SRE/PSD/4GD Group Researc'i & Devclopment C-ntrc,
Britis'lli-7~j,::2zicz,ri Tonacco Cn.
Southaapton.
760~ Y-rch, 3975
LIFE SCIE:.T'C7_ RE6-ZIARCH
STATUS REPORT: nLa',CH 1975
Th-*-.s review generally covers th~.- past 12
months but also
includes the final results of earlier experiments
which have
become available.
During the year the major expcrirnents on the
B12/B!3 J_znus
samples were started at Life Science Rc3earch
Limited and W~Jckram
Research Laboratories Limited. One of these PRT
samples is a?.z;o
being ex_=mined, together with ths control, in an
inhalation
(promotion) experimenL at Battelle. This
inhalat4.on expeL-iment
-ent (WRL) will terminate
and the skin painting promotien exper4n
shortly.
A major change, which occurred in August 1974, was
tre
decision that the L.S. group should undertake
short-term
inhalat.-*-.n studies with the aim of making a
submission to the
Hunter Ccmimittee an foamed B;~TFLAKE
inc:orporated into both flue-
cured and blended cigarettes.
The outline below i3 given in terms of the major
studies
rather than under the variuus researc-h centres.
MOUSE SKIN PAItTTING
The results of the B11 experiments (PC], v.;~ SRTJ
are of
considerable interest because of the differences
found between the
long-term and the promotion studies (Battelle).
In the long-term experiment the interim results
(to week 108)
show that:
(a) PCL condensate is significantly less active
than that from
C-D
L_n
r,-)
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the tobacco control (TR 0.82)*:
(b) -~RT condensate has a much lower acti-ity than
that ot the
conLrol (TR 0.42) and is alsu significantly less
active than
condensate frcm the PCL (TR 0.52).
In contrast, the tumour incidence ir. the
promotion study did
not suggest that there was a meaningful difference
between these
samples. Nevertheless, statistical analysis of the
final results
shows that:
(a) the tumour promoting activity of PCL
condensate is slightly,
but not s-4.,rificantly, higher than that of the
control
tobaccol
(b) the tumour promoting activity of the SRT
condensate is lolver
than that of the control: the difference is again
not
sianificant;
(c) the comparison between SRT and PCL condensates
is significant
at the 91.7% level: this level of significance
would probab-ly
have increased if more mice had been used or if
the test had
been extended past 40 weeks.
on balance therefore it is clear that, in terms of
its
tumorigenicity to mouse skin, SRT has advantages
over PCL due to
a much lower overall effect which is in part a
result of a lower
pramot-ing activity. At the same time it is
apparent that the !on,-]-
term results for PCL are offset by the promoting
activity which is
slightly higher t,an that of the original tobacco.
In the light of the importance of product solubl3s
(see belc-..,)
it is interesting to speculate whether the
differences between PCL
and PRT in the promotion experir-ent could be
accounted for in terms
o,: this variable. The solubles 3-evel of SRT
(B11/3) is about 40%
(glycerol free) while that of comparable PCL
(B10/2 which was made
Footnote
*TR = tumorigen.-c ratio which is thF6 ratio of
the weights of
condensate to produce equivalent tumorigenic
effects. Ratics leis
than 1.00 indicate that the activz-tv of the
samule di--cussed is
lower than that of the concrc-'L oL uLj"=!.L
N.)
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from an almost identical blend) is about 60?1.1,
but this value
includes nearly 8% glycerol.
Experimental wcrk started on the B12/B13 samoles
last suirriter.
This "PRT variants" e...--eriment is designed to
examine differences
between base grarrsnage, base ASV and product
solubles.
The long-term experiment at LSR Limited has been
in progress
for 35 weeks but to date no skin-tumours have been
recorded:
mortalities are low and reasonably uniform. The
slow in-ftial
response is probably due to the use of a maximum
dose level of
40 mg rather than to other causes, i.e. the
response of the
animals or unexpectedly low activity of the
condensate.
The results from the promotion (skin-painting)
experiment at
WRL Limited have been analysed to week 24. This
analysis, based
on provisional data which took no account of
regressions, has
demonstrated the power of the balanced block
experimental deS4gn
since the tumour incidence in all aroups %-.-as
ver7 low. The
interim conclusions reached are:
(a) promotion activity is lower at the higher base
weight;
(b) promotion activity is higher at the higher
level of product
solubles;
(c) difference in ASV (apparent specific volume)
has no
significant effect on promotion activity. %'NOTE;
This
lack of significance could be due t-) the lack of
difference
in the ASV level in the PRT samples produced).
The experiment is being terminated netween week 28
and week 32.
A new long-term -=eriment (1314) has been planned
in detail.
The experiment is designed to examine the possible
effect on
tu;,iorigenicity of adding nicotine to a tobacco
blend. This
approach should separate the effect of nicotine
when it is pyroly%-ed
in a cigarette from the effects of other variables
which may
influence the result if use %.,,as made of
different tobaccos having
a range of nicotine levels.
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Preliminary work is currently in progress to
cxamine a new
approach to the manuf-cture of the samples. A
toxicity trial on
nicotine enriched condensates will be conunenced-,
at LSR Limited
irturic-diately after Ed6tur.
SEBACEOUS GLAND TEST
LSR Limited have also been attempting to set up
the sebaceous
gland test to the protocol developed by B-A.T.,
Hamburg. The
project has taken much longer than anticipated.
