2
21.1 0.6
Application of the Painting Solutions
V
(%)
7.2
4.o
19.0
12.2
8.7
6.6
7.8
6.0
5.7
2.7
AV.
(mg)
0.681
0.679
0.697
0.7o4
0.691
0.692
0.655
0.650
0.574
0.577
a V
(mg) W
0.023 3.4
m3 i 4.5
0.028 4.0
m23 3.3
0.023 3.4
0.019 2.8
0.024 3.6
0.021 3.2
0.025 4.3
0.017 2.9
Luring the first six months of the experiments,
the solutions were
applied to the mice by a hand-operated syringe.
Since January 1967,
however, an automatic dispensing pipette has been
used. The volume (0-3 ml)
delivered by the pipette is regularly checked.
At the lower dose levels (25 al and 50 mg) the
mice receive a con-
stant dose over the complete experiment. With the
highest dose level
(75 mg), however, the mice receive a dose of onlY
50 mg during the first
four weeks. Thereafter a constant dose of 75 mg is
applied for the
remainder of the experiment. This "training
period" is thought to be
advisable to prevent an excessive number of mice
being killed in the
early weeks of the experiment.
c::>
CrD
4 :_
(z:D
CC.
BATCo document for Province of BritiSh Columbia 5
November 1999


-24-
Ibxicity Trial
Eight weeks toxicity trials were carried out in
Experiments B2 and
B_3. Groups of 50 mice were painted with dose
levels of 25, 50, 75 and
100 mg of condensate. She mice receiving the 75 mg
and 100 mg doses were
given the four week training period of a 50 mg
dose level. Me number
of mice which died were very few in number and it
was concluded that it
was pex-missible in Experiments B2 and B3 to match
the dose levels used
in Experiment BO.
CD
C:::)
BATCo document for Province of British Columbia 5
November 1999


-25-
APPENDIX III
CALIBRATION OF THE MICE
It is inevitable that during the period of the
experiments the mice
delivered in the various quarterly shipments will
not be identical.
However, the degree to which the response of the
mice to tumour formation
will change is not known.
Two approaches are being used to monitor any
changes which occur in
the mice. The first of these is to repeat
Experiment BO after a period
of 15 months (i.e., Experiment B5). The second
approach is to 'calibrate,
each shipment of mice In their response to the
known skin carcinogens,
benzo[a]pyrene and dibenz[a,h]anthracene.
Doses of each carcinogen (20 pg in 0.1 ml
acetone:water (9:1)) are
appplied to groups of 27 mice for periods of 1-3
weeks (39 applications)
and 26 weeks (78 applications). Several
precautions are taken to ensure
that contamination of the main experiments by
small amounts of benzpyrene
or dibenzanthracene does not occur. The
calibration mice are based in a
separate Animal Room (Room D) and the cages are of
a different size from
those used in the main experiment.
The results of the calibration experiments to date
are given in
Table 12.
BATCo document for Province of BritiSh Columbia 5
November 1999


1
1
X 2 X '. x
A- - -11
- -I t- A,pL t
.- - .
-
Ix 1~ (2
9 1 10
3 11 25 3 W 3 2L
1 ' ,
1
~
, 2 1
6
36 T) 5 53 M b 27
55 21 15
BATCo document for Province of British Columbia 5
November 1999


-27-
The calibration experiments are, to date, running
as might have
been expected. For example, in Experiment BO, the
26 week treatment
with the skin carcinogens is giving rise to more
tumours than is the
13 week treatment whilst benzpyrene appears to
lead to more tumours than
does dibenzanthracene.
BATCo document for Province of BritiSh Columbia 5
November 1999


-28-
APPENDIX IV
TUMORIGENIC INDEX
Sir Charles Ellis (5) has recently introduced a
new approach to the
analysis of results from long-terTn tests and this
approach is being used
in the current experiments.
Consider an imaginary mouse-painting experiment in
which groups of
identical mice continuously enter the population
at small intervals of
time.
In infinite time a "steady state" will be achieved
in which the
mice entering the experiment will be exactly
balanced by those leaving
the experiment. Mice leave the experiment either
because they die, or
because they get tumours.
This "steady state" population can be
characterised by
(a) The number of healthy mice (NH), i.e., the
number of mice
which are alive and which do not have tumours;
(b) The number of mice (rr) which develop tumours
in unit time, i.e.,
AP
TT AT,
where AP is the number of mice which develop
tumours in time AT;
(c) The average age of the healthy mice;
(d) The average age of the mice which develop
tumours.
It is proposed that a "steady state" experiment is
the logical
basis on which to compare the tumorigenicity of
condensates.
It is clearly not possible to carry out a steady
state experiment
in practice. However, it is suggested that it is
possible to synthesise
the steady state experiment from the results which
are obtained from the
usual bioassay experiment. r-
C.D
BATCo document for Province of BritiSh Columbia 5
November 1999


