Document 6R4X6graxkV31nz3O119KnDGo

FUNDAMENTAL AND APPUED TOXICOLOGY 5, 327-340 (1985) Downloaded from toxsci.oxfordjoumals.org at HAM-TMC Library on February 2, 2011 Chronic Inhalation of Short Asbestos Fibers1,23 S. Frank Platek, David H. Groth, Charles E. Ulrich,* Lloyd E. Stettler, Myra S. Finnell, and Margrit Stoll Division ofBiomedical and Behavioral Science, National Institute for Occupational Safety and Health, Cincinnati, Ohio 45226, and *International Research and Development Corporation, Mattawan, Michigan 49071 Chronic Inhalation of Short Asbestos Fibers. Platek, S. F., Groth, D. H., Ulrich, C. E., Stettler, L. E., Finnell, M. S., and Stoll, M. (1985). Fundam. Appl. Toxicol. 5, 327-340. An animal inhalation study was initiated to study the chronic biological effects of inhalation of short chrysotile asbestos fibers. Rats and monkeys were exposed for 18 months, 7 hr/day, 5 days/week to a specially prepared, chrysotile asbestos aerosol. Based upon daily chamber measurements, the mean concentration of fibers in the chamber air was 1.0 mg/m13. 2By phase contrast microscopy, the number of fibers > 5 pm in length was determined to be 0.79 fiber per cubic centimeter. Rats were autopsied for pathological and histochemical examination at 1, 3, 6, 12, 18, and 24 months after initiating exposures. No significnt differences in the histochemical data were seen between the exposed and control groups. Gross and histopathologic examination of exposed and control groups of rats indicated no compound-related lesions, including fibrosis. Open lung biopsies were performed on the chrysotile-exposed and the control monkeys 28 months after initiating exposures. Histopathologic evaluation of the lung biopsy tissue showed the presence of asbestos bodies adjacent to the terminal bronchioles of the asbestos-exposed monkeys. There was no observed fibrosis in pulmonary tissue. All monkeys are being maintained for an indefinite period and observed for signs of latent pulmonary disease. Asbestos contains numerous insulating as well as chemical- and wear-resistant properties which make it an extremely valuable mineral in the production of textiles, cement and tile products, gaskets and friction materials, coat ings and automotive brake linings (Hendry, 1965). However, asbestos has been shown by numerous investigators to cause the debili tating human diseases of asbestosis, broncho genic carcinoma, and mesothelioma (Newhouse and Thompson, 1965; Kannerstein et 1 A preliminary report of this material was presented at the First NCI/EPA/NIOSH Collaborative Workshop at Rockville, Md., May 7, 1980. 2 This study was conducted under contract with The International Research and Development Corporation, Mattawan, Mich, under NIOSH Contract 210-77-0151. 3 This study was supported by the National Cancer Institute (NCI) via an Interagency Agreement between NCI and NIOSH. al., 1977; McDonald and Liddell, 1979). Results of rather extensive animal research have shown that fiber length and diameter are the most important determinants in the induction of mesotheliomas. Fibers that are >8 /tm in length and <1.25 pm in diameter are much more potent than those of other sizes (Stanton et al., 1981). It is not known, however, and little research has been done to prove whether fiber size is an equally important determinant in the induction of asbestosis or lung cancer. A few authors have mentioned the potential hazards and prob lems associated with short fiber asbestos (Yeager et al., 1983; Gross, 1974; Selikoff et al., 1972; Holt et al., 1965). The present Occupational Safety and Health Administration (OSHA) standard for airborne asbestos fiber exposure is not to exceed two fibers longer than 5 pm per cubic 327 0272-0590/85 $3.00 328 PLATEK ET AL. centimeter of air over an 8-hr, time-weighted average period (OSHA, 1978). Although the OSHA standard regards airborne asbestos fibers less than 5 jim as a nuisance dust with exposure to 5 mg/m3 permissible, it has been shown that for every fiber greater than 5 ^m there may be more than 100 fibers less than 5 pm in length (Holt et al., 1965). Of these smaller fibers, most are probably beyond the resolution of the optical light microscope (~0.25 ^m) and may be seen only by electron microscopy. Thus, the several million Amer icans employed in the use of asbestos-con taining products, including