LIFE SCIENCE RESEARCH
READING FILE
CIRCULATED
PROGRESS REPORT : SEPTEMBER-DECEMBER, 1975
AUTHOR: S.R. EVELYN
ISSUED BY: F. HASLAM
DISTRIBUTION:
Dr. S.J. Green
Dr. I.W. Hughqs
Dr. R.A. Sanford
R.M. Gibb, Esq.
R.S. Wade, Esq.
Herr H. Sottorf
Dr. F. Seehofer
R.G. Nicl~olls, Esq.
Dr. C.J.P. de Siqueira
Dr. D.G. Felton
Dr. F. Haslam
File 38A
~
Copv No. . 2, 3
4
5
6
7, 8
9
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12
13
14
15. 16
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CIN
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Group Research & Development Centre,
British-American Tobacco Co. Ltd.,
SOUTHAMPTON.
SRE/PSB/38A 5th January, 1976
LIFE SCIENCE RESEARCH
PROGRESS REPORT : SEPTEMBER-DECEMBER, 1975
INTRODUCTION
The final reports of the studies on BATFLAKE Mk.
II in a
flue-cured blend were presented as a Stage I
submission to the
secretariat of the Hunter Committee in November.
The work was
.well received and we have since been informed 'in
confidence' that
the Committee was satisfied with the smoke
chemistry and acute
inhalation studies and that we may now proceed to
Stages II and
III.
The evaluation of the study of BATFLAKE Mk. II in
blended
cigarettes is nearing completion although the
detailed assessment
of some aspects of the pathology remains to be
reported. It is
anticipated that this submission will be made in
January.
in the meantime 'in-house' experimental work has
continued
with a 13-week study to investigate changes in
lung structure over
this longer period, the regression of the changes
found after a
6-week exposure and to examine related changes in
lung biochemistry.
The smoke inhalation system has been uprated with
prototype
development of a 20-port exposure system which is
being evaluated.
It has now been decided that no further
experimental work
will be undertaken at Battelle, Frankfurt, until
the backlog in
reporting has been eliminated. Battelle have been
asked to
prepare a new contract for the completion of the
various reports.
The mouse-skin. painting programme at Life Science
Research
has been expanded by the start, in October, of the
second long-
QrN
term experiment. This experiment is designed to
examine the 110
effect, if any, of added nicotine on condensate
tumorigenicity.0111
_Ij
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PART I - PROGRESS REVIEW
Battelle Institut, Frankfurt.
As already indicated, Battelle have been advised
that no new
experimental work will be considered until the
backlog in reporting
completed experiments has been eliminated.
Battelle have also
been asked to prepare, within the next three
months, a new contract
to cover the completion of these reports.
The report on two long-term experiments (B6 and
B7) which
examined the effects of fermentation and cpi on
tumorigenicity
should be completed early in 1976. Similarly,
reports on two skin-
painting promotion studies (PCL vs PRT and
BATFLAKE Mk. I variants)
are being prepared. The report on a third
promotion study (Gerlach
sheet vs control at various dose levels) has been
delayed for
further statistical evaluation in G.R. & D.C.
Provisional results are now available for the
inhalation
promotion experiment which compared a PRT with the
tobacco control.
The results are given in Part II although their
assessment will
obviously require detailed analysis.
Life Science Research, Essex.
Two long-term mouse skin-painting experiments are
in progress
at LSR. The B12/B13 (PRT variants) experiment has
been in progress
for over 72 weeks but as yet the tumour incidence
is very low.
In the period under review the death rate has
increased, however,
so that about/40% 8f the mice in most groups have
died while
the figure i:s 32% for the solvent control group.
With the increase in mortality, it is apparent
that the
tumorigenicity of the condensates being examined
will be low. -
The small numbers of tumours have given rise to
some concern -
c::)
because of the shortage of cigarettes.
Neverthelqss, for most c::)
C7 \
groups, supplies are available to continue to
experiment for
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COLI
L11
almost six months.
The second experiment at LSR started in october;
this is
designed to examine the effect, if any, on
condensate tumorigenicity
of nicotine added to the tobacco at three close
levels. The
nicotine was added as the citrate'~a-that there
i.,j no
,%*~pgf u1
change in the pH of the condensates. In the f irst
eight weeks
only a f ew mice died and thus it appears that
excessive nicotine
toxicity should not affect the course of the
experiment.
British Industrial Bioloqical Research
Association, Surrev.
