Document V3gxD5Kwy4dB6BJo89oLQprpg

J^SSBrnSK wBm '; ^aMENTAL AND APPLIED TOXICOLOGY 5, 327-340 (1985) ^j;:^ (- 37 Chronic Inhalation of Short Asbestos Fibers1 A3 ; frank Plater, David H. Groth, Charles E. Ulrich,* Lloyd E. Stettler, Myra S. FInnell, and Margrit Stoll pjr&ion ofBiomedical and Behavioral Science, National Institutefor Occupational Safety and Health, Cincinnati, 45226. and *lmemmional Research and Development Corporation. Mattawan, Michigan 49071 ... . Chronic Inhalation of Short Asbestos Fibers. Plater, 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 chrysolilc asbestos fibers. Rats and monkeys were exposed for 18 months, 7 hr/day, 5 days/wcck to a specially prepared, chrysolite asbestos aerosol. Based upon daily chamber measurements, the mean concentration of fibers in the chamber sir was 1.0 mg/m3. By 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 bistochemical examination at 1, 3, 6, 12, 18, and 24 months after initiating exposures. No signifiant differences in the htstocbemkal 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 chrysotUe-exposed and the control monkeys 28 months after initiating exposures. Histopathologic evaluation of (he lung biopsy (issue 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 bang maintained Tor an indefinite period and observed for signs of latent pulmonary diseaseT Asbertos contains numerous insulating as well as chemical- andweanresistant properties ^Hich make it an extremely valuable mineral iggbe. production of textiles, cement and tile Igoducts, gaskets and friction materials, coatipgsfrand automotive brake linings (Hendry, ^SS^However, asbestos has been shown by numerpus investigatoesSto cause the debilit^dig human diseases of asbestosis, bronchogcnipTcardnoma, and mesothelioma (Newbouierand Thompson, 1965; Kannerstein el _ K preliminary report of this material was presented tfafce-Rm Nd/EPA/NIOSH Collaborative Workshop j&nckvfife, Md., May 7, 1980. faThis study was conducted under contract with The taouatiooal Research and Development Corporation, Mutxwan, Mich, under NIOSH Qrairacl 210-77-0151. -^This study was supported by the National Cancer ksftute (NCI) via an Interagency Agreement between NCI and NIOSH. a/., 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 pm in length and <1.25 pm in diameter are much more.potent than those of other sizes (Stanton el 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 sshestos (Yeager el al,, 1983; Gross, 1974; SefikofF el ai:, 1972; Holt el at., 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 a, 0272-059<yS5 $3.00 327 -111 i I VjV' HWBUI0009541 * 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 S jim as a nuisance dust with exposure to 5 reg/m3 permissible, it has been shown that for every fiber greater than 5 Mm there may be more than 100 fibers less than 5 Mm in length (Holt et al., 1965). Of these smaller fibers, most are probably beyond the resolution of the optical light microscope (~0.25 pm) 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 fim) asbestos fibers. The purpose of this study was to determine the chronic biological effects after inhalation of chrysotile asbestos fibers less than 5 jtm in length in laboratory rats and monkeys. MATERIALS AND METHODS Chrysotile (shortfiber) preparation. Type 7TF1 chrys otile was obtained from the Johns-ManviUe Sales Cor poration in Denver, Colorada Four-hundred-para batches of the chrysotile were dried in an oven for24 hri. at 191"C milled in a ceramic tall mill for 24 then dried again for 24 hr at 191C. Each batd&u^j. examined by scanning electron microscopy to dttritsb* it fiber size. Five batches were randomly sdectedpad i| quantitatively analyzed by induction couplediph&na y emission spectroscopy for their elemental contents^ %, Inhalation chambers, dust generation, and diara/tp-lS nation. Experimental exposures were coaducted'nr!tao 16-ra5 glass and stainless steel, dynamic air flow exposure'! chambers. Air for both control and exposure-chauer j ventilation was filtered with HEPA filters to-rcmoire particulates and controlled for temperature and biMnSSty. ? The mean temperature wrthia the two exposure chamteas t was 23.6C (74.5F) with a mean relative humiiiity.es 6 61.5%. The chamber air flow rate for both study groups s was maintained at 1500 liters per minute (55 cfropThe j control inhalation duunber was supplied onfywilSibe / filtered and temperature-and- humidity-controSedhir. : The asbestos inhalation chamber used a specialty designed dust-feed elutriator system (International Research :nd ; Development Corporation, Matawan, Midi) to generate . the short fiber asbestos atmosphere (Fig. I). The opera- i`. tional characteristics of the device were as follows!* known weight of prepared asbestos was placed iolfie reservoir of the geheralor and stirred by a stirring mech anism. A disk containing cups around the perimeter w rotated through the reservoir permitting the cups lithe filled with the chrysotile dust The filled cups then passed over a blowout port where a metered Bow ofdesicased air "puffed" the asbestos into a vertical elutriator cofimra fitted between the generator and the chamber air inlet. ^. Fig. 1. Diagram of system used to generate short asbestos fibers. ...i HWBUI0009542 INHALATION of short asbestos fibers 329 jtatbarobcr's incoming air Sow diluted the aerosol to aa m^earei concentration. The elulriaior column only b ^3thedesired smaller fibers (<5 yim) while a sediment ^gjlgef.fibers and particles was collected on the bottom `ZJ Jfttfe convolutions of the column. in; j^ng'rtbe experiment, the inhalation chamber atJi4 p^feres were routinely characterized by three different as ptjf^Afc'Mass concentrations were determined three ^ffifatyd'iring exposures by drawing known volumes . (ftSunberair through preweighed glass fiber filters. The j5?vere'then reweighed and the mass concentration an ^ggJisiSlatoti as the difference in weight divided by the bj yjfffefiiinc of sir sampled. 8 jj^rairemeots of the chamber concentration of fibers hr. .5 pm in length were also made three times as "`'Wing exposures. In this procedure, chamber samof jinown volume were collected on 0.8-pm porew luiose ester membrane filters. The filters were lie nted and counted by phase contrast light lb fcjpsoppy.ai a magnification of 400X using NIOSH tt puyMl'a'nd Chemical Analysis Method No. 239 (Taylor, < s fwtBe size distributions were also determined daily de&gtllx exposures utilizing as Andersen cascade tra- n- pjt^Aodersen Samplers, Atlanta, Ga.) operating at . i IXSfisn per minute. The weight of particles on each !b & floras.determined gravimetrically. &*amng electron microscopy was also used to monitor teSsilistrttosion ofpartides in the exposure chambers. b sitf&>estos chamber samples r$ueh were collected on w ''a! 8.f5tsh';pBre-size Nudepore filters were sized. Scanning 51 etra,oricrograph enlargements of individual fields of e *y|tniagdificatioa of 5000X were used in the sizing freodure.' Nonfibrous partides were sized using a Zeiss MnHjICG-3 particle size counter (Carl Zeiss, Germany). TKgpgtiB and widths of fibrous partides were deter- w&acuslly using a ruler---- -....... aSouI&tudy. Three hundred male Sprague-Dawley aafCferks River Breeding Laboratories, Wilmiogton, *>||Mth- an average body weight of 109 g were '"wjjjljfdjvided into two groups of ISO each. The rats : :.*'Plusrt.iadivjdually in suspended wire-mesh stain1 ^*K^3!S and given food (Purina Laboratory Chow) -**k[ierW libitum throughout the study except during Twenty male cynomolgus monkeys. Macaco fascicu- laris (Primate Imports Corporation, Port Washington, N.Y.), weighing from 3.8 to 3.