automotive brake and clutch relining as well as reinforcing pipe and tile manufacturing (DHEW, 1978; Bruckman, 1978; Lynch, 1968), are exposed to potentially high levels of short (<5 Mm) asbestos fibers. The purpose of this study was to determine the chronic biological effects after inhalation of chrysotile asbestos fibers less than 5 Mm in length in laboratory rats and monkeys. MATERIALS AND METHODS Chrysotile (short fiber) preparation. Type 7TF1 chrys otile was obtained from the Johns-Manville Sales Cor poration in Denver, Colorado. Four-hundred-gram batches of the chrysotile were dried in an oven for 24 hr at 191C, milled in a ceramic ball mill for 24 hr, and then dried again for 24 hr at 191C. Each batch was examined by scanning electron microscopy to determine fiber size. Five batches were randomly selected and quantitatively analyzed by induction coupled plasma emission spectroscopy for their elemental content. Inhalation chambers, dust generation, and character ization. Experimental exposures were conducted in two 16-m3 glass and stainless steel, dynamic air flow exposure chambers. Air for both control and exposure chamber ventilation was filtered with HEPA filters to remove particulates and controlled for temperature and humidity. The mean temperature within the two exposure chambers was 23.6C (74.5F) with a mean relative humidity of 61.5%. The chamber air flow rate for both study groups was maintained at 1500 liters per minute (53 cfm). The control inhalation chamber was supplied only with the filtered and temperature- and humidity-controlled air. The asbestos inhalation chamber used a specially designed dust-feed elutriator system (International Research and Development Corporation, Mattawan, Mich.) to generate the short fiber asbestos atmosphere (Fig. 1). The opera tional characteristics of the device were as follows: a known weight of prepared asbestos was placed in the reservoir of the generator and stirred by a stirring mech anism. A disk containing cups around the perimeter was rotated through the reservoir permitting the cups to be filled with the chrysotile dust. The filled cups then passed over a blowout port where a metered flow of desiccated air "puffed" the asbestos into a vertical elutriator column fitted between the generator and the chamber air inlet. Downloaded from toxsci.oxfordjoumals.org at HAM-TMC Library on February 2, 2011 Fig. 1. Diagram of system used to generate short asbestos fibers. 330 PLATEK ET AL. Downloaded from toxsci.oxfordjoumals.org at HAM-TMC Library on February 2, 2011 At all sacrifice periods, the left lungs from five randomly selected rats from each exposure group were analyzed for the presence of an inflammatory response by mea suring the following enzymes: lactic dehydrogenase (Zugjbe, 1970), acid phosphatase (Barka and Anderson, 1963), and /3-glucoronidase (Thompson, 1966). At the 3-, 6-, 12-, and 18-month and terminal sacrifices, the left lungs from 10 randomly selected rats from each exposure group were taken for subsequent hydroxyproline and elemental silicon analyses. These lungs were sectioned longitudinally along the main stem bronchi such that each contained portions of the apex and base. One section was then analyzed for hydroxyproline and the other for elemental silicon by plasma emission spectros copy. In addition, blood was taken from these same rats for elemental silicon determination. Blood was also drawn from all monkeys at these sacrifice intervals for elemental silicon quantitation. Hematoxylin and eosin stained sections of the remain ing lung tissue and of each of the above tissues were prepared for and examined by light microscopy. Twenty-eight months after the initiation of exposures (10 months after completion of exposures), open lung biopsies were performed on all monkeys for histopathological evaluation and for lung tissue fiber content deter minations. Approximately 2 g of tissue was taken from the anterior margin of the right lower lobe of each monkey. The procedure for determining lung fiber content is as follows. Pieces of lung tissue were freeze-dried to constant weight using a Labconco Freeze Dry 5 freeze dryer and then ashed in a low-temperature asher (Inter national Plasma Corporation, Model 1PC 1005-148AN) using an oxygen plasma. The ash from each lung was then added to 50 ml of a 0.05% solution of Aerosol OT (Fisher Scientific) in filtered, deionized water and then sonicated for 10 min in an ultrasonic bath. The resulting suspensions were then diluted to 100 ml and stirred magnetically for 10 min. Aliquots of the suspensions were then filtered through 0.1 -nm pore-size Nucleopore filters. The filters were then carbon coated in a vacuum evaporator (Edwards High Vacuum, Model 306). Pieces of these filters were then placed on 200-mesh transmission electron microscope grids and cleared with chloroform using a modified Jafle-Wick technique (Jaffe, 1948; Chatfield and Dillon, 1978). The resulting grids were examined in a transmission electron microscope (JEOL, Model 100CX) equipped with an energy dispersive X-ray spectrometer system (EG&G Ortec, Model EEDS II). All of the fibers found in 10 grid spaces for two grids of each sample were counted and sized. Every 10th fiber was analyzed by energy dispersive X-ray analysis and selected area electron diffraction to confirm that it was chrysotile. Lung fiber concentrations were calculated from the fiber densities on the filter preparations and the original dry lung weights. All of the monkey lung biopsies, both exposed and controls, were analyzed for fiber content In addition, lungs from five of the 18- and 24-month (terminal sacrifice) rats from each exposure group were also analyzed for fiber content RESULTS Five batches of the ball-milled chrysotile preparations used in the exposure chamber were chemically analyzed. The results of these elemental analyses are summarized in Table 2. Of interest are the relatively high concentrations of aluminum (0.88-1.1%). Each batch of the ball-milled asbestos was also examined using the scanning electron microscope (JEOL, JXA 50A) at magnifica tions of 400X and 5000X to ensure that the vast majority of the prepared fibers were less than 5 /tm. An unavoidable by-product of the ball-milling procedure was the preparation of agglomerated asbestos "balls" or bundles of asbestos fibers. Figure 2a is a scanning electron micrograph showing a typical ballmilled preparation. A high magnification en largement of one of the asbestos "balls" is shown in Fig. 2b. The results of the exposure chamber mon itoring were as follows: the mean mass con centration as determined by gravimetric sam pling was 1.0 mg/m3 with a standard devia tion of 0.28. The range of mass values over the entire experiment was 0.4 to 1.8 mg/m3. The mean number of asbestos fibers greater than 5 /im in length as determined by light microscopy (Taylor, 1977) was 0.79 fiber/ cm3 with a standard deviation of 0.41. The values ranged from 0.08 to 1.5 fibers/cm3. The mean equivalent aerodynamic diameter for the particles in the exposure chamber as determined by using the Andersen cascade impactor was 5.0 /tm with a geometric stan dard deviation of 2.9. A total of 14,558 particles (fibrous and nonfibrous) in 138 fields of view from nine samples of the asbestos chamber atmosphere collected on Nucleopore filters were counted and sized by SEM. Of these particles, 6940 Downloaded from toxsci.oxfordjournals.org at HAM-TMC Library on February 2, 2011 332 Downloaded from toxsci.oxfordjoumals.org at HAM-TMC Library on February 2, 2011 334 PLATEK ET AL. TABLE 3 Mean Values for Rat Lung and Body Weights Exposure interval (months) Group Body weight (g) Lung weight (6) 1 Control 330 + 33.5" 1.58 0.25 Asbestos 332 21.9 1.66 0.13 3 Control 454 45.4 1.65 0.11 Asbestos 463 47.0 1.81 0.22* 6 Control 558 50.5 1.88 0.15 Asbestos 565 59.0 1.92 0.16 12 Control 634 61.4 2.01 0.20 Asbestos 656 85.3 2.03 0.16 18 Control 710 95.4 2.09 0.19 Asbestos 693 101.1 2.07 0.19 24" Control 765 185.2 2.34 0.22 Asbestos 756 152.4 2.31 0.20 " Mean SD. * Statistically different from the control group (p < 0.05). c Six months postexposure. concentration of fibers less than 5 pm in length was 272 31 X 106 (range, 241-308 X 106), and the mean concentration of fibers greater than 5 pm in length was 23 4.9 X 106 (range, 16-28 X 106). The mean percentage of fibers greater than 5 pm in length in these lungs was 7.7% (range, 5.8- 9.6%). A scanning electron micrograph of a typical ashed lung preparation is shown in Fig. 5. Note the individual fibers as well as one of the asbestos "balls." In the 6-month postexposure group (Fig. 6), a total of 919 fibers were sized. The mean number of fibers per gram of dried lung for these rats was 192 27 X 106 (range, 143-- 209 X 106). This value is significantly lower than the same value obtained at 18 months (p < 0.001). The mean concentration of fibers less than 5 fim in length was 164 28 X 106 (range, 117-189 X 106). This value is significantly lower than the same value ob tained at 18 months (p = 0.001). The mean concentration of fibers greater than 5 fim in length was 27 9.3 X 106 (range, 18-41 X 106). This value is not significantly different from that seen at 18 months (p > 0.05). The mean percentage of fibers greater than 5 fim in length in the 6-month postexposure lungs was 12.6%. Only one fiber was found in the lung of one out of four control rat lungs at the 18month sacrifice. The concentration of fibers in the lung of that rat was calculated to be 0.16 X 106 per gram of dried lung. Only one fiber was found in the lung of one out of five control rats at the 24-month sacrifice. The concentration of fibers in the lung of that rat Group ConL Exp. ConL Exp. ConL Exp. ConL Exp. ConL Exp. mg/g. TABLE 4 Silicon Concentrations in Rat Lungs and Blood" Sac. interval (months) Range Dry lungs Mean SD Range 3 60-300 115.0 98.6 2-4 3 60-200 79.0 43.6 2-30 6 40- 90 59.0 13.7 2-270 6 40- 70 54.0 10.8 2-5 12 70-450 86.1 142.02 0.8-120 12 20-110 35.9 28.90 0.8-240 18 70- 80 36.0 19.55 1-13 18 20-110 54.6 28.25 1-3 24 20-120 67.7 27.7 1-24 24 30-130 59.5 31.91 1-485 Serum Mean 2.9 6.4 32.8 2.8 14.6 24.9 2.8 1.9 5.4 5.9 SD 0.60 8.44 83.70 1.03 37.18 75.60 3.61 0.60 7.42 6.34 Downloaded from toxsci.oxfordjoumals.org at HAM-TMC Library on February 2, 2011 INHALATION OF SHORT ASBESTOS FIBERS 335 TABLE 5 Hydroxyproune Determination in Experimental Rat Pulmonary Tissue was determined to be 327 53 X 106. The values for the 10 biopsy samples ranged from 228 to 356 X 106 fibers/g. Exposure interval- (months) Group Hydroxyproline (mg/g dry lung tissue) 3 Control Asbestos 28.38 2.29 28.84 3.24 6 Control Asbestos 31.08 2.48 32.20 + 2.84 12 Control Asbestos 35.39 2.81 33.69 6.92 18 Control Laboratory error--sample lost Asbestos Laboratory error--sample lost 24* Control Asbestos 24.53 + 7.38 26.73 10.63 Mean SD. * Six months postexposure. was calculated to be 0.19 X 106/gram of dried lung. The results of the fiber size analyses for the fibers recovered from the lungs of the 10 monkeys at the 28-month biopsy are sum marized in Fig. 7. A total of 4124 fibers were sized. Of these fibers, 239 or 5.8% were greater than 5 /im in length. The number of .fibers per gram of dry lung for these monkeys DISCUSSION The results of our study show that inha lation exposures of male rats to ball-milled chrysotile at a concentration of 1 mg/m3 and 0.79 fiber > 5 /im in length/cm,3 7 hr/day, 5 days/week for 18 months is insufficient to produce pulmonary fibrosis or tumors within 24 months. It also shows that a concentration of 23 X 106 chrysotile fibers > 5 /im in length/g of dry lung, 272 X 106 chrysotile fibers < 5 fim in length/g of dry lung, or a combination of the two are insufficient to product pulmonary fibrosis or tumors in a 6-month period (18-24 months after initiat ing exposures) in male rats. There are no other comparable studies reported in the literature. However, there are several inhalation stud ies reported which have shown that a variety of types of asbestos can produce pulmonary fibrosis and tumors in rats. In 1977, Gross et al. reported the induction of lung tumors in male rats (10/41) exposed to ball- and hammer-milled Canadian chrysotile at an average weekly concentration of 86 mg/m3, Fig. 4. Fiber size distribution for fibers extracted from five rats of the 18-month sacrifice. 336 PLATEK ET AL. TABLE 6 Numbers and Lengths of Chrysotile Fibers in Rat Lungs by TEM" Fiber length Rat No. 80-217 80-218 80-219 80-220 80-221 Mean (SD) All lengths 302 291 257 294 327 294 (31)6 <5 /im 277 263 241 269 308 272 (31)* >5 |im >8 nm 18 Months of exposure 25 14 28 14 16 7.3 25 8.8 19 11 23 (4.9) 11 >10 nm 5.6 6.0 2.4 5.1 5.0 4.8 >15 iim 3.2 3.0 0.61 2.2 2.9 2.4 80-554 80-555 80-556 80-557 80-558 Mean (SD) 201 209 143 201 204 192 (27) 183 189 117 170 163 164 (28) 6 Months postexposure 18 11 20 12 26 9.5 31 9.8 41 24 27 (9.3) 13 5.8 4.8 1.9 4.4 8.4 5.1 5.0 2.4 1.9 2.2 4.8 3.3 " Values are No. of fibers/g dry lung/106. 