Biopersistence of In haled Organic andInorganic Fibers in the Lungs of RatsDavid B. Warheit, Mark A. Hartsky, Tim A. McHugh, andKristen A. KellarCentral Research and Development, Haskell Laboratory for Toxicology and Industrial Medicine, E . 1. du Pont deNemours and Co., Newark, Delaware

Fiber dimension and durability are recognized as important features in influencing the development of pulmonary carcinogenic and fibrogeniceffects. Using a short-term inhalation bioassay, we have studied pulmonary deposition and clearance patterns and evaluated and compared the pul-monary toxicity of two previously tested reference materials, an inhaled organic fiber, Kevlar para-aramid fibrils, and an inorganic fiber, wollastonite.Rats were exposed for 5 days to aerosols of Kevlar fibrils (900-1344 f/cc; 9-11 mg/m3) or wollastonite fibers (800 f/cc; 115 mg/m3). The lungs ofexposed rats were digested to quantify dose, fiber dimensional changes over time, and clearance kinetics. The results showed that inhaled wollas-tonite fibers were cleared rapidly with a retention half-time of <1 week. Mean fiber lengths decreased from 11 pm to 6 pm over a 1-month period,and fiber diameters increased from 0.5 pm to 1.0 pm in the same time. Fiber clearance studies with Kevlar showed a transient increase in the num-bers of retained fibrils at 1 week postexposure, with rapid clearance of fibers thereafter, and retention half-time of 30 days. A progressive decreasein the mean lengths from 12.5 pm to 7.5 pm and mean diameters from 0.33 pm to 0.23 pm was recorded 6 months after exposure to inhaled Kevlarfibrils. The percentages of fibers >15 pm in length decreased from 30% immediately after exposure to 5% after 6 months; the percentages of fibersin the 4 to 7 pm range increased from 25 to 55% in the same period. These data suggest that both inhaled Kevlar and wollastonite fibers have lowdurability in the lungs of exposed rats, and this may be responsible for the measured differences in toxicity between Kevlar and wollastonite on theone hand, and durable dusts such as silica or crocidolite asbestos fibers on the other. - Environ Health Perspect 102(Suppl 5):151-157 (1994)

Key words: wollastonite, Kevlar, biopersistence, durability, rat lung, lavage, BrdU labeling

IntroductionFiber dimensional characteristics and bio-persistence are two of the most importantfactors in the development of fiber-inducedlung disease. Several studies have shownthat long, thin, durable fibers are moretoxic in vitro and in vivo than short, thinfibers (1). A Syrian hamster embryo (SHE)in vitro cell system was used to comparenormal glass fibers measuring 15 pm with asimilar preparation of milled fibers, 2 pmin length (2). The longer glass fiberswere cytotoxic to SHE cells and increasedthe transforming frequency, but theeffects disappeared after milling. In con-trast, both long and short crocidoliteasbestos fibers were toxic to macrophagesin vitro via an oxidant and iron-dependentmechanism (3).

This paper was presented at the Workshop onBiopersistence of Respirable Synthetic Fibers andMinerals held 7-9 September 1992 in Lyon, France.

This study was supported by Du Pont Fibers.The authors thank L. Stanickyj, D. P. Kelly, andDr. G. Oberdoerster for their advice and technicalexpertise.

Address correspondence to Dr. David B. Warheit,Central Research and Development, HaskellLaboratory for Toxicology and Industrial Medicine,E. I. du Pont de Nemours and Co., Newark, DE19714. Telephone (302) 366-5322. Fax (302)366-5207.

In vivo studies have also demonstrated adependence upon fiber length to inducepulmonary pathological effects. When longand short sample preparations of eitherfiberglass or asbestos were instilled into thelungs of guinea pigs, long fibers of eithertype produced severe pulmonary fibrosis,while the shorter sample preparations pro-duced no significant effects (4). In a seriesof experiments using inhalation exposures,rats were exposed for 1 year to aerosols ofspecially prepared "short" amosite orchrysotile asbestos fibers <5 pm in length,and the pulmonary fiber-induced effectswere compared with preparations of longamosite or chrysotile asbestos fibers,>20 pm long, at similar gravimetric con-centrations. One-third of rats exposed tothe "long" amosite fibers developed pul-monary tumors or mesotheliomas, and vir-tually all animals had pulmonary fibrosis.In contrast, the shorter fiber-types pro-duced no significant pulmonary effects(5). Similar results were reported in thechrysotile study. Following a 1-year inhala-tion exposure, the long-fiber chrysotileproduced a 3-fold increase in the numbersof pulmonary tumors and six times moreadvanced interstitial fibrosis, compared tothe effects produced by the shorter fibers(6). In contrast to these results, cytotoxiceffects were observed following repeated

injections of short crocidolite fibers intothe peritoneal cavities of mice, whenthe clearance of these fiber-types wasprevented (3).

Fiber clearance studies following short-term exposures either to inhaled chrysotileor crocidolite asbestos fibers in rats, havebeen reported, in which fibers were recov-ered from digested lung tissue and analyzedfor dimensional changes at several postex-posure times. Following exposure to croci-dolite, there was a progressive increase inmean fiber length over time postexposure,but no significant change in the meandiameter of fibers retained in the lung (7).In the chrysotile-exposed rats, there was asimilar progressive increase in mean fiberlength but a significant reduction in meanfiber diameter (8). It appeared that thelonger fibers of chrysotile and crocidolitewere selectively retained in the lungs ofexposed rats, whereas longitudinal splitting,with a corresponding decrease in meanfiber diameter, occurred only with thechrysotile asbestos fibers. These resultshave been supported by the results of a 2-year intratracheal instillation/fiber clear-ance study, wherein the lung clearance ofshort crocidolite fibers was slow and thenumbers of crocidolite fibers longer than5 pm did not decrease over a period of oneyear. In contrast, the numbers of retained

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Table 1. Kevla r and wollastonite fiber exposure data and inhaled dose.

Duration of MMAD (8g), Mean gravimetric Mean fiber dose, Number of retained Count median Count medianFiber type exposure, days um concentration, mg/m3 f/cc fibers, f/g DLTa length, pm diameter, pm

Wollastonite 5 4.3 (2.2) 59 123 ND ND ND5 2.6 (2.0) 114 835 1.3 x 105 ND ND

KevlarExperiment lb

5 4.5 (2.7) 4.4 1073 3.16 xl06 9.9 0.35 3.4(2.7) 8.5 1344 3.5x 106 9.9 0.3

Experiment 2c5 3.2(2.9) 2.9 613 1.4x106 10.0 0.35 4.7 (3.2) 11.1 877 1.3 x 106 10.0 0.3

abAbbreviations: MMAD, mass median aerodynamic diameter; bg, standard geometric deviation; ND, not determined. Number of retained fibers/gram dry lung tissue. b Used for BAL,cell-labeling and fiber clearance/retention studies. c Used for fiber clearance/retention studies.

chrysotile fibers >5 jim in length increasedcontinuously over a 2-year period, due pri-marily to longitudinal splitting of thefibers.

The current studies were developed toelucidate pulmonary clearance patterns andto evaluate the pulmonary toxicity of a

selected organic fiber, Kevlar para-aramid,and wollastonite, an inorganic fiber, rela-tive to other reference materials, using ashort-term inhalation bioassay. The resultsindicate that the low durability of thesefiber types may be responsible for the

Table 2. Pulmonary inflammation and cell labeling responses in Kevlar and wollastonite-exposed rats.

Time after 5-day exposure 0 hr 24 hr 1 week 1 month 3 monthsNumbers of lavaged granulocytesControl 8.0x103 7.8x103 7.0x103 7.3x103 8.6x103Wollastonite 1.3 x105 9.2 x1048 3.0 x 106a 3.0 x 104 1.2 x 104Kevlar 4.1 x105" 4.4x 10Oa 3.3x 1058 2.6x 104 8.7x 103

Pulmonary cell labeling: % of labeled terminal bronchiolar cellsControl 1.0 ± 0.1 0.9 ± 0.03Wollastonite 1.4± 0.1 0.9 ± 0.03Control 0.8±0.2 0.9± 0.1 1.1 ±0.1Kevlar 2.3 ± 0.4a 1.3 ± 0.1 1.2 ± 0.1

Pulmonary cell labeling: % of labeled pulmonary parenchymal cellsControl 1.0 ± 0.1 0.8 ± 0.1Wollastonite 1.1 ± 0.1 0.8 ± 0.02Control 1.0 ± 0.1 1.1 ± 0.2 1.1 ± 0.1Kevlar 1.4 ± 0.2 1.1 ± 0.6 1.0 +-.

