VISIT TO ROSWEL~ PJAIM WIDIUAL USTITUTE. BUFFALO.
V-1-
ON ArRIL 29th ArD 30th. 1958.
This visit was made by 11.4. Raid and D.G. Felton
with the
primnry purpose of seei-.g the work which fir.
F.G. Bock is carrying out
on the biologicnl tostiie of cig?rette smoke.
Wn Pnintin&. 11x. BoA uses skin painting of mice
an his main test
nrd rdorted this technique becnuso it wen the only
test which gave
consistently positive results. The work was
started as a result of
Wynder's experDnents am confirmed these
quPlititively, n1though not
quantitativ-1y. Mien vhole ter was used, at a
pyeater done rate than
used by 1-1.ynder, rpproximately 25~ of the mice
develop papillomata and
evontunlly 5 - 10A yiele- carcinomata. This is a
lower tumour response
thnn Wynder fourd.
For the skin painting experiments, whole tar
(prepared as
described below) was dissolvad in acetone to yield
en spproximately
2014- solution, such that 25 cc. containcd the ter
from one carton, i.e.
200 cigarettes. The dose per application vpa 0.25
cc. and mice were
painted morning and evening for five drys per week
(i.e. 10 applications
per week). Tumours generally appeared at the end
of one year, during
which time 25 - 30 g. of tar had been applied to
each mouse - rather more
than the body weight of the animal.
Bock has studied the effect of trauma, produced by
repeated
sh,-ving and by ebrssion with srnd prper, on the
tumour yield, and found
no effect. However, irrediation with X-rays (2,500
r.) on a small areaL
of the dorarl region, followel by painting with
whole trrs appeared to
trigger the cnrcinogenic process by an indirect
effect. For this
CZD
BATCO document for Province of BritiSh columbia 2
November 1999


-2 -
experiment a stronger solution or tnrp
corresponding to 3 co. per cartong
was used; the application or 0.25 cc. per mouse
vaS made once dailY
only for five days per weak. ;he tumours again
required about a year
for appearance nnd most were carcinomata. They did
not, howevorp appear
at the site of irrndistion.
lie thinks that the active constituents in
cigarette smoke tare
are aromatic polycyclic hydrocarbons and he
postulates that the presence
of other mnteri,,ln in smoke slows up the
absorption and detoxification
of these compoanda. Ile bsoes his bolier on tho
correlations he has
found between nctivity, an determined by, skin
painting, and the potency
of similnr frrctions nnd pure hydrocnrbons in
suppressing sebaceous
glrnd nctivity in mouse skin.
Sebrecous Glrnd 'red.
Bock defines a unit of activity in this test as
the i-mount of
mrterinl which will dostroy ono half the sebaceous
glands in an area on
the bpek of the nouse. Each pure oubstsnoe cnn be
given n Suppreasion
Index, defined as the number of units of rctivity
per gram of pure
materinl epplied. Cigeretto smoke tnr hqa a
suppression index of
40 - 60 unito por carton or 10 - 15 units per grvm
of tar. Bock believes
thia is wholly due to polycyclic hydrocarbons. Ile
has prepnred a list
of suppression ifidicis which showed that only
carcinogenic polyoyolio
hydroonrbons structurnlly rolated to
benznnthrneeno had high values*
Other classos of compounds, which, olthough
cnuoing tumour formation in
particular orgnns when administered by other
rouLes ' 6.9. parenterally,
are non-cRrcinogenic when painted on mouse skin,
grve nogetive results
CC,
BATCo document for Province of British Columbia 2
November 1999


in the sebmaeous gland test. Exrmples of these
Vero Butter Tellow,
2-naphthylamine and 2-4,-cetamidofluorene.
Dock, therefore, uses the sobaceous gland ts~st to
follov the
activity during the fr.-setionation or eignrette
smoke condensate in
various ways. Activity always seems to follow the
polycyclic
hydrocarbon frnetion. It can be separated into
fractions up to a point,
but further frnctionation leads to a complete loss
of activity and
recombination of all the fractions fails to
restore the activity. lie
is of the opinion that this mpy be due to
oxidation and that until this
crucial stnge the presence of entoxidants protects
certain hydrocarbons.
Whole tar does not appenr to be sensitive.
Activity as measured by the
sebaceous gland test seems to be constant during
cold storage. Tar
stored in a solvent at room tempernture poscesnes
a hnlf-life of about
one month. The half-life of solutions in either
acids of bases in a
very little less than this.
314:9t10 - Dibenzpyrene is not A very patent
suppressor-of
sebaceous gland ectivity; Bock quoted Kensler as
saying it had a
Suppression Index of about 3,001, about equal to
3t4 benzpyrene. The
amounts of the various carcinogenic hydrocarbons
reported by Bonnot, and
7'euko-=, recording to Book, add up to a
Sl~.-]ppression Index of 0.12 units
per gram. Consequently Back suspects tbat other,
more Potent,
fractions exist.
He is rot altogether hoppy that the sebeceous
glpnd test is
accurpte erd would prefer to rely on skin painting
alone, except for its
slowness in response. When he hns succeeded in
evoking quantitative
CD
BATCo document for Province of British Columbia 2
November 1999


