Short, Fine and WHO Asbestos Fibers in the Lungs of Quebec Workers With an Asbestos-Related Disease

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1 AMERICAN JOURNAL OF INDUSTRIAL MEDICINE 56: (2013) Brief Report Short, Fine and WHO Asbestos Fibers in the Lungs of Quebec Workers With an Asbestos-Related Disease Georges Adib, MSc, 1 France Labrèche, PhD, 2,3 Louise De Guire, MD, MSc, 1,3 Chantal Dion, PhD, 2,3 and André Dufresne, PhD 3 Background The possible role of short asbestos fibers in the development of asbestosrelated diseases and availability of lung fiber burden data prompted this study on the relationships between fiber characteristics and asbestos-related diseases among compensated workers. Methods Data collected between 1988 and 2007 for compensation purposes were used; lung asbestos fibers content of 123 Quebec workers are described according to socio-demographic characteristics, job histories and diseases (asbestosis, mesothelioma, lung cancer). Results Most workers (85%) presented chrysotile fibers in their lungs, and respectively 76%, 64%, and 43% had tremolite, amosite, and crocidolite. Half of the total fibers were short, 30% were thin fibers and 20% corresponded to the World Health Organization definition of fibers (length 5 mm, diameter 0.2 and <3 mm). Chrysotile fibers were still observed in the lungs of workers 30 years or more after last exposure. Conclusion Our findings stress the relevance of considering several dimensional criteria to characterize health risks associated with asbestos inhalation. Am. J. Ind. Med. 56: , ß 2013 Wiley Periodicals, Inc. KEY WORDS: chrysotile; amphiboles; lung fiber burden; short fibers; fine fibers Abbreviations: CSST, Commission de la sante et de la se curite du travail; EDS, energy dispersive spectrometer; WHO fibers, fibers corresponding to the World Health Organization (WHO) counting criteria; EPA, Environmental Protection Agency; AFSSET, Agence française de se curite sanitaire. 1 Institut national de sante publique du Que bec (INSPQ),Montreal,Quebec,Canada 2 Institut de recherche Robert-Sauve en sante et en se curite du travail du Que bec (IRSST), Montreal,Quebec,Canada 3 De partement de sante environnementale et sante au travail, Universite de Montre al, Montreal,Quebec,Canada Contract grant sponsor: French Agency for Food, Environment and Occupational Health and Safety (ANSES); Contract grantnumber: 2008_CRD_28. Disclosure Statement: The authors report no conflicts of interests. Disclaimer: The findings and conclusions in this report are those of the authors and do notnecessarily represent theviews of INSPQ,IRSST, or Universite de Montre al. *Correspondence to: Georges Adib, MSc, Institut national de sante publique du Que bec, 190 Cremazieblvd, East Montreal, Quebec,Canada H2P1E2. georges.adib@inspq.qc.ca Accepted 4 February 2013 DOI /ajim Publishedonline 26 March 2013 inwiley Online Library (wileyonlinelibrary.com). ß 2013 Wiley Periodicals,Inc.

2 1002 Adib et al. INTRODUCTION A careful analysis of lung fiber content is valuable to characterize prior exposure to asbestos [Baker, 1991; Dodson and Atkinson, 2006]. The presence of asbestos fibers and of ferruginous and asbestos bodies in lung tissues confirms exposure [Dodson et al., 2008; Gibbs and Pooley, 2008] and a lung content analysis provides an estimate of fiber burden [Baker, 1991]. Much of the debate on pathogenicity of asbestos fibers rests on the difference in potency between fiber types and their dimensional and physicochemical characteristics, which are often associated with their biopersistence [Aust et al., 2011]. Asbestos fibers are classified in two major types: serpentines, represented by chrysotile, and amphiboles, in particular amosite and crocidolite (that have industrial applications), tremolite, actinolite, and anthophyllite [NIOSH, 2011]. Regarding fiber dimensions, the relative innocuousness of short fibers has been questioned, owing to the lack of data on the subject [Dodson et al., 2003]. A previous study reported that the concentration of short tremolite fibers significantly predicted lung fibrosis among Quebec asbestos mine workers [Nayebzadeh et al., 2006]. On the other hand, asbestos fibers biopersistence in the lungs, generally inferred from animal studies, is considered a marker of the failure of the lungs clearance mechanisms: the longer a fiber remains in the lungs, the more potent it is presumed to be [Davis, 1994]. However, it is suspected that animal models underestimate fiber retention in humans because of several anatomical and physiological differences [Maxim and McConnell, 2001]. Rodents breathe only through the nose and thus filter dusts more efficiently, whereas humans breathe through the nose and mouth (possibly resulting in larger intake of fibers). Humans have larger airways and higher ventilation rates than rodents (possibly leading to larger deposition of fibers). On the other hand, the number and size of human alveolar macrophages are larger (allowing for a better clearance capacity for larger fibers). Persistent questions regarding the dimensional characteristics of fibers and the availability of mineralogical analyses of lung tissues from workers with an occupational asbestos-related disease prompted this study. Our objective was to explore the relationships between fiber characteristics and asbestos-related diseases among compensated workers, according to their socio-demographic characteristics, their disease and their occupational history. MATERIALS AND METHODS Study Population and Asbestos-Related Diseases Quebec legislation designates asbestosis, mesothelioma and lung cancer associated with asbestos exposure as occupational lung diseases [Government of Quebec, 2012a]. Each worker s claim is studied by a special medical committee reporting to the Quebec Workers Compensation Board (Commission de la Santé et de la Sécurité du Travail, CSST) that may request an analysis of lung fiber content, if available, to inform its decision-making process. In Quebec, no biopsy is performed purely to establish attribution, as the expert chest physicians ask for a lung fiber burden only when pathological material is readily available (through diagnostic biopsies or autopsies). This paper reports on Quebec workers whose asbestos-related disease was recognized as an occupational lung disease and whose lung fiber content was analyzed between 1988 and For this purpose, access to the CSST data was authorized under section 175 of the Act Respecting Occupational Health and Safety [Government of Quebec, 2012b]. Obtaining written consent from the study subjects was not required because the data were depersonalized. From 135 cases with an asbestos-related disease for whom a mineralogical analysis had been requested during that period, 123 were retained in the study after excluding one duplicate case, eight for whom no mineralogical data could be traced and three with no fiber counts. The data used in this study were extracted from the workers medical record summary kept by the CSST: gender, age, diagnosis made by the special medical committee (based on the