The toxicity study of coumarin at several dose
levels in the
baboon has been assessed in some detail during the
period under
review. To assist the assessment based on liver
biopsy samples,
as indicated in the last progress report, animals
from three
different dose levels were sacrificed and
subjected to detailed
histopathology. This study indicated that
treatment at a dose
of 67.5 mgAg/day leads to dilation of the hepatic
smooth
endoplasmic reticulum, together with reductions of
glucose-6-
phosphatase and aniline 4-hydroxylose. Although
this is
considered as a toxic manifestation, no
histological evidence of
cell damage was found. . At the lower dose level
(22.5 mg/kg/day)
there were no differences in the liver from the
control in terms
of histological appearance, histochemistry or
ultrastructure.
As a result of these studies, it has been decided
to kill
some of the baboons and to continue the experiment
with four animals
at each of the three higher dose levels (7.5, 22.5
and 67.5 MgAg/day)
until June 1976, thus completing two years of
treatment.
Group R. & D. Centre, Southampton.
The evaluation of the study on blended cigarettes
containing
BATFLAKE Mk. II in varying proportions is
virtually complete. \0
- L,-.
In general, the results of the study confirm the
trends established a\.
\0
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in the earlier experiments on flue-cured/BATFLAKE
cigarettes.
There may be minor differences in response to the
two cigarette
types which need to be examined further. The
blended cigarette
study gives added information (e.g. the
identification of a
"no-effect" threshold level for some
characteristics) which
should make it possible to define more exactly the
exposure
conditions appropriate for bioassay work.
The report on blended cigarettes with BATFLAKE
will be
ready for presentation to the Hunter Committee in
January.
The submission on BATFLAKE Mk. II in cigarettes
with flue-cured
tobacco, which included work carried out by the
Life Sciences
Group, was reviewed by the Hunter Committee in
November. All
aspects of the work were well receiv d and,as a
result, Stage
_IG_: _r_e
I clearance was given f.0"r'Ithe substitute
material.
The first stage of a 13-week study to investigate
changes
in lung structure in rats in response to smoke
exposure has
been completed. The evaluation of responses, to
clarify
characteristics of respiratory pathogenesis, will
begin in the
New Year and will take several months. This study
is considered
important'because it should give information on
the rate of
development of pulmonary lesions used to compare
relative
toxicity of different smokes, and indicate the
total exposure
time necessary for, say, short-term in-house
Hunter-style studies.
A further dosimetry study on rats has also been
underta-ken.
The work was designed to give further information
on TPM "dose"
in rat lungs. variations in exposure time, smoke
concentration
and exposure sequence were used in an attempt to
determine
optimal exposure conditions for smoke toxicity
studies.
Development of the smoke inhalation machine has
been carried a
stage furtlier with the production of the
prototype 20-port system
with modified animal tubes. Essentially, this
machine is a higher
capacity version of the 6-port exposure system
which we have used
for toxicity studies. The larger machine should
make it possible
to undertake more comprehensive studies within the
same laboratory
space and without the need to increase staff
numbers substantially.
ON
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The 20-port system incorporates various
improvements,
including better control of smoke movement in the
system, changes
in chamber and restraining tube design. Smoke
exposures have
already been done in a study designed to compare
dosimetry
characteristics in rats exposed at three smoke
dilution levels
in the new 20-port and the old 6-port systems. If
the new
machine appears satisfactory from this point of
view, existing
6-port systems will be converted to the up-rated
larger capacity
machine. The results of this dosimetry comparison
should be
available by the end of February 1976.
The first studies on lung biochemistry in relation
to smoke
exposure have been completed and evaluated.
Changes in lung
biochemistry have been induced, particularly in
the guinea pig.
In this respect, the rat seems less responsive. In
some cases,
the responses to smoke exposure seem to be
dose-related and
some biochemical effects can be linked to the more
extensive
pathological observations which have already been
observed in
animals exposed to smoke. It seems likely at this
stage that
bioassay of smoke in terms of lung biochemistry
may be a useful
addition to the methods which are already in use
in the Southampton
laboratories.
In inhalation studies carried out so far, changes
in the
structure of the larynx have been an important
index of relative
smoke toxicity. Because of this, work has been
started on the
detailed morphology and microanatomy of this
organ. Improved
knowledge of structure and cell population
characteristics will
allow us to interpret more confidently for
bioassay work smoke-
indiced changes in the larynx. Conventional
histological methods are
being used and, for this project, increasing use
is also being made
of the scanning electron microscope facility.