,not determined. ap< 0.05.

Table 3. Kevlar and wollastonite fiber dimensional and lung clearance data.

Calculated massTime after Mean length, Mean diameter, concentration, pg/g

5-day exposure Fiber type pm pm lung tissue

Ohr Kevlar 12.5 ± 2 0.33 ± 0.03 21.4Wollastonite 11.2 ± 3.9 0.53 ± 0.32 63.5

72hr Kevlar 11.1 ± 5 0.30 ± 0.06Wollastonite

1 week KevlarWollastonite 6.9 ± 2.5 0.68 ± 0.35 20.5

1 month Kevlar 9.0 ± 2 0.295 ± 0.04 10.1Wollastonite 6.0 ± 1.2 0.95 ± 0.3 4.9

3 months Kevlar 8.1 ± 2 0.27 ± 0.09 5.6Wollastonite

6 months Kevlar 7.45± 3 0.23 ± 0.02 2.7Wollastonite

,not determined.

observed transient pulmonary inflamma-tory effects.

Materials and MethodsGeneral Experimental DesignGroups of 8-week-old male Crl:CD BRrats (Charles River Breeding Laboratories,Kingston, NY) were exposed 6 hr/day for 5days to aerosols of Kevlar in concentrationsranging from 877 to 1344 f/cc (9-11mg/m3), or to wollastonite fibers at 835f/cc (114 mg/m3). Following exposure,groups of 3 to 4 animals and aged-matchedcontrols were evaluated at 0, 24, or 72 hr,1 week, 1, 3, or 6 months postexposure.Additional groups of three to four ani-mals/time point were used for lung diges-tion studies.

Fiber PreparationsUltrafine respirable-sized Kevlar para-aramid fibrils (DuPont Fibers, E. I. duPont de Nemours, Willmington, DE), pre-pared for a 2-year inhalation study (11),were utilized for this study. A preparationof Wollastonite NYAD-G fibers (NYCO,Willsboro, NY), measured using scanningelectron microscopy (SEM), had diametersranging from 0.2 to 3.0 pm.Inhalation EFxposureDust generation techniques for Kevlar andwollastonite exposures have been describedin detail elsewhere (12,13). Atmospheresof Kevlar fibrils or wollastonite fibers weregenerated with a K-tron bin feeder (K-tronCo., Glasboro, NJ) equipped with twinscrews. Baffles were inserted into the gen-eration apparatus and increased the res-pirability of the sample. Fibrils or fiberswere metered into a plastic funnel con-nected to a cyclone where high-pressure airtransferred the test material into a microjetapparatus. Chamber concentrations were

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maintained by controlling the dust-feedrate into the generation apparatus or byvarying the air-flow rate. The methods fordetermining gravimetric concentrations,particle/fiber size and fiber numbers havebeen previously described (12,13).

0F

SHAM

C0NTR0L

Pulmonary LavageBronchoalveolar lavage procedures (14)were repeated five times or until 50 ml offluid was collected. Lavage fluids recoveredfrom control and dust-exposed rats werecentrifuged at 250g, and the supernatant

---O- SHAM--o- ALKALINE

PHOSPHATASE-------A LACTATE

DEHYDROGENASE--V-- PROTEIN

VALUES--o-- N-ACETYL

GLUCOSAMINIDASE

f1 3b 90(1WK) (1MO) (3MO)

DAYS AFTER 5-DAY EXPOSURE

Figure 1. Pulmonary biomarkers in lavage fluids of Kevlar-exposed rats. Alkaline phosphatase, protein, lactate dehydro-genase (LDH), and N-acetyl-p-glucosaminidase (NAG) values in BAL fluids of rats exposed to Kevlar fibrils. Values givenare expressed as percent response (± SD) of corresponding control values. Transient increases in BAL protein, alkalinephosphatases, NAG, and LDH levels were measured through 1 week following 5-day exposure to Kevlar fibrils, but werenot different from controls after 1 week of recovery.