Initially the
delay was due to the lack of suitably experienced
staff but
subsequently it has proved, and still is proving
quite difficult
to duplicate the test. It is not possible, at
present, to be
certain of the reasons for all the difficultieF,,
but is is apparent
that there are two major factors. These are (a)
the close similarity
of partially suppressed sebaceous glands with
small portions of
healthy glands, and (b) a large animal to animal
variation. LSR
LI'mited are currently assessing slides from an
experiment in which
four condensate samples were examined. These
samples have previously
been submitted to the sebaceous gland test in
Hamburg and examined
in long-term experiments in Frankfurt.
I14HALATION STUDTES
in an initial e:cperiment it has been shown that
tumours can
be produced in the hamster larynx by pre-treating
the larynx .-.rith
Y-methyl-N-nitroso-urea (NMU) 'followed by
exposure to smoke
(Dontenwill machine) for six months. This study
has been reported
in "Experimental Lunq Cancer; Carc-4nogenesis and
Bioassays" edited
by E. Karbe and J.F. Park; Springer-Verlag.
In mid-1974 a second inhalation promotion
experiment based
oil this method was started. In this experiment
twu levels of
exposure (to see if" there is a dose response) are
being used for
the examination of smoke from a control tobacco
blend and from
PRT produced from this tobacco. If eifferences
between the sm,-Ae '-0
from these cigarettes can be shown, then a
worthwhile inhal.tion U-1
technique which produces laryngeal tumours in a
relatively short r\-)
time will have been developed. Somc of the animals
will be
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sacrified in March at week 24, while the remainder
will be exposed
to week 32.
in a different_ Lype of expezinteat tlx(--
pQs~.sible effect uf
'Vitamin A deficient diets on the development of
laryngcal lesions
in hamsters is being examined. The rationale for
this experiment
is based on the observation that enhanced dieta-ry
levels of Vitamin
A lead to a reduction in the incidence of tumours
in mouse skin
experiments while it is 31so known that Vitamin A
deficiency leads
to metaplastic changes in the respiratory
epithelium of hamsters.
The experimznt 'has been in progress for almost
two years but,
unfortunately, up to .,reek 82 the most severe
lesicn i..,hich bad be-en
noted was in Class 3. This result is disappointing
despite the
overall logical pattern in the results which
sh.cws that mortal iti es,
the time of appearance and the severity of the
lesions appear to be
related to (i) the level of smoke exposure, and
(ii) the level c.L-
Vitamin A in the diet. It had been decided to
terM4na-e the
experiment at about week 106.
'IN-HOUSE' STUDIES
Temporary facill''Ities for in vivo studies were
obtained a~.
Wickham late in 1973 and a research prograirne was
undertaken in the
first half of 1974. Apart from training staff in
the measurement.
of respiratory monitoring techniques, an initial
assessment of a
simoke inhalation machin- was made. The machine
was developed in
Southampton by coupling smoke 'transfer and smoke
exposure char-bers
onto the back of a 24-port Mason smoking machine.
The initial
assessment of the machine and its subsequent
rr~utine use has
demonstrated that the machine is very successful.
Tc%vards the
end of this period the machine was used to examine
the dose ot
particulate material retained in the respiratory
tract of the
hamster and the rat. Decachlorobiphenyl was
employed as the
particulate marker and the results show that about
80% of the
particulate material retained in the respiratory
tract was present C=)
in the lungs.
In mid-1974 the L.S. group was asked to
undo,_,,ta!:Q studies
(JI
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to produce biological eata on cigarettes
cortaining foamed SATFLAIC"
for a Phase 1 submission to the Hunter Committee.
The Committee
requires data on a substituLe material ~.ncluded
at not lesz tnan
four levels (0, 30%, 50~~ and 75%. in this case)
and in the blends
which it is proposed to use7 in our case date nn
both flue-cured
and a cased and flavoured blend are required.
Although the data
required is basically that from a six-weeL,.
inhalation study in thl:
rat, this has taken much lonqer than anticipated
only partly due
to the limited resources available. T'he stages
involved (for each
series) are as follows: (a) preliminary inhalation
toxicity study
including settina of various smoke exposure
levels, i.e., equal.-L551.mj
smoke concentrations in the exposure chariber for
the var-4ou3
cigarettes, (b) smoke dos-imetr, stud, to
demonstrate adequ_r-te
penetration of smoke into the lungs, (c) riain
6-week study using
at least two exposure levels for each cigarette,
and (d) pathoicay
and reporting.
These studies arc still in progress and the
detailed assess-
ment of the pathology is awaited. Nevertheless,
the initiaJ
results indicate that the studies have been
satisfactory. For
example, the results show that (a) high levels of
particulate
material were retained in tne lungs, (b)
significant differences
in carboxyhaemoglobin levels were noted between
the groups
(effect of puff number?), and (c) preliminary
counts of lung
macrophages are related to smoke exposure levels
and to cigdrette
types. Other changes in pathology have been noted
(e.g., goblet
cells, hyperplasia and retaplasia) but it is not
yet known whether
these can be related to either exposure level or
to the level of
BATFLAKE inclusion.
The new Life Sciences Research laboratory is
rapidly nearing
COMpletion. The building Is being taken over from
the contractors
on 27th March and, following the r,--)ve from
Wickham, the final part
of the BATFLAKE studies will be undertaken in tha
new laboratory.
C)
-'D I
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November 1999