toxicologicalinvestigations on silicon carbide. 1. inhalation studies · bruch, rehn, song,...

British J7ournal ofIndustnial Medicine 1993;50:797-806

Toxicological investigations on silicon carbide.1. Inhalation studies

J Bruch, B Rehn, H Song, E Gono, W Malkusch

AbstractThe question of lung damage as a result ofexposure to silicon carbide (SiC) was investi-gated by inhalation experiments to obtaininformation on the qualitative response oflung tissue to the test substance (SiC). Forcomparison, quartz, kaolinite, and temperedclay dusts were used. The indices for theeffects of the dusts studied were organweights, numbers of bronchoalveolar cells,lung surfactant phospholipid concentrationsincluding subfractions, and lung clearance.Exposure to the test samples was carried outaccording to the Essen inhalation model intwo independent series. The results of the twoseries were similar: Compared with shamcontrols, exposure to SiC did not affect theindices studied. Even at a low dose (a quarterof the SiC dose) quartz gave pronounced devi-ations in all indices. In particular, an increasein granulocytes indicated toxic properties ofthe dust. The long term elimination of quartzfrom the lung was worse than that of SiC. Thekaolinite and tempered clay dusts were inter-mediate between SiC and quartz based onseveral of the indices studied. It is concludedthat SiC is deposited practically inert in thelung.

(British Journal ofIndustrial Medicine 1993;50:797-806)

Old occupational studies showed that employeesinvolved with synthetic abrasives had an up totwofold increase in the incidence of tuberculosis.This was considered to be the result of a silicogeniceffect. On the other hand, it was also shown thatsuch workers developed significantly less fibrosesthan those working with sandstone.I

In a more recent epidemiological study based onmedical examinations and lung radiographs, noincrease in lung disease was found in workers thatused synthetic abrasives in conjunction with pre-manufactured silicon carbide (SiC) abrasivewheels. There was, however, a limited pneumoco-niosis hazard in those concerned with SiC produc-tion.2

Only few reports are available on the causes.Open lung biopsies from two employees workingfor a fireproof brick manufacturing plant foundsubstantial amounts of SiC and other dusts but notquartz.3 An interstitial fibrosis in lung tissue wasdiagnosed in a further case after only a one yearexposure to various non-quartz abrasive dusts. Asthe patient was a heavy smoker and also subjectedto other inhalative noxious compounds in his earli-er working life, this could not be unequivocallyrelated to the abrasive dusts.

Significant increases in pulmonary diseases havealso been reported by SiC manufacturing plants. Inthe most comprehensive study to date, 171 menfrom SiC industrial manufacturing branches wereexamined radiologically and by means of lung func-tion tests; 35% of workers showed shadows on theradiographs with average profusion greater than 0/1and 14% had average profusion readings of greaterthan 1/1. In this report the highest quartz concen-tration amounted to 0-1 mg/m3 whereas that of SiCwas 057 mg/m3.4 Exposures to quartz and othercrystalline SiO2 compounds in the manufacturingplants must be taken into account as competingcauses; according to our findings these can be pre-sent in significantly greater concentrations than thementioned values for SiC and quartz.5 6 Moreover,manufacture related exposures to SiO2 also exist.7The recent study by Durand et al 8 concerningalterations in chest radiographs was based on acohort of 128 workers exposed to SiC in a manu-facturing plant. The authors reported that the work-ers were exposed to crystalline SiO2 during variousmanufacturing stages. Also, SiC fibres were presentin the respired air. Both must be considered to beconfounders during exposure to SiC dust.The results from animal experiments are contra-

dictory: older studies with ill defined experimental

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Institute for Hygiene and Occupational Medicine,University of Essen, Medical School (Univer-sitditskiinikum), Hufelandstrasse 55, 4300 Essen 1,GermanyJ Bruch, B Rehn, H Song, E Gono, W Malkusch

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Bruch, Rehn, Song, Gono, Malkusch

conditions have reported fibrogenic effects.1Another group has reported negative results inanimal experiments and concluded that SiC dust isinert.9

After an inquiry, the commission for establishingthreshold values for dusts of the DeutscheForschungsgemeinschaft (DFG) concerned itselfwith the setting up of hygiene standards for SiC in1985. After assessing the available experimentalanimal and epidemiological findings, a fibrogeniceffect could not be absolutely ruled out and athreshold value for SiC was introduced as a precau-tionary measure.As accompanying quartz admixtures could not

be excluded with certainty in the considerationswhen establishing the threshold value, the questionof pneumoconiotic effects of SiC need to bechecked in animal studies taking more recentinhalatory toxicological procedures into account.According to general medical opinion, pneumoco-nioses are pulmonary diseases caused by dusts andare characterised by chronic inflammatory alter-ations to the lung tissue with scarring of the organstructure. Clinical symptoms such as shortness ofbreath and tightness of the chest are found; in gen-eral, it is assumed that there is a progression of thedisease after sufficient exposure. The pathogenesisof pneumoconioses can be attributed simplisticallyto an inflammatory stimulation of alveolarