Document zo3e33e1OoOxMb6yx9OZ699xz
FILE NAME State of the Art Literature SAL
DATE 1957
DOC SAL076 DOCUMENT DESCRIPTION Journal Article - The Effects of the Inhalation of Asbestos in Rats
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Br J. Cancer 1974 29 252
THE EFFECTS OF THE INHALATION OF ASBESTOS IN RATS
J. WAGNER G. BERRY W. SKIDMORE and V. TIMBRELL
From the Medioni Research Council's Pneumoconiosis Unit Llantough Hospital Penarth Glamorgan
.
Received 5 September 1973 Accepted 29 November 1973
Summary experiments in which SPF Wistar rats were exposed by inhalation
to dust clouds of the UICC standard reference samples for periods of between on
day and 2 years are described All the samples of asbestos produced asbestosi which continued to progress after removal from exposure but only a little fibrosi
was observed in control rats Lung tumours ranging in severity from adenomata to squamous carcinomata were produced by all 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 11 mesotheliomata occurred 4 of which were with
crocidolite and 4 with Canadian chrysotile Two of the mesotheliomata occurred
only with
one day's exposure to asbestos
asbestosis and lung tumours
There was a positive association between
.
WAGNER Berry and Timbrell 1973
The asbestos samples used consisted of
reported the results of experiments in the 5 UICC standard reference samples
e
which rats were inoculated intrapleurally Timbrell Gilson and Webster 1968 which
with samples of asbestos In the discussion it was mentioned that two experiments in which rats had been exposed
e to dust clouds of the reference
samples had been carried out and the results of these are now presented Some preliminary results of one of these experi-
re. ments were reported by Wagner 1972
were prepared following recommendations of the UICC These samples are of 3 amphibole amosite anthophyllite and crucidos and 2 chrysotiles Canadian and an Rhodesian sample
The rats were exposed in 4 minhalation chambers Timbrell et al 1970 which
contained 8 cages each of which could hold
6 rats or for a short period with young
In both experiments the rats were rats there was room for a seventh Pive
exposed to similar dust concentrations chambers were used one for each type of
and the dose varied by exposing rats for asbestos The chambers were constructed
various lengths of time The main aimi so that the rats could be tended without the
was to establish the relationship between _ chambers being opened
a.
the development of malignant tumours
The dust clouds were generated using a
specially devised dispenser Timbrell Hyett
in the lungs and the dose and type of and Skidmore 1968 The clouds were
asbestos dust but additionally the generated for 7 hours a day and 5 days per
amount of fibrosis was assessed
week The respirable dust concentrations
MATERIALS AND METHODS
Caesarean derived rats of the Wistar strain were used which had been bred at
the Unit from SPF stocks given to us by the Imperial Chemical Industries Pharmaceutical Division at Alderley Edge Cheshire
in 1964 and 1968
were measured using size selective gravimetric dust samples Cassella Type 114A The collected samples were evaluated at the end of each daily session In order to achieve the required dosage calculated as the product of concentration and time
variations occurring in the concentrations
were corrected on the following days At
the end of exposure the rats were left in the
chambers for a few days to allow time for
;
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ert Ung
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ite PALO al ar ere
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EFFECTS OF INHALATION OF ASBESTOS IN RATS
253
their fur to become clear of asbestos before
transferring them to a clean environment
for the remainder of their lives At transfer
Sections of both lungs were examined without knowledge of the duration or type of asbestos
exposure and with the animals in rando~-n
some rats generally 2 or 3 of each sex order The sections were observed on 1
were killed and the lungs histological examination and
of dust content
removed for determination
viewing screen of a Projectina Microscope 4013 BK using a ^ objective At this magnification a large proportion of the lung
As each type of ashestos has a considerable silicate content about 50 the amount
could be assessed in a single field and as it was not possible to observe asbestos fibres
of dust in the lungs was determined by first Assessing its silica content and referring this value to that of the respirable dust to which
at this magnification the results were not biased by knowing the type of asbestos to which the animal had been exposed
the rat had been exposed This method
was used in previous experiments Morris et
al 1967
_
After exposure the rats were caged in threes or fours isolated in a special unit supplied with filtered air The inhalation
chambers were also in this unit but in a
Experiments
In both experiments and for all doses there were groups which were exposed to all 5 reference samples there were also control groups which were not exposed
woe separate
prietary
room
brand
They were fed on a pro-
of autoclaved cubes and
Rats were allocated to treatinents at random
At the start of the exposure the inajority
water ad libitum Except for the scheduled of the rats were between 5 and 7 weeks old
killings each rat was allowed to live until a few being slightly older or younger and
it died or appeared to be distressed and a there were approximately equal numbers of
full necropsy examination was carried out
males and females
.
Histological preparation staining and
Experiment There were 2 time inter-
microscopic methods 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 following exsanguination and in the second groups of rats were
the thorax opened and the lungs removed exposed for 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 right lung were taken for electron microscopic
exposure in the month group there were also intermediate sacrifices at 5 8 and 10
examination and the remainder of the lung
dilated with neutral buffered formalin Slices
weeks
were
Experiment There were 3 time inter
were taken from both lungs for histological examination After the histological sections
had been cut the embedded tissue and the
vals of exposure months 12 months and 24 months The experiment started in January 1969 and after 6 months half of
o
trimmings were dewaxed and added to the the rats were removed from the cabinets
:
remainder of the lungs which were used for They were replaced and a year later these
the chemical estimates
replacement animals were in turn removed
For other animals at post mortem the They were replaced by animals to be used
lungs were dilated with neutral buffered
formalin and after fixation were sliced
for special electron microscopy examinations which will not be reported in this paper
sagittally routine sections being taken of the whole left lung and the upper and lower lobes of the right lung In addition any
The remaining animals were removed in January 1971 after 2 years exposure In
the month groups in addition to the
other suspicious lesions from the lungs or other organs were taken for histological
killings at the end of exposure rats were
also killed after 2 years i.e. 18 months
examination
after removal from exposure
In all cases sections were stained with
The numbers of rats are given in Table I.
haematoxylin and eosin and the lung sections
were stained for elastin reticulin and col-
In the 24.month groups overcrowding
amongst the males tended to occur due to
}
lagen Special stains were used in some increase in size after about a year and
cases as an aid to diagnosis of the tumours
some were removed prematurely after 13
|
Assessment of the severity of asbestosis.- months For analysis these rats have been
.
*
.!
ne
Fto Slight asbestosis Grade 4 Thickening of the walls of the alveoli arising directly from
the respiratory bromchioles with replacement of epithelium by type cells H. & E.E.
oeas an % oo hLy ig
S
-"/-
Fig Slight asbestosis Grade ) Higher in the alveolt of a rosquiratory bronchiole
tibres
Illumination reduced to illustrate the tibres
power showing showing nuit^-erous refinettle erveidoute ibres
Asbestos fundies new
500
H. &E. E.
< 500
murely
seen
the lungs of tits
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236
C. WAGNER WAGNER G. BERRY J. W. SKIDMORE AND V. TIMBRELL
Od
4
+2
mend
:
ee eanpee?
vragen Cie PA eA LOS
54t.at
7
sty
LA
MS
lower power jae Fig Moderate asbestosis
AY
OS, 1
although still
Grado 6 A lower power projection illustrating that the desput
mainly involving the respiratory bronchiole is now diffuse
v7
pneumbcytes are seen in the lumina as well as
Aggregations of type 11
investing the walls H. & E. ^ 80
ae
ectean
bell dedh aD
5S
St
rare v
we
2
Fic Severe Severe asbestosis Grade 8 There is a generalized interstitial fibrosis H. & E.
SU
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EFFECTS OF INHALATION OF ASBESTOS IN RATS
257
diffuse interstitial fibrosis with gradual increase in the density of the fibrous tissue ultimately resulting in the replacement of most of the lung parenchyma by a dense collagen network surrounding distorted air spaces many of which contain large clumps of granular pneumocytes which in some
areas have lysed leaving foci of alveolar
proteinosis
The assessment of these lesions WAS
based on + grades of minimal slight moderate and severe Typical examples of slight moderate and severe asbestosis are shown in Fig 14. In ackli tion when carrying out the assessment it
was found convenient to introduce the
adenomatosis In contrast to this a few
control animals were seen to have solitary
adenomata which were small in size The
adenocarcinomata were of the same type
and origin as the alveolar adenocarcino mata described by Shabad and Pyley 1970 many of these tumours were papillary
carcinomata The squamous car
cinomata appeared to originate from foci of squamous metaplasia occurring in the asbestotic lesions in the respiratory bronchioles Fig 7. 8 As far as can be ascertained all these tumours were peripheral in origin and not bronchial papillomata
The classification of these tumours was
discussed in some detail in Session VI at
intermediate categories minimal slight slight the Gatlinburg Conference on the Morphology
moderate and moderate The 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 IARC Monograph on Pathology
asbestosis was scored 1 and the assessments of Tumours in Laboratory Animals contains
were averaged for each group of rats The detailed descriptions of the tumours that we
repeatability of this form of assessment was have illustrated
tested on 154 sections reassessed in a
The rats used in this experiment are
=
different random order and 90" were from a caesarvan derived barrier maintained
~
assigned to within one category of the first colony and fortunately they have been kept
reading
free of rat bronchitis therefore the squamous
Classification of tumours The tumours metaplasia was not associated with bronchi
found in the lungs of rats after exposure to ectasis
the various types of ashest^s dusts were
peripheral adenomata widespread adeno-
Metustases in the thoracic cavity to the chest wall diaphragm pericardium or the
matosis adenocarcinomata and squamous bronchial lymph glands were seen
carcinomata The rarity of pulmonary tu in 14 animals the majority had lesions
mours in rats has been stressed in the invading 3 of the sites and in only 2 animals
reviews by Kuschner and Laskin 1970 and Shabad and Pyler 1970 Further these authors have described the development and
were metastases observed in the tracheo-
bronchial glands In one animal secondary deposits were seen in sections from a kidney
morphological features of the tumours that
we are reporting All these tumours were peripheral and appeared to arise from the region of the respiratory bronchioles in which
Eight adenocarcinomata and 6 squamous tumours had metastasized
RESULTS
the asbestos fibre had accumulated
The origin of the adenomata appeared to be from accumulation of type if epithelial
cells that proliferated in the alveoli of the respiratory bronchioles Fig 5. 6 In many
of the exposed animals these tumours were multiple and in a number of animals
particularly those with the more grades of asbestosis there seemed
severe
to be
All except 2 of the rats in the groups with exposure of 12 months or less survived for the whole of their planned
exposure In the month group there
was appreciable mortality before the end
of exposure and only 53 survived for the full period Out of 1013 rats it was impossible to obtain adequate histological
adenomata arising from numerous adjoining material in only 8 because of cannibalism
respiratory bronchioles giving an impression
of contiguous adenomata invading large Dust retention
areas of the lung Dr Harold Stewart
personal communication suggested that
The mean weights of asbestos dust
.. this type of lesion should be referred to as in the lungs of animals killed at the
Bah
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ie ve ee
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wer fe
Fio Puquillary adenoma H. & E. ^ 320
he
ane
%
tha
ral2
.
[ye
RA
% tk
en
cf
i ficy 9
:
Se
pe Bs
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Fra micrograph of an adenoma showing type [ pneminocytes
^'
Fitt Squatuous metaplasia metaplasia superimposed on moderate asbestosis H. & E.
290
Fu Early squainous carcinoma in un animid with severe asisestasis H.
E. JIXI
me.Ce
ot
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Ob
=
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et denwe ooo
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am hmmm
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ote te
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SET TBINV
260
C. WAGNER G. BERRY J. W.
SKIDMORE AND V. TIMBRELL TIMBRELL
Length of
exposure
5 weeks
8 weeks 10 weeks
3 months 6 months 12 months 24 months 8 months
after 18 months
non exposure
TABLE Dust Retained in Lungs mg
TITTTT TITTTT
TIT T
TITTTT hours
TITTTT TITTTT 3290 TITTTT TITTTT TITTTT TITTTT
TITTTT
TIT T Amosite
TITTTT TITTTT TITTTT TITTTT TITTTT TITTTT TITTTT TITTTT
TITTTT
TITTTT TITTTT TITTTT TITTTT TITTTT TITTTT TITTTT TITTTT
Crocidolite
TITTTT TITTTT TITTTT TITTTT TITTTT TITTTT TITTTT TITTTT
TITTTT
TITTTT
TITTTT TITTTT TITTTT TITTTT
TITTTT TITTTT TITTTT TITTTT
.
TITTTT TITTTT
TITTTT TITTTT TITTTT TITTTT TITTTT TITTTT
TITTTT
TITTTT TITTTT
:
Length of TABLE IVMean Survival after First Exposure days
exposure exposure
Chrysotile Chrysotile dav
3 monthmosnths
6 months
12 months
Ml 24 months
Amosite
804 771 763 692 807
Anthophyllite
806 823 896 759 778
Crocidolite
795 817 788 776 736
Canadian
783 790
778
595
Rhodesian
753 857 766 826
753
Control 803
793
754
*
Adjusted to be independent of sacrifices
me
chrysotile scheduled times are given in Table III the
More dust was
day the rats were first
usually found in males than in females and on average the female
given in Table
have been
IV
The
exposed are survival times
lungs contained only % as much dust
estimated so as to be inde
as the male lungs The values in Table pendent of the sacrifices The short
III are the averages of the male and female
survival of the month group exposed
'
means
For the 3 amphiboles there
to Canadian chrysotile was largely due
a similar pattern with an almost was to 8 rats dying before Day 400 in the
portional
increase
of lung dust
prowith
close The 2 chrysotiles were similar
other four month
2
died before
Day 400
groups These
only
rats
early deaths
one
another
but
much
less
dust
to
was
were not due to exposure since 5 died
due to an infection in
found than with the amphiboles also the were killed in
one cage and 2
chrysotile figures did not show the 8
a fight Discounting these
clear increase with dose
same
deaths the mean survival was 698
features are summarized in The main days which was still the lowest of the
dust in the lungs of the Fig 9. The month groups When the
.
animals which had 6 months exposure had been
partially
taken over all the lengths the Canadian chrysotile
mean was
of exposure
eliminated 18 months after removal from least
groups showed
exposure The proportions eliminated were 74 for amosite % for crocidolite
than
survival but only a month less
the control groups
The amosite
but only 41 for anthophyllite How- and anthophyllite groups had mean sur-
ever the lower elimination of antho-
vivals only a few days less than the
controls while the crocidolite
phyllite was not significantly different
from the
desian
and Rho-
amosite and crocidolite figures
groups had longer sur-
vivals of 2 and 3 weeks
Hence there is
respectively
very little indication that the exposure had any effect
Survival
on the overall survival of the animals This
The mean lengths of survival from is in marked contrast to our intrapleural
inoculation experiments in which injection
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ak magi te
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15 4
EFFECTS
EFFECTS
EFFECTS
OF
OF
INHALATION
INHALATION
INHALATION
INHALATION
OF
ASBESTOS
ASBESTOS
ASBESTOS
ASBESTOS
IN
IN
RATS
RATS
22661 1
10 4
S Pr.
Amphiboles
Tse Tse
After removal from exposure
C
Chrysotiles
eta)
6 4
Q
TT NSS
Te
Re
mm ene wee ew -0
3
6
12
24 Time months
he L 1
7
T T
T
t
q
10000
20000
30000
Fig 9
Cumulative dose mg hours
Mein weight of dust in lungs of ents in relative ter clusas astad kinne
of asbestos reduces the expectation of life by several months Wagner et al
1973
Asbestosis
The amount of asbestosis was assessed for all the rats killed at scheduled times
in Experiment 2 and after 8 weeks and 3 months exposure in Experiment .
There were 5 or 6 rats per treatment for each exposure except that there were
only 3 after 8 weeks Overall the 2
sexes had similar amounts of asbestosis
and they have therefore been combined to give the mean asbestosis scores in Table V which are summarized in Fig 10 Except for some inconsistency between
the 3- and month means there was an increase of asbestosis with exposure for
all the dusts Also following 6 months
exposure there was progression during
the following 18 months without exposure for all the asbestos types but these rats did not fare as badly as those which continued exposure There were signi-
ficant differences between the asbestos
types P < 0.01 amosite invariably gave the least asbestosis throughout anthophyllite and Canadian chrysotile showed most asbestosis after 6 months or longer exposure crocidolite and Rhodesian chry-
sotile were intermediate
The mean asbestosis scores of the rats
which were allowed to live out their
lives are given in Table VI Those rats
=
~
yT: maseigite
ey Ee
%
Sere kag eee Fe SARS
ge rad
Satna
y
.
po bee rer fg EX
Pa _;
at
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ea
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cae
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tate
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waste a
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rae
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trast
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127016
127016i
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127016127016 127016
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C. WAGNER G. BERRY J. W. SKIDMORE AND V. TIMBRELL
Asbestosis grade
Moderate
Slight 4
4
Exposed to
asbestos
. -*
waeet
ne
_-
After removal from exposure
Minimal =
Nil
rc
0
Controls 7-7
ee ee
ee
ee
ee
~~
n ~~?
weet
ee
,
j T
6
i
T
10000
12
,
T
T
20000
24 Time months
1
r
T
*
30000
Frz 10.
Cumulative dose mg hours
Asbestosis in sacrificed nuts in relation to close and time
Length of exposure exposure exposure
8 works works 3 months months 6 months months 12 months months 18 montles montles 24 months
6 months months
after 18 1onths 1onths
TULL ONPOSITIV
V
TABLE .--
Mean
Asbestosis Scores
Asbestosis Scores
Suerificed Suerificed
of Suerificed
Rats
Amosite
eb 265 2:2
4.11
-
AnthopAnthophylite yllite
11 2.7 3.2 3-2
-~
Crocidolite
2.0 2.8 2h 4.3
Chrysotile
Camudinu
2.0 2.7 3.0 +3
ne
Chrysatile Rhodesian
2.7 cheb 2.4
4 63
$e) 1g
H-2 3.0
4
.
tied 5.5
5-8 3.7
1 nil 2 minimal 4 slight fiz monterate 8 sovYTY
Control 1 1.3 1
1.8
:
scheduled for 24 months have been divided
into those that died before completion
of exposure and those that survived for
a period of exposure The amount of asbestosis found in the rats exposed for one day was no more than that found in control rats Comparing Tables V and VI for the rats which completed their exposure progression had occurred be-
tween the end of exposure and death with
all dusts the single exception being the
3 months exposure of Rhodesian chryso
tile The rats which died before com
pleting 24 months exposure had more
asbestosis than those sacrificed after 24 months exposure for amosite antho-
phyllite and Rhodesian chrysotile This
was not the case for crocidolite and
Canadian chrysotile for which those rats that died during exposure had shorter
mean survivals than for the other dusts
Meaned over all dusts those rats that
nia.
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EFFECTS OF INHALATION OF ASBESTOS IN RATS
TABLE VI Mean Asbestosis Scores of Survivors Mean Survival in Months
Length of
exposure
Amosito
Anthophyllite Crocidulite
Chrysotiln Canadian
Chrysatile hodesian
day
months 6 months 12 months
Up to 24
months
1-3 2.9 3.3 4-8 6-0
26 25 24 23 23
1-3 3-2 4.2 6.0 4
26 27 20 25 22
1.2 3.1 3-2 3.6 4.2
26 27 24
25 14
1-2 3.3 3-7 5 5.1
25 26 20 25 16
1.4 2.8 4.2 + BL * Het
23 28 23 27
22
24 months
6-3 28
7.0
6-6 29
_
6.8 28
'
nil 2 minimal 4 slight , di moderate 8 severe
died during exposure had 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
control rats out of 4 survivors in Experiment 1 with adenomata but in Experiment 2 there were no lung tumours out of 42 control rats There were slightly
more male than female rats with tumours
was very slight and as observed earlier -128 compared with but the only
tha
the exposure did not affect survival to 2 tumour types for which there was any
any extent In Table VI there is again major difference between the sexes were
less asbestosis for amosite than the other adenocarcinoma and squamous carein-
dusts although the difference is not as oma Out of 50 adenocarcinomata 35
large as in Table V. For rats which occurred in males whereas 30 of the 40
completed their exposure the difference
between amosite and the other 4 asbestos
squamous carcinomata were in females Metastases occurred in 20 rats 10 of
types had a mean of 0.7 for rats sacrificed
and 0-5 for survivors The results in
each sex There were also 11 meso-
theliomata Table VII 7 in males Two
Table VI do not support the findings in Table V that anthophyllite and Canadian
of the mesotheliomata occurred with
only one day's exposure t with 3 months
chrysotile produce more asbestosis than crocidolite and Rhodesian chrysotile and
none with 6 months 6 with 12 months and 2 with 24 months The meso-
we conclude therefore that there were thelioma which occurred with 3 months
no important differences in the amount of asbestosis produced by these 4 samples
exposure to crocidolite was a peritoneal tumour the others were all of pleural
Tumours of the lung
origin The distribution of the lung tuinours
with time after first exposure are shown
Lung tumours were observed in 247 of in Fig 11 for all dusts and all lengths of
the rats exposed to asbestos The total exposure except one day In the 5
numbers of each kind for each dust are groups exposed to asbestos for one day
shown in Table VII where for those there were 14 adenomata and compared
rats with more than one tumour of the with the 4 in the corresponding controls
lung classification is by the more severe condition No tumours of the lung were
there was clearly no evidence that these
adenomata were a consequence of expo-
observed within 300 days of the start of sure to asbestos There were 5 more
exposure and therefore only rats which serious tumours 2 of these were meso-
survived this initial period are considered theliomata one with amosite after 715
'
to have been at risk Apart from the days and the other with crocidolite after
i
scheduled killings only 13 rats died 551 days There were also 3 adeno-
| within the first 300 days There were 7 carcinomata one with crocidolite after
1 20
'
i
PTE
Lee
I eeri bae nal
heAne
nett ee baOEE
264
J. WAGNER G.
BERRY J. W. SKIDMORE AND V. TIMBRELL
TABLE VII Number of Animals with
Lung Tumours or MesotheliomataMesotheliomata
Exposure
Exposure
Amosite
day
3 months 6 months 12 months 24 months
Total
Anthophyllite
No.
mais at
risk
45 37
18
25 21 140
with Jung
.tummar
_
3 30
2 10 13 38
,
Adenoma
Type of lung tumour
n
Adenomatosis
Adeno
carcinomat
" "
MITTWO
11011
i
MITTWO
11011
MIT WO 1
1 01
5
MITTWO
1 01
3
MIT WO
11011
19
MITTWO
5
Squamous carcinomat
0 ooocoo ooocoo ooocoo ooocoo ooocoo
No. with
-00940 |
thelioma -
.
-000 -000 , -000 -000
| 0.
I
1 day
44
200823
NOMANE
3 months
37
200823
6 months 18 200823 NONMOAMANNEE
12 months
28
200823
NOMANE
24 months 18 200823 NOMANE
Total
145
200823
NOMANE
Crocistolite
:
001052 0 111110
001052 0 111110
0 1052
J
1 1 10
001052
4
1110
001052
3
111110
001052
8 1
8
=
eo.
(
Oo. 4,
}
and
1
3
day
3 months months 6 months months 12 mouths 24 months
Total
Chryzofile Canadian day
3 months 6 months 12 months 24 months
Total
Chrysotile Ithodesian dny
3 montlas months months 12 months 24 months
Total .
Control
43 36 ts 26 18 141
*
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SENATO
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SENATO
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589-93
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589-93
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137
45
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5 +
36
16
-2000
19 8 -2000
27
19
-2000
17
Yt
-2000
144
39
19
SOLIDNO SOLIDNO SOLIDNO SOLIDNO SOLIDNO SOLIDNO
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Total
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Rats which survived at least 300
Numbers in brackets
days after start start
are those with inetastases
of exposure
0
0
Q
0
0
0
)
6
807 days another with sotile after 719 days
Rhodesian chry
and one which
metastasized with
after 838 days
Canadian
chrysotile
In interpreting Table VII and Fig 11
it has to be borne in mind that there was
a greater tendency for rats to develop
adenomata in Experiment 1 than in
Experiment 2 as shown in the controls
Therefore the higher proportion of ani-
mals with adenomata after 3 protin months
exposure than after 6 months
is
exposure
probably an artefact We do not
know why adenomata occurred in the
3 MC AMOSITE
sy
a
|
i 7
_
m
500
ANTHOPHY
ROCIDOL
utroi utroi ol!
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ne
ASSIS
R LI
LE AR ONY TN MO
eco a
bn
AEE
EE
EFFECTS OF INHALATION OF ASBESTOS IN RATS
265
3 MONTHS AMOSITE
100 750 1000 ANTHOPHYLLITE
6 MONTHS
Af a 3
300 750 1000
12 MONTHS
24 MONTHS
;
i
500 [2 8
500 750 1000
t t
a "
500
750
1000
h
500 750 CROCIDOLITE
_
=
h
1000
G
Seen
500 750
1000
CHRYSOTILE Canadian
Dig pin
500
750
1000
bal bal
500
bal
bal
c *Exom
500
750 1000
300
750
1000
.
1000
CHRYSOTILE Rhodesian
300
750
1000
500
1000
500
1000
ei .
4 Ke
Om
500 750
x000
'
6 Squamous carcinoma
8 Adenocarcinoma
@ Adenoma adenomatosis
Other causes
Fra 11. Distribution of survival times in days after first exposure
i
i
i
|
~
controls in one experiment and not in the other but as the finding is significant P = 0.06 in its own right and is supported by the results from the exposed animals it is unlikely to be due to
chance
There was a higher incidence of
tumours with 12 months exposure than with 6 months but little difference
between the 12 and 24 months exposure Half of the 8 mesotheliomata in
Experiment 2 occurred with Canadian hrysotile so that in total crocidolite and
Canadian chrysotile produced 4 meso-
theliomata each Of the 20 tumours which metastasized 16 were after expo-
sure to a chrysoti1l0e with the Rhodesian
sample and 6 with the Canadian Three
others were with crocidolite and -- ----
with anthophyllite Two of the mesotheliomata in the month groups occurred within 400 days after first exposure one with crocidolite after 399 days and one with Canadian chrysotile after 335 days the only rat which failed to survive for its scheduled 12 months exposure
+ a .
ass
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C. WAGNER G. BERRY J. W. SKIDMORE AND V. TIMBRELL
Asbestosis and lung tumours
An analysis was carried out to deter-
mine whether there was
any relationship between the grade of asbestosis and the
presence of lung tumours Since the asbestosis grade depends on survival it
was necessary to standardize to a constant
survival time This was achieved by
calculating the regression coefficients of
least 600 days the mean survival tir
of those with and without lung tumog
were very similar There were 17 lunes tumours in 201 rats only 6 of these occurred in 157 rats without asbestosis
3-8 while 11 occurred in the 44 ruts
with minimal or slight asbestosis 25
t highly significant difference 0.001)
asbestosis grade on survival time for
rats without lung tumours exposed for
3 months or more and with survival of
Tumours at sites other than lung
A total of 412 tumours other
at least 400 days after first exposure lung tumours or mesothelioma of
Differences in these coethicients between pleura or peritoneum were observ
the 5 types of asbestos and the 4 lengths The majority of these were adenomaka
of exposure were not significant and tho of the breast or pituitary adenomata
e
pooled coefficient of 0-00304 -0-00065 which were common mortem findl
grade units per day was used Tho ings Both of these adenomata occurred
Pe
we asbestosis grade of each rat was then
adjusted using this slope to an arbitrary
4 times as frequently in females as in
males The numbers of these tumours
survival The adjusted mean asbestosis other benign tumours and malignand
grades were then calculated for each of tumours for each type of asbestos are
the 20 groups for those with and without shown in Table VIII For none of
lung tumours In 15 of these groups the
mean asbestosis grade was higher in the animals with lung tumours in the other
5 groups the opposite occurred but only slightly so in 4 cases The accuracy of
an estimate of the difference in asbestosis
between those with and those without a
tumour is dependent on the number of
animals between
to take
in each category which
groups and weighting each
account of this gave a
varies group
mean
difference of 0-710-13 0-710-13 0-710-13 Hence overall
the animals with lung tumours had
TABLE Number of Tumours at Other than the Lang
Benign tumours
Amosire Aurbophyllite Aurbophyllite Croeulolite
Chrysatile Canneliari
Chrysotile Rhodesian Rhodesian
Control
Brest
HEAA HEAA HEAA HEAA
E
&
Pituit ary
40 38 31 26
37
31+
Other
4 4 4
rs
Madne
taseeel
TuZLIKATE TuZLIKATE TuZLIKATE
"
tes tes
:
$
bd
significantly < 0-001 more asbestosis
than those without Differences between
the dusts were not significant but the
wide range in anthophyllite 0-25 Canadian chrysotile 0-42 crocidolite 0.66
amosite 0-85 and Rhodesian chrysotile
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
There was very little asbestosis in these
groups Table VI and restricting atten-
tion to animals which survived for at
tumour types was the difference betwe the control and the asbestos frealesi
significant In Table IX more detail is
given of the sites of the tumours with all
types of asbestos combined The largest
differences between the treated and con
trol rats were for tumours of the ovary 10 in treated and none in controls sund
tumours of male urinary organs 1)
in treated and none in controls How
neither difference was significant A few rats had multiple mali
tumours 2 of the rats with mesothelioma
of the pleura also had a lung
and one rat with a squamous
carcinoma carcinomis carcinomis
EFFECTS OF INHALATION OF ASBESTOS IN RATS
267
TABLE IX
Sites of Tumours Other than
Ashiestos tratedl
we
ee
--
Site Tuniour type
Digestive organs and peritumwum
Bono and skin Breast
Ovary
Other female urinary organsi Male urinary organs Intracraniat
Thyinoima
Lyinphoma leukaemin
Others
Benign
Malignanst
4
ay
4
4
Leste
"
ik
7
o
*
3
*
173
]
7
\
_
N
5
33
) thyroid mediastinum
netrymui 4 salivary gland Thyroid
Lung
Control
vee ee
Benign
I
"
ay
( " 1 314
"
.
1
Malignant
3 2 l } + = 1
011 011
superervnal
of the lung had a mesothelioma tunica
vaginalis Mesotheliomata of the vagin-
alis were scen in this and one other 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 cases tumours have
Fem
z
classified by the primary site and few cases of multiple malignant
ours there was no difficulty nizing the distinct types i.r. not a secondary of the other
in recog-
one was
from the worn hatumer of 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 hypothesis to explain the high
rate of lung tumours The amount of chrysotile retained in
the lungs did not show any clear increase
with dose in rats exposed for longer
than 3 months In two earlier experi-
ments
Morris
Wagner and Skidmore 1965 et al 1967 a higher airborne
dust concentration was used to give a
cumulative dose in 6 weeks similar to
DISCUSSION
Our finding that the asbestosis produced by exposure progressed after cessa-
tion of exposure is in agreement with
human experience but contrasts with the early inhalation experiments reported by Vorwald Durkan and Pratt 1951 in which progression did not occur Wagner 1963 reported more asbestosis with amosite than with chrysotile in
guinea rats and monkeys but our experiments show that of the UICC standard reference samples amosite is the
least fibrogenic in rats
Gross et al 1967 found lung cancers
in
in 25 of 72 rats which survived 16 months
exposure to chrysotile dust at a mean concentration of 86 mg for 30 hours & week They considered that contami-
nation of the asbestos by trace metals
that given in the present experiment over 3 months The weight of asbestos found in the lungs of rats exposed to amphibole was 3 times greater than in those exposed to chrysotile In the present experiments the ratio was 6 to 1 after 3 months but increased with
continuing exposure as the weight of amphibole in the lungs continued to increase but the amount of chrysotile did not The previous experiments had
shown that the rate of elimination of
dust from the lungs was much greater
for chrysotile than for the amphiboles The present results may be explained on this basis the weight of chrysotile having
reached equilibrium level i.e. the rate of
elimination equalling the rate of retention
There are a number of features of the
results presented above which we found
a
tog
OU
mer
arpnoes 2
Nvasts
PUPes
2
af
HF
he
es
qa
MEWS q
Di
AROEE
rT Pe
Gat
7
=. 4') Bi
FIRE SIE
:
iqcg
a
IE
oe
ie
GE ae
it ,
| RCE
ult i
ROR ae
1gi[M|
Aviwd a
~ fh
Al)
pes
aie im Snr
4 lll
be tH
268
J. WAGNER G. BERRY J. W. SKIDMORE AND V. TIMBRELL
surprising First in Experiment 1 two
mesotheliomata occurred with the one
day exposure compared with only one
with the 3 months exposure which had a dosage mare than 50 times greater
If the incidence of mesotheliomata was
proportional to dose as is indicated for
the inoculation experiments Wagner et al 1973 then the probability of such au extreme result occurring by chance
would be about 2 in a 1000
Secondly there was no evidence of
either less carcinogenicity or less asbestosis in the groups exposed to chrysotile th^n those exposed to the amphiboles even
though the amounts of dust in the lungs
were so different In particular the UICC Canadian chrysotile produced as
many mesotheliomata as the UICC croci-
dolite The 2 UICC samples of chrysotile
produced 12 of the 14 tumours with
metastases However much less dust
was retained in the lungs of rats exposed to chrysotile than amphiboles Fig 10 Moreover after intrapleural inoculation the risk of a mesothelioma occurring
with UICC crocidolite is 3 times the risk
with chrysotile Wagner et al 1973 Therefore allowing for the greater retention of crocidolite after inhalation we might have expected the risk with croci-
dolite to have been of the order of 20
times that of chrysotile
Two of the mesotheliomata occurred
within 400 days of the start of exposure This may be compared with our injection experiments in which only 20 out of 803 occurred within 400 days Wagner and Berry 1969 Wagner et al 1973 Also
the earliest mesothelioma occurred after
355 days and we observed only 3 within
this period in our injection experiments
The positive association between as-
bestosis and lung tumours which we have
established in the animals is in agreement
with epidemiological findings e.g. Minister
of Labour and National Service 1949
Knox et al 1968 Elmes and Simpson
1971
The failure to establish any association between asbestos exposure and tumours
of sites other than the lung is equivoca '
Although
intestinal
an association with gast tumours has been found e
demiologically it is not yet regarded a.
clearly established Selikoff Hammond
and hurg 1972 Newhouse 1974 and
is of lower magnitude than the extess
lung cancer risk Our experiments pro vide no support for such an association The experimental work of Graham and
Graham 1967 suggested that intra
peritoneal injection of tremolite ashoste
could produce ovarian tumours but : follow of women asbestos work
reported by Newhouse et al 1972 n
duced no definite conclusions on th
question because of the rarity of the tumour Our experiments do give sotme
support to an association between asbestos asbestos
exposure and ovarian tumours as well
as tumours of the male urinary system Although neither was significant this could be because of the relatively
small size of the control group T
overcome this we have included i
control rats from some of our oth
experiments thus increasing the Dola
treated group to 403 rats of the same
strain This larger group contained 2
malignant tumours of the ovary and 5 tumours of the urinary tract in males In over 700 rats exposed to
asbestos there were 10 ovarian tumours
7 of which were malignant and i!
tumours of the urinary tract in
males Hence based on the larger of controls the association between ash
tos exposure and ovarian tumours 1-
weak and significant whereas there is
no support for an association with tumours
of the male urinary system The UICC chrysotile samples are finer
than the chrysotile which has been used
in industry in the past However there is a trend for industry to use finer chry sotile Wright 1969 and so the experi
mental results may be more relevant to
the current situation than to the patst
We are investigating the effects of inhala tion of chrysotile in more detail in ar
experiment involving UICC Canadian
#
in
508
|
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OTE
EPA
RUE SSSR
<>
eyo
p
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a
ra eaebots
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oe
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whe.
hoe
PS
s
Ep Ae
ES WN,
ore
oe
NS
RA
wal ork
Seno ane.
es
Y
,
eS ng
pean errs g it
ee4 e. 1
bre
PEER art i oe oP
L wf
EFFECTS OF INHALATION OF ASBESTOS IN RATS
269
| chrysotile a grade 7 sample from a
| Canadian mine and the superfine sample
G. W. 1967 Comparison of Dust Retention in
Specific Pathogen Free and Standard Rats
In Inhaled Particles and Vapoura II Ed C. N.
| proved which
the most carcinogenic of
the materials which we inoculated intra-
Davies Oxford Pergamon p 205 N whouseM. L. 1974 Cancer Among Workers
in the Asbestos Textile Industry In Biological
| pleurally Wagner et al 1973 The experiments we report have given results which in several respects cor-
Effects of Ashentos Lyon 2-5 October 1972 Ed P. Bogovaki C. Gilson V. Timbrell and C. Wagner IARC Scientific Publications No.
;
8 In print
respond to those found in man Thus this experimental method is established
as a valid tool for the investigation of
NEWHOUSE M. L Berry G. Wagner C. &
Turok M. E. 1972 A Study of the Mortality of Female Asbestos Workers Br J. ind Med
29 134
the biological effects of asbestos
Selikoff I. J. HAMMOND E. C. & Chung J.
1972 Carcinogenicity of Amosite Asbestos
Archs envir Hith 25 183
Shadad M. & PYLEY L. N. 1970 Morphological
;
'
:
anid
v-
}
We are grateful to all our colleagues
who over a number of years were res-
ponsible for the daily attention necessary
in carrying out the experiments We would also like to acknowledge our thanks
to Dr Harold Stewart of the National
Cancer Institute who advised us on the
classification of tumours We are also
grateful to our former colleague Dr A. Walter who had classified the lung tumours occurring in some of our earlier experiments which we mentioned for
comparison
Lesions in Rat Lungs Induced by Polycyclic
Hydrocarbons In Morphology of Experimental Respiratory Carcinogenesis Proc Conf Gatlinburg 13 16May 1970. Ed P. Netteshein M. G. Hanna Jr. and J. W. Deatherage Jr.
U.S. Atomic Energy Commission Symposium
Series No. 21 p 227
STANTON M. F. 1974 In Pathology of Tumours in Laboratory Animals Vol 1. Tumours of the
Rat Part 2. I.A.R.C. Scientific Publications
No. 6. Lyon International Agency for Research
Cancer In preparation
TIMARELL V. Gilson J. C. & Webster I. 1968
UICC Standard Referenco Samples of Asbestos
Int J. Cancer 3 406 Timbrell V. HYETT A. W. & Skidmore J W.
1969 A Simple Dispensor for Generating Dust Clouds from Standard Reference Samples of
Asbestos Ann occup Hyg 11 273 TIMBRELL V. Skidmore J. W. HYETT A. W.
an
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~
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GRAHAM J. & GRAHAM R. 1967 Ovarian Cancer
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Gross P. De Treville R. T. P. TOLKER E. B.
KASCHAK M. & Babyak M. A. 1967 Experi-
ft
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| Chrysotile coy +2
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Asbestos Dust Archa envir Hith
it
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M. G. Hanna Jr. and J. W. Deatherage Jr.
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| for Series No. 21. p 203 MINISTER OF LABOUR AND
NATIONAL
SERVICE
1949 Annual Report of the Chief Inspector of Factories the Year 1947 Cmd 7621 London
HMSO
MORRIS {
T. G. ROBERTS W. H. SILVERTON R. E
)
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_
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1951 Experimental Studies of Asbestosis A.M.A. Archa ind Hyg 3 . Wags rJ. C. 1963 Asbestosis in Experimental
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Wagner C. 1985 The Sequelae of Exposure to Asbestos Dust Ann N.Y. Aend Sci 132 691
Wagner J. C. 1972 The Significance of Asbestos
in Tissuo In Recent Resulta in Cancer Reseurch
Vol 39 Current Problems in the Epidemiology
of Cancer and Lymphomas Ed E. Grundmann and H. Tulinius New York Springer
P. 37
WAGNER J. C. & BERRY G. 1969 Mesotheliomas in Rats Following Inoculation with Asbestos Br J. Cuncer 23 567
WAGNER J. C BERRY G. & TIMBRELL V. 1973 Mesotheliomas in Rats Following Inoculation with Asbestos and Other Materials Br J. Cancer
28. 173 WAGNER J. C. & SKIDMORE J. W. 1965 Asbestos
Dust Deposition and Retention
N.Y. Acad Sci 132 77 WRIGHT G. W. 1969 Asbestos
1969. Am Rev. resp Dia 100
in Ruts 1an
and Health in 467
e
Saye Re
tent
.
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