1

0F

SHAM

C0NTR0L

-0--O-0--O

.......

-V-

SHAMALKALINEPHOSPHATASEPROTEINVALUESLACTATEDEHYDROGENASE

(1WK) (iMO) (3MO)DAYS AFTER 5-DAY EXPOSURE

Figure 2 Pulmonary biomarkers in lavage fluids of wollastonite-exposed rats. Alkaline phosphatase, protein, and lactatedehydrogenase values in BAL fluids of rats exposed to wollastonite fibers. Values given are expressed as percent response(± SD) of corresponding control values. Transient increases in BAL protein and LDH levels were measured through 1 weekfollowing 5-day exposure to wollastonite fibers, but were not different from controls after 1 week of recovery.

was removed and concentrated for bio-chemical studies. The cell pellet was resus-pended in Eagles Minimal EssentialMedium (Eagles MEM F-I1; pH 7.2,GIBCO, Grand Island, NY) supplementedwith penicillin and streptomycin. Themethods of quantitation of cell numbers,viabilities and differential counts have beenpreviously described (15).

Biochemical Assays onBronchoalveolar Lavage FluidLavage fluids from the first two washeswere centrifuged and the supernatant con-centrated 10-fold in an Amicon concentra-tor (cut-off mw=10,000). All biochemicalassays were performed on concentratedbronchoalveolar lavage (BAL) fluids at30°C, using a semiautomated clinicalchemical analyzer (Encore II, BakerInstruments, Allentown PA). Lactate dehy-drogenase (LDH), alkaline phosphatase(ALP), and N-acetyl-13-glucosaminidase(NAG) were measured using commerciallyavailable reagent kits (Baker Instruments,Allentown, PA, for LDH and ALP;Boehringer Mannheim Biochemica,Indianapolis, IN, for NAG). Lavage fluidprotein was measured using a commerciallyavailable reagent kit based on CoomassieBlue dye binding (QuanTtest, Quanti-metrix, Hawthorne, CA). Statistical meth-ods have been previously reported (15).For statistical analysis of the biochemicaldata, a one-way analysis of variance(ANOVA) and Bartlett's test were calcu-lated for each sampling time. Statisticalanalyses for fiber dimensions and 5-bromo-2'deoxyuridine (BrdU) cell labeling werecarried out using Student's t-test.

Pulmonary Celi-Laheling StudiesBrdU cell labeling (12) was designed tomeasure the effects of Kevlar or wollas-tonite fiber inhalation on airway and lungparenchymal cell turnover in exposed rats.Groups of Kevlar fiber-exposed rats andcontrols were pulsed immediately afterexposure and one week or one month laterwith an intraperitoneal injection of BrdUdissolved in a 0.5 N sodium bicarbonatebuffer solution at a dose of 100 mg/kgbody weight. Groups of wollastonite-exposed rats and controls were similarlypulsed. The animals were sacrificed 2 hrlater by pentobarbital injection. It has beenshown that the single intraperitoneal pulseleads to efficient and sufficient labelingin the peripheral lung to differentiatesignificant differences between exposuregroups (12).

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NO. FIBERSPER GRAM 2X10 ----- 613 fibers/cc

-0--- 877 fibers/cc

(1M) (3M)RECOVERY PERIOD AFTER 5-DAY EXPO

were counted. Three animals per Kevlarexposure group/time period and four perwollastonite group/time period were mea-sured in this manner and recorded. Fiberdimensional analysis was carried out byrandom selection by SEM of fibers >4 pmin length. Fiber mass concentrations werecalculated using the formula of mass =volume x density.ResultsThe Kevlar and wollastonite studies werecarried out at several exposure concentra-tions and for several time periods. Only thedata from the 5-day, high-exposure con-centrations will be reported here.

Chamber Atmosphere Analysis24w The exposure generation data for

aerosolized Kevlar fibrils and wollastoniteISURE fibers are summarized in Table 1.

Figure 3. Clearance of fibers from Kevlar-exposed rat lungs following a 5-day exposure. Values given are mean numbersof fibers/gram of lung tissue ± SD. Clearance studies demonstrated a transient increase in the numbers of retainedKevlar fibrils at 1 week postexposure, with rapid clearance of fibers thereafter. The lung retention half-time was 30 days(Table 3). From Warheit et al. (12); reprinted with permission.

