Non-asbestiform elongate mineral particles and mesothelioma risk: Human and experimental evidence.

The presentations in this session of the Monticello II conference were aimed at summarizing what is known about asbestiform and non-asbestiform elongate mineral particles (EMPs) and mesothelioma risks based on evidence from experimental and epidemiology studies. Dr. Case discussed case reports of mesothelioma over the last several decades. Dr. Taioli indicated that the epidemiology evidence concerning non-asbestiform EMPs is weak or lacking, and that progress would be limited unless mesothelioma registries are established. One exception discussed is that of taconite miners, who are exposed to grunerite. Drs. Mandel and Odo noted that studies of taconite miners in Minnesota have revealed an excess rate of mesothelioma, but the role of non-asbestiform EMPs in this excess incidence of mesothelioma is unclear. Dr. Becich discussed the National Mesothelioma Virtual Bank (NMVB), a virtual mesothelioma patient registry that includes mesothelioma patients' lifetime work histories, exposure histories, biospecimens, proteogenomic information, and imaging data that can be used in epidemiology research on mesothelioma. Dr. Bernstein indicated that there is a strong consensus that long, highly durable respirable asbestiform EMPs have the potential to cause mesothelioma, but there is continued debate concerning the biodurability required, and the dimensions (both length and diameter), the shape, and the dose associated with mesothelioma risk. Finally, Dr. Nel discussed how experimental studies of High Aspect Ratio Engineered Nanomaterials have clarified dimensional and durability features that impact disease risk, the impact of inflammation and oxidative stress on the epigenetic regulation of tumor suppressor genes, and the generation of immune suppressive effects in the mesothelioma tumor microenvironment. The session ended with a discussion of future research needs.

The health effects of short fiber chrysotile and amphibole asbestos.

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.

Final results from a 90-day quantitative inhalation toxicology study evaluating the dose-response and fate in the lung and pleura of chrysotile-containing brake dust compared to TiO2, chrysotile, crocidolite or amosite asbestos: Histopathological examination, confocal microscopy and collagen quantification of the lung and pleural cavity.

The final results from this multi-dose, 90-day inhalation toxicology study in the rat with life-time post-exposure observation have shown 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. The groups exposed to brake dust 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. Slight alveolar/interstitial macrophage accumulation of particles was noted. Wagner grades were 1-2 (1 = control group), similar to the TiO2 particle control group. Chrysotile was not biopersistent, exhibiting in the lung a deterioration of its matrix which results in breakage into particles and short fibers which can be cleared by alveolar macrophages and which can continue to dissolve. Particle-laden macrophage accumulation was observed, leading to a very-slight interstitial inflammatory response (Wagner grade 1-3). There was no peribronchiolar inflammation, occasional very-slight interstitial fibrosis (Wagner grade 4), and no exposure-related tumorigenic response. The pathological response of crocidolite and amosite compared to the brake dust and chrysotile was clearly differentiated by the histopathology and the confocal analysis. Crocidolite and amosite induced persistent inflammation, microgranulomas, persistent fibrosis (Wagner grades 4), and a dose-related lung tumor response. Confocal microscopy quantified extensive inflammatory response and collagen development in the lung, visceral and parietal pleura as well as pleural adhesions. These results provide a clear foundation for differentiating the innocuous effects of brake dust exposure from the adverse effects following amphibole asbestos exposure.

Standardized methods for preparation and bi-variate length & diameter counting/sizing of aerosol and tissue digestion fiber samples.

Most fiber length distributions fit a log-normal distribution with their being many more shorter fibers present as compared to the longer fibers. As the longer fibers have been suggested to be more important for possible pathogenesis giving equal weight to all fiber lengths when sizing fibers will under sample the longer fibers. The methods described here, are based upon the optimization of fiber counting/sizing rules over a number years of experience and have been developed to provide a stable estimate of the mean number of particles and fibers present in the size ranges: particles, fibers < 5 µm; 5-20 µm; and >20 µm. These methods were first applied using TEM, however, with the development of high resolution SEM, it was found that higher reproducibility could be obtained with SEM.

Evaluation of the deposition, translocation and pathological response of brake dust with and without added chrysotile in comparison to crocidolite asbestos following short-term inhalation: interim results.