6 Significantly higher than the same-sized fiber concentration obtained 6 months postexposure (p 0.001). Downloaded from toxsci.oxfordjoumals.org at HAM-TMC Library on February 2, 2011 6 hr/day, 5 days/week for 62 weeks and observed for up to 34 months following initiation of exposures. He also observed pulmonary fibrosis and one mesothelioma in the exposed rats. No information on the number of fibers per cubic centimeter in the chambers or fibers per gram of lung tissue was given. In 1974, Wagner et al. reported the induction of pulmonary fibrosis, lung tumors, and mesotheliomas in rats exposed for a variety of intervals ranging from 3 to 24 months to amosite, anthophyllite, crocidolite, Canadian chrysotile, or Rhodesian chrysotile. The exposures were for 7 hr/day, 5 days/week. The mean respirable dust con centrations varied from 10.1 to 13.5 mg/m3. No information on the number of fibers per cubic centimeter in the chambers or the concentrations of fibers in the lung tissue was given. Davis et al. (1978) reported the induction of pulmonary fibrosis, lung tumors, and two mesotheliomas in rats exposed to concentra tions of UICC chrysotile, crocidolite, and amosite ranging from 2 to 10 mg/m3, 7 hr/ day, 5 days/week for 1 year and sacrificed 16.5 months postexposure. The highest inci dence (15/40) of lung tumors and the most severe fibrosis occurred in the group exposed to chrysotile (10 mg/m3). The concentration of fibers greater than 5 ym in length in the chamber air by phase contrast light micros copy was found to be 1950/ml. The rats exposed to chrysotile at 2 mg/m3 (390 fibers > 5 ym in length/ml) also developed lung tumors (8/42) and pulmonary fibrosis. The incidence of lung tumors in the rats exposed to crocidolite and amosite ranged from 2.5 to 4.7% and the extent of fibrosis was less than that seen in the groups exposed to chrysotile even though the mass and number of fibers > 5 ym in length per milliliter in the aerosol were comparable to those seen with the chrysotile-exposed animals. Exami nation of the fiber preparations by scanning electron microscopy, however, revealed many INHALATION OF SHORT ASBESTOS FIBERS 337 Downloaded from toxsci.oxfordjoumals.org at HAM-TMC Library on February 2, 2011 Fig. 5. A scanning electron micrograph of an ashed lung preparation for an 18-month rat. Note that an asbestos "ball" (arrow) is still intact. Bar = 1.0 /m>- more long fibers in the chrysotile preparation, length per milliliter, whereas the crocidolite It was estimated that the chrysotile aerosol and amosite aerosols at the same mass con(10 mg/m3) contained 360 fibers > 20 jim in centrations had only 34 and 6 fibers > 20 Fig. 6. Fiber size distribution for fibers extracted from five rats of the 24-month (terminal) sacrifice. 338 PLATEK ET AL. Downloaded from toxsci.oxfordjoumals.org at HAM-TMC Library on February 2, 2011 (Mm) FlO. 7. Fiber size distribution for fibers extracted from 10 monkeys at the 28-month biopsy. fim in length per milliliter. Concentrations of fibers in the lungs of the animals were not measured. The most probable explanation for why pulmonary fibrosis and lung tumors were produced in rats by the above investigators, but not in our experiment, is the large differ ence in the concentration of long fibers in the aerosols. One chrysotile aerosol (10 mg/ m3) in the Davis et al. (1978) study contained 2538 times more fibers greater than 5 fim in length than the aerosol in our experiment and their other chrysotile aerosol (2 mg/m3) contained 500 times more fibers greater than 5 fim in length. It is interesting to note also that Davis et al. (1978) found that the pul monary fibrosis and tumor incidence corre lated better with the number of fibers >20 fim in length when the preparations were examined by scanning electron microscopy. Other methods of administering asbestos to animals have also supported the concept that fiber lengths are most important in the induction of biological effects. Wright and Kuschner (1977) injected guinea pigs intra tracheally with long and short fibers of crocidolite, as well as synthetic fluoramphiboles and glass fibers. Animals were sacrificed at intervals up to 24 months. One crocidolite preparation in which 80% of the fibers were greater than 10 /im in length produced ex tensive interstitial pulmonary fibrosis, whereas the crocidolite preparation, in which 99% of the fibers were less than 5 fim in length, produced no