clinical picture and lung burden data), date of recognition as an occupational respiratory disease, smoking status and history; and for each job ever held by the worker: dates of start and of termination of employment, occupation, economic sector, and duration of asbestos exposure. The occupational history had been collected from an interview or from documents produced by the worker or his family and, in some cases, from surveys of the work environment carried out by public occupational health teams. Lung burden data are used to confirm asbestos fiber exposure according to reference counts of fibers and of ferruginous bodies [Dufresne et al., 1994] when the workers asbestos exposure is not clear or does not appear sufficient in duration or intensity to induce an asbestosrelated disease, or among certain smokers with lung cancer. Twenty-three workers had received a diagnosis of asbestosis, 27 of mesothelioma, and 58 of lung cancer, while three workers had been diagnosed with both asbestosis and mesothelioma, and 12 with both asbestosis and lung cancer. In the following analysis, we considered each disease independently and a worker with two diagnoses is considered twice, that is, once for each disease. Lung Fiber Data and Analysis The lung fiber raw data were made available to us by the CSST. Lung tissue preparation and analysis have been

3 Asbestos Fibers in Lung Tissues of Quebec Workers 1003 previously described [Dufresne et al., 1995; Nayebzadeh et al., 2006]. In summary, after digestion of lung tissue in a sodium hypochlorite solution and vacuum filtration on a Millipore 1 (EMD Millipore Corporation, Billerica, MA) cellulose ester membrane filter (25 mm diameter and 0.45 mm porosity), the filter was ashed in an oxygen plasma furnace (LFE 1 Corp., Model 500, Waltham, MA, 15 ml/min of oxygen, 150 W) for 4 hr. The ashes were recovered with distilled and deionized water, and the suspension filtered on a polycarbonate Nuclepore 1 membrane filter (25 mm diameter and 0.2 mm porosity). All solutions used during sample preparation were filtered on 0.45 mm pore size cellulose membrane filter (Millipore) and residues on these filters were regularly analyzed. Blanks were analyzed every seven samples. On average one or two particles, usually less than 1 mm in diameter, were observed per grid opening [Dufresne et al., 1994]. Four microscope grids were prepared from each sample and were observed in transmission mode under an electron microscope (Model 100 CX, JEOL, Inc., Peabody, MA) equipped with an X-ray energy dispersive spectrometer (EDS; PGT System IV, Princeton Gamma Tech, Inc., Model 4000T, Rockyhill, NJ) with an accelerating voltage of 80 kv and a magnification of 10,000. From those grids, 30 openings (or less if 30 fibers were counted) with an area of 6,400 mm 2 were randomly observed for fibrous particles less than 5 mm in length. A similar counting procedure was applied for fibers longer than or equal to 5 mm. Fiber types were recognized from morphological features and EDS spectrum. We did not distinguish between cleavage fragments and fibers but rather decided to use the World Health Organization (WHO) and the Occupational Safety and Health Administration (OSHA) definition of a respirable fiber (e.g., length to diameter ratio > 3). The length and diameter of all fibers were measured to the nearest mm (0.5 mm on the screen) with two concentric circles (10 and 50 mm diameters) printed on the fluorescent screen [Dufresne et al., 1995; Nayebzadeh et al., 2006]. Millipore cellulose ester membrane filters, with a 25 mm diameter and a 3.0 mm pore size, were used for identification and numeration of ferruginous bodies. The filters were cleared and fixed on a glass slide with dimethyl formamide/acetic acetic/water and Eukitt 1 (O. Kindler GmbH & Co, Freiburg, Germany) solutions; counting was done with a phase contrast microscope at a magnification of 312 [Dufresne et al., 1996b]. The same methods were used to establish fiber burden and ferruginous bodies reference values for a non-exposed Quebec population [Dufresne et al., 1994]; consequently, any artifact introduced by these methods would not have impaired comparisons to be drawn between the study population and the non-exposed Quebec reference population. Chrysotile, amosite, crocidolite, and tremolite fibers have been identified based on their elemental composition. Anthophyllite fibers were not counted because of the difficulty in differentiating this type of asbestos due to its chemical similarity with talc fibers. Although these two types of fibers may be differentiated by electron diffraction analysis, this technique had not been used on fiber counts available for this study. The length, diameter and asbestos fiber type were recorded for every observed fiber. Fibers longer than 0.5 mm, with a diameter smaller than 3 mm and having a length to diameter aspect ratio 3:1 were counted. An absence of fiber was noted when none was observed after viewing 60 grid openings (30 for fibers <5 mm and 30 for fibers 5 mm), for each of the four asbestos fiber types; for the analysis of concentrations, absence of fiber was replaced by half the detection limits of the laboratory performing the analyses (i.e., 35 or 53 fibers per milligram [f/mg] of dry lung tissue), and the same was done for undetected results of ferruginous bodies (20 or 35 ferruginous body per gram of dry lung tissue) [Hornung and Reed, 1990]. A detailed report was produced for each worker, stating the concentrations and types of fibers observed, by fiber length. For most workers (97/123), fiber metrics were available, as well as their concentrations by type of fiber; for the 26 remaining workers, only fiber concentrations were available. No information was available on the exact location of the excised tissue in the lung, and for the same worker, different lung tissue blocks showed a wide variation in fiber concentrations. Hence, the mean concentrations values were derived from the pooled lung tissue blocks obtained for each of the 123 workers rather than from individual blocks. Two laboratories conducted the lung fiber burden analysis: a university laboratory between 1988 and 2000 and a private one between 2000 and 2007, always under supervision by the same chemist (AD). Data Analysis The descriptive analyses were performed using SPSS 1 version 19.0 (SPSS, Inc., Chicago, IL). Workers characteristics (age, sex, occupation, etc.) are presented as proportions, arithmetic means and standard deviations. Lung fiber burden results are presented per fiber type, as geometric means, minimum and maximum values, counts, concentration and dimensions, according to the asbestosrelated disease and occupational asbestos exposure history (main industry/main job). As the focus of this study was placed on fiber characteristics, ferruginous body counts were only considered for descriptive purposes. For this study, we did not have access to lung tissues from an unexposed reference population of workers. We could only compare our results with reference counts from a Quebec unexposed population [Dufresne et al., 1996a].

4 1004 Adib et al. This was true for all workers. We thus tried to overcome this by looking at fiber characteristics according to a rough marker of exposure, namely duration of work in an exposed job (presuming that a longer duration results in a larger lung burden). We also explored clearance by looking at the lag between the last exposure and the lung fiber burden analysis (presuming that the longer the lag, the lower the burden, because of clearance time). Statistical significance of the differences between geometric mean fiber concentrations according to median duration of exposure or median lag were tested using the non-parametric Mann Whitney U-test. Asbestos Fibers Classification In this study, fibers are defined as particulates with a length (L) to diameter (d) aspect ratio (L/d) 3:1. Asbestos fibers were classified into three broad categories: short asbestos fibers are fibers with 0.5 < L < 5 mm and d < 3 mm; fine asbestos fibers are fibers with L 5 mm and d < 0.2 mm, whereas fibers corresponding to the World Health Organization (WHO) counting criteria (WHO fibers) are those with L 5 mm and 0.2 mm d < 3 mm. Industry and Job Categories A main industry and job category was assigned to each of the 123 workers based on their occupational history. The main industry (or job) was defined as the one in which the worker spent at least 60% of his asbestosexposed working years. Some categories were created by combining different industries and jobs to better represent asbestos exposure. The categories are: (1) Chrysotile asbestos mining (2) Construction: work on construction sites with asbestos containing materials (3) Maintenance and repair: all activities of maintenance and repair involving asbestos products, asbestos containing materials or structures that may contain asbestos (4) Manufacturing of asbestos products (5) Other manufacturing industries (6) Ship building and repair (7) Miscellaneous industries (8) Mixed: when no industry represented at least 60% of the asbestos-exposed working years Five job categories were created, again to better reflect asbestos exposure: (1) Skilled workers (miners, electricians, plumbers and pipefitters, etc.) (2) Machine operators and drivers (stevedores, machine operators, truck drivers, etc.) (3) Laborers (construction helpers, general laborers, etc.) (4) Executives, office workers, foremen, natural and applied sciences related occupations (5) Mixed jobs: when the worker never occupied one of the previous categories for at least 60% of his asbestos exposed working years. When the occupational history was incomplete or inaccurate, workers were assigned to an unknown industry or occupation category. RESULTS Table I describes the subjects included in this study by asbestos-related disease. Most of these workers were male, with an average age of 69 years at disease recognition by the special medical committee, and 57% of them had lung cancer. For 70% of the sample, the main industries (in which workers had at least 60% of their asbestos exposure) were chrysotile asbestos mining, maintenance and repair and construction; for 79% of the workers, their main job was skilled worker, laborer, or machine operator and driver. The exposure characteristics of these workers varied with their disease and their main industry. The longest average duration of exposure and the longest average latency periods were found in workers with asbestosis and in workers whose main industry was chrysotile asbestos mining, whereas the longest average lags between last exposure and lung fiber analysis were for workers with a mesothelioma and in construction workers and those in other industries (Table II). Globally, chrysotile fibers were found in the lungs of 85% of the workers, tremolite in 76%, amosite in 64% and crocidolite in 43%. Chrysotile and tremolite fibers were observed in almost all workers whose main industry was chrysotile asbestos mining, whereas amosite fibers were identified in more than 80% of workers in all industries except chrysotile asbestos mining, and crocidolite fibers were found in 61% of workers in construction (Table II). Chrysotile fibers were found in more than 85% of workers with asbestosis or lung cancer, whereas amosite, chrysotile and tremolite were each found in 70% of workers with mesothelioma. Table III presents geometric mean concentrations, counts and dimensions of asbestos fibers, as well as geometric mean concentrations of ferruginous bodies, by main industry, disease and fiber type. Concentrations of short (<5 mm) chrysotile and tremolite fibers were generally two times higher than those of fibers 5 mm. The highest concentrations of these two fiber types were found in the lungs of workers in the chrysotile asbestos mining

5 Asbestos Fibers in Lung Tissues of Quebec Workers 1005 TABLE I. Demographic Characteristics, Smoking Habits and Occupational History of Study Subjects, According toasbestos-related Disease, Quebec, Canada, 1988^2007 Asbestosis (N ¼ 38) Pleuralmesothelioma (N ¼ 30) Lungcancer (N ¼ 70) Total (N ¼ 123) Male:N(%) 38(100.0) 30(100.0) 69(98.6) 122(99.2) Averageage: years(sd) 69.7(8.4) 68.3(11.7) 69.0(8.3) 68.7(9.4) Smokingstatus a : NeversmokerN(%) 2(6.7) 3(27.3) 4(6.2) 8(8.4) Smokerorex-smokerN(%) 28(93.3) 8(72.7) 61(93.8) 87(91.6) Averagepack-years(SD) 44.5(32.4) 41.9(31.9) 41.5(23.1) 41.0(26.1) Mainindustry b :N(%) Chrysotileasbestosmining 13(34.2) 5(16.7) 30(42.9) 42(34.1) Maintenance andrepair 9(23.7) 10(33.3) 8(11.4) 26(21.1) Construction 7(18.4) 4(13.3) 10(14.3) 18(14.6) Other manufacturing 3(7.9) 4(13.3) 4(5.7) 9(7.3) Manufacturingofasbestosproducts 1(2.6) 2(6.7) 5(7.1) 8(6.5) Shipbuildingandrepair 2(5.3) ö 8(11.4) 8(6.5) Miscellaneous 1(2.6) 3(10.0) 4(5.7) 8(6.5) Mixed 1(2.6) 1(3.3) ö 2(1.6) Unknown 1 (2.6) 1 (3.3) 1 (1.4) 2 (1.6) Mainoccupation b :N(%) Skilled workers 17(44.7) 11(36.7) 34(48.6) 58(47.2) Laborers 9(23.7) 5(16.7) 13(18.6) 22(17.9) Machine operatorsanddrivers 2(5.3) 9(30.0) 7(10.0) 17(13.8) Executives,officeworkers,foremen, 2(5.3) 2(6.7) 4(5.7) 7(5.7) natural andappliedsciencesrelatedoccupations Mixed 7(18.4) 2(6.7) 9(12.9) 15(12.2) Unknown 1 (2.6) 1 (3.3) 3 (4.3) 4 (3.3) N, number of subjects; SD, standard deviation. Asbestos-related diseases are those that have been recognized as occupational respiratory diseases forcompensation purposes by a special medical committee.each disease was considered independently and a worker with two diagnoses is considered once foreach disease (12 workers had both asbestosis and a lung cancer and three had both asbestosis and a mesothelioma). a Missing data: eight cases with asbestosis,19 cases with pleural mesothelioma,five cases with lungcancer, and 28 cases with at leastone asbestos-related disease. b Main industry oroccupation: industry oroccupation in whichthe subjects spent 60% or more of their occupational asbestos-exposed years. sector. Workers in the maintenance and repair sector and in construction had the highest concentrations of amosite fibers in their lungs, while the highest concentrations of crocidolite fibers were found in the lungs of workers whose main industry was chrysotile asbestos mining or other industries and of workers with mesothelioma. Average concentrations of ferruginous bodies were the highest for workers with lung cancer and for those in the construction sector, with values as high as 2,782,200 ferruginous bodies per gram of dry lung tissue observed for workers in this industry. In Quebec, a count of 1,000 ferruginous bodies per gram of dry lung tissue is considered indicative of occupational asbestos exposure [Dufresne et al., 1994]. In general, amosite and crocidolite fibers were the longest and chrysotile fibers, the shortest; tremolite fibers also had the largest diameters and chrysotile fibers, the smallest. These characteristics result in tremolite fibers presenting the smallest length to diameter aspect ratio (L/d) regardless of the disease or the industry; chrysotile fibers had the largest L/d in asbestos chrysotile mining, whereas crocidolite fibers had the largest L/d in construction (Table III). For all subjects, all diseases and all fiber types together, 50% of the total fibers observed in the lung tissues were short, 30% fine and 20% WHO fibers (data not shown). However, this categorization of asbestos fibers revealed certain peculiarities, depending on the disease, main industry and fiber type (Table IV). Workers with asbestosis had mostly short fibers in their lung, regardless of fiber type, whereas those with mesothelioma or lung cancer had higher proportions of short

6 1006 Adib et al. TABLE II. Exposure Characteristics of the Study Subjects, According to Asbestos-Related Disease and Main Industry, Quebec, Canada, 1988^2007 Asbestosis (N ¼ 38) Asbestos-relateddisease a Pleural mesothelioma (N ¼ 30) Lung cancer (N ¼ 70) Chrysotile asbestosmining (N ¼ 42) Mainindustry b Maintenance andrepair (N ¼ 26) Construction (N ¼ 18) Other c (N ¼ 37) Allcases (N ¼ 123) Exposureto asbestos(duration inyears) N Mean (SD) 26.6(13.4) 17.7(15.8) 24.9 (12.4) 27.8(11.4) 25.0 (13.6) 15.2(12.7) 21.4 (15.3) 23.5 (13.8) Latency(yearsbetweenfirstexposureto asbestos andrecognitionof the disease) N Mean(SD) 46.2(9.9) 43.3(13.0) 44.8(9.5) 45.4(10.4) 43.2(9.6) 43.0(7.7) 44.6(11.2) 44.3(10.1) Lag(yearsbetweenlastexposureto asbestos andlungfiberanalysis) N Mean (SD) 17.1 (12.9) 28.9 (15.3) 19.5(13.6) 17.5(11.5) 19.3 (15.4) 23.8(15.8) 23.0 (16.1) 20.4 (14.2) Typeofasbestosfoundin lungtissues Amosite N (%) 24 (63.2) 21 (70.0) 43(61.4) 11 (26.2) 21 (80.8) 17 (94.4) 30(81.1) 79 (64.2) Chrysotile N(%) 33(86.8) 21(70.0) 64(91.4) 42(100.0) 17(65.4) 15(83.3) 31(83.8) 105(85.4) Crocidolite N(%) 20(52.6) 15(50.0) 25(43.1) 17(40.5) 9(34.6) 11(61.1) 16(43.2) 53(43.1) Tremolite N(%) 26(68.4) 21(70.0) 60(76.4) 41(97.6) 15(57.7) 12(66.7) 26(70.3) 94(76.4) N, number of subjects; mean, arithmetic mean; SD, standard deviation. a Asbestos-related diseases are those that have been recognized as occupational respiratory diseases for compensation purposes by a special medical committee. Each disease was considered independently and a worker with two diagnoses is considered once foreach disease. b Main industry: industry in whichthe subjects spent 60% or more of their occupational asbestos-exposed years. c Other includes mixed and unknown industries, and all otherindustries except chrysotile asbestos mining,construction and maintenance andrepair (seetable I fordetails). chrysotile and tremolite fibers, as well as higher proportions of fine crocidolite fibers. Regardless of the industry, chrysotile and crocidolite fibers were mostly short and fine fibers, while tremolite fibers were mostly fine and WHO fibers. Amosite fibers were generally distributed equally among the three dimensional categories except in construction where the proportion of short fibers was the highest. Of interest are the relatively low proportions of WHO chrysotile fibers, regardless of the disease or the industry. Lung asbestos concentrations of chrysotile, crocidolite and tremolite were statistically significantly higher for workers with a longer median duration of exposure (Fig. 1). Concentrations were statistically significantly lower for chrysotile fibers of all lengths and for crocidolite fibers <5 mm with increasing median number of years between last exposure and lung fiber burden analysis (Fig. 2). A cross-tabulation of concentrations by fiber type according to median duration of exposure (categorized as less than 25 years and 25 years or more) and median number of years since last exposure (less than 15 years and 15 years or more) was done. There was no significant decrease in concentration with time since last exposure for amphiboles, and for chrysotile fibers, a statistically significant decrease for fibers of both lengths (<5 mm and 5 mm) only in workers with a shorter duration of exposure (less than 25 years; data not shown). Table V describes the 24 workers with a lag of 30 years or more between cessation of exposure and lung fiber analysis, and their lung concentration of different asbestos fibers. Chrysotile fibers were observed in the lungs of two out of three with asbestosis (with or without a cancer), of four out of eight with mesothelioma and of all 13 workers with lung cancer. Workers whose lungs still contained chrysotile fibers had been working mainly in chrysotile mining. DISCUSSION The relationships between fiber characteristics and asbestos-related diseases among compensated workers are discussed below according to their asbestos-related disease and their occupational history.

7 Asbestos Fibers in Lung Tissues of Quebec Workers 1007 TABLE III. Geometric Mean Concentrations, Counts and Dimensions ofasbestos Fibers According to Asbestos-Related Disease, Main Industry, and Fiber Type, Quebec, Canada, 1988^2007 Asbestosis (N ¼ 38) Asbestos-relateddisease a Pleural mesothelioma (N ¼ 30) Lung cancer (N ¼ 70) Chrysotile asbestosmining (N ¼ 42) Mainindustry b Maintenance andrepair (N ¼ 26) Construction (N ¼ 18) Other c (N ¼ 37) Allcases (N ¼ 123) Geometric meanfibersconcentration(fibers/mgdry lung) Amosite L < 5 mm L 5 mm Chrysotile L < 5 mm , L 5 mm Crocidolite L < 5 mm L 5 mm Tremolite L < 5 mm , L 5 mm , Fibercountsanddimensions N Amosite N f ,457 L g (mm) d g (mm) (L/d) g Chrysotile N f ,212 2, ,932 L g (mm) d g (mm) (L/d) g Crocidolite N f L g (mm) d g (mm) (L/d) g Tremolite N f ,790 2, ,510 L g (mm) d g (mm) (L/d) g Ferruginousbodiesconcentrations(FB/gdry lung) N Mean g 4,362 4,271 7,813 4,446 8,448 10,825 6,789 6,425 % undetected ö Range ND-2,782,200 ND-98,350 ND-529,800 ND-76,800 ND-828,400 40^2,782,200 ND-284,000 ND-2,782,200 N, number of subjects; mg, milligrams; L, length; mm, micrometers; N f, number of fibers; g, geometric mean; d, diameter; L/d, length to diameter aspect ratio; FB: ferruginous bodies; g,gram; Mean g,geometric mean; ND, non-detected. a Asbestos-related diseases are those that have been recognized as occupational respiratory diseases for compensation purposes by a special medical committee. Each disease was considered independently and a worker with two diagnoses is considered once foreach disease. b Main industry: industry in whichthe subjects spent 60% or more of their occupational asbestos-exposed years. c Other includes mixed and unknown industries, and all otherindustries except chrysotile asbestos mining,construction and maintenance andrepair (seetable I fordetails).

8 1008 Adib et al. TABLE IV. Number and Proportions of Short, Fine, and WHO Fibers According to Asbestos-Related Disease, Main Industry, and FiberType, Quebec, Canada, 1988^2007 Asbestos-relateddisease a Mainindustry b Asbestosis (N ¼ 38) Pleural mesothelioma (N ¼ 30) Lungcancer (N ¼ 70) Chrysotile asbestosmining (N ¼ 42) Maintenance andrepair (N ¼ 26) Construction (N ¼ 18) Other c (N ¼ 37) Allcases (N ¼ 123) N Amosite N tot ,457 Shortfibers d N f (%) e 210(45.6) 48(31.8) 336(35.9) 19(26.4) 211(39.1) 184(42.3) 145(35.3) 559(38.4) Finefibers d N f (%) e 130(28.2) 50(33.1) 259(27.7) 31(43.1) 171(31.7) 104(23.9) 106(25.8) 412(28.3) WHOfibers d N f (%) e 121(26.2) 53(35.1) 340(36.4) 22(30.6) 157(29.1) 147(33.8) 160(38.9) 486(33.4) Chrysotile N tot ,212 2, ,932 Short fibers N f (%) 431(56.6) 176(61.3) 1,141(51.6) 1,052(50.9) 124(72.5) 120(81.6) 309(56.5) 1,605(54.7) Finefibers N f (%) 319(41.9) 103(35.9) 1,046(47.3) 994(48.1) 42(24.6) 26(17.7) 224(41.0) 1,286(43.9) WHO fibers N f (%) 9(1.2) 5(1.7) 15(0.7) 12(0.6) 2(1.2) 1(0.7) 12(2.2) 27(0.9) Crocidolite N tot Short fibers N f (%) 126(47.9) 110(35.9) 166(39.7) 107(50.5) 36(39.1) 11(30.6) 205(37.2) 359(40.3) Finefibers N f (%) 108(41.1) 160(52.3) 207(49.5) 88(41.5) 37(40.2) 20(55.6) 287(52.1) 432(48.5) WHO fibers N f (%) 29(11.0) 35(11.4) 43(10.3) 17(8.0) 18(19.6) 5(13.9) 57(10.3) 97(10.9) Tremolite N tot ,790 2, ,510 Short fibers N f (%) 436(51.7) 192(49.5) 1,470(52.7) 1,547(51.8) 23(74.2) 47(38.5) 208(56.2) 1,825(52.0) Finefibers N f (%) 134(15.9) 53(13.7) 451(16.2) 486(16.3) 1(3.2) 11(9.0) 51(13.8) 549(15.6) WHO fibers N f (%) 274(32.5) 143(36.9) 867(31.1) 953(31.9) 7(22.6) 64(52.5) 110(29.7) 1,134(32.3) N,numberof subjects; N tot,total number of fibers,per fiber type,regardless of theirdimensions; N f,numberof fibers. a Asbestos-related diseases are those that have been recognized as occupational respiratory diseases for compensation purposes by a special medical committee. Each disease was considered independently and a worker with two diagnoses is considered once foreach disease. b Main industry: industry in whichthe subjects spent 60% or more of their occupational asbestos-exposed years. c Other includes mixed and unknown industries, and all otherindustries except chrysotile asbestos mining,construction and maintenance andrepair (seetable I fordetails). d Short fibers: 0.5 mm < L < 5 mm, d < 3 mmandl/d 3:1; Fine fibers: L 5 mm, d < 0.2 mmandl/d 3:1; WHO fibers: fibers counted according to the WHO criteria (L 5 mm,0.2 mm d < 3 mmandl/d 3:1). e The proportions of short, fine and WHO fibers are calculated within each cell for a given fiber type, based on the total number of fibers, which includes fibers of other dimensions that are not shown in this table.

9 Asbestos Fibers in Lung Tissues of Quebec Workers 1009 FIGURE 1. Variation in geometric mean concentration of asbestos fibers by fiber length and type of asbestos, according to median duration of exposure. Fiber Length, Diameter, Length to Diameter Aspect Ratio and Concentration This analysis of lung tissue data obtained between 1988 and 2007 from workers whose disease has been recognized as asbestos-related in Quebec showed that the length and diameter of the different types of asbestos fibers were consistent with previous findings. For chrysotile and tremolite fibers, the geometric mean concentrations of fibers <5 mm were twice those of fibers 5 mm, whereas the difference was less pronounced for amosite and crocidolite fibers; this is similar to what is reported in the literature [Langer and Nolan, 1994; Dodson et al., 2003; AFSSET, 2008]. Amosite and crocidolite fibers were the longest, chrysotile fibers had the smallest average diameter and tremolite fibers showed the lowest average length to diameter aspect ratio (L/d), in FIGURE 2. Variation in geometric mean concentration of asbestos fibers by fiber length and type of asbestos, according to median lag between last exposure and lung fiber burden analysis.

10 1010 Adib et al. TABLE V. Characteristics of WorkersWith a Lag of 30 Years or More Between Last Exposure and Lung FiberAnalysis Geometricmeanconcentration(fibers/mgdrylung) Age Amosite Chrysotile Crocidolite Tremolite Disease (years) Mainoccupation/industry a Lag b <5 mm c 5 mm <5 mm 5 mm <5 mm 5 mm <5 mm 5 mm Asbestosis 72 Skilledworker/construction ö ö ö ö ö ö Asbestosis/lungcancer 71 Mach. operator-driver/other manufacturing 40 ö 105 ö 105 ö ö Asbestosis/Meso 79 Laborer/mining 52 ö ö 6,045 3,735 3,326 1,379 18,130 19,813 Mesothelioma 63 Skilledworker/construction 30 5,500 2,620 1,045 ö ö ö ö ö 65 Skilledworker/maintenance 30 3,141 1,680 ö ö ö ö ö Skilledworker/construction 36 ö 273 ö ö ö , EOWF/maintenance 37 ö ö 1,066 ö ö ö 105 ö 80 Skilledworker/mixed 38 2, ,570 ö ö ö 625 ö NA Skilledworker/construction 44 ö 315 ö ö ö ö ö ö 73 Laborer/othermanufacturing 46 ö ö ö Mach. operator-driver/maintenance ö ö ö ö Lungcancer 76 Skilledworker/shipbuilding 32 2,236 2,321 ö 183 ö ö 1, Mach. operator-driver/mining 32 ö ö 390 ö ö ö 5,992 2, Skilledworker/construction 33 1,242 1, ö ö Skilledworker/Shipbuilding , ö ö Laborer/mining 38 ö ö ö ö 4,190 1, Skilledworker/mining 39 ö ö 9,384 3,547 ö ö 52,549 27, Laborer/mining 40 ö 450 2, , ,752 8, Skilledworker/othermanufacturing 40 ö ö 2, ö ö 13,688 3, Skilledworker/construction ö Laborer/mining 44 ö ö 2, ö ö 1, Laborer/Asb. products 48 1, ö 420 ö ö 3, Laborer/Asb. products , ö ö ö ö 81 EOWF/Asb. products 54 3,381 2, ö ö ö mg, milligram; mm, micrometers; Meso, pleural mesothelioma; mining, chrysotile mining; maintenance, maintenance and repair; Asb. products, manufacturing of asbestos products; ship building, ship building and repair; mach. operator-driver, machine operators and drivers; EOWF, executives, office workers and foremen. a Main occupation/industry: occupation/industry in whichthe subjects spent 60% or more of theiroccupational asbestos-exposed years. b Lag: years between last exposure andlung fiberanalysis. c Fiberlength. agreement with findings of earlier studies [Warnock, 1989; Dufresne et al., 1996a,b; Suzuki and Yuen, 2001]. For tremolite, the L/d was always less than 20:1, which would correspond to a non-asbestiform fiber or a non-asbestiform elongated particle, according to the Environmental Protection Agency (EPA) [Perkins and Harvey, 1993]. Dufresne et al. [Dufresne et al., 1996a,b] have also reported tremolite fibers shorter than 10 mm and having a L/d < 20:1, while Churg et al. [1984] showed a geometric mean aspect ratio of 11 for tremolite/actinolite/anthophyllite fibers of all lengths, in six mesothelioma cases. For each disease, chrysotile and tremolite fibers had the highest average concentrations in the workers lungs. The picture varied by main industry: chrysotile and tremolite were in larger concentrations in chrysotile asbestos mining, whereas amosite and chrysotile predominated in the remaining industries. This is consistent with the literature, although high crocidolite fibers concentrations were also generally reported [Langer and Nolan, 1989; Dodson et al., 2005]. On the other hand, tremolite fibers were observed in the lungs of practically all of these workers but in lower concentrations, except for asbestos mining industry, possibly as a consequence of tremolite being a contaminant of the chrysotile ore [McDonald et al., 1997; Normand, 2001; Roggli and Vollmer, 2008]. Workers whose main industry was chrysotile asbestos mining had higher average concentrations of chrysotile and tremolite, but crocidolite and to a lesser extent

11 Asbestos Fibers in Lung Tissues of Quebec Workers 1011 amosite fibers were also found in their lungs, as previously reported in Quebec miners [Dufresne et al., 1995, 1996a,b; McDonald et al., 1997; Nayebzadeh et al., 2006]. The presence of crocidolite and amosite in the lung tissues was suggested to be related to employment in a military gas mask filters assembly plant that used these amphiboles [McDonald et al., 1997]; however, work in this plant is mentioned in the occupational history of only one of the 42 workers employed in chrysotile asbestos mining and milling in our study. Given that some of these workers had a skilled trade (e.g., welder, tinsmith, etc.), it is possible that their work on structures involving materials containing amphiboles (e.g., insulation, sprayed asbestos, etc.) may explain the presence of these types of asbestos in their lungs. In addition, employment for the remainder of their work history in another industry entailing exposure to commercial amphiboles cannot be ruled out totally. Chrysotile fibers in the lungs of workers with lung cancer who worked outside the chrysotile asbestos mining industry were shorter in this study than in workers with lung cancer from several industries in the U.S. [Dodson et al., 2004], whereas crocidolite fibers were longer in this study. However, the diameters of both fiber types are similar in both studies, resulting in smaller length to diameter aspect ratios in our study. Tremolite fibers observed in the present study were also shorter, with smaller diameters, than in the U.S. study [Dodson et al., 2004]. Differences between the two studies could be attributable to exposures of dissimilar nature and to different analytical methods. Short, Fine, and WHO Fibers Altogether, asbestos fibers found in the lung tissues of 97 workers of this study were mostly distributed as short asbestos fibers (50%), fine asbestos fibers (30%) and WHO fibers (20%). We could not find publications on the distribution of fibers in lung tissues that used dimensional definitions comparable to ours. However, in a recent review on short asbestos fibers and fine asbestos fibers, the Agence Française de Sécurité Sanitaire de l Environnement et du Travail [AFSSET, 2008] reported results of fiber analysis in air samples carried out in mining and other industries. They showed that percentages of short asbestos fibers varied from 40% to 100%, and of fine asbestos fibers, from 0% to 20%. Thus, results in the lungs of the workers of this study are consistent with what is found in the air of mining and other industries as reported by AFSSET. Distribution of the different fiber dimensional categories shows that, with few exceptions, (1) chrysotile and tremolite had the highest proportions of short fibers, (2) crocidolite and chrysotile had the highest proportions of fine fibers, and (3) amosite and tremolite had the highest proportions of WHO fibers. Regardless of the asbestosrelated disease, the gradation of fibers in relation to their dimensions can be generally summarized as follows: Short asbestos fibers : chrysotile tremolite > crocidolite > amosite Fine asbestos fibers : crocidolite chrysotile > amosite > tremolite WHO fibers : amosite tremolite > crocidolite > chrysotile The gradation of short fibers in this study differs from that observed in series of lung cancer and mesothelioma cases reported in the literature, where the proportions of short crocidolite and tremolite fibers predominate [Dodson et al., 2004, 2005]. The gradation of fine fibers also differs in the lung cancer cases, for whom Dodson et al. [2004] report that chrysotile and amosite fibers are prominent; however, the sequence is identical in their series of mesothelioma cases [Dodson et al., 2005]. For WHO fibers, gradation is the same as in the present study [Dodson et al., 2004, 2005]. It is worth mentioning that Dodson et al. attribute tremolite exposure in their cases to products containing contaminated talc, whereas in our population it appears to be associated with chrysotile exposure. Differences between the results reported by Dodson et al. [2004, 2005] and our results could be explained by variations in the calculation methods of short and fine fibers proportions. Although short fibers are generally found in larger proportions than fine fibers or WHO fibers, the different dimensional categories of fibers appear to vary according to the disease (Table IV). For the asbestosis cases, the proportion of short fibers is somewhat larger than the proportion of other fiber types. Not many studies report data on fibers shorter than 5 mm in relation to asbestosis, because it is generally believed that short fibers are not fibrogenic [Roggli and Vollmer, 2008; Schneider et al., 2010]. Fiber Concentration and Exposure or Clearance Proxies As stated earlier, we did not have access to lung tissues from an unexposed reference working population but only to published reference fiber counts. We used duration of work in an exposed job as the exposure proxy (assuming that a longer duration resulted in a larger lung burden), and lag between the last exposure and the lung fiber burden analysis as a clearance proxy (assuming that the longer the lag, the lower the burden). In our study population, the lung concentration of fibers was statistically significantly larger when the exposure duration was longer, for all fiber types except amosite. This increasing burden with increasing duration of exposure has been reported consistently for amphibole fibers, but not for chrysotile fibers. Albin et al. [1994] and Churg and Wright [1994] reported no relationship between duration or intensity of exposure to chrysotile fibers and lung chrysotile fiber

12 1012 Adib et al. concentrations, whereas Finkelstein and Dufresne [1999] showed a significant association with duration of exposure for chrysotile and for tremolite fibers. The few published studies on the clearance of asbestos fibers from human lungs report inconsistent findings. For example, Churg and Wright [1994] concluded that commercial asbestos fibers clearance tends to be measured in years or decades, whereas chrysotile fibers halftimes are expressed in months. Churg et al. [1992] showed that cigarette smoke could increase the retention of short fibers in animal models; as the proportion of smokers is quite large among our study population, it may have influenced the proportion of short fibers remaining in the lungs. On the other hand, we did not have information on the latency period between the last time the worker smoked and the data of diagnosis of the asbestos-related disease, which could have helped in interpreting the data. Finkelstein and Dufresne [1999] indicated that lung fiber burden studies are not useful for examining the behavior in rapidly clearing compartments of the lung, but may provide insight concerning the more slowly clearing compartments. Churg and Wright [1994] also mention that most studies fail to show a correlation between chrysotile concentrations and time since last exposure, whereas Finkelstein and Dufresne [1999] reported a decrease in concentration of chrysotile fibers with time since last exposure; based on their modeling, they suggested that retained chrysotile concentrations tend to plateau after an accumulation of about 35 years of exposure, while tremolite concentrations continue to increase. They also indicated that chrysotile concentrations may begin to increase again after 40 years, suggesting that an overload is eventually reached for chrysotile as well. Our observation that chrysotile concentrations were not significantly reduced after more than 15 years since last exposure in workers exposed for 25 years or more is consistent with this latter statement. Although based on a limited number of subjects, our results suggest different retention patterns according to duration of exposure and time since last exposure between fiber types, which may be interpreted as an overload phenomenon. Other mechanisms by which asbestos fibers are eliminated from the lungs are translocation of fibers into surrounding tissues, which explains the occurrence of mesothelioma outside the lungs, and dissemination through the lymphatic system. These mechanisms, although demonstrated in animal models and reported in a few case studies, are not fully understood [Boutin et al., 1996; Broaddus et al., 2011] and some studies may have underestimated differences attributable to analytical procedures [Roggli, 2006]. In the present study, we observed chrysotile fibers in the lungs of workers who stopped being exposed some 30 years or more prior to lung fiber analysis, which is in agreement with the overload hypothesis by Finkelstein and Dufresne [1999] and not inconsistent with the observation of Churg [1994] that some chrysotile fibers may persist in a sequestered compartment. Non-occupational exposures to chrysotile fibers cannot be ruled out for these workers, and it is possible that living in a mining town [Rees et al., 2001] or close to an asbestos products manufacturing plant [Magnani et al., 1998] could have resulted in chrysotile exposures after cessation of occupational exposures. However, chrysotile fibers concentrations were still much higher in the lungs of these workers than the geometric mean upper 95% confidence interval values from a reference autopsy series population: Dufresne et al. [1996a] reported upper 95% confidence interval values of 228 fibers/mg of dry lung for short chrysotile fibers (<5 mm), and of 79 fibers/mg dry lung for chrysotile fibers 5 mm. A very large environmental exposure would have to have taken place to explain such large concentrations, which is unlikely given that chrysotile fiber levels in a mining town in Quebec are around 4 fibers/l of air [Bourgault and Belleville, 2010], whereas in occupational settings, such as chrysotile asbestos mines, levels were around 1,000 fibers/l in the late 1990s [Lajoie et al., 2005]. Methodological Considerations This study describes the lung fiber content of 123 workers and we did not have access to lung tissues from an unexposed reference population of workers, which is a noteworthy limitation. The lung fiber burden data used in this study were collected to further document and support compensation claims; as already mentioned, lung fiber analysis may be requested by the special medical committee studying claims when the claimant s occupational history reveals no obvious asbestos exposure, when the exposure history is not considered sufficient to induce an asbestos-related disease, or, for certain smokers with lung cancer. Hence, our study population probably represents a less exposed subgroup of workers than all workers with a recognized asbestos-related disease, the proportion of lung cancer cases is larger than usual (and consequently influences the pooled results of the study group), and the lung cancer cases are, or were, mostly smokers. Thus, the results of this study cannot easily be extrapolated to all workers who submitted a claim or whose disease was recognized, or to all Quebec workers exposed to asbestos. Finally, fiber burden analysis has several inherent limitations, many of which were previously summarized by Baker [1991]. As mentioned earlier, the lung fiber data presented herein had already been collected. Their availability made them useful, but without much control over certain parameters, such as the lung tissue biopsy site, the number of lung tissue blocks obtained or the analytical

13 Asbestos Fibers in Lung Tissues of Quebec Workers 1013 protocol. We therefore cannot conclude on the representativeness of the lung tissue blocks compared to the total lung burden. Moreover, we did not have detailed exposure data (e.g., initial exposure intensity, specific tasks, etc.) to work with. These parameters could therefore not be studied in conjunction with the lung fibers concentrations at hand. Despite these limitations, our study is among the few describing lung fiber content in relation to occupational history and to several asbestos-related diseases. Moreover, the analysis carried out by electron microscopy allowed us to shed more light on the distribution of fine and short fibers, compared to WHO fibers in the lungs of workers exposed to asbestos, according to fiber type. CONCLUSION Our results are generally consistent with the published literature. Some new observations are however noteworthy. First, chrysotile fibers were observed in the lung tissues of 85% of Quebec workers with an asbestos-related disease for whom a lung fiber analysis was available. Second, most asbestos fibers remaining in the lung tissues were short (length < 5 mm) or fine (length 5 mm and diameter <0.2 mm) fibers. Consequently, the low proportion of WHO fibers compared to the proportions of short and fine fibers in the lungs of workers with an asbestos-related disease strengthens the relevance of taking into account other dimensional criteria to characterize the health risks associated with asbestos inhalation. And finally, chrysotile fibers were still found in the lungs of some workers 30 years or more after they stopped being exposed, in concentrations above those published for a Quebec unexposed population. These observations show that short fibers still remain in significant proportions in the lungs of workers with occupational asbestos exposure. 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