Recovery ofKevlar or WollastoniteFibers from Digested Lung ThssueTo recover Kevlar fibers from the lungs ofexposed rats, the fixed lung tissue wasdigested in an 11% KOH solution (inethanol and water; pH>14) and pouredinto a grinding tube. Wollastonite-exposedlungs were digested with a 5.25%hypochlorite (bleach) solution (pH 10.6).All samples were then ground and incu-bated in a shaking water bath at 600C for 4hr. Following incubation, the samples weresonicated, vacuum-filtered onto Milliporefilters (pore size = 0.20 pm or 0.45 pm),and placed in an oven overnight. The fil-

ters were then mounted and preparedeither for phase contrast light microscopy(PCOM: for counting), or for scanningelectron microscopy (SEM: for fiberdimensional analysis). It has been demon-strated that these techniques did not affectthe dimensions of the retained fibers(16,17).

The numbers of fibers/area of filterwere counted by PCOM using theNational Institutes for Occupational Safetyand Health (NIOSH) 7400B countingmethod (NIOSH Manual of AnalyticalMethods). Only fibers with an aspect ratioof 3:1 (length:width), and >5 pm in length

Analyses of Celiular Constituents inBALExposures to Kevlar fibrils did not alter thetotal numbers of cells recovered by lunglavage; increased numbers of cells wererecovered 1 week post exposure from thelungs of wollastonite-exposed rats but thenumber of cells returned to control levelsafter one month. The viability of cellsrecovered in exposed or control rats wasgreater than 94% at all time periods.

Cell differential analyses of lavaged cellsrecovered from the lungs of exposed ratsdemonstrated that fiber inhalation pro-duced an early but transient pulmonaryinflammatory response, as evidenced byincreased numbers of neutrophils in BALfluids (Table 2). This effect was measuredthrough a 1-week postexposure period butwas short-lived, since neutrophil numbers

a

Figure 4. Scanning electron micrographs of Kevlar fibrils (arrows) recovered from the digested lungs of an exposed rat: (A), immediately after a 5-day exposure; (B), 3-months postex-posure. Rat lungs were digested with a KOH solution.

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10^

NUMBER OF

FIBERS /GRAM 10 ^5--\OF LUNGTISSUE

103- I\

10^1- *IWK iMO

TIME AFTER 5-DAY EXPOSURE

Figure 5. Clearance of fibers from wollastonite-exposed rat lungs following a 5-day exposure. Values given are meannumbers of fibers/gram of lung tissue ± SD. Clearance studies demonstrated a rapid decrease in the numbers of retainedfibers postexposure, with an estimated clearance half-time of 1 to 2 weeks.

were not significantly different fromcontrol values after 1 month or at anylater time.

Enzyme and Protein Analyses inBAL FluiidsTransient increases in BAL LDH, protein,alkaline phosphatase and NAG values weremeasured in the rats exposed to fibers for 5days. However, no significant increases in

these parameters were measured after oneweek (Figures 1,2).

Cell-labeling Studies andHistopathogyNo significant differences in the labelingindex of lung parenchymal cells weredetected between Kevlar or wollastonite-exposed rats and their corresponding con-trols at any time period (Table 2).

Increased BrdU-labeling of terminal bron-chiolar cells in Kevlar-exposed rats wasmeasured immediately after exposure.However, no significant differences wereobserved later, indicating that this effectwas transient. Histopathologic analysis at 3months postexposure indicated that neitherfiber produced pulmonary lesions.

Fiber Clearance StudiesClearance studies demonstrated a transientincrease in the numbers of retained Kevlarfibrils at 1 week postexposure, with rapidclearance of fibers thereafter (Figure 3).The retention half-time was <30 days(Table 3). Mean fiber lengths and diame-ters decreased progressively with time overa 6-month postexposure period. Meanfiber lengths decreased from 12.5 pm to7.5 pm and mean fiber diameters, from0.33 pm to 0.23 pm (Table 3) (Figure4A,B). Wollastonite fibers were clearedrapidly from the lungs of exposed animalswith a retention half-time of <1 week(Figure 5). Mean fiber lengths decreasedfrom 11 pm to 6 pm over a 1-monthperiod, but mean fiber diameters increasedfrom 0.5 to 1.0 pm (Table 3) (Figure6A,B).

