Document 5x1eY1Ew3eLxDXDq3ynZ7220

JUN 13 2002 15:56 FR CISTI ICIST FUNDAMENTAL AND APPLIED TOXICOLOGY S, 327-340 (1985) TO 12145201181 P.03 PLAINTIFF'S EXHIBIT Chronic Inhalation of Short Asbestos Fibers1,2,3 S. Frank Platek, David H. Groth, Charles E. Ulrich,* Lloyd E. Stettler, Myra S. Finnell, and Margrit Stoll Division ofBiomedical and Behavioral Science. National Institutefor Occupational Safety and Health. Cincinnati, Ohio 45226, and International Research and Development Corporation, Mattawan, Michigan 49071 Chronic Initiation of Short Atbesto* Fibers. Platek, S. F., Groth, D. H., Ulrich, C. E., Stettler, L. E-, FINNELL, M. S., and Stoll, M. (1985). Fundam. Appi. Toxicol. 5, 327-340. An animal inhalation study was initiated to study the chronic biological effects of inhalation of short chrysotite 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 morn concentration of fibers in the chamber air was 1.0 mg/m13. 2By phase contrast microscopy, the number of fibers > 5 iaa in length was determined to be 0.79 fiber per cubic centimeter. Rats were autopsied for pathological and histochemicxl examination at 1, 3, 6, 12, 18, and 24 months after initiating exposures. No signifient 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 lining;: (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, 1971; 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 ftm in length and <1.25 ftm 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 ura per cubic RECEIVED "iME JUN. 3. 2:58PM 327 PRINT TIME vUN. 13. 0272-0590/85 S3.00 3:06PM JUN 13 2002 15:57 FR CISTI ICIST TO 12145201181 P. 04 328 PLATER 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 pm as a nuisance dust with exposure to 5 mg/m3 permissible, it has been shown that for every fiber greater than 5 pm 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 pin) 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), arc exposed to potentially high levels of short (<5 nm) asbestos fibers. The purpose of this study was to determine the chronic biological effects after inhalation of chrysotile asbestos fibers less than 5 ftm in length in laboratory rats and monkeys. MATERIALS AND METHODS Chrysotile (short fiber) preparation. Type 7TF1 chrysotile 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 191 "C, milled in a ceramic ball mill for 24 hr, and then dried again for 24 br at 191 "C. 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 gla and stainless steel, dynamic air flow exposure chamber*. 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 23.6*C (74.5'F) with a mean relative humidity of 61.546. The chamber air flow rite for both study groups was maintained at 1500 liters per minute (S3 efin). 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. DECEIVE) TIME JUIv. 13. 2:58PM PRINT TIME JUIU3. 3:06PM JUN 13 2002 15:57 FR CISTI ICIST TO 12145201181 P. INHALATION OF SHORT ASBESTOS FIBERS 329 The chamber's incoming air flow diluted the aerosol to the desired concentration. The elutriator column only passed the desired smaller fibers (<5 fim) while a sediment of larger fibers and particles was collected on the bottom and side convolutions of the column. During the experiment, the inhalation chamber at mospheres were routinely characterized by three different methods. Mass concentrations were determined three times daily during exposures by drawing known volumes of chamber air through preweighed glass fiber filters. The filters were then reweighed and the mass concentration was calculated as the difference in weight divided by the total volume of air sampled. Measurements of the chamber concentration of fibers greater than S /im in length were also made three times daily during exposures. In this procedure, chamber sam ples of known volume were collected on 0.8-fim poresize cellulose ester membrane filters. The filters were then mounted and counted by phase contrast tight microscopy at a magnification of 400X using NIOSH Physical and Chemical Analysts Method No. 239 (Taylor, 1977). Particle size distributions were also determined daily during the exposures utilizing an Andersen cascade impactor (Andersen Samplers, Atlanta, Ga.) operating at 28.3 liters per minute. The weight of