Chronic Inhalation Study of Fiber Glassand Amosite Asbestos in Hamsters:Twelve-month Preliminary ResultsT.W. Hesterberg,l C. Axten,2 E.E. McConnell,3G. Oberdorster,4 J. Everitt,5 W.C. Miiller,1 J. Chevalier,6G.R. Chase,1 and P. Thevenaz71Schuller International, Inc., Littleton, Colorado; 2North AmericanInsulation Manufacturers Association, Alexandria, Virginia; 3Consultant,Raleigh, North Carolina; 4University of Rochester, Rochester, New York;5Chemical Industry Institute of Toxicology, Research Triangle Park, NorthCarolina; 6Experimental Pathology Services, Muttenz, Switzerland;7Research and Consulting Company, Fullinsdorf, Switzerland

The effects of chronic inhalation of glass fibers and amosite asbestos are currently under study inhamsters. The study includes 18 months of inhalation exposure followed by lifetime recovery.Syrian golden hamsters are exposed, nose only, for 6 hr/day, 5 days/week to size-selected testfibers: MMVF10a (Schuller 901 insulation glass); MMVF33 (Schuller 475 durable glass); amositeasbestos (three doses); or to filtered air (controls). Here we report interim results on airborne fibercharacterization, lung fiber burden, and pathology (preliminary) through 12 months. Aerosolizedtest fibers averaged 15 to 20 pm in length and 0.5 to 1 pm in diameter. Target aerosolconcentrations of World Health Organization (WHO) fibers (longer than 5 pm) were 250 fibers/ccfor MMVF10a and MMVF33, and 25, 125, or 250 fibers/cc for amosite. WHO fiber lung burdensshowed time-dependent and (for amosite) dose-dependent increases. After a 12-month exposure,lung burdens of fibers longer than 20 pm were greatest with amosite high and mid doses, similarfor low-dose amosite and MMVF33, and smaller for MMVF10a. Biological responses of animalsexposed for 12 months to MMVF10a were limited to nonspecific pulmonary inflammation.However, exposures to MMVF33 and each of three doses of amosite were associated with lungfibrosis and possible mesotheliomas (1 with MMVF33 and 2, 3, and 1 with amosite low, mid, andhigh doses, respectively). Pulmonary and pleural changes associated with amosite werequalitatively and quantitatively more severe than those associated with MMVF33. As of the 12-month time point, this study demonstrates that two different fiber glass compositions with similarfiber dimensions but different durabilities can have distinctly different effects on the hamster lungand pleura after inhalation exposure. (Preliminary tumor data through 18 months of exposure and 6weeks of postexposure recovery became available as this manuscript went to press: No tumorswere observed in the control or MMVF1 Ca groups, and no additional tumors were observed in theMMVF33 group; however, a number of additional mesotheliomas were observed in the amositegroups.) - Environ Health Perspect 1 05(Suppl 5):1223-1229 (1997)

Key words: fiber glass, amosite, asbestos, hamster, inhalation

IntroductionSynthetic vitreous fibers (SVF) are a class rock/slag wool, and refractory ceramicof inorganic fibrous materials made pri- fibers (RCF). Recently, a series of chronicmarily from rock, clay, slag, or glass. The inhalation studies was conducted to evaluatethree major classes of SVF are fiber glass, the biological effects in rodents of fibers

This paper is based on a presentation at The Sixth International Meeting on the Toxicology of Natural and Man-Made Fibrous and Non-Fibrous Particles held 15-18 September1996 in Lake Placid, New York. Manuscriptreceived at EHP26 March 1997; accepted 1 April 1997.

The authors gratefully acknowledge the invaluable contributions of the following people: H. Fleissner forhamster studies; B. Whitehead, A. Neuerberg, P. Wilson, and M. McPherson for aerosol and lung fiber analy-ses; and G. Hart for assistance in writing the manuscript. This research was funded by the North AmericanInsulation Manufacturers Association (NAIMA).

Address correspondence to Dr. T.W. Hesterberg, Health, Safety and Environmental Department, JohnsManville International, Inc., P.O. Box 625005, Littleton, CO 80162-5005. Telephone: (303) 978-3831. Fax: (303)978-2358. E-mail: hesterbe@jm.com

Abbreviations used: RCF, refractory ceramic fiber(s); SVF, synthetic vitreous fiber(s); WHO, World HealthOrganization.

from each of the major classes of SVF andof two forms of asbestos. Results from theseand previous studies suggest that the rat isa useful laboratory model for assessing bothfibrotic and tumorigenic properties ofairborne fibers (1-3). The maximum SVFexposure concentration used in these studieswas 30 mg/m3 air (approximately 200-300fibers/cc longer than 5 pm). In the rat, thisconcentration of RCF was associated witha significant increase in lung and pleuraltumors, but similar concentrations of insu-lation fiber glass or rock/slag wool were notassociated with an increase in tumors.Lower RCF concentrations did not inducefibrosis or elevate tumor incidence in rats.