Chrysotile has been frequently used in the past in manufacturing brakes and continues to be used in brakes in many countries. This study was designed to provide an understanding of the biokinetics and potential toxicology following inhalation of brake dust following short term exposure in rats. The deposition, translocation and pathological response of brake dust derived from brake pads manufactured with chrysotile were evaluated in comparison to the amphibole, crocidolite asbestos. Rats were exposed by inhalation 6 h/day for 5 days to either brake dust obtained by sanding of brake-drums manufactured with chrysotile, a mixture of chrysotile and the brake dust or crocidolite asbestos. No significant pathological response was observed at any time point in either the brake dust or chrysotile/brake dust exposure groups. The long chrysotile fibers (>20 µm) cleared quickly with T(½) estimated as 30 and 33 days, respectively in the brake dust and the chrysotile/brake dust exposure groups. In contrast, the long crocidolite fibers had a T(½)>1000 days and initiated a rapid inflammatory response in the lung following exposure resulting in a 5-fold increase in fibrotic response within 91 days. These results provide support that brake dust derived from chrysotile containing brake drums would not initiate a pathological response in the lung following short term inhalation.

The health risk of chrysotile asbestos.

PURPOSE OF REVIEW: The word asbestos is a poorly attributed term, as it refers to two very different minerals with very different characteristics. One is the serpentine mineral of which the white asbestos, chrysotile, is the most common. The other is the amphibole asbestos, which includes the blue asbestos crocidolite and the brown asbestos amosite. Although today chrysotile is the only type used commercially, the legacy of past use of amphibole asbestos remains. This review clarifies the differences between the two mineral families referred to as asbestos and summarizes the scientific basis for understanding the important differences in the toxicology and epidemiology of these two minerals. RECENT FINDINGS: Biopersistence and sub-chronic inhalation toxicology studies have shown that exposure to chrysotile at up to 5000 times the current threshold limit value (0.1 fibers/cm) produces no pathological response. These studies demonstrate as well that following short-term exposure the longer chrysotile fibers rapidly clear from the lung and are not observed in the pleural cavity. In contrast, short-term exposure to amphibole asbestos results quickly in the initiation of a pathological response in the lung and the pleural cavity. SUMMARY: Significant progress has been made in understanding the factors that influence inhalation toxicology studies of fibers and epidemiological studies of workers. Evaluation of the toxicology and epidemiology studies of chrysotile indicates that it can be used safely under controlled use. In contrast, even short-term exposure to amphibole asbestos can result in disease.

Product stewardship and science: safe manufacture and use of fiber glass.

This paper describes a proactive product stewardship program for glass fibers. That effort included epidemiological studies of workers, establishment of stringent workplace exposure limits, liaison with customers on safe use of products and, most importantly, a research program to evaluate the safety of existing glass fiber products and guide development of new even safer products. Chronic inhalation exposure bioassays were conducted with rodents and hamsters. Amosite and crocidolite asbestos produced respiratory tract cancers as did exposure to "biopersistent" synthetic vitreous fibers. "less biopersistent" glass fibers did not cause respiratory tract cancers. Corollary studies demonstrated the role of slow fiber dissolution rates and biopersistence in cancer induction. These results guided development of safer glass fiber products and have been used in Europe to regulate fibers and by IARC and NTP in classifying fibers. IARC concluded special purpose fibers and refractory ceramic fibers are "possibly carcinogenic to humans" and insulation glass wool, continuous glass filament, rock wool and slag wool are "not classifiable as to their carcinogenicity to human." The NTP's 12th report on carcinogens lists "Certain Glass Wool Fibers (Inhalable)" as "reasonably anticipated to be a human carcinogen." "Certain" in the descriptor refers to "biopersistent" glass fibers and excludes "less biopersistent" glass fibers.

The toxicological response of Brazilian chrysotile asbestos: a multidose subchronic 90-day inhalation toxicology study with 92-day recovery to assess cellular and pathological response.

Inhalation toxicology studies with chrysotile asbestos have in the past been performed at exceedingly high doses without consideration of fiber number or dimensions. As such, the exposures have exceeded lung overload levels, making quantitative assessment of these studies difficult if not impossible. To assess the cellular and pathological response in the rat lung to a well-characterized aerosol of chrysotile asbestos, a 90-day subchronic inhalation toxicology study was performed using a commercial Brazilian chrysotile (CA 300). The protocol was based on that established by the European Commission for the evaluation of synthetic vitreous fibers. The study was also designed to assess the potential for reversibility of any such changes and to permit association of responses with fiber dose in the lung and the influence of fiber length. Wistar male rats were randomly assigned to an air control group and to 2 CA 300 exposure groups at mean fiber aerosol concentrations of 76 fibers L > 20 microm/cm3 (3413 total fibers/cm3; 536 WHO fibers/cm3) or 207 fibers L > 20 microm/cm3 (8941 total fibers/cm3; 1429 WHO fibers/cm3). The animals were exposed using a flow-past, nose-only exposure system for 5 days/wk, 6 h/day, during 13 consecutive weeks (65 exposures), followed by a subsequent nonexposure period lasting for 92 days. Animals were sacrificed after cessation of exposure and after 50 and 92 days of nonexposure recovery. At each sacrifice, subgroups of rats were assessed for the determination of the lung burden; histopathological examination; cell proliferation response; bronchoalveolar lavage with the determination of inflammatory cells; clinical biochemistry; and for analysis by confocal microscopy. Through 90 days of exposure and 92 days of recovery, chrysotile at a mean exposure of 76 fibers L > 20 microm/cm3 (3413 total fibers/cm3) resulted in no fibrosis (Wagner score 1.8 to 2.6) at any time point. The long chrysotile fibers were observed to break apart into small particles and smaller fibers. In vitro modeling has indicated that these particles are essentially amorphous silica. At an exposure concentration of 207 fibers L > 20 microm/cm3 (8941 total fibers/cm3) slight fibrosis was observed. In comparison with other studies, chrysotile produced less inflammatory response than the biosoluble synthetic vitreous fiber CMS. As predicted by the recent biopersistence studies on chrysotile, this study clearly shows that at that at an exposure concentration 5000 times greater than the U.S. threshold limit value of 0.1 f(WHO)/cm3, chrysotile produces no significant pathological response.

Combat and warfare in the early paleolithic and medically unexplained musculo-facial pain in 21st century war veterans and active-duty military personnel.

In a series of recent articles, we suggest that family dentists, military dentists and psychiatrists with expertise in posttraumatic stress disorder (especially in the Veterans Health Administration) are likely to see an increased number of patients with symptomatic jaw-clenching and early stages of tooth-grinding (Bracha et al., 2005). Returning warfighters and other returnees from military deployment may be especially at risk for high rates of clenching-induced masticatory muscle disorders at early stages of incisor grinding. The literature we have recently reviewed strongly supports the conclusion that clenching and grinding may primarily be a manifestation of experiencing extreme fear or severe chronic distress (respectively). We have recently reviewed the clinical and paleoanthropological literature and have noted that ancestral warfare and ancestral combat, in the early Paleolithic Environment of Evolutionary Adaptedness (EEA) may be a neglected factor explaining the conservation of the archaic trait of bite-muscle strengthening. We have hypothesized that among ancestral warriors, jaw clenching may have rapidly strengthened the two primary muscles involved in biting, the masseter muscles and the much larger temporalis muscles. The strengthening of these muscles may have served the purpose of enabling a stronger, deeper, and therefore more lethal, defensive bite for early Paleolithic humans. The neuroevolutionary perspective presented here may be novel to many dentists. However, it may be useful in patient education and in preventing progression from jaw-clenching to chronic facial pain.

Synthetic vitreous fibers: a review toxicology, epidemiology and regulations.

This review addresses the characteristics which differentiate synthetic vitreous fibers (SVFs, e.g., fiber glass, stonewool, slagwool, refractory ceramic fibers, etc.), how these influence the potential biopersistence and toxicity, the most recent epidemiological results and the integration of these findings into the health and safety regulations in Europe and the United States. Also presented is the historical basis for the European classification directive. The use and equivalence of the chronic inhalation toxicology and chronic intraperitoneal injection studies in laboratory rodents for evaluation of fiber toxicology is assessed as well as the impact of dose selection and design on the validity of the study. While synthetic vitreous fibers can span a wide range of chemistries, recognition and understanding of the importance of biopersistence (ability to persist in the lung) in fiber toxicity has led to the development of more and more biosoluble fibers (that break down rapidly in the lung). Still, the epidemiological data available which are largely based upon the use of fibers in past decades, indicate that the SVF do not present a human health risk at current exposure levels. The animal toxicology and biopersistence data provide a coherent basis for understanding and evaluating the parameters which affect SVF toxicity. The current regulations are based upon an extensive knowledge base of chronic studies in laboratory rodents which confirm the relationship between chronic adverse effects and the biopersistence of the longer fibers that can not be fully phagocytised and efficiently cleared from the lung. The amorphous structure of synthetic vitreous fibers facilitates designing fibers in use today with low biopersistence. Both the epidemiological data and the animal studies database provide strong assurance that there is little if any health risk associated with the use of SVFs of low biopersistence. IARC (2001) reclassified these fibers from Category 2b to Category 3 (with RCF and special purpose fibers remaining in 2b) an event which has not been common in the history of these monographs.

The health effects of chrysotile: current perspective based upon recent data.

This review substantiates kinetically and pathologically the differences between chrysotile and amphiboles. The serpentine chrysotile is a thin walled sheet silicate while the amphiboles are double-chain silicates. These different chemistries result in chrysotile clearing very rapidly from the lung (T(1/2)=0.3 to 11 days) while amphiboles are among the slowest clearing fibers known (T(1/2)=500 days to infinity). Across the range of mineral fiber solubilities chrysotile lies towards the soluble end of the scale. Chronic inhalation toxicity studies with chrysotile in animals have unfortunately been performed at very high exposure concentrations resulting in lung overload. Consequently their relevance to human exposures is extremely limited. Chrysotile following subchronic inhalation at a mean exposure of 76 fibers L>20 microm/cm(3) (3413 total fibers/cm(3)) resulted in no fibrosis (Wagner score 1.8-2.6), at any time point and no difference with controls in BrdU response or biochemical and cellular parameters. The long chrysotile fibers were observed to break apart into small particles and smaller fibers. Toxicologically, chrysotile which rapidly falls apart in the lung behaves more like non-fibrous mineral dusts while response to amphibole asbestos reflects its insoluble fibrous structure. Recent quantitative reviews of epidemiological studies of mineral fibers have determined the potency of chrysotile and amphibole asbestos for causing lung cancer and mesothelioma in relation to fiber type have also differentiated between these two minerals. The most recent analyses also concluded that it is the longer, thinner fibers that have the greatest potency as has been reported in animal inhalation toxicology studies. However, one of the major difficulties in interpreting these studies is that the original exposure estimates rarely differentiated between chrysotile and amphiboles. Not unlike some other respirable particulates, to which humans are, or have been heavily occupationally exposed, there is evidence that heavy and prolonged exposure to chrysotile can produce lung cancer. The value of the present and other similar studies is that they show that low exposures to pure chrysotile do not present a detectable risk to health. Since total dose over time decides the likelihood of disease occurrence and progression, they also suggest that the risk of an adverse outcome may be low if even any high exposures experienced were of short duration.

Comparison of Calidria chrysotile asbestos to pure tremolite: final results of the inhalation biopersistence and histopathology examination following short-term exposure.

Calidria chrysotile asbestos, which is a serpentine mineral, has been shown to be considerably less biopersistent than the durable amphibole mineral tremolite asbestos, which persists once deposited in the lung. The initial results of this inhalation biopersistence study in rats that demonstrates this difference were reported in Bernstein et al. (2003). This article presents the full results through 1 yr after cessation of the 5-day exposure. This study was based upon the recommendations of the European Commission (EC) Interim Protocol for the Inhalation Biopersistence of synthetic mineral fibers (Bernstein & Riego-Sintes, 1999). In addition, the histopathological response in the lung was evaluated following exposure. In order to quantify the dynamics and rate by which these fibers are removed from the lung, the biopersistence of a sample of commercial-grade chrysotile from the Coalinga mine in New Idria, CA, of the type Calidria RG144 and that of a long-fiber tremolite were studied. For synthetic vitreous fibers, the biopersistence of the fibers longer than 20 microm has been found to be directly related to their potential to cause disease. This study was designed to determine lung clearance (biopersistence) and the histopathological response. As the long fibers have been shown to have the greatest potential for pathogenicity, the aerosol generation technique was designed to maximize the number of long respirable fibers. The chrysotile samples were specifically chosen to have 200 fibers/cm3 longer than 20 microm in length present in the exposure aerosol. These longer fibers were found to be largely composed of multiple shorter fibrils. The tremolite samples were chosen to have 100 fibers/cm3 longer than 20 microm in length present in the exposure aerosol. Calidria chrysotile has been found to be one of the most rapidly cleared mineral fibers from the lung. The fibers longer than 20 microm in length are cleared with a half-time of 7 h. By 2 days postexposure all long fibers have dissolved/disintegrated into shorter pieces. The fibers between 5 and 20 microm in length were cleared with a half-time of 7 days. This length range represents a transition zone between those fibers that can be fully phagocytosed and cleared as particles and the longer fibers that cannot be fully engulfed by the macrophage. The fibers/objects shorter than 5 microm in length were cleared with a half-time of 64 days, which is faster than that reported for insoluble nuisance dusts such as TiO2. By 12 months postexposure, 99.92% of all the remaining chrysotile was less than 5 microm in length. Following the 5 days of repeated exposure to more than 48,000 chrysotile fibers/cm3 (190 fibers L > 20 microm), histopathological examination revealed no evidence of any inflammatory reaction either after the cessation of the last exposure or at any time during the subsequent 12-mo period. This is in marked contrast to the amphibole tremolite, which was also investigated using the same inhalation biopersistence protocol. The long tremolite fibers, once deposited in the lung, remain over the rat's lifetime with essentially an infinite half-time. Even the shorter fibers, following early clearance, also remain with no dissolution or further removal. At 365 days postexposure, there was a mean lung burden was of 0.5 million fibers L > 20 microm and 7 million fibers 5-20 microm in length with a total mean lung burden of 19.6 million fibers. The tremolite exposed rats, even with exposure to 16 times fewer total fibers than chrysotile, showed a pronounced inflammatory response with the rapid development of granulomas as seen at day 1 postexposure, followed by the development of fibrosis characterized by collagen deposition within these granulomas and by 90 days even mild interstitial fibrosis. With the short exposure, this study was not designed specifically to evaluate pathological response; however, it is quite interesting that even so there was such a marked response with tremolite. These findings provide an important basis for substantiating both kinetically and pathologically the differences between chrysotile and the amphibole tremolite. As Calidria chrysotile has been certified to have no tremolite fiber, the results of the current study together with the results from toxicological and epidemiological studies indicate that this fiber is not associated with lung disease.

The biopersistence of brazilian chrysotile asbestos following inhalation.

With the initial understanding of the relationship of asbestos to disease, little information was available on whether the two different groups of minerals that are called asbestos were of similar or different potency in causing disease. Asbestos was often described as a durable fiber that if inhaled would remain in the lung and cause disease. It has been only more recently, with the development of a standardized protocol for evaluating the biopersistence of mineral fibers in the lung, that the clearance kinetics of the serpentine chrysotile have been shown to be dramatically different from those of amphibole asbestos, with chrysotile clearing rapidly from the lung. In addition, recent epidemiology studies also differentiate chrysotile from amphibole asbestos. The biopersistence studies mentioned have indicated that chrysotile from Canada and California clear rapidly from the lung once inhaled. However, variations in chrysotile mineralogy have been reported depending upon the region. This is most likely associated with variations in the forces which created the chrysotile fibers centuries ago. In the present study, the dynamics and rate of clearance of chrysotile from the Cana Brava mine in central Brazil was evaluated in a comparable inhalation biopersistence study in the rat. For synthetic vitreous fibers, the biopersistence of the fibers longer than 20 microm has been found to be directly related to their potential to cause disease. This study was designed to determine lung clearance (biopersistence) and translocation and distribution within the lung. As the long fibers have been shown to have the greatest potential for pathogenicity, the chrysotile samples were specifically chosen to have more than 450 fibers/cm(3) longer than 20 microm in length present in the exposure aerosol. For the fiber clearance study (lung digestions), at 1 day, 2 days, 7 days, 2 wk, 1 mo, 3 mo, 6 mo, and 12 mo following a 5-day (6 h/day) inhalation exposure, the lungs from groups of animals were digested by low-temperature plasma ashing and subsequently analyzed by transmission electron microscopy (at the GSA Corp.) for total chrysotile fiber number in the lungs and chrysotile fiber size (length and diameter) distribution in the lungs. This lung digestion procedure digests the entire lung with no possibility of identifying where in the lung the fibers are located. A fiber distribution study (with confocal microscopy) was included in order to identify where in the lung the fibers were located. At 2 days, 2 wk, 3 mo, 6 mo, and 12 mo postexposure, the lungs from groups of animals were analyzed by confocal microscopy to determine the anatomic fate, orientation, and distribution of the retained chrysotile fibrils deposited on airways and those fibers translocated to the broncho-associated lymphoid tissue (BALT) subjacent to bronchioles in rat lungs. While the translocation of fibers to the BALT and lymphatic tissue is considered important as in cases of human's with asbestos-related disease, there has been no report in the literature of pathological changes in the BALT and lymphatic tissue stemming from asbestos. Thus, if the fibers are removed to these tissues, they are effectively neutralized in the lung. Chrysotile was found to be rapidly removed from the lung. Fibers longer than 20 microm were cleared with a half-time of 1.3 days, most likely by dissolution and breakage into shorter fibers. Shorter fibers were also rapidly cleared from the lung with fibers 5-20 microm clearing even more rapidly (T1/2 = 2.4 days) than those < 5 microm in length (T1/2 weighted = 23. days). Breaking of the longer fibers would be expected to increase the short fiber pool and therefore could account for this difference in clearance rates. The short fibers were never found clumped together but appeared as separate, fine fibrils, occasionally unwound at one end. Short free fibers appeared in the corners of alveolar septa, and fibers or their fragments were found within alveolar macrophages. The same was true of fibers in lymphatics, as they appeared free or within phagocytic lymphocytes. These results support the evidence presented by McDonald and McDonald (1997) that the chrysotile fibers are rapidly cleared from the lung in marked contrast to amphibole fibers which persist.

The biopersistence of Canadian chrysotile asbestos following inhalation.

Chrysotile asbestos is often included with other asbestos materials in evaluation and classification. However, chrysotile is a serpentine with markedly different physical and chemical characteristics in comparison to amphiboles (e.g., crocidolite, amosite, tremolite). In contrast to amphiboles, which are solid, rodlike fibers, chrysotile is composed like a rope of many fine fibrils, which tend to unwind. In order to quantify the dynamics and rate by which chrysotile is removed from the lung, the biopersistence of a sample of commercial chrysotile from the Eastern Townships area of Quebec, Canada, labeled QS Grade 3-F, which is the longest commercial grade intended for textile use, was studied. As the long fibers have been shown to have the greatest potential for pathogenicity, the chrysotile samples were specifically chosen to have more than 200 fibers/cm3 longer than 20 micro m present in the exposure aerosol. This publication presents the results of this study through 3 mo postexposure. The study design included: (1) Fiber clearance (lung digestions): At 1 day, 2 days, 7 days, 14 days, 1 mo, 3 mo, and 12 mo (to be reported) following a 5-day (6 h/day) inhalation exposure, the lungs from groups of animals were digested by low-temperature plasma ashing and subsequently analyzed by transmission electron microscopy for total chrysotile fibers number in the lungs and chrysotile fiber size (length and diameter) distribution in the lungs. (2) Fiber distribution (confocal microscopy): This procedure was included in order to identify the location of the fibers in the lung. At 1 day, 2 days, 7 days, 14 days, 1 month, and 3 months (to be reported) postexposure, the lungs from groups of animals were analyzed by confocal microscopy to determine the anatomic fate, orientation, and distribution of the retained chrysotile fibrils deposited on airways and in the parenchymal region. Chrysotile was found to be rapidly removed from the lung. Fibers longer than 20 micro m were cleared with T(1/2) = 16 days, most likely by dissolution and disintegration into shorter fibers. The shorter fibers were also rapidly cleared from the lung, with fibers 5-20 micro m clearing even faster (T(1/2) = 29.4 days) than those <5 micro m in length. The fibers <5 micro m in length cleared at a rate (T(1/2) = 107 days) that is within the range of clearance for insoluble nuisance dusts. The breaking apart of the longer fibers would be expected to increase the short fiber pool and therefore could account for this difference in clearance rates. The short fibers were not found clumped together but appeared as separate, fine fibrils, occasionally unwound at one end. Short free fibers appeared in the corners of alveolar septa, and fibers or their fragments were found within alveolar macrophages. The same was true of fibers in lymphatics, as they appeared free or within phagocytic lymphocytes. Neutrophil-mediated inflammatory response did not occur in the presence of chrysotile fibers at the time points examined. Taken in context with the scientific literature to date, this report provides new robust data that clearly support the difference seen epidemiologically between chrysotile and amphibole asbestos.

Comparison of Calidria chrysotile asbestos to pure tremolite: inhalation biopersistence and histopathology following short-term exposure.

The differences between chrysotile asbestos, a serpentine mineral, and amphibole asbestos have been debated extensively. Many studies have shown that chrysotile is cleared from the lung more rapidly than amphibole. In order to quantify the comparative clearance of chrysotile and the amphibole asbestos tremolite, both fibers were evaluated in an inhalation biopersistence study that followed the European Commission recommended guidelines. In addition, the histopathological response in the lung was evaluated following the short-term exposure. This article presents the results of this study through 90 days after cessation of exposure. Following the termination of the study, a subsequent article will provide the complete results through 12 mo after cessation of exposure. In order to quantify the dynamics and rate by which these fibers are removed from the lung, the biopersistence of a sample of commercial grade chrysotile from the Coalinga mine in New Idria, CA, of the type Calidria RG144 and of a long-fiber tremolite were studied. For synthetic vitreous fibers, the biopersistence of the fibers longer than 20 microm has been found to be directly related to their potential to cause disease. This study was designed to determine lung clearance (biopersistence) and the histopathological response. As the long fibers have been shown to have the greatest potential for pathogenicity, the aerosol generation technique was designed to maximize the number of long respirable fibers. The chrysotile samples were specifically chosen to have 200 fibers/cm3 longer than 20 microm in length present in the exposure aerosol. These longer fibers were found to be largely composed of multiple shorter fibrils. The tremolite samples were chosen to have 100 fibers/cm3 longer than 20 microm in length present in the exposure aerosol. Calidria chrysotile fibers clear from the lung more rapidly (T1/2, fibers L > 20 microm = 7 h) than any other commercial fiber tested including synthetic vitreous fibers. With such rapidly clearing fibers, the 5-day exposure would not be expected to result in any pathological change in the lung, and the lungs of animals that inhaled Calidria chrysotile showed no sign of inflammation or pathology and were no different than the lungs of those animals that breathed filtered air. Following this 5-day exposure to tremolite, the tremolite fibers once deposited in the lung parenchyma do not clear and almost immediately result in inflammation and a pathological response in the lung. At the first time point examined, 1 day after cessation of exposure, inflammation was observed and granulomas were already formed. By 14 days postexposure these microgranulomas had turned fibrotic, and by 90 days postexposure the severity of the collagen deposits had increased and interstitial fibrosis was observed in one of the rats. These findings provide an important basis for substantiating both kinetically and pathologically the differences between chrysotile and the amphibole tremolite. As Calidria chrysotile has been certified to have no tremolite fiber, the results of the current study together with the results from toxicological and epidemiological studies indicate that this fiber is not associated with lung disease.

Indices of fiber biopersistence and carcinogen classification for synthetic vitreous fibers (SVFs).

It is generally accepted that the biopersistence of a synthetic vitreous fiber (SVF) is an important determinant of its biological activity. Experimental protocols have been developed to measure the biopersistence of an SVF from short-term inhalation experiments with rats. Clearance kinetics of long (>20 microm) fibers (those believed to have greatest biological activity) have been approximated by one- or two-pool models. Several measures or indices of biopersistence have been proposed in the literature of which three, the weighted half-time (WT(1/2)), the time required to clear 90% of long fibers (T(0.9)), and the so-called slow-phase half-time (T(2)), have been investigated in some detail. This paper considers both one- and two-pool models for long fiber clearance, characterizes the properties of these candidate indices of fiber biopersistence, identifies measures with potentially superior statistical properties, suggests possible cutoff values based on the relation between biopersistence and the outcome of chronic bioassays, and offers comments on the selection of efficient experimental designs. This analysis concludes that WT(1/2) and T(0.9) are highly correlated, are efficient predictors of the outcome of chronic bioassays, and have reasonable statistical properties. T(2), although perhaps attractive in principle, suffers from some statistical shortcomings when estimated using present experimental protocols. The WT(1/2) is shown to be directly proportional to the cumulative exposure (fiber days) after the cessation of exposure and also the mean residence time of these fibers in the lung.

Experience from a long-term carcinogenicity study with intraperitoneal injection of biosoluble synthetic mineral fibers.

The carcinogenic potential in the intraperitoneal cavity of three newly developed biosoluble insulation glass wool fibers (M, P, and V) and one newly developed biosoluble insulation stone wool fiber (O) was investigated and compared to that of a previously developed soluble glass fiber (B). The in vitro dissolution coefficient of the three glass wool fibers ranged from 450 to 1037 ng/cm(2) x h and was 523 ng/cm(2) x h for the stone wool fiber. The in vitro dissolution coefficient of the B fiber was 580 ng/cm(2) x h. Groups of female Wistar rats (strain Crl: Wi BR) were exposed by repeated injections to doses of 0.5, 2, and 5 x 10(9) WHO fibers, which corresponds to between 41 mg to 724 mg fiber injected. In addition, 2 groups of crocidolite were used as positive controls at doses of 0.1 x 10(9) and 1 x 10(9) WHO fibers (0.5 and 5 mg). The in vitro dissolution coefficient of crocidolite is estimated to be approximately 1 ng/cm(2) x h. The protocol of the study and the size distribution of the test samples conformed to the European Commission Protocol EUR 18748 EN, and the study was executed under Good Laboratory Practice conditions. Two of the new insulation wools, fibers M and 0, showed no statistically significant tumorigenic response even at the very high dose of 5 x 10(9) WHO fibers injected. Fibers P and V showed a small tumorigenic response in the ip cavity similar in magnitude to the B fiber, which has been declared in the German fiber regulations as a noncarcinogenic fiber. The response to the soluble insulation fibers was notably different from that of the known carcinogen crocidolite, which produced 53% tumors at a comparatively low dose of 0.1 x 10(9) WHO fibers. The incidence of mesothelioma was found to be highly correlated to the incidence of intra-abdominal nodules and masses at different sites. The incidence of abdominal nodules and masses was highly correlated to the number of animals with ascites. The incidence of chronic peritonitis with fibrotic nodules at different organs also correlated with the incidence of mesotheliomas. Differences in etiology were observed between the massive doses of the highly soluble insulation wools when injected directly into the ip cavity and the lower doses of the extremely insoluble fiber crocidolite. The variability in this reaction and the impairment of animal health put into question the value of these massive doses in evaluating the carcinogenic response of soluble insulation wools. All of the fibers tested fulfilled the exoneration criteria with respect to carcinogenicity according to the European Directive 97/69/EC ("an appropriate intra-peritoneal test has not expressed signs of excessive carcinogenicity"). The dose as defined in the EC-Protocol EUR 18748 EN was 1 x 10(9) WHO fibers with a defined geometric spectrum. The influence of fiber dimensions on the ip tumor response and the difficulty in assessing the influence of the difference in background levels between this and previous studies make direct application of the German TRGS 905 criteria difficult; however, by comparison to fiber B, which in the TRGS 905 is considered as a noncarcinogenic fiber, all of the synthetic mineral fiber types tested in this study also appear to meet the intended German criteria for exoneration.

Calibration study on subchronic inhalation toxicity of man-made vitreous fibers in rats.

This 3-mo inhalation study investigated the biological effects of a special-purpose glass microfiber (E-glass microfiber), the stone wool fiber MMVF21, and a new high-temperature application fiber (calcium-magnesium-silicate fiber, CMS) in Wistar rats. Rats were exposed 6 h/day, 5 days/wk for 3 mo to fiber aerosol concentrations of approximately 15, 50, and 150 fibers/ml (fiber length >20 microm) for E-glass microfiber and MMVF21. For the CMS fiber only the highest exposure concentration was used. During a 3-mo postexposure period, recovery effects were studied. In the highest exposure concentration groups, gravimetric concentrations were 17.2 mg/m3 for E-glass microfiber, 37 mg/m3 for MMVF21, and 49.5 mg/m3 for the CMS fiber. After 3 mo of exposure, lung retention of fibers longer than 20 micro m per lung was 17 x 10(6) for E-glass microfiber, 5.7 x 10(6) for MMVF21, and 0.88 x 10(6) for CMS. After 3 mo of recovery the concentration of the long fiber fraction was decreased to 38.4%, 63.9%, and 3.0% compared to original lung burden for the E-glass microfiber, MMVF21, and CMS, respectively. Biological effects measured included inflammatory and proliferative potential, histopathology lesions, and the persistence of these effects over a recovery period of 3 mo. Generally, observed effects were higher for E-glass microfiber when compared to MMVF21. The following clear dose-dependent effects on E-glass microfiber and MMVF21 exposure were observed as main findings of the study: increase in lung weight, in measured biochemical parameters and polymorphonuclear leukocytes (PMN) in the bronchoalveolar lavage fluid (BALF), in cell proliferation (BrdU-response) of terminal bronchiolar epithelium, and in interstitial fibrosis. The values observed in the proliferation assay on the carcinogenic E-glass microfiber indicate that this assay has an important predictive value with regards to potential carcinogenicity. Surprisingly, for the biosoluble CMS fiber, fibrogenic potential was detected in this study. The results of the CMS exposure group indicate that effects may be dominated by the presence of nonfibrous particles and that fibrosis may not be a predictor of carcinogenic activity of fiber samples, if the fiber preparation contains a significant fraction of nonfibrous particles. In summary, this study demonstrates the importance of fiber dust contamination by granular components. For future subchronic studies a longer posttreatment observation period would be advisable.