The health effects of short fiber chrysotile and amphibole asbestos

Abstract

The potential toxic effects of short chrysotile and amphibole asbestos fibers with lengths <5 to ∼10 µm have been debated over the years. This stems from the large database of epidemiology, toxicology, and in-vitro studies, each of which often provides different information in understanding and differentiating the effects of short fibers. The epidemiology studies in which the cancer potency estimates were based upon relatively high exposure concentrations provide a conservative assessment that shorter fibers would have little if any effect, especially under controlled exposure or environmental conditions that may occur today. The QSAR models have shown that fiber aspect ratio and Mg content are excellent predictors of cancer potency and that short fibers/particles of amphibole would have no effect. The studies of motor vehicle mechanics and in particular workers who serviced chrysotile containing brakes with the majority of the fibers being short provides evidence that motor vehicle mechanics, including workers who were engaged in brake repair, are not at an increased risk of mesothelioma. Several inhalation toxicology studies clearly differentiated that short chrysotile and amphibole asbestos fibers did not produce a significant carcinogenic effect in the lung or pleural cavity. Because of dosing and lack of sensitivity to biosolubility, in vitro studies can be difficult to interpret; however, a number have differentiated short chrysotile and amphibole asbestos fibers from long fibers. Integral to understanding the importance of fiber length in determining possible health effects is an understanding of the biological and physiological function of the respiratory system. Short asbestos fibers, like innocuous dust, can be cleared through the tracheobronchial ciliated mucous transport, phagocytized by macrophages and cleared via the bronchial tree, and can also be removed through the lymphatic system. While the first two methods can remove them from the lung, with lymphatic transport through one-way valves, fibers are removed from the active area of the lung where the fiber-related disease has been shown to develop and can accumulate in lymphatic sumps and lymph nodes. While short asbestos fibers are present in most occupational or environmental exposures, the large body of studies strongly supports that they do not contribute to the health effects of asbestos exposure.

Keywords:

• Short fiber
• chrysotile
• amphibole asbestos
• elongated mineral particles
• crocidolite
• amosite
• toxicology
• human-studies
• in-vitro

1. Introduction

The potential toxic effects of short elongated mineral fibers (EMF) of chrysotile and amphibole asbestos have been debated over the years. While there appears to be general agreement that long fibers are more toxic than short fibers, the potential effects of short fibers remain debated. This stems from the large database of epidemiology, toxicology, and in-vitro studies, each of which often provides different information in understanding and differentiating the effects of short fibers.

This review examines in detail the biological activity of short chrysotile and amphibole asbestos EMF as reported in publications and reports on short fiber asbestos that were identified through either a Google or PubMed search using the terms: “asbestos toxicity,” with either “epidemiology,” “animal,” “in vitro,” and “short or long.” Where possible, asbestos was further differentiated into chrysotile and amphibole asbestos. These studies were evaluated not only for assessing the effects of short fiber chrysotile and amphibole asbestos but also examining where possible the exposure and dosing regimes that were used, which often provide further insight on the applicability of the results reported.

The term asbestos is in itself ambiguous. Classically, it referred to what was termed regulated asbestos fibers, which included chrysotile, and the amphibole asbestos amosite, crocidolite, actinolite anthophyllite, and tremolite. Initially, there was little, or no differentiation between the two minerals named asbestos – chrysotile (serpentine) and amphibole asbestos.

Chrysotile was first described as being decomposed by acid by von Kobell (1834). Chrysotile has the approximate composition Mg₃Si₂0₅(OH)₄ and is a sheet silicate composed of silicate and brucite layers (magnesium hydroxide octahedra). The different dimensions of these two components result in a structural mismatch in which the layers curl concentrically or spirally (Pauling 1930). The fiber walls are made up of ∼12–20 of these layers in which there is some mechanical interlocking. However, there is no chemical bonding as such between the layers. Each layer is about 7.3 A° thick, with the magnesium hydroxide part of each layer closest to the fiber surface and the silicon-oxygen tetrahedra “inside” the curl (Whittaker 1957, 1963; Tanji 1984; Titulaer et al. 1993, Table 2). Hargreaves and Taylor (1946) reported that if fibrous chrysotile is treated with dilute acid, the magnesia can be completely removed. The hydrated silica, which remains, though fibrous in form, had completely lost the elasticity characteristic of the original chrysotile and had a structure that was “amorphous” or “glassy” in type.

In contrast, the amphibole asbestos class of fibers is formed as solid rods/fibers (Skinner et al. 1988; Whittaker 1960). The structure of amphibole is a double chain of tetrahedral silicate (Si₄O₁₁)⁶⁻ with the silica on the outside of the fiber, which makes it very strong and durable. Crocidolite and amosite were the only amphiboles with significant industrial uses (Virta 2002). Tremolite, while not used commercially, has been found as a contaminant in other fibers or in other industrial minerals (e.g. chrysotile and talc). Due to the structural matrix of amphibole fibers, they have negligible solubility at any pH that might be encountered in an organism (Speil and Leineweber 1969).

A distinction is often made between asbestiform and non-asbestiform amphibole. In the asbestiform habit, fibers grow almost exclusively in one direction and exhibit narrow widths (on the order of 0.1 μm). Bundles of individual fibers can often occur. The most common occurrence of asbestos is in cross-fiber or slip fiber veins. In the former, the fiber axes are perpendicular to the walls of narrow openings in the host rock; in the latter, they are parallel. Asbestos rarely occurs as mass fiber bundles in which fibrillar growth is in many directions.

Non-fibrous amphibole asbestos is also sometimes referred to as cleavage fragments. When mechanical stress is applied to non-asbestiform minerals, as when rock extracted from quarries is ground up to produce aggregate, for example, they can split and release particles of different lengths called “cleavage fragments.” These particles can sometimes be counted as asbestos fibers, especially because of their dimensions.

With non-asbestiform amphibole, the mineral crystal growth tends not to grow with parallel alignment but to form multi-directional growth patterns instead. When pressure is applied in crushing the rock, the crystals fracture easily, fragmenting into prismatic particles called cleavage fragments. Some particles or cleavage fragments are acicular or needle-shaped as a result of the tendency of amphibole minerals to cleave along two dimensions but not along with the third (Langer et al. 1991).

In terms of the assessment of short fibers, there is no scientific basis for an absolute cutoff. In addition, as is discussed below, the available studies that address short fibers refer to a range of fiber lengths often up to 10 µm and often use a variety of size distributions having a broader distribution. Most long fiber samples include fibers longer than 15–20 µm, which, as discussed below, cannot be cleared by macrophages. Due to the log-normal distribution of fiber length in most aerosols, the long fiber samples will also include short fibers. There were no studies identified which examined uniquely the effects of intermediate length fractions of 5–15 µm.

Short fibers may also be defined based on physiological deposition and clearance mechanisms in the respiratory tract. As discussed below, the deposition, cellular interaction, and clearance pathways are different depending upon fiber length, diameter, and biosolubility.

While epidemiology studies are important in determining the relationship of exposure to effect, the distribution of fiber length in most situations is broad, which impacts the ability of epidemiology studies to differentiate effect by length. In addition, populations were often exposed to mixtures of chrysotile and amphibole asbestos. Meta-analysis and quantitative structure-activity relationships (QSAR) models of epidemiology studies that have characterized the fiber dimensions provide evidence for differentiating effect by length and diameter. The toxicology of short fibers can be assessed more quantitatively through inhalation toxicology studies where the exposure conditions are well-characterized. In-vitro studies have also investigated differential effects, however, these systems are static and not sensitive to dissolution and the dose to the cell was often high and based on mass rather than fiber number making extrapolation more difficult.

The more inclusive term elongated mineral particles (EMP) has been used to describe a wide variety of mineral fibers without differentiation of composition or dimensions (IOM 2009; NOISH 2011). While the use of the term EMP has become more common, many of the studies reviewed predate this terminology and refer to fibers. To avoid confusion in referencing these studies, the term fiber is used differentiating whenever possible amphibole asbestos fibers from chrysotile. As such, the following terminology is used:

Short fibers (EMP) (<∼5–10 µm) of chrysotile: SFC

Short fibers (EMP) (<∼5–10 µm) amphibole asbestos: SFA

Longer fibers (EMP) >10–20 µm of chryostile: LFC

Longer fibers (EMP) >10–20 µm of amphibole asbestos: LFA

While due to the nature of mining, production, and use, there is often some overlap, this review address primarily asbestiform fibers.

2. Fiber distribution

Asbestos is a natural material that is mined. The mined ore is then milled, which is primarily carried out by fiberizers or crushers, which frees the fibers from the rock and separates them from each other. The separation process typically involves a jaw-type crusher. Following processing, the fibers are separated into numerous standard grades and cleansed further in this rock circuit. The fibers are then machine packaged either by compressing the material into a dense bundle or by blowing the material into bags (EPA 2020a).

Fibers are essentially cylindrical particles that have a length longer than the diameter. This length to diameter ratio is referred to as the aspect ratio. As asbestos fibers are produced through a milling or crushing procedure, the distribution of the length and diameter is usually best described by a bivariate (length and diameter) lognormal distribution (Cheng 1986). The mean values of such a bivariate lognormal distribution are described by geometric mean diameter (geometric standard deviation) and the geometric mean length (geometric standard deviation) (Ramachandran and Cooper 2011). Figure 1 shows a typical plot of fiber number concentration against fiber length showing the skewed nature of the lognormal distribution.

Figure 1. Typical plot of fiber number concentration against fiber length showing the skewed nature of the lognormal distribution.

With the log-normal distribution of length, in most exposure situations, there would be considerably more shorter fibers present than long fibers. The question to be addressed is whether the larger number of short fibers is causing an effect or the fewer long fibers.

3. Human studies

Due to the sampling techniques available for measuring exposures during the first half of the 20th century when asbestos exposures were high, there was no systematic measurement of fiber length and usually not of fiber type. Fiber concentration measurements were initially performed using the midget impinger sampling and later by phase-contrast optical microscopy (PCOM) measurements (WHO 1997). Most studies that have assessed fiber potency from occupational exposure cohorts have relied upon exposure measurements made using the PCOM method. The PCOM method presents limitations as it does not measure the specific length or diameter of the fiber and does not identify the mineral composition of the fiber. In terms of resolution, as it is based upon optical microscopy, it does not assess fibers with a diameter ≤0.2 μm. As mentioned above, to facilitate counting, only fibers >5 µm in length with an aspect ratio of L/D > 3 and diameter <3 µm are counted. Finally, the method specifies that 100 fibers are counted without differentiating length. Because of the larger number of shorter fibers in the log-normal distribution of the aerosol, there will likely be a bias toward counting short fibers as opposed to long fibers.

Subsequent simulations of work environments or reanalysis of available filters, where available, by transmission electron microscopy (TEM) (e.g. Dement et al. 2009) have provided information for assessing the influence of fiber size. Conversion factors from PCOM to TEM have been proposed as well (Cherrie et al. 1989). As usually, 100 fibers were counted and sized by TEM, without differentiation of size range categories (Bernstein and Kunzendorf 2018), the sensitivity of finding fibers longer than 20 µm is often limited.

In a review of epidemiological morbidity or mortality studies, an expert panel set up by ATSDR (2003) identified no studies specifically of fibers shorter than 5 µm. Studies of human/epidemiological data which assess the relationship of fiber length to cancer potency are based upon meta-analysis of studies with fiber size and mineral type information, on quantitative structure-activity relationships (QSAR) models, and on studies in which the vast majority of the airborne fibers were reported to be <10 µm in length.

3.1. Mixed exposures (chrysotile and amphibole)

Berman and Crump (2008) performed a meta-analysis of asbestos-related cancer risk that addresses fiber size and mineral type. In their analysis, the authors expanded the U.S. Environmental Protection Agency (Nicholson 1986) models for asbestos-related lung cancer and mesothelioma to allow the potency of fibers to depend upon their mineralogical types and sizes.

To address the major obstacle of the lack of data on the types and size destructions of fibers to which the study populations were exposed, concentrations were estimated using surrogate data, which included fiber size distributions obtained from air samples collected in various occupational environments and analyzed using TEM. The data was matched to the studies based on factors, such as location (in a limited number of cases), type of operation, and major fiber type. In addition to uncertainty as a result of the matching process, the size distribution measurements were limited to 10 µm, which, as the authors state, precluded assessing the importance of fibers longer than 40 µm, which have been described by Berman et al. (1995) as especially potent. An additional constraint of the size distribution data was the absence of information on morphological types of fibers (e.g. fibrils or single-crystal fibers, bundles, clusters, or matrices). As the counting rules in the original studies (Dement and Harris 1979; Gibbs and Hwang 1980; Hwang and Gibbs 1981) were not clearly documented, it is neither known to what extent such complex fibers were included in the reported distributions nor whether such considerations were applied consistently across the studies. Thus, consideration of the effects of fiber morphology on potency cannot be addressed with the available data.

In addition, none of the studies from which the fiber size distributions were obtained contained information on the relative amounts of chrysotile and amphibole asbestos in each environment. It was necessary to assess this separately.

As there are few studies for which such surrogate data are available, the meta-analysis was limited in the number of studies included. The following studies were identified as potential sources of such data: Cherrie et al. (1979), Dement and Harris (1979), Winer and Cossette (1979), Gibbs and Hwang (1980), Hwang and Gibbs (1981), Roberts and Zumwalde (1982), Marconi et al. (1984), Snyder et al. (1987), Rood and Scott (1989), and Dement et al. (2008).

Berman and Crump (2008) reported that “Although the best estimates of the potency of shorter fiber (5 < length <10 µm) is zero for the “all widths” and widths <0.4 µm metrics (or a small fraction of that of longer fibers for the widths >0.2 µm metric for mesothelioma), the hypothesis that these shorter fibers were non-potent could not be rejected for any of these metrics. Expansion of these metrics to include a category for fibers with lengths <5 µm did not find any consistent evidence for any potency of these shortest fibers for either lung cancer or mesothelioma.”

Korchevskiy et al. (2019) developed a model that reconstructed the mesothelioma potency factors for various types of fibers based on chemical composition and dimensionality. The mesothelioma potency factors were modeled based upon the following data:

Chrysotile, Quebec – Hodgson and Darnton (2000)

Amosite, South Africa – Darnton (2010)

Crocidolite, South Africa – Hodgson and Darnton (2000)

Crocidolite, Australia – Hodgson and Darnton (2000)

Libby amphibole – Darnton (2010)

Russian anthophyllite – Calculated based on Korchevskiy et al. (2013)

Turkish erionite – Calculated based on Simonato et al. (1989)

The following variables were included in the modeling analysis: Silicon oxide content, Ferrous, Ferric oxide content, Iron content expressed as the sum of Fe₂O₃ and FeO, Aluminum oxide content, Magnesium oxide content, Manganese oxide content, Potassium as potassium oxide content, Sodium as sodium oxide, Calcium as calcium oxide, and the Median aspect ratio minus 3.

The model which best predicted the mesothelioma potency factor included silicon oxide content, median aspect ratio minus 3, ferric oxide content, and magnesium oxide content (R = 0.998, R² = 0.996, adjusted R² = 0.988, p = 0.0077). There was only a slight difference in including the Ferric oxide content.

The authors reported that a combination of the aspect ratio of fibers and Mg fraction by itself predicted the mesothelioma potency factor with good accuracy, though with a slightly lower correlation coefficient of R = 0.988.

This analysis further supports the role of dimensionality in fiber toxic effects and, through the observed relationship with magnesium, the effect of biopersistence.

3.2. Amphibole exposure

Korchevskiy and Wylie (2021, 2022) assessed the carcinogenic potency of elongated amphibole particles using quantitative structure-activity relationships (QSAR) models. The mesothelioma and lung cancer potency of amphibole particles were modeled based on their dimensional characteristics and mineral habit (asbestiform vs. non-asbestiform) using epidemiological data and detailed size information. This model was then applied to a large database that they established on dimensional and other relevant characteristics of elongate mineral particles based on dimensional and other relevant characteristics of elongate mineral particles. Included in this analysis was particle-by-particle information for 77 datasets, including dimensionality information for 114,098 particles, covering data for 45 mineral type categories. In the applied database, only particles longer than 2 µm were considered.

To assess the lung and mesothelioma carcinogenic potency from the dimensional characteristics of this dataset, the authors created a set of reference datasets that contained 19,509 particles (82% were simulated from published data, and 18% derived directly from raw laboratory data). The validation datasets included 738 particles, all of them from raw laboratory data. The carcinogenic potency was derived from lung (RL %) and mesothelioma (RM %), potency factors defined by Hodgson and Darnton (2000) for crocidolite from Australia and South Africa, amosite from South Africa, and Na-Ca amphiboles from Libby, MT. Three additional types of particles (asbestiform fluoro-edenite, asbestiform anthophyllite, and non-asbestiform grunerite) were used as validation sets. The mesothelioma potencies for asbestiform anthophyllite and asbestiform fluoro-edenite were estimated as described in Korchevskiy et al. (2020): lung cancer potencies were calculated by regressing the log-log relationship between mesothelioma and lung cancer potency from Hodgson and Darnton (2000). Zero potencies for both mesothelioma and lung cancer was assumed for non-asbestiform grunerite from the Homestake mine based on Gamble and Gibbs (2008) and Garabrant and Pastula (2018).

Table

The reference datasets included:
Asbestiform riebeckite (crocidolite)	South Africa, Cape Province
Asbestiform riebeckite (crocidolite)	Australia, Wittenoom
Asbestiform grunerite (amosite)	South Africa, Transvaal
Na-Ca amphiboles (Libby amphiboles)	USA, Montana
While the validation datasets data from:
Asbestiform fluoro-edenite	Biancavilla, Italy
Non-asbestiform cummingtonite-grunerite (mineral fragments)	Homestake mine, Lead, South Dakota, USA
Asbestiform anthophyllite	Ekaterinburg, Russia

The datasets used for the comparison of various habits of asbestiform minerals included 31,444, non-asbestiform 5058, and mixed 33,379 particles; 22.9% of the particles were from the simulation datasets, and 77.1% from raw data. These included data on dimensional characteristics from the following asbestiform, non-asbestiform, and mixed habit datasets (shown in parenthesis are the country/region from which they were sampled) (from Korchevskiy and Wylie 2021, Table 2; repeated names indicates either difference in TEM/SEM or simulation measurements of either bullk ofr airborne samples):

3.2.1. Asbestiform

Cummingtonite-grunerite (Transvaal, SA; Transvaal, SA; Electric company Shipyard; Transvaal, SA; Transvaal, SA)

Riebeckite (Kuruman Hills, Cape SA; Cape Province, SA; Cape Province, SA; Cape Province, SA; Cape Province, SA; Wittenoom, Australia)

Anthophyllite (Finland; Russia; India)

Tremolite/Actinolite (Lone Pine, CA; Lone Pine, CA; Korea Metsovo, Greece; Udiapur, India; Fairfax, VA; Transvaal, SA)

Na-Ca Amphiboles (Libby, MN; Libby, MN; Libby, MN; Libby, MN)

Fluoro-edenite (Biancavilla, Italy; Biancavilla, Italy; Basilicata, Italy)

Hornblende (Goolwa, Australia)

3.2.2. Non-asbestiform

Cummingtonite-grunerite (Peter Mitchell mine, MN; Homestake, SD; Homestake, SD; Portugal)

Riebeckite (Long Valley, CA; St. Peter's Dome, CO)

Anthophyllite (Sweden)

Tremolite/Actinolite (Miners Bay, Canada; Gouverneur, NY; Balmat, NY; San Bernadina, CA; Enoree, SC; Shadwell, VA)

Fluoro-edenite (Sicily, Italy (Set 1); Sicily, Italy (Set 2))

3.2.3. Mixed

Tremolite/Actinolite (Barstow, CA; El Dorado, CA; Ontario, Canada; El Dorado, CA; Boulder City, NV; Boulder City, NV; Boulder City, NV; Rockhill Quarry, PA; Rockhill Quarry, PA; Calaveras Dam, CA)

Korchevskiy and Wylie (2022) derived regression equations from the reference and validation datasets, which were applied to the data from the asbestiform, non-asbestiform, and mixed selections listed above to estimate the predicted potency factors for each category.

This analysis found that dimensional parameters and morphological habits are the main drivers for differentiating the cancer potency of amphibole elongated mineral particles. The mesothelioma potency was found to correlate best with specific surface area and lung cancer potency correlated best with particle aspect ratio. In addition, through a statistical evaluation of various criteria for carcinogenic potency in the scientific literature, Korchevskiy and Wylie (2022) found that the Stanton criteria (fraction of particles longer than 8 μm and thinner than 0.25 μm) were a relevant predictor of the carcinogenic potency of EMPs, along with recently introduced EMPA and EMPB parameters¹ (Wylie et al. 2020).

The authors also reported that the results suggest that asbestiform amphibole particles are typical of the particles found in the lung burden of mesothelioma patients and that non-asbestiform amphiboles, as a rule, are irrelevant for cancer risk assessment (non-asbestiform particles refer to “mineral fragments,” or “cleavage fragments”) and have negligible potency factors.

3.3. Chrysotile exposure

As shown in the toxicology study by Bernstein et al. (2021) the chrysotile present in brake dust is largely short fiber <5 µm in length. Garabrant et al. (2016), Goodman et al. (2004) presented the results of a meta-analysis of the association between work as a motor vehicle mechanic and mesothelioma. This analysis was based on studies that reported relative risk estimates for mesothelioma among motor vehicle mechanics (in general), and those who were engaged in brake repair (specifically) and included ten case-control studies, one cohort study, and five proportionate mortality ratios (PMR)/standardized mortality odds ratio (SMOR) studies. The results of this meta-analysis provide evidence that motor vehicle mechanics, including workers who were engaged in brake repair, are not at an increased risk of mesothelioma.

The specific studies that examined mesothelioma risk among workers involved in brake dust repair are summarized in Figure 2 (reproduced from Garabrant et al. 2016). All four studies reported odds ratios (ORs) below 1.0, which is indicative of no statistical difference in mesothelioma risk between the workers involved with brake dust repair and the corresponding control group not involved in brake dust repair.

Figure 2. Meta-analysis of mesothelioma risk among workers involved with brake repair (reproduced from Garabrant et al. 2016).

4. Biopersistence

The most standardized study assessing the biopersistence of mineral fibers is the five-day inhalation or intratracheal installation study following a protocol similar to that established by the European Commission (Bernstein and Riego 1999). However, there are no biopersistence studies that have specifically examined samples of short fiber vs. long fiber asbestos.

As discussed below, the clearance of short and long fibers follows different pathways. Fibers that are sufficiently long and insoluble can frustrate the ability of the macrophage to dissolve, break apart or transport them, leading to inflammation and disease. Long fibers which remain in the lung are too large to be cleared by the lymphatic system. The biopersistence of these longer fibers has been associated with carcinogenic potential.

With short fibers, the lymphatic system is one of the major routes of clearance. However, in the standard biopersistence study, the entire lung is digested with no differentiation of where the fibers are in the lung. Short fibers can accumulate in the lymphatic system. With amphibole asbestos, there is often a difference in short-term clearance between long and short fibers. However, with the presence of long fibers, the developing inflammatory response has been shown to, in effect lock up the remaining short fibers as well (Bernstein et al. 2020a, 2020b). With chrysotile, the long fibers have been shown to be considerably less biopersistent and to quickly break apart into shorter fibers which augment the number of shorter fibers present (Bernstein et al. 2013; Bernstein and Kunzendorf 2018). Thus following lung digestion and quantification, the apparent biopersistence of the shorter chrysotile fibers is a function of the increase in numbers from the breakdown of the longer fibers and the accumulation in the lymphatic system. In these inhalation toxicology studies, such accumulation has not been associated with disease.

There are two primary factors that would likely influence the biopersistence of asbestos fibers. The first is the difference in biosolubility between the two mineral types chrysotile and amphibole asbestos. Amphibole asbestos is encased by a crystalline silica shell which has been shown to have little solubility at either pH 7.4 (lung surfactant) or ∼4.5 (macrophage phagolysosome), the two pH environments encountered in the lung. In contrast, chrysotile has been shown to dissociate into individual fibrils at pH 7.4 and to break down into amorphous silica particles at pH ∼4.5.

Chrysotile inhalation biopersistence studies performed with aerosol exposure concentrations at a few orders of magnitude higher than past human exposures have shown that even with significant long fiber (>20 µm) exposure, chrysotile breaks down into smaller particles and fibers which are cleared from the lung with half-times of ∼1 day to 15 days, following a 5 day exposure, depending on the source.

Bernstein et al. (2020a) has determined the clearance half-times of short chrysotile following 90 days of exposure (6 h/d, 5 d/wk) at concentrations of 665 f/cm³ and 753 f/cm³. At the end of the 90 days exposure, the chrysotile fibers with length <5 µm were found to clear with half-times of 29 and 32 days and for fibers 5-20 µm in length with half-times of 28 and 42 days. This is faster than insoluble dust which are considered innocuous (Muhle et al. 1990; Stober et al. 1993; Oberdoester 1994; Yu et al. 1994; ECETOC 2013; Bevan et al. 2018).

There are no biopersistence studies of short fibers of amphibole asbestos. From their composition and the physiological mechanisms of macrophage and lymphatic clearance, they would likely clear at rates similar to insoluble dust. However, with amphibole asbestos aerosols which contain fibers longer than ∼20 µm, Bernstein et al. (2021) has shown using confocal imaging that the intense inflammation caused by the long fibers, once inhaled, quickly locks up the shorter fibers that may deposit in the same regions. In those regions of the lung without such long fiber-initiated inflammation, short fibers have not been observed to accumulate.

5. Toxicology studies

Several inhalation toxicology studies have been designed to determine the differential response of short vs. long fibers. In general, the short fiber samples were produced by milling or grinding the original sample. In many early studies, the exposure concentration was set gravametrically at 10 mg/m³. Fiber concentrations were rarely reported using TEM at the time. A rough estimate can be determined based on a fiber’s cylindrical dimensions and density. For example, at 10 mg/m³, chrysotile (density 2.4 g/cm³) with a mean length of 3 µm and diameter of 0.1 µm would have ∼175,000 fibers/cm³ in the exposure atmosphere.

Vorwald et al. (1951) reported on several inhalation and injection studies on guinea pigs, rabbits, cats, clogs, rats, and mice that were performed at Saranac Laboratory. The inhalation studies were whole body in a cubical dust room with aerosol generation using a rotating dust hopper. The injection studies used either dry asbestos or asbestos suspended in fluid with administration by intravenous, intraperitoneal, intratracheal, or another route.

The short fiber chrysotile samples were ball milled. In the inhalation studies with short fibers asbestos dust, a small quantity of unground short fiber asbestos was mixed with the ball mill product to generate a suitable dust cloud. The authors reported that “When that experiment failed to produce an accelerated tissue reaction, in comparison with the response initiated by Kings floats (a chrysotile which contained fibers from 1 mm to 1 micron and less in length as well as much particulate matter), it became apparent that the biological activity of asbestos is not increased by a reduction of fiber size. Thus the possibility arose that the tissue reaction observed was due solely to the relatively few long fibers of the unground asbestos and that the short fibers of asbestos had no more than a very insignificant role in the production of asbestosis, a concept not in accord with previous experiments concerning pneumonoconiosis.”

Another experiment used white rats and mice exposed to 100% ball-milled chrysotile with rats exposed for periods of up to 20 months and mice for periods of up to 12 months. The exposure concentration was 100–150 mppcf = 3530–5295 particles/cm³.² Fiber size was determined at a magnification of 1300× and showed that 90 percent of the particles/fibers seen were smaller than ∼3 µm with only 1% >10 microns in length. The authors reported for this experiment that “In neither species, it even a suggestion of asbestosis develop and the reaction was limited to phagocytosis of inhaled particles by widely scattered dust cells which remained free in air spaces or were transported to the tracheobronchial lymph nodes. No asbestos bodies were found in the rats, but in the mice, there were a very few small, non-haustrated forms within phagocytes.”

Stanton and Wrench (1972), Stanton (1974), and Stanton et al. (1977, 1981) were the first to systematically assess the effect of fiber length on mesothelioma incidence through the implantation of fibers on gelatin-coated glass substrates in the pleural cavity of rats. Over the course of the studies, more than 70 samples were evaluated, ranging from crocidolite to talc. Stanton (1974) reported that the samples of asbestos and glass evaluated produced a tumor incidence that may be related to fibers below 2.5 µm in diameter and between 10 and 80 µm in length. The 1977 and 1981 publications included a wider variety of materials. The 1981 publication presented a summary of more than 10 years of particle characterization-implantation research. For many of the samples, the fiber measurements showed few fibers with diameters <0.25 µm and lengths longer than 8 µm (Table-Figure 1, Stanton et al. 1977 and Text-Figure 1, Stanton et al. 1981). When repeated samples from the same original fibers were analyzed the authors reported that considerable variation in counts occurred and stated that “Clearly, the method is subject to several errors; calibration of the electron microscope, deviation of particles from the assumed cylindrical shape, and sampling errors, especially where large particles are concerned, represent the major problems. Nevertheless, the estimates are probably valid to within one order of magnitude. Consequently, the counts are reported as the common log with the characteristic of the log representing the probable limit of accuracy (Text-Figure 1).” The log of the counts smooths out some of the variations to facilitate correlation. To include as many samples as possible, the correlation coefficients were only presented in the range <4, >4–8, and >8 µm. It would have been interesting to have presented correlations for subsets of the samples that had a larger number of longer fibers present.

Table 1. In vitro studies that differentiate the effects of short versus long fiber asbestos.

Cell culture model	Asbestos type	Fiber sizes	Exposure concentrations and duration	Type of assays	Results	Publication
Rat phagocytic ascetic tumor cells	AMOSITE; CHRYSOTILE; CROCIDOLITE	Ultrafine: 3 µm Fine: 8 µm Modified: 50 µm	30 µg/ml 27–216 × 10^6 fibers/ml 72 h	DNA and protein synthesis; cytotoxicity	It was possible to demonstrate clear-cut relationships between fiber length and cytotoxicity. At the 27 × 10^6 fibers/ml dose, the short fiber fraction (3 µm) was not different than the controls.	Tilkes and Beck 1980
Mouse peritoneal macrophages	AMOSITE; CHRYSOTILE; CROCIDOLITE NOTE: Same samples as used by Tilkes and Beck (1980)	UICC: ultrafine; medium; long	100 µg/1.5 × 10^6 cells 72 h	Erythrosin uptake, the release of lactate dehydrogenase (LDH), and uptake of 3H amino acids	Long-fiber asbestos dusts proved more cytotoxic than the corresponding short fibers when used on the basis of equal mass.	Kaw et al. 1982
Hamster tracheal organ culture	CHRYSOTILE, ANTIGORITE, the amorphous analogue of chrysotile	Chrysotile: Short: 100% <5 µm 59% < 1 µm Long: 0% <5 µm 50% > 30 µm Antigorite 100% < 3 µm 47% < 0.2 µm	1, 4, and 16 mg/ml 1 h	Assessment of metaplasia	While short fibers did not stimulate cell proliferation, long chrysotile fibers induced cell distortion and sloughing. Attapulgite (palygorskite) and both “long-” and “short-“ fiber preparations of chrysotile-asbestos had similar but less marked effects. Authors reported that the results suggest that the ability of inorganic particulates to induce squamous metaplasia depends upon their fibrous geometry and not on any particular chemical or structural property unique to asbestos.	Woodworth et al. 1983
Guinea pig lung macrophages	CHRYSOTILE; CROCIDOLITE	Chrysotile: Short Munroe: 0.4 9.99 µm Long Munroe: <3–80 µm Jeffrey 1: 1–19 µm Jeffery 2: 0.1–39 µm Crocidolite: Short: 0.1–19 µm Short S. African: 0.4–29 µm Long: 3–129 µm	100 µg/10^6 cells 20 h	Cytotoxicity; phagocytic activity	Long, very thin fibers of glass, chrysotile, crocidolite, and synthetic fluoro-amphiboles were all toxic in the test system	Tilkes and Beck 1983
Rat peritoneal macrophages	AMOSITE	Amosite: Short: 0.5% >5 µm Mean ∼ 0.5 µm 1.9 × 10^7 fibers/µg UICC: 9.5% >5 µm Mean ∼ 1.5 µm 6.2 × 10^6 fibers/µg Long: 31% >5 µm Mean ∼4 µm 2.3 × 10^6 fibers/µg	200 µg/ml with 3–4 × 10^6 macrophages/ml 48 or 96 h	Collagen protein synthesis	All fiber types induced production or release of fibrogenic factor, however, short fibers were more active than long fibers. The short fiber AM sample had ∼1000 fibers/macrophage while the long fiber samples had ∼100 fibers/macrophage	Aalto and Heppleston 1984
Syrian hamster embryo cells	CHRYSOTILE	Experiment 1: Length unmilled: 9.5 ± 0.7 µm Length milled: 1.7 ± 0.1 µm Experiment 2: Length unmilled: 16.0 ± 1.7 µm Length milled: 0.95 ± 0.12 µm	Asbestos-induced Cytotoxicity and Morphological Transformation: 0.25–2 µg/cm^27 days	Cytotoxicity; morphological transformation	After milling chrysotile asbestos in a mortar and pestle, its cytotoxic potency was greatly reduced. More importantly, milling of chrysotile asbestos completely eliminated its transforming potency at all doses tested.	Hesterberg and Barrett 1984
Hamster tracheal epithelial cells	UICC CHRYSOTILE UICC CROCIDOLITE RIEBECKITE, the non-fibrous mineral analogue of crocidolite ANTIGORITE, the amorphous analogue of chrysotile	Chrysotile Short: <2 µm Long: >10 µm Riebeckite ∼1–2 µm Antigorite ∼1–2 µm	0.36–11.6 µg/cm^2CH Short, Long and antigorite 24 h	Induction of ornithine decarboxylase: ODC activity	Maximal elevation of ODC activity was observed after exposure of cells to very low concentrations (0.72 µg/cm l dish) of long fibers, whereas a 4-fold higher concentration (2.9 µg/cm^2 dish) of short fibers was required to induce similar effects. Potency of ODC induction was directly related to fiber length. Riebeckite and antigorite did not increase ODC activity significantly.	Marsh and Mossman 1988
Peritoneal macrophages	CROCIDOLITE	Short: 1.1% > 5 µm (mean 1.2 µm) 4.64 × 10^9 fibers/mg Mixed: 8.8% > 5 µm (mean 2.8 µm) 2.93 × 10^9 fibers/mg Long: 27% >5 µm (mean 5.4 µm) 1.22 × 10^9 fibers/mg	Doses of 100, 300, 500, 700, and 1000 µg of long crocidolite correspond to 1.2, 3.7, 6.1, 8.5, and 12.2 (×10^8) fibers, respectively Doses of 100, 300, 500, 700, and 1000 µg of short crocidolite correspond to 4.6, 13.9, 23.2, 32.5, and 46.4 (×10^8) fibers, respectively Up to 22 h	Cell viability; H202 pro duction; mitochondrial membrane potential	The release of reactive oxygen species was stimulated by both long and short crocidolite; however, long fibers were found to be more toxic than short fibers. The fiber dose administered per cell for the short fibers ranged from 0.9 to 9 × 10^4 fibers/cell	Goodglick and Kane 1990
Rat alveolar macrophages	AMOSITE NOTE: The same fiber samples were used by Davis et al. (1986)	Short: 100% <10 µm Long: 40% >10 µm and 15–20% >20 µm	10, 25, 50 µg/ml Approximately 10–50 fibers/cell 24 h	Cytotoxicity; cell viability: presence of TNF in macrophage supernatants	Short fibers were not effective at stimulating release of TNF at any dose while high doses (50 µg/ml) of long fibers significantly increased the release of TNF following exposure to amosite.	Dogra and Donaldson 1995
Chinese hamster ovary cells	AMOSITE NOTE: The same fiber samples were used by Davis et al. (1986)	Short: 100% <10 µm Long: 40% >10 µm and 15–20% >20 µm	10 µg/ml of long or short amosite asbestos 48 h	Chromosomal aberrations, hyperploidy, and mitotic index	The incidence of all parameters in cells treated with the short amosite fiber sample was similar to control levels. Long amosite fibers significantly increased the incidence of chromosomal aberrations and hyperploidy. Cells exposed to long amosite also demonstrated significantly less mitotic index.	Donaldson and Golyasnya 1995
Rat alveolar macrophages	AMOSITE NOTE: The same fiber samples were used by Davis et al. (1986)	Short: 100% <10 µm Long: 40% >10 µm and 15–20% >20 µm	1 mg/ml 30 m	Assay of superoxide anions	A dramatic enhancement of super oxide anion release was observed with long, but not short amosite fibers following opsonization with immunoglobulin. Unopsonized short amosite seemed to inhibit superoxide release. Opsonization short fibers induced a small superoxide anion release above background. There was no obvious toxicity to the alveolar macrophages with any small fiber treatment, as assessed by the percentage of total cellular lactate dehydrogenase released into the culture medium during incubation.	Hill et al. 1995
Primate models human alveolar macrophages	CHRYSOTILE	UICC A Rhodesian: 75% <5 µm Unmilled RG 144 (Calidria) chrysotile: 98% <5 µm Milled RG 144 (Calidria) chrysotile: 99–100% <5 µm As milling time for RG 144 increased, the particle number per unit mass increased as well.	50–100 µg 24 h	Cytotoxicity	Viability of the cells following treatment was 67% for UICC chrysotile, 47% for un-milled RG 144 chrysotile, and 24–40% for milled RG 144 chrysotile. Doses were based on equal mass, thus the more the RG 144was milled the more particles were administered per cell. With milled RG 144, based on decisions and dose, there were at least 2,500 fibers administered per cell.	Yeager et al. 1983
Human type 2 alveolar epithelial cells	AMOSITE NOTE: The same fiber samples were used by Davis et al. (1986)	Short: 100% <10 µm Long: 40% >10 µm and 15–20% >20 µm	10, 25, 50, 75, and 100 µg/ml 4 h	Epithelial cell injury assay	The long amosite sample was found to cause a rapid detachment of the epithelial cells live from their substratum compared to the short fiber treatment.	Donaldson et al. 1993
Human lung epithelial cells	AMOSITE NOTE: The same fiber samples were used by Davis et al. (1986)	Short: 100% <10 µm Long: 40% >10 µm and 15–20% >20 µm	0.35, 3.5, and 35 µg/cm^2 24 h	Measurement of PPP activity; lipid peroxidation assay; cytotoxicity; enzyme activity assay	Long amosite fibers initiated free radical reactions, significantly inhibited glucose-6-phosphate dehydrogenase (G6PD) and PPP activity, decreased intracellular glutathione and increased thiobarbituric acid reactive substances and leakage of lactase dehydrogenase more effectively than short amosite. Surface reactivity: despite the higher surface exposed, short amosite did not exhibit significant activity in the homolytic cleavage of the carbon–hydrogen bond of format, while the activity of long amosite in the same conditions was very high.	Riganti et al. 2003
Human lung epithelial cells	AMOSITE NOTE: The same fiber samples were used by Davis et al. (1986)	Short: 100% <10 µm Long: 40% >10 µm and 15–20% > 20 µm	0.35–35 μg/cm^2 for LFA and SFA 24 h	NO synthesis and NO synthase (NOS) activity	LFA is more effective than SFA in causing activation of the NO synthesis in human lung epithelial cells after a 24 h incubation.	Tomatis et al. 2010
Macrophages: Human monocytic, monomac-6 (MM6) mouse macrophage (J774A.1) cell lines BAL cells Sprague–Dawley (SD) rats	AMOSITE NOTE: The same fiber samples were used by Davis et al. (1986)	Short: 100% <10 µm Long: 40% >10 µm and 15–20% >20 µm	7.5–125 µg/ml 4, 24, and 48 h	Cytotoxicity induced in J774A.1 cells was assessed via LDH release Pro-inflammatory mediators release	The morphology in J774A.1 cells following treatment with SFA was comparable to NPCB. Cell morphology was considerably different with exposure to LFA; with increased membrane ruffling associated with phagocytosis and increased lamellipodia from cells migrating toward and along fibers. LFA exposed cells were found to “spear” themselves along fibers that were too long for successful phagocytosis, while some cells attempted to phagocytose multiple LFA fibers simultaneously	Boyles et al. 2015

Table 2. Median diameter (and range) of the alveolar macrophage population and interstitial macrophage population for never smokers, smokers and individuals with COPD (from Dewhurst et al. 2017).

	Never smokers	Smokers	COPD	p-Value
IM diameter	13.2 (4.6–15.8)	11.5 (8.7–15.8)	11.7 (7.6–16.9)	0.0035
AM diameter	17.1 (2.9–27.4)	23.7 (14.1–25.6)	23.6 (13.4–25.7)	<0.0001
p-Value	<0.0001	<0.0001	<0.0001

This concept was stated by Wylie et al. (1987), who reexamined using STEM and optical images seven crocidolite samples used by Stanton et al. (1981). Figure 3 reproduced from Wylie presents the tumor probability as a function of the log of the number of fibers per microgram for those samples that had sufficient fiber measurement data to determine what was called the index number – that is, the log of the number of particles longer than 8 µm with widths equal to or <0.25 µm. This figure illustrates the wide variation in the relationship with tumor probability. The correlation coefficient was r = 0.53 (r² = 0.281). While significant, this represents a little more than 25% of the variation in the data. Stanton et al. (1981), including all index numbers, reported an r = 0.80. As Wylie states, “Stanton was aware of the limitations in the characterization of the mineral particulates.”

Figure 3. Regression curve showing the relationship of Station criteria to tumor probability (reproduced from Wylie et al. 1987).

Stanton was instrumental in indicating the importance of fiber dimensions in tumor production. What has become known as the Stanton criteria of fibers L > 8 µm and W ≤ 0.25 µm should be viewed in context with the understanding of the limitations.

Davis et al. (1986) investigated the pathogenicity of long vs. short fiber samples of amosite asbestos administered to rats by inhalation and intraperitoneal injection. The amosite sample was produced so that almost all fibers were <5 µm in length. This sample was compared to a raw amosite that contained a very high proportion of long fibers. The total dust concentration was 1.9 mg/m³ for the long fiber amosite and 11.6 mg/m³ for the short fiber amosite. The authors reported that “At the end of 12 months of dust inhalation, significantly more short fiber amosite was present in the lung tissue compared to the long, but while the long fiber dust caused the development of widespread pulmonary fibrosis, no fibrosis at all was found in animals treated with short fiber. One-third of animals treated with long fiber dust developed pulmonary tumors or mesotheliomas, but no pulmonary neoplasms were found in animals treated with short fiber dust.”

Davis et al. (1986) also exposed animals by interpersonal injection. “Following intraperitoneal injection, the long fiber amosite produced mesotheliomas in 95% of animals with a mean induction period of ∼500 days. With short fiber dust, only single mesothelioma developed after 837 days.”

Davis and Jones (1988) also performed a similar study on long and short chrysotile again prepared by ball milling. The exposure concentration was 13.9 mg/m³ short fiber chrysotile and 11.81 mg/m³ long fiber, with for each exposure group, 10 mg/m³ being respirable. However, ball milling chrysotile was more difficult than amosite, and while the short aerosol had more short fibers than the long aerosol, more than 5% of the fibers were longer than 5 µm with some longer than 20 µm. The fiber length distribution curves appeared parallel with no difference in slope (as did occur in the amosite study). The results did differentiate the long and short exposures; however, the short fiber group still showed a tumorigenic response.

Ilgren and Chatfield (1997, 1998) reported on a chronic inhalation toxicology study that was performed by the US National Institute of Environmental Health Science (NIEHS) that included a short fiber chrysotile Coalinga. Fisher 344 rats were exposed 7 h/day, 5 days/week for 12 months with the mean exposure concentration of 7.78 ± 1.46 mg/m³. The fiber size of the aerosol as measured by TEM and reported as 63% <5 µm (dia 0.39 µm) and 77% <10 µm (dia 0.71 µm) as reported by Pinkerton et al. (1983). Parenchymal fibrosis was scored with the average Wagner score of 1.9 (males) and 2.0 (females). Wagner grade 2 is defined as a few macrophages in the lumen of the terminal bronchioles and alveoli (McConnell and Davis 2002). The incidence of adenomas, carcinomas, or mesothelioma in the Coalinga exposed animals was not statistically different from the controls.

Platek et al. (1985) and Stettler et al. (2008) reported on a chronic inhalation toxicity study to evaluate the effects following exposure to short chrysotile asbestos fibers. Sprague-Dawley rats and Cynomolgus monkeys were exposed for 18 months, 7 h/day, 5 days/week to a specially prepared, chrysotile asbestos aerosol at a mean exposure concentration of 1 ± 0.28 mg/m³. The chrysotile was type 7TF1 obtained from the Johns Manville Sales Corporation (Denver, Colorado). Fibers were prepared by grinding for 24 h in a ceramic ball mill. The count median length of the fibers determined by SEM was 0.67 µm (geometric standard deviation = 1.87), the count median diameter was 0.09 µm, and the median aspect ratio (length:width) was 12. Only 46 (0.66%) of the 6940 fibers were >5 µm in length. The mean number of fibers >5 µm in length was 3.0 fibers/cm³. Through 24 months, the rats showed no fibrosis or pulmonary tumors compared to the controls. With the monkeys, the mean chamber exposure concentration was 1.0 mg/m³ with an average of 0.79 fibers/ml >5 μm in length. Fifteen monkeys (9 exposed and 6 controls) were maintained for 11.5 years following exposure, after which the lungs were examined grossly and microscopically with no lesions attributable to the inhalation exposure noted.

Wagner et al. (1984, 1985) and Wagner (1988) reported on inhalation and intrapleural inoculation studies of long and short erionite and crocidolite asbestos. The short fiber samples were milled to be <5 µm, with the majority <3 µm in length. The inhalation exposure concentration for the erionite was 10 mg/m³, mean gravimetric respirable dust concentration. None of the short fiber samples administered either by inhalation produced tumors following 12 months of exposure and a further 12 months of observation. By intrapleural inoculation, with long fiber crocidolite, there were over 90% tumors, while with short crocidolite, there was a single tumor. No tumors occurred in animals exposed to short erionite.

Ilgren and Chatfield (1998) reported on the results of a lifetime investigation of F344 rats exposed by inhalation to Coalinga chrysotile that was performed at the National Institute of Environmental Health Sciences (NIEHS) and the National Toxicology Program (NTP) between 1978 and 1980. The Coalinga chrysotile exposure used in this study was unique in that almost all of the fibers were <5 µm in length. The exposure concentration was 7.78 ± 1.46 mg/m³. The study showed that animals exposed to these short fibers displayed no tumors above control levels.

Bernstein et al. (2021) reported on the results from a multi-dose, 90-day inhalation toxicology study in the rat with lifetime post-exposure observation which showed a significant fundamental difference in pathological response and tumorgenicity between brake dust generated from brake pads manufactured with chrysotile or from chrysotile alone in comparison to the amphiboles, crocidolite and amosite asbestos. Three doses of brake dust containing chrysotile were used 0.20, 0.34, and 0.67 mg/m³. For the three doses, aerosol exposure concentration of the total number of fibers and those <5 µm (fibers/cm³) were: Low dose 36.44/34.02; mid-dose 48.83/43.98 and high dose 78.44/71.81 fibers/cm³. The chrysotile fibers present in the brake dust were largely smaller than 5 µm and showed no significant pathological, or tumorigenic response in the respiratory track compared to the air control group at exposure concentrations and deposited doses well above those at which humans have been exposed. Two doses of similar chrysotile to that used in the brakes were also exposed at similar gravimetric concentrations of 0.27 and 0.64 mg/m³. The total number of fibers/no fibers L < 5 µm were: Low dose 665/546 and High dose 753/520 fibers/cm³. At these exposure concentrations, the authors reported that there was no peribronchiolar inflammation, occasional very-slight interstitial fibrosis, and no exposure-related tumorigenic response.

6. In-vitro studies

In vitro cell culture studies of fibers are accessible, and these testing methods are valuable to elucidate possible mechanisms involved in pathogenesis. In vitro, cellular systems are most often static systems and not sensitive to differences in fiber solubility. There are usually performed with a single layer of a specific immortalized cell line. An immortalized cell line is a population of cells from a multicellular organism that would normally not proliferate indefinitely but, due to mutation, have evaded normal cellular senescence and instead can keep undergoing division. Immortalized cells are primary cells whose telomeres and/or tumor suppressor genes have been altered to facilitate continued cell division. The cells can therefore be grown for prolonged periods in vitro. This is in contrast to in-vivo inhalation studies where the fibers are exposed to the dynamic behavior of the respiratory system with systematic interaction and signaling between multiple cell types. In addition, the effect of lung surfactants and the fluid flows of the lung are not present.

Fiber number and dimensions are frequently not or are poorly characterized with the in vitro dosing often stated gravimetrically. Many in vitro studies were performed at concentrations to each cell that were significantly higher than that which would occur in either occupational or environmental exposures. High doses of fibers are often used to obtain a positive response, and it is difficult to extrapolate from these large short-term cellular exposures to the considerably lower-dose chronic exposures that occur in vivo.

Most important, however, is that these endpoints have not been validated as screening assays that are predictive of long-term pathological effects in vivo. While in-vitro tests may be useful tools to identify and evaluate possible mechanisms with fibers, these in-vitro test systems are of limited use in differentiating different fiber types (IARC 1999; Bernstein et al. 2005; Griesinger et al. 2016).

Several in vitro studies have been performed that compared short and long samples of chrysotile, amosite, and crocidolite asbestos fibers. As presented in Table 1 (adapted and updated from Barlow et al. 2017), most of these studies exposed cell cultures on an equal mass basis, which resulted in a large difference in fiber dose for the short as compared to the long fibers.

All studies differentiated short from long fibers of either chrysotile or amphibole asbestos except that reported by Aalto and Heppleston (1984). In this study, the amosite dosing was based on an equal mass of fibers. All fiber length fractions induced the production or release of fibrogenic factors; however, short fibers were more active than long fibers. Based on the fiber dimensions, the approximate amosite fiber dose was calculated from the dimensions (and density) presented in the publication. The short fiber amosite sample resulted in a dose of ∼1000 fibers/macrophage, while the long fiber samples had 100 fibers/macrophage.

One of the more recent studies on short and long amosite by Tomatis et al. (2010) provided an evaluation of amosite fiber number per µg of the sample with a determination of the number of fibers administered per cell, which ranged between 0.1 and 10 for long fibers and between 1.5 and 150 for short fibers amosite, and was 2 f/cell for a synthetic glass fiber MMVF10. The dose-response range of this study was unique in that it was based upon the number of fibers administered per cell and was within plausible ranges of dosing. However, the in-vitro system was static with little sensitivity to dissolution effects. The authors stated that in terms of the number of fibers per cell, the short fiber amoiste at a dose of 0.35 μg/cm² may be compared to long fiber amosite at 3.5 μg/cm² and MMVF10 at 35 μg/cm². The authors reported that long fiber amosite shows a higher free radical yield and stimulates more than short fibers the NO production by cells and reacts with ascorbic acid. This difference was associated with the presence of Fe2+ ions poorly coordinated to the surface. The short fibers showed only loosely bound Fe3+ ions as the pristine Fe2+ ions were oxidized during the grinding process converting long fibers into short fibers. Both length and these differences were considered determinants in the differential toxicity.

Donaldson et al. (1993), Dogra and Donaldson (1995), Donaldson and Golyasnya (1995), Hill et al. (1995), Riganti et al. (2003), Tomatis et al. (2010), and Boyles et al. (2015) all used the same short and long fiber amosite samples that were prepared for the amosite short fiber chronic inhalation study (Davis et al. 1986). These studies included rat alveolar macrophage (cytotoxicity), Chinese hamster ovary cells (chromosomal aberration), rat alveolar macrophages (superoxide anions), human type 2 alveolar epithelial cells (epithelial cell injury assay), human lung epithelial cells (measurement of PPP activity; lipid peroxidation assay; cytotoxicity; enzyme activity assay), human lung epithelial cells (NO synthesis and NO synthase (NOS) activity), macrophages (cytotoxicity), respectively and all differentiated short from long fibers. Tilkes and Beck (1980) and Kaw et al. (1982) used the same short and long samples of amosite, chrysotile, and crocidolite in rat phagocytic ascetic tumor cells (cytotoxicity) and mouse peritoneal macrophages (cytotoxicity), also differentiating effects from short from long fibers for each. Woodworth et al. (1983) used a hamster tracheal organ culture-related squamous metaplasia to assess fiber geometry. Tilkes and Beck (1983) used guinea pig lung macrophages with short and long (chrysotile and crocidolite and reported a length- and dose-dependent cytotoxicity with long, very thin fibers of glass, chrysotile, crocidolite, and synthetic fluoro-amphiboles were all toxic in the test system. Hesterberg and Barrett (1984), using Syrian hamster embryo cells, reported that milling chrysotile greatly reduced its cytotoxicity and eliminated its transforming potency. Marsh and Mossman (1988), using hamster tracheal epithelial cells using chrysotile and crocidolite, reported that the potency of ODC induction was directly related to fiber length. Goodglick and Kane (1990), using peritoneal macrophages using crocidolite at relatively high exposures, reported that the release of reactive oxygen species was stimulated by both long and short crocidolite; however, long fibers were found to be more toxic than short fibers.

7. Inhalation and pulmonary response to short asbestos fibers

Humans can breathe through their mouth as well as their nose. Rodents that are often used for inhalation toxicology studies, however, are mandatory nasal breathers. In humans, inhaled fibers can be inhaled through the larynx and trachea and enter the tracheal, bronchial airways which are designed to conduct air to and from the alveoli for respiration. The primary (main) bronchi divide into secondary lobar bronchi. The airways continue to fan out into 17–21 generations of bronchioles, with the terminal bronchioles being the last generation of conducting airways that give rise to respiratory bronchioles, which ultimately lead to the alveoli (Hyde et al. 2009). Fibers that land on the bronchial tree can be cleared through ciliated mucous transport and eventually swallowed or expectorated. Fiber movement in this region is largely by diffusion. Each respiratory bronchiole divides into two through eleven alveolar ducts; each duct gives rise to five through six alveolar sacs.

Depending on aerodynamic diameter, <∼20% of the fibers inhaled by nose-breathing and <∼30% by mouth breathing will deposit in the alveoli (Dai and Yu 1998). The alveoli are lined by a dynamic layer of surfactant which is secreted by the type II alveolar epithelial cells, which have as well microvilli on their surface (Mason and Lewis 2011; Crouch and Wright 2001). Fibers that deposit in the alveoli will come into contact with pulmonary surfactant and will likely be coated by surfactant.

Our understanding of the roles of fiber dose, dimension (length and diameter), and durability (biopersistence) on fiber toxicology has evolved largely through the study of asbestos and synthetic vitreous fibers (SVF). Numerous groups and publications have detailed the importance of the 3 parameters, dose, dimension, and durability associated with potential fiber-related pathology. These include Hammad (1984), Hart et al. (1994); USEPA (1995), Bernstein et al. (1996); ATSDR (2003, 2004, 2018), Donaldson et al. (2006, 2011), and SCOEL (2012).

The differential paradigm of short and long fiber inhalation, deposition, and response is summarized in Figure 4. The aerodynamic diameter (which is ∼3 times the fiber’s width) determines whether and where in the respiratory tract the fiber can deposit.

Figure 4. Differential paradigm of short and long fiber inhalation, deposition, and response.

The fiber length determines in conjunction with biodurability the potential clearance within the respiratory tract. Two transitional ranges are important. The first is whether the fiber is short and thin enough to be cleared by the lymphatic system, and the second is whether it is short enough to be phagocytized by the alveolar macrophages.

For longer fibers, the biopersistence determines whether the longer fibers which have the potential of frustrating phagocytosis by the macrophage will dissolve or disintegrate into shorter fibers and particles or persist. This differentiates chrysotile from amphibole asbestos. Amphibole asbestos is relatively insoluble at either acid or neutral pH at body temperature (Speil and Leineweber 1969).

Choi and Smith (1972) reported on the kinetics of chrysotile dissolution in water (neutral pH) at ambient and body temperatures. They found that the magnesium cations may be continuously liberated from chrysotile fibers leaving behind a silica skeleton that has the elements of the original structure and that the chrysotile dissolves congruently in water. The experimental evidence suggested that the rate-controlling step is the removal of the brucite layer from the fiber surface of chrysotile and that the smaller the particle size, the more magnesium is liberated from the chrysotile in water suspension. Chrysotile has been shown to be acid soluble (Kobell 1834; Whittaker 2009; Gualtieri et al. 2019). The macrophage phagolysosome produces an acid environment which is considered instrumental in the biosolubility of chrysotile. However, these studies are ex-vivo, and mineral dissolution rates alone do not seem not to fully model the dissolution/breakage that has been observed in-vivo (see biopersistence section). It is likely that a combination of the dissolution of the brucite layer combined with the effect of the macrophage acid environment weakens the thin rolled structure of the chrysotile fibril resulting in breakage into shorter fibers and particles. Finally, biopersistence will also have an impact on the number of shorter fibers remaining.

With fibers long enough to frustrate the ability of the macrophage to clear them and which are not bio-soluble, will persist in the lung and initiate an inflammatory cascade. Chronic inflammation has been shown to be a precursor in the pathogenesis of lung cancer and is associated with the development of cancer (Hanahan and Weinberg 2011; Conway et al. 2016). In contrast to acute inflammation, which is important for clearing infection, healing wounds, maintaining tissue homeostasis, and clearing short fibers, inflammation associated with tumor development is often low in grade and chronic (Yang and Lin 2017). With fiber-related inflammation and carcinogenesis, long durable fibers, such as the crocidolite and amosite fibers, once inhaled, are present as physical impediments, which the macrophage fails to successfully phagocytize and clear. With such fibers, the frustrated phagocytosis releases the proteolytic enzymes, which can break down the connective tissue of the lung (Fels and Cohn 1986). Grecian et al. (2018) and Michel et al. (2012) have reviewed the relationship of the cross-talk between macrophages and natural killer cells, such as neutrophils, and the role of neutrophils in cancer. In the subchronic inhalation study discussed above (Bernstein et al. 2021), has shown through collagen quantification and histopathological examination the important role of inflammation in association with tumorigenesis of the long fibers of amosite and crocidolite asbestos. The inflammation also extended to the visceral and parietal pleura with significant increases in thickening and collagen formation.

The lack of inflammation and pathogenesis in the control and chrysotile containing brake dust groups of this study and the lack of persistence in response in the chrysotile groups, which largely resolved by 24 months, provides a further explanation as to why the exposures in these groups did not produce a carcinogenic response in this study. The chrysotile in the brake dust groups was largely short fibers.

7.1. Macrophage clearance

The alveolar macrophage in humans is larger than that found in rats and hamsters. Krombach et al. (1997) reported the following diameters for alveolar macrophages:

• Hamster 13.6 ± 0.4 μm (n = 8)

• Rat 13.1 ± 0.2 μm (n = 12)

• Monkeys 15.3 ± 0.5 μm (n = 7

• Human volunteers 21.2 ± 0.3 μm (n = 10)

The authors stated that the alveolar macrophages (AM) from humans were significantly larger (p < 0.05) than those from all other species studied, corresponding to a 4-fold larger cell volume of human AM (4990 ± 174 μm³) compared to hamster (1328 ± 123 μm³) and rat (1166 ± 42 μm³) AM.

More recently, Dewhurst et al. (2017) examined the characteristics of lung macrophage subpopulations in COPD patients and controls. They differentiated as well macrophages located in the alveolar spaces [alveolar macrophages (AM)] and macrophages located in the alveolar walls or peripheral tissue [interstitial macrophages (IM)]. The median diameter of the alveolar macrophage population was significantly larger than the interstitial macrophage population for never smokers. As shown in Table 2, alveolar macrophage size is increased in COPD patients compared to controls.

7.2. Lymphatic clearance

The peri-bronchoalveolar lymphatics wind around the airways to the level of the respiratory bronchioles and also surround the pulmonary arteries and veins. The lymphatics are involved in removing fluid and particles from the alveolar lumina. Open intercellular junctions composed of the over-lapping and free edges of neighboring endothelial cells are considered to act as “one-way inlet flap valves” together with the help of the anchoring filaments, thus allowing fluid or particulate matter to enter and not to leave the lymphatic capillary lumen (Lauweryns and Baert 1977).

Most important is the existence throughout the lung and pleural lymphatic system of one-way valves which direct the flow. The visceral pleura is abundantly endowed with lymphatic vessels. These lymphatics form a plexus of intercommunicating vessels that run over the surface of the lung toward the hilum and also penetrate the lung to join the bronchial lymph vessels by passing through the interlobular septa. The larger lymphatic vessels in the visceral pleura are equipped with one-way valves directing flow toward the hilum of the lung. All lymph draining from the visceral pleura, therefore, reaches the lung root, either through the lymphatic vessels of the lobular and lobar lung septae, all by flowing along the visceral pleural surface to the lung hilum (Hallifax and Rahman 2016; Bernstein and Pavlisko, 2017).

Bernstein et al. (2004) quantified, using 3D confocal microscopy imaging, the uptake and clearance of chrysotile fibers following a 5 day (6 h/day) exposure to Brazilian chrysotile at an exposure concentration of 2098 WHO³ fibers/cm³. Quantified were the numbers of fibers observed as a function of time in the airspace, bronchial and alveolar epithelium, interstitium, lymphatics, and alveolar macrophages (Figure 5). The lymphatics had the highest clearance rate surpassing that of alveolar macrophage clearance. The authors also demonstrated using confocal imaging that chrysotile fibers of ∼5 µm in length were also observed in the distal lymphatics, both free and in phagocytic lymphocytes (Figures 9(b,e), Bernstein et al. 2004). Fibers <5 µm were not reported; however, these results suggest that the shorter fibers would also be cleared similarly.

Figure 5. Quantification of the numbers of chrysotile fibers observed as a function of time after a 5-day exposure in the airspace, bronchial and alveolar epithelium, interstitium, lymphatics, and alveolar macrophages (reproduced from Figure 11, Bernstein et al. 2004).

7.3. Pleural clearance

7.3.1. Parietal pleura

On the parietal pleura, lymphatic channels communicate with the pleural space via stomas measuring 2–12 μm in humans that are found predominantly along the mediastinal parietal pleura and intercostal surfaces, particularly in the lower chest. These stomas are the primary pathway for the elimination of fluid and material from the pleural space. The flow through the parietal pleura lymphatic channels is aided by one-way valves that prevent fluid from flowing back into the pleura (Wang 1985; Miura et al. 2000; Bertin and Deslauriers 2011; Negrini and Moriondo 2013, Light and Lee 2016). The pleural liquid is conveyed to the regional lymph nodes, including the intercostal, parasternal, diaphragmatic, and posterior mediastinal lymph nodes (Broaddus et al. 1988; Yalcin et al. 2013). From there, lymph is carried to the thoracic duct and right lymphatic duct (Krenke and Mierzejewski 2020).

7.3.2. Visceral pleura

The visceral pleura does not have stomata and thus does not act in the clearance of pleural fluid.

The visceral pleura has an extensive lymphatic plexus that drains from the lung surface toward the hilum. The plexus also penetrates internally to join lymph channels along the interlobular septa. All drainage from the visceral pleura eventually reaches the lung root (Finley and Rusch 2011).

Alveolar structure and the lung lymphatic system: Around the alveoli, the lymphatic ducts wind around the blood vessels surrounding the alveoli. The surfactant in the alveoli enters into the lymphatic ducts through a system of one-way valves, which are designed to assure that the flow of fluid is into the lymphatic ducts then up through lymphatic channels exiting into the bloodstream.

In addition, there is an extensive lymphatic network below the visceral pleura. Again one-way valves assure that the lymphatic fluid flow is away from the visceral pleura and leads eventually to the hilar lymph node region.

Riquet (2007) described the lymphatics of the lung as a visceral pleural network and a parenchymatous peri-bronchovascular plexus, located mainly in the connective tissue surrounding the airways and their bronchial artery vascular supply. Lymphatics originate from capillaries located between the alveolar walls and the interlobular, pleural, peribronchial, or perivascular connective sheets. They form lymphatic collecting vessels called “collectors.” The lymphatic collectors contain smooth muscles and one-way valves every 2–10 mm along their length. The segments within two valves, the lymphangions, act as a small pump, with smooth muscle contraction in one lymphangion forcing fluid into the next. The valves prevent retrograde flow. The visceral pleura lymphatic collectors course over the surface of the lung toward the lung hilum, where they generally anastomose with the lymphatic collectors of the peribronchovascular plexus (the latter, strictly speaking, being the bronchial lymphatics). They also can travel further along the bronchi and the lower part of the trachea without anastomosing and enter directly into the mediastinum (Lauweryns 1970; Lauweryns and Baert 1977; Riquet et al. 1989; Drake et al. 1991).

Bronchus-associated lymphoid tissue (BALT) describes small clusters of cells found near large airways, usually bronchi. BALT is especially prominent in rodents following inhalation exposure which accumulate and likely transport particles cleared by the lymphatic system. These cellular accumulations are not present in the healthy lungs of rodents or humans but can be induced following infections or other antigenic events. In humans, a similar pattern of cells has been described and is called IALC for isolated aggregations of lymphoid cells (Macklin 1955; Semmler-Behnke et al. 2007; Randall 2010; Van Winkle et al. 2018, Margaris and Black 2012).

8. Discussion

8.1. Reviews and reports

The Agency for Toxic Substances and Disease Registry (ATSDR 2004) reported on the findings of an expert panel that was convened to evaluate the influence of fiber length on the health effects of asbestos and synthetic vitreous fibers. The panel of experts concluded 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 shorter than 5 μm are unlikely to cause cancer in humans.”

When considering the non-cancer effects of short fibers, the panel concluded that laboratory animal studies, epidemiologic studies, and in vitro studies generally suggest that asbestos and SVF pathogenicity increases with fiber length, but there are several notable exceptions. In laboratory animals at sufficiently high doses, which may not be relevant to human environmental exposures, inflammation and fibrosis were observed. Four epidemiological studies (Churg et al. 1989; Churg 1990; Nayebzadeh et al. 2001) suggested that short fibers in the lung at death were associated with fibrosis. These associations in these publications were fairly weak and largely attributed to short amphibole fibers. As an example, Nayebzadeh et al. (2001) found a correlation of tremolite fibers <5 µm with R = 0.44, thus explaining <20% of the association with fibrosis. Churg et al. (1989), examining 21 cases, found a positive correlation of tremolite grade with fibrosis and a negative association with length. However, Churg et al. (1989) counted by TEM, 100 sequentially encountered chrysotile and/or tremolite asbestos fibers which preferentially count short fibers, which were larger in number than long fibers. In addition, the correlation coefficient was R = 0.46, accounting for 21% of the association with fibrosis.

The US Environmental Protection Agency (EPA) has recently conducted an assessment to evaluate the dangers associated with high-density products made with chrysotile and, in particular, brake shoes and sealing gaskets which could be found on the US market in which the EPA identified cancer risks from inhalation exposure to chrysotile asbestos (EPA 2020a, 2020b, 2020c, 2020d). While the Science Advisory Committee on Chemicals (SACC), peer-reviewed the document, many questions remained unanswered. As reviewed by Paustenbach et al. (2021), the EPA did not consider more than 15 epidemiology studies as discussed above, which indicated that exposures to encapsulated chrysotile asbestos in brakes and gaskets, which were generally in commerce from ∼1950 to 1985, did not increase the incidence of any asbestos-related disease. Instead, the EPA based their evaluation of the chrysotile mesothelioma hazard based on populations in two chrysotile textile facilities where mixed exposure to chrysotile and commercial amphibole asbestos (amosite and crocidolite) occurred.

As Paustenbach et al. (2021) has shown, ∼96% of the person-time accrued for the North Carolina textile cohorts was from workers at plants three and four (Garabrant 2020), which also had documented use of amosite and crocidolite asbestos (UNARCO 1954; UNARCO 2012).

It also remains unclear why exceedingly high exposures 1940s–late 1970s to longer textile fibers (Dement et al. 2009; Loomis et al. 2009; Elliott et al. 2012; Loomis et al. 2012, 2019) were used to assess lifetime doses of chrysotile asbestos that were around 2–5 f/cc-years as occurred for auto mechanics in the 1950s–1985 and as presented above was exposed predominantly to short fibers (Paustenbach et al. 2003, 2004; Finley et al. 2007). The resulting suggested inhalation unit risk for chrysotile was not greatly different than that for amosite even though evaluation of mesothelioma potency has shown that amphibole asbestos is considerably more potent than chrysotile (Berman et al. 1995; Hodgson and Darnton 2000; Berman and Crump 2008; Garabrant and Pastula 2018; Wylie et al. 2020). The importance of length and diameter has been stated by many researchers (Pott 1978; Berman et al. 1995; Berman and Crump 2008; Loomis et al. 2010; Lippmann 2014; Wylie et al. 2020).

Korchevskiy et al. (2019) reported on the development of an empirical model that would reconstruct mesothelioma potency factors for various types of fibers based on their chemical composition and dimensionality. The authors modeled the mesothelioma potency factors estimated by Hodgson and Darnton method based upon the chemical composition and dimensionality metrics (aspect ratios) for Quebec chrysotile, South Africa amosite, South Africa and Australian crocidolite, Russian anthophyllite, Libby amphiboles, and Turkey erionite. The mesothelioma potency factor was highly correlated with a function of the aspect ratio, silicon dioxide, iron, and magnesium (R² = 0.994). However, a combination of the aspect ratio of fibers and Mg fraction also predicted the model mesothelioma potency factor with good accuracy (R = 0.988). Through this analysis, the authors proposed the possibility that the biopersistence of asbestos fibers in the human respiratory system is approximately inversely proportional to magnesium content.

Barlow et al. (2017) has reviewed asbestos fiber length in relation to disease and found that studies reported over the last several decades have consistently supported the conclusions that there is very little if any risk associated with exposure to fibers <5 µm and that exposure to fibers longer than 10 µm and perhaps 20 µm are required to significantly increase the risk of developing asbestos-related disease in humans.

Boulanger et al. (2014) also reviewed fiber size toxicity through the quantification of short and long asbestos fibers to assess asbestos exposure. Included was a review of air sampling measurements collected in the city environment and buildings with asbestos-containing materials (ACM) and reported that the majority of fibers were <5 µm in length with concentrations sometimes ≥ 0.01 f/cm³. The authors concluded that the pathogenicity of short asbestos fibers could not be completely ruled out, especially in high exposure situations. There was no consistent differentiation between chrysotile and amphibole asbestos but rather the generalized use of the term asbestos.

Gualtieri et al. (2019) presented a structure model to explain the toxic action of chrysotile in terms of structural, chemical, and physical properties using in vitro cytotoxicity tests and alkaline comet assay tests. The cytotoxicity effects of acid-leached chrysotile fibers were tested on THP-1 (monocytes) and Met-5A (benign immortalized mesothelial cells) cell lines. Cells were dosed at concentrations of 25–100 µg/ml without any details provided of the number of fibers administered to the cells in these static systems. The authors reported that the in vitro tests indicate a significant cyto/genotoxicity of the acid-leached chrysotile in both investigated cells lines. There was no discussion of the limitations of using static in-vitro methods or of the fiber dose to the cells.

Gualtieri (2021) presented a novel approach to correlate how and to what extent the physical/crystal‐chemical and morphological parameters (including length, chemistry, biodurability, and surface properties) of mineral fibers are associated with major adverse effects with an emphasis on asbestos. The authors state that the model assumes that chrysotile is less potent for the induction of MM and low exposures to chrysotile do not present a detectable risk to health compared to amphibole asbestos (Roggli 1995; Hodgson and Darnton 2000; Roggli and Vollmer 2008; Mossman et al. 2011; Bernstein et al. 2013; Garabrant and Pastula 2018). The model fiber characteristics include that chrysotile is not biodurable and easily leached in vivo in the lungs, whereas amphibole asbestos fibers are biodurable (Jaurand et al. 1977; Morgan 1994; Bernstein et al. 2013) and induce chronic inflammation responsible for adverse effects. The authors viewed this as an initial model and plan to verify the model predictions with in vitro toxicity tests and eventually in vivo testing. However, the use of in-vitro testing due to the difficulties and biases described in this review may not be the best validation. A recent exchange between Gualtieri and Di Giuseppe 2022 and Wylie and Korchevskiy (2022) concerning the letter to the editor by Wylie and Korchevskiy (2021) on the carcinogenicity of fibrous glaucophane has highlighted this. Wylie and Korchevskiy (2021, 2022) emphasized that epidemiology should be the basis for the validation of structural models and pointed out that the model developed by Gualtieri (2021) does not correctly predict the potency of fibrous glaucophane.

8.2. In vitro

In vitro studies can provide valuable information depending upon how they are performed. Many in vitro studies, however, have been reported with little if any information on the number of fibers actually dosed per cell in the cell cultures. Most often, dosing is stated as µg/cm² applied to the cell culture dish. As is illustrated below, this can result in very high dosing to each cell far above that which would be expected to occur in human occupational or environmental exposures.

The number of fibers applied can be estimated based on the cylindrical dimensions and density of the fiber. If fiber has an average diameter of 0.1 µm and length of 3 µm, with a density of 2.4 g/cm³ (chrysotile), there would be more than 10 million fibers per µg.

In the publication by Huang (1979), the authors stated that they used a Flacon cell dish (100 mm) with 10⁵ cells per dish and applied 10 µg/cm² to the dish. As shown in Table 3, ∼500,000 fibers were applied to each cell.

Table 3. In-vitro dosing and extrapolated fiber exposure in Huang (1979).

1.77E + 07	Number fiber in 1 µg	Density 2.4
	In vitro
314	Area in cm^2 Falcon cell dish 100 mm
10	µg/cm^2 applied in vitro
1.77E + 08	Total no fiber applied per cm^2
1.00E + 05	No of cells per dish
3.18E + 02	No cells/cm^2
5.55E + 05	No fibers/cell

In a more recent study by Levresse et al. (2000), the authors stated that “Normal RPMC from Sprague–Dawley rats were cultured according to a method described elsewhere (Jaurand et al. 1981).”

Jaurand et al. (1981) state “5 × 10⁵ cells were then put into a Falcon tissue culture flask (25 cm²).”

The authors in this study reported the length and diameter were 1.7 ± 2.2 and 0.05 ± 0.04 μm, respectively, for chrysotile and 2.1 ± 3.6 and 0.19 ± 0.12 μm for crocidolite.

The cells were exposed to 0.5, 2, 5, or 10 μg/cm² chrysotile, stating that “Briefly, cells were plated in 75 cm² tissue culture flasks (Costar, Dutscher, Brumath, France).”

For the low dose of 0.5 µg/cm², as shown in Table 4, ∼70,000 fibers were applied per cell.

Table 4. In-vitro dosing and extrapolated fiber exposure in Levresse et al. (2000).

	In vitro
75	Area in cm^2 cell dish
0.5	µg/cm^2 applied
9.75E + 07	Total no fiber applied per cm^2
1.00E + 05	No of cells per dish
1.33E + 03	No cells/cm^2
7.31E + 04	No fibers/cell

Huang et al. (2011) summarized a large series of over 80 in vitro studies. The greatest detail provided in dosing was by µg/cm² applied. The above analysis of two of these studies chosen at random shows that the number of fibers applied per cell is certainly excessive and far exceeds the few fibers per cell that are occasionally seen following exposure to ∼250 WHO fibers/cm³ of chrysotile (Bernstein et al. 2021).

At these concentrations, dispersion and agglomeration can also be important. Cohen et al. (2014) reported on evidence of the effect of agglomeration on the dose delivered to the cell.

8.3. Clearance of short fibers from the lung

In contrast to longer fibers (>∼20 µm length) which are too long to be fully engulfed by the macrophage or enter the lymphatic system and can only be removed either by dissolution or breakage, short thin chrysotile or amphibole asbestos fibers, especially those shorter than 5 µm in length can be removed by these routes. If a short fiber is phagocytized by a macrophage, it can be transported to the ciliated bronchi and cleared by the ciliated mucous bronchial clearance. An equally important route of clearance is by entry through the lymphatic valves into the lymphatic system. As presented above, the study by Bernstein et al. (2004), which quantified using confocal microscopy the differential clearance between these routes, has shown that lymphatic clearance is the most important for short, thin fibers.

Distributed throughout the lymphatic system are 1-way valves (every 2–10 mm), which assure that the lymph flow is in the direction of the hilum. Distal lymph nodes located within the lobes along the segmental bronchi lymphatics probably develop during the years following birth, as does bronchial and bronchiolar lymphoid tissue (Riquet 2007).

Bronchus-associated lymphoid tissue (BALT) in rodents and isolated aggregations of lymphoid cells (IALC) in humans describe small clusters of cells found near large airways, usually bronchi. BALT and IALC increase following inhalation exposure which accumulates and likely transport particles cleared by the lymphatic system. Through the one-way valves, the lymphatics reach the larger peri-tracheobronchial lymph nodes. The lymphoid tissue and lymph nodes can accumulate the short fibers. Dodson et al. (2000) reported that even in non-occupationally exposed individuals, short non-commercial amphibole asbestos fibers could be found.

Short fibers in both inhalation toxicology studies and human tissue burden studies may be cleared from the active area of the lung but may be stored in the lymphatic tissue. Lung digestion studies which will include the lymphatics, cannot differentiate wherein the lung the fibers were located, and thus the attribution of shorter fibers remaining in the lung to possible disease should not be based on such studies.

The role of lymphatics is also important when determining fiber concentration in the pleural cavity and associating this with the possible disease. The extensive lymphatic network just below the visceral pleura can be an important source for short fibers. Upon necropsy, if the visceral pleura of the lung is dissected, which is often the case to reach the pleural cavity, the extensive network of one-way valves and the pumping action of the lymphangions will no longer be present, allowing the draining of the fluid into the pleural cavity effectively contaminating the area with short fibers.

Barlow et al. (2017) has summarized human studies which document the abundance of short fiber compared to longer fibers. “Several groups have documented fiber concentration and size retained in the lung tissue of those occupationally exposed to asbestos (Churg and Wiggs 1984, 1986, 1987; Langer and Nolan 1994; Tossavainen et al. 1994; Suzuki and Yuen 2002). These groups reported that shorter fibers were more abundant in the human lung of those occupationally exposed to asbestos than longer fibers, regardless of fiber type. One group suggested, based on these data, that either the carcinogenic size range is much broader than thought or a small number of fibers in certain size ranges can induce tumors in humans (Churg and Wiggs 1984).”

However, these authors did not assess where in the lung the short fibers were located. Lung digestion studies cannot differentiate fiber location in the lung. In addition, as presented above, biopsy and especially autopsy samples will have a very high likelihood of disturbing the homoeostatic pressure balances in the lung. As presented above, the lymphatic system is important in the removal of short fibers from the active area of the lung, where the fiber-related disease has been shown to develop. The extensive network of lymphatic sumps and nodes in the human lung in which fibers have been shown to accumulate the shorter fibers that enter the lymphatics. Confocal microscopy as discussed above does provide the means to differentiate the routes of fiber clearance in the lung without producing artifacts through the cross-contamination of fluid and tissues through surgical procedures.

8.4. Assigning causality based upon fiber number

As discussed above, because of the log-normal distribution of fiber length, there are usually many more short fibers than longer fibers present. This is augmented through the use of fiber counting/sizing protocols in which 100 fibers are counted without differentiating size categories (that is, individual counting rules for fibers <5 µm, 5–20 µm, >20 µm).

As an example, Suzuki et al. (2005) reported pathological evidence that short, thin asbestos fibers contribute to the development of human malignant mesothelioma. The authors reported that the samples were taken at autopsy or biopsy. Suzuki et al. state (in Table 1 of the publication) that for the digested tissues 3.7% of the chrysotile found and ∼50% of the amphiboles (amosite, crocidolite, tremolite, and anthophyllite) were longer than 5 µm in length. By ashing 0.3% of the chrysotile and between 43 and 75% of the amphiboles were longer than 5 µm. However, the authors provided no basis other than fiber numbers to associate cause and effect.

As mentioned above, Bernstein et al. (2021) reported on the results of a 90-day inhalation exposure which included chrysotile and amosite and crocidolite asbestos. At 12 months following the 90-day exposure, the chrysotile exposed rats had 5% of fibers in the lung >5 µm, similar to what Suzuki et al. reported 63% of amosite and 77% of crocidolite were longer than 5 µm. With chrysotile, there was only a very-slight interstitial inflammatory response, peribronchiolar inflammation, occasional very-slight interstitial fibrosis, and no exposure-related tumorigenic response. Crocidolite and amosite induced dose-related lung tumor response, which correlated to the number of fibers L > 20 µm. Confocal microscopy quantified extensive inflammatory response and collagen development in the lung, visceral and parietal pleura, as well as pleural adhesions. The assigning of cause and effect based solely on fiber numbers can be very misleading.

In addition, concerning the Suzuki studies, the ATSDR report (2003) stated that they must always be regarded with suspicion, particularly when the study lacks controls and the finding is in what is in fact, tissues of new growth, such as plaques and tumors (Case 1994).

9. Conclusions

Epidemiology, toxicology, and in-vitro studies provide strong support that short chrysotile and amphibole asbestos fibers with lengths <5–10 µm do not contribute to the health effects of asbestos. The epidemiology studies in which the cancer potency estimates were based upon relatively high exposure concentrations provide a conservative assessment that shorter fibers would have little if any effect, especially under controlled exposure or environmental conditions that may occur today.

The QSAR models have shown that fiber aspect ratio and Mg content are excellent predictors of cancer potency and that short fibers/particles of amphibole would have no effect. The studies of motor vehicle mechanics and in particular, workers who serviced chrysotile containing brakes with the majority of the fibers being short provides evidence that motor vehicle mechanics, including workers who were engaged in brake repair, are not at an increased risk of mesothelioma. Several inhalation toxicology studies clearly differentiated that short chrysotile and amphibole asbestos fibers did not produce a significant carcinogenic effect in the lung or pleural cavity. Nearly all these studies were performed at exposure concentrations far exceeding those that occurred with uncontrolled human exposures in the past. Because of dosing and lack of sensitivity to biosolubility, in vitro studies can be difficult to interpret; however, a number have differentiated short chrysotile and amphibole asbestos fibers from long fibers.

Integral to understanding the importance of fiber length in determining possible health effects is an understanding of the biological and physiological function of the respiratory system. Short asbestos fibers, like innocuous dust, can be cleared through the tracheal-bronchial ciliated mucous transport, phagocytized by macrophages and cleared via the bronchial tree, and can also be removed through the lymphatic system. While the first two methods can remove them from the lung, with lymphatic transport through one-way valves, fibers are removed from the active area of the lung where the fiber-related disease has been shown to develop and can accumulate in lymphatic sumps and lymph nodes.

While short asbestos fibers are present in most occupational or environmental exposures, the large body of studies strongly supports that they do not contribute to the health effects of asbestos exposure.

Acknowledgments

I am pleased to acknowledge the anonymous reviewers the Editor selected. Besides these anonymous reviewers and the author, no individual has reviewed any draft material included in the paper and any drafts of the paper.

Declaration of interest

This study was initiated and funded by the International Chrysotile Association (ICA) to promote a further update on the science of short fibers. David Bernstein is an independent consultant specializing in inhalation toxicology. He has appeared twice as an expert witness in litigation concerned with alleged health effects of exposure to chrysotile and has been deposed seven times. ICA does not intend to use this publication for litigation purposes. Dr. Bernstein has published more than 20 peer-reviewed papers on asbestos over the past 20 years. The work product, including the conclusions drawn, is exclusively those of the author, Dr. Bernstein, and has not been influenced by anyone other than the author. Aside from the author, no one, including the International Chrysotile Association or its members, has reviewed, commented on, or revised this paper before its submission.

Notes

1 The EMPA index is defined as the fraction of elongated mineral particles (EMPs) with aspect ratio (AR) ≥ 3, length > 5 μm and width ≤ 0.15 μm to total EMPs with AR ≥ 3 and length > 5 μm. The EMPB index is the fraction of EMPs with AR ≥ 3, length >5 μm and width ≤ 0.25 μm to all EMPs with AR ≥ 3, length >5 μm (Wylie et al., 2020).

2 mppcf × 35.3 = million particles per cubic meter = particles per c.c. (Occupational Safety and Health Admin., Labor § 1910.1001).

3 Fibers with length ≥5 μm, diameter ≤3 μm (WHO, 1985).