The hamster model was also used in aprevious study in this series, in which theinhalation effects of a refractory ceramicfiber (RCF1) and chrysotile asbestos wereevaluated (2). While both RCF1 (at 30mg/m3, 215 fibers/cc longer than 5 pm)and chrysotile (at 10 mg/m , 3000 fibers/cclonger than 5 pm) induced fibrosis, neitherfiber induced any lung tumors. However,RCF1, but not chrysotile, induced pleuralmesotheliomas [in 42 of 112 hamsters (4)].

In the present hamster inhalation study,hamsters are exposed to SVF at an averageaerosol concentration of 30 to 37 mg/m3(approximately 250 fibers/cc longer than 5pm). This concentration was chosen as themaximum exposure based on results from aprevious 13-week range-finder study thatestimated that this exposure would be themaximum tolerated dose for hamsters (5).The present paper reports interim results onairborne fiber characterization, lung fiberburden, and pathology (preliminary)through 52 weeks of exposure. A later paperwill present findings from other assays andfrom the remaining time points.

Materials and MethodsExperimental Design

Syrian golden hamsters (125-140 males perexposure group, 13-15 weeks of age at onsetof exposure [Charles River Laboratories,Quebec, Canada]) are exposed to test fiberaerosol or to filtered air in nose-onlyinhalation chambers, 6 hr/day, 5 days/week, for 18 months. The original protocoldesignates five time points (after 3, 6, 12,and 18 months of exposure and after a post-exposure recovery period to be terminatedat 10-20% survival) at which hamstersfrom each of the six exposure groups are tobe euthanized to evaluate pulmonary and

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pleural responses and lung burden. Herewe report interim findings through12 months of exposure.

Because of increased mortality (see"Results"), the planned 26-week (6-month)sacrifice was not conducted; however, lungburdens and pathology were analyzed infive animals per exposure group that diedspontaneously or were euthanized inextremis after 26 ± 1 week of exposure.

Test FibersThe present study evaluates the chronicinhalation effects of three fiber composi-tions, each of which was size-selected to besimilar to the dimensions of fibers in work-place air (6) and to be rodent respirable(Table 1). MMVF1Oa was derived fromSchuller 901, which is representative ofcommercial insulation glass and is compara-ble in composition to the MMVF10 testfiber used in previous rat inhalation studies(1). MMVF33 was derived from Schuller475 glass, one of the more durable glassfibers used commercially. Because 475 glassis used in high efficiency air filtration prod-ucts (HEPA filters), it has been engineeredto have greater environmental durability,including resistance to high humidity, thaninsulation glass. The 475 glass differs from901 glass in composition as well as in man-ufacturing process: 475 glass contains zincand barium for greater durability, and it ismanufactured using a flame attenuationprocess. The 901 insulation wool fromwhich MMVFlOa was derived is not flameattenuated and does not include zinc orbarium in its formulation.

Amosite asbestos was included in thisstudy because it is a known human car-cinogen and because a test fiber was avail-able that is longer and thicker than mostother asbestos test fibers. After size-selectionfor the longer fibers, the amosite testfiber dimensions were comparable to the

dimensions of the SVF (Table 1). A primarygoal was to have dimensions for the threetest fibers be as similar to each other as pos-sible to achieve comparable lung dosingand thus focus on any possible pathogenicdifferences due to fiber composition orsurface reactivity.

Fiber Aerosol xposureThe study included an air control group(exposed to filtered air) and five fiber expo-sure groups: MMVF1Oa and MMVF33(both at target concentrations of 250 WorldHealth Organization (WHO) fibers/cc) andthree doses of amosite asbestos (25, 125,and 250 WHO fibers/cc, respectively)(Table 1). WHO fibers are defined as hav-ing a length/diameter ratio > 3, diameter< 3 pm and length > 5 pm (7). Nose-onlyinhalation exposure was conducted accord-ing to the method of Bernstein et al. (8).Techniques for nondestructive aerosoliza-tion and aerosol monitoring have beendescribed previously (9).

Aerosol samples were collected on filtersplaced in animal exposure ports for 5 hr.Fiber mass concentrations (mg/m3) weredetermined for each fiber aerosol once perexposure day throughout the study. Theconcentrations of fibers per cubic centimeterand their bivariate dimensions were deter-mined at least once every 2 weeks usingscanning electron microscopy described byHesterberg et al. (10). Counting proce-dures were conducted according to WHOMonograph 4 counting rules (7) modifiedfor use with electron microscopy.

