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BATCo document for Province of British Columbia 5
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DISTRIF-RurioN:
A.D. McCormick, Esq.
Sir Charles ElliS
-Dr-S.i. Green
T.W. Burgard, Esq.
DI. R.A. Sanford
L.C. Laporte, Esq.
Dr. W.B. Fordyce
Herr H. Sottorf
Dr. D.G. Felton
Dr . I - W. Hu&hes
AUTHOR: C.I. :~yrcs
Copy INC. I
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BATCo document for Province of British Columbia 5
November 1999


PROJL)CT JANUS
ANNUAL REPORT 1968/69
As with the previous years, the major portion of
the work on
Project JA14US at the Battelle Institut,
Frankfurt-am-Miain, is concerned
with the examination of smoke condensate by a
series of long-term skin-
painting experiments. All the mice in the first
experiment are now
dead and the detailed histological examination of
the sections will be
finished in the next few weeks so that the
complete data should be
available for statistical analysis by the end of
1969.
Samples of cigarettes continue to be examined on a
routine basis
by the Hyperplasia Test, but the use of the
Ciliastasii Test was termin-
ated in March.
Mid-way through the year, Dr. Miedreich, the
leader of the Project
JANUS team at Battelle, left Frankfurt. His
departure necessitated a
degree of re-organisation of the staff at
Battelle. Dr. Hofmann took
over as leader of the team and has overall
responsibility for the Project.
Dr. Kiendl is responsible for the skin-painting
programme (long-term testa)
and Dr. Kramer joined the team to assist Dr.
Konigsmann with the patholo97.
Dr. Militzer continues to operate the Hyperplasia
Test.
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LONG-T-1--r~':"i TESTS
Eaz-;h ta'-.ez 22 3 years to be completed
and an in-d-lic"rion is Olvan ~_olow of .'..c
ri:z~in, ~--I',-.~_.~.n~'.; T.L) date, toge7her
with the length of time zhe exp,~-_-imenz has
beE::i in progress.
Exper-J.-nant M (152 wceks) - !A.1
'2 he smoke condensate from a flue-cured larina
(C:~102) blend was
examined. The m_-jor, but ;;erhaps
s.J*,-..!)le,_ain. of this first experiment
was to demonstrate -that a lor-Z-tuerm, test could
be carried out succes-s-
lly at Battelle. At -u',,.e same rime,
'no-...,ever, the tu:morigenic response
_u
lo this condensate provides a useful "bench-mark"
or reference point for
comparisin with the results from other long-term
tests carried out under
Project JANJUS, and, to a derree, for comparison
with experimental results
obtained elsewhere.
The high tumour response and the long life
expectancy of the mice in
the Battelle experiments means that we have a
sensitive test procedure
compared, for example, with T.R.C. Harrogate.
E7 periment Bl (136 wee%s) - All mice d-oad
In thi.-3 experiment, the ci,--aret.,~ ..:~ed ias
idontical with that. in the
first experiment. Mrieren_.~ i:i DO, -3.
rruff-vol-umc of 1`15 ml
was used to smoke the cigarette, in Zx-.eriment Bl
a range uf puff-volun-_s
(10, 25 and 50 ml) was used.
The indication from the resultz, Which have
recently been confirmed
by a T.R.C. experiment ;it Harrogate. is that the
larger the puff -v0 lume,
the lower is the activity of the condcnoate.
302 (11410 wreks' - 711
This experiment is an ex,=inatio,i of a "typical"
U.S. X.S.r.T. eigarptte.
The current i-ndication frcm the reoults is that
the activity of the condensate
C=>
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is similar, but probably less than that from the
cigarette containing
flue-cured lamina.
Experiment B) (128 weeks) - In progress
A cigarette made from 100% PCL is being examined
in this experiment.
The PCL was made in Montreal: the flour was
prepared from CNI02 lamina
and the binder from Canadian stem.
It is clear from the current results that the
activity of this con-
densate is less than that from flue-cured lamina.
This is an encouraging
and imoortant finding.
Experiment B4 (92 weeks) - In nrogress
The cigarette for this experiment was made from
equal portions of
flue-cured lamina (CN102 blend) and Canadian stem
(as CRS). As an
approximation, therefore, it contains the
"starting tobaccos" in the
proportions which are used to made PCL.
The current results indicate two important
findings:
(a) Increasing the level of CRS leads to a
reduction in the specific
activity of the condensate (Experiment B4 vs.
Experiment BO).
(b) The processing of tobacco into PCL leads to a
reduction in the
specific activity of the condensate (Experiment B3
vs. Experiment B4).
Experiment B5 (92 weeks) - In progress
As there is no guarantee that the response of
different batches of
mice is the same, the condensates used in
Experiments BO, B2 and B3 are
being re-examined in this experiment. There is no
indication that a
-large change in the response of the mice has
occurred.
A second point being examined is the effect of
changing the puffing
frequency from once per minute to three times per
minute. The current
results indicate that this change in puffing
frequency has little, if
BATCo document for Province of BritiSh Columbia 5
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any, effect on the specific activity of the
condensate.
