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A Review of Fiber Biopersistence
as a Potential Mechanism of Asbestos Tumorigenicity
David Bernstein
Thomas Hesterberg
Ken Donaldson
Gunther Oberdörster
Abstract:
It is well accepted that the dose, dimensions, durability of respirable fibers in the lung, and in some
cases the surface characteristics of the fibers, are the critical determinants of their potential adverse
health effects. This mechanistic understanding of fiber toxicology is based primarily on the results of
well conducted chronic inhalation toxicology studies of synthetic vitreous fibers. More recently,
studies have extended these concepts to the two major classes of asbestos; serpentine asbestos
(chrysotile) and amphibole asbestos (e.g., crocidolite, amosite, etc.). Chrysotile asbestos is a very
thin rolled sheet silicate, which can be dissolved and broken apart by the acidic environment inside
the lysosomes of macrophages. In contrast, amphibole asbestos is a chained silicate, which is
formed in a solid cylindrical shape with a quartz like external surface and is insoluble at all pHs. The
existing database of fiber toxicity studies strongly suggests that human exposure to respirable fibers
that are biopersistent in the lung and induce significant and persistent pulmonary inflammation, cell
proliferation, and fibrosis should be viewed with concern.
Introduction:
‘Asbestos’ is not a mineral in itself. Instead, it is a collective term given to a group of minerals having
crystals that occur in fibrous forms. The term ‘asbestos’ was adopted for commercial identification.
The six minerals commonly referred to as asbestos come from two distinct groups of minerals. One
group is known as serpentines (chrysotile, white asbestos); while the other group is termed the
amphiboles (amosite, brown asbestos; crocidolite, blue asbestos; anthophyllite; tremolite; and
actinolite). While both are silicate minerals, the two groups are chemically and mineralogically
distinct.
Recent studies have shown that these two classes of asbestos are very different in their toxicological
potential. A greater knowledge of the differences in mineralogy and chemistry of these two classes
of mineral fibers has provided a mechanistic basis for understanding the differences in their toxicity.
Many of the earlier studies of these two classes of asbestos did not demonstrate such differences in
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toxicity and suggested that serpentine asbestos (chrysotile) was of similar potency to amphibole
asbestos. This paper provides a review of not only the more recent studies, which demonstrate a
marked difference between chrysotile and amphibole asbestos, but also examines on a quantitative
basis the earlier toxicological studies in order to determine whether there is coherence in the
results.
THE DIFFERENCES IN SERPENTINE AND AMPHIBOLE ASBESTOS:
SERPENTINE ASBESTOS (CHRYSOTILE)
Chrysotile is a cylindrical fibrous silicate, which is formed as a very thin rolled sheet, as illustrated in
Figure 1.
The sheet, which is composed of a sandwich of magnesium and silica, is about 8
angstroms (0.8 nanometers) thick and the magnesium is on the outside of the role. Magnesium is an
essential element in the body (the adult human body contains approximately 20-28 gm of
magnesium, WHO, 2002) and is soluble in the lung surfactant (NAS, 1980). In the lung, the
magnesium dissociates and the crystalline structure of this sheet silicate is readily attacked by acid,
such as occurs upon contact with or when phagocytized by the macrophage (pH 4 - 4.5). This
process causes the rolled sheet of the chrysotile fiber to break apart and decompose into small
pieces (Figure 2). These pieces can then be readily cleared from the lung by macrophages through
muco-cilliary and lymphatic clearance. If the fiber is swallowed and ingested it is attacked by the
even stronger acid environment (hydrochloric acid, pH 2) of the stomach.
Figure 1
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Figure 2
AMPHIBOLE ASBESTOS (CROCIDOLITE, AMOSITE, TREMOLITE, ETC.):
This is in contrast to the amphibole asbestos class of fibers, which are formed as solid rods/fibers as
illustrated in Figure 3. The structure of an amphibole is a double chain of silicate tetrahedral which
makes it very strong and durable. The external surface of the crystal structures of the amphiboles is
quartz-like, and has the chemical resistance of quartz. The amphibole fibers have negligible
solubility at any pH that might be encountered in an organism.
Figure 3
The characteristics of these two classes of mineral fibers, serpentines and amphiboles, have been
long described in the scientific literature. The differences in chemistry and solubility would imply
that within the milieu of the pulmonary system that these two minerals would behave differently.
Pundsack (1955) described the colloidal and surface chemistry of chrysotile and how it behaves in
suspension. Hargreaves and Taylor (1946) indicated that “If fibrous chrysotile is treated with dilute
acid the magnesia may be completely removed, and the hydrated silica remaining, though fibrous in
form, has completely lost the elasticity characteristic of the original chrysotile fibre and gives an Xray pattern of one or perhaps two diffuse broad bands indicating that the structure is 'amorphous' or
'glassy' in type.” The acid insolubility of amphiboles in comparison to chrysotile has been confirmed
experimentally by Speil and Leineweber (1969).
FACTORS WHICH INFLUENCE FIBER TOXICITY:
Mineral fiber toxicology has been associated with three key factors: dose, dimension and durability.
One determinant of the dose to the lung is the fiber exposure level, which is determined by the
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fiber’s physical characteristics/dimensions, how the fibrous material is used, and the industrial
hygiene control measures that are implemented. In addition, the thinner and shorter fibers will
weigh less and thus can remain suspended in the air longer than thicker and longer fibers. Most
asbestos fibers are thinner than commercial insulation fibers, and depending upon type are thicker
or in the range of the new nano-fibers which are currently being developed.
The fiber dimensions (length and diameter) also determine whether a fiber is respirable, i.e.,
whether it will be deposited in the alveolar region of the lung. Fiber length is also a factor in
determining toxicity in the lung milieu once inhaled. Shorter fibres, of the size which can be fully
engulfed by the macrophage, will be cleared by mechanisms similar to those for non-fibrous
particles. These include clearance through the lymphatics and macrophage phagocytosis and
clearance via the mucociliary escalator. It is only the longer fibers, which the macrophage cannot
fully engulf, which if they are biopersistent can lead to disease.
This leads to the third factor, that of durability. Those fibers whose chemical structure renders them
wholly or partially soluble once deposited in the lung are likely to either dissolve completely, or
dissolve until they are sufficiently weakened locally to undergo breakage into shorter fibres. The
remaining short fibres can then be removed though successful phagocytosis and mucociliary
clearance.
Synthetic vitreous fibers (SVFs) such as fiber glass are amorphous silica structures. In the lung, these
fibers have been found to dissolve by two principal mechanisms either through congruent or
incongruent dissolution. Congruent dissolution of SVFs leads to complete dissolution of the fibers
over time. In contrast, incongruent dissolution leads to leaching of metal oxides from the fiber,
leaving a weakened silicon dioxide matrix, which can lead to fiber breakage. The shortening of fibers
resulting from fiber breakage can enhance macrophage-mediated clearance from the lung, thus
minimizing toxicity, even from long fibers, if they are biosoluble.
The association of longer fibers (20–50 µm) with pathological effects in the lung compared to the
lack of toxicity of shorter fibers (3 µm or less) was reported as early as 1951 by Vorwald et al. (1951).
Vorwald concluded that “The mode of action of the long asbestos fiber in the production of
asbestosis is primarily mechanical rather than chemical in nature” and that “Long asbestos fibers are
essential in the production of the peribronchiolar fibrosis; short fibers are incapable of producing
this reaction.”
A fiber is unique among inhaled particles in that the fiber’s aerodynamic diameter is largely related
to three times the fiber diameter (Timbrell, 1982). This is because fibers tend to line up with the air
flow within the lung, making diameter the most important determinant of deposition (Bernstein,
2006). Thus long thin fibers can penetrate into the deep lung. Within the lung, shorter fibers, which
can be fully engulfed by the macrophage, can be removed as with any other particle. However,
those fibers, which are too long to be fully engulfed by the macrophage, cannot be cleared by this
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route. Zeidler-Erdely et al. (2006) have shown that 20 µm fibers can be fully engulfed by human
alveolar macrophages. Longer fibers will remain in the lung and can be cleared from the lung only if
they can dissolve or break apart.
In the lung, extensive work on modeling the dissolution of mineral fibers, using dynamic in-vitro
dissolution techniques and inhalation biopersistence, has shown that the lung has a very large
buffering capacity (Mattson, 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 SVFs at pH 7.4 as that which
occurs in the lung. (at ph 4.5?)
In humans, the normal amount of fluid entering the lung’s tissue
which is removed through the lymphatic flow has been estimated as 0.2 ml/min in non-exposed
lungs and can increase by more than 15 fold in response to injury (Taylor, 2006).
The relationship between chemical composition and dissolution and subsequent breakage was first
reported by Hammad (1984). Synthetic mineral fibers <5 µm in length had the longest retention in
the lung following short-term inhalation, with longer fibers clearing more rapidly and fibers >30 µm
in length clearing very rapidly. He proposed that clearance of SVFs is the result of biological
clearance and the elimination of fibers by dissolution and subsequent breakage. He proposed that
the long fibers were leaching and breaking into shorter fibers, which explains the rapid
disappearance of these fibers from the lung. The shorter fibers appear to have a longer retention
time, because these fibers were added to the pool via breakage of longer fibers over time. The
importance of this relationship to fiber toxicity has been quantified for synthetic mineral fibers
(Hesterberg et al. 1998a & b; Miller et al. 1999; Oberdörster, 2000; Bernstein et al 2001a & b) and
more recently in differentiating the toxicity of serpentine asbestos, e.g., chrysotile, from amphibole
asbestos, i.e., amosite and crocidolite (Bernstein & Hoskins, 2006).
Chronic inhalation toxicity studies of chrysotile
Early chronic inhalation studies of fibers were often performed without consideration of the
respirability of the fibers in the rat lung and without preserving the length distribution of the fibers.
In addition, they were often performed at very high total particle/fiber exposure concentrations. As
asbestos fibers often occur in bundles of long strands, investigators would grind the fibers to
produce a more respirable fraction instead of separating the long thin fibers from the bulk material.
This process frequently pulverized the long respirable fiber fraction producing excessive particles
and shorter fibers, sufficient to cause lung overload in the rats.