Lung Burden AnalysisTo allow upper airway clearance, animalswere euthanized for histopathological andlung burden analyses 48 hr after the lastexposure. Thus, the measured lung fiberburdens are assumed to reflect fibers thatwere essentially deposited in the alveolar

region. At necropsy, the left lung (withoutthe bronchi) of each animal was removed,stored frozen, dried, ashed by a low temper-ature method, and suspended in water. Thelung fibers were recovered from the suspen-sion by filtration and analyzed using elec-tron microscopy according to proceduresdescribed by Hesterberg et al. (10).PathologHamsters were observed daily for clinicalsigns, morbidity, and mortality throughoutthe study. Euthanasia was performed usingpentobarbital and exsanguination asdescribed by McConnell et al. (11). Eachhamster was necropsied upon death. Thelungs were removed, weighed, and exam-ined under a dissecting microscope. Theright lung and the diaphragm were pre-pared for histopathological examinationaccording to the method of McConnell etal. (2) and graded for inflammatory change,fibrosis, and neoplasms by the study pathol-ogist (11). This paper focuses on thepathology observed at three time points(13, 26, and 52 weeks) and provides pre-liminary results of the study pathologist. Atthe termination of the study, a panel ofpathologists will independently review thepulmonary and pleural lesions. After discus-sions with this panel, the study pathologistwill then determine the final diagnoses.

ResultsTest Aerosols

Because of differences in fiber dimensionsand in specific gravity between the threetest fibers, the mass concentration targetsfor MMVF1Oa (30 mg/m3), MMVF33(37 mg/m3), and high dose amosite(7.5 mg/m3) were set at different levels inan attempt to achieve similar aerosol con-centrations ofWHO fibers (fibers longerthan 5 pm). TheWHO fiber concentration

Table 1. Fiber concentrations and dimensions, averages through 12 months.

Fiber dimensionsAerosol concentrationsa Aerosol, AMa Aerosol, GMb Lung, GMb

Test fibers Mass, mg/m3 WHOC fibers/cc Fibers >20 pm/ccd Diameter, pm Length, pm Diameter, pm Length, pm Diameter, pm Length, pmInsulation glassMMVF1 Oa 30 ± 2 323 ± 57 151 ± 22 0.95 ± 0.46 19.5 ± 20.9 0.85 ± 1.65 12.6 ± 2.4 0.50 ± 1.61 6.4 1.9

Durable glassMMVF33 37 ± 2 283 ± 42 106 ± 20 0.91 ±0.75 17.4 ± 16.6 0.70 ± 2.00 12.2 ± 2.3 0.44 ±1.51 7.6 2.1

Amosite asbestosLow 0.8 ± 0.2 33 ± 23 9 ± 8 0.60 ± 0.24 13.7 ± 17.0 0.56 ± 1.45 8.9 ± 1.7 0.57 ± 1.41 8.9 2.1Mid 3.7 ± 0.6 157 ± 59 37 ± 16 0.58 ± 0.24 12.5 ± 16.0 0.54 ± 1.48 8.1 ± 1.7 0.60 ± 1.38 8.9 2.0High 7.3 ± 1.0 255 ± 89 67 ± 27 0.59 ± 0.24 14.0 ± 17.1 0.55 ± 1.46 9.0 ± 2.0 0.58 ± 1.36 8.4 2.1

aArithmetic mean ± SD. bGeometric mean ± the mean of the SD. CWHO respirable fibers (fibers having an aspect ratio . 3 and length > 5 pm, as defined by the WHO. dFiberslonger than 20 pm.

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of MMVFlOa (323 WHO fibers/cc) wassignificantly greater than that of eitherMMVF33 (283 WHO fibers/cc) or amositehigh dose (255 WHO fibers/cc), but the lat-ter two were not significantly different(Table 1). The concentration of fibers percubic centimeter longer than 20 pm forMMVFlOa (151 fibers/cc > 20 pm) was alsosignificantly greater than that of MMVF33(106 fibers/cc >20 pm), which was signifi-candy greater than that of amosite high dose(67 fibers/cc > 20 pm) (Table 1).

MortalityAn increased mortality rate occurred in allexposure groups during weeks 17 to 26 dueto an infectious disease diagnosed as wettail, a common disease of hamsters. Becausemortality rates during this time were highin air controls and in unexposed sentinelhamsters, the mortality was judged to beunrelated to fiber exposure. Tetracyclinewas administered to all animals at two dif-ferent time points (400 mg tetracycline/literof drinking water for 17 days and 13 days,respectively). After the second tetracyclinetreatment the diet was also modified toinclude higher roughage content (from5.2-7% with compensatory decrease in car-bohydrates). After the 26-week time point,mortality rates returned to levels similar tothose of previous hamster studies.

Body and LungWeightsAverage body weights of the five fiberexposure groups did not differ significandyfrom sham-exposed controls. After 13 and52 weeks of inhalation, lung weights forthe mid and high doses of amosite weresignificantly elevated compared to the aircontrols (Figure 1). In contrast, lungweights for the other fiber exposure groupswere similar to those of air controls.