% 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 animats 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 cleaned. The monkeys were then returned to the chambers while the rats remained in the adjacent holding room until tire 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-montb postexposure period and then transferred to a contractor for long-term observation. - ' Pathological analyses. Rat sacrifice microtis and on- study 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 following organs were also fixed its 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 ' T- ' ' ' lung - r: liver ' '-> kidney ;-v pancreas .'v"'.'V- '''-- spleen ' mesenteric lymph nodes '." tracheobronchial lymph node stomach ' "' colon (ascending and >`v'r descending) .. testis (2) ' prostate ...i.-.. .. y&i . SBfc: !|r rf-To : - TABLE l ' . Experimental Rat Sacrirce and Mortality Schedule - Sacrifice (study month) Mortality" (month interval) 36 12 18 24 1-3 3-6 6-12 12-18 -18-24'-f Total 15 15 15 20 38 15 15 IS 20 45 .SirDied in extremis or sacrificed moribund (unscheduled deaths). 12 23 . 150 14 15 ' 150 ,1 !-.fe>` W,i-- KEf"' SS-i; --$U .Mir' I: ss-v.j.'r - \w-'m,,: 330 PLATEK ET AL At all sacrifk* periods, the left lungs from five randomly All of the monkey lung biopsies, both exposed inf selected rats from each exposure group were analyzed controls, were analyzed for fiber content In addition, for the presence of ao inflammatory response by mea lungs from five of the 18- and 24-month (terrianti. suring the following enzymes; lactic dehydrogenase (Zu- sacrifice) rats from each exposuregroup were also anafytaj.'. gibe, 1970), acid phosphatase (Barks and Anderson, for fiber content 1963), and gluooroaidase (Thompson, 1966). At the 3-, 6>, 12-. and 18-month and terminal sacrifices, the left lungs frost 10 randomly selected cats from each exposure group were taken for subsequent hydroxyprotoe RESULTS and elemental sifioon analyses. These lungs were sectioned longitudinally along the main stem bronchi such that Five batches of the ball-milled cl each contained portions of the apex and base. One preparations used in the exposure cbaml ' section was then analyzed for hydroxyproiine and the other for detnental silicon by plasma emission spectrm' 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. were chemically analyzed. The resjfli these elemental analyses are sumra; Table 2. Of interest are the rclativelyglgjii; concentrations of aluminum (O.S8-i:l%] Each batch of the ball-milled asbestos^n* . Hematoxylin and cosin stained sections ofthe remain also examined using the scanning electron ing lung tissue and of each of the above tissues were prepared for and examined by light microscopy. Twenty-eight months after the initiation ofexposures (10 months after completion of exposures), open lung biopsies were performed on all monkeys for histopatho- microscope (JEOL, JXA 50A) at magnifies, tions of 400X and 5000X to ensure thagihe vast majority of the prepared fibers werejess: than 5 pm. ftp-unavoidable by-productfof logical evaluation and for lung tissue fiber content deter the ba!i-milfing'praxdure was the preparaSoB minations. Approximately 2 g of tissue was taken from the anterior margin of the right tower lobe of each monkey. . - The procedure for determining lung fiber content is as follows. Pieces of lung tissue were freeze-dried to of agglomerated asbestos "balls" or bunfflo of asbestos fibers. Figure 2a is a scanning electron micrograph showing a typical.mDmilled preparation. A high magnificatioiTai- constant weight using a Labconco Freeze Dry S freeze Iargement of.one of the asbestos "ballsj'ij dryer and then ashed in a low-temperature asher (Inter national Plasma Corporation, Model I PC 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 shown in Fig. 2b. ^|| . The results of the exposure chamber mon itoring were as follows: the mean mass con centration as determined by gravimetriesam- sonicated for to min in an ultrasonic bath. The resulting pling was 1.0 mg/m3 with a standard dera suspensions were then diluted to 100 ml and stirred magnetically for 10 min. Aliquots ofthe suspensions were then filtered through 0.!