1 J10T.1S11-AM1-'R.rGAN TOBACCO COMLIANY L'ID.
PROJECT JANUS
PROGRESS REPORT: SY. PTE"M B PR -DEC EM 11.1-,'IZ
1971
23. 12-71
AUTHOR: S.R. Evelyn
DISTRIBUTION:
A.D. McCormick, Esq. Copy No. 1
Sir Charles Ellis 2
j)r. S.J. Groon 3. 4. 5
Dr. I.W. litighos 6
Dr. R.A. Sanford 7
R.S. Wade, Esq. 819
R&D Manager, BTCo. 10
Herrn Ii. Sottorf 11
Dr. F. Seehorer 12
Dr. D.G. Foltuti 13
Dr. F. Haslam 14 COPY NO:
BATCO document for Province of British Columbia 5
November 1999


T.IrM.S
PROGNEISS III-T(MT: 1 ')7 1
LONG-TERM EXPERTMENT-1;
Experillien t B8 .
The latest resul ts for
week 108 are given ill Table
sonic groups there are already
results are very similar to
exjwrimciit 116 (X-IICI,-5 and 1-:303) to
1 , fro= which it will be noted that ill
xio healthy mice left alive. The
those in the last Progress Report.
TA R LE I
EXPERIMENT BS - POSITION Al' WBE-K 108
Dose i lical thy
Code Sample Level Mice
(mg) M
B8- I Controlq 4o 3
50/50, lamina/CRS 60 1
B8-2 X-PCL-5 60% 40 ill
20/20, laniina/CRS 6o 11
B8-3 X-PCL-5 100% 110 21;
6 o 14
B8-4 i-3o8 6o% 40 1 3
20/20, lamina/CRS 60 ! 1
B8-5 1-308 100% 4o 6
6 o
0
B8-6 Silica gel filter I Ito 0
Control tobacco i 6o 0
'Puniour Ra Lo
-1 -,~
01 Std
I Pr.di c ted
(I'll I PSIZ
67 72 74
78 80 80
43 4:3 51
519 52 59
25 23 32
39 40 49
67 70 72
71
79 81
73 69 72
78 1 84 84
76 85 86
82 1 90 go
C-7,
ON
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-2-
Al L I IOU gli t] I cre ;11',- al,111-ci; 1 1) 10
1) T-1) (I I 'i i M IS 0 1' h 0,1. 1 11 IN' IIIJ
('(-- i-11
the Groups treated with X-IM-5 coiid(~it.~;tlo, it
i~-, ck!ar 1.11;iL Hw
c arc i no go nici ty of those con deji5~1. LOS is
I I) p reciabl y I owor Ll I, I I) Ll la
of the sample 138-1.
At both dose lovcls the pre,licted 1.LIIIJUIIr
i.:; redliced
in relation to the proportion of' X-JICI,-5 iii
the lAt!ii(j.
The results for 1-308 indicate fliat the specific
activity of
the condensate from this material is very simil'ar
to that of the
control blend. The yield of dry condensate is
reduced, compared
with the control (DS-1), by about 55% for sample
138-4 and almost
90% for sample B8-5. In contrast, the silica gel
filter reduces
condensate yield by about 40% but leads to a
somowhaf; hialier tuillori-
genicity.
Experiment BI) (Ethanol extraction, Gerlach)
In this experiment, the following are being
examined: (a) the
effect of extractine tobacco with ethanol wid of'
adding the extract
back, (b) the use of Gerlach additives in the PCL
process, (c) the
effect of the Gerlach process, and (d) the
possibJe additive effect
of ethanol extraction and the Gerlacli process. An
earlier sample
manufactured from PCL (133) is incorporated at one
dose level only.
The results are presented in Table 2. It is
omphasised that,
although the experiment has been in prugress for
64 wec],Ts, many of
the groups have a high proportion (60-80c'.) of
healthy animals.
Closer examination and comparison of' the observed
tumour rate
and the predicted values indicates that the 13SR
values incorporate
a considerable multiplyini, factor (x5 in many
casus).
