RB/LP/46D 4 October, 2979
BIOLOGICAL STUDIES
NOTES ON SOME KEY CO.',1110.NENT TASKS
INTERNAL STUDIES
1) LIFETIME RAT STUDY
In an earlier series of short-term studies,
experimentally
Induced changes in rat lungs exposed to smoke have
been determined.
Subsequently a test system has been established
for examining the
comparative irritancy of smoke from different
cigarettes. The
techniques used have established the biology group
as a leader in
this aspect of testing. To date, short-term
studies have been used
to investigate a potential substitute, alternative
sheet materials,
major blend types, experimental filters (see
below), particulate/
vnpour phase effects.
. As an extension of this successful. relatively
short-term
work, a pilot study on longer term inhalation of
tobacco smoke has
been completed and is now being evaluated. The
objective is to
determine whether a satisfactory long term assay
system can be
developed, based on any relevant changes In lung
structure induced by
lifetime exposure of animals. At this stage, no
attempt has yet been
made to compare biological response to different
cigarettes on a long
term test. Whether such a test will be attempted
will depend on the
outcome of the lifetime study on rats.
2) PRODUCT EVALUATION
Comparative techniques have been developed for
assessing
biological response to smoke from different
cigarette types. The tests
are intended for evaluation of significant product
features or possible
future developments rather than, for example,
large-scale brand comparison
work.
a) Evaluation of 4 filters
Smoke-induced pathology was least in the lungs of
rats inhaling smoke
from the control cigarette (cellulose acetate
filter) .Animals retained
in the lungs relatively more smoke particulates
from cigarettes with
vapour phase triple filters (carbon,
carbon/silicate, Duolite resin).
Consequently, more marked lung pathology was
induced In rats exposed to
smoke from cigarettes with reduced vapour phase
delivery. Presumably, c0
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smoke from these experimental cigarettes was more
readily inhaled. Vapour
phase alone produced minimal change in lung
structure.
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Preliminary tests by a panel of smokers which
would support the
general thesis raised as a result of the animal
study. Reduction of the
levels of some smoke constituents (suggested by
pressure to extend league
tables), need not necessarily result in an
improvement in smoke characteristics
as assessed biologically.
b) Ventilated cigarettes
An evaluation of cigarettes is being made to
determine whether the
degree or method of ventilation used affects
'biological activity of 'smoke.
C) Nicotine/TPM ratio
Nicotine/TPN ratios are of interest inside and
outside the industry
(Lancet; 25.8.79). We have no information on the
effects of altering this
ratio on biological activity of inhaled smoke. An
experiment to produce
some infor6ation on this topic Is planned for
1980. The cigarettes to be
used are those made for Experiment B14 (see
below).
3) IRRITANCY ASSAYS
(a), (b).
On-going method development is required to extend
or improve the
ranged of biological tests, to allow better
assessment of cigarettes. A
lung enzyme (Cathepsin D) and the mccnanism and
measurement of mucus
production arc beine investigated as possible test
system end-points.
(C)
Some research is being planned to study the
relative importance of
smoke particulate and vapour phase on overall
biological activity of smoke.
Work to date has shown that the vapour phase way
not alone produce marked
tissue damage, but its sensory effects may
influence the extent to which
smoke particles are inhaled. The relationship
between vapour phase and
particulate phase in determining overall smoke
irritancy is th erefore of
interest.
4) SMOKE MONITORING
(a), (b) & (c)
Reliable inhalation studies require controlled
exposure conditions,
information on the concentration of key chemical
constituents of smoke and
the measurement of dose of material (e.g.
nicotine, TPM, CO) to which animals
are exposed.