Lung Fiber BurdenSeveral lung burden findings are noteworthy:a) The average dimensions of MMVFlOaand MMVF33 fibers in the lung aresmaller than in the aerosols, which sug-gested that the larger fibers of the aerosolwere not able to penetrate to the lowerlung (Table 1). However, amosite lungfiber dimensions are similar to aerosoldimensions, probably because the vastmajority of amosite aerosol fibers were inthe respirable range. b) The dimensions ofthe three test fibers in the lung were moresimilar to each other than the dimensionsin the aerosols (Table 1). In contrast to theaerosol fiber dimensions, the average lungfiber dimensions for the three test fibers

2-

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3-

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AAir control

E3 MMVF1Oa* MMVF33* Amosite, low* Amosite, mid* Amosite, high

co

X 200-C)a

=LE 150LOA

L1 100-8*Lr

50

Exposure, weeks

Figure 1. Lung weights of hamsters after 13 and 52weeks of inhalation exposure. Statistically significantdifferences comparing inhalation-exposed rats andcontrols were determined using the two-tailed t-testand were found to be **p<0.01. (Note: Lung weightsafter 26 weeks of exposure were not included becausemost of these animals died spontaneously and werenot exsanguinated; thus, lung weights for this timepoint were affected by variable amounts of residualblood and were not comparable to lung weights of ani-mals euthanized for the 13- and 26-week time points).

did not differ significantly from each other(geometric mean dimensions after 13weeks of exposure). c) Lung burden datashowed both time- and dose-dependence.The number ofWHO fibers per lung forMMVF1Oa and MMVF33 was timedependent from 26 to 52 weeks of inhala-tion (but not from 13-26 weeks) (Figure2A). WHO fiber burdens for amosite wereboth time- and dose-dependent with theexception that the mid and low doses didnot differ statistically from each other atthe 13-week time point. d) WHO fiberlung burdens were similar for the two fiberglasses and low-dose amosite and for RCF1(Figure 2) [RCF1 data is from a previousstudy (2)]. e) The lung burdens of fiberslonger than 20 pm for amosite also tendedto be dose- and time-dependent; in con-trast, the number of long glass fibers/lungtended either to decrease (MMVFl1a) orremain the same (MMVF33) from 13 to52 weeks (Figure 2B). In contrast to thefiber glasses, the long fiber lung burden ofRCF1 (2) showed time-dependent increasesthrough 52 weeks of exposure (Figure 2B).f) Lung fiber retention in the amositehigh-dose group was greater than in theSVF groups even though aerosol concen-trations were comparable. At 52 weeks, theWHO fiber lung burden for amosite highdose was 8-fold greater than for MMVF 1Oa

* Am, highA Am, midA Am, lowo RCF1 ** MMVF33o MMVF1Oa

13 26Exposure weeks

39

B40 1

COC 30-xcma

E 20-

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Exposure weeks

Figure 2. Lung burdens. (A) Fibers longer than 5 pm(WHO fibers). Am, amosite asbestos. *RCF1 data fromMcConnell et al. (2). (B) Fiber longer than 20 pm/lung.Am, amosite asbestos. *RCF1 data from McConnellet al., with permission (2).

and 5-fold greater than for MMVF33(Figure 2B).

HistopathologyAir Controls. Air controls showed nosignificant macroscopic pulmonary changes.Microscopically, a few pulmonary macro-phages were noted scattered randomlythroughout the parenchyma. There was noprogression in severity during the course ofthe study. No changes were observed in thepleura at any time point.MMVFlOa (323 WHO Fibers/cc).

Pulmonary changes seen at 3 months werelimited to a slight to mild excess in thenumber of pulmonary macrophages andthe presence of microgranulomas and a fewmultinucleated giant cells concentrated

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Table 2. Severity of changes in hamster lungs and pleura: average scores.ab

Lung PleuraExposure Macrophage Alveolar Interstitial Mesothelial Fibrosis

Fiber time, weeks infiltration bronchiolization Microgranuloma Giant cells fibrosis Wagner gradeb hyperplasia (collagen deposition)

Air controls 13 0 0 0 0 0 1 0 026 0 0 0 0 0 1 0 0.452 0 0 0 0 0 1 0 0

MMVF1 Oa 13 2.0 0 0.6 0.4 0 2.0 0 026 1.0 0 0.4 0.2 0 2.0 0 0.452 0.6 0.5 1.3 0 0 2.3 0 0

MMVF33 13 2.0 0.8 1.6 2.0 0 2.6 0 026 2.0 0.4 2.0 0 0 3.1 0.2 1.452 2.3 2.0 2.3 2.0 0.8 4.0 0 2.0

Amosite, low 13 2.4 1.4 2.0 2.0 0 3.2 0 0.626 2.4 1.6 2.6 2.0 0 4.0 0.8 2.052 2.8 2.3 3.0 2.0 0.8 4.0 1.5 1.5

Amosite, mid 13 3.0 2.2 3.2 2.0 0 3.6 0.6 1.026 3.0 2.6 3.0 4.0 0.8 4.1 1.6 2.852 3.8 3.0 4.0 4.0 2.3 5.3 1.8 3.3

Amosite, high 13 3.0 2.8 3.6 2.0 0 3.8 1.6 1.626 3.2 3.2 3.4 2.8 1.6 4.3 0.6 2.652 3.8 2.3 4.0 3.0 2.3 6.0 2.8 3.3