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'PAIII.F; ;1
EXPFITZ I.-MENT 110SI 'I I () R AT W I
Do s Tum" I, r RZI to
1 Beal I'lly
Love Mi C
Code Sawl) I e
0 bs SLO Prcdic Lc,t
M
M I)s1z
139- 1 Con trol 'to 63 20 21 65
60/40, laininn/ s tent 00 .37 113 117 83
D9-2 Control 'to 22
10
1 1
48
Eth an o 1 extracted 6o 66 16 16i 6o
D9-3 As 139-2 /to 67 16 17 61
Extract returned 6o 1 59 17 is 63
B9-4 PCL + Gerlach 'to
72 8
8
43
additives 6o 70 10 10 49
139-5 Gerlach sheet 'to 72 4 28
f rom Bq- I 6o 73 8 8 43
B9-6 Gerlach shect 4o 78 1 1 11
from B9-2 60
75
5
5
31
Bg/B3 Repeat B3 'to 75 8 8 43
Very tentative conclusions would indicate tliat
(1) ethanol
extraction reduces activity, (2) Gerlach additives
in theinselvcs
do not affect the activity of PCL condensate, (:3)
the Gerlach
process reduces activity considerabl.-,- and
possibly more than the
PCL process, and (4) ethanol oxtraction and the
Gerlach process
may be additive.
Experiment BIO (Noll-combil.-tiblo Fillors)
There has been no recurrence of' the earl),
problems with res-
piratory infection. 'Flic 1-ritest restitts For
wook 28 show t11rk(: a
number of tumours have berin recorded. It. will
Stil.l. be S01110 til1w,
however, before oven t0111;1tiV0 j)1-V(JLC-tiQ1)S
C;M bO 111ildo.
C7,
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F',xj)cj-im(-ttL III I
This experiment h;is been desijjwd so th;it i.)w
,FJ',!c-.L )f*
reconstitutirij.; tobacco vi;t the 1'(',L wid
SOtweitzer 1);,por 1).rocc,~iscs
can be compared with Lhe control tob~wco in a
sitir,.-Je exporimonL.
Arrangements have now been nuide for procc-sing
UJO tobacco at Le
Mans in December. AlthOL11,11 HIO Cij-,A]'OtLos
samplos wiLl bo
available early in 1972, Lhe st;~j-(-- wi.11- ho
de-Laycd itiltil a cloan
stock of mice can be obtaiiied froill Car%%!01-th
EUI-01)0.
SHORT-TERM TESTS
(a) 11vPerpl.asia Tests
1. Effect-of-Shape
In the last progress report, results were
presented for various
ciGarettes of different circumference. Two of
these cigarettes
have been re-examined to assess the possible
effect of oval manii-
facture: the same effect is examined usinU
cigarettes manufactured
from Schweitzer reconstituted tobacco (SIZT). The
samples are
described in Table 3.
TABLE 3
SAMPLE DESCRIPT.1-ON
Circumference Pressurn
Code and Shape Tobacco Weight Drop
No.
(cm WG)
257 2_5
258 19
262 11-75 x 6.75
263 11.7-5 -, 6.75 HP
272 11-75 x 6.75
S7 11 25
Flue- 0.98 6.3
cured 0.58 12.2
.1.11"J Ila I . 211 5.6
blond (119)
0.95 2. It
SRT 1.2o 3.7
1.02 11.9
hialL poro!~.Lty paper
c::)
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T1 I e hyperpl.a.,ia 1' 1 1 Is ;tj-(' 1-i v,, I I
of both tests are comi);-ii-o(I J-ii Table
Comparison of' the t6 in Tab-1c )I wi LIt tliuso
cjven pr-oviousl)-
show-5 that there is goorl agreelljoilt f'ot,
t1lose salilpjes (258, P-57, 262)
ex-amined in the two hypc.-i-ljla.~i-a tc-st.-3.
All ilie r-sttll-s have 1j,2eii
_5 mm CirCL1111f IT-01 CO, FlUO-CUred
norma.lised againsL sample 257 (2 " -I
lamina blend) in Table 5.
The results for botli samples (272 ai)(I S711)
manufacLured froill
Schweitzer rocorisLituted tobacco arch
sir-iiiFicantly lower thall those
for the equivalenL flUC-CUrod samples. The value
obtained for
sample S74 (AAR 74, table 4) is similar to tljat
(AAR 70) found much
earlier for sample R272 (document 133/6). Direct
comparison of
these results is, of coti-rse, not strictly valid
because of dj.f'f'cr-
ences in starting materials.