-|im pore-size Nudeopore filters. The filters were then carbon coated in a vacuum evaporator (Edwards High Vacuum. Model 306). Pieces tion of 0.28. The range of mass values ora the entire experiment was 0.4 to 1.8 rag/tir. The mean number of asbestos fibers greater than 5 pm in length as determined byJgfct ofthese filters were then placed on 200-mesh transmission microscopy (Taylor, 1977) was 0.791fitcr/ electron microscope grkb 'and cleared with chloroform cm3 with a standard deviation of 0.41351* using a modified jaffe-Wkk technique (lafie, 1948; Chalfidd and Dillon, 1978). The resulting grids were examined in a transmission electron microscope (JEOL, Model 100CX) equipped with an energy disprove X-ray spectrometer system values ranged from 0.08 to 1.5 fiberS/aS*. The mean equivalent aerodynamic diari||er for the particles in the exposure chambays determined by using the Andersen casaxfc (EG&G Ortec, Model BEDS II). AU of the fibers found impactor was 5.0 pm with a geometricstan- in 10 grid spaces for two grids of each sample were dard deviation of 2.9. - 'Jiff counted and sized- Every 10th fiber was analyzed by A total of 14,558 particles (fibrousftad energy disprove X-ray analysis and selected area election nonfibrous) in 138 fields of view fromitffl* diffraction to confirm that it was chrysotile. Lung fiber concentrations were calculated from the fiber densities samples of the asbestos chamber atmosptet on the filter preparations and the original dry lung collected on Nudeopore filters were counW weights. and sized by SEM. Of these partides/c HWBUI0009544 ) it in >1k n INHALATION OF SHORT ASBESTOS FIBERS TABLE 2 Concentration of Inorganic Elements in 7TF1 Chrysotile" Batch No 33 J 21 29 ~ 39 61 84 Meats lik h 1 1 JSm ^; .. i jEjip. llSS~ 1 W^' . 0.880 0.425 0.086 3.65 .001 18.3 0.068 0.005 0.466 0.132 0.018 . ' 0.039' 0.003 0.003 0.004 0.003 ' 0.960 0.396 0:079 3.33 0.001 16.4 0.063 .001 0.279 0.102 0.017 0.034 0.002 0.003 0.004 0.005 0.904 0.416 0.095 3.59 .001 15.5 .. 0.067 0.005 0.544 0.132 0.018 0.034 0.003 0.003 0.006 0.005 _ 1.10 0.429 0.088 3.59 0.001 14.4 0.067 0.005 0.614 0.131 0.019 0.036 0.003 0.003 0.00S 0.006. 0.909 0.420 0.093 3.64 .001 16.9 0.068 0.00$ 0.515 0.134 0.021 0.034 0.003 0.003 0.005 0.004 0.95 0.42 . . 0.088' 3.56 . .001 16.3 0.067 ' 0.004 0.483 ` . 0.126 . . 0.019 .. 0.035 0.003 0.003 .____ ` 0.005 0.005 folfewsrsg ekraants were less than 0,001% in sS of the samples: Ag, As, Be, Cd, Co, Cu, Pb, Pi, Sc, So, Y. Stats are percentages. .were fibers and 76l8-(52%) were nonStjrirSs>The majority of the nonfibrous par- were the "asbestos balk" as shown in b). The fiber size distribution is shown |f*3 for eight of the nine samples. The jeognt^median length of the. fibers was 0.67 f ptnj(geoirietrip - standard ^deviation = 1.87), unt median diameter was 0.09 pm, he median aspect ratio (lengthwidth) ^J&^Only 46 (0.66%) of the .6940 fibers |*W|gireater than 5 pm in length." Only 0.35% were bothjgreater than 5 pm in luand greater"tHan'0.3 pmjp diameter. J^itlie-volume of chamber air pumped te."