'Scoring system for all but Wagner: 0 = normal; 1 = minimal; 2 = mild; 3 = moderate; 4 = marked; 5 = widespread and severe. Scores for five animals/time point/exposurewere averaged. bWagner grades: 1 = normal; 2 = minimal cellular change (macrophage response); 3 = mild cellular change (macrophages and bronchiolization); 4 = minimalfibrosis (restricted to bronchoalveolar junctions); 5 = mild fibrosis (interlobular linking); 6 = moderate fibrosis (early consolidation); 7 = severe fibrosis (marked consolidation);8 = very severe fibrosis (complete obstruction of airways). [McConnell et al. ( 16)]. n = 4-5 hamsters.

near the bronchoalveolar junctions (Table2). There was no progression in severityfrom 3 to 6 months, but after 12 monthsof exposure, the number of macrophageshad increased and clusters of macrophagesappeared. These macrophage clustersappear to be a response to high dosing; inprevious studies, high doses of nonfibrousparticles induced lung overload (12).Many fibers were observed, most of whichwere within these macrophages and micro-granulomas. Overall, the pulmonary changesin most of the hamsters was consistent withWagner grade 2 (Table 2, footnote b). Notreatment-related changes were found inthe pleura through 12 months.

MMlVF33 (283 WHO Fibers/ce). Theonly macroscopic abnormalities noted at thescheduled time points were at 12 months: a2-mm diameter white focus on the lung sur-face of one hamster and variable sized finetranslucent granular patches on the surfaceof the rib cages (only discernible with a dis-secting microscope) of several of the ham-sters. One hamster that died spontaneouslyat 7.5 months showed variably sized whiteflat foci on the surface of the lung, rib cage,and diaphragm, which were subsequentlydiagnosed as mesothelioma.

The primary microscopic lung changeat 3 months was a slight to mild excess ofmacrophages randomly scattered through-out the parenchyma but concentrated inthe bronchoalveolar junctions (Table 2).

Microgranulomas and occasional multi-nucleated giant cells were also seen at thebronchoalveolar junctions. Many fibers andfiber fragments were found within theinflammatory lesions and free in the alveoli.A slight amount of alveolar bronchioliza-tion (change from flat to cuboidal cells) wasnoted in the alveoli adjacent to the terminalbronchiole. The overall severity at 3 monthswas consistent with Wagner grade 2 to 3(Table 2). The inflammatory changes pro-gressed in severity by 6 months of exposureand were accompanied by the presence ofcollagen (fibrosis) in the proximal portionof occasional alveolar ducts and were classi-fied as Wagner grade 4. The lesions hadfurther progressed by 12 months whenmost of the alveolar ducts and the terminalbronchioles showed evidence of mild inter-stitial fibrosis, but the lesions were still clas-sified at Wagner grade 4 because nointerlobular linking was observed.

Pleural changes were observed at 6months; all hamsters exhibited a slightamount of collagen deposition appearing assmall focal accumulations in the pleura justsubjacent to the mesothelial covering. Themesothelial cells overlying these foci attimes showed change to a spherical (nor-mally flat) appearance that was interpretedas hypertrophy. By 12 months, the lesionshad progressed to include microvillous-likehyperplasia of the pleura at the edge ofthe lobes.

AmositeAsbestos (33 WHO Fibers/cc).Three macroscopic abnormalities appearedin this group after 12 months of exposure:failure of the lungs to collapse normallywhen the thorax was opened, pleuralthickening, and irregular lung margins.Microscopically, at 3 months a moderatenumber of neutrophils (not seen with SVF),macrophages, clumps of macrophages,many well-defined microgranulomas (somecontaining fibers) and a few multinucleatedgiant cells were found, primarily in the areaof the bronchoalveolar junctions (Wagnergrade 3, Table 2). In most of the hamstersexposed for 3 months, a minimal to slightamount of bronchiolization was observed, aswell as minor collagen deposition in thewalls of the alveoli subjacent to microgranu-lomas. After 6 months of exposure thelesions had progressed in severity. Theinflammatory response was much moreintense, and pulmonary fibrosis (found in allof the animals), while slight, affected nearlyall of the bronchioles and extended periph-erally along the alveolar duct and adjacentalveoli (Wagner grade 4). Fibers, some quitelong (> 40 pm), were numerous within thelesions and occasionally penetrated thepleura. Occasional fibers had a knobbyappearance comparable to classic asbestosbodies. The lesions had further progressedby 12 months and were more severe thanwith MMVF33, although they were stillinterpreted as Wagner grade 4.

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Table 3. Pleural mesotheliomas in hamsters during 12 months of inhalation exposure.

MesotheliomasHyperplasia-borderline Early to advanced

Test fibers No. at risk mesothelioma* mesotheliomas Incidence, %

Air controls 63 0 0MMVF1 Oa 63 0 0MMVF33 50 0 1 2.0AmositeLow 42 2 0 4.8Mid 43 1 2 7.0High 56 1 0 1.8

*Tentatively diagnosed as early mesotheliomas. Final diagnoses to be made by a panel of pathologists at termina-tion of study approximately April 1997.