Previously it was also noted that, on the basis of
the hyper-
plasia test, the effect of oval construction
appeared to be stnall.
This comment is confirmed by the present results
and, in the case
of the SRT sample, the activity rating is
increased.
The results indicate that the use of high porosity
paper for
the oval cigarette (sample 263 vs. 262) reduces
activity by an
appreciable amount. The weight of this cigarette
is, however, about
30% lower than that of the control. and this may
al.Ler the conditiolis
of combustion. In a previous case, the
hypcrj-)1as:i.;t test indicated
that a high porosity paper increased activity
(T119 vs. T1111, doctmicjit
174/8). In a long-tortit skin-painting experiment,
terminated at 57
weeks, no siGnificant dil'Yerence was FouiLd
beLwuen the TRG samples
T44 (control) and T49 (Iti-r-h porosity paper). CD
C
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TA I 11,1-;
A I I,TS
Codc
No.
DO
257
258
262
263
272
S74
Ac Llvi I y )?;, t-ing
(., i. d; t y!;
and Sliapc Tobtcco
(ijo = 100)
25 FC lam-i.ii;l 8.7 1 Oo
2-5 F I n 81 93
19 calrud 79 91
11-75 x 6.75 1,11.1i.na 1 79 91
11-75 x 6.75 111' b1clic]: 139 7-3 84
11-75 x 6.75 SFT 69 79
25 6/1 74
TA)ILE 5
COMPAIU SON OF JIVII J-1,11LASTA. RF'SULTS
Circumference Arr--a Actj.-%,it)r IZ;t(-.j.lla
Code Nonnalise d (Sample 257 = 100)
No and Sha c Tobacco
. (mill)
Test I Te s 1-1 2
258 19 96 98
259 22 914
Flue-
257 25 (100) (100)
cured
26 o 28 96
lainina
261 31 911
b.1 erld
262 11-75x6.75 100 98
263 11-75x6.75 iii, go
S71, 25 SRI, 79
272 11-75x6.75 SI Z T 85
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Since the paper poi-,~.-i 1-~- and 1-1'tl-c'
1-1.11 F')r
sample 263, it is not: po.,sibf~~ to di'~Ilv I
C011C)LISJO11 I'J-0111 1,11jS C0111-
parison, buL it is siieL,,osted Lhat the po:isible
(.-f 1'(!(-L of
density should not be ignored.
2. Non:~oLijL)~stible-YJ
The samples prepared For long-term axlierimenL
1310 wore submitted
for a hyperplasia teril.. It ,hould be jic)Lad
that iii the long-term
skin-painting experimcrit, sample. 1310-?l was
replaced by J310-6; both
have the same target level of NCF but thp 1310-6
cigaxeLte5 were
closer to the tarcet weicht alid firmiloss.
A brief description of Lho samples, which were all
based on a
flue-cured blend (CN102) and lamina/stcin ratio of
60/1i0, and tho
Battelle analytical values, are givLn below;
hyperplasia. results
are tabulated in Table 6.
Code Sample Descript:io)) Ind ']';srj~ut Levels
B10-1 Control. flue-cured blend, 60/40
laminik/stem
B1072 PCL manufactured from control. tobaccos
B10-3 NCF 25%. coiitrol blend 75%
Bio-4 NCF 50%, control blend 50%
B10-5 CF* 50%, control blend 50%
*The Cr, sample contains about 25%
carboxymethy1cellulose. The
actual levels of NCF were about 25-30% lower than
the target level,
while that for CF (BIO-5) was just below the
target.
The results o.f this test confirm (.hose of t.lj(,
previous Ityper-
plasia. evaluation of non-combustible fillers
(docimiciit 209/11), iii
that the addition of this malcrizi.l. has jio
signil*Jcant offoct on
short-term activity of Clio condensate. A
comparable conellision J s
CC
BATCO document for Province of BritiSh Columbia 5
November 1999


apparent for CF which cont.;iins twice the
prc)porLi.01L 0C C,11_'1,(Jxy111Q 1111%, 1 -
cellu.1050.
It should also be noted that the condonsatc,
activiA.), for samplo
B10-1 is comparable with that of sample 130, i.e.
the inclu.,ion of
40% stein does riot lead to a rechiced
act-.ivi(.y. Simi.Jrir rcsull.s were
found for sample B9-1 on two occasions (documents
200/17, 200/6).