*s2?8^-'tbese sine fillers was measured at Se'jof collection (2.1 liters/min for 60 jhe number of fibers per cubic centl' Chamber air was also"'calculated. Jean number of fibers greater than 5 Qgth found by this method was 3.0/ aur (range, 0.4-7.5). This is 3.8 times number pf fibers found by phase blight microscopy. Since the number of fibers greater than 5 fim in length and greater than 0.3 pro in diameter represented about 52% of all fibers greater than 5 pm 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 "ton 5 pm 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 ^-glucuronidase, acid phosphatase. Sit ig.mg, s, ... HWBUI0009545 HWBUI0009546 INHALATION OF SHORT ASBESTOS FIBERS 333 K FlO. 3. Aerosol chamber fiber size distribution. The data shown are (he sum of nine filter analyses. tc dehydrogenase, indicated no dif ference between the control and exposed jjSup. The silicon content of die experimental rxfuurig and blood serum for each sacrifice i interyal-is shown in Table 4. Because of the bread range of silicon values in both the rat andononkey groups as well as varying limits (^detectability, no conclusions may be drawn tjjhlLrtgard to silicon content! bydroxyproline determination of the cgSerimcntal rat lung as seen in Table 5 revealed no significant difference between the exposed and control groups at sacrifice inter rupts well as oyerJthe.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 ofthe 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 - upon gross and microscopic observSKinsrthe onlypuffionary alterations seen i|ptsithat could be attributed to exposure tagetrysotije were few and scattered macroPhagesmthe pulmonary alveoli. Scanntngitgmmission electron microscopy of these n*Seraj)hages revealed the presence ofehryso- 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 postexposuie 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 10* (range, 257-327 X iO5). The mean fr-EtC. 2. (a) Scanning electron. micrograph of a typical ball-milled asbestos preparation. Bar = 2.0 jun. i) A scanning electron micrograph showing a typical asbestos "ball." Note that individual short fibers aggregated together, to form the ball. Bar = 1.0 pm. HWBUI0009547 mm 334 PLATE1C ET AL. ISWL* i. f : i * ! ; 1- . t '! . j i ' TABLE 3 Mean Values for Rat Lung and Body Weights Exposure interval (months) Group Body weight (8) Lung weight (8) 1 ... Control Asbestos 3..:^~' Control i 1 Asbestos '6':'..'.. Control Asbestos 12:.-' Control Asbestos 18 Control V Asbestos 24* . Control Asbestos 330 33.5* 1.58 0.25 332 21.9 ' 1.66 0.13 454 45.4 1.65 0.11 463 47.0 . 1.81 0.22* 558 50.5 565 59.0 1.88 0.15 1.92 0.16 634 61.4 656 85.3 2.01 0.20 2.03 0.16 710 95.4 693 101.1 2.09 0.19 207 0.19 765 185.2 756 152.4 2.34 0.22 231 0.20 * Mean SO. * Statistically Afferent from the control group ip < 0.05). Six months postexposure. concentration of fibers less than 5 pm in length was 272 31 X 10* (range, 241-308 X 10*), and the mean concentration of fibers greater than 5 fim in length was 23 4.9 X 10s (range, 16-28 X 10s). The mean percentage of fibers greater than 5 jim in length in these lungs was 7.7% (range, 5.8- 9.6%). A scanning electron micrograph^* typical ashed lung preparation is showxria Fig. 5. Note the individual fibers as wdla* one of the asbestos "balls." In the 6-month postexposure group*] 6), a total of 919 fibers were sized. TherSoHL number of fibers per gram of dried hit" these rats was. 192 ..27 X 10* (rane_ 209. X 10s). This value is significant^] titan the same value obtained at lgsga (p < 0.001). The.-mean concentrate/ fibers less than 5 |im in length wasTl X 10s (range, 117-189 X 10s). Tfais'i significantly lower than the same vaii tained at 18 months (p = 0.001); Th^jmean concentration of fibers greater than-frifnSips length wag. 2\ 9.3 X 10s (tange,-.184^C 10s). This value is not significantly diflajm from that seen a| A8Imoafts (p> 0.01 mean percentage of fibers greater thanrjSfgS in length in the 6-month was 12.6%. Only one fiber was found in the lunggof one out of four control irat lungs at thejllmonth sacrifice." lAte concentration of. fibers in the lung of that rat was caiculatedvto|be 0.16 X 10s per gram of dried lung. Ohlywe fiber was found in the lung of one out of.five control rats at the 24-month sacrific&!tnr concentration of fibers in the lung ofthatA iri'.