Slight pleural collagen deposition similarto that described with MMVF33 wasobserved as early as 3 months. By 6 monthsit had progressed in severity and was accom-panied by mesothelial hypertrophy andhyperplasia. By 12 months pleural fibrosishad become more diffuse and thicker, andmesothelial hypertrophy and hyperplasiawere common features. Similar, but some-what more prominent, changes wereobserved in the pleura lining the rib cageand diaphragm. Two hamsters showedmesothelial hyperplasia with dysplasticchanges that were given the preliminarydiagnosis of early, borderline mesotheliomas(Table 3).

AmositeAsbeswos (157 WHO Fibers/cc).Macroscopic abnormalities were observedin this group after 6 months of exposure:lungs failed to collapse normally when thethorax was opened, and a few small gray-ish-white foci were scattered on the surfaceof the lungs of some hamsters. After 12months these macroscopic abnormalitieswere more severe and were noted in allhamsters. Microscopically, the lung lesionswere qualitatively similar to those of thelow-dose amosite group but more wide-spread and severe. Although the lesionwas certainly more severe than with 25fibers/cc, by definition it was still consis-tent with a Wagner grade 4, which is a lim-itation of this grading system. At 6 monthsof exposure, the overall lesion had pro-gressed in severity, although it was still inthe Wagner grade 4 category. The fibroticlesion had extended peripherally along thealveolar duct and into the adjacent alveoli,almost to the level of the pleura.Numerous fibers could be found inmacrophages, granulomas, and giant cellsat this time. At 12 months the fibroinflam-matory lesions were much more apparentthan at 6 months. Fibers and asbestos bod-ies were abundant within the lesions. Inaddition, interstitial fibrosis had progressed

to the point of interlobular linking, whichis the essential feature for classifying theoverall lesion as Wagner grade 5.

By 3 months, collagen deposition inthe pleura was evident in most hamsters.After 6 months it was more extensive inall hamsters and was accompanied bymesothelial hypertrophy and hyperplasia.By 12 months, the pleural fibrosis hadbecome thicker and more diffuse andmesothelial hypertrophy and hyperplasiawere common features. Again, theselesions were more prominent in the pleuralining the rib cage and diaphragm, wherea fine granular appearance was observed atnecropsy. Occasional multinucleated giantcells, comparable to those observed in thelung, were found in hyperplastic lym-phoid tissue at the costophrenic junction.In two hamsters, small mesotheliomas ofthe papillary type were noted. Additionally,one hamster had mesothelial hyperplasiawith dysplastic changes characteristic ofearly mesothelioma.

AmositeAsbestos (255 WHO Fibei/cc).Macroscopically and microscopically,the lesions in this group were similarto those described for the low- and mid-dose amosite groups through 3 months.(Wagner grade 4). By 6 months, the pul-monary lesions had progressed in severityand appeared comparable to those in themid-dose group. The most apparent differ-ence was that interstitial fibrosis had pro-gressed to a fibrotic linking of the lobulesin a majority of the hamsters, which placedthe lesion in Wagner grade 5 category.After 6 and 12 months of exposure, thelungs failed to collapse and appeared evenmore stiff than in the mid-dose group.Focal (2-6 mm diameter) reddish solidareas resembling liver (hepatization) wereseen in all of the lungs. Microscopically, thefibroinflammatory lesion had progressedfurther and the lungs were severelycompromised, even more so than in the

mid-dose group. Fibers, as well as asbestosbodies, were again commonly observedwithin the lesions. Areas of consolidationwere a prominent feature in most lobes(Wagner grade 6). Pleural collagen deposi-tion was seen in all hamsters after 3months and was advanced and accompa-nied by moderate mesothelial hypertrophyby 6 months. By 12 months the pleuralfibrosis had progressed and mesothelialhypertrophy and hyperplasia were com-mon features. In some cases the fibroticlesion was thicker than the diaphragm itselfand was just as prominent in the rib cage.One hamster had mesothelial hyperplasiawith dysplastic changes that are characteristicof early mesothelioma (Table 3).

Preiminary Resultsthrothe End ofthe StudyPreliminary tumor data through the end ofthe study (18 months of exposure and 6weeks of postexposure recovery) werereceived just before this manuscript went topress. No additional tumors were observedin any of the air controls or fiber glass-exposed hamsters; however, a number ofadditional mesotheliomas were observed inthe amosite-exposed groups.

DiscussionEven though exposure concentrations anddimensions were comparable for the twofiber glasses and high-dose amosite, laterlung burdens differed considerably, espe-cially for fibers longer than 20 pm. Forexample, the long-fiber lung burden forMMVF33 was significantly greater thanfor MMVF1Oa (by an order of magnitude)even though the long-fiber aerosol concen-tration for MMVF33 was 30% less thanfor MMVFlOa. High-dose amosite (67fibers/cc> 20 pm) resulted in a 50-foldgreater lung burden than MMVF1Oa(aerosol concentration of 150 fibers/cc > 20pm). Differences in lung burdens couldresult from differences in lung deposition,differences in lung biopersistence, or both.To determine whether lung depositionswere similar for each of the fibers adminis-tered at similar aerosol concentrations, abrief lung deposition test was conductedtoward the end of the 18-month exposureperiod. Hamsters were exposed to testaerosols for 1 day (6 hr), held for an addi-tional day, then sacrificed. The data, whichbecame available just before this manu-script went to press, indicate that lungdepositions were very similar; the numbersof fibers longer than 20 pm/lung (x 105)were 1.6 ± 0.6 (MMVF1Oa), 2.2 ± 0.6

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(MMVF33), and 2.0 ± 0.5 (amositehigh dose). These data suggest that theobserved differences in the 12-month lungburdens of the three fibers were related todifferences in biopersistence rather than inlung deposition.