High concentrations of insoluble dusts [GO: Yes, but overload is defined for poorly soluble particles
of low cytotoxicity: crystalline SiO2 is not part of this; are amphiboles? Are described earlier as
having quartz-like surface], when administered by inhalation in the rat, have been shown to
overload the lung by compromising the clearance mechanisms, which can then result in chronic
inflammation, fibrosis and a tumor formation (Bolton et al., 1983; Muhle et al., 1988; Morrow, 1988
& 1992; Oberdorster, 1995a&b).
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As illustrated in Figure 4, inhalation toxicology studies of asbestos were performed well above the
levels to which humans have been exposed. However, when the exposure level is elevated to more
than 100,000 times human exposure, as occurred in most older inhalation studies using chrysotile
and amphibole asbestos, lung overload occurs. [GO: I did not associate asbestos-induced effects
with overload in the PSP sense. If you do, you need to show that short asbestos is of low
cytotoxicity, like carbon or TiO2. Thus, it should be presented differently: Effects seen in rats with
inhaled high concentration PSPs occur with cytotoxic particles (e.g., crystalline SiO2) already at much
lower concentrations, and obviously even more at PSP-like higher concentrations/doses. Fig. 4
shows the concept, for cytotoxic particles it is just shifted to the left. If text is left as is, will be cause
for criticism, unless there are data shown that short amphibole is low cytotoxic.] [ DB: Davis et al.,
1986, showed that short fiber amosite did not produce disease following chronic inhalation. (Davis,
J.M.G., et al., "The Pathogenicity of Long Versus Short Fibre Samples of Amosite Asbestos Administered to Rats by
Inhalation and Intraperitoneal Injection," British Journal of Experimental Patholog, 67:415-430, 1986.)
Figure 4
Problems in Interpretation of Past Inhalation Studies
In reviewing the scientific literature of animal studies, it is often difficult to evaluate the exposure or
lung dose administered in comparison to human exposures, the bivariate size distribution of the
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fibers, whether dose-response was evaluated, and the route of exposure used in the study. The
expression of fiber dose to the deep lung is often the weakest component in these earlier studies.
Many studies reported only gravimetric concentration of the aerosol with no information provided
on fiber number or bivariate fiber size distribution. This is of particular importance in differentiating
the effects of various types of fibers, as fiber length is related to potency. Where fiber number is
reported, it was often assessed using phase contrast optical microscopy (PCOM) or scanning electron
microscopy (SEM), which for asbestos fibers, can detect only a fraction of the total fibers present.
Only transmission electron microscopy (TEM) can accurately detect all of the fibers in the exposure
aerosol or deposited in the lung. When conducting fiber toxicology studies, it should be standard
practice to use TEM to determine the bivariate length and diameter of fibers in the exposure aerosol
and lung. These data can then be used to determine exposure levels in fibers/cm3 and lung
deposition.
Dose-response assessments are lacking in most in vivo and in vitro studies. The large majority of
earlier inhalation exposure studies and many of the more recent studies have been performed at an
apparently arbitrary dose of 10 mg/m3. As an example, the fiber number exposure attained at a 10
mg/m3 exposure to chrysotile has been reported as measured by phase contrast light microscopy
(PCOM) to be approximately 2000 WHO fibers/cm3 (length greater than 5 µm). When a similar mass
concentration of another chrysotile was measured by SEM, a total fiber count of 100,000 fibers/cm3
was observed (Mast et al., 1995; Hesterberg et al. 1993). TEM analysis would show as much as 17
times more fibers than SEM analysis on the same aerosol samples (Breysse et al., 1989). Thus, TEM
measurement of the 10 mg/m3 exposure to chrysotile would likely correspond to more than
1,000,000 fibers/cm3. This is 500 times the number of fibers/cm3 measured using PCOM. Also, there
are few quantitative data presented in past publications on nonfibrous particle content of test fiber
preparations. Since nonfibrous particles can contribute to lung overload (Oberdörster et al., 1995),
particle/fiber numbers and dimensions should be comprehensively evaluated in materials used in
future work. The historical chrysotile chronic inhalation studies are summarized in Table A1
(Appendix 1). The exposure concentration in all studies were based upon gravimetric determination.
Of the 16 studies, 6 did not report the fiber concentration, 8 reported estimates by PCOM and 3 by
SEM. The gravimetric exposure concentrations ranged from 2 to 86 mg/m3, which based upon the
extrapolation described above (Mast et al., 1995; Breysse et al., 1989), corresponds to between
200,000 and 8,600,000 fibers/cm3. The large majority of these earlier studies targeted 10 mg/m3.
The single study performed at the lowest concentration of 2 mg/m3 had a comparative
concentration group of 10 mg/m3. In this study, the author’s reported “With a 2 mg/m3 cloud the
percentage retention of chrysotile is almost double that for a 10 mg/m3 cloud,” which reflects the
difficulty of evaluating dose response at these overload conditions.
Exposure of rats to high aerosol concentrations of fibers creates a very different dose profile in the
lung in comparison to human exposures. The rat is considerably smaller than humans and
correspondingly the rat’s lungs are more than 300 times smaller than the human lungs. While the
rat inhales proportionally less air per minute, the doses administered in some toxicology studies can
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result in unrealistic fiber lung burdens as compared to human exposure. In addition, for the rat
which is mandatory nasal breather, alveolar deposition is largely limited to fibers less than
approximately 1 µm in diameter, while in humans, this limit is approximately 3 µm (Morgan, 1985).
For most asbestos fibers, however, this difference is less important than for MMVF.
Bernstein (2007) has compared the respiratory and physiological parameters of the rat and human
lung and their influence on fiber deposition (Table 3). It should be noted that 100 % [GO: why not
using realistic deposition fraction?] deposition was assumed. Deposition is a function of fiber
dimension and species. It is estimated that the actual deposition for such fibers is between 10 and
20 %.
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Table 3: Respiratory and physiological parameters in the rat and human and their influence of fiber
deposition (Reproduced from Table 4 in Bernstein (2007))
Parameter
Minute ventilation* (ml/min)
Volume inhaled/6 h day (ml)
Lung Volume* (ml)
Alveolar Surface Area* (µm2)
Number Alveoli/lung*
Number Alveolar Cells/lung*
FIBER EXPOSURE CONCENTRATION (f/ml)
Number Fibers Inhaled/day [WHO? Total?]
Number fibers/alveoli/day
Number fibers/alveolar cell/day
Number fibers/ µm2 of alveolar surface/day
Assuming
100%
deposition
Rat
Human
Rat/Human
Ratio
100 [GO: default:
200]
36,000
13
463,000,000,000
40,000,000
130,000,000
7,000
0.014
2,520,000
4341
143,000,000,000,000 1
1,000,000,000
56,000,000,000
0.014
0.003
0.003
0.04
0.002
100
3,600,000
0.09 ( see 2)
0.03 (see 2)
0.0000078
0.2
504,000
0.0005
0.000009
0.000000004
500
7
179
3,077 (see 3)
2,206
1.
FIBER EXPOSURE CONCENTRATION (f/ml)
Number Fibers Inhaled/day
Number fibers/alveoli/day
Number fibers/alveolar cell/day
Number fibers/ µm2 of alveolar surface/day
1,000
36,000,000
0.9
0.28
0.000078
0.2
504,000
0.0005
0.000009
0.000000004
5,000
71
1,786
30,769
22,061
2.
FIBER EXPOSURE CONCENTRATION (f/ml)
Number Fibers Inhaled/day
Number fibers/alveoli/day
Number fibers/alveolar cell/day
Number fibers/ µm2 of alveolar surface/day
100,000
3,600,000,000
90
28
0.0078
0.2
504,000
0.0005
0.000009
0.000000004
500,000
7,142 (see 3)
178,571
3,076,923
2,206,109
* Pinkerton et al. (1992)
1 – 143,000,000,000,000 --- that is TLC, should use FRC. EPA’s Particle Health Criteria Document has
rat and human normal values.
2 – there are only 3 cells per alveolus?
3 – these are just proportionally higher, no need to bring all three. Point is made well with first
example. However, it would be better to use lung parameters as in EPA, and add assuming 100%
deposition.
In the chronic inhalation study mentioned above using NIEHS chrysotile (Mast et at, 1995;
Hesterberg et al. 1993), in which total fiber aerosol exposure was reported by SEM as 100,000 f/cm 3
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and which by TEM would have been more than 1,000,000 f/cm3, the total chrysotile lung burden
following 24 months of exposure was by SEM observed to be 5.5 X 1010 fibers/lung (Bernstein, 2007),
however, with extrapolation to that which would have been observed by TEM (Breysse et al., 1989)
the lung burden in this study would be 9.4 X 1011 fibers/lung. From Table 3, this would correspond to
an average of 2,338 fibers reaching each alveoli (assuming 10 % deposition).
Similar results were reported in the chronic inhalation study on crocidolite asbestos (McConnell et
al., 1994). In this study, exposure was stopped after 26 weeks (instead of the planned 104 weeks)
because of the intense inflammatory response in the lung that was causing the death of animals.
The aerosol exposure was 1,610 WHO fibers/cm3 with a total fiber concentration of 4,214fibers/cm3.
At 24 months (after 26 weeks of exposure and 78 weeks of no exposure) there were 2.03 x 109 fibers
per lung reported with 88 x 106 fibers longer than 20 µm per lung (Bernstein et al., 2007). Using the
number of alveoli/lung (Table 3), this fiber lung burden corresponds to 50 total fibers per alveoli of
which there were 2 fibers/alveoli longer than 20 µm following only 26 weeks of exposure and 78
weeks of clearance.
The percent tumors in these studies corresponds very closely to the percent of tumors in chronic
inhalation studies that were reported as overload when the fiber number and dimension parameters
are modelled as described by Oberdörster (2002) Figure 6.
An asbestos exposure concentration of 10 mg/m3 corresponds to more than 18 million times the
ACGIH Threshold Limit Value (TLV) of 0.1 f/cm3 for asbestos fibers. At an exposure level 18 million
times the TLV, it would be reasonable to expect that the lung would be overloaded and have
difficulty clearing even the shorter fibers deposited there. These lung overload conditions [GO: It
would be best to give the estimated mass lung burden under these conditions, e.g., x mg/rat lung.
Again, “overload” with more cytotoxic particles occurs at much lower lung burdens – this was not
reported in these studies] would be sufficient to severely reduce the normal clearance of the
chrysotile fibers or any poorly soluble particle (PSP) from the lung (Oberdörster 1995, 2002). While
some shorter fibers may still be cleared through the lymphatic system, the clearance of intermediate
and longer fibers, which require macrophage mediated clearance, would be significantly
overwhelmed as a result of the impairment of macrophage motility at this exposure level
(Oberdörster 1995, 2002). In the future, well-designed inhalation studies should be performed in the
range of concentrations of human exposures or within one or two orders of magnitude. In addition,
it would be important to include dose-response studies using multiple exposure levels.
In 1988, a series of chronic inhalation studies of synthetic mineral fibers (SMF) were conducted,
which took into account the respirability of the fibers in rats and the importance of fiber length in
both the preparation of the fibers and the exposure techniques (Hesterberg et al., 1993, 1995; Mast
et al., 1995a, 1995b; McConnell et al., 1994, 1995). The results of these studies indicated that the
more biosoluble fibers tested showed little or no pathogenic response, while less biosoluble fibers
showed a greater response. Unfortunately, these studies did not use the same selection and
exposure criteria for the chrysotile and crocidolite asbestos control groups that were included.
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These positive control groups were included in this series of inhalation studies primarily to show that
asbestos could induce fibrosis and tumors in the rat model that was being used at an exposure
concentration of 10 mg/m3.
To further investigate the importance of biopersistence to fiber pathogenicity, a 5 day inhalation
protocol was developed for the evaluation of the biopersistence of SVF (Musselman et al., 1994;
Bernstein et al., 1994) with numerous fibers analyzed using this protocol (Bernstein et al., 1996;
Hesterberg et al., 1998). This 5-day inhalation exposure protocol was adopted by the U.S.
Environmental Protection Agency (EPA, 1996) for evaluating biopersistence of inhaled fibers and was
deemed to be an accurate predictor of the pathologic potential of fibers.
The biopersistence protocol was also adopted by the European Commission (European Chemicals
Bureau “Ispra Protocols”, EUR 18748 EN, 1999) for fiber-containing product cancer warning labelling
as part of the European Commission’s synthetic fiber directive (European Commission, 1997).
The difficulty of designing chronic inhalation toxicology studies with fibers:
As mentioned above while many chronic inhalation toxicology studies of fibers, ranging from
amphibole asbestos to soluble glass fibers and to organic fibers have been performed, their design
and subsequent interpretation have been often confounded by incomplete description of the fiber
aerosol exposure, relying upon only the gravimetric concentration with little or no information
provided on fiber number or bivariate fiber size distribution. This is of particular importance as fiber
length is related to pathogenicity. Where fiber number is reported, it is often determined by light
microscopy or scanning electron microscopy, which as previously discussed, detects only a small
fraction of the total number of asbestos fibers present.
The fiber size distribution and the ratio of longer fibers to shorter fibers and non-fibrous particle
content are essential in determining the dose response relationship to these fibers. Thus, it can
become very difficult to use these studies for human risk assessment or even to compare the effects
of one study with those of another.
The issue of using equivalent fiber number for exposure was approached in a study reported by
Davis et al. (1978) where chrysotile, crocidolite and amosite were compared on an equal mass and
equal number basis. However, the fiber number was determined by phase contrast optical
microscopy (PCOM) and thus the actual number of especially the chrysotile fibers was probably
greatly underestimated.
There is little quantitative data presented in these publications on the non-fibrous particle
concentration of the test substances to which the animals were exposed. Pinkerton et al. (1983)
presents summary tables of length measurements of Calidria chrysotile using SEM in which the
number of non-fibrous particles counted is stated, however, from the data presented, the aerosol
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exposure concentration of non-fibrous particles cannot be extracted. In all studies, the asbestos was
ground prior to aerosolisation, a procedure which would produce a lot of short fibers and nonfibrous dust. Most of the studies prior to Mast et al. (1995) used a Timbrell aerosol generator for
aerosolisation of the fibers, which employed a rotating steal blade to push/chop the fibers from a
compressed plug and into the airstream. As some of the authors have noted, the grinding of the
asbestos and the flow through the aerosolisation apparatus often abraded the steel surfaces,
resulting in considerable contamination of the exposure aerosol with metal fragments. These
factors further contribute to the difficulty in interpreting the results of these earlier asbestos
inhalation studies.
These deficiencies in study design bring into question the value of the existing chronic inhalation
study database on asbestos, especially chrysotile asbestos. As an example, in the chronic inhalation
study of refractory ceramic fibers (RCF1) reported by Mast et al. (1994), the exposure aerosol had a
total of 234 fibers/cm3 (WHO criteria) of which there were 101 long fibers (Length>20 µm)/cm3. This
exposure resulted in a total fiber number in the lung after 24 months of approximately 1 million
fibers. In the chrysotile study reported by Hesterberg et al. (1994), SEM analysis showed the
exposure aerosol had a total of 102,000 fibers/cm3 (The number of fibers longer than 20 µm/cm3
was not reported). This resulted in a total fiber number in the lung after 24 months of exposure of
approximately 55 million fibers (again extrapolating to TEM analysis).
While well-designed chronic inhalation toxicology studies of synthetic vitreous fibers have been
performed, nearly all chronic inhalation toxicology studies of asbestos have not been designed in a
similar fashion. McConnell, et al. (1999) reported on perhaps the only well designed multiple-dose
study of any asbestos where amosite particle and fiber number and length were chosen to be
comparable to the SVF exposure groups. In this hamster inhalation toxicology study the amosite
aerosol concentration ranged from 10 to 69 long fibers (> 20 µm)/cm3 with exposure levels selected
based upon a previous, multi-dose 90-day subchronic inhalation study (Hesterberg et al., 1999). In
the chronic study, the high dose amphibole amosite asbestos exposure resulted in 19.5 %
mesotheliomas. In well-designed short-term exposure study in the rat (6 hours/day, 5 days) with
the amphibole tremolite asbestos at an exposure concentration of 100 long fibers (> 20 µm)/cm3,
interstitial fibrosis developed within 28 days after cessation of the 5 day exposure (Bernstein et al.,
2005). While no chronic inhalation toxicology studies of chrysotile using similar fiber selection
techniques and without exceeding lung overload doses have been performed, as discussed below, a
well designed 90-day sub chronic inhalation toxicity study has been performed.
THE USE OF SUB CHRONIC INHALATION TOXICOLOGY STUDIES IN THE EVALUATION OF FIBER
TOXICITY:
The 90-day sub chronic toxicity study has been used extensively in regulatory evaluation. The use of
this and other shorter term studies for the evaluation of the toxicity and potential carcinogenicity of
fibers was reviewed by an ILSI Risk Science Institute Working Group (ILSI, 2005). This working group
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was sponsored by the ILSI Risk Science Institute and the U.S. Environmental Protection Agency Office
of Pollution Prevention and Toxics.
The objectives of the working group were:
1. To summarize the current state of the science on short-term assay systems for assessing potential
fiber toxicity and carcinogenicity of natural and synthetic fibrous materials.
2. To offer insights and perspectives on the strengths and limitations of the various methods and
approaches.
3. To consider how the available methods might be combined in a testing strategy to assess the
likelihood that particular fibers may present a hazard and therefore may be candidates for further
(e.g., long-term) testing.
Current testing methods were reviewed and testing strategies were recommended for prioritizing
fibers for chronic testing.
The working group reiterated the importance of dose, dimensions,
durability in the lung, and in some cases surface reactivity of the fibers as critical parameters related
to adverse health effects (ILSI, 2005).
The working group stated that current short-term testing methods, defined as 3 months or less in
exposure duration, evaluate a number of endpoints that are considered relevant for lung diseases
induced by fibers such as asbestos. Subchronic studies to assess biomarkers of lung injury (e.g.,
persistent inflammation, cell proliferation, and fibrosis) are considered to be more predictive of
carcinogenic potential than in vitro measures of cellular toxicity.
Of particular importance in the evaluation of fiber toxicity using the 90 day sub chronic inhalation
toxicity study is the finding that:
“All fibers that have caused cancer in animals via inhalation have also caused fibrosis by 3 mo.
However, there have been fibers that have caused fibrosis but not cancer. Therefore, in vivo studies
that involve short-term exposure of rat lungs to fibers and subsequent assessment of relevant
endpoints, notably fibrosis, are probably adequately conservative for predicting long-term
pathology— that is, will identify fibers that have a fibrogenic or carcinogenic potential.”
The working group also recommended that specific parameters should be measured in 90 day fiber
inhalation studies, which have been noted in the U.S. EPA Guideline for Combined
ChronicToxicity/CarcinogenicityTesting of Respirable Fibrous Particles (U.S. EPA, 2001). These
parameters should include lung weight, fiber lung burden and clearance, cell proliferation,
inflammatory response markers, and histopathology. The European Commission guideline for
subchronic inhalation toxicity testing of synthetic mineral fibers in rats (European Commission,
1999b) specifies similar parameters.
Bellmann et al. (2003) reported on a calibration study to evaluate a number of endpoints in a 90-day
subchronic inhalation toxicity study, which compared the toxicity of a number of SVFs having a range
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of biopersistences with that of the very biodurable amosite asbestos. One of the SVFs tested was a
calcium-magnesium-silicate (CMS) fiber, a relatively biosoluble fiber, for which the stock preparation
had a large concentration of non-fibrous particles in addition to the fibers. In this study, due to the
method of preparation, the aerosol exposure concentration for the CMS fiber was 286 fibers/cm3
length < 5µm, 990 fibers/cm3 length > 5µm, and 1793 particles/cm3, a distribution which is not
observed in manufacturing.
The total CMS exposure concentration was 3,069 particles &
3
fibers/cm . The authors pointed out that “The particle fraction of CMS that had the same chemical
composition as the fibrous fraction seemed to cause significant effects.” For the CMS fiber, the
authors reported that the number of polymorphonuclear leukocytes (PMN) in the bronchoalveolar
lavage fluid (BALF) was higher and interstitial fibrosis was more pronounced than had been expected
on the basis of biopersistence data. In addition, the interstitial fibrosis persisted through 14 weeks
after cessation of the 90-day exposure. This effect was attributed to the large number of nonfibrous particles in the exposure aerosol--50% of the aerosol was composed of non-fibrous particles
and short fibers.
By comparison, after chronic inhalation exposure of rats to another CMS fiber, X607 fiber, which had
considerably fewer non-fibrous particles present, no lung tumours or fibrosis was detected
(Hesterberg et al., 1998b). This provides support for the argument that it was the large non-fibrous
component of the CMS used in the Bellmann study and the resulting lung overload that caused the
pathogenicity observed with this relatively biosoluble fiber. A similar overload mechanism might
explain the results of earlier chrysotile inhalation studies, in which animals were exposed to much
higher levels of non-fibrous particles and short (< 5 µm) fibers.
Bernstein et al. (2006) reported on the toxicological response of a commercial Brazilian chrysotile
following exposure in a multi-dose sub chronic 90 day inhalation toxicity study, which was
performed according to the protocols mentioned above as specified by the U.S. EPA (2001) and the
European Commission (1999b).
In this study, male Wistar rats were exposed to two chrysotile levels at mean fiber aerosol
concentrations of 76 fibers L>20 μm/cm3 (3,413 total fiber/cm3; 536 WHO fiber/cm3) or 207 fibres
L>20 μm/cm3 (8,941 total fiber/cm3; 1,429 WHO fiber/cm3). The animals were exposed using a flow
past, nose only exposure system for five days per week, 6 h/d, during 13 consecutive weeks followed
by a subsequent non-exposure period of 92 days. Animals were sacrificed after cessation of
exposure and after 50 and 92 days of non-exposure recovery. At each sacrifice, the following
analyses were performed on sub-groups of rats: lung burden; histopathological changes; cell
proliferation; inflammatory cells in the broncho-alveolar lavage ; clinical biochemistry; and confocal
microscopic analysis.
Exposure to chrysotile for 90 days and 92 days of recovery, at a mean exposure of 76 fibres L>20
μm/cm3 (3,413 total fiber/cm3) resulted in no fibrosis (Wagner score 1.8 to 2.6) at any time point.
The long chrysotile fibers were observed to break apart into small particles and smaller fibers. At an
exposure concentration of 207 fibres L>20 μm/cm3 (8,941 total fiber/cm3) slight fibrosis was
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observed. In comparison with other studies, the lower dose of chrysotile produced less inflammatory
response than the biosoluble synthetic vitreous CMS fiber referred to above and considerably less
than amosite asbestos (Bellmann et al. 2003).
In contrast, tremolite (amphibole asbestos) exposure for five days (6 h/d) at an aerosol
concentration of 100 fibers L> 20 µm/cm3 (2,016 total fiber/cm3) resulted in extensive inflammatory
response with interstitial fibrosis observed within 28 days after cessation of exposure (Bernstein et
al. 2005).
SHORT FIBER CLEARANCE and EFFECTS:
For all fiber exposures, there were many more fibers less than 20 µm in length and even more that
were less than 5 µm in length. Because of the mechanical processes of breaking the fibers, fiber
length usually follows a log-normal distribution. The clearance of the shorter fibers in biopersistence
studies has been shown to be either similar to or faster than the clearance of insoluble nuisance
dusts (Muhle et al., 1987; Stoeber et al., 1970). In a report issued by the Agency for Toxic
Substances and Disease Registry entitled ‘Expert Panel on Health Effects of Asbestos and Synthetic
Vitreous Fibers: The Influence of Fiber Length’, the experts 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; EPA, 2003).
Bernstein et al. (2008) compared the clearance of chrysotile alone with a mixture of chrysotile and
non-fibrous joint compound particles in a five-day biopersistence study. The aerosol exposure was
in the range of 5,000 WHO fibers/cm3 and 12,000 to 18,000 total fibers/cm3. The joint compound
particle exposure concentration was 1.3 mg/m3. Across all size ranges there was approximately an
order of magnitude decrease in the mean number of each size category of fibers remaining in the
lungs of animals exposed to the mixture of chrysotile and joint compound particles compared to
animals exposed to chrysotile alone, even though the fiber aerosol exposures were similar. This
study demonstrated that at this short-term non-overloading exposure, the addition of non-fibrous
particles to an aerosol containing chrysotile asbestos fibers results in a greater rate of clearance of
fibers from the lung, which is likely due to an increase in the recruitment of macrophages in the
lung, which degrade the longer fibers. The increased number of macrophages was confirmed
histologically, with this being the only exposure-related finding reported.
THE CORRELATION OF FIBER LENGTH AND BIOPERSISTENCE TO CHRONIC TOXICITY:
In the series of SVF chronic inhalation studies performed at the Research and Consulting Company
Ltd., in the 1990s, the relationship between exposure to more durable fibers and lung disease
became apparent and resulted in the design of the inhalation biopersistence studies as described
above. The importance of fiber length to the potential of a fiber to induce a pathogenic effect has
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been well documented (Lippmann, 1990: McClellan et al., 1992; WHO, 1988; Goodglick & Kane,
1990).
To evaluate the importance of fiber length on clearance, Bernstein et al. (1996) examined the
biopersistence of 9 different fibers types, using this inhalation biopersistence protocol to determine
the clearance of fibers in 3 length categories: fibers with length <5 µm, 5-20 µm, and > 20 µm. The
length categories were chosen based upon an understanding of the impact of fiber length on
macrophage-mediated clearance. Fibers <5 µm in length can be completely phagocytized by
macrophages and treated by the lung essentially as non-fibrous particles. The length fraction >20 µm
was considered as greater than the size of the macrophage and represented a fiber length that the
macrophage most likely could not fully phagocytize. It was pointed out that these length fractions
were not considered as strict cutoffs but rather representative of mechanistic categories with the
transition between categories thought to occur over a range of lengths. The fiber clearance halftimes for each of these length fractions was then correlated with the fiber durability as determined
using an acellular in-vitro flow though system using a modified Gambles solution. This analysis
provides a good indication that the disappearance from the lung (including fiber breakage) of fibers
longer than 20 µm may be mediated by their dissolution rate at pH 7.4. While not reported in the
publication, the authors chose the 20 µm cutoff for the longer fibers by performing iterative
analyses starting at a fiber length of 10 µm and greater and then increasing the minimum fiber
length to determine the lower cutoff that provided the best correlation with the dissolution rate
observed using the in-vitro durability test (Personal communication). The authors found that the
best association between in-vitro dissolution and lung clearance in the rat was found for fibers
having lengths of approximately 18 to 20 µm. At fiber lengths > 20 µm, this correlation diminished as
a result of fewer and fewer fibers being available in the longer size ranges.
Subsequently, in an analysis that provided the basis for the European Commission’s Directive on
synthetic mineral fibers, Bernstein et al. (2001a&b) reported on the correlation between the
biopersistence of fibers longer than 20 µm and the pathological effects following either chronic
inhalation or chronic intra-peritoneal injection studies. As summarized in Table 1, this analysis
showed that it was possible, using the retention half-time of SVFs longer than 20 µm, which was
determined from the inhalation biopersistence studies, to predict 1) the number of fibers longer
than 20 µm remaining in the lung following a 24 month chronic inhalation exposure; 2) the early
fibrotic response (collagen deposition) observed after 24 months of exposure in the chronic
inhalation toxicology studies; and 3) the number of tumours and fiber dose in the chronic
intraperitoneal injection studies. These studies, however, only evaluated SVFs and not asbestos
fibers.
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Table 1
Summary of the correlation between the biopersistence of fibers longer than 20 µm and the
pathological effects following either chronic inhalation or chronic intra-peritoneal injection studies.
(Bernstein et al., 2001a&b).
THE BIOPERSISTENCE OF FIBERS
LONGER THAN 20 µm
CORRELATES WITH:

The number of fibers L> 20 µm remaining in chronic
inhalation toxicology studies following 2 years of
exposure.
 The early fibrotic response (collagen deposition)
observed after 24 months of exposure in the
chronic inhalation toxicology studies.
 The number of tumours in the chronic
intraperitoneal injection studies.
For SVFs, the clearance half-time of fibers longer than 20 µm ranged from a few days to less than
100 days. This is illustrated in Table 2. Also shown in this table are the results from biopersistence
studies performed on serpentine (chrysotile) and amphibole asbestos using the same protocol. In
these studies, chrysotile asbestos was much more biosoluble than the amphibole asbestos types
(amosite and crocidolite)—in fact chrysotile was even more biosoluble than most of the SVFs. For
synthetic vitreous fibers, the European Commission has established a Directive, which states that, if
the inhalation biopersistence expressed as retention half-time of a fiber is less than 10 days, then it
is not classified as a carcinogen (European Commission. 1997).
Table 2 Clearance Half-Times for Synthetic Vitreous Fibers and Asbestos
FIBER
TYPE
T1/2
FIBERS L>20
REFERENCE
µm*
(days)
Calidria
chrysotile
Brazilian
chrysotile
Fiber B
Fiber A
Fiber C
Fiber G
MMVF34
MMVF22
17
Serpentine asbestos
Serpentine asbestos
B01.9
Glasswool
Glasswool
Stonewool
HT stonewool
Slagwool
0.3
Bernstein et al., 2003a
2.3
Bernstein et al., 2004a
2.4
3.5
4.1
5.4
6
8
Bernstein et al., 1996
Bernstein et al., 1996
Bernstein et al., 1996
Bernstein et al., 1996
Hesterberg et al, 1998
Bernstein et al., 1996
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Fiber F
MMVF11
Fiber J
MMVF 11
Fiber H
Canadian
chrysotile
MMVF10
Fiber L
Stonewool
Glasswool
X607
Glasswool
Stonewool
Serpentine asbestos
8.5
9
9.8
13
13
Bernstein et al., 1996
Bernstein et al., 1996
Bernstein et al., 1996
Bernstein et al., 1996
Bernstein et al., 1996
11.4
Bernstein et al., 2004b
Glasswool
39
Bernstein et al., 1996
Stonewool
45
Bernstein et al., 1996
Special purpose
MMVF33
49
Hesterberg et al, 1998
glass
RCF1a
Refractory ceramic
55
Hesterberg et al, 1998
MMVF21
Stonewool
67
Hesterberg et al, 1998
Special purpose
MMVF32
79
Hesterberg et al, 1998
glass
MMVF21
Stonewool
85
Bernstein et al., 1996
Amphibole
Amosite
418
Hesterberg et al, 1998
asbestos
Amphibole
Crocidolite
536
Bernstein et al., 1996
asbestos
* When analysing the results non-linear exponential regression was used. If a single
exponential explained at least 80 % of the variance then the T1/2 determined from the single
exponential was reported. Otherwise, a double exponential was used to fit the data and the
Weighted T1/2 determined from the double exponential was reported (EUR 18748 EN, 1999).
Berman et al. (1995) statistically analyzed the results of 13 separate animal inhalation studies, which
exposed animals to 9 different asbestos types. Due to limitations in the characterization of asbestos
structures in the original studies, new exposure measures were developed from samples of the
original dusts, which were re-generated and analyzed by transmission electron microscopy. The
authors reported that, while no univariate model was found to provide an adequate description of
the lung tumor responses in the inhalation studies, the measure most highly correlated with tumor
incidence was the concentration of structures (fibers) > or = 20 µm in length. However, using
multivariate techniques, measures of exposure were identified, which adequately described the lung
tumor responses. The authors reported that “Structures contributing to lung tumor risk appear to be
long (≥5 µm) thin (0.4 µm) fibers and bundles, with a possible contribution by long and very thick (≥5
µm) complex clusters and matrices. Potency appears to increase with increasing length, with
structures longer than 40 µm being about 500 times more potent than structures between 5 and 40
µm in length. Structures <5 µm in length do not appear to make any contribution to lung tumor
risk.”
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This analysis found no difference in the potency of chrysotile and amphibole regarding the induction
of lung tumors. However, the authors stated that the mineralogy appears to be important in the
induction of mesothelioma, with chrysotile being less potent than amphibole. These results,
however, should be viewed in the context of the inhalation toxicology studies evaluated by Berman
et al. (1995, Table 1), the majority of which were performed at very high concentrations (10 mg/m3).
As discussed above, the overload effect from these very high exposure concentrations would be
expected to produce similar tumouregenic response in the lung for chrysotile and amphibole.
Recent studies on the serpentine asbestos, chrysotile, have shown that it is not very biopersistent in
the lung. As serpentine is a naturally occurring mined fiber, there appear to be some differences in
biopersistence depending upon from where it is mined. However, chrysotile lies on the soluble end
of this scale and ranges from the least biopersistent fiber to a fiber with biopersistence in the range
of glass and stonewools. It remains less biopersistent than refractory ceramic fibers and special
purpose glasses and more than an order of magnitude less biopersistent than amphibole asbestos.
A 90 day sub-chronic inhalation toxicity study of chrysotile in rats showed that at an exposure
concentration 5,000 times greater than the US-American Conference of Governmental Industrial
Hygienists (ACGIH) Threshold Limit Value (TLV) of 0.1 f(WHO)/cm3, chrysotile produced no significant
pathological response or sustained iinflammatory response.
Some earlier studies have shown chrysotile to clear less rapidly than in the studies performed using
the EC protocol. An example is the study by Coin et al. (1992) in which rats were exposed for 3 h to a
NIEHS chrysotile aerosol of 10 mg (respirable)/m3 and then followed for a period of 29 days. The
authors reported that through 3 weeks after cessation of exposure, fibers greater than 16 µm in
length were cleared slowly, if at all.
While a brief description is provided, the details of the aerosol exposure to the NIEHS chrysotile
which was used in the Coin et al. (1992) study are not described directly in the publication.
However, the characteristics of the exposure aerosol and the preparation methods can be derived
from an earlier publication by Pinkerton et al. (1983) referenced by Coin and a non-published report
by Campbell et al. (1980) referenced by Pinkerton.
From these, we find that the chrysotile used by Coin et al. (1992) was prepared from a Grade 4
chrysotile used in the plastics industry which was prepared by passing the material through a
hurricane pulverizer. The hurricane pulverizer is an industrial high-speed impact hammer mill with a
size classifier which recycled larger fibers/particles back into the device for continued milling (Perry
& Chilton, 1973; Work, 1963).
The aerosol used in the Coin et al. (1992) study was generated from this ground material as
described by Pinkerton et al. (1983) using a Timbrell generator (Timbrell, 1968). The stainless steel
blades of this generator are known to further pulverize fiber samples. While the original chrysotile
sample had 13.9 % fibers longer than 19.9 µm (Campbell, et al., 1980), the final aerosolized sample
used in the Coin et al. (1992) study had 1.8% fibers longer than 19.9 µm (Pinkerton et al., 1983). For
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fibers ≥ 16 µm in length, Coin only presents the data graphically. Visual extrapolation from Figure 5
of Coin indicates that there were approximately 2, 2, 5 and 4 x 10 5 fibers L ≥ 16 µm (measured by
SEM) present at 1, 8, 15 and 29 days post exposure, respectively (no error bars were indicated and
no tables of the values given).
The following summarises differences in study design with those performed following the EC
protocol.

Study length:
Coin, following a single exposure, examined sub-groups on animals for 3 weeks. In the
chrysotile studies performed following the EC protocol, animals were exposed for 5 days and
then followed for 1 year pose-exposure.

Fiber counting statistics:
Coin counted 20 fibers per animal in the size range L ≥ 16 µm. In the EC protocol studies, 100
fibers (200 fiber ends) were counted for fibers with a length >20 μm. From statistical
sampling theory, assuming as Coin stated that there are ~ 100,000 fibers L ≥ 16 µm in each
lung sample, then 383 fibers L ≥ 16 µm should have been counted to have 95 % confidence
in the mean (Hamburg, 1974, Lang & Secic, 1997). By counting only 20 fibers per lung, the
confidence is 35 %. Thus, by sampling only 20 fibers, there is a less than a 1 in 3 chance that
the mean value reported by Coin is actually correct.
While the counting statistics alone are probably sufficient to demonstrate the difficulty of
comparing the results of these studies, other features of Coin et al., deserve mention:
20

Fiber diameters: 0.2 – 0.1 vs 0.05 µm
In humans, the mean diameter of chrysotile fibers recovered from lung samples is
approximately 0.05 µm (Dufresne et al., 1996). This corresponds with the diameters of
chrysotile fibers reported mineralogically (Naumann & Dresher, 1966). In the EC protocol
studies, the geometric mean diameter ranged from 0.04 to 0.06 µm. Coin et al., however,
reported that the fibers longer than 16 µm that deposited in the lungs had average
diameters of ~ 0.2 µm. This suggests that the fibers used in the Coin study were largely in
bundles as illustrated in Figure 3 of Coin et al. (1992).

Control levels:
It is standard practice in such studies to include a control group of animals exposed to
clean/filtered air. Coin states that the mean number of fibers found in the control animals
was 7 x 105 WHO fibers per animal and 3 x 103 fibers ≥ 16 µm per animal, indicating
contamination. No standard deviation is given, however, so the extent of this contamination
remains unknown. Coin does not state how this contamination occurred. In the EC protocol
studies, no WHO fibers (including fibers L> 20 µm) were observed in the lungs of any of the
control animals.
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
SEM – TEM difference
The fiber lung burden results reported by Coin were determined using SEM while the results
in the EC protocol studies were determined using TEM. TEM analysis could show as much as
17 times more fibers than SEM analysis (Breysse et al., 1989).
Fiber translocation to the pleural cavity
Within the lung, shorter fibers, which can be fully engulfed by the macrophage (< ~ 15 μm length in
the rat), are cleared at a similar or faster rate than non-fibrous insoluble particles. Such particles,
once deposited in the lung parenchyma, can be phagocytised by alveolar macrophages and
transported either or to the bronchial airways, where they are cleared from the lung by mucociliary
transport, or to a smaller extent by the pulmonary lymphatics which drain to the hilar lymph nodes.
While the translocation of fibers to the lymphatic tissue is an important route of clearancein cases of
human’s with asbestos related disease, there has been no report in the literature of pathological
changes in the lymphatic tissue stemming from asbestos (Sampson and Hansell, 1992, Gross et al.,
1971).
Longer fibers have the potential to pose a problem for the lung if they cannot be removed by the
macrophage. If the longer fibers are biosoluble and can dissolve or break apart in the lung, then
they can be cleared. If they are durable, macrophages can only partially engulf them and are
activated (frustrated phagocytosis), so they can persist in the lung and cause a chronic inflammatory
response, which can result in lung fibrosis and tumors.
The lung tissue, however, is in constant movement as it inflates and deflates during each respiration.
The alveoli expand non-uniformly and increase in size by a mean of 14 % during respiration (Perlman
& Bhattacharya, 2007). While the actual mechanism facilitating fiber transport in unknown, such
non-uniform expansion during breathing could possibly facilitate the penetration of long durable
fibers
out of the lung alveoli and into the adjacent interstitium. When this occurs in those alveoli adjacent
to the visceral pleural surface, the long biopersistent fibers could then translocate into the pleural
cavity…
Donaldson et al. (2010), in a recent review, explained that there exists a body of literature which
shows that a portion of all particles deposited in the lung reaches the pleura, pass through the
pleural space and exit through the stomata which in humans range from 0.5 to >20 µm (see
discussion below) of the parietal pleura, and are then cleared to the mediastinal lymph nodes rather
than hilar lymph nodes.
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The pleural space is approximately 10 to 20 µm wide and extends completely around the lung to the
hilar root (La-Fook, 2004; Wang, 1998; Albertine et al., 1991). The visceral pleura covers the lung and
interlobar fissures; the parietal pleura covers the chest wall, diaphragm and mediastinum. Each
membrane is comprised of a single cell layer of mesothelial cells and each is nourished by the
systemic circulation.
The visceral and parietal pleural membranes differ from one another in one important respect: only
the parietal pleura has lymphatic stomata that open directly into the pleural space which provide
drainage of the fluid and any particles entering the pleural cavity. Current concepts of pleural liquid
turnover have stemmed in large part from considering the differential anatomy of the two pleural
membranes (Staub et al., 1985).
The visceral pleura in humans is approximately 20 to 80 µm thick and consists of a mesothelial layer
and a sub-pleural connective tissue layer. The alveoli and the pulmonary circulation lie beneath the
visceral pleural membrane (Wang, 1998; Albertine, et al., 1982).
As described by Lai-Fook (2004) and Zocchi (2002) and as illustrated in Figure 4 (reproduced with
permission from Zocchi, 2002) pleural fluid is mainly an infiltrate from the capillaries in the parietal
pleura lining the chest wall. This fluid filters into the pleural space driven by Starling forces through
the parietal pleura. The pressure differential across the visceral pleura is in favour of liquid
absorption (Agostoni, 1972, 1986, 1991, 1998; Miserocchi & Negrini, 1997; Negrini, 1995).
Figure 5: Pleural fluid is mainly an infiltrate from capillaries in the parietal pleura lining the chest wall.
This fluid filters into the pleural space driven by Starling forces through the parietal pleura. The
pressure differential across the visceral pleura is in favour of liquid absorption (Figure and caption below
reproduced with permission from Zocchi, 2002).
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Figure reproduced with permission from Zocchi (2002): Fig. 2. – Schematic
representation of transpleural liquid flows (large arrows) under physiological
conditions in the rabbit. Mesothelial cells and adjoining interstitia, with embedded
capillaries and lymphatics, are shown on each side; the mesothelial cell at the top
displays cellular mechanisms involved in pleural liquid turnover (microvilli, vesicles,
electrolyte transporters (grey circles)). Arrow size is not proportional to
corresponding flow magnitude. 1: Starling filtration at the parietal pleura (tentative
estimate:~0.15– 0.20 mL.h-1.kg-1); 2: Starling absorption at the visceral pleura
(tentative estimate: ~0.1 mL.h-1.kg-1); 3: direct drainage through lymphatic stomas
(not known; tentative estimate of total pleural lymphatic flow (through
stomaszinterstitial): ~0.07 mL.h-1.kg-1); 4: electrolyte-coupled liquid outflow
(measured [Agostoni et al., 1993]: 0.07 mL.h-1.kg-1); 5: vesicular flow of liquid
accompanying protein transcytosis (not known; recent estimates have shown it to
be 0.02 mL.h-1.kg-1). Pairs of large numbers are hydraulic (above) and colloidosmotic
(below) pressures (cmH2O) involved in Starling balance, pertaining to (from left to
right): parietal capillary, parietal interstitium, pleural liquid, visceral interstitium,
visceral capillary. Hydraulic pressures refer to heart level; they are functional mean
values for capillaries, and averaged value over a breath for pleural liquid, while
interstitial values are indirect and uncertain. Interstitial colloidosmotic pressures are
uncertain.
As summarized by Zocchi (2002), direct in vitro measurements of the biophysical properties of the
pleura appear unreliable, because the mesothelium is very labile (Agostoni, 1998; Fentie et al., 1986;
Peng et al.¸ 1994), and the procedures required to obtain the specimens are likely to damage it
(Agostoni, 1998).
Studies performed on pleura specimens stripped from the underlying tissues (Kim et al., 1979; Payne
et al., 1988) provided extremely high values of permeability to water and solutes, which are
regarded as being due to the experimental conditions (Zocchi et al., 1991, 1992, 1998; Agostoni &
Zocchi, 1993; Agostoni et al., 1999; Bodega et al., 2000; Miserocchi & Negrini, 1997; Negrini, 1995;
Negrini et al., 1994).
The pressure differentials described above cease to exist post mortem. The visceral pleural is
permeable and as illustrated in Figure 5, underlying the mesothelium is a sub-pleural layer
containing the blood and lymph vessels (Bernaudin, 1985). Although the larger lymphatic vessels of
the visceral pleura are equipped with one-way valves, the lymph flow in the smaller vessel is
governed by pressure gradients which are no longer present post-mortem. With the visceral pleura
in humans being approximately 20 to 80 µm thick, this barrier can be easily breached during
intervention at autopsy, thereby releasing shorter fibers into the pleural cavity which would not be
present in-vivo without intervention.
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Figure 6: The visceral pleural is permeable and, as illustrated, underlying the mesothelium is a subpleural layer containing the blood and lymph vessels (figure and caption reproduced with permission
from Bernaudin, 1985).
Figure reproduced with permission from Bernaudin (1985). Schematic
drawing of the human visceral pleura according to Naigaishi (1972).
The different constitutive layers are (1) mesothelium; (2) sub
mesothelial tissue; (3) external elastic layer with elastic fibers (E); (4) a
subpleural layer containing the blood (B) and lymph (L) vessels; and (5)
internal elastic layer.
Short fibers and particles which enter the pleural cavity are quickly cleared through the stomata.
Some of these particles will accumulate below the parietal pleural surface adjacent to the stomata in
lymphatic tissue. In urban dwellers, these areas appear as black spots on the parietal pleural surface
and appear to be due to the accumulation of black carbon particles inhaled from urban air and
translocated to the pleura. Mitchev et al. (2002) suggested that these areas appeared to be related
to the structures responsible for lymphatic drainage. Electron microscopic analyses of these black
spots further revealed that they also can accumulate shorter asbestos fibers (Mitchev et al., 2002).
Donaldson et al. (2010) have summarized very clearly the mechanisms of particle and fiber pleural
transport and clearance: “So from the above there is good evidence to support the contention that
a fraction of all deposited particles reach the pleura by an obscure pathway and that short fibres and
compact particles leave the pleura through the stomatal openings. Most of the particles are
transported to lymph nodes and some enter the interstitium at the mouth of the stoma to form a
‘black spot’ or equivalent. Long fibres that reach the pleural space, however are an exception to this,
since they have the potential to physically block the stomata due to their difficulty in negotiating the
bend into the stoma which would result in interception of the ends of the fibre with the walls of the
stomatal openings and with the lymph vessels walls themselves. This is likely to lead to mesothelial
and endothelial cell damage at this site, inflammation and the accumulation of pleural macrophages
attempting to phagocytose these retained fibres. The macrophages are likely to undergo frustrated
phagocytosis in attempting to enclose the long fibres and so release cytokines and oxidants. This
24
IMPORTANCE OF BIOPERSISTENCE IN THE PATHOGENICITY OF ASBESTOS
Draft 17 January 2011
would lead to further inflammation, fibrosis and genotoxicity in the bystander mesothelial cells in
these areas of congestion around the stomatal entrances. Direct interaction between retained long
fibres and mesothelial cells around the stomata could also result in direct genotoxicity. Eventually
some stomata are likely to be entirely blocked by cells and fibres. Figure 9 demonstrates
diagrammatically the difference between formation of black spots and pleural plaques with particles
and short fibres (A), compared to the response to long fibre retention at parietal stomata (B).”
[GO: Cite also paper by Miserocchi- Which one ? His 1997 paper is cited below. Do you mean his
2008 paper ?? My preference is not to give the 2008 paper any credibility …..
Pleural physiology and understanding the use of intraperintoneal injection (IP) studies:
The pleural fluid turns over rapidly (Lai-Fook, 2004), continuously exiting through stomata in the
parietal (not the visceral) pleura via lymphatic capillaries. In humans, Miserocchi & Negrini (1997)
reported that the diameter of stomatas are quite variable, ranging from approximately 0.5 µm for
tiny stomatas grouped in cluster to >20 µm in case of large isolated stomata.
In the rat, however, the stomata are smaller and have been reported by Wang et al. (1997) to be less
than 2 to 7 μm in diameter with a mean diameter of approximately 3 µm. The distribution is shown
in Figure 7. The density of the stomata varies with location and ranges from 400 to 1000
stomata/mm2. The net effect is that the parietal pleural surface is like a sieve which allows drainage
of excess fluid and particles and shorter fibers that can pass through the stomata.
Figure 7: Reproduced from Wang et al. 1997.
Left plate: Sizes of the pores in the anterior costal pleura (white bars) and in the sub-mesothelial collagen
sheet (grey bars) in the rat. Right plate: Densities of the pore distribution in the costal pleura (white bars) and
in the sub-mesothelial collagen sheet (grey bars) of the rat anterior thoracic wall.
Intraperitoneal injection studies use the peritoneal cavity as a surrogate for the pleural cavity, which
because of the relatively thin space between the visceral and parietal pleural surfaces, is difficult to
repeatedly dose with fibers via injection. Wang et al. (2010) have recently reviewed the advances in
understanding the function of the lymphatic stomata. They explain that the peritoneal cavity,
25
IMPORTANCE OF BIOPERSISTENCE IN THE PATHOGENICITY OF ASBESTOS
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pleural cavity, and pericardial cavity are all connected with the lymphatic system via these small
openings, which have the function of active absorption. Although the stomata of the peritoneal
cavity have not been characterized as comprehensively as the parietal pleura surface in the rat, AbuHijleh et al. (1994) reported that the stomata on the diaphragm of rats of the peritoneal surface
were between 4 – 10 µm in diameter, thus providing an indication of similar size.
In 1987, Pott et al. reviewed a series of intraperitoneal injection studies, which employed a wide
variety of different fiber types injected into the peritoneal cavity of the rat at a dose of 5 x 109 WHO
fibers. However, there was no standardization of fiber diameter or length injected with the mean
diameter of fibers injected ranging from 0.06 to 7.4 µm. This clearly will affect the outcome of the
study. For very thin fibers, such as asbestos, the shorter fibers can be cleared through the
stomata/lymphatic drainage system accounting for up to 90 % of injected fibers (because of the
method of preparation and the log normal distribution of the lengths). The much smaller number of
long fibers present in the injection bolus cannot be cleared and become the biologically effective
dose. However, when synthetic mineral fibers were injected, they generally had much larger
diameters, often reported with a mean diameter greater than 3 µm, the same as the mean diameter
of the stomata reported in the pleural cavity. In this situation, the likelihood of even a shorter fiber
being cleared through the stomata is considerably reduced. This difference becomes even more
pronounced with the injection of chrysotile asbestos, which is a fiber known to form relatively thick
bundles. If injected directly, these bundles may not be cleared through the stomata. However,
following inhalation, it is highly unlikely that such bundles would ever reach the pleura or
peritoneum. Individual fibril which might could be ready cleared through the stomata. The doseresponse relationships in the IP studies reported in the literature are meaningless if the size of the
fiber and its ability to be readily cleared through the peritoneal lymphatic stomata are not taken into
account. Furthermore, because of the filtration which occurs in the lung airways during inhalation,
many of the long and thick fibers injected in these IP studies would not reach the deep lung and thus
would not reach the pleural or peritoneal mesothelium under normal conditions. As well, the dose
of fibers deposited in the lung by inhalaton and translocating to the pleural cavity would be orders of
magnitude lower.
MECHANISMS OF ACTION OF ASBESTOS
Understanding the mechanism of action of asbestos-related disease has been an underlying goal
since even early studies, nearly 60 years ago. Using the data from inhalation exposures, Vorwald
(1951) proposed that the mode of action of long asbestos fibers in the induction of asbestosis was
primarily mechanical rather than chemical in nature, with the fiber length being the most critical
determinant of pathogenicity. With the understanding developed subsequently that SVFs appear to
be generally less toxic than the more durable asbestos fibers, there have been a plethora of
biological models developed to understand the mechanism that might explain these differences,
including the development of in vivo biopersistence protocols, the use of in vitro cellular toxicity
studies, and the development of models to assess solubility of fibers in vitro.
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IMPORTANCE OF BIOPERSISTENCE IN THE PATHOGENICITY OF ASBESTOS
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The use of in vitro assays is advantageous in that they allow the isolation of specific biological and
mechanistic pathways under very controlled conditions that are difficult to achieve in vivo.
However, caution needs to be emphasized when comparing or extrapolating from in vitro to in vivo
findings. In vitro testing with fibers can produce results that suggest hazards that may or may not be
present after in vivo exposures. For example, in vitro tests on fiberglass suggest far greater toxicity
(i.e., false positives) than what is observed in vivo (Hart et al., 1994; Hesterberg and Hart, 2001). On
the other hand, in the case of single-walled carbon nanotubes, in vitro tests suggest low toxicity,
while in vivo bioassays have indicated otherwise (i.e., false negative) (Donaldson et al., 2009).
Nearly all in vitro studies are performed in a static environment and do not have the ability of
differentiating fibers based upon their solubility.
Threshold for the effects of asbestos. Carthew et al. (1992) reviewed several studies of asbestos and
erionite carcinogenicity in rats and identified dose levels of asbestos and erionite that do not result
in carcinogenicity. Interestingly, these suggestive thresholds for dose effects in rats appeared to
vary by fiber type. However, these studies were mostly by injection [intrapleurally or
intraperitoneally], which also presents challenges in extrapolating to human exposure by inhalation,
even when assuming equivalent internalized fiber dose across species. While the animal-to-human
extrapolation is of course difficult, the extrapolation from in vitro study findings to humans is
dramatically more challenging. The authors cited the study by Lippmann (1994) which examines
inhalation studies of asbestos carcinogenicity–the study by Carthew et al. (1992) should also be
considered.
Another issue with in-vitro assays is fiber dose and dose-response relationships. Most of the studies
provide the dose gravimetrically without any specification of the number of fibers per cell exposed
or the physical dimensions of the fibers. There is often no differentiation in studies of effects by
fiber type, fiber length or dose level. As with the intracavity injection studies, target cells are often
exposed to fiber doses and dimensions that would never reach the target cells in the lower lung by
inhalation exposure.
It can be calculated that when humans continuously breathe 10 times the asbestos TLV of 1
fiber/cm3 through an 8 hour work day, approximately 1 fiber will deposit in every 10,000 lung
alveoli. Thus, the likelihood of a single cell encountering more than one fiber at a time is extremely
small, even with long term exposure.
To compare this dose to the exposure of target cells in the lung following inhalation, the cellular
doses used in cell culture studies can be calculated. Assuming that 2 x 106 cells/dish are exposed to
between 1 to 300 ug of asbestos in a 35 mm-diameter Petri dish, depending upon fiber length
(which is rarely reported in these studies), 1 µg of asbestos would correspond to about 1 x 108 fibers
(as measured by TEM), and the average dose to each cell in culture would therefore range from 50
fibers/cell to 15,000 fibers/cell. This in vitro cellular dose is 500,000 to 150 million times the cellular
dose in the lung after inhalation exposure, even at 10 times the TLV. These huge cellular in-vitro
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IMPORTANCE OF BIOPERSISTENCE IN THE PATHOGENICITY OF ASBESTOS
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doses bring into question their utility for human hazard or risk assessment and also question their
relevance for understanding the mechanisms of asbestos toxicity because the mechanism is dosedependent (Slikker et al., 2004 ) From experience with particle overload studies, particle dose not
only determines toxicity, but can also determine the mechanism of toxicity.
CONCLUSIONS:
Recent well-designed inhalation toxicity studies have shown that the fiber toxicity paradigm of dose,
dimension, and durability in the lung are applicable to the two fibrous asbestos types,
serpentine/chrysotile and amphibole asbestos.
Chrysotile is a very thin rolled sheet silicate, which can be leached and broken apart by the acidic
environment of the lysosomes located inside macrophages of the lung. In contrast, amphibole
asbestos is a chained silicate, which has a solid cylindrical shape with a quartz-like external surface,
and is insoluble at all pHs. The existing database of fiber toxicity studies strongly suggests that
human exposure to respirable fibers that are biopersistent in the lung and induce significant and
persistent pulmonary inflammation, cell proliferation, or fibrosis should be viewed with concern.
Recent studies have shown that some forms of serpentine chrysotile asbestos, are not very
biopersistent in the lung. Serpentine asbestos is a naturally occurring mined fiber, and there appear
to be some differences in biopersistence depending upon the source. However, chrysotile lies on
the soluble end of this scale and ranges from the least biopersistent fiber to a fiber with a
biopersistence in the range of some of the more soluble SVFs, such as fiber glass and stonewools.
On the other hand, amphibole asbestos fibers are highly biopersistent in the lung, with the fibers
longer than 20 µm having little clearance once deposited in the deep lung. Chronic inhalation
studies have shown this asbestos fiber type to be carcinogenic.
GO: Chrysotile studies by Roggli show longer T ½ ‘s, these should be discussed to give a balanced
view. They still show much shorter T ½ than those for amphiboles. Have included above discussion
of Coin et al. (Roggli) paper.
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REFERENCES
ATSDR. 2003. Report on the Expert Panel on Health Effects of Asbestos and Synthetic Vitreous
Fibers: The Influence of Fiber Length. Atlanta, GA.: Prepared for: Agency for Toxic Substances and
Disease Registry Division of Health Assessment and Consultation.
Bellmann, B., Muhle, H., Creutzenberg, O., Ernst, H., Muller, M., Bernstein D.M., Riego Sintes, J.M.,
2003. Calibration study on subchronic inhalation toxicity of man-made vitreous fibers in rats. Inhal
Toxicol 15 (12),1147-1177.
Berman, D.W., K.S. Crump, E.J. Chtfield, J.M. Davis, and A.D. Jones. 1995. The sizes, shapes, and
Minerology of abestos structures that induce lung tumors or mesothelioma in AF/HAN rats following
inhalation. Risk Anal 15(a): 181-95.
Bernstein DM, Mast R, Anderson R, Hesterberg TW, Musselman R, Kamstrup O, Hadley J. 1994. An
experimental approach to the evaluation of the biopersistence of respirable synthetic fibers and
minerals. Environ Health Perspect 102 Suppl 5:15-8.
Bernstein DM, Morscheidt C, Grimm H-G, Thévenaz P, Teichert U. 1996. Evaluation of soluble
fibers using the inhalation biopersistence model, a nine-fiber comparison. Inhalation Toxicology
8(4):345-385.
Bernstein, D. M., J. M. Riego Sintes, B. K. Ersboell, and J. Kunert. 2001a. Biopersistence of synthetic
mineral fibers as a predictor of chronic inhalation toxicity in rats. InhalToxicol. 13(10), 823-849.
Bernstein, D. M., Chevalier, J., Smith, P. 2005. Comparison of Calidria Chrysotile Asbestos to Pure
Tremolite: Final Results of the Inhalation Biopersistence and Histopathology Examination Following
Short-Term Exposure. Inhalation Toxicology. 17 (9) 427-449.
Bernstein, D.M. & Hoskins, J.A. 2006. The health effects of chrysotile: current perspective based
upon recent data. Regulatory Toxicology and Pharmacology 45/3 pp. 252-264.
Bernstein, D.M., Riego Sintes, J.M., Riego-Sintes, J.M., Ersboell, B.K., Kunert, J., 2001b.
Biopersistence of synthetic mineral fibers as a predictor of chronic intra-peritoneal injection tumor
response in rats. Inhal. Toxicol. 13(10), 851-875.
Bolton RE, Vincent JH, Jones AD, Addison J, Beckett ST. 1983. An overload hypothesis for
pulmonary clearance of UICC amosite fibrefibers inhaled by rats. Br. J. Ind. Med. 40:264-272.
Davis JM, Beckett ST, Bolton RE, Collings P, Middleton AP. 1978. Mass and number of fibres in the
pathogenesis of asbestos-related lung disease in rats. Br J Cancer. 37(5):673-88.
EPA. 2003. Report on the Peer Consultation Workshop to discuss a proposed protocol to assess
asbestos-related risk. Prepared for U.S. Environmental Protection Agency, Office of Solid Waste and
Emergency Response, Washington, DC 20460, EPA Contract No. 68-C-98-148, Work Assignment
2003-05. Prepared by Eastern Research Group, Inc., 110 Hartwell Avenue, Lexington, MA 02421.
Final Report May 30, 2003.
EUR 18748 EN, 1999. Methods for the determination of the hazardous properties for human health of
29
IMPORTANCE OF BIOPERSISTENCE IN THE PATHOGENICITY OF ASBESTOS
Draft 17 January 2011
man made mineral fibers (MMMF). Bernstein, D.M., Riego-Sintes, J.M.R. Vol. EUR 18748 EN,
April. 93, http://ecb.ei.jrc.it/DOCUMENTS/Testing- Methods/mmmfweb.pdf: European Commission
Joint Research Centre, Institute for Health and Consumer Protection, Unit: Toxicology and Chemical
Substances, European Chemicals Bureau.
European Commission. 1997. O.J. L 343/19 of 13 December 1997. Commission Directive 97/69/EC
of 5 December 1997 adapting to technical progress for the 23rd time Council Directive 67/ 548/EEC
on the approximation of the laws regulations and administrative provisions relating to the
classification, packaging and labelling of dangerous substances.
Goodglick, L.A. and A.B. Kane. 1990. Cytotoxicity of long and short crocidolite asbestos fibers in
vitro and in vivo. Cancer Res 50(16): 5153-5163.
Hammad Y, Simmons W, Abdel-Kader H, Reynolds C, Weill H. 1988. Effect of chemical
composition on pulmonary clearance of man-made mineral fibers. Ann Occup Hyg 22:769-779.
Hesterberg TW, Chase G, Axten C, Miller WC, Musselman RP, Kamstrup O, Hadley J, Morscheidt
C, Bernstein DM, Thevenaz P. 1998a. Biopersistence of synthetic vitreous fibers and amosite asbestos
in the rat lung following inhalation. Toxicol Appl Pharmacol 151(2):262-75.
Hesterberg, T.W., Hart, G.A., Chevalier, J., Miiller, W.C., Hamilton, R.D., Bauer, J., Thevenaz. P.,
1998b. The importance of fiber biopersistence and lung dose in determining the chronic inhalation
effects of X607, RCF1, and chrysotile asbestos in rats. Toxicol. Appl. Pharmacol., 153(1),68-82.
in the Rat Lung," Annals of Occupational Hygene, 43(3): 167-1 79, 1999.
Latexaggregaten und Asbestfassern. Staub Reinhalt Luft 30, 277-285.
Lippmann, M. 1990. Effects of fiber characteristics on lung deposition, retention, and disease.
Environ Health Penpect 88: 31 1-317.
Mast, R.W., McConnell, E.E., Anderson, R., Chevalier, J., Kotin, P., Bernstein, D.M., Thevenaz, P.,
Glass, L.R., Miiller, W.C., Hesterberg, T.W., 1995. Studies on the chronic toxicity (inhalation) of four
types of refractory ceramic fiber in male Fischer 344 rats, Inhal Toxicol 7(4):425-67
McClellan, R.O., FJ. Miller, T.W. Hesterberg, D.B. Warheit, W.B. Bunn, A.B. Kane, M. Lippmann,
R.W. Mast, E.E. McConnell, and C.F. Reinhardt. 1992. Approaches to evaluating the toxicity and
carcinogenicity of man-made fibers: summary of a workshop held November 11-13, 1991, Durham,
NC. Regul Toxicol Pharmacol 160): 321-364.
Miller, B.G., et al., "Influence of Characteristics of Inhaled Fibres on Development of Turnours
Morrow, P.E., 1988. Possible mechanisms to explain dust overloading of the lung. Fundament. Appl.
Toxicol., 10, 369-384.
Morrow, P.E., 1992. Dust overloading of the lungs: update and appraisal. Toxicol. Appl. Pharmacol.
113, 1-12.
Muhle H, Bellman B, Heinrich U. 1988. Overloading of lung clearance during chronic exposure of
experimental animals to particles. Ann. Occup. Hyg. 32(suppl. 1)::141-147.
30
IMPORTANCE OF BIOPERSISTENCE IN THE PATHOGENICITY OF ASBESTOS
Draft 17 January 2011
Muhle H, Pott F, Bellmann B, Takenaka S, Ziem U. 1987. Inhalation and injection experiments in rats
to test the carcinogenicity of MMMF. Ann Occup Hyg 31(4B):755-64.
Musselman RP, Miiller WC, Eastes W, Hadley JG, Kamstrup O, Thevenaz P, Hesterberg TW. 1994.
Biopersistences of man-made vitreous fibers and crocidolite fibers in rat lungs following short-term
exposures. Environ Health Perspect 102 Suppl 5:139-43.
Oberdörster G. 2002. Toxicokinetics and effects of fibrous and nonfibrous particles. Inhal Toxicol.
Jan;14(1):29-56.
Oberdorster G. 2000. Determinants of the pathogenicity of man-made vitreous fibers (MMVF). Int
Arch Occup Environ Health 73 Suppl:S60-8.
Oberdörster, G., 1995a. Lung particle overload: implications for occupational exposures to particles.
Regul Toxicol. Pharmacol. 21(1):123-35.
Oberdörster, G., 1995b. The NTP talc inhalation study: A critical appraisal focused on lung particle
overload. Regul. Toxicol. Pharmacol., 21, 233–241.
Pinkerton KE, Brody AR, McLaurin DA, Adkins B Jr, O'Connor RW, Pratt PC, Crapo JD. (1983).
Characterization of three types of chrysotile asbestos after aerosolization. Environ Res. Jun;31(1):3253
Stoeber, W., Flachsbart, H. and Hochrainer, D. (1970) Der Aerodynamische Durchmesser von
Vorwald AJ, Durkan TM, Pratt PC. 1951. Experimental studies of asbestosis. A M A Arch Ind Hyg
Occup Med 3(1):1-43.
WHO. 1988. Environmental Health Criteria 77 Man-Made Mineral Fibres. Vol. 77. World Health
Organigation Geneva.
WHO. 2002. Human Vitamin and Mineral Requirements. Report of a joint FAO/WHO expert
consultation, Bangkok, Thailand. World Health Organization, Food and Agriculture Organization of
the United Nations. Rome, 2002
Zeidler-Erdely PC, Calhoun WJ, Ameredes BT, Clark MP, Deye GJ, Baron P, Jones W, Blake T,
Castranova V. 2006. In vitro cytotoxicity of Manville Code 100 glass fibers: effect of fiber length on
human alveolar macrophages. Part Fibre Toxicol. 28;3:5.
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IMPORTANCE OF BIOPERSISTENCE IN THE PATHOGENICITY OF ASBESTOS
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Appendix 1:
Table A1: Chronic inhalation studies with chrysotile.
Fiber type
Chrysotile
Canadian
Exposure
Time
h/d,
d/wk,
total
months
6, ,5, 14
Fiber
Concentration
f/cm3
Fiber
Mass
Concentration
Equivalent fiber
concentration/cm3
TEM
Type
&
Total
no. of
rats
Number
Pulmonary
Tumours
%
Pulmonary
Tumours
Number
of
mesotheliomas
References
8,600,000
NS,
41
10
24
1
Gross et al., 1967
mg/m3
Nd
86 mg/m3
42-106 mg/m3
1st year
67-146 mg/m3
2nd year
(Nickel, cobalt,
chromium and lead
contamination)
Chrysotile UICC
Canadian
7, 5, 24
Nd
9.7 mg/m3
970,000
W,
21
10
48
1
Wagner et al., 1974
Chrysotile UICC
Rhodesian
7, 5, 24
Nd
14.7 mg/m3
1,470,000
W,
17
11
65
0
Wagner et al., 1974
Chrysotile
Canadian 714-7D
5, 5, 24
1.7 x 105 SEM
9978 > 5µm
15 mg/m3 Total
5.2 mg/m3 Resp
1,500,000
W,
45
9
20
0
Le Bouffant et al.,
1984-1987
7, 5, 24
430 > 5µm PCOM
669 particles
10.8 mg/m3
1,080,000
W,
22
8
36
1
Wagner et al., 1980
10.8 mg/m3
1,080,000
W,
24
3
13
0
Wagner et al., 1980
(friction linings)
SFA chrysotile
Grade 7 chrysotile
7, 5, 24
PCOM
1020 > 5µm PCOM
745 particles
PCOM
IMPORTANCE OF BIOPERSISTENCE IN THE PATHOGENICITY OF ASBESTOS
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10.8 mg/m3
1,080,000
W,
23
5
22
0
Wagner et al., 1980
241 by SEM
131>5 µm reported
as “thick bundles”
6 mg/m3
600,000
W,
50
0
0
0
Muhle et al., 1987
1170 >5µm PCOM
33 > 20 µm PCOM
12 % > 5um SEM
10 mg/m3
1,000,000
W,
20
50
2
Davis et al., 1988
7
17
1
Davis et al., 1988
UICC chrysotile
7, 5, 24
3750 > 5µm PCOM
338 particles
Chrysotile
Calidria
5, 5, 12
Chrysotile long
7, 5, 12
PCOM
Chrysotile short
7, 5, 12
5510 >5µm PCOM
670 > 20 µm
PCOM
7 % > 5um SEM
2560 >5µm PCOM
0.5 % > 20um
SEM
10 mg/m3
40
1,000,000
0.2% > 20um
SEM
W,
40
10 mg/m3
1,000,000
1.02 x 105 SEM
1.06 x 104 >5µm
10 mg/m3
1,000,000
F,
69
13
18
1
7, 5, 12
1950 >5µm PCOM
360 >20µm PCOM
10 mg/m3
1,000,000
W,
40
15
38
0
Mast et at, 1995
Hesterberg et al.
1993
Davis et al. 1978
7, 5, 12
390 >5µm PCOM
72 >20µm PCOM
2 mg/m3
200,000
W,
42
8
19
1
Davis et al. 1978
4
0
Ilgren & Chatfield
1997, 1998;
Pinkerton et al.
1983
Chrysotile UICC A
7, 5, 12
Chrysotile NIEHS
6, 5, 24
Chrysotile
Chrysotile
Included for comparative fiber numbers without animal
exposure
Davis et al., 1988
Author’s reported “With a 2 mg/m3 cloud the percentage retention of chrysotile is almost double that for a 10 mg/m3 cloud.”
Chrysotile Calidria
7, 5, 12
Nd
7.78 mg/m3
778,000
F,
51
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IMPORTANCE OF BIOPERSISTENCE IN THE PATHOGENICITY OF ASBESTOS
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Chrysotile Jeffrey
7, 5, 12
Nd
11.36 mg/m3
1,136,000
F,
49
11
22
0
Ilgren & Chatfield
1997, 1998;
Pinkerton et al.
1983
Chrysotile UICC/B
7, 5, 12
Nd
10.99 mg/m3
1,099,000
F,
54
13
24
0
Ilgren & Chatfield
1997, 1998;
Pinkerton et al.
1983