Document YnGDbabgvLm0gKZqZ4oxRNrO
FILE NAME: Kaiser Gypsum (KG) DATE: 1974 KG091 DOC#: KG091 DOCUM ENT D ESCR IPTIO N : Journal Article - The Effects of the Inhalation of Asbestos in Rats
v/ H r -L dancer (l!)74) Sti2
TH E EFFECT S OF TH E INH ALATIO N OF A SB EST O S IN RATS
( \ W A l.N E IL U . H K H H V , .1 .W . S K ID M O K E an V. T l.M B K K L L
From the M edical Itw nrch (UtuoriV* FtieumwoHiotiH U n it, IJfwrfouffh Hospital, /$ewtrth, (lamorgnn
Iiccvtvr<f R K<jiti*mho! Iif73. Ac<sojM*<l 2tt Xovomluu* 1072
Sum m ary.--Two experiments in which S P F Wistar rats were exposed by inhalation
to dust ciouds of the U IC C standard reference samples for periods of between one
day and 2 years are described. All the samples of asbestos produced asbestosis which continued to progress after removal from exposure but only a little fibrosis was observed in control rats. Lung tumours, ranging in severity from adenomata to squamous carcinomata, were produced by ail samples but in the controls there were only a few adenomata and none of the more serious tumours. Of the 20 tumours which metastasized, 16 occurred after exposure to one or other of the 2 chrysotile samples. In addition, a total of It mesotheiiomata occurred, 4 of which were with crocidolite and 4 with Canadian chrysotile. Two of the mesotheiiomata occurred with only one day's exposure to asbestos. There was a positive association between asbestosis and lung tumours.
XVao n ku , Berry and Timbrell {I973) reported the results of experiments in which rats wore inoculated Ultra pleura Il\ with samples of asbestos. In the dis cussion it was mentioned that two experi ments in which rats had been exposed to dust clouds of the U IC C reference samples had been carried out, and the results of these are now presented. Some preliminary results of one of these experi ments were reported by Wagner (11)72)
In both experiments the rats were exposed to similar dust conecul rat ions and the dose varied by exposing rats for various lengths of time. The main aim was to establish the relationship between the development of malignant tumours in the lungs and the dose and type of asbestos dust but, additionally, the amount of fibrosis was assessed
.WATK1UAUS AND MKTIIODh
Caesarean derived rats of the Uistar strain were, used which had hern bred at the. Unit from SPF stocks given tu us by the Imperial Chemical Industries. Pharma ceutical Division at Alderlev Edge Cheshire in I9(U and 1968
The asliestox samples used consisted of the 6 UICC standard reference samples (TimbrcH, Gilson and Webster, 1968) whicli were prepared following recommendations of Ihe l :10(!. These samples are of 3 amphibolt* types--amosite. anthophyliite and crocidolite--ami 2 chrysotile*--a Canadian and a
Pvhodesian sample. The rats were exposed in 1*4 m3 inhala
tion chamlicrs (Timbrell et a}.. 1970) which
contained 8 cages, each of which could hold
t> rats. or. for a short fieriod with yountr ruts, there utis room for a seventh. Kiu chandlers were used, one for each typo *<1 ishextos The chambers were constructed so that the rats could he tended without the chandlers licing opened.
The dust clouds were genet ated using a
specially devised dispenser (Timbrell, Hvett and .Skidmore. 1968). The clouds wen* generated for 7 hours a day and 5 days per week. The respirable dust concentration' were measured using size selective gummetric dust samples (Cassella Type 114Ai The collected samples wore evaluated ftt the cmi of each daily session. In order > achieve the loquiml dosage, calculated a*
the product of concentration and tin variations occurring in the concentrationwere corrected on the following days,
the end of exposure the rats weie left m tie chandlers for a few days, to allow tune fot
- W
KFKBITS OF INHALATION OF AXHKSTOS IN KATS
253
their fur to become clear of asbestos liefote Sections of both lungs were examined without
transferring them to a clean environment for the remainciet of then lives. At transfer
some rats, generally 2 ti 3 of eaeh sox, were killed and the lungs renuned for histological examination and determination of dust, content.
As each type of asbestos has a eonsider-
able silicate eonton! (about 50%) the amount <f dust in the lungs was determined by first
assessing its silica content and referring this
knowledge of the duration or t\'|e of asbestos exposme and with the animals in random
order The sections were olwerved on a viewing screen of a Projection .Microscope 4013 BK using a x 7 objective. At this
magnification a large projwuliou of the lung
could ! assessed in a single field and, as it was not possible lo oliserve usliestos fibres
at this magnification, the results were not biased by knowing the type of usliestos to
value to that of the respirable dust to which which flu* animal Imd lieen exposed.
the rat had been exposed. This method
was used m previous expetintents (Morris a l, 1967).
E xperinut*
After exposure the rats were caged in
in IKith experiments and for all doses
threes or fours isolated in a special unit there were groujvs which wen* exposI to
supplied with filtered air, The inhalation all 5 reference samples; there were also
chambers were also in this unit but in a control groups which were not exposed.
separate room. They were fed on a pro Rats were allocated to treatments at random.
prietary brand of autoclaved cubes, and At the start of the exposure the majority
wrater
adlibitum. Kxeept for the scheduolefdthe rats were between 5 and 7 weeks old.
killings, each rat was allowed to live until a few Iicing slightly older or younger, and
it died or appeared to he distressed, and a there were approximately equal numbers of
full necropsy examination was carried out. males and females.
Histological
preparation,siat tuny
Experiment I.--There were 2 time, inter
microscopic methods.-- Foi the scheduled vals of exposure: in the first, rats were
killings, animals were killed by chloroform exposed for 3 months starting in .May 1967
anaesthesia and follow'ing exxangumation and in the second, groups of rats were
the thorax opened and the lungs removed. exposed foi one day only in August 1967.
The left lung was air inflated and suspended In addition to the killings at the end of
in formalin Representative portions of the exposure, in the 3-month group there wore
right lung were taken for electron microscopic also intermediate sacrifices at 5, 8 and 10
examination and the remainder of the lung weeks.
dilated with neutral buffered formalin. Slices
Experiment 2 --There were 3 time inter
were taken from both lungs for histological vals of exposim-- 0 months. 12 months
examination. After the histological sections had been cut. the embedded tissue amt the trimmings wore dewaxed and added to the
and 24 mouths. The experiment started tu Januaiy 1969 and after 6 months half of
the rats were, removed from the cabinets
remainder of the lungs which wore used for They were replaced and a year later these
vthe chemical estimates.
replacement animals were m turn removed.