particles on each stage was determined gravimetricaUy. Scanning electron microscopy was also used to monitor the size distribution of partides in the exposure chambers. Nine asbestos chamber samples which were collected on 0.1 -ftm pore-size Nuckpore filters were sized Scanning electron micrograph enlargements of individual fields of view at * magnification of5000X were used in the sizing procedure. Nonfibrous partides were sized using a Zeiss MoOel TCG-3 partide size counter (Carl Zeiss, Germany). The lengths and widths of fibrous partides were deter mined manually using a ruler. Animal study. Three hundred male Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington, Mass.) with an. average body weight of 109 g were randomly divided into two groups of ISO each. The rats were housed individually in suspended wire-mesh stain less-steel cages and given food (Purina Laboratory Chow) and water ad libitum throughout the study except during the daily exposures. Twenty male cynomolgus monkeys, Macaca fascicularis (Primate Imports Corporation, Port Washington, N.Y.), weighing from 3.8 to 3.96 kg were randomly divided into exposure and control groups of 10 each. The monkeys were housed individually in suspended stainless-steel cages and given food (Purina Monkey Chow) and water ad libitum (except during the daily exposures) plus supplementary fruit throughout the study. All animals were exposed in the inhalation chambers for 7 hr/day, 5 days/week for 18 months. At the end of each daily exposure, all animals were removed from the chambers. The chambers were then droned. The monkeys were then returned to the chambers while the tats remained in the adjacent holding room until the next exposure period. At the end of the 18-months exposure period, all surviving rats were maintained for an additional 6-month postexposure observation period prior to final sacrifice. The monkeys were maintained for a 10-month postexposure period and then transferred to a contractor for long-term observation. Pathological analyses. Rat sacrifice intervals and onstudy mortality are indicated in Table 1. At each sacrifice period, the rats were killed with an overdose of sodium pentobarbital administered by intraabdominal injection and then necropsied for gross and histopathological evaluation. The lungs were weighed and then inflated with phosphate-buffered, neutral formalin. Sections of the Mowing organs were also fixed in phosphate-buffered, neutral formalin: brain pituitary trachea nasal turbinates larynx adrenal (2) thyroid urinary bladder skin sternal bone marrow salivary gland mammary gland any other grossly abnormal tissue mammary gland lung liver kidney pancreas spleen mesenteric lymph nodes tracheobronchial lymph node stomach colon (ascending and descending) testis (2) prostate TABLE 1 Experimental Rat Sacrifice and Mortality Schedule Treatment 1 Sacrifice (study month) Mortality* (month interval) 3 6 12 18 24 1-3 3-6 6-12 12-18 18-24 Asbestos 5 15 15 15 20 38 2 2 3 12 23 Control 5 15 15 15 20 45 0 4 2 14 15 * Died in extremis or sacrificed moribund (unscheduled deaths). Total .150 150 RECEIVED TIME JJN. 13. 2:58PM PKIF time JUN. 13. 3:06PM JUN 13 2002 15:58 FR CIST1 ICIST TO 12145201181 P. 06 330 PLATER ET AL. 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 (Zugibe, 1970), acid phosphatase (Barka and Anderson, 1963), and 0-gIucoronidase (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 (Fisber 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 Q.l-pan 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 Jaffe-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 pm. 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 pm 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 pm 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 RECEIVE) -:ME JIN. `3. 2:53PM 0R!NT "lME JUN. 13. 