DiscussionThe finding of increased numbers ofretained fibers at 1 week postexposure inKevlar-exposed rats may relate to fibershortening during the first week after expo-sure. Mean fiber lengths recovered fromdigested lung tissue decreased from12.5 pm to 7.5 pm, over a 6-month postexposure period, and mean fiber diameters,

Figure 6. Scanning electron micrographs of wollastonite fibers (arrows) recovered from the digested lungs of an exposed rat: (A), immediately after a 5-day exposure; (B), 1 monthpostexposure. Rat lungs were digested with a hypochlorite solution.

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from 0.33 pm to 0.23 pm. These resultssuggest that Keviar fibrils are shortened inthe lungs of exposed rats, indicating a dif-ferent pulmonary clearance mechanismfrom that associated with either chrysotileor crocidolite asbestos; in both of thesefiber-types the mean lengths of inhaledfibers was progressively increased (7,8).

The wollastonite clearance data pre-sented here confirm the recently reportedresults of studies using intratracheal instil-lation methods of exposure (18). In thatstudy, the durability of instilled wollas-tonite, crocidolite asbestos, and glass fiberswere evaluated in lung tissue from exposedrats. The retention half-times for three wol-lastonite samples were 10, 1 1, and 12 days.The retention half-times for different glassfiber samples ranged from 38 to 238 days;and the clearance of crocidolite wasinsignificant, with a half-time rate of 1000days. When a correlation between durabil-ity of fibers in the lung and carcinogenicpotency was sought in the intraperitonealtest, the data supported the hypothesis thatlong, thin, and durable fibers are capable ofinducing tumors.

Wollastonite fibers are composed pri-marily of calcium silicates, which are solu-ble in lung fluids and within cells.Conceivably, the thinner calcium-contain-ing wollastonite fibers were quickly solubi-lized and cleared following inhalation,while the thicker wollastonite "stumps"(Figure 5B) were more difficult to clearfrom the lung.

Fiber dimension and durability gener-ally have been recognized as important fea-tures in influencing the development ofcarcinogenic and fibrogenic effects in thelungs of exposed animals. Stanton et al.(19) have proposed that fibers >10 pm inlength and <0.25 pm in diameter have thegreatest potential for producing lungtumors. Similar conclusions can be drawnfrom recent studies with silicon carbidewhiskers and fibers (20,21). Inhalation ofthese durable materials produces severepulmonary fibrosis and lung tumors, whilenonfibrous silicon carbide particles areregarded merely as nuisance dusts (22).A short-term inhalation bioassay has

been developed to assess the potential forinhaled particles or fibers to produce pul-

monary fibrosis. This screen has utilized aseries of biomarkers that may predict theprogression of fiber-induced pulmonaryinjury following chronic exposures. In pre-vious studies, the efficacy of this short-terminhalation screen was tested by exposingrats to several concentrations of various ref-erence materials, including known fibro-genic dusts such as ao-quartz silica (15) andcrocidolite asbestos (13), as well as materi-als with minimal or moderate biologicalactivity such as titanium dioxide, carbonyliron particles (15), or carbon fibers (23).Short-term exposure of rats to silica or cro-cidolite asbestos produced sustained pul-monary inflammatory responses. Incontrast, exposures to wollastonite orKevlar fibrils, which have low biopersis-tence, produced only transient inflamma-tory effects. In summary, short-terminhalation of durable fibers produces sus-tained pulmonary inflammatory effectsalong with consistently elevated indicatorsof cytotoxicity and consequent pulmonarylesions, while only transitory effects areproduced by fibers of low biopersistence.

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2. Hesterberg TW, Barrett JC. Dependence of asbestos and mineraldust-induced transformation of mammalian cells in culture on fiberdimension. Cancer Res 44:2170-2180 (1984).

3. Goodglick LA, Kane AB. Cytotoxicity of long and short crocidoliteasbestos fibers in vitro and in vivo. Cancer Res 50:5153-5163(1990).