For other animals, at post mortem the They were replaced by animals to he list'd
lungs wore dilated with neutral buffered for special electron microscopy examinations
formalin and after fixation were sliced which will not he reported in this paper
sagittally, routine sections being taken of the The remaining animals were removed m whole loft lung and the upper and lower .January 1971 alter 2 years* exposure*-- fu
lobes of the right lung. In addition, any other suspicious lesions from the lungs oi other organs were taken for histological
tile 6-month groups, in addition to the killings at the end of exposure. tuts were also killed after 2 years, i f 18 months
examination.
after removal from exposure.
In all cases, sections were stained with
The numbers o f rats are given in 'fable l.
luiematoxylin and eosin and the lung sections In the 24-month groups, overcrowding
were stained for elastin. reticulin ami col amongst tin* males tended to occur due to lagen. Special stains were used in some increase in size after about a year, anti
rases as an aid to diagnosis of the tumours. some were removed prematurely alter 131
> Assessment of
theseverity mofonatshttsexto*Fio<.r--analysis these rats have been
*v
254
** <* W A C SK M . li. liK ItU V . .1. W . SKIll.M OKK A N D V. Tl.M KUKfd.
it tl ` N |h
) <lm m onths
it m o n th s 12 im M iilis 21 month**
T aulk l.
Am* silt-
-It
52 24
Zl
<\nl iu>{>li\ lin e
4Si i52
`24 2h Ml
i *11h*ii Jifill c*
4ii 72 24 2U 20
Xftmittr o ffCxpoM*/
rh*vil i(f*
W r>2
24
2:1 24
H u VS(tl tie ( H ImmIc - ih i))
49
52
is
't
27
>
-n J
t 'niit nil 4H r>H
4
T ahi, b II .'- - M m v
Ift'itpitable
{uK jjur1)
<tn<l ('hwttlulivo.
Do (uir/ftH*
s of I <In\ li nvmf It* i>miml h* 12 month 24 months
AlltfNlIt1 14* i SHI 12 4 ranmi 11*2 Mm 10-8 17000 10- .1351X1
Ant iioj li\ llifi* 12 X Sill 13 -ft 52411 10 8540 11*4 171110 10-0 3371X1
(V m'kIoIiIi* 12 r XX 12*1 5n3u 10 7 K430 1 0 l7<)oo 0*3 33200
rhrjsofilr antolnu) U*7 ox 12*1 4)30 t o i 8240 10-7 17100 10* l 33200
(iirywtf tk* {iilitxlrsmn) 14 7 103 1**1 507(1 ID* 7 8590 io 0 17100 10 t 33000
fr& jn m
included in the 12-monlh groups and hence
The lesions consist initially of a deposi
the numtxu of rats in these groups is slightly tion ot asbestos fibres, alveolar macrophage
higher, and m the 24-month groups slightly and cell dehri< in the alveoli ansing directly
lower, than planned.
from the respirufoiv bronchioles. These
In Table li the mean respirable dust deposits Iwcomc organized firstly by heing
concentat ions and the cumulative doses enmeshed m a thin retieulin network which
the products of concentration and time, are coarsens with time and becomes replaced In
shown. The one-day exposuie. was 7 hours collagen fibres, 'flic alveolar epithelium
for ail dusts Kor the other 4 time intervals reacts with replacement of the type I cells
ol e.vposine. there were slight vaiiations in and becomes completely lined by grantilui
the munliei of horns required to achieve pneiimocvtes. In some of the alveoli the
approximately equal doses hut the mean epithelium is shed into the lamina, m other*
times were 402. 788. 1574 and 3287 hours there is a walling of the alveoli by these
respectively. The mean concent rat ions were cells. This usually occurs at the bifurcations
usually higher in l\f>enmcnt 1 than Kxpeii* where groups of alveoli are closed off from men! 2. and the 3-month group had an the luinma of the lespuntoiv bronchiole*,
avetage dos<* ot UO', ol that of the (i-montli givinc the so-called pseudo-acinar ap* group Keasonabie equality of dose between peaianee In the guinea-pigs the** sm.ilt
the dusts was achieved for all the lengths of exposure, except for the one-day which was loo short to allow am adjustments
e\*tie spaces contained asbestos hbre and degeueratmg maciophages, but ill the Ml
numerous granular pnouinocvtes were nl-o pieseot. The initial lesions were eoiilim'd
io occasional discrete individual lespitatniv
/ ilU' t'jil r ln lio ii o j h i.slolnr/H'iff fu nlu/i/s
bronchioles scattered thioughout the lung
( 'lawujivo/ion
o f t t textosxuibss--tanTchee leAslitoenr- turthei exposure. more and
seen m tin* lungs of i.iis-iixpused to nil types more rvspiratoi\ brum luole- bec<mic invoke!
of asbestos weie similar to those deseritied and all the ospiMtoiv hioochtolcs ili'MIV.
m guinea-pigs (Wagner liMW. ItHiA) There Irom leinunai bioiuluoles become thicken"
were 2 mam dilietcucex* (irsllv. asbestos as the libious tis-ue net woil* extends mi<*
Iindies were never seen in the lung tissue the wall ol tin* ifsp n a io iy Inoimhiole. aed
of tin* tat although they me frequently tin* inteistitial lead ion spnad* down nit*
seen m the pleural granulomata which follow the jieriphend elements of the piiotuiv
the intra-pietnal inoculation ot amphifmlc mut., involving the alveol.il dint* .on*
fibres; secondly. there was sv fur greater and finally tlie au sic* and alveoli Win
production of gratmlai ptieumncvles {t>jm* ptOifl* ssloll tie 1 nuliv dual lesions tend
II) alveolar epithelial cells in the tat
coalesce leading to the development ot
j
ICKKKtTS OK INHALATION' OK ASMCSTOX IN HATS
2.*r>
l"K! 1 - -Slight usb<`htoms (Ornile 4) ThioUoiiiMg <1 the* wtills of tiw* tlv*h nrwi11< <Ih.t i )v Iron fho n sp im tn ry hmnohioli"., with teplnremcnt of no in m lo jm li.'lnmi In n jn I i (111. II < H so
f:
*
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' V ."