3:06PM JUN 1 1 3 2002 15:58 FR CISTI ICIST TO 12145201181 INHALATION OF SHORT ASBESTOS FIBERS P. 07 331 TABLE 2 Concentration of Inorganic Elements in 7TF1 Chrysooue* Batch No. 21 29 39 61 84 Mean At 0.880 0.960 0.904 1.10 0.909 0.95 Ca 0.425 0.396 0.416 0.429 0.420 0.42 Cr 0.086 0.079 0.095 0.088 0.093 0.088 Fe 3.65 3.33 3.59 3.59 3.64 3.56 U .001 0.001 .001 0.001 .001 .001 Mg 18.3 16.4 15.5 14.4 16.9 16.3 Ma 0.068 0.063 0.067 0.067 0.068 0.067 Mo 0.005 .001 0.005 0.005 0.005 0.004 Na 0.466 0.279 0.544 0.614 0.515 0.483 Ni 0.132 0.102 0.132 0.131 0.134 0.126 P 0.018 0.017 0.018 0.019 0.021 0.019 Ti 0.039 0.034 0.034 0.036 0.034 0.035 n 0.003 0.002 0.003 0.003 0.003 0.003 V 0.003 0.003 0.003 0.003 0.003 0.003 Zn 0.004 0.004 0.006 0.005 0.005 0.005 Zr 0.003 0.005 0.005 0.006 0.004 0.005 Note. The following elements were less than 0.001% in all of the samples: Ag, As, Be, Cd, Co, Cu, Pb, Pt, Se, Sn, Te, W, and Y. . .. .. * Values are percentages. (48%) were fibers and 7618 (52%) were nonfibrous-^Xhe majority of the nonfibrous par ticles were the "asbestos balls" as shown in Fig."2(b). The fiber size distribution is shown in Fig. 3 for eight of the nine samples. The count median length of the fibers was 0.67 jim (geometric standard deviation = 1.87), the count median diameter was 0.09 jim, and the median aspect ratio (length:width) was 12. Only 46 (0.66%) of the 6940 fibers were greater than 5 jam in length. Only 0.35% of the fibers were both greater than 5 jim in length and greater than 0.3 jim in diameter. Since the volume of chamber air pumped through these nine filters was measured at the time of collection (2.1 liters/min for 60 min) the number of fibers per cubic centi meter of chamber air was also calculated. The mean number of fibers greater than 5 fim in length found by this method was 3.0/ cm3 of air (range, 0.4-7.5). This is 3.8 times the mean number of fibers found by phase contrast light microscopy. Since the number of fibers greater than 5 /tm in length and greater than 0.3 jim in diameter represented about 52% of all fibers greater than 5 jtm in length, their concentration in the chamber air was estimated to be 1.6 fibers/cm3 of air. This is about two times the mean number of fibers found by phase contrast light micros copy. The mean number of fibers less than 5 jim in length in these nine samples was 493/cm3 of air (range, 144-1226). Table 1 shows the on-study mortality of the experimental rats between scheduled sac rifice intervals. Table 3 shows the rat mean body and lung weights at the designated sacrifice intervals. Both tables illustrate that no significant difference was observed in group mortality or body and lung weights over the course of the experiment. No signif icant body weight differences were observed in the monkey groups at regular weighing intervals except at the 3-month period. Results of histochemical tests, which in cluded /3-glucuronidase, add phosphatase, RECEIVED TIME JUS. 13. 2:58PM PRINT TIME JUN. 13. 3:06?M Jl'\ 1 3 2002 IS:58 FR CI ST I ICIST TO I 2145201181 P- 08 i 332 .1 >.. \ . v *J. 2 V Jt'N 13 2002 15:59 FR CISTI- ICIST TO 12145201181 P. 09 INHALATION OF SHORT ASBESTOS FIBERS 333 Fig. 3. Aerosol chamber fiber size distribution. The data shown are the sum of nine filter analyses. and lactic dehydrogenase, indicated no dif ference between the control and exposed group. The silicon content ofthe experimental rat lung and blood serum for each sacrifice interval is shown in Table 4. Because of the broad range of silicon values in both the rat and monkey groups as well as varying limits of detectability, no conclusions may be drawn with regard to silicon content. The hydroxyproline determination of the experimental rat lung as seen in Table 5 revealed no significant difference between the exposed and control groups at sacrifice inter vals as well as over the course of the study (analysis of variance, p > 0.5). tumors were seen. An unexpected finding was gastritis in 12/38 exposed and 2/45 con trol rats at the terminal sacrifice. Microscop ically the majority of these lesions consisted of focal mucosal ulcerations and submucosal inflammation in the nonglandular portion of the stomachs. Microscopic examination of the lung biop sies from the monkeys revealed a few scat tered macrophages in alveoli and a few as bestos bodies adjacent to the terminal bron chi. No fibrosis or tumors were seen in these biopsies. Lung Fiber Analyses Pathology Based upon gross and microscopic obser vations the only pulmonary alterations seen in rats that could be attributed to exposure to chrysotile were few