-.-i- 'V"r (Group ConL Exp. ConL Exp. ConL Exp. ConL Exp. ConL Exp. TABLE 4 Silicon Concentrations in Rat Ujngs and Blood* Sac. interval (months) 3 3 6 6 12 12 18 18 24 24 Dry lungs Range Mean 60-300 60-200 40- 90 40- 70 70-450 20-110 70- 80 20-110 20-120 . 30-130 115.0 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 14202 28.90 19.55 28.25 27.7 31.91 Serum -m Range 2-4 2-30 2-270 2-5 0.8-120 0.8-240 1-13 i-3 1-24 1-485 ` Mean sill 2.9 ' !?>gg 6.4 32.8 2.8 14.6 24.9 ^7568 2.8 1.9 .'.ViSjst ,:;:0j8) 5.4 5.9 'rag/g. * rC HWBUI0009548 " ' : v - --.in.......... ,-- INHALATION OF SHORT ASBESTOS FIBERS TABLES j HVW-OXYPROLINE DEIERMnUTlON IN EXPERIMENTAL ft.-' 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 |mterval Smooths) Group Hydroxyproline' . (mg/g dry lung tissue) Control Asbestos 28.38 2.29'' 28.84 * 3.24 Js?TiVr ' - Control Asbestos 31.08 2.48 32.20 2.84 flippy j p-V Control . ` 35.39 2.81 Asbestos 33.69 6.92 Control - Laboratory error--sample lost Asbestos Laboratory error--sample lost W Control, Asbestos 24.53 738 26.73 10.63 lean SO. months postexposure. was calculated to be O.l 9 X 10*/gram of dried i^g-.-'. j^Tbe results of the fiber size analyses for Ibe fibers recovered from the lungs of the 10 tffpnkeys at the 28-month biopsy are suml,, tnanzed in Rg. 7. A total of 4124 fibers were roedi^Of these fibers, 239 or 5.8% were grater, than 5 pm in length. The number of m 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 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 pm in length/g of dry lung, 272 X 10s chrysotile fibers < 5 pm 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 el a!, 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/m}. L" riven uxfm fie., (PB1 3-e. ~ . t .5 2 2.5 3 A 5 FIBER LENGTH 10 >tQ - A 1 Fk#. 4.'Fjber size distribution for fibers extracted from five mis of the l$-montb sacrifice. *rn?v.' HWBUI0009549 irfe-' si I^SrtS**' js *;~i . ,; .f.- 336 PLATER. ETAL TABLE 6 Numbers and Lengths of Chrysotile Fibers in Rat Lungs by TEM* Fiber length % m Rat No. All lengths *..80-2!7..fv: ' : -80-218 -- 80-219 80-220 '80-22! 302 291 257 294 327 Mean (SD) . 294 (31)* <5 pm 277 263 . ' 241 269 308 272 (31)* >5 ftm >8 pm >10 JOB v 18 Months of exposure 25 28 16 25 19 14 > -..V . 5.6 . 14 ' 6.0 7.3 V i 2.4 8.8 - . .5.1 II . ' =: / 5.0 . *-- -O*.2k0tiX -zrrSF 23 (4.9) 11 .. .4.8 .. - 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 20 . 26 31 41 11 - -|2-- 5.8 . 4.8 9.5 1.9 _____-;| 9.8 -------------='4.4 24 8.4. _ ------'44# 3jH27 (9.3) 13 5.1 ` Values are No. or fibors/g dry lungMtf. * Significantly higher than die same-sized fiber concentration obtained 6 months postexposure (p. 0.001). ;^ts - ' ' - 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- lions of UICC chrysotile, crocidolite\at>d amosite ranging from 2 to 10 mg/m3,. 7 hr/ day, 5 days/week for I year and sacrificed 16.5 months postexposure. The highest inct deuce (15/40) of lung tumors and the modi severe fibrosis occurred in the group exposed to chrysotile (10 mg/m3). The concentration of fibers greater than 5 pm in length in the chamber air by phase contrast light micros copy was found to be 1950/mL The rate exposed to chrysotile at 2 mg/m3 (390 fiben > 5 tim in length/ml) also developed-long tumors (8/42) and pulmonary fibrosis- The incidence of lung tumors in the rats exposed to croridolite and amosite ranged from 2s5 to 4.7% and the extent of fibrosis was. l*eiuaj than that seen in the groups exposed to chrysotile even though the mass and numfe of fibers > 5 pm in length per milliliter in the aerosol were comparable to those seen with the chrysotile-exposed animals. Exatnfc nation of the fiber preparations by scanning electron microscopy, however, revealed many HWBUI0009550 HWBUI0009551 338 PLATEJt ET AL iiliiil.nWl<tnW>l.n`afc>l &'l * i ' pim 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/ ra3) in the Davis el 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 el d. (1978]T found that the pul..