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V
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r> < t .^ v T5
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fV W A i ?- i'll Ntighf (kIcwIii^i^. (<Jia<ir 4} hihtM jxiuot shim m y muni m ih if h a i'iiV
m tho ttivivili ot a rvN|iim iorv bionehio]*. A 'sb^'uiw Iiu<u *n tiro m io k * \% m i Ih* htn^-
tlluinmatmn iwit^cul u> ilUiM roto H ' tibivs H iV K <M.
` jlnt > <ii nU^
AU
(i. W A C N K H . ( J . H KHKY . .1. W . SK1DM ORK A N D V. TIM H R K LL
VU>^V. i r - . .
,. -
Km
a
-Milimite <iheM<wi'* ((nule (>) A louer pottei* firojeclion ithiMnnm^ lhat the iesmu igh Mill mnml\ involving the re^jnrniorv bionolwole ih nmv flifltiwe Aggtejcatinn*' of type JL
although Mill pntMunoeytes
mmmv nle\eninivnotlvhienglutmheinrae*n-jn>rnu--rlhiryas .mwvvoiininmg
t.t.h.h.e.*.'..U.w..t..ai.l.lUl*.,
HH,. && KK..
Xx KHOO,.
KKKKCTK OK INHALATION' <iH ASHKSTOS IX HATS
ihffu.se interstitial fibrosis with gradual in. adenomatosis In contrast to this, a few
eicase in the density of tin* fihron% i Kmic, control animals were seen to have solitary
ultimately resultin'; in the replacement of adenomata which were small in mw*. The
most of the, lung parenchyma by a dense iuh*rioeareinoiuufa w'ere of the same type
collagen netwoik .surtoundmg disputed air and origin as the " alveolar adeimcuremo-
spaces muny
ofwhich contain hugineatc.liu*m' pd-,eserilied by Shabad and Pvlev
of giatiulai pneunioevtrs which, m .some- (1970): many ofthese tumours wen* pajnllary
areas, have h'srd leaving foci of hpo-u!vco!at adenoma-carcinomata. Tltc squamous car
proteinosis.
cinomata appeared to originate Irom foci
The assessment of these lesions was of squamous metaplasia occurring in the
hast'd on 4 grades of fibrosis---minimal, as)M*stotic lesions in the respiratory bron-
slight, moderate and severe. Typical ex cbiolcs (Fig. 7, 8) As far us can lie ascer
amples of slight, moderate and severe tained, all these tumours were peripheral
asbestosis are shown in Fig. 1-4, In uddi- ill origin and not bronchial papillomata.
lion, when carrying out the* assessment it The classification of these tumours was
was found convenient to introduce' the discussed in some detail in Session VI at
intermediate categories mmimal/slight, slight/ the ('at linburg Conference on the Morphology
moderate and moderate/severe. Tho 7 re of Experimental Respiratory Carcinogenesis
sulting categories were scored 2-8 and the in 1970: and the chapter by M. F Stanton
normal lung, in which there was no sign of (1974) in the [ARC Monograph on fttthofoyy
asbestosis, was scored 1, and the assessments of Tumour# in. Ijuborttlory Auimuh contains
were averaged for cacti group of rats. The detailed descriptions of the tumours timt we
repeatability of this form of assessment was have illustrated.
tested on 154 sections, reassessed in a
'Hu* rats used in this experiment are
different random order and 181% were from a caesarean derived, harrier maintained
assigned to within one category of the first colony and fortunately they have been kept
reading.
free of rat bronchitis; therefore the squamous
ClaMfiJi&thoH
of --(mio Themteutmapoluarssia was not associated with bronchi-
found in the lungs of rats aftei exposuic to eetosis.
the various typos of asbestos dusts were
Aletaxtases in the thoracic cavity to the
peripheral adenomata, widespread adeno chest wall, <liaphragm, pericardium or the
matosis, adenoearcinonmta and squamous tracheo-bronchial lymph glands were seen
carcinomata The rarity of pulmonary tu in 14 animals; tho majority hud lesions
rnout's m rats has been stressed in tin* invading 8 of the sites and in ordv 2 animals
reviews by Kuschnet* and bask in (1970) and were metustases observed in the tracheo
Slmhad and Pvlev (11)70) Further, these bronchial glands. In one animal secondary
authors have described the de\elopment and morphological features of the tumours that
we are reporting All these tumours were jieripheral and appeared to arise from the
deposits were seen in sections from a kidney Fight ndenoenreinomata and (> squamous
tumours had metastasized
region of the respiratory bronchioles m which the asbestos fibre Had accumulated
The origin of the adenomata appeared to he front accumulation of type 11 epithelial cells that proliferated m the alveoli of the respiratory bronchioles (Fig 5 <5) In many of the exposed annuals Ihe-r lumouts were
RESULTS
Ail except 2 of the rats in the groups with e..\|K)Sure of 12 months or less survived for the whole qf their planned exposure In the 24-month gioup theio was appreciable mortality before the etui
multiple, and in a nmnber of animal-, particularly those with the more seven* crudes of asbestosis, there seemed to be ndmomata arising from numerous adjoining nwjuratory bronehioli's. giving an impression
o{ exposure and only 58'b survived for the full period. Out of 1018 rats it was
impossible to obtain adequate histological material in only S because of cannibalism
<>f contiguous adenomata invading large urns of the lung. Dr Harold Stewart (personal conmnmieathm) suggested that
tins type of lesion should be referred to as
bust
rbonlioH
'Pile mean weights ol asbestos dust in the lungs of animals killed at the
K K K K C T s O K I X I IA I.A T I O X O K A .S H K S T O S IN' KA'f'S
2ti
**
v
'v r t - .r - * ,* ''o ' c i v - V i C
<
: v*u
*\>.