4. Wright G, Kuschner M. The influence of varying lengths of glassand asbestos fibers on tissue responses in guinea pigs. In: InhaledParticles IV, Part 2 (Walton WH, ed). Oxford:Pergamon Press,1977; 455-474.

5. Davis JMG, Addison J, Bolton RE, Donaldson K, Jones AD, SmithT. The pathogenicity of long versus short fibre samples of amositeasbestos administered to rats by inhalation or intraperitoneal injec-tion. BrJ Exp Pathol 67:415-430 (1986).

6. Davis JMG, Jones AD. Comparisons of the pathogenicity of longand short fibres of chrysoti[e asbestos in rats. Br J Exp Pathol69:717-737 (1988).

7. Roggli VL, George MH, Brody AR. Clearance and dimensionalchanges of crocidolite asbestos fibers isolated from lungs of rats fol-lowing short-term exposure. Environ Res 42:94-105 (1987).

8. Roggli VL, Brody AR. Changes in numbers and dimensions ofchrysotile asbestos fibers in lungs of rats following short-term expo-sure. Exp Lung Res 7:133-147 (1984).

9. Bellmann B, Konig H, Muhle H, Pott F. Chemical durability ofasbestos and of man-made mineral fibres in vivo. J Aerosol Sci17:341-345 (1986).

10. Bellmann B, Muhle H, Pott F, Konig H, Kloppel H, Spurny K.Persistence of man-made mineral fibers (MMMF) and asbestos inrat lungs. Ann Occup Hyg 31(4B):693-709 (1987).

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Evidence for biodegradability of inhaled fibrils. Toxicol ApplPharmacol 116:225-239 (1992).

13. Warheit DB, Moore KA, Carakostas MC, Hartsky MA. Acute pul-monary effects of inhaled wollastonite fibers are dependent on fiberdimensions and aerosol concentrations. In: Mechanisms in FibreCarcinogenesis (Brown R, Hoskins J, Johnson N, eds). NATO ASISeries A: Life Sciences, Vol 223, New York:Plenum Press,1991; 143-156.

14. Warheit DB, Chang LY, Hill LH, Hook GER, Crapo JD, BrodyAR. Pulmonary macrophage accumulation and asbestos-inducedlesions at sites of fiber deposition. Am Rev Respir Dis 129:301-3 10(1984).

15. Warheit DB, Carakostas MC, Hartsky MA, Hansen JF.Development of a short-term inhalation bioassay to assess pul-monary toxicity of inhaled particles: Comparisons of pulmonaryresponses to carbonyl iron and silica. Toxicol Appl Pharmacol107:350-368 (1991).

16. Kelly DP, Williams SJ, Kennedy GL Jr, Lee KP. Recovery andcharacterization of lung-deposited Kevlar aramid fibers in rats.Toxicologist 5:129 (1985).

17. Warheit DB, Hwang HC, Achinko L. Assessments of lung diges-tion methods for recovery of fibers. Environ Res 54:183-193(1991).

18. Muhle H, Bellmann B, Pott F. Durability of various mineral fibresin rat lungs. In: Mechanisms in Fibre Carcinogenesis (Brown R,Hoskins J, Johnson N, eds). NATO ASI Series A: Life Sciences,Vol 223, New York:Plenium Press, 1991;181-187.

19. Stanton MF, Layard M, Tegeris A, Miller E, May M, Morgan E,Smith A. Relation of particle dimension to carcinogenicity inamphibole asbestos and other fibrous minerals. J Natl Cancer Inst67:965-975 (1981).

20. Birchall JD, Stanley DR, Mockford MJ. Toxicity of silicon carbidewhiskers. J Mater Sci Lett 7:350-352 (1988).

21. Bogoroch R, Luck SR. Workplace handling requirements and pro-cedures for ACMC silicon carbide whiskers based on a subchronicinhalation study in rats. In: International Conference on Whiskersand Fiber-Toughened Ceramics, 7 June 1988, Oak Ridge, TN.

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22. American Conference of Industrial Hygienists. Threshold LimitValues and Biological Exposure Indices for 1988-1989, ACIH.

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tal carbon fiber: pulmonary biochemical and cellular effects.Proceedings of the VII International Symposium on InhaledParticles, September 1991, Edinburgh, Scotland. Ann Occup Hyg(in press).

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