The three fiber types in the presentstudy induced a range of pathogenicitiesthat paralleled differences in lung burdensof fibers longer than 20 pm (Table 2;Figure 2B). As in previous studies, the ini-tial response induced by each of the fiberexposures appeared at the level of thebronchoalveolar junction, the area of high-est fiber deposition, and consisted of aninflux of macrophages, similar to thatinduced by most inhaled foreign particu-lates, followed by microgranulomas. In theMMVF1Oa hamsters, the lung responsedid not progress further through 12 monthsof exposure. However, in the MMVF33animals, the lesions did progress with time/exposure, with more intense inflammation,interstitial fibrosis, pleural collagen deposi-tion, mesothelial hypertrophy, and hyper-plasia, and a single mesothelioma. Thepulmonary and pleural lesions in theamosite-exposed hamsters were more severeand differed qualitatively from those asso-ciated with MMVF33. Previous studies inhamsters of RCF at approximately equalexposures (200-300 WHO fibers/cc) andwith chrysotile asbestos at much higherexposures (3000 WHO fibers/cc) failed toshow lesions as severe as those induced byamosite, even after 20 months of exposure(2). The present study suggests that inthe hamster, long-fiber amosite asbestos isnot only more toxic than the two fiberglasses, but also more toxic than chrysotileasbestos. Average aerosol concentrationsof fibers/cc longer than 20 pm were

comparable for amosite and chrysotile (67and 77, respectively).

Differences in long-fiber lung burdensbetween MMVFlOa and MMVF33 andbetween the fiber glasses and amosite wereapparently related to differences in fiberbiopersistence and not to differences inlung deposition. Fibers that are relativelysoluble or leachable could either dissolve inthe lung or break into shorter segments,allowing more rapid removal from the lungby clearance mechanisms. Differences inthe biopersistence of long fibers in the lungwould, in turn, be reflected in the qualityand severity of the pathological responsesof the lung and pleura to the fibers.However, as mentioned above, withoutdata on initial lung deposition, it couldalso be argued that long-fiber depositionefficiency could have been greater foramosite because of its smaller diameterscompared to the two glass test fibers.

As for the differences in pathogenicitybetween amosite asbestos and chrysotileasbestos, there are at least three possibleexplanations. First, there were very fewchrysotile fibers longer than 20 lim, andlonger fibers are thought to be more toxicthan shorter fibers (13). In contrast, 25%of the amosite aerosol fibers were greaterthan 20 pm. Second, chrysotile is more sol-uble and therefore less persistent in the lungthan amphibole types of asbestos, althoughchrysotile is several orders of magnitudemore durable than fiberglass (14). Finally,chrysotile is a relatively soft serpentine(curly) fiber, while amosite, an amphibole,is a straight (needlelike) fiber. It could bethat this latter feature allowed more of thelong amosite fibers to reach the pleura thanwas possible for chrysotile, and this couldexplain the differential pleural response.

The pathogenesis associated with 475glass in the present study does not agreewith five previous rodent inhalation studiesof this composition, all of which reportedno fibrosis or increase in tumor incidenceafter 1 or 2 years exposure to 3 to 10 mg/m3(15-19). The lack of reported effects couldbe explained by the facts that, in all but theSmith et al. study (19), the 475 fibers wererelatively short and the test system was rats;furthermore, the first three studies usedwhole-body exposure rather than nose only.Smith et al. (19) exposed hamsters for up to2 years to 5 mg/m3 (530 fibers longer than10 pm/cc), compared with the present studythat exposed hamsters to 283 fibers longerthan 5 pm and 106 fibers longer than 20pm. There is no obvious explanation for thedifferences in the effects of 475 glass in thetwo studies. Two possibilities are offered: a)Although the Smith study aerosol had morefibers/cc longer than 5 pm, it may not havehad as many fibers/cc longer than 20 pm asthe present study--the report by Smith etal. (1987) does not provide these data. b)Lung burden data were not as thoroughlyreported in the Smith study, so it is notknown whether target tissue dosing oflonger fibers was as high in the Smith studyas in the present study.

In conclusion, it is clear from theseand other studies that not all types ofasbestos have the same pathogenic poten-tial. It is equally clear that not all SVF oreven all glass fibers have the same biologi-cal reactivity after inhalation exposure. Thefact that strikingly different pathogenicitieswere induced by each of the three fibercompositions, even when aerosols werecomparable in fiber dimensions and con-centrations, points to the importance offiber biopersistence and surface reactivity.