Kijtliltnou*
kH|X*I
nil moth-mU1U>fm*s|<
II Jt; K
Ol
%'t^Sf
m v c a M eat& Li
2i>0
\. V . WACXKK, (5. ItKRHY. J . W. KKfDMOKIC AMO V. TtMIUtKLb
%
T.UIUC i l l . -- D m i
in Lvtujs(niff)
l*f*tt}fh of
M.MUI (lose
1>!,%oi !<-
<*x|KKtm*
iHji/in4hoiii'K Aittosih* Anlhophylhte (YMM4iito (Cniuutmn) (llhntlrHtnn)
5 uvek*
1880
I*U
I 3
l-l
0- 1
0- 1
8 wi't'k*
2450
(hi)
! ft
1
0- 1
0-3
HJ ttcvk.M
n 20
2H
2 1
0-6
0-5
O41^-
3 months
50.10
:\1
3*fi
no
0-6
0-7
i
ft months
8470
4 1
4*4
4-5
0-4
0-4
{
12 months
171 (HI
h :\
i) ft
*>
0-8
1-4
f
24 months
.1X401
10 8
13*8
14 9
on
0-6
ft month*
1*3
2* ft
1-2
0-0
0-1
4f
{*%fIi*r IM months non-c\postin')
if
Vt.
T u si.b I V . - Mran Kin viral* afire Find
f i x insure(dai/s)
/ J
IdHtf'lh of
(`lirysof ilo riiryoth*
f
o\post!IV Amusin* Anthophyliitc i'f<W*l<lollf* (('aiJHtluut) {Hh{loian) (.mlml
1
1 ilay
804
800
70.1
76-*l
75.
80.
[
3 moot h*
771
82
817
790
857
793
ft months
76
686
788
669
706 *1[
r
12 months
6112
760
776
778
826 } 754
{
24 month
807
778
760
.78.1
758 j1
* Arijuxtwi to bn md<*|X`tulrnf af surnhctw.
scheduled times are given in Table i l l More dust was usually found in males than in females and on average the female lungs contained only 70% as much dust, as the male lungs. The values in 'fable 111 are the averages o f the male and female means. For the 3 amphiboles there was a similar pattern, with an almost propoitiomd increase o f lung dust with dose The 2 ehrysotiles were similar to one another but much less dust was found than with the am pinholes, also the chrysotilc figure- did not show the same clear increase with dose. The main features are summarized in Fig. H. The dust in the lungs o f the animals which had <5months' exposure had been partially eliminated IS mouths after removal from exposure. The proportions eliminated were 74% fm am ositc, 73% foi orocidolite but oni\ 4 1 l o r anlhopln Ibte llo u e v e i. the lower elimination o f anthophyllite was not significantly different irom the amositc and orocidolite figures.
S n retro/
'flu* mean lengths of survival from
the day the rats were iivst exposed are given in Table IV . The survival times have been estimated so as to be inde pendent o f the sacrifices. The short survival o f the 24-month group exposed to Canadian chrysotilc was largely due to 8 rats dying before Day 400 (in the other four 24-month groups only 2 rats died before D ay 400). These early deaths were not due to exposure, since 5 died due to an infection in one cage and 2
wore killed in a fight. Discounting these s <(catIts, the mean survival wou? 09,s
days, which was still the lowest of the 24-month groups. When the mean was taken over all the lengths o f exposure, the Canadian eh rysotile groups showoil least survival, but only a month lcs< than the control groups. The amositc and anthophvHite groups had mean sur vivals only a few days less than tliF controls, while the eroeidolito and Him desian chrysolite groups had longer sui vivals of 2 and 3 weeks respectively. Hence, there is very little indication that the exposure hud any effect on the m erall survival o f the animals. This* is in marked contrast to our intraplem.il inoculation experiments in which injection
f i
KKKK( Ts UK 1XMA LATluX UK V.sKK.xTus J \ |{.\TS
ii<; i
Weight duel in lungs (rngl
Ft*.. il
-- ,--
-- ,--
10000
20000
Cumulative dose (mg/ro* hours)
MiMj'lit t>i tliiu in Iuiiih I .t*in n I.iIkhi
-- ,--
10000
anil t itii
ul asbestos reduces the e x p e ifa tio n of the 3- and (-month means, there was an
life b\ several m onths (W agner <I
merea.se of asbestosis with exposure for
1!*73)
all the dusts. Also, following ( months'
exposure, there was progression during
.Ufa sin#itt
tin* following IS months without exposure lor all the asbestos I \ pes, hut these rats
The amount of ashes}om> was assessed did no! fare as badly as those which
ft all the tats killed at scheduled tim es cnid lined exposine There were signi
in Kxperimenl
2and after n fwiceaenkts daifnfderences between the asbestos
.{ months' exposine in experim ent I t\pes(/J ' m il) amosite in\ariabh gave
There u eie .7 or <> rats pet treatm ent for the feast ishestosis throughout; aulho-
f.tcli exposure, exeept that theie were ph\ llite and Canadian eh n sotile showed
only 3 aftei X weeks O verall the li most ashestosis after (i months' or longer
"(xos lutd sim ilar am ounts of ashestosis ex|Hstire, erooidolite and Khodesian chrv-
.utd they leave, therefore, lwen com bined sotile were intermediate.
to give the mean ashestosis scores in
The mean ashestosis semes of tin* rats
T.thle V, whieh are summarize! in Fig. H). which were allowed to lu e out their
r.veepl for some ineoiiMsfencs between lives are given in Table VI Those tats
202
J. (*. WACXKR. 0 . UEHRV, J. W. KKlDMOltK AND V. TI.MHKEU
Abeaiois
grade
V
Moderate
*
Slight 4
i
i X
Minimal -4
t
Nil -*
1m * m
--. j
12
-nr
10000
20000
30000
Cumulative dose (mg/m3hours)
A s IhM ohIn tn Miieniicrd imK hi ivlntimi to ijonr >hmI jum .
Time (month)
T ahle V
M o an A xbcxtoxix S co res* o f S a crificed H ats
mirth t,f
( `hrysotiir
C h n v it lie
i
**AIIO
\monht* Ant hopin' Hit e ( 'men Soli to (Cnnmhiin) (Rhoilosian) I'nnti*!
f
*2 11
J`<>
2 a
2 0
2-7
i 1
t
H \\. U
!i mantls
4 *
2-7
2K
m* 1
vu
1 'I
u mtimhs
>, *
.1-2
2 li
Ml
i ft
i J
*
12 M onth*
4 0
Vi
-4 .*t
4 1
4 a
lK llliHHh*
-
-
< J
24 m onth*
4 :t
ft 2
4 S
(I Ml
.V S
|M
(1 month**
2
.7 <1
tt 7
5n
:-7
{nftm IK m onth*
!
r
I * ml, 2 tnmtmal.