fibrosis. The fiber diameters in both preparations were between 0.1 and 0.3 fim. The fact that only 4 mg of the long fibers compared to 25 mg of the short fibers had been injected per animal enhanced the significance of the results. The most detailed studies relating fiber sizes to biologic effects were those reported by Stanton et al. (1981). They tested fibers of differing lengths and of several different chemical compositions. These included cro cidolite, various types of fibrous glass, alu minum oxide, dawsonite (NaAl(0H)2C03), wollastonite, tremolite, amosite, attapulgite, hallyosite, silicon carbide, and potassium octatitinate. The fibers were injected intrapleur ally in rats. They found that fibers >8 fim in length and <1.25 in diameter were much more potent in inducing mesotheliomas than other fibers (i.e., those <8 fim in length and >1.25 fim in diameter). In all of the above studies, asbestos was tested alone; that is, no other carcinogen was administered with the asbestos. Therefore, no comment can be made on the relative importance of short fibers versus long fibers INHALATION OF SHORT ASBESTOS FIBERS 339 Downloaded from toxsci.oxfordjoumals.org at HAM-TMC Library on February 2, 2011 in acting as cocarcinogens for substances like cigarette smoke, which is most important in the occupational setting (Hammond et al., 1979). There are two other factors that might have influenced the results of our study. These are the rather high aluminum content (0.88-1.1%) and the fact that the chrysotile was ball milled. The aluminum content was about twice as much as that reported to be present in the UICC Rhodesian chrysotile (Timbrell, 1969) tested by Wagner et al. (1974). Aluminum compounds have been reported to modify the fibrogenic potency of silica (LeBouffarit et al., 1977), and it is possible that they could also modify the fibrogenic potency of silicates, e.g., asbestos. Other investigators have reported that ball milling may also cause a degradation of the crystal structure of the asbestos fibers (Spumy et al., 1980) which may affect the biological activity of the asbestos. The modification of chrysotile, e.g., by leaching in 1 N HQ (with out apparently modifying fiber length), has been shown to modify the biological effects both in vitro and in vivo (Morgan et al., 1977; Evans et al., 1983). ACKNOWLEDGMENTS The authors express their appreciation to David Brewer, JeAnne Burg, Ph.D., Richard Carlson, D. Gayle Cecil, Charles Gorski, Kathy Hicks, Susan Kaelin, Lea Kalejs, Hazel Patterson, Randall Smith, Allen Stein, William D. Wagner, and Glenda White of the National Institute for Occupational Safety and Health (NIOSH) and Dr. Basil Leong, Thomas Moore, David Pydlek, and contributing staff of the International Research and Development Corporation (IRDC) for their contribution to this study. Research was performed under NIOSH Contract 210 77-0151. REFERENCES Barka, T., and Anderson, P. J. (1963). Histochemistry: Theory, Practice and Bibliography. Harper & Row, New York. Bruckman, L. (1978). A Study of Airborne Asbestos Fibers in Connecticut, Workshop on Asbestos: Defini tions and Measurement Methods. U.S. Department of Commerce, NBS Special Publication 506, 179-191. Chatfield, E. J., and Dillon, M. J. (1978). 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U.S. Department of Labor, 1910.1001, 545-550. Selikoff, I. J,, Nicholson, w. j., and Langer, A. M. (1972). Asbestos air pollution. Arch. Environ. Health 25, 1-13. Spurny, K. R., Stober, W,, Opiela, H., and Wess, G. (1980). On the problem of milling and ultrasonic treatment of asbestos and glass fibers in biological and analytical applications. J. Amer. Ind. Hyg. Assoc. 41, 198-203. Stanton, M. F., Layard, M., Tegeris, A., Miller, E., May, M., Morgan, E., and Smith, A. (1981). Relation of particle dimension to carcinogenicity in amphibole asbestoses and other fibrous minerals. J. Natl. Cancer Inst. 67(5), 965-975. Stanton, M. F., Layard, M., Tegeris, A., Miller, E., May, M., and Kent, E. (1977). Carcinogenicity of fibrous glass: Pleural response in the rat in relation to fiber dimension. J. Natl. Cancer Inst. 58(3), 587 597. Taylor, D. G., ed. (1977). NIOSH Manual ofAnalytical Methods. Method No. P&CAM 239, 2nd ed. DHEW (NIOSH) Publication No. 77-157-A, 1:239-1-21. Thompson, S. W. 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