Experiments B6 and B7 (48 weeks) - In progress
Two experiments were combined so that the effects
could be examined
of:
(a) A change in strand width (_30, 60 and 120 cpi)
using two types of
tobacco.
(b) Yeast treatment of flue-cured tobacco. The
process was based upon
the earlier work under Project AIRFERM.
Whilst it is too early in the experiments to draw
firm conclusions,
and it must be emphasised that the picture could
change in the next few
months, the initial results indicate that:
(i) The effect of a change in strand width is such
that a minimum
activity is associated with 60 epi. The
differences, however,
are only small.
(ii) Yeast treatment might lead to a small
reduction in the specific
activity of the condensate.
HYPERPLASIA TEST
During the year, over forty samples of cigarettes
were examined by
the Hyperplasia Test. Several of these were
semi-commercial samples
examined at the request of Monzreal, Louisville
and Hamburg. The coating
of cigarette paper with aluminium was shown to
have no effect on the
response in the Hyperplasia Test. As part of a
joint exercise with Harrogate
and Battelle, Geneva, a series of eight T.R.C.
samples were examined.
These samples involved changes in circumference,
cpi and paper porosity,
but no clear trends were apparent in the
Hyperplasia results.
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A series of of co:,.taining PCL or Batex
made from different tobaccu6 was - An important
inference from the resultz was t.^at not only does
the nrocess affect the
activity of the condensa-~e but the nazure of the
starting tobaccos is
also of significz_nce.
CILIASTASIS TESTS
Before the work was --arminated in 'March.,
.-_'--ttelle had examined 24
samples of cigarettes by Clr-.-.i Gil! and Rabbit
Trachea tests. In no
case, however, was a loa tzxicity observed. This,
perhaps, was
largely to be expected since the of the samples
were plain
cigarettes which had been examined '_-Y the
Hyperpla2ia test and their evaluation
by the Ciliastasis tests mainly to complete the
biological
Even the few fil~&r-tipped samples (e.g. bondod
polyurethane)
assay.
examined were not expected to have a low smoke
toxicity but were examined
as a safeguard against !:.issing the "unforeseen"
result.
MAST CELLS
There is a claim in "the literazure that, prior to
the appearance of
a papilloma, there is an increase in the number of
mast cells in the
epidermis of a mouse. it was possible, therefore,
that a monitor of mast
cell concentration might form the basis of a
useful short-term test.
Battelle examined a number of skin-sections from
the mice which had died
during the long-term Experiment BO, 'out the
results were not encouraging
and the approach was terminated.
FREI AND SrEPHrNS
Frei and Stephens have publis.-Cc, a papar. on the
response in the
mouse ear to the applicaticn of chemic.-___s
,fnich are known to be promoters
of carcinogenesis. 3attall-a undertook a
preliminary examination of the
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effect of painting the ears of mice with smoke
condensate. Again,
however, the results obtained were not encouraging
and this work was
also terminated.
GOBLETI C=- TEST
In the early part of the year discussions were
held with Professor
Lynne Reid and Dr. Mawdes ley -Thomas on the
proliferation of goblet cells
which occurs when a rat is exposed to an irritant.
At that time it
seemed feasible to develop a Goble7; Cell Test for
the examination of
cigarette smoke.
Subsequent work by the Huntingdon Research Centre
indicated that
the initial promise of the work was not being
maintained. This, coupled
with the failure of the "Harrogate Smoker" to
provide a satisfactory
system for exposing rats to smoke, caused further
work at Battelle on
the development of a test procedu.-e to be
suspended. It is possible,
however, that the approach might be re-opened if
further work by the
Huntingdon Research Centre indicates that it is
worthwhile.
CZ)
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November 1999


-7-
ourLINE 196c/7o
GENERAL
The current contract with the Battelle Institut
expires in March
1970, and to continue the work it will be
necessary to negotiate an
extension.
For the next few years, the long-te-nn mouse test
is likely to
remain a major test by which modified cigarettes
are judged. It is
possible that, in time, this test might be
replaced by Inhalation Tests
in which animals are exposed to cigaret-;e smoke.
However, inhalation
tests are not yet developed. For example, a degree
of publicity, parti-
cularly in the German press, has been g~~7en to
the inhalation work of
Dontenwill at Lokstedt, in which he has exposed
hamsters to cigarette
smoke. At present, however, he has onl7 produced
"tumour-like" changes
and not malignant tumours in the hamsters, and it
will be some considerable
time before a test is developed which cculd be
used to compare the effects
of the smoke from different cigarettes.
It seems essential, therefore, that the long-term
skin-painting
test should be available to B-A.T. . As
specialised facilities and personnel
are required for this type of work, it would be
difficult to switch from
Battelle in the near future and undertake the work
elsewhere.
It would seem worthwhile to allocate approximately
20% of the costs
involved in a new contract to allow Battelle to
continue to examine samples
of cigarettes on a routine basis by the
1~rperplasia test, and also to
undertake feasibility studies of other tests of
interest to B-A.T.
