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Accepted for Publication in the Journal Inhalation Toxicolo
g
y
Running Title: Biopersistence of Canadian Chrysotile Page 1 of 35
The biopersistence of Canadian chrysotile asbestos
following inhalation.
David M. Bernstein1*, Rick Rogers2, Paul Smith3
1Consultant in Toxicology, Geneva, Switzerland;
2Rogers Imaging Corporation, Needham, Massachusetts;
3Research & Consulting Company Ltd., Füllinsdorf, Switzerland
_____________________________________________________
Abstract
Chrysotile asbestos is often included with other asbestos materials in evaluation and
classification. However, chrysotile is a serpentine with markedly different physical and
chemical characteristics in comparison to amphiboles (e.g. crocidolite, amosite, tremolite). In
contrast to amphiboles which are solid rod-like fibers, chrysotile is composed like a rope of
many fine fibrils which tend to unwind.
In order to quantify the dynamics and rate by which chrysotile is removed from the lung, the
biopersistence of a sample of commercial chrysotile from the Eastern Townships area of
Québec, Canada, labelled “QS Grade 3-F” which is the longest commercial grade intended
for textile use was studied. As the long fibers have been shown to have the greatest potential
for pathogenicity, the chrysotile samples were specifically chosen to have more than 200
fibers/cm3 longer than 20 µm present in the exposure aerosol. This publication presents the
results of this study through 3 months post exposure.
Study Design: 1) Fiber clearance (lung digestions): At 1-day, 2-days, 7-days, 14-days, 1-
month, 3-months, and 12-months (to be reported) following a 5 day (6 hours/day) inhalation
exposure, the lungs from groups of animals were digested by low temperature plasma ashing
and subsequently analysed by transmission electron microscopy for total chrysotile fibers
number in the lungs and chrysotile fiber size (length and diameter) distribution in the lungs.
2) Fiber distribution (confocal microscopy): This procedure was included in order to identify
where in the lung the fibers were located. At 1-day, 2-days, 7-days, 14-days, 1-month and
3-months (to be reported) post exposure, the lungs from groups of animals were analysed by
confocal microscopy to determine the anatomic fate, orientation and distribution of the
retained chrysotile fibrils deposited on airways and in the parenchymal region.
Chrysotile was found to be rapidly removed from the lung. Fibers longer than 20 µm were
cleared with a T1/2 = 16 days, most likely by dissolution and disintegration into shorter fibers.
The shorter fibers were also rapidly cleared from the lung with fibers 5-20 µm clearing even
faster (T1/2 = 29.4 days) than those < 5 µm in length. The fibers <5 µm in length cleared at a
rate (T1/2 = 107 days) which is within the range of clearance for insoluble nuisance dusts. The
* This study was supported by grants from the Government of Québec and The Asbestos Institute, Montréal,
QC, Canada
Address correspondence to Dr. David Bernstein, Consultant in Toxicology, 40 chemin de la Petite-Boissière,
1208 Geneva, Switzerland, E-mail : davidb@itox.ch
Running Title: Biopersistence of Canadian Chrysotile Page 2 of 35
breaking apart of the longer fibers would be expected to increase the short fiber pool and
therefore could account for this difference in clearance rates. The short fibers were not found
clumped together but appeared as separate, fine fibrils, occasionally unwound at one end.
Short free fibers appeared in the corners of alveolar septa, and fibers or their fragments were
found within alveolar macrophages. The same was true of fibers in lymphatics, as they
appeared free or within phagocytic lymphocytes. Neutrophil-mediated inflammatory
response did not occur in the presence of chrysotile fibers at the time points examined.
Taken in context with the scientific literature to date, this report provides new robust data
which clearly supports the difference seen epidemiologically between chrysotile and
amphibole asbestos.
Running Title: Biopersistence of Canadian Chrysotile Page 3 of 35
The biopersistence of Canadian chrysotile asbestos
following inhalation.
David M. Bernstein1†, Rick Rogers2, Paul Smith3
1Consultant in Toxicology, Geneva, Switzerland;
2Rogers Imaging Corporation, Needham, Massachusetts;
3Research & Consulting Company Ltd., Füllinsdorf, Switzerland
_____________________________________________________
Asbestos has been implicated in disease through both epidemiological and animal toxicology
studies. However, the serpentine asbestos chrysotile is very different chemically and
mineralogically from amphibole asbestos such as amosite, crocidolite or tremolite. This has
resulted in some researchers suggesting that chrysotile may not be of the same potency as the
amphiboles and may clear faster from the lung (Howard, 1984; Churg & DePaoli, 1988;
Mossman et al., 1990; Morgan, 1994; Churg, 1994; McDonald, 1998; Rodelsperger, et al.,
1999; McDonald et al., 1999).
To examine the dynamics and rate of clearance of chrysotile from the lung an inhalation
biopersistence study in the rat was initiated using a sample of commercial chrysotile from the
Eastern Townships area of Québec, Canada. The protocol for this study was designed to
meet the specific recommendations of the European Commission (EC) Interim Protocol for
the Inhalation Biopersistence of synthetic mineral fibers (Bernstein & Riego-Sintes, 1999).
For synthetic mineral fibers, the biopersistence of the fibers longer than 20 µm has been
found to be related to their potential to cause disease (Bernstein et al., 2001). As described
below, the specifications in the protocol for counting and sizing the fibers were modified to
accommodate the finer dimensions of the chrysotile fibers in comparison to mineral fibers.
In addition, the disposition of fibers within the lung was also determined using confocal
microscopy. This paper presents the results through 90 days after cessation of exposure. A
subsequent paper will present further results through 1 year after cessation of exposure.
The exposure and in-life phases of the study were performed at the Research and Consulting
Company Ltd., Füllinsdorf, Switzerland. Fiber Counting and sizing was performed under
subcontract to RCC at Gesellschaft für Schadstoffmessung und Auftragsanalytik (GSA),
Neuss, Germany. The confocal microscopy analysis was performed by Rogers Imaging
Corporation, Needham, Mass., USA.
Methods
Chrysotile sample characteristics
The chrysotile fiber is monoclinic in crystalline structure and has a unique rolled structure
described below. The chrysotile used in this study was labelled “QS Grade 3-F” on the
† Address correspondence to Dr. David Bernstein, Consultant in Toxicology, 40 chemin de la Petite-Boissière,
1208 Geneva, Switzerland, E-mail : davidb@itox.ch
Running Title: Biopersistence of Canadian Chrysotile Page 4 of 35
Canadian Quebec Screening Scale (QSS) and is a commercial textile grade which is the
longest grade intended for textile use (Cossette & Delvaux, 1979).
The chemical composition and the structure of chrysotile are markedly different from that of
amphiboles such as tremolite or amosite (Hodgson, 1979).
Table 1 Typical chemical composition (percent)
Compound Chrysotile1Tremolite2Amosite2
SiO240.6 55.10 49.70
Al2O30.7 1.14 0.40
Fe2O32.3 0.32 0.03
FeO 1.3 2.00 39.70
MnO -- 0.10 0.22
MgO 39.8 25.65 6.44
CaO 0.6 11.45 1.04
K2O 0.2 0.29 0.63
Na2O -- 0.14 0.09
H2O+-- 3.52 1.83
H2-- 0.16 0.09
CO20.5 0.06 0.09
Ignition loss 14.0 -- --
Total 100 99.93 100.26
1. Typical chemical analysis of Canadian chrysotile from the Quebec Eastern Townships
(LAB Chrysotile, Inc., Quebec, Canada)
2. Hodgson (1979) ; pp. 80-81
Table 1 summarizes the chemical composition of typical serpentine and amphibole asbestos.
The chemistry of chrysotile is composed of a silicate sheet of composition (Si2O5)n-2n, in
which three of the O atoms in each tetrahedron are shared with adjacent tetrahedra and a non-
silicate sheet of composition [Mg3O2(OH)4]n+2n. In chrysotile the distances between apical
oxygens in a regular (idealized) silicate layer are shorter (0.305 nm) than the O-O distances in
the ideal Mg-containing layer (0.342 nm) which may account for the curling of the layers
which results in the rolling up like a carpet to form concentric hollow cylinders (Skinner, et
al., 1988). This structure is illustrated in Figure 1 (adopted from Skinner et al., 1988) and
transmission electron micrographs of chrysotile are shown in Figure 2 (Kiyohara, 1991). The
Mg molecule is on the outside of the curl and is thus exposed to the surrounding
environment.
Running Title: Biopersistence of Canadian Chrysotile Page 5 of 35
Figure 1
Figure 2
In contrast, with amphiboles such as tremolite, the basic structure is in the form of an I-beam
with corner-linked (SiO4)-4 tetrahedra linked together in a double-tetrahedral chain that
sandwiches a layer with the Ca2Mg5. In contrast to chrysotile, with tremolite, the Mg is
locked within the I-beam structure. This is illustrated in Figure 3.
Running Title: Biopersistence of Canadian Chrysotile Page 6 of 35
Figure 3
* Adapted with permission from: Department of Geology and Geophysics, University
of Wisconsin, Crystal Structure Movies, http://www.geology.wisc.edu
Experimental Design
The results of the lung digestion measurements through 3-months after cessation of exposure
and of the confocal microscopy examination through 1 month after cessation of exposure are
presented. Following termination of the study at 12 months post exposure the remaining
results will be presented in a separate publication.
The experimental design of the in-life and biopersistence analysis has been presented in detail
previously (Bernstein, et al., 1994) and is summarized below. In particular, details of the
counting and sizing procedures are reiterated as these are considered essential to the
successful interpretation of these studies.
Animal Exposure: Groups of 56 weanling (approximately 9 weeks old) male Wistar rats
(Specific Pathogen Free quality) were exposed by flow-past nose-only exposure to a target
fiber aerosol concentration of 200 fibers L>20 µm/cm3 for 6 hours/day for a period of 5
consecutive days. This concentration corresponded to two times that required by the EC
Biopersistence Protocol in order to assure that there was no question of sufficient long fiber
exposure. In addition, a negative control group was exposed in a similar fashion to filtered
air. Wistar rats (HanBrl:WIST, SPF), obtained from RCC Ltd, Biotechnology and Animal
Breeding Division, CH-4414 Füllinsdorf, Switzerland were used.
Exposure System: The fibre was prepared for the exposures prior to the technical trials by
grinding it in a Cyloctec® Sample Mill (Tecator, Sweden) which grinds samples by a high-
speed action, rolling the sample against the inner circumference of a durable grinding surface
and then passes it through a fine mesh screen. The fiber aerosol generation system was
designed to loft the bulk fibers without breaking, grinding or contaminating the fibers
(Bernstein, et al., 1994). The animals were exposed by the flow-past nose/snout-only
Running Title: Biopersistence of Canadian Chrysotile Page 7 of 35
inhalation exposure system. This system was derived from Cannon, et al. (1983) and is
different from conventional nose-only exposure systems in that fresh fiber aerosol is supplied
to each animal individually and exhaled air is immediately exhausted.
Fiber clearance: At 1-day, 2-days, 7-days, 2-weeks, 1-month, 3-months and 12-months (to
be reported) post exposure, the lungs from groups of animals were digested by low
temperature plasma ashing and subsequently analysed by transmission electron microscopy
(at the GSA Corp.) for total chrysotile fibers number in the lungs and chrysotile fiber size
(length and diameter) distribution in the lungs. This lung digestion procedure digests the
entire lung with no possibility of identifying where in the lung the fibers are located.
Fiber distribution: This procedure was undertaken to determine the distribution of fibers
within various pulmonary compartments. At 1-day, 2-days, 7-days, 14-days, 1-month and 3-
months (to be reported) post exposure, the lungs from groups of animals were prepared and
analysed by Confocal microscopy. The locations of chrysotile fibrils deposited on conducting
airways, respiratory airways and parenchyma was quantified.
Lung Digestion for Fiber/Particle Analysis:
From 5 out of the 7 rats per group per time point the lungs were thawed and the entire lung
was prepared for analysis. The tissue was initially dehydrated by freeze drying (Edwards
EF4 Modulyo freeze dryer) and dried to constant weight to determine the dry weight of the
tissue. The dry tissue was plasma ashed in a Plasma Systems 200 (Technics Plasma GmbH)
multiple chamber plasma unit at 300 watts for approximately 16 hours. Upon removal from
the ashing unit, the ash from each lung was weighed and suspended in 10 ml of methanol
using a low intensity ultrasonic bath. The suspension was then transferred into a glass bottle
with the combustion boat rinse and the volume made up to 20 ml. An aliquot was then
removed and filtered onto a gold-coated polycarbonate filter (pore size of 0.2 µm).
Counting rules for the evaluation of air and lung samples by transmission electron
microscopy:
All fibres visible at a magnification of 10,000x were taken in consideration. All objects seen
at this magnification were sized with no lower or upper limit imposed on either length or
diameter. The bivariate length and diameter was recorded individually for each object
measured. Fibers were defined as any object that had an aspect ratio of at least 3:1. The
diameter was determined at the greatest width of the object. All other objects were considered
as non-fibrous particles.
The stopping rules for counting of each sample were defined as follows: For Non-fibrous
particles, the recording of particles was stopped when a total of 30 particles were recorded.
For fibres, the recording was stopped when 500 fibres with length ≥ 5 µm, diameter ≤ 3 µm
(often referred to as a WHO fiber (WHO, 1985)) or a total of 1000 fibres and non-fibrous
particles were recorded. If this number of fibres was not reached after evaluation of 0.15 mm²
of filter surface, additional fields of view were counted until either 500 WHO-fibres were
reached or a total of 5 mm² of filter surface was evaluated, even if a total of 500 countable
Running Title: Biopersistence of Canadian Chrysotile Page 8 of 35
WHO-fibres were not reached. The evaluation of short fibres (length < 5 µm) was stopped
when 100 short fibres were reached.
Confocal Imaging of Fibers and Lung Tissue.
The lungs of animals designated for confocal microscopy analysis were fixed in Karnovski's
fixative by gentle instillation under a pressure of 30 cm H2O with simultaneous immersion in
fixative. The trachea was then ligated and the inflated lungs were stored in the same fixative.
Following fixation, apical lobes were divided into five pieces (10 mm2 x 5 mm thick) cut
parallel to the hilum, dehydrated in graded ethanolic series to absolute, stained with 0.005%
lucifer yellow, and embedded in Spurr plastic for microscopic analysis (Rogers et al., 1999).
Flat surfaces were prepared from hardened plastic blocks containing embedded lung pieces.
Confocal Fiber Quantification.
Confocal microscopy was performed on three randomly selected animals from each time
point using Sarastro 2000 (Molecular Dynamics, Inc.) laser scanning microscopes fitted with
25 mW argon-ion lasers and an upright microscope (Optiphot-2; Nikon, Inc., or Zeiss
Axiophot) modified for reflected light imaging. These confocal microscopes were used to
record image data in dual channel reflected and fluorescent imaging mode. Optical bench
settings for the Sarastro 2000 CLSMs were: excitation - 488 nm (Lucifer yellow), emission
>510 long pass filter, laser power 12-15 mW, 30% transmission, photomultiplier voltage set
between 500-800 volts. Fluorescently labeled cellular constituents and reflective/refractive
fibers (and particles) were imaged simultaneously with this arrangement. Each “exposure”
produced two digital images in perfect register with one another.
An image recorded in either mode was a two-dimensional (x,y), 512 x 512 array of pixels,
each with an intensity value from 0 to 254 gray scale units (a value of 255 indicated
saturation of the intensity scale). Optical (x,y) sections, individually and in depth series, were
recorded at various positions along the z-axis by adjusting the stage height using stepper
motors under computer control. Images and image series were analyzed and prepared for
presentation by employing specialized computer software.
Images were recorded through 40X objectives. The dimensions of voxels in the recorded
volume were (x, y, and z dimensions, respectively) 0.13 µm, 0.13 µm, and 0.3 µm.
Morphometric Methods
In the case of three-dimensional microscopical methods, strategies for specimen examination
and image sampling are strongly determined by the selection of questions to be addressed.
Although it was realized that questions regarding the size distribution of fibers retained
within the entire lung would be more efficiently answered by the conventional
ashing/electron microscopical technique, questions about the numbers (not sizes) of fibers
within various anatomical compartments would be answered most effectively by confocal
microscopy employing serial optical section techniques. In many instances, the true length of
Running Title: Biopersistence of Canadian Chrysotile Page 9 of 35
individual fibrils was captured within the volume recorded in serial section stacks. This
occurs if the fibril profile is oriented such that two free ends are present.
Sampling strategy for parenchyma.
As parenchyma provides about 90% of the lung’s volume and varies little if at all from one
region of the lung to another, it is readily possible to acquire random fields-of-view of
parenchyma from which quantitative data may be obtained. Our procedure was to place the
microscope objective at random over the lung specimen exposed at the surface of the epoxy
embedment, collect a depth series of images, return to the initial starting depth, move two
field widths in the positive x-direction, and repeat the process. Twenty five depth series per
piece of lung (for a total of 100 fields-of-view per animal) were obtained in this way. (If the
perimeter of the lung section was encountered, the objective was moved two field widths in
the positive y-direction, and the stepping was continued in the negative x-direction.) At each
location, if the profile of a conducting airway was in the volume to be recorded by the depth
series, the field-of-view was skipped, and another step was made, until a volume was found
which did not contain an airway. Each volume was recorded by obtaining 25 optical sections
separated by 0.3 µm along the z-axis. The real-world dimensions of a volume, therefore,
were 61.6 µm x 61.6 µm x 7.5 µm in x, y, and z, respectively. More than 75,000
micrographs of the parenchyma region were recorded to obtain the necessary quantitative
information.
The number of fibers in each volume was counted by a human operator who was able to
move up and down through the depth series of images while looking for the characteristic
bright points or lines which indicated a reflective or refractile particle or fiber. The person
counting fibers did not know which experimental group the images were drawn from, that is,
the counting was done under “single blind” conditions. These counts provided data with
units of (number of fibers / volume of parenchyma in cubic micrometers). Knowing the
volume represented by each depth series and the volume of parenchyma (including airspaces)
in the animal’s entire lungs, as fixed, the fiber load in the lung’s parenchyma could be
calculated.
Whenever a fiber was detected, the anatomic compartment in which it occurred was also
noted. In instances where free ends of the fiber were observed, fiber length was recorded
using three dimensional measurement techniques. Fibers in parenchyma were classified as
occurring: in alveoli, alveolar ducts, or respiratory bronchioles, in contact with the surface of
tissue; in ducts or alveoli, but not in contact with tissue in the recorded volume; and wholly or
partly inside alveolar macrophages. Fibers were observed in other parenchymal contexts such
as interstitium or BALT were noted in the “other” category. These counts made it possible to
estimate the fraction of fiber present in the different categories.
Sampling strategy for airways
Airways occupy only 10% of total inflated lung volume and exist as a tree-like structure that
is relatively coarse compared to parenchymal structures. Therefore, a field-of-view
positioned at random on a lung sample has a rather low probability of containing any airway
Running Title: Biopersistence of Canadian Chrysotile Page 10 of 35
wall profile. Instead, it was efficient and valid to proceed along a randomly positioned line
on the lung sample’s surface and record volumes whenever the line encountered an airway
whose local axis was nearly enough parallel to the sample surface’s normal that the tissue
layers in the airway wall were readily discerned.
Ten depth series (dimensions identical to parenchymal depth series) were recorded from each
of 4 samples per animal, and these stacks held, on average, 75 µm of airway wall profile
each. More than 30,000 micrographs of the airways were recorded and quantified from the
airway category.
The average airway diameter in these lungs is estimated at 300 µm, and airway volume, as
noted, is ca. 10% of lung volume. These numbers allow a further estimate of the length of an
equivalent cylinder and its wall area, which is an estimate of the total airway wall area in the
lungs.
Having measured the number of fibers per area of airway wall, the total fiber burden in the
airway compartment was estimated.
Inflammatory cells
Inflammatory cells were identified by morphologic recognition in serial section image data.
The nuclear morphology of the cells and the surrounding pulmonary tissue are distinguished
due to variations in fluorescent staining. Mononuclear cells, such as alveolar macrophages
were easily distinguished from neutrophils which exhibit poly-morphonuclear profiles.
RESULTS:
Inhalation biopersistence:
The EC Inhalation Biopersistence Protocol specifies that the exposure atmosphere to which
the animals are exposed should have at least 100 fibers/cm3 longer than 20 µm. In this study,
the number of fibers longer than 20 µm in the exposure atmosphere was purposely increased
to a mean of 200 fibers/cm3 longer than 20 µm, in order to maximise any potential effect of
these long fibers on clearance from the lung. The number, concentration and size distribution
of the air control and chrysotile exposure group are shown in Table 2.
Running Title: Biopersistence of Canadian Chrysotile Page 11 of 35
Table 2: Number and size distribution of the fibers in the chrysotile exposure aerosol.
Exposure Group
Gravimetric
Concentration
m
g
/m3
Number of Fibers
evaluated
Number of total
fibers/cm3
WHO Fibers/cm3
Percent WHO fibers
Number of fibers ≥20
µm/cm3
Percent of WHO
fibers ≥20 µm/cm3
Diameter Range (µm)
Length Range (µm)
GMD (µm) (Std. Dev.)
GML (µm) (Std. Dev.)
Mean Diameter (µm)
Std. Dev.
Mean Length (µm)
Std. Dev.
Length weighted
arthm. dmeter µm)
Length weighted
geom. diameter (µm)
Aspect ratio
Air
Control
0 2 0.3 0 0 0 0
0.05 -
0.13 1.5 -
3.5 0.08 2.29 0.09 2.5 0.07 0.07 40.8
Canadian
Chrysotile
4.32
(0.36) 2482 14805 1849 13 200 1 0.02-1 0.5-
110 0.12 2.42 0.14 3.32 0.16 0.12 36.1
As illustrated in Figure 4, all of the longer fibers (L>20 µm) in the exposure atmosphere were
less than 1 µm in diameter (99.6 % less than 0.8 µm) and thus potentially respirable. Figure
5 shows the bivariate length and diameter distribution of the fibers recovered from the lung at
1 day following cessation of exposure. The mean concentrations and dimensions of the fibers
recovered from the lungs at each time point are presented in Table 3.
Photomicrographs of the original bulk sample and an aerosol sample taken using scanning
electron microscopy (SEM) are shown in Figures 6 and 7. SEM was used for these
micrographs in order to provide a visual overview of the fiber size distribution. As described
above, transmission electron microscopy (TEM) was used for all quantification of fiber size.
Running Title: Biopersistence of Canadian Chrysotile Page 12 of 35
Figure 4
Chrysotile Fibers in the Exposure Atmopshere
Bivariate Length-Diameter Histogram of WHO Fibers
Running Title: Biopersistence of Canadian Chrysotile Page 13 of 35
Figure 5
Chrysotile Fibers in the Lung at 1 Day after Cessation of Exposure
Bivariate Length-Diameter Histogram of WHO Fibers
Figure 6 Chrysotile from the bulk sample
Running Title: Biopersistence of Canadian Chrysotile Page 14 of 35
Figure 7 Chrysotile sampled from the aerosol exposure atmosphere
Running Title: Biopersistence of Canadian Chrysotile Page 15 of 35
Table 3: Summary data of the mean lung burden results as determined by transmission electron
microscopy (Fiber Concentrations: Means ± Standard Deviation).
Sacrifice time point 1 2 7 2 1 3 12
(time since cessation of last exposure) day days days weeks month months months
Number of fibres evaluated 328.7±
12.1 320.9±
10.8 319.6±
7.8 314.0±
2.8 308.3±
2.6 226.1±
21.3
Number of total fibres per lung lobes (million) 95.68±
13.6 92.72±
21.2 93.94±
19.6 71.38±
3.7 56.76±
5.4 41.20±
2.9
Number WHO fibres per per lung lobes (million) 11.0±
3.3 9.7±
4.2 12.1±
2.5 8.2±
1.1 5.6±
1.3 1.4±
0.2
NumberWHO fibres of total fibres (%) 11.48 10.14 12.92 11.52 9.86 3.42
Number of fibres L >20 µm per lung lobes
(million) 0.4±
0.2 0.3±
0.1 0.2±
0.09 0.1±
0.03 0.1±
0.03 0.02±
0.01
Fibres L > 20 µm of total fibres (%) 0.36 0.28 0.24 0.18 0.14 0.06
Number of fibres L 5 - 20 µm per lung lobes
(million) 10.7±
3.2 9.4±
4.1 11.9±
2.5 8.1±
1.1 5.6±
1.3 1.4±
0.2
Fibres L 5-20 µm of total fibres (%) 11.12 9.88 12.68 11.32 9.76 3.36
Number of fibres L =<5 µm per lung lobes
(million) 84.7±
12 83.0±
17.6 81.8±
17.2 63.1±
2.8 51.1±
4.8 39.8±
2.7
Fibres L =<5 µm of total fibres (%) 88.52 89.86 87.08 88.48 90.14 96.58
Diameter Range (µm) 0.02-1.3 0.02-1.1 0.03-1 0.03-0.09 0.03-0.09 0.02-0.8
Length Range (µm) 0.07-62 0.9-43 0.8-46 0.8-42 0.8-40 0.7-41
Mean Diameter (µm) 0.17 0.15 0.16 0.14 0.11 0.10
Std. Dev. 0.17 0.17 0.12 0.11 0.12 0.20
Mean Length (µm) 2.93 2.70 3.11 2.87 2.73 2.33
Std. Dev. 8.84 7.34 7.22 6.37 5.79 10.77
GMD (µm) 0.14 0.12 0.13 0.11 0.09 0.07
Std. Dev. 2.11 2.25 1.99 1.98 2.23 2.51
GML (µm) 2.29 2.17 2.51 2.33 2.23 1.85
Std. Dev. 3.44 3.30 2.99 2.99 2.97 3.59
Length weighted arthm. diameter (µm) 0.21 0.19 0.18 0.16 0.14 0.11
Length weighted geom. diameter (µm) 0.17 0.15 0.15 0.13 0.11 0.09
Mode diameter (µm) 0.16 0.10 0.12 0.11 0.06 0.05
Mode length (µm) 1.62 1.46 2.44 1.90 1.86 1.84
Median diameter (µm) 0.16 0.14 0.15 0.11 0.09 0.07
Median length (µm) 2.14 1.94 2.40 2.16 2.16 1.82
Aspect ratio mean 22.15 25.06 26.88 26.86 30.65 31.84
Number of particles evaluated 0.2 0 0 0 0 0.2
Mean Number of particles per lung lobes
(million) 0.002 0 0 0 0 0.002
<= 1µm particles per lung lobes (million) 0 0 0 0 0 0.002
> 1µm - <= 3µm particles per lung lobes
(million) 0 0 0 0 0 0
> 3µm particles per lung lobes (million) 0.002 0 0 0 0 0
Running Title: Biopersistence of Canadian Chrysotile Page 16 of 35
Fiber Clearance:
The fibers longer than 20 µm which deposit in the lung rapidly ‘disappear’ from the lung as
shown in Figure 8 with a clearance half-time of the fibers longer than 20 µm of 16 days. The
clearance half-times (Table 4) were determined using the procedures specified in the EC
Inhalation Biopersistence protocol (Bernstein & Riego-Sintes, 1999). The clearance curve
was fitted to the data using non-linear regression techniques with a double exponential
(StatSoft, Inc., 2003).
Figure 8
Clearance of Canadian Chrysotile from the Lung
Fibers with lengths > 20 µm
0 20406080100
Time since cessation of exposure (days)
0
20
40
60
80
100
120
140
Percent remaining of fibers L>20 µm (day 1 = 100 %)
Running Title: Biopersistence of Canadian Chrysotile Page 17 of 35
Table 4
Double exponential fit to the data
Fibers L > 20 µm
R=.80119 Variance explained: 64.190%
a1 a2 T1 T2 WT
Estimate 65.96 30.38 6.16 38.04 16.22
Std.Err. 19.78 19.04 3.21 15.56 2.67
t(26) 3.33416 1.59552 1.918053 2.44419 6.06857
p-level 0.00258 0.12268 0.066150 0.02161 0.00000
At 30 days post exposure a mean of 1.9 fibers was measured microscopically on the filter of
the aliquot taken from the digestion of the whole lung. As shown in Table 3 this corresponds
when extrapolated to the whole lung to 20,000 fibers L>20 µm/lung at 30 days. After similar
exposure of the insoluble amphibole fiber amosite, there would be approximately 1,000,000
fibers L>20 µm /lung remaining at 30 days (Extrapolated from Hesterberg et al., 1998).
Table 5
Fiber length Clearance Half-time T1/2
> 20 µm WT1/2 = 16 days
5 – 20 µm T1/2 = 29.4 days
< 5 µm T1/2 = 107 days
As seen in Table 5, the fibers 5 to 20 µm in length also clear rapidly from the lung although
slower than the fibers L>20 µm. The clearance curves for the 5 – 20 µm fibers and the
objects < 5 µm were best fit using a single exponential also fit to the data using non-linear
regression techniques (StatSoft, Inc., 2003). While, the objects with lengths < 5 µm have the
slowest clearance half-times, this may be strongly influenced by the breaking apart of the
longer fibers which serve as a replenishing source for the shorter fibers.
Confocal Microscopic analysis:
The classic biopersistence study as defined by the EC protocols involves the digestion of the
entire rat lung for the determination of the fiber number and size distribution at each time
point. The TEM analysis of ashed samples provides a measure of only the total number and
size of fibers. It can not detect where the fibers are located within the lung.
In order to determine the disposition of those fibers remaining in the lung, confocal
microscopic analysis was performed on lobes of lungs embedded in plastic. A lens such as
the one on a microscope that is closest to the sample to be examined (the objective lens)
Running Title: Biopersistence of Canadian Chrysotile Page 18 of 35
brings light to a focus at a certain fixed distance. If the lens is well designed and constructed,
there will be a plane where objects will be in focus. To use a conventional microscope
effectively, it is necessary to cut a very thin slice of material to avoid tissue above and below
the plane of focus from degrading the quality of the final image. The confocal microscope
goes beyond the conventional microscope in this regard, because it excludes out-of-focus
light using a light-limiting aperture to form a sharp, high-quality image even if there is
material present that is not at the plane-of-focus. This means that specimens do not have to
be thin sectioned before they can be examined. Instead, it is possible to obtain an image of
the material at the plane-of-focus even if that plane lays tens of micrometers deep within the
specimen.
Microscopic appearance of the chrysotile fibers retained in lung.
The limit of detection of the confocal method used to quantify the disposition of the
chrysotile fibers in the lung was approximately 150 nm point to point resolution. Since most
fibers were oriented to present various oblique profiles in longitudinal orientation, most
fibrils greater than 150 nm in diameter were detected. This provided an accurate account of
all fibers longer than 20 µm in the lung on day 1 as shown in Figure 9 as all such fibers
observed by TEM were thicker than 0.15 µm. Shorter fibers with diameters greater than 0.15
µm were present in considerably larger number than thinner fibers (diameter < 0.15 µm) as to
provide an excellent account of the disposition of these length fibers as well. Similar results
were seen throughout the study with the results from 3 months shown in Figure 10. It is
interesting to note that the shorter fibers (especially below 5 µm in length) with diameter
> 0.15 µm are decreasing in number which suggests that the remaining chrysotile continues
to be removed by macrophages and/or dissolves in the lung.
Figure 9
Fib er Len gth in th e Lun g at 1 D ay aft er cessatio n of exposu re
Fi bers wi th diameter < 0.15 µ m and >=0.15 µm*
*D etermined thr ough the l ung di gesti on procedur e using Tr ansmissi on Elect ron Mi cros copy
0 5 10 15 20 25 30 35 40 45
Leng th µm
0
5000
25000
30000
35000
No of F iber s Counted
Di ameter >= 0.15 µm
Di ameter < 0. 15 µm
Running Title: Biopersistence of Canadian Chrysotile Page 19 of 35
Figure 10
Fib er Leng th in the L ung at 3 Mo nth s aft er cessatio n of exposur e
Fi bers with diameter < 0.15 µm and > =0.15 µm*
*Det ermined thr ough the l ung dig esti on procedur e using Trans mission El ectron M icr oscopy
0 5 10 15 20 25 30 35 40
Lengt h µm
0
2000
4000
6000
8000
28000
No of F ibers Counted
Di ameter >= 0.15 µm
Di ameter < 0. 15 µm
Parenchymal fiber load.
At all exposure time points, the parenchyma contained 99% of the total fiber load.
Sixty-eight percent of the fibers and particles seen at 1 day after cessation of exposure were
found within alveolar macrophages with the remainder occurring on epithelia of alveoli,
alveolar ducts, and terminal bronchioles. This represents a substantial clearance of fibrils
from the respiratory region at this time point. Similar observations were made at 2-day, 7-
day and 14-day after exposure time points.
At 1-month after cessation of exposure, less than one-half (42%) of the fibers were detected
inside alveolar macrophages, a decrease from that found at earlier exposure time point (68%,
60%, 69% and 64% at 1, 2, 7, and 14-days respectively). The balance of fibers observed at
the 1-month post exposure time point was located on the airway epithelia surfaces. It is
important to note that compared to the 1-day post exposure group, 92% of all fibers have
been cleared from the lungs by 1-month post exposure.
Airway fiber load.
Taking the airway fiber load in the 1-day post exposure group as 100%, the fiber loads in the
2-day, 7-day, 14-day and 1-month post exposure groups are respectively 69%, 45%, 54%,
and 29%.
Partitioning of fiber load within airway compartment.
In the airways of the 1-day post exposure animals, 31% of the particles were found in airway
macrophages in contrast to that seen in the 14-day post exposure animals where 58% were
Running Title: Biopersistence of Canadian Chrysotile Page 20 of 35
found in airway macrophages. With the exception of the 14-day post exposure group, higher
numbers of fibrils were observed on the surface of airway epithelia. The fibers on the airway
epithelium and free in the airway lumen accounted for 62% of the fiber load, about 2 times
that of the fiber load carried by airway macrophages. This indicates a steady, long term
clearance pathway by ciliated epithelium within conducting airways, supplemented by airway
macrophage clearance.
Lung fiber lengths in parenchyma and airways
Fiber length within parenchyma, by time point.
The mean fiber length was between 5.5 to 7.4 microns. The number of fibers longer than
20 µm rapidly decreased by a factor of 5 from 1-day post exposure to 2-days post exposure.
No fibers greater than 20 µm were observed in parenchyma at 1-month post exposure. These
data strongly support that fibers, upon contact with lung surfaces and cells, rapidly
disintegrate into shorter fibrils.
Fiber length within airways, by time point.
Fiber lengths in the airways were on average, longer than those seen in the parenchymal
region. In general, average fiber lengths were in the range of 7 to 9 microns. Very few fibers
longer than 20 µm were observed in any animal and by 1-month after cessation of exposure,
no fibers greater than 20 µm were observed in randomly collected image data.
Inflammatory cells
Neutrophils were not observed free in alveolar spaces in any of the data volumes collected.
Neutrophil-mediated inflammatory response did not occur in the presence of chrysotile fibers
at the time points examined to date.
Time course – Confocal Imaging
The disposition of the chrysotile fibers in the lung from 1-day through 30-days after cessation
of exposure is shown in the confocal micrographs which were image processed to identify
through colour coding of the different refractive indices the profiles of the Canadian
chrysotile fibrils in context with lung tissue.
At 1-day after cessation of exposure, the chrysotile fibers appeared to have been well
distributed throughout the lung in both airway and parenchyma. As shown in Figure 11,
fibers appeared as separate, fine fibrils, occasionally unwound at one end and were not found
clumped together. Fibers were found on the surface of a ciliated airway (Figure 11a), in the
alveoli (Figure 11b), phagocytosed by alveolar macrophages (Figure 11c), and the shorter
fibers were found to be transported to the distal pulmonary lymphatics (Figure 11d).
Running Title: Biopersistence of Canadian Chrysotile Page 21 of 35
Figure 11
By 1 month after cessation of exposure, Figure 12 shows four similar regions as shown above
at 1 day (Figure 11). These are the airway wall (Figure 12a), the alveoli (Figure 12b), fibers
in the alveolar macrophages (Figure 12c) and the distal pulmonary lymphatics (Figure 12d).
While some fibers are found in each of these regions, what is notable is that number and
length of fibers observed is greatly reduced.
Running Title: Biopersistence of Canadian Chrysotile Page 22 of 35
Figure 12
The numbers of fibers quantified using confocal examination are shown as a function of time
for the parenchyma (Figure 13) and for the airway (Figure 14) regions of the lung. The
parenchyma and the airway were each subdivided into four compartments as defined below.
In addition the inflammatory cells present were also identified.
Running Title: Biopersistence of Canadian Chrysotile Page 23 of 35
Definitions:
Parenchyma:
1. Touching alveolar, ductal epithelium: Those fibrils observed in serial section series to have
at least one end, or portions in direct contact with alveolar, of alveolar duct epithelium.
2. In alveolar macrophage: Fibrils observed in serial section series to have been completely
internalized by phagocytic engulfment, or portions along the length in direct contact with
alveolar macrophage. This also includes fibrils with at least one end in contact with alveolar
macrophages.
3. Seen in alveolar, ductal airspace: Those fibrils observed in serial sections to have no direct
contact with alveolar, of alveolar duct epithelium.
4. Another location in parenchyma: Those fibrils observed in serial section series to have at
least one end, or portions in direct contact with other structures in parenchyma, such as sub-
epithelial cells, interstitial spaces, lymphatic ducts, or indeterminate structures in the alveolar
region.
Airways:
1. On surface of or intercalated within ciliated epithelium of conducting airway: Those
fibrils observed in serial section series to have at least one end, or portions in direct contact
with epithelium of conducting airways, or observed to be in contact with cells immediately
subjacent to the epithelium of conducting airways.
2. Within airway macrophage: Fibrils observed in serial section series to have been
completely internalized by phagocytic engulfment, or portions along the length in direct
contact with a macrophage in a conducting airway. This also includes fibrils with at least one
end in contact with airway macrophages.
3. In airway lumen; portion of fiber visualized not touching tissue: Those fibrils observed in
serial section series to have no direct contact with conducting airway epithelium.
4. Another location in airway: Those fibrils observed in serial section series to have at least
one end, or portions in direct contact with other structures in conducting airway structures,
such as interstitial spaces, broncho-associated lymphoid tissue, lymphatic ducts, or other
indeterminate structures.
5. Identification of inflammatory cells: Inflammatory cells were identified by examination in
serial section image data the nuclear morphology of cells and surrounding pulmonary tissue.
Mononuclear cells, such as alveolar macrophages were easily distinguished from neutrophils
which exhibit poly-morphonuclear profiles.
For both the parenchyma and airway regions, from 1-day to 30-days after cessation of
exposure there is a marked reduction in the number of fibers observed in all compartments.
As mentioned above, the large majority of fibers are found in the parenchyma with most of
Running Title: Biopersistence of Canadian Chrysotile Page 24 of 35
those found in the alveolar macrophages (Figure 11). By 1 month post exposure, fibers were
no longer observed in the interstitium.
While these confocal images provide quantification of where the chrysotile fibers are in the
lung, they do not show the surrounding lung surfactant which is very important in mediating
dissolution in the lung. Nearly all the fibers observed are within the surfactant layer and
therefore more readily subject to dissolution with the biodegradation of chrysotile being
diffusion-dependant (Atkinson, 1973). In addition, Etherington et al. (1981) has shown that
macrophages can generate low pH at the surface of the macrophage membrane and, in
particular, within the macrophage phagolysosome which surrounds the particles, pH values as
low as 3.5 are encountered. Chrysotile is most soluble at such acid pH. When in contact
with dilute acids or even aqueous media at pH<10, magnesium has been shown to readily
dissociate from the fiber’s surface (Hargreaves & Taylor, 1946; Atkinson, 1973; Nagy &
Bates, 1952) resulting in a leached fiber (Atkinson, 1973). The results of our study are
consistent with these proposed mechanisms leading to the breakup of longer chrysotile fibers
into shorter pieces.
Figure 13
Distribution of Fibers (all lengths) in the Airways
Percentage of the total number of fibers in the lung
12 7 14 30
Time since cessation of exposure (days)
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
Percent of the total number of fibers in the lung
On surface of or intercalated within ciliated
epithelium of conducting airway
Within airway macrophage
In airway lumen; portion of fiber visualized
not touching tissue
All other locations in the airway
Running Title: Biopersistence of Canadian Chrysotile Page 25 of 35
Figure 14
Distribution of Fibers (all lengths) in the Parenchyma
Percenta
g
e of the total number of fibers in the lun
g
12 7 14 30
Time since cessation of exposure (days)
0
10
20
30
40
50
60
70
80
Percent of the total number of fibers in the lung
Touching alveolar, ductal epithelium
In alveolar macrophage
In alveolar ductal airspace
All other locations in parenchyma
Discussion :
Fiber structural chemistry and rapid dissolution:
In chrysotile, the magnesium hydroxide part of each layer is closest to the fiber surface and
the silica tetrahedral is within the structure (see Figure 1). In water, the dissolution of
chrysotile has been shown to be effected by the buffer capacity of the leach solution with the
amount of extractable Mg and SiO2 increasing with increasing buffer strength (Smith, 1973).
This reaction has been determined to be diffusion controlled through a layer of water near the
mineral’s surface.
In the lung, extensive work on modeling the dissolution of synthetic mineral fibers (SMF)
using in-vitro dissolution techniques and inhalation biopersistence has shown that the lung
Running Title: Biopersistence of Canadian Chrysotile Page 26 of 35
has a very large buffer capacity (Matson, 1994). These studies have shown that an equivalent
in-vitro flow rate of up to 1 ml/min is required to provide the same dissolution rate of SMF as
that which occurs in the lung. In addition, chrysotile is more soluble at acid pH and thus
may be affected by complete or even partial phagocytosis of fibers by macrophages.
With the chrysotile tested, it appears that as the magnesium dissolves, the fiber breaks apart
into smaller pieces. Thus, while the rats were exposed to a very large number of long
respirable fibers (200 fibers L>20 µm/cm3, GMD=0.12 µm), it was observed that by the 6th
day of the study (1 day after cessation of exposure) already a large number of fibers had
dissolved/disintegrated.
Dose delivered and comparative clearance:
To assess how much of the deposited dose had been cleared from the lung by the first time
point of analysis (day 1 after cessation of exposure) and what the relative lung burdens are of
chrysotile in comparison to an amphibole and a highly soluble fiber, we compared the data
from this study to that from a inhalation biopersistence study of amosite asbestos and the
soluble stonewool fiber MMVF 34 (25). In the Hesterberg et al. (1998), study, the aerosol
exposure concentrations were 150 fibers L>20 µm/cm3. The GMD of the WHO fibers was
reported as 0.48 µm and 0.73 µm for amosite and MMVF 34, respectively (the GMD of the
long fibers was not reported). As described above, the chrysotile exposure concentrations
were 200 fibers L>20µm/cm3 with GMD of these fibers of 0.12 µm and thus were also rat
respirable.
To compare the two studies, we multiplied the lung burdens of the fibers L> 20 µm given by
Hesterberg et al. (1998) by two to provide ‘calculated’ equalized doses as the chrysotile
exposure of long fibers in our study was twice that of the Hesterberg study. The numbers of
fibers in the lung as a function of time since cessation of exposure is shown in Figure 13. If
chrysotile was insoluble, on day 1 after cessation of exposure, approximately 5 x 106 fibers
with L > 20 µm would have been found in the lung as seen for amosite and MMVF 34. The
chrysotile, however, is so soluble that approximately 3 x 105 fibers L> 20 µm or 6% remain
in the lung at 1 day after cessation of the 5 day exposure. The clearance of MMVF 34 which
has a reported half-time of 6 days (Hesterberg et al., 1998) is seen to quickly diverge from
that of amosite most of which remains in the rat for its life-time. By 90 days, more than 1 x
106 long amosite fibers remain. In comparison, the long MMVF 34 and chrysotile fibers are
reduced by approximately 100 fold. This result is interesting as MMVF 34 was tested in a
chronic inhalation study at high exposure concentrations and produced neither fibrosis nor
tumors (Kamstrup, et al., 1998). As shown in panel insert in Figure 15, at 30 days after
cessation of exposure, using the extended counting procedures described above, only 1 to 2.5
fibers longer than 20 µm were observed on the filter from the digested lung for each animal,
thus the levels present are rapidly approaching the background level.
Running Title: Biopersistence of Canadian Chrysotile Page 27 of 35
Figure 15
Comparison with other chrysotile biopersistence studies:
This study provides the first application of the EC biopersistence protocol to chrysotile.
Ilgren & Chatfield (1998) reviewed a number of studies which provided estimates of
clearance of chrysotile based upon the silica content of the lung. These studies reported
clearance half-times considerably longer than that found here. This analysis however did not
differentiate clearance as a function of fiber length or compartment within the lung. As seen
in our study, the long chrysotile fibers clear most rapidly while some of the shorter fibers
accumulate in the lung and lymphatics. Nearly all of these previous studies involved
exposure periods of from 3 to 12 months and used a range of exposure concentrations from 2
to 10 mg/m3. With an exposure concentration of 10 mg/m3, the total fiber concentration was
more than 1 x 106 fibers (Mast et al., 1995). The number of non-fibrous particles was not
reported although from the current study this could equal the number of fibers. With these
very high exposure concentrations it is likely that rat specific lung overload occurred which
would present a serious bias in any lung clearance measurements (Oberdoester 1995a & b,
2002). In addition, in these studies there was no reported investigation of the presence of
other silicates in the aerosol and especially of amphiboles fibers such as tremolite. Wagner
et al., (1980) stated that “all materials contained impurities” in the chrysotile samples that he
studied although he did not identify these impurities.
Running Title: Biopersistence of Canadian Chrysotile Page 28 of 35
Fibers Remaining
We have seen that chrysotile clears with a half-time of 16 days for fibers L> 20 µm.
However, as stated above, some long fibers are observed at 30 days. The question, of course,
remains of whether these few remaining fibers are biologically relevant in producing a
possible pathological response.
The question of the possible effect of shorter chrysotile fibers has been addressed by the
chronic inhalation studies reported by Ilgren and Chatfield (1997, 1998a, 1998b). In these
studies, 7 hours/day, 5 days/week for 12 months to a mean exposure concentration of
7.8 mg/m3 of Coalinga chrysotile. The Coalinga chrysotile was reported as being relatively
short with the majority of fibers less than 5 µm in length. No fibrotic or tumorigenic
response was observed following exposure to this fiber. Similar results were reported in
another study with the Coalinga fiber by Muhle et al. (1987). In addition, the Coalinga fiber
was tested in four chronic IP studies of up to 3 mg dose with tumor levels in the reported
background range of up to 10 % (Muhle et al., 1987; Pott et al., 1987; Rittinghausen et al.,
1992). These studies provide support that shorter chrysotile is not carcinogenic following
both inhalation and IP exposure at relatively high concentrations. In addition, in a Report on
the Expert Panel on Health Effects of Asbestos and Synthetic Vitreous Fibers: The Influence
of Fiber Length, issued recently by the Agency for Toxic Substances and Disease Registry
(ATSDR) it was stated that “Given findings from epidemiologic studies, laboratory animal
studies, and in vitro genotoxicity studies, combined with the lung’s ability to clear short
fibers, the panelists agreed that there is a strong weight of evidence that asbestos and SVFs
(synthetic vitreous fibers) shorter than 5 µm are unlikely to cause cancer in humans”
(ATSDR, 2003).
Conclusion:
Taken in context with the scientific literature to date, this report provides new robust data
which clearly supports the difference seen epidemiologically between chrysotile and
amphibole asbestos.
Running Title: Biopersistence of Canadian Chrysotile Page 29 of 35
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Running Title: Biopersistence of Canadian Chrysotile Page 33 of 35
Figure Captions:
Figure 1: Schematic representation of the chemical structure of chrysotile showing the Mg
molecule is on the outside of the curl (adopted from Skinner et al., 1988).
Figure 2: Transmission electron micrographs of chrysotile showing the curled sheetlike form
of the fibers (Kiyohara, 1991)
Figure 3: Schematic representation of the chemical structure of tremolite showing the Mg
which is locked within the I-beam structure (Adapted with permission from: Department of
Geology and Geophysics, University of Wisconsin, Crystal Structure Movies,
http://www.geology.wisc.edu).
Figure 4: Bivariate length and diameter histogram of the chrysotile WHO fibers in the
exposure atmosphere.
Figure 5: Bivariate length and diameter histogram of the chrysotile WHO fibers recovered
from the lung at 1 day following cessation of exposure.
Figure 6: Photomicrographs of the original bulk chrysotile sample taken using scanning
electron microscopy (SEM). SEM was used for these micrographs in order to provide a
visual overview of the fiber size distribution. As described above, transmission electron
microscopy (TEM) was used for all quantification of fiber size.
Figure 7: Photomicrographs of chrysotile fibers from an aerosol sample taken using scanning
electron microscopy (SEM). SEM was used for these micrographs in order to provide a
visual overview of the fiber size distribution. As described above, transmission electron
microscopy (TEM) was used for all quantification of fiber size.
Figure 8: Graph showing the clearance of the Canadian chrysotile fibers longer than 20 µm
from the lung following cessation of the 5 day exposure period. The diamonds indicate the
percent remaining of the individual lungs. The solid line is the clearance curve fitted to the
data using non-linear regression techniques with a double exponential (StatSoft, Inc., 2003).
The regression coefficients are presented in Table 4. (Note that 5 lungs were analyzed at each
time point, however, in the Figure some points are superimposed on each other.)
Figure 9: Length histogram of fibers recovered from lungs at 1 day following cessation of
exposure for those fibers which were < 0.15 µm in diameter and > 0.15 µm in diameter. This
confocal microscopy measurements which has a detection limit of 150 nm thus provides an
accurate account of all fibers longer than 20 µm in the lung as all such fibers observed by
TEM were thicker than 0.15 µm. Shorter fibers with diameters greater than 0.15 µm were
present in considerably larger number than thinner fibers (diameter < 0.15 µm) and provide
an excellent account of the disposition of these length fibers as well.
Figure 10: Length histogram of fibers recovered from lungs at 3 months following cessation
of exposure for those fibers which were < 0.15 µm in diameter and > 0.15 µm in diameter.
Running Title: Biopersistence of Canadian Chrysotile Page 34 of 35
This confocal microscopy measurements which has a detection limit of 150 nm thus provides
an accurate account of all fibers longer than 20 µm in the lung as all such fibers observed by
TEM were thicker than 0.15 µm. Shorter fibers with diameters greater than 0.15 µm were
present in considerably larger number than thinner fibers (diameter < 0.15 µm) and provide
an excellent account of the disposition of these length fibers as well.
Figure 11: Confocal micrographs recorded from 1 Day post exposure group. All images
were digitally processed from original optical sections, approximately 0.5 microns thick
recorded from undisturbed regions of the lung. Pulmonary tissue appears as a grayscale
composite with profiles of test article (red). Fibers were found on the surface of ciliated
airways. Panel A (upper left) shows a cross section of a large airway with a thin fibril resting
on ciliated epithelial cells. Scale bar is 5 microns. Fibers were observed in the alveoli. Panel
B (upper right) shows a typical field of view in the parenchymal region and reveals numerous
fibrils next to an alveolar wall and a few fibrils appearing free in the alveolar space. Scale bar
is 10 microns. Fibers were frequently observed to have been phagocytosised by alveolar
macrophages. Panel C (lower left) shows fiber clearance by alveolar macrophages.
Numerous alveolar macrophages appear to surround fibrils in the alveolar space. These cells
are known to carry foreign material from the gas exchange region to the airways, ultimately
to be swept up to the trachea and cleared from the pulmonary compartment altogether. Scale
bar is 10 microns. The shorter fibers were found to be transported to the distal pulmonary
lymphatics. Panel D (lower right) shows a distal pulmonary lymphatic duct containing short
chrysotile fibrils as well as a cell containing numerous chrysotile profiles. It is clear the
pulmonary lymphatics are a clearance pathway for material deposited via inhalation
exposure. Scale bar is 10 microns.
Figure 12: Confocal micrographs recorded from the 1 Month after exposure group. All
images were digitally processed from original optical sections, approximately 0.5 microns
thick recorded from undisturbed regions of the lung. Pulmonary tissue appears as a grayscale
composite with profiles of test article (red). At this time point, very few micrographs actually
contain profiles of the test article. These particular images were selected to show the
appearance of chrysotile, when present. Fibers were found on the surface of ciliated airways.
Panel A (upper left) shows a cross section of a large ciliated airway near a bifurcation with a
sort, thin fibril inside a cell adjacent to the epithelium. Scale bar is 10 microns. Fibers were
observed in the alveoli. Panel B (upper right) shows a typical field of view in the
parenchymal region showing an alveolar wall, and alveolar space. Scale bar is 10 microns.
Occasional fibers were observed phagocytosed by alveolar macrophages. Panel C (lower
left) shows fiber clearance by an alveolar macrophage. Scale bar is 10 microns. Very few
fibrils were observed in alveolar interstitial spaces, including distal pulmonary lymphatics.
Panel D (lower right) shows a distal pulmonary lymphatic duct (lower center of the image)
devoid of any chrysotile fibrils. Calcified structures were occasionally found on the alveolar
epithelium, but no adverse tissue response has been observed associated with these structures.
Scale bar is 10 microns.
Figure 13: The numbers of fibers quantified using confocal examination are shown as a
function of time for the parenchyma region of the lung. As defined in the text, the
parenchyma was subdivided into the following four compartments: Fibers Touching
Running Title: Biopersistence of Canadian Chrysotile Page 35 of 35
alveolar, ductal epithelium; In alveolar macrophage; Seen in alveolar, ductal airspace; and
Another location in parenchyma:
Figure 14: The numbers of fibers quantified using confocal examination are shown as a
function of time for the airway region of the lung. As defined in the text, the airway was
subdivided into the following four compartments: Fibers On surface of or intercalated within
ciliated epithelium of conducting airway; Within airway macrophage; In airway lumen -
portion of fiber visualized not touching tissue; and Another location in airway.
Figure 15: The number of fibers in the lung as a function of time since cessation of exposure
is shown for Canadian chrysotile, MMVF 34 and amosite. If chrysotile was insoluble, on day
1 after cessation of exposure, approximately 5 x 106 fibers with L > 20 µm would have been
found in the lung as seen for amosite and MMVF 34. The chrysotile, however, is so soluble
that only approximately 3 x 105 fibers L> 20 µm remain in the lung at 1 day after cessation
of the 5 day exposure. The clearance of MMVF 34 which has a reported half-time of 6 days
is seen to quickly diverge from that of amosite most of which remains in the rat for its life-
time. By 90 days, more than 1 x 106 long amosite fibers remain. In comparison, the long
MMVF 34 and chrysotile fibers are reduced by approximately 100 fold. The panel insert
shows that at 30 days after cessation of exposure, using the extended counting procedures
described in the text, from 1 to 2.5 fibers longer than 20 µm were observed on the filter from
the digested lung for each animal.