Only a ina-rainal reduction iii coridensato
activity was found for-
sample BIO-2. Previous results for sinjilay-
fluo-curod PCT. samples
have given a significalitLy lowel- value in this
test; at this
no explanation can be given for this apparetit.
anomaly.
']'A 11 rr, 6
HYPERPLASTA RESUL'I'S
Doscriptioji
Code (TaracL Levels)
DO Flue-cured lamina blend
B10-1 Control PC blend lantina/stem Who
BIO-2 PCL manufactured from B10-1
B10-3 NCF 25,"-, control blend 75',10'
1310-4 NCF
50%, control bleric]
)0%
BIO-51 CF 50%, control blond 50%
91,
102
89 97
92 100
92 100
94 102
3. DATEX Incorporating Additives
The effect of incorporating carbon and clialk into
13ATEX has
been examined usixl,,,, the byperplasin test.
I'lie based on a
flue-cured blond (CV112) urith a 20% CRS coiAcnt,
were manufactured
00
L)j
I'D
01-
00
Area AcLiviLy Rciting
(microii-days)
Observed No rmali sed
(130 = loo)
92 100
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into 84 initt filter-Lipped cil-arottes. Thc,
b.Lc,[ILI r~oii La Liiod Pa ki. .9 Lan
Indian and Korean (11 HO lo~rit' (20"11) ~tjitl th
i -, pot-L.Lon o1' Llic blond
was made into DATEX. The sample.5 wore:
Al Control flue-curod blend
A2 Control. blond wi 1.1i I'll K I)o i-tj on jimde
j n I-o DATEX
A3 As A2 with 20% CZLI.-IM11 :Lii TIATEIX
A4 As A2 with 40% cliaLk in BATJ-"X
The percentages of' carbon and cl),alk in DATE'X
were calculated
on an "add-on" basis so thn.L the tobocco
composition wa.5 substall-
tially constant in all samples.
The samples were sinoked to an overal.l. butt
Lengtl~ of* 23 mm,
filter plus 8 mm. The hyporp-1 a sia resu I I s
are V, i-ve), i), 'Pal-,J U 7 -
TPOILE 7
BATEX 11YRERPLASIA RESULTS
Area Activity Rating
(11licron-days)
Code Sample.
Observed Normalised
(Bo = 100)
BO 87 100
Al Control blend (CV42) 88 101
A2 'Normal' DATEX in blend 92 1o6
A3 tCarbon' BATFX in blend 83 95
A4 'Chalk' DATEX in blend 78 go
The area activity ratinas indicate that form.ing a
portion of'
the flue-cured tobacco iziLo DATEX increaz;es the
specific activity
(Z--.
<ZD
1-0
00
LAJ
,NO
Coll
1-0
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of the condensat.e. A simi.kil- Micil
Durley tobacco w;l.s 111acle i1)['., 13A'J'J--'X
;t111) 5(),.'
level into a flue-cured blf,ijd. 'Ill tile saillo
expol-1111011t, (docurliolit
17 VIO , a decreased activi ty was fouiid
tts:iiig- 50',"" Flue-cured DATEX
and it was concluded tll;-Lt 111,01Cilla
f*1110-CLIVEd tobacco :hito DATUX hod
little OffL-Ct OTL -,;j)cciA'ic ac.LiviLy.
In terms Of UW carbon and chalk ii-irorporatod
into the BATEX
the results are encoural.~ii)?-.. In both czuses,
thv acLiviLy of 4.1ic
condensate, as jUdaed by this hyperplasia test, is
reduced sigriifi-
cantly relative to uiifillcd UATEX aiirJ is lower
thnii that of tbe
control blend. For the saml)le with adcled chalh.
the reduction in
the hyperplasia rating, compared wi(Ii the
coi)tj-ol,, is probably
significant. The appreciable reduction ill
activity is surprising
ill View Of the relatively low Jovel .,F DAT.EX
atid chalh ii-i tile blend
and it would also be interestilia to speculate why
the addition of'
chalk to BATDX reduced activity while
incorporation into sheet
material (NCF) has no significant effect on
tli(-,, result.
(b) Promotion (Skin-PairitintO Test
As stated in the -last Progress Rcj)ort, a further
test of B9
samples is being undertaken at Battelle. Samples
D9-2 to Bq-5
inclusive are being examined at one dose level of
initiator (180
DMBA) and condensate (40 mg). Re-examination of
samples B9-1 and
B9-6 has been designed so that tile following
effects can be examined:
(1) the level of WIDA, (2) tile level of
condensate, and (3) iliter-
actions between these factors. Incitidint; tlic~
zvro controls, Fotir
levels of initiator arid four levels of condensate
are be-iijIg used.
CD.
110
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Because of the very low r,_,sponu %,rioi
additional high dose lovej ( 80 till-) hos I)nell
j)lcoj:pol-~Itod For IAILS
sample. Althoutdi this wilt lead 1-1) ZI
-iAl(:jA(_1llCC, il,
may not reduce sij,,n:i.f'j.CM)tly thC Li.1)10 01'
01' W1110111-S.
This additional group couLd well. fielp iii
plaiiiiiiij, VuLurc, pr-omotion
tests with condensates expected to have Jow
Promotion activity.
The healthy animals ill C3'OL1136 troaLed w_iCh
139-1 and. 139-6 condensates
will not be sacrifice(l al'ter six montlis so thaL
th,.-! offects of'
longer time periods can be studiod.
INHALATTON STUDIES
~Iutagcnesis
Long-term samples 138-1 and J18-3 have been used
for this study.
These cigarettes were chosen on the basis of an
initial toxicity
trial, levels of carbon moiioxidu and because tljo
puff numbers could
be equated. Despite Vic relativcl.v low toxicity,
the male mice had
to be acclimatized to the smoke; very short
exposurcs were used at
first and the maximum (lose was a daily exposure
to 7 Puffs from each
of 30 cigarettes. The inhalation exposure, which
has been completed,
caused the mice to lose weight compared with the
control aniina15
which were 11sham" smoked.
The first batch of females have now been
sacrificed and the
initial examination indicates that fertilization
has boon satis-
factory.
2. Promotion sturlies: hamstor larvnx
Pro-treatmcjit of the hamsters with the initiator,
N-methyl.-N-
nitrosourea (MNU) has been completed and tljo main
group of aiiinials
Cp
.0
.-JZ)
00
BATCo document for Province of BritiSh Columbia 5
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is being exposod to :,m(,k(! i1111.11;11-ioll
111ochil"'..
'llic whole experimerititL LreatmeiiL plan is
riveii is Tj1)1(_. 8
TA131,F, 8
1101STER r.\m,i1.AT1'0N
MNU Applic aLion
Groups
Frequency I
To La. I
(ma)
8 x 36 2.88
8 x 36 2.88
8 x 36 2.88
IV
8 x 9 0-72
V 4
9 0
36
X .
VI 2 x 36 3.60
VII 1 8 x 36 1 o
VIII
Ix
X 4x with 1%
probe
XI 1 4x with 0%
probe
Ntimber of AnimaJs
So I veil t
At start i At 1.11-71
sa I j. i i e 26
bu f, fc. r 26 19
niuCLIS rC11111"C'
C,
buffer 21
buffer 21 19
buff'or 22 16
e ther 22 11
buFfer 22 17
20 20
closed nostril _30 25
Lille 6 6
saline 6 6
1 228 171
The animals in Croups I VIII inclusive will be
exposed to
smoke with groups III, IV, V and VII forminG the
main experiment
using four dose levels of initiator. Alternative
solvents or tech-
niques for applyina the MNU were employed iii
Groups 1, 11 and-VI,
and it call be seen that the use of ether results
in a higfi pro-
portion Of mortalities.
C=)
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It should be noted Hiat t1j(-, a(j(jiti()jjjLj c,
groups
were not includod at the request uf D-AT. In
ofCocl., althouj.-.-Ii
they were coupled with the JANUS experimonL, they
fonn part of'
the Battelle researcli proaramme. Group IX is xn
experimental
attempt to see whet1jer ftamster5 survive and
devolop a reg-111-ar
respiratory pattern when they are forced to
bl'O~Lt]]V throuj,-h We
mouth; if this proved possible, it could provido a
mv;irts of
changing the pattern of cloposition in the
respiratory tract.
Groups X and X1 are furthoi- attempts to develop
saLi.-factory in,,-ttlods
of applying MNU to the respiratory epithelium.
SRE/SEW/3.5
21 st December, 1971
C=)
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BATCo document for Province of British Columbia 5
November 1999