and scattered macro phages in the pulmonary alveoli. Scanningtransmission electron microscopy of these macrophages revealed the presence ofchryso tile. No pulmonary fibrosis or pulmonary The results of the fiber size analyses for the fibers recovered from the lungs of five rats from the 18-month exposure group and five rats from the 6-month postexposure group (terminal sacrifice) appear in Figs. 4 and 6 and Table 6. In the 18-month group, a total of 2141 fibers were sized. The mean number of fibers per gram of dried lung for these rats was 294 31 X 10s (range, 257-327 X 106). The mean Fig. 2. (a) Scanning electron micrograph of a typical ball-milled asbestos preparation. Bar * 2.0 pm. (b) A scanning electron micrograph showing a typical asbestos "ball." Note that individual short hbers arc aggregated together to form the ball. Bar 1.0 pm. RECEIVED TIME M. 13. 2:58PM PRINT TIME JUN. 13. 3:05PM JUN 13 2002 16:00 FR CISTI ICIST TO 12145201181 ~ " P. 1 334 PLATEK ET AL. TABLE 3 Mean Values for Rat Lung and Body Weights Exposure interval (months) Group Body weight <g) Lung weight (g) l 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 10U 2.07 0.19 24' Control 765 185.2 2.34 0.22 Asbestos 756 152.4 2.31 0.20 * Mod SD. k 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 10 (range, 241-308 X 106), and the mean concentration of fibers greater'than $ pm in length was 23 4.9 X JO6 (range, 16-28 X 10). 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 10 (range, 143 209 X 106). This value is significantly lower than the same value obtained at 18 months ip < 0.001). The mean concentration of fibers less than 5 pm in length was 164 28 X 10 (range, 117-189 X 10). 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 pm in length was 27 9.3 X 10 (range, 18-41 X 10). This value is not significantly different from that seen at 18 months (p > 0.05). The mean percentage of fibers greater than 5 pm 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 10s 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 Corn. Exp. ConL Exp. ConL Exp. Coat Exp. Cone Exp. * mg/g. TABLE 4 Silicon Concentrations in Rat Lungs and Blood* Sac. interval (months) 3 3 6 6 12 12 18 18 24 24 Range 60-3Q0 60-200 40- 90 40- 70 70-450 20-110 70- 80 20-110 20-120 30-130 Dry lungs Mean 115.0 79.0 59.0 54.0 86.1 35.9 36.0 54.6 67.7 59.5 SD 98.6 43.6 13.7 10.8 142.02 28.90 19.55 28.25 27.7 31.91 Range 2-4 2-30 2-270 2-5 0.8-120 0.8-240 1-13 1-3 1-24 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 RECEIVED TIME Jl'V. :3. 2:53PM PRINT TIME JUN. 13. 3:05PM JUN 13 2002 16:00 FR CISTI ICIST TO 12145201181 P. INHALATION OF SHORT ASBESTOS FIBERS 335 TABLES Hydroxyproune Determination dm 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 (ing/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. lOVgram ofdried 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 siged. Of these fibers, 239 or 5.8% were greater than 5 fan in length. The number of fibers per gram ofdry 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 pm 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 in length/g of dry lung, 272 X 106 chrysotile fibers < 5 ftm 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-raonth sacrifice. RtCEiVTD TIME JN. 13. 2:58PM PRINT TIME JUN. 13. 3:05PM JUN 13 2002 16:00 FR CISTI ICIST TO 12145201181 P. 336 PLATEK ET AL. TABLE 6 Numbers and Lengths of Chrysotile Fibers in Rat lungs by TEM* Fiber length Rat No. All lengths <5 pat >5 pm >8 pm >10 pm >15 pm. 80-217 80-218 80-219 80-220 80-221 Mean (SD) 302 291 257 294 327 294(31)* 277 263 241 269 308 272(31)* 18 Months of exposure 25 14 28 14 16 7.3 25 8.8 19 11 23 (4.9) 11 5.6 6.0 2.4 5.1 5.0 4.8 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 12 4.8 3.3 * Values are No. of fibcra/6 dry lung/106. . . . 6 Significantly higher than the same-sized fiber concentration obtained 6 months postexposure (p < 0.001). 