-29-
At the present time, attention is focussed on the
characterisation
factors (a) and (b), and a useful Tumorigenic
Index is suggested as
IT
NH'
The age factors ((c) and (d)) can be considered at
a later date and
may prove to be equally valuable.
At present, therefore, consider in more detail the
imaginary steady
state experiment.
Suppose.the experiment started at time T = 0, and
consider the
group of mice m6t in number which enter the
experiment in the time inter-
val between t and t + 6t.
Ls the number of healthy mice surviving (6N H ) is
given by
6 N H -m6tf(T - t),
where f is the mortality function.
Consequently the total number of healthy mice at
time T is given by
T
NH(T) = S mf(T - t)dt
0
T
= m S F(t)dt.
0
Thus the stationary value of NH is obtained by
integrating over
infinite time, i.e.,
NH m F(t)dt. .... (1)
0
The next stage is to consider the stationary state
during an
interval AT.
CL
BATCo document for Province of BritiSh Columbia 5
November 1999


-30-
The total number of healthy mice is made up by
mice of all age
groups. The number of healthy mice of age t is
given by m6tF(t).
Let PWAT represent the probability of a healthy
mouse of age t
developing a tumour in time AT.
Therefore the number of mice from the group of age
t which will
develop tumours in time AT is
AF = m6tF(t)p(t)6T.
For the total population, therefore, the number of
mice which
develop tumours in time AT is given by
TT = ~
j YTI
0
Tr = m S_ F(t)p(t)dt. .... (2)
0
It was suggested that a valuable tumorigenic
index-is -I!-. Thus by
NH
combining equations (1) and (2) we obtain:
F(t)p(t)dt
Tumorigenic Index = Tr 0 (3)
RH F(t)dt
The problem, therefore, is to synthesise this
tumorigenic index
from the data which is obtained from the usual
bioassay experiment.
In the long-term test we have:
N0 = number of mice at the start of the
experiment.
N - number of healthy mice surviving at the
beginning of each four-
weekly period.
AF = number of previously healthy mice which
develop tumours in the
four-week period.
BATCo document for Province of BritiSh Columbia 5
November 1999


Therefore
N = NOF(t), (4)
where F(t) is as defined in the steady state
experiment and
AP - NOF(t)p(t)AT, .... (5)
where P(t) is as defined in the steady state
experiment and AT - four
weeks.
Thus from equations (3), (4) and (5) we obtain the
tumorigenic
index as
1 S' Ap d t
iF AT
0
TT 0
RH ,
73 Ndt
0
0
AP dt
AT
0.
S Ndt
0
The numerator of expression 6 is the sum of all
the tumour bearing
animals obtained In the experiment whilst the
denominator depends upon
the mortality curve of the mice. It is possible to
calculate values for
the tumorigenic index as the experiment is
progressing the longer the
experiment has been operating, the more reliable
will be the value
obtained.
In handling the data from the long-term tests, it
is convenient to
allow minor approximations in expression (6).
Also, there is danger of
some confusion as the definition of N Hhas been
changed. Thus in the
data obtained from the Battelle experiments:
CD
BATCo document for Province of BritiSh Columbia 5
November 1999


-32-
6 P - number of healthy mice which develop tumours
in a four-week
period.
NH = number of healthy mice at end of four-week
period.
Tumorigenic Index EAP
EN H*
Examination by this ratio of some long-term
experiments which have
been completed at the TRC Laboratories, Harrogate,
indicates that the
value of the ratio is a useful guide to the
progress of an experiment
and perhaps even of the relative tumorigenicity of
the condensates.
BATCo document for Province of British Columbia 5
November 1999


-33-
REFERENCES
1. B-A.T. R. & D.E. Report No. RD.384-R, 28.,3.66.
2. B-A.T. R. & D.E. Report No. RD.480-R, 19-b-67.
3. Day, T. D. :Brit. J. Cancer. 1967, 21, 56.
4. Elmenhorst, H. : Beitrage Tabakforschung 1965,
3 (2), 101-7.
5. Sir Charles Ellis: 117he Analysis of Mouse
Painting Experiments"
December, 1966.
C.
C-
BATCo document for Province of British Columbia 5
November 1999


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01
1 ~0
(P
4 >
C>
I'D
BATCo document for Province of British Columbia 5
November 1999


BATCo document for Province of British Columbia 5
November 1999