I
K
scheduled for *J4 months have born divMrrt" info ({lose i foil died before e,c>mplot ion of exposure and those that survived for a )humoil of non-exposure. The amount o f asbestosis found in the rate exposed for one da3 was no more flam that found in control rats. Comparing Tables V and V I for 4ho- rats which completed their exposure, progression had occurred be tween the end of exposure and death witli If dust.-*, tho single exception being the
~tj months' exposure of Rhodesian elirw* tile The r.its which died before com
plofiiig 21 months' exposure had man ushcsfosis than those .sacrificed aftei IN months' exposure for amosite, until'* phvllite and Rhodesian ehrysotilc. The
was not the ease for croeiilolife ami
Canadian ehrvsotilo, for which tluw: rat that died during exposure had siaaM mean survivals than for the other <l*eiMeanni over all dusts, those, rats that
0 )e d<
l,i e< hi
an 'J'a
r ,1 , i, T we no o f.
Tu,
tlie nun vim
.if V
lum. 1 out
I >i >M
e\pi Min
to I,
s<`hi`*
it it
T a h i, K V I .-- ( Mro tt
Me an
A sites!onia Sof Survivor#
S tu vivaiin
L r f f j ' t h 1
1 it i .y 3 in o n ib s U ttm n tlw 12 tlM M tlb s U p to 24 m on t Ih 24 m o n th s
A m * -in
1 2 * !) :iii 4 *S fi*0
(20) (2.*>) (4 )
ti ) (2 b
0 : 1 (-' )
A iilh o p h y ilitv
i a a 2 4 2 ii-ll O
(20) (27)
(20) (2-7) (22)
7 0 (2S)
( Y tr fl4 * iilu
1 -2 :t-1 :t 2 .(> 4
(2) (27) (24) <2f>) (14)
U -G (20)
(('itiiH ilm iiJ
1 -2 :> H -7 8 1 r>- i
(2.7) (2) (20) (*.) (io )
< * h ,v s o l ih* ( H I i . m I1* HUtil
1 4 2 -K 4 -2 1 o l
( 2 :* ) (2S)
(23) (27) (22)
0*8 m
* ltui, 2: ruminiti!, 4: sitato* ti: modrait*, S w x n v .
died during exposure hud slightly more asbestosis than would be expected from the sacrifice rats, consistent with the more severely affected animals having the shorter survivals. However, the effect was very slight and, as observed earlier, the exposure did not affect survival to any extent. In Table V I there is again less asbestosis for amosite than the other dusts although the difference is not as large as in Table V. For rats which completed their exposure, the difference between amosite and the oilier 4 asbestos t\ pcs had a mean of 0-7 for rats sacrificed and 0*5 for survivors. The results in Table V I do not support the findings in Table V that anthophyllite and ('auadian chrvsotilc produce more asbestosis than <mcidolite and Rhodesian ehrysotile, atwl \u* conclude, therefore, that there were no important differences in the amount of asbestosis produced by these 4 samples
Tumour# of the httitj
hung tumours were observed in 247 of tlit* rats exposed to asbestos. The. total numbers of each kind for each dust are damn in Table V II, where for those r.its with more than one tumour of the luuir, classification is by the more severe onulitton. No tumours of the lung were *.Wrved within 300 days of the start of xposure and therefore only rats which arrived this initial period arc considered m have been at risk. Apart from the cladulod killings, only 13 rats died within the first 300 days. There were 7
control rats out of 84 survivors in txpri ment I with adenomata, but in Experi ment 2 there were no lung tumours out of 42 control rats. There were slightly more male than female rats with tumours -- 128 compared with Hi)--but the only 2 tumour types for which there was any major difference between the sexes were
adenocarcinoma and squamous carcin oma. Out of 50 ndenoearcinomuta, 3 occurred in males whereas 30 of the 40
squamous carcinomata were in females Mtastases occurred in 20 rats, 10 of each sex. There wore also II meso thelioma la (Table VII), 7 in males. Two
of the mesotheliomata occurred with only one day's exposure, 1with 3 months',
none with 0 months', 0 with 12 months'
and 2 with 24 months'. The meso
thelioma which occurred with 3 months'
exposure to crocklolite was a peritoneal
tumour; the others were all of pleural origin.
The distribution of the lung tumours with time after first exposure are shown in Fig. 11 for all dusts and all lengths of exposure except one day. In the. 5 groups exposed to asbestos for one day there were 14 adenomata ami, compared
with the 4 in the corresponding controls, there was clearly no evidence that these adenomata were a consequence of expo
sure to asbestos There were 5 more serious tumours; 2 of these were meso-
thcliomata, one with amosite after 715 days and the other with vroddoiile after 551 days. There were also 3 adenocarchumuitu, one with crocidolite after
2(i4
,. (. WAG N K R, <. JtK K K Y , .1. W. SKiDMORK A N D V . TJ MH UE LL
T a u le
lOvjuwuiV' Amostfa
dny 8 mont l
( month* 12 mont hs 4 months
Total
V U . -- X u m k r r o f A n im a is with L u n g T u m o u r s or M esothelioni a la
rats al
risk*
Iutl{ tumour
Typo of lung tumour
f-------- "---
Ailonoma A<lc*tvfiiiitciMv
A<lcno* ruremomat
iSnuumous caromomaf
iW , w1111
moo*
lioiiorufl
4fi
3
8
0
0
87
SO
7
8
18
2
0
i
25
JO
r>
4
1
21
13
8
i
3
I4(i
38
to
8
5
0
1
0
fi
0
0
0
fi
fi
0
fi
i
A Hthophijlliic
1 day
44
2
2
a
0
0
0
8 months
a 7
6
0
0
0
0
6 months
18
(i
3
1
J
1
0
I months
2
20
fi
<)
4(1)
1
\
24 months
IH
1(5
2
r*
3
fi
Total
145
ro
22
12
8 (1)
8
2
Crondolih
1 day
4:i
0
5
0
3 mont h
:u
14
JO
2
(> months
1S
4
2
2
i 2 muni Jim
20
IS
5
4
24 months
IH
18
4
a
Total
141
a a
2<>
18
1 1
0
3
2 (1)
7 (1)
0
l
t G)
0
0
fi
a
2 (1)
0
fi (2)
4
Vhr^ohU*
(('a)i<uh<in)
day
42
i
u
*1moni hs
:(4
IH
in
0 month
17
f>
2
12 months
21
1 I
1
24 mont )w
21
10
2
Total
137
4*
20
0
1 U)
(1
0
0
3
0
0
2
0
1
0
8
<> (l)
l (1)
:i
3
1 (I)
4 (2)
l
K
11 (3)
(S)
4
( `hitf&ofttr
(tih&h som)
] <lav
4
4
8 mont lis
II#
Iti
H
0
2
0 moni h*
10
8
a
8
12 mont lis
27
Hi
*>
4
24 months
n
n
0
l
Ti >!nl
144
.V)
10
io
t'oohl
\ &*\\
44
4
4
O
* month>
h
8
4
0
f 24 inontlw
42
o
0
O
Toi al
12<>
7
7
0
* Hat^ \\ltu`li Niifnhns ht him
ni 1o rtliil ia\s jiilm ^J;tiri o o\|niMiif' no* tl)*""' with m< i ^t
1
a (I) 3 (l) 7 (2j
C2>
lt (fi)
o 0 0 0
0
n
(1
(i
0
0
fi (4)
0
0
0
li (4)
li
0
0
0
u
1)
0
"0
0
.SO" t<i\ s , a n o th e r w'Ui R h o d e siati ehrvsotile after 7 IL da,vs and one wliieh m etastasized with ('anadian ehrysotile
after SUS dav?h In in te rp re tin g T a L le V i l and h'ig I I ,
il has t<> Lu Lni no in m im i (h a i tlioro \v:us a greater tendency for rats to develop
m le n o in a ta ni i-:x p crim o n t l than in U xiM M im eni *2, as show n ut the controls. T h e re fo re , the higher proportion o f am-
tun 1m w ith a d e n o m a ta a fte r 3 months e x p o s u re th a n u tte r (> m o n th s' ex p o m i li is p ro lu d ile Ut a ri e fa et. \Ve do noi know wl\ a d e n o m a ta occurred in the
i iKHn
3 MONTHS
AMOSnt
K F F F O T S O F IN H A L A T IO N O F A.SHKNTOS IN RATS
6 MONTHS
12 MONTHS
0
no >000
ANTHORHYUIT
n i
L L B -O L ,--------
500
750
WOO
CROCIOOUTE
n n i % I------L
loo no kx
nr
500
750
>000
500 710 WOO
>00 750
J3
WOO
CHRYSOTItT Canodtan
500
750
WOO
CHRYSOTllE Rbodeiton
-a. ) ;>o kw
-H V ,750 WOO
4 ~mr ei,,
500
750
00
8 S<$uamovi carcinoma
SB Adenocarcinoma
0 Adenoma / odenomafo**
O Other couiet
l` K>. II.- J)<u ilmiton <1 m iu u rl times m tlil\'> alter- lust <-\po-uiii
controls in one experiment and not in the other but as the finding is significant {/* = 0-Oii) in its own right and is sup ported by the results from the exposed animals, it is unlikely to be due to chance.
There was a higher incidence o f tumours with 12 months' exposure than *ith 0 months' but little difference between the 12 and 24 months' exposure.
Half of the 8 mcsotheliomata in
K\jH,*riincnt 2 occurred with i 'anadian thry-utile, so that in total crocidolite and
Can adian chrysotile produee<l 4 mesotheliom ata eaeh (){ the 2U tum ours which m etastasized, JO were after e x p o sure to a ( hrvsolile ( lo with the Rhodesian sam ple ami 0 with the Canadian). Throe others were with eroeidolile and one with anthophyllite. Two o f the mesothehom ata in the 12-month groups occur red w ithin 400 d ays after first exposure, one w ith crocidolite after 800 d ays and one. with Can ad ian chrysotile ifter 8o.> days (the only rat which failed to survive for its scheduled 12 m on ths' exposure)
$ '1 * f: * *s
2(56
.}. <!. WAONKR, ft. i'.KKKY, J . W. SKIDM ORE A M ) V. TLMliRKM,
A $!)?.$(ox i s a m i lu n g Im m h u m
An analysis was carried out to deter mine whether there was any relationship between the grade of asbestosis and the peesenco, o f lung tumours. Since the asbestosis grade, depends on survival, it
was necessary to standardize to a constantsurvival time. This was achieved by calculating the regression coefficients o f asbestosis grade on survival time for rats without Jung tumours, exposed for 3 months or more and with survival o f at least 4-00 flays after first exposure. Differences in these, coefficients between the 5 types of asbestos and the 4 lengths of exposure were not significant and the pooled coefficient o f 0*00304 ;J-; Q*000fio grade units per day was used. The asbestosis grade of each rat was then adjusted using this slope to an arbitrary s u n iv a l. The adjusted mean asbestosis grades were then calculated for each o f t he 2f> groups, for those with and without
lung tumours In 15 of these, groups the mean asbestosis grade was higher in the anim als with lung tum ours: in the other 5 groups the opposite occurred but only slightly so in 4 eases. The accuracy o f an estimate o f the difference in asbestosis between those will and those without a tumour is dependent on the number o f animals in each category which varies between groups, and weighting each group to take account, of this gave a mean difference o f 0*71 ; 0*13. Hence overall the animals with lung tumours had significantly (/* *: o-oo)) more asbestosis than those without Dilfereue.es between the dusts were not significant but the wide range m means - anthophxHite o 25, Canadian elnysotile 0*42, erocidnlite 0*00, amosde u-K5 a m i lUmdesiau ehiysolile 1*22 - shows that there are insufficient data to reach any firm conclusions on this question.
The. groups exposed for only one day provide supporting evidence of a relation ship between lung tumours and asbestosis T h cie was vor\ little asbestosis in these groups {'fable VI) and lestiicting atten tion to animals which survived for at
least 000 days, the mean survival times
o f those with and without lung tumours were, very similar. There were. 17 lung tumours in 201 rats; only 0 of these occurred in 157 rats without asbestosis
while 11 occurred in the 44 rats with minimal or slight asbestosis (23%). a highly significant, difference (PcO-OOf)
Tumours of
silosother (firm lung
A total o f 412 tumours, other than
lung tumours or mesothelioma o f the pleura or peritoneum, were observed.
The m ajority o f these were adenomata of the breast or pituitary adenomata, which were common post-mortem find ings. Both o f these adenomata occurred 4 times as frequently in females as in males. The numbers of these tumours, other benign tumours and malignant tumours for each type o f asbestos are
shown in Table V I I I . Kor none of tin*
T/UitK V III.- -A
1mulicrTumours al >Sil< Ofh e r ( h n n
s _Hrni,,ign i iim o u i . %
INt uit
lim a
JJlD lW t
try O th e r tum o ui-
A mi h<>|>h\ liit*
40
4
:is
4
in
{ Hnruloht*
0
Hi
4
10
( Hu \ il<*
SA
Jh
7
7
(( a n a ilm ii)
( `hi \
10
H 7
0
a
M.4U}
<\ m l rot
20
.14
2
i:i
tumour typ<*s was the difiercnco between the control and the asbestos treated significant. In 'fable IX more detail igiven o f the sites o f fbe tumours with all l\pes o f asbestos combined The large**!
differences between the treat oil and con trol rats were for tumours of the ovaiv, 10 in treated and none in controls, ami tumours of male genito-m iuary organs, 11 m I rented and none in c o n t r o l s . However,
noil her difference was significant A few rats had multiple maligna it
t umours. 2 o f the rats with mesothelioma o f the p in n a also had a lung carcinoma and one rat with a squamous carcinoma
U inani
rlimn ` Hi1'in i mom.*
KFFKiTiS OP INHALATION OF ASUKSTOS IN HATS
2 In
T aklk IX . Site
' of TiUiumns Other t h o u L m u /
A-Itfwlris lli.lfcil
('ntllidl
Njtf/Tuinoui tyjjt
Miitljii
mid
4
\
Hoiim iuh! dvin
;i
4
n
2
Hivnsl
H HI
h
1*0
1
Ovury
:<
7
IWmih*
mmy ot^mm
a
K
o
o
4
Mid* 'iitiMJMhury *m
4
S
o
O
Intiootmiuil
174
t
f
Thymoma
7
\
0
l
Lymplmuui l<nkat*NiM
X
CMIiim*'*
3
A
f
u
1liyroiii in* hiiMiimm
mln'iiid f4) nalivary dund
I hvrowl
of the lung ha<! a mesothelioma tunica vaginalis. Mesotheliomafcn of the vagin alis were seen in this and one oilier rat; there is no evidence to suggest an associa tion with exposure to asbestos. Also, in some cases there were secondaries, for example, a synovioma had spread to the lung. In all such eases tumours have lnvn classified by the primary site and in the few eases of multiple malignant tumours there was no difficulty in recog nizing the distinct tyjics, t>. one was not a secondary of the other.
D IM T'SSlO N
Our finding that the ashestosis produml by exposure progressed after eessatit'ii of exposure is in agicement with human experience but contrasts with the early inhalation experiments reported 1>\ Yonvald, Durkau and Pratt (1951) m which progression did not occur W.iLMier (1903) report oil more ashestosis \wih amosite than with ehrysotile in guinea-pigs, rats and monkeys but our
ivjiciiments show' that, of the UH'C tamlard reference samples .unosite is the 1<ms| fibrogeuic in rats.
(iross et uf. (1907) found lung cancers :i of 72 rats which survived Hi months' xpiMirc to eluysotih* duvt at a mean mm cut rat ion of 30 mjr/m3 for 30 hours v week They eoiisuleml th.d eontumit nion of tin* asbestos bv trace metals
from the worn hammer o f the mill used
to produce the respirable fibre could
have been a factor in the causation of
the tumours. However, our results now
show that there is no need to invoke
such a Its pothesis to explain the high
rate of lung tuiimiih
The amount of ehiysotde retained in
the lungs did not show any clear increase
with dose in rals exposed for longer
than 3 months. In two earlier expet i-
ments (Wagner and Skidmore. 1905,
Morris
NuL, 1907) a higher airborne
dust (.oneentration was used to give a
cumulative dose in 0 weeks similar to
that given n> the picsenl experiment
over 3 months The weight of asbestos
found in the lungs of tats exposed to
amphihole was 3 times greater than in
those exposed to ehrvsotile. In the
present experiments the ratio was (> to I
alter 3 months, hut increased with
continuing exposure as the weight of
amphihole in the lungs continued to
increase, hut the amount of ehrvsotile
did not The previous experiments had
shown that the rate of elimination of
dust from the lungs was much greater
for ehrysotile than for tin* amphiholes.
The present results mav be explained on
this basis, the weight of ehiysotde having
reached cquthhtium level, i.e. the rate of
elimination equalling the late of retention.
There are a number of features of the
results presented above uhih we found
'-9 S
.i. r . W VSXK!. ISKRUW .1. W. SK1DMOKK AND V. TfMItKKM*
1
surprising. First, in Kxpeiimcnl. I two of sites other than the lung is equivocal.
mcsol hehomata ociuued wit It (ho one Although an association with gastro
el 11 \
day c.\|h>suc
(ompare,I withinteosntliynalontenniouis has been touml epi*
%
f `.1 it
with ilir 3 months' exposuie, which had .1 demiologicallv. it is not yet tegarded as
w III.
dosage nene (han .10 timos greater. eleaih established (Selikoll. Hammond
the
II the incidence oftnesnl heliomata was and Clung, 1972, New house, 1974), and
l
j
J>l< a
* |
proportional to lose-, .is is nuheated for is of lower magnitude than the excess
1
tin iiio* hIkI in o\pt'iimonls (Warner c/ lung 1.meet* risk Our experiments pro
til., 1973), then the prohahditv of such vide no suppoit for such an association
\
j >(
an extremo result occurring liy chance The expeiimeulal um k o f Craham and
(Ins
would he about 2 in a Inon
Craham (19(57) suggested that jntra-
t
as ,,
Secondly, there was no evidence o f peritoneal injection <d' tremolile asbestos
the I
I either less carcinogenicity or lessasbestosis could pio<luce ovarian tumours, but the
in the groups exposed to chrysolite than follow-up of women asbestos workers i L
those exposed to the amphiholes, even reported by New house fl a! (1972) pro
w ho
though the amounts o f dust in the hums duced no definite conclusions on this
were so different In particular, (he question because ol the rarity of (lie
(Oils,
U K 'C Canadian ehrysotile piodueed as tiiimiiii Our experiments do give some
i
m <
many meant heliomata as the l Tl(*C eroci- support to an association between asbestos
woof.
it
dolile. The 2 I' K V samples of ehrysotile exposure and ovarian tumours ns well
f
to h
l!