6 hr/day, 5 days/week for 62 weeks and tions of UICC chrysotile, croddolite, and observed, for -up to 34 months following amosite ranging from 2 to 10 mg/m3, 7 hr/ initiation of exposures. He also observed day, 5 days/week for 1 year and sacrificed 1 i pulmonary fibrosis and one mesothelioma in 16.5 months postexposure. The highest inci the exposed rats. No information on the dence (15/40) of lung tumors and the most number of fibers per cubic centimeter in the severe fibrosis occurred in the group exposed chambers or fibers per gram of lung tissue to chrysotile (10 mg/m3). The concentration was given. In 1974, Wagner et al. reported of fibers greater than 5 /im in Length in the the induction of pulmonary fibrosis, lung chamber air by phase contrast light micros tumors, and mesotheliomas in rats exposed copy was found to be 1950/ml. The rats for a variety of intervals ranging from 3 to exposed to chrysotile at 2 mg/m3 (390 fibers 24 months to amosite, anthophyllite, crocid- > 5 Mm in length/ml) also developed lung oiite, Canadian chrysotile, or Rhodesian tumors (8/42) and pulmonary fibrosis. The chrysotile. The exposures were for 7 hr/day, incidence of lung tumors in the rats exposed 5 days/week. The mean respirable dust con to crocidolite and amosite ranged from 2.5 centrations varied from 10.1 to 13.5 mg/m3. to 4.7% and the extent of fibrosis was less No information on the number of fibers per than that seen in the groups exposed to cubic centimeter in the chambers or the chrysotile even though the mass and number concentrations of fibers in the lung tissue of fibers > 5 Mm in length per milliliter in was given. the aerosol were comparable to those seen Davis et al. (1978) reported the induction with the chrysotile-exposed animals. Exami of pulmonary fibrosis, lung tumors, and two nation of the fiber preparations by scanning mesotheliomas in rats exposed to concentra electron microscopy, however, revealed many RECEIVED TIME JUN. 13. 2:58PM PRINT TIME JUN. 13. 3:05FM 002 1u;0[ PH CISTI ICTST TO 12145201181 INHALATION OF SHORT ASBESTOS FIBERS P. 13 337 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. Bax -- 1.0 ftm. 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 p.m in centrations had only 34 and 6 fibers > 20 :g. 6. Fiber size distribution for fibers extracted from five rats of tha 24-raonth (terminal) sacrifice. ~ A " U !!' ;v JUN 13 2002 16:01 FR CISTI. ICIST 338 PLATEK ET AL TO 12145201181 P. 14 Ftc. 7. Fiber size distributioa For fibers extracted from 10 monkeys at the 28-month biopsy. pm 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 tb* Davis et al. (1978) study contained 2538'times more fibers greater than 5 pm in length than the aerosol in our experiment and their other chrysotile aerosol (2 mg/m3) contained 500 times more fibers greater than 5 pm 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 pm 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 pm in length produced ex tensive interstitial pulmonary fibrosis, whereas the crocidolite preparation, in which 99% of the fibers were less than S pm in length, produced no fibrosis. The fiber diameters in both preparations were between 0.1 and 0.3 pm. 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 (NaA^OH^COj), wollastonite, tremolite, amosite, attapulgite, hallyosite, silicon carbide, and potassium octatitinate. The fibers were injected intrapleur ally in rats. They found that fibers >8 pm in length and <1.25 pm in diameter were much more potent in inducing mesotheliomas than other fibers (i.e., those <8 pm in. length and >1.25 pm 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 RECEIVED TiME JUN. 13. 2:58PM PRINT TIME JUN. 13. 3:05PM JUN 13 2002 16:02 FR CISTI ICIST TO 12145201181 P. 15 INHALATION OF SHORT ASBESTOS FIBERS 339 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 feet 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 (LeBouffant 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 HC1 (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 er al., 1983). " ACKNOWLEDGMENTS The authors express their appreciation to David Brewer, JeAnne Burg, Ph.D., Richard Carlson, D. Gayle Cecil, Charles Gorslci, Kathy Hides, Susan Kaeiin, Lea Kalejs, Hazel Patterson, Randall Smith, Allen Stein, William D. Wagner, and Glenda White of the National Institute fot Occupational Safety and Health (N10SH) 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 St 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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