REFERENCES

1. Hesterberg TW, Miiller WC, McConnell EE, Chevalier J,Hadley J, Bernstein DM, Thevenaz P, Anderson R. Chronicinhalation toxicity of size-separated glass fibers in Fischer 344rats. Fundam Appl Toxicol 20:464-476 (1993).

2. McConnell EE, Mast RW, Hesterberg TW, Chevalier J, KotinP, Bernstein DM, Thevenaz P, Glass LR, Anderson R. Chronicinhalation toxicity of a kaolin based refractory ceramic fiber(RCF) in Syrian golden hamsters. Inhal Toxicol 7:503-532(1995)

3. Mast RW, McConnell EE, Anderson R, Chevalier J, Kotin P,Bernstein DM, Thevenaz P, Glass LR, Miiller WC, HesterbergTW. Studies on the chronic toxicity (inhalation) of four typesof refractory ceramic fiber in male Fischer 344 rats. InhalToxicol 7:469-502 (1995).

4. Hesterberg TW, Chase GR, Versen RA, Anderson R. Studiesto assess the carcinogenic potential of man-made vitreousfibers. In: Toxicology of Industrial Compounds (Thomas H,

Hess R, Waechter I, eds). Bristol, PA:Taylor and Francis, 1995.5. Hesterberg TW, McConnell EE, Miiller W, Chevalier HJ,

Everitt J, Thevenaz P, Fleissner H, Hart G, and Oberdorster,G. Subchronic inhalation study of fibrous glass in the Syriangolden hamster [Abstract]. Toxicologist 36(1):7 (1995).

6. Hesterberg TW, Hart GA. A comparison of human exposuresto fiber glass with those used in a recent rat chronic inhalationstudy. Regul Toxicol Pharmacol 20:S35-S46. (1994)

7. WHO. WHO Reference Methods for Measuring Man-madeMineral Fibers (MMMF). Copenhagen:World HealthOrganization, 1985.

8. Bernstein DM, Thevenaz P, Fleissner H, Anderson R,Hesterberg T, Mast R. Evaluation of the oncogenic potential ofman-made vitreous fibers: the inhalation model. Ann OccupHyg 39(5):661-672 (1995).

9. Bernstein DM, Mast R, Anderson R, Hesterberg TW,Musselman R, Kamstrup 0, Hadley J. An experimental

1228 Environmental Health Perspectives * Vol 105, Supplement 5 * September 1997

CHRONIC INHALATION OF FIBER GLASS IN HAMSTERS

approach to the evaluation of the biopersistence of respirablesynthetic fibres and minerals. Environ Health Perspect102(Suppl 5):15-18 (1994).

10. Hesterberg TW, Miiller WC, Musselman RP, Kamstrup 0,Hamilton RD, Thevenaz P. Biopersistence of man-made vitre-ous fibers and crocidolite in the rat lung following inhalation.Fundam Appl Toxicol 29:267-279 (1996).

11. McConnell EE, Wagner JC, Skidmore JW, Moore JA. A com-parative study of the fibrogenic and carcinogenic effects ofUICC Canadian chrysotile B asbestos and glass microfibre UM100). In: Biological Effects of Man-made Mineral Fibres, Vol2. Copenhagen:World Health Organization, 1982;234-252.

12. Morrow PE. Possible mechanisms to explain dust overloadingof the lungs. Fundam Appl Toxicol 10:369-384 (1988).

13. Stanton MF, Layard M, Tegeris A, Miller E May M, MorganE, Smith A. Relation of particle dimension to carcinogenicityin amphibole asbestos and other fibrous minerals. J NatlCancer Inst 67:965-975 (1981).

14. Scholze H, Conradt R. An in vitro study of the chemicaldurability of siliceous fibres. Ann Occup Hyg 31:683-692(1987).

15. Wagner JC, Berry GB, Hill RJ, Munday DE, Skidmore JW.Animal experiments with MMM(V)F-effects of inhalation andintrapleural inoculation in rats. In: Biological Effects ofMan-madeMineral Fibres, Vol 2. Copenhagen:World Health Organization,1984;209-233.

16. McConnell EE, Wagner JC, Skidmore JW, Moore JA. A com-parative study of the fibrogenic and carcinogenic effects ofUICC Canadian chrysotile B asbestos and glass microfibre JM100). In: Biological Effects of Man-made Mineral Fibres, Vol2. Copenhagen:World Health Organization, 1984;234-252.

17. Le Bouffant L, Daniel H, Henin JP, Martin JC, Normand C,Thiehoux G, Trolard F. Experimental study on long-termeffects of inhaled MMMF on the lung of rats. Ann Occup Hyg31:765-790 (1987).

18. Muhle H, Pott F, Bellmann B, Takenaka S, Ziem U.Inhalation and injection experiments in rats to test the carcino-genicity ofMMMF. Ann Occup Hyg 31:755-764 (1987).

19. Smith DM, Ortiz LW, Archuleta RF, Johnson NF. Long-termhealth effects in hamsters and rats exposed chronically to man-made vitreous fibers. Ann Occup Hyg 31:731-754 (1987).

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