CZ)
C:11
1-Z
OC
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LONG-TERM TESTS
A main aim of undertaking the long-term tests is
to provide infor-
mation to assist in the design of a modified
cigarette which has a low
mouse-skin response.
From a review of the factors involved in the
design of a modified
cigarette, it appeared that the work in -the
immediate future should be
concentrated on the examination of sheet
materials.
A new experiment (Experiment BS) will be starting
in October and
involves an examination of FCL from Louisville
(X-PCL-5), and a Celanese
smoking material (1-308). Cigarettes containing
100% of the sheet materials
will be examined, together with cigarettes
containing a blend of sheet
material and tobaccos.
Also included in Experiment B8 is an examination
of the effect of
a silica gel filter. A forthcoming publication
from Harrogate will show
that when a solution of a purified fraction of
smoke condensate is passed
through a bed of silica gel, then a loss in
mouse-skin activity of the
solution occurs. Consequently, the silica gel
filter is being examined
to determine whether a reduction in activity
occurs when smoke is passed
through a bed of silica gel.
The experiment, therefore, involves six samples:
(i) Control tobacco
(ii) Blend of 40% control tobacco 60% X-PCL-5
(111) 100% X-PCL-5
(iv) Blend of 40% control tobacco 6C% 1-308
(v) 100% 1-308
(vi) Control cigarette with silica gel filter.
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An inference which can be drawn from a comparison
of the long-term
results from Battelle, Harrogate, and Lokstedt, is
that the activity of
the condensate from a Gerlach sheet is less than
that of the condensate
from PCL. It is proposed to confirm this inference
in a test which will
start in March, 1970. At the same time, a PCL
sheet incorporating the
"Gerlach additives" will be also examined to
determine whether these
additives lead to an additional reduction in the
activity of the condensate.
A second feature of this March experiment is an
examination of the
effects of an ethanol extraction of tobacco.
Lokstedt have shown that
extraction with ethanol of a German blend leads to
a 35% reduction in the
activity of the condensate and it is of interest
to determine whether a
similar reduction in activity occurs when a
flue-cured blend is extracted.
An important extention of this work is planned.
Extraction with ethanol
removes a major portion of the nicotine from the
tobacco. It is intended,
therefore, to concentrate the extract and add the
concentrate back to the
tobacco. It is possible that the simple process of
"separating" and
"recombining" tobacco constituents leads to a
reduction in the activity of
the condensate.
Finally, it is intended to determine whether the
beneficial effect
of "extraction" and of "Gerlach process" 'can be
combined, i.e. make a
Gerlach sheet from extracted tobacco. In this way
it might be possible
to obtain a conden-s-a-Te with very low activity.
The samples involved, therefore, in the March
experiment are:
(i) Control tobacco
(ii) Gerlach sheet from control tobacco
(iii) PCL sheet from control tobacco, but
incorporating the Gerlach additives
(iv) Control tobacco extracted with ethanol
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_10-
(v) Extracted tobacco with the addition of the
concentrated ethanol
extract
(vi) Gerlach shec~L_from extracted tobacco
The Animal House at Battelle has limited capacity
and a major factor
which determines the starting date of a new
experiment is the length of
time taken for sufficient mice to die and thereby
create space to house
~ new intake of animals. At the present time, it
appears feasible that
~ third experiment can be started in the later
part of 1970.
Experiments completed elsewhere have shown that
the addition of
sodium nitrate to tobacco leads to a marked
reduction in activity, i.e.
the incorporation of chemical additives provide
the possibility of a
break-through" in the search for a low activity
condensate.
The third experiment, therefore, will probably
involve the examination
of a series of chemical additives. It may be
desirable in this experiment
to examine the mode of addition of the chemical,
e.g. the effect of adding
the chemical to the surface of cut tobacco may be
different from that
produced when the chemical is incorporated into a
"PCL mix."
Possible alternatives for this third experiment
are a further
examination of stem (e.g. WTS on different types
of stem) or an examination
of an annular cigarette, i.e. a cigarette in which
PCL or other material
is lo-calised in the axial portion of the
cigarette.
HYPERPLASIA TEST
The tests in hand include the examination of a
series of filters
containing water and samples of cigarettes from
Sydney and Louisville. It
is anticipated that in the coming year, the heavy
demand for samples to
be examined will continue.
CZ)
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REPORTS
Title Report No.
The Tumorigenic Index. B-11
Hyperplasia Test: Part IV - Progress Report,
October 1968. B-12
Skin-Thickening Test: A Report to T.R.C. B-13
Further results of work aimed at the development
of a
Goblet Cell Test B-14
Mast Cell counts in the skin of mice treated with
tobacco
smoke condensate. B-15
Ciliastatsis tests: Part IV - Progress Report,
January 2969. B-16
Ciliastasis tests: Part V - Progress Report,
September 1969. B-17
C-r)
BATCo document for Province of BritiSh Columbia 5
November 1999