-4-
responses to skin painting, he intends to use this
method as his indioator
In chemical fractionation. Meanwhile, he is
seeking to extend the
sebsceous gland suppression test to other species.
He in studying the
hPir-growth cycle in hamsters and is dyeing the
hair of albino hamsters
with this in view. Three cycles have, so far,
proved insufficient to
replace the dye codpletely. Ile Is bopePal that
the he.-noter ear will
prove suitable.
His technique with mouse skin is to remove the
skin sometime
after trertment hr.9 ceased and to scrape away the
top layer of epithelial
tissue. The nkin is then sb.,ined with Vile Blue,
with which lipids
stain red while nll else steins blue. Sections are
then cleared and
fixed. Normally sebaccous glond.!, occur in pairs
In the mouse skin,
one gland boing associated with the old hair and
one with the new growth.
A fixed area is taken al3d both hairs and glands
Are counted. The
number of hairs less the number of glands observed
elves the number of
glands destroyed. This technique is conducted with
serial dilutions of
frnctions or compounds until approximately the 50%
destruction level
is found.
Trr FreDarption.
Bock prepares his amoks tars by using two
different smoking
machines.
(a) A 20-unit Ecusta smoking machine. In this are
smoked
eig,arettes carefully selected by weiglat and draw
resistance. Dynamic
Pressure dop -jen-murements are made during
smoking by the use of a
tambour and kymograph. The response time vna
considerably lose than
C:D
BATCo document for Province of British Columbia 2
November 1999


- 5 -
0.2 sec. This is used to study the effect of
extremes of cigarette
parameters or. the activity of smoke taro. So fer
no significant
difference has been domonstrnted using the
sebaceous gland test. This
instr=nont hns Aso been used for the evaluation of
tars from filter-
tipped cigarettes both with filter intset and
removed. The amount of
trr is assessed by the optical absorption at 360
m.,- . Activity in
the sebRaeous plrrd test pirallels exretly the
smoulit of tar passing
the filter.
(b) An automatic 600-unit manifold smoking
machine. built by
Process end InstruTiOnt3 Inc., 2 Itore Avenueq
nrookl~m, r,,Y,p to the
desi,7n of a 111r. Flornnt. nis consists of 5
barks of 120-unit manifolds
which rotete clowly. Cigexettes from a hopper
are'loaded by compressed'
air into the Teflon mouthpieces and are lit by
passing through a gas
jet. There are five take-offs per brnk and the
units rotate twice
before the cigarette butts are rejected. Each
cigarette is therefore
puffed 10 timesp the waction being produced by R
vecuum pump At the
end of the collection train. Smoke pnoses into the
train of collection
vessels which are hold in a rofrigerntea airbath.
It in first cooled
by slow pnsqige through largo anpreity vessels and
is then precipitated
by impingement by passing through many U-tubea
which cause rapid velocity
changes, The tars Lure then removed by solvent
waahingo stored in deep
freeze and,uhan sufficient hes been collected, the
bulked solvents are
removed in a rot-try fil-n flesh-eveporntor. Some
tarn are frpctionated
che.,ically; nearl.;- all are denicotinised. This
apparr-tus is used for
C=
the preprrntion of bulk supplies for i,nirting
e7periments. In one week
c:-::
BATCo document for Province of BritiSh Columbia 2
November 1999


-6
three people cmn -moke 400 cartons or cigarettes
(808000) and thereby
produce 1,600 g. tar.
The tnr in stored at -30 0 or lower' and none is
used for skin
paintirg wUch hra been prepared more than a month
ago.
Ficotino toxicity is sonewhat of a problem when
whole tar is
used. Dock used to smoke JOJITI ALDEJ: cigarettes
for his experiments
a th, -,ve ft very low nicotine wioke, although
the nor-nicotine was
no 17
ralstively hir
,Ii. nie detoxicgtion of nor-rLicotine occurs much
more
readily however.
Ir))-InUon rxncr4menta.
Bock, in conjunction with Dr. George Moore (the
Director of
Roswell 1,-.r!7 1'emorinl Irstitute) who is
particularly interested in this
aspect, hrus nttomptod to induce bronchinl
carcinomata in mice by
subjecting them to the intermittent snoke from
cig.-irettes. The mice
arc confined in rlnss tubes throur)i which smoke
from cigarettes can be
drrwn; the technique is described in their pnper
in "'urrerv, 1956,
Vol. 12, p 120-130. All efforts have boon
uniformly negative.
Cnrcinorcnic rfft?ct or Tobecco 77xtr!,ct
Bock has tested extracts of leaf tobacco for
carcinogenic
properties using shin peinting of mice and has
obtained a pspilloma an
one mouso only.
Cprcino-cnic r"foct oC "nuff
noth flock ourl Dr. Hoore are originFilly from
Minnesota, where
snuff-cliewliq; is prnctiDed. This is claimed to
be a factor in the
incidence of bucc!,l c:-reer w!-Ich in prevalent
in t his etptc. The
cnrcinogeric propcnnity or rrrufr hrr b-en
investiflited by I)lacing
CI-)
BATCo document for Province of British Columbia 2
November 1999