For the studies on particulate phase, vapour
phase, nicotine/TP.11 ratio
referred to above, careful measurement and
monitoring of experimental para-
meters will improve the final interpretation of
biological data. CD
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5) METABOLIC EFFECTS OF SNIOKE/DIET
a) Inhalation toxicity studies with animals have
shown consistently
that exposure to smoke is accompanied by changes
in growth rate, food intake,
food utilisation, degree of obesity. Similar
observations have been
extensively recorded for smokers compared with
non-smokers. Information on
the interaction of effects due directly to smoke
and those produced
indirectly by changes due to dietary
considerations is Important if
inhalation toxicity studies, particularly long
term experiments, are to
be interpreted correctly.
b) Smoking has been implicated in the development
of cardio-
vascular disease in man. Overall, the project
involving the use of special
diet will be used to investigate the potential
toxicity of smoke and diet
to the circulatory system, with a view to
establishing comparative
bioassay procedures for examining different
cigarette types or smoke
components.
6) BIOCHEMICAL INDICATORS
a) Methods are required for measurement of
biochemical
responses to experimental treatment of animals. In
thi3 area methods
are beinE developed particularly for the work to
be done on particulate/
vapour phase; nicotine/TPM ratio; smole/diet
interaction.
b) An accurate method is required for the
dotection in
biological samples of nicotine and cotiftine, its
major metabolite.
The method is intended for use initially in the
study of nicotine/TPl
ratios. Further use of the technique will be made
in other studies,
particula-ly concerning specific biochemical
effects of nicotine.
Store widespread use could be made of the assay
method by
groups interested in, say, smoking behaviour,
dosimetry etc.
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EXTERNAL STUDIES
Cigarettes with added nicotine (B14)
A study has been made of condensate from
cigarettes made of tobacco
with nicotine added in the form of nicotine
citrate solution during
manufacture.
Throughout the study there has been a consistent
increase in tumours
related to the level of nicotine added to tobacco,
which is reflected in
differences in nicotine levels in the condensates
used.
Complete analysis of the study is dependent on the
results of the
final examination of tissues, which is now in
progress.
Whether the interim results of the experiment can
be related to
peculiarities in physical or chemical
characteristics of condensates
produced from the special cigarettes prepared for
this particular study is
not known. From this study, and from the
literature, it is not yet
possible to reach a clear conclusion on the
carcinogenicity of nicotine.
At this stage, the possibility of increasing
nicotine content of
cigarettes by spraying during processing should be
dismissed.
2) Ventilated cigarettes (1315)
The study is designed to examine the effects of
various combinations
of tippirZ papers and varying porosity papers on
the tumcrgenic activity of
condensates.
3) Cocoa (B16)
Work to determine long-term biological activity of
smoke condensates
is carried out routinely in an established
programine of contract research.
7 be work has been done by three major biological
reseurch companies in
collaboration with GR&DC.
Since the work started, various studies have been
carried out to
examine.the comparative biological effects of, for
examole, stem, lamina,
PCL, U.S. blend, fermented flue-cured tobacco,
extracted tobacco, sheet
materials, potential substitutes, added nicotine,
ventilated cigarettes.
a) At this stage we need to change our supplier of
the mice used
for this work. Before continuing with any major
experiments, we propose to
test the suitability of animals from different
sources. 7bere are quite
marked differences in the sensitivity of different
strains of mouse. The
proposed experiment will allow us to optimise the
test system and to avoid
the dangers of proceeding without the necessary
information, which this
study will give. Previous experience indicates
that the proper selection ofCO
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a supplier and a strain of animal, rather titan an
unreasoned switch, will beC-::)
worthwhile.
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b) The next important long-term study, which has
already
been planned and for which cigarettes have been
made, will investigate
the effects of cocoa as an additive to tobacco.
4) Bioassay development
The need for short term biological tests which are
predictive of
carcinogenic potential of a wide range of
materials is well recognised
in many major industries.
A project at the University of Surrey will be
started to investigate
the development of such a short-term test, based
upon a study of
cellular biochemistry. The objective is to examine
specific changes in
biochemistry induced by known carcinogens and
non-carcinogens, with a
view to distinguishing their mode of interaction
with specific cellular
enzymes. The possibility of eventually
establishing a short-term assay
on the basis of this work will be explored.
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BATCo document for Province of BritiSh Columbia 9
November 1999