Some-of the basic work which has been done on the
B-A.T.
animal exposure sy.stem and on smoke deposition
characteristics
in rats and other small laboratory animals has
been documented
in internal reports. Amended versions of these
reports have been
circulated via the Tobacco Research Council prior
to their
submission for publication. Because the work
reported is fairly
fundamental in the field of inhalation toxicology,
copies have
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also been requested by the secretariat to the
Hunter Committee.
PART II - DETAILED RESULTS
Battelle Institut, e.V.-, Frankfurt.
Mouse-Skin Paintin
The final provisional results for experiment B11
(PCL vs SRT)
were reported in the last progress review. As this
was the last
long-term experiment undertaken at Battelle no
further results
will be available under this heading.
Llyp2Epl~!Eia-Test (PRT variants)
Results have been obtained for seven of the PRT
variants
produced at Glory Mill for the B12/B13 long-term
experiments.
The results for these samples are set out in Table
1.
CN
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TABLE 1
Hvperplasia Results
Details of Variants Area Activity Rating
Code Nominal Nominal Product Observed Normalised
Granunage ASV Solubles (BO = 100)
BO 83 (100)
B13/1 Light Low --Low 87 105-
B13/2 Light Low High 84 101
B13/3 Light High Low 81 98-
B13/4 Light High High 84 101
B13/5 Heavy Low Low 88 106
B13/6 Heavy Low High 89 107
B13/7 Heavy High Low 85 102
The results indicate that there are no significant
differences
between any of the condensates in this short-term
test. This
conclusion has been confirmed by statistical
evaluation taking into
account the balanced block design of the samples.
CZ)
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Inhalation_ExE?2Eiments
As indicated previously, the assessment of
laryngeal lesions
from the vitamin A experiment has been delayed so
that assessment
of the inhalation promotion experiment could be
completed.
Results from the latter experiment have now become
available and
are given in Table 2 which shows ihe incidence of
grade 3 and
grade 4 lesions. It will be recalled that the
hamsters in this
experiment were pre-treated with
N-methyl-N-nitrosourea (NNU) at
two dose levels and were subsequently exposed to
one or two rounds
(30 cigarettes/round) of cigarettes per day;
surviving animals
were sacrificed at weeks 24 and 32.
The results given in Table 2 show the incidence of
hamsters
.with the most severe laryngeal lesion in grade 3
(endophytic growth
with cellular differentiation, with or without
parakeratosis) and in
grade 4 (epithelial endophytic growth with lack of
cellular
differentiation). Very few animals exposed to
smoke had lesions
of grades 0, 1 and 2 although these were common in
hamsters not
exposed to smoke; in the latter groups the
indidence of lesions
in these lower grades appear to be related to the
NMU dose level.
It is clear from the results that the incidence of
grade 3
lesions is related to the level of NMU
pre-treatment and also to
the level of exposure (1 or 2 rounds per day).
Similar relations
can be observed in the incidence of grade 4
lesions but because
of the small numbers the position is less clear.
In attempting to draw preliminary conclusions
regarding the
two cigarettes, it is apparent that smoke from the
tobacco control
(B12/1) leads to an appreciably higher incidence
of grade 3
lesions than exposure to smoke from the 100% PRT
cigarette (B12/2).
In contrast it appears that smoke from PRT leads
to a greater
incidence.of grade 4 lesions although it must be
pointed out that
the number of animals with such lesions is small.
It is obviously important that a more
detailed-analysis of
these results should be undertaken to take into
account the lower
grade lesions and, perhaps more importantly the
date of occurrence
C-D
01-1
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of the higher grade lesions. It must be
remembered, however,
that the analysis of inhalation experiments is
still at a
relatively elementary stage.
The computer system, developed by Dr. D. Langbein
at
Battelle, is now virtually complete and a draft
system/programme
description has been writtenj no further
development will be
undertaken at Battelle. The system will be used to
provide
information on the transition rates between lesion
grades for
the various groups in the experiment.. This type
of information.
is useful in following the progression of lesions
and in examining
some of the assumptions which have to be made in
developing
(proposed) alternative analysis. Nevertheless, the
method does
not produce a single statistic unless the lesion
grades are given
an arbitary weighting. Even so the value obtained
is not analagous
to the tumorigenic ratio used to give an overall
assessment of
mouse-skin painting experiments.
ON
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