^nonary 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 intratracheally with long and short fibers of crocidolite, as well as synthetic fluorampbiboles 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-pcf tensive interstitial pulmonary fibrosis, whereas the crocidolite preparation, in which 995T1aF' the fibers were- less than S pm .in length^ produced no fibrosis. The fiber diameters iir both preparations were between 0.1 aad.&S' pm. The fact that only 4 mg of the-4obgFfibers compared to 25 mg of the short fibcnr had been injected per animal enhanced the- I significance of the results. - 4- The most detailed studies relating fiber! sizes to biologic effects were those reported? by Stanton el al. (1981). They tested, fibeti j of differing lengths and of several different* j chemical compositions. These included cn& f ddolite, various types of fibrous glass,1 alu-f' minum oxide, dawsonite (NaAlfOHhCQiJJf wollastonite, tremolite, amosite, attapulgite^ hallyosite, silicon carbide, and potassium oo|. tatitinate. The fibers were injected intrapleura ally in rats. They found that fibers >8 pm length and <1.25 pm in diameter were muefcj 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 fiber^r \j HWBUI0009552 INHALATION OF SHORT ASBESTOS FIBERS 339 acting as cocardoogens for substances like Cigarette smoke, which is most important in jbe occupational setting (Hammond el al. mi9y. There are two other factors that might Chatfield, E. J., and Dillon. M. J. (1978). Some aspects of specimen preparation and limitations of particulate analysis by SEM and TEM. Scanning Elec tron Microsc. I, 487--496. Davis, J. M. G., Beckett, S. T., Bolton, R. E; Collings, P.. and Middleton, A. P. (1978). Mass Sre riafluenced the results of our study. and number of fibers in the-pathogenesis of asbestosSltese are the rather high aluminum content related lung disease in tats. Brit. J- Cancer 37, 6730.88-1.1%) and the fact that the chrysotiteDe6p8a8r.tment of Health, Education and Welfare (DHEW) Jas;t>a31 milled. The aluminum content was (1978). Asbestos: An Information Resource. Publication ' Jjixiut twice as much as that reported to be (NIH)78-1681, Washington, D.C. . ' . ' present' in the UJCC Rhodesian chrysotile inbrcll, 1969) tested by Wagner et al. g174).. Aluminum compounds have been reported to modify the fibrogenic potency of 4sica-. (LeBouSant et al., 1977), and it Is Documentation ofthe Threshold Limit Ka/ue 4th cd., " pp. 27-30. (1980). American Conferenoe of Govern mental Industrial Hygienists, Inc. Evans, P. H., Brown, R. C, and Poole, A; (1983). Modification of the in vitro activities of amosite asbestos by surface dcrivatization. /. Toxicol. Environ. possible that they could also modify the JiiSrbgemc potency of silicates, e.g., asbestos, jpiber. investigators have reported that- ball Smiling may also cause a degradation of the Crystal structure of the asbestos fibers (Spumy feal^-1980T which may affect the biological Health 11.535-543. Gross, P. {1974). Is short-fibered asbestos dusy* bioloyai hazard? Arch. Environ. Health 29, 115-117. Gross, P., deTreville, R. T. P,, Tolker, E B., Kaschak, 'M., and Babyak, M. A. (1977). Experi mental asbestosis. The development of lung cancer is rats with pulmonary