-7 -
glycorino-gelatin oupposltori~-n containing snuff
and powdered tobacco in
thc hamstcr cheek, pouch. The nn:L-lal is e.Vozed
in this way for 5 days
por week, the suppository beirg wired in. It is
replaced each week
by c fresh supronitory. Yo positive results have
beer observed.
However, control atperiments in Vhich te-3 was
used as an innocuous
vcr_etable matter led to rapid nnd complete
arosior of the cheek pouch.
hxd-il Heise ~~:-intcr:,nce.
The m3cs ard hamsters, which are uned in tobacco
smoke
invostigatinn, rre housed soprrately from the
Institute in a converted
lv!relloure. TI-As is rather unsuitnblo for the
purpose ard tho standard
of ho-is2keepinC, while rdolurte, is n~)t of a
very high level. There
wz!n somic Ovidercr of extrnreaus rcdcnt nctivity.
The wooden warehouse
floor is difficult to mnintain in n clc3r- stnte.
The nnimals appeared
to be renzonably licrilthy And efforti Are made to
quarnntine the colorq
in the event of apidemics in nearby colonlas.
Dock was ahkod for his opinion of Vyndor and
daacribed him as
i "sincere nvEngelistll. WyTAer wao very upset
tbat no-one would confirm
his results rubliely elthough it wns knoini that
both Kensler and Bock
lied obtnined s1miltir findings. renslor could
only give confidential
confl=.ition as lie wrn bound by A.D. Little, Inc.
Doak wao not
satisfied that his controls were rigorous enough.
Bock wns a little
perturbed Vint WYnder implied nore then lie hnd
proved and cited the opse
of V13-nder's work with extrscted tobneco.
Originnlly, this was presented
in ouch r way as to infer tlint tobacco from which
the woxco had been C.7->
c::>
BATCo document for Province of British Columbia 2
November 1999


extrected More recently, Wynder had
.,ivo P non-cnrcinogenio tor.
been unhappy Rbout his results and at a meeting of
the A.A.C.Ro at
Philadelphin at the beginning of Aprilp ha.4
admitted that the specific
activity of tpr from extrncted tobacco was little
li-so theh that of
normal smoke tnrs.
Dock pointed to n f.,ct which eppeared to have
escaped marq
people, nsmoly Lhrt Wynder's results fit n normnI
dose response curvoy
and that consequently, no they lie on the signoid
pqr'.tion of the curve,
n amr-11 rh,-n[,.e in dose mry lend to a profound
change in effect.
m. 0 ~
C-
A - 131 the area covered by 14yrder's r-1-ort
C - D: n smell chnnge in dose will greatly reduce
biological
actIvity.
Dock wvs not impressed by the newt tent u1sed by
floukomm as
lie foresrw grnat difficulty in putting t1iie on
nn atiourntely quantitative
basis. lie was not fVIly conversant with the
chemical work which Donnet
lind done, but he felt thnt the importnnco of
3v4'9110 - dibenspyrene
had been disprorortionately mr-pnified by the
French workers.
Despite tho reliance which lie plnccs on mouse
akin painting
C=
e)Teriments, Dock was unwilling to extrrpolate the
results to other
species of nnimpl.s. lie feels t1int the
stntiotical evidence is the only
CC
BATCo document for Province of BritISh Columbia 2
November 1999


- 9-
strong point connecting moking and lung cancer.
Consequently, he
would not be willing to place reliance upon m
diminished animal activity
of cigarette amoke (Pfter sulteble treatment of
the tobacco, say) As an
indiertor of reduced ri!e-- to humans.
He mentioned, in pnsqir!T,, tho work of Dr.
Kushner at Sow lork
University who hnd blown methyleholanthrene dust
into the lungs of mice,
demonstrating its presence in the bronchial tree
by fluorescence
microicopy. This fluorercence is destroyed
entirely in 24 hours.
Bock impronred us Ps ,n intelligent, hprd worker
who had
accomplished much with limited ansistence. Pc- was
very helpful in
all he told us rnd e very friendly relntionship
was established. It
was a profitable visit in that it enabled Rn
essesment to be made
simultareously of the relativp merits or skin
pointing and of the
sebaceous glnnd count as a bioloFical test.
CC
cx_-
BATCo document for Province of British Columbia 2
November 1999