produced 12 of' the 14 tumours with as tumours of the male gcnito-uriiiary
f.iiii. el.issji
Sfc metastases. However, much less lust s\ stem Although neither was significant l grater
was retained in the lungs o iats exposed this <mild be bccnu.sc o f the relatively
Walt,
to chiysotilc than amphiholes (Fig. 10) small si'/.e of the control group. To
1him ,
Moreover, after intrapleural inoculation oveteomc (Ins, u <' have included the
<` Ypei
the risk o f a mesothelioma occurring condo! rats from some of our oilier
<ouip >
with l'IC C cioeidohte is 3 times tin* risk experiments, thus increasing the nun
S
with chiysotilc (Wagner <f <tl., 1973). tieated group to 403 rats of the .sunn
Theicfore, allowing for the greater reten strain This larger group contained
S
tion of' croculolite after inhalation, we malignant Honours of the ovary ami .1 f
(.1 Ml s U.Ml
might have expected the risk with '-roei- turnouts of (ho genito-urmary tract in
/ ,,
dohte to have been of th<` ordei of 2U males. In over 700 rats exposed t
**1 M
times that of <hiysotile
asbestos there weie lo ovaiian turnout*. *
Two of the mesotheliomata occurred 7 of which wen* malignant, and II
(X ,s.
within 4<K) days of the stait of exposuie tumours o f the genii urinary tract 111
*M< f|1
This may be compared with our injection males Hence, based on the, laigcr *t
* Hilt . * lit V
experiments in winch only -0 out. of >*<)3 of controls, the association between ad* *
1^ n
occurred within 400 day's (Wagner and to.s exposuie and ovarian tumours 1* ,
* 1,H\.
Berry, 1909: Wagner
el <tl, 1973w) eakAlasnod, non-significant, whereas then* 1* V
His
the earliest mesothelioma occurred after nosuppoit for an association with tumour*
1 Imi
*< lf\,
3.1.1 days ami we observed only 3 within of the male genito-urmary system
*-1*0 ll.
this period in our inject ion experiments.
The U K V ehrysotile samples are law
>e !>
The positive association between ax- Ilian tlic ehrysotile which has been in i
>: fd|j J
hestosis and lung turnouts which we have m industry m (In* past. However, tlum
M i,
established in the animals 1., in agreement is a trend for imlusliy to use liner Huy
>
with epidemiological findings j/. Minister sotde (Wright-, I!M59) and so the oxpeu
S` tDs ^ M mi i
ol Labour and National Service, 1949, mental results may he more relevant (>
f I'll.
Knox ft al , 190S: Limes and Simpson, the cunent situation than to flic |u*t
^ */ fu t t
M\|m 1
1971)
We are investigating the effects of I 111 1.111
Mm.. ;
The failure to c-tablKh any association lion ol chiysol'de m mote detail in
between asbestos exposure and tumours expei iment involving U K 'C Canadiet
K K FK ( r x U P i M l \l. \ T l O X 1)1' AxlSKST is | \' II Vi's
2iil
chisot lie ,i goule 7 sample from a Camidian mow* and (he supcilme sample wiiieh p r o x od (he m o st e.neinogeuto ui
i * W . ( I *1(17) ( umpui i **<*n 1 Du'st Ua< m ton in
SjMfMtH' t*atho^tn t*'< t ind Stu*kud Hat^
In /nf/afoii I 'urfttfts 4nf*i t
// l*!d (* X
Davit
O \ io id Hotgumon p 0,7
the materials xvlmh xxe morul.ded intrajtfeuraliy (Wagnei t i n ! , I!*7Ti)
'I'he experiments we teport base gixen
X t'W o o i si , M \4 { f 074) ( a n m Am ong W o ik o ts
in (lit \^}n/-Hto- T\iil< liuJtfNtiv* In (iioftuftt td
of , I x/ sft** i A on* 2 A ()<*l/>fni li)?2
Ivf I* Hogiv^ki%J . (*
\' Tnnlni'U and
results whieh in s< vet.d respects (or lespotul to those found tn man Thus,
4 * * VV 1^1M I K), In pnnl N iu iu n xi\ M
(| \ H( vK< t* tit iflf Pu
'dlix, Vi#
!, , M m u;v. C . W \ :n i :h , .1,
tV
this evpctimental method is established
*I*t h iik , M K (IU72) A ^fndv ( Hu* Morfahty
as a vili<l tool
U mthe i n v eos( tFioimoani* ol
\Viki^* />/*. / /###/
29. 1 N
the biological effects of asbestos
S l t l h d l I*, \ .1* I I vMMOMi* IX C <V (*Mi i{(,, .)
{ I!>7J) i dtemogh tiiriiy *i` Anto'sii Vsln^fos,
, I k /u ttntt, fllflt, 25, 1st
We are grateful to all our colleagues
L M A F\ i i \ , L X ( fM7n) Motphtikijrical in Hat Lung' I n d u e d H\ HoHcycliO
who over a number of xear.s were res ponsible for the <Lni\ atten tion neecssai y m carrying out the experiments. \\V
H\ tluHMi lois In Mot jdtttlotf*/ o f h t t*n nneulul ft* sjotafto *j ( t<rt tnotft m st * Pint ( * o n f f (iatim I'tog* K> H> M a \ , H470 Kil. I* X V M o s h m n , M D H a n n a , J i tin! -I W l)t <itIn tago J r .
would also like to aekuoxx ledge our than ks to Dr Harold Stew art of the N atio nal
l f.S Atomic S r u o s X't* 21 iS r w r o v , M l*`
KiM*iLiv DofuuuvM<n, XympoKoim p. 227
(1*171) In I'udtolotftf of lUttuoutn to
Cancer Institute who advised us on the classification ol' turnouts Wo are also jjwiteful to our former colleague. Dr A .
L fth nn tfot// I mtWitss \f*4 1 M'uiocmOh of the
Rat !\ut 2 l A H <* vnrnlilM* ISddicm No. In Lyon lot* *nat 101191I Vgrurv fu R**^uiroh (7nu*<*r In pi 'para tmu
Walter, xvho laid classified the non-lung tumours occurring m some of our earlier experiments which xxe mentioned for
'j'tM H IiK lI, V . , ( i l l s o v , .} ( ` A W l 'O M K K , I 1 HUS)
L H H 1 Statolai! lodinoucr \t|ii|il*x ol Asia
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