deposits-of chrysotile asbestos activity of the asbestos. The modification of |&rysotile, e.g,j byleaching in I N HCJ (with- igit^pparentJy modifying fiber length), has tjbeen shown to- modify the biological effects dusL Arch. Environ. Health 15, 343-355. Hammond, E C, Seukoff, L 1., and Seidman, H. (1979). Asbestos exposure, cigarette smoking and death rates. Ann. N. Y. Acad. Sci. 330,473-490. " Hendry, N. W. (1965). The geology, occurrences and $both in vitro and in vivo (Morgan et at.. |197I; Evans et al., 1983). major uses of asbestos. Biological effects of asbestos. Ann. H.Y. Acad. Sci. 132, 12-22. . Holt, P. F., Mills, }., and Young, D. Kl (1965).' Experimental asbestosis with four types of fibers: Im- AGKNOWLEDGMENTS poitance ofsmall particles. Biological effects ofasbestos.. , Ann. N.Y. Acad. Sci. 132, 87-97. . ' I^The-authors express their appreciation to David Brewer, ' f&noe-Buig. Ph.D., Richard Carlson, D. Gayle Cedi, .CbzdesGarsb', Kathy Hides, Susan Kaelin, Lea Kalejs, %Zt-if3<terspQ, Randall Smith, Allen:Stein, William 0. #iSaer,:EQd Glenda White of the National Institute for ^Sjntional Safety and Health (NIOSH) and Dr. Basil Jaffe, M. S. (1948). Handling and washing fragile replicas. J. Appi. Physio!. 19, 1187. . '. Kannerstein, M.. Churg, J., McCauohey. W. T. E, ' and Seukoff. I. J. (1977). Pathogenic effects of asbestos. Arch. Pathol. Lab. Med. 101, 623-627. ' . LeBouffant, L., Daniel, H.. and Martin, J. C IfpsSi^omas Mooier-Slvid Pydlefc and contributing (1977). The therapeutic action of aluminum com gbf'lthe International' ResearchVand Development ' don (IRJDC) for their cootributicm to this study. iva$ performed under NIOSH Contract 210- pounds on the development of experimental lesions produced by pure quartz or mixed dust fir Inhaled Particles (W. H. Walton, ed.), VoL TV,-Part-1, pp. 389-401. Petgamon, New York. .- ; ".\vi-SjC-- LYNCH, J. R. (1968). Brake lining decomposition prod- ; REFERENCES nets. J. Air Pollut. Control Assoc. IE 824-826. ". McDonald,-J.-C, and Liddell, F. D. K. (1979). r,AND Anderson, P. J. (1963). Histochemistry: y.'-Pmaiee and Bibliography. Harper & Row, York. t_ (1978). A Study of Airborne Asbestos I in.Connecticut. Workshop on Asbestos: Definiand Measurement Methods. U.S. Department of roerce,.NBS Special Publication 506, 179-191. Mortality in Canadian miners and millers exposed to chrysotile. Health hazards of asbestos exposure. Ann. H.Y. Acad. Sci. 330, 1-9. Morgan, a., Davies, P., Wagner, J. C., Berry, G., and Holmes, A. (1977). The biological effects of magnesium-leached chrysotile asbestos. Brit. J. Exp.' Pathol. 58. 465^473. PLATER ET At, Taylor, D. G., cd. (1977). NfOSH Manual ofAnalytic Methods. Method No. P&CAM 239. 2nd ed. DHEy (NIOSH) Publication No. 77-157-A'; (:239-l-21^ Thompson, S. W. (1966). Selected HisttxHamqf and Histopathological Methods. Charles C Tbcmne, Springfield, 11L ' . Timbreul, V.(1969). Characteristics ofthe InteraaskS Union Against Cancer Standard Reference Samples of Asbestos. In Pneumoconiosis Proc. Ira. ' Shapiro, ed.), pp. 28-36. Johannesburg. Wagner, J. C., Barry, G, Skidmore, J. * Timbreu, V. (1974). The effects of the iahalatioa^f asbestos in rats. Brit. J. Cancer 29, 252-269.'i|2*|j|| Wright, G. W,, and Kuschner; M. (t977jirjpe influence of varying lengths of glass and asbestos fibs on tissue response in guinea pigs, tn Inhaled Partides (W. H. Walton, ed.), Vol. IV, Part 2. pp.;95^fc Peigamon, New York. ....... Yeager, H, Russo, D. a.. Yanez, ML, GerardcSI Nolan, R. p.-, Kagan, E., and Langes, Al-ife (1983). Cytotoxicity ota short-fiber chrysotile ssbtsts for human alveolar macrophages: Preliminary obser vations. Environ. Mes. 30, 224-232. ' Zugibe, F.T. (1970). Diagnostic Histochemistry: Modje' St. Louts. ` . 'S'St ---- J HWBUI0009554