RESTRICTED
STATUS REVIEW NOTE
Work Area: 01 Biological
Author: R.E. ThorntL-n
Period Coveredt January - june, 1982
OBJECTIVE:
To assess, in quantitative terms, the biological response to cigarette
smoke and its constituents and to develop accectable cigarettes with
minimum blological activity.
PROGRESS:
There are five areas of study:
1. Direct measurement of biological activity in products and the assess-
ment of changes in product variables such as ;aper permeability, etz.
2. Smoke Inhalation Studies.
3. Development and use of short-term tests related to cancer.
4. Other short-term tests.
5. Bioussay of nicotine.
The work going on in these areas is as follows:
1. Products of reduced biological activity
(a) Empirical approach
The main activity of the year was quite unexpected twelve months
ago. Product variables such as paper permeability, tipping ventila-
tion, filter pressure drop and blend composition had been investi-
gated in two skin-painting experiments (814 and 815). Unfortunately,
subsequent to an analysis of the results, errors in the records
kept by the contract research laboratory carrying out the study,
were detected. 53 animals in study 314 and 146 animals in B15 were
incorrectly recorded. These errors have now been corrected and
re-analysis is proceeding as a matter of urgency, and is scheduled
for completion in July.
(b) Fundamental studies
Studies into the role of tobacco leaf proteins as precursors of
mutagenic compounds of high activity are being planned: assaYs
will be carried out by the Ames Test.
2. Smoke inhalation studies
(a) Investigation of product variables
A major project has been the adaption of -experimental protocols to
permit the examination of low tar products. For many such products,
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chan=ss -',% the concentration. of smoke components (suc- as nicotine
and -:nr!:-_n moncxide) relative to tar, precluded examination by
earlier Protocols. This work has been concluded and a series of
low cigarettes has been examined. Cigarettes ccnta_@:-.ing
ex=an@_ed tobacco are beina studied by a number of protoc::s,
a one day test developed by Battelle fGaneva) for Gallaner
Ltd. --e activity of sidestream smoke is also under examinazi,,n:
prelin`nary results suggests different sites in the respirazzry
trac': are affected in comoarison to mainstream smoke.
(b) Devel-cz:7ent of immroved techniques for investigating the response
of rcdents to inhaled cigarette smoke
This zro@ect has been abandoned. We came to the view that an ei=`t
wee.- rcdent (hartster) study was unlikely to give useful Lnformat:..-n:
a tw= :e2r study would be required. This was considered to be an
ineffec:lve way .of utilising our limited resources.
3. Shor,:@ torn test related to cancer
(a) Deve:cznent of nutaqenicity assay
The t_-es' Test using salmonella typhimurium has now been establis'-ed
at Different smoke condensates have beer. examined in
a ccl'a-orative test involving the Hamburg, Montreal and Southar.;t;n
labc:atories: the results from the different group were in exce-l-ent
agreement.
In ;:e1i'minary experiments, fresh sidestream condensate has @:een
exa--4ned' for mutagenic activity and has been found to possess
sin4lar activity to that observed for mainstream smoke. However,
it is exnected that this relationship between sidestream and -Ain-
strean smoke may vary depending upon smoking conditions, and tnls
is undar investigation.
(b) Develz=nent of a chromsome damage assay
To the minimum test battery to ensure an optimu= system for
detecting mutagens, an in vitro assay to investigate the ability
of cc-t=oLnds under investigation to Induce chromosome-damage in
mamr.a14-an cells is recommended. The in vitro human peripheral
blocd 1-Yno'nocyte assay system is now under development and will be
able to detect the ability of compounds to induce chromosome muta-
ticns.
(c) DNA-Sindincr
In v_'e- of the progress that has been made in project (d) belzw,
work cn Z@I:A-binding has been given low priority and this pro:e,:t
will te reviewed in the Autumn when a biochemist (Dr. P.I.M. Godden)
is @ed-.:led to join the staff.
(d) Bioc`erlstrv of chemical carcinogens
Studies -Involving the University of Surrey.
it haa been demonstrated that the enzyme ethoxyresorufin de-athylase
(ER::', 2 ::yto-P448 linked enzyme and part of the microsoral detoxi!_-
cat-,-,,. s-Ystern, is frequently involved in the activation of chemlc3l
comcz-.@7-.4_- to potential ly active carcinogens. The general response
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of this enzyme fzllowing in vivo administration of a sPectrum of
stru,cturall-, re'.ated known carcinogens or non-carcinocens has been
studied at Surrev. The preliminary findings indicate that the
indiction of r.--.D 4 s h, ghly correlated with known carcinogen exposure -
Collaborative s=,_,!_;es between GR&DC and Surrey have been concerned
with study; ng the changes in lung levels of ERD together with changes
in its kin;tic ;rcoerties following smoke exposure. Initial studies
indicate the f@_!Iowing relatively short periods of smake exposure
ERD can be induced considerably (6-10 fold) with changes In its
kinetic properties. These studies are continuing to investigate
(I) the longer t;.-le course of smoke exposure (II) the effect of
exposure to d4:ferent smoke dilution as well as different cigarette
types and (iii) z@,anaes in the lung ERD activity following exposure
to known carcinczens.
4. Other short-tern tests
(a) Test related to enphysema
Efforts have L-een concentrated on studying the retention of nitrogen
dioxide, a cc==o-,;.-.d that has been associated with the development
of emphysema, -wcr@z being carried out under contract for the verband
der Cigarettenin&--atrie. We have also kept in informal contact with
similar wor% Ice-,ng carried out at Papworth hospital by Dr. T. Higen-
bottam.
(b) Test related to cronchitis
This test was averaged by Dr. F.J.C. Roe in September 19S1. I.R e
concluded that this technique had merit as a method for quantifying
irritation, but --@;as not necessarily related to bronchitis. work has,
therefore; conci:-ued and encouraging results obtained. The time
course of the rasnonse has been established and high sensitivity
(to smoke) established. Currently, the dose response curve is being
established.
(c) Development of =ciels for atherosclerosis
Work has progressed on the development of a physico-chertical model
for modelling at .nerosclerosis, based on the notion t hat athero-
sclerosis occ-zrs in regions of low shear (contract stud-es, Imperial
College, Londzn' -
S. Bioassay of ni:-_tina
Demand "or this ;cc--Iar assay continues to be brisk. Currently we are
attempting to ext;nd the assay to include measurement of a second
nicotine metabolite, NI'cotine N-Oxide.
6. ZORBONXTE
The filter mater-lal n1trosobenzene, ('ZORSOMITE'), was examined by a
battery of tests closer% in consultation with the Group's consultants.
Unfortunately, sig-nificant biological activity was found in certain
tests, notably s%in sensitization, and the use of this material was not
recommended.
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FUTURE WORK
Sidestream smoke will be stzdied in greater detail, with attention being
given to the development of better methods for collecting sidestream
smoke. it is also likely that some further work on expanded tobacco will
be required, if only to clarify differences in results reported by
ourselves and Gallaher Limited.
Now studies being considered include work aimed at examining the effects
of smoking on weight gain and metabolic rate in humans and collaborative
studies into the effects of smoking in animals and humans on lung permea-
bility, the so-called 'lea., lungs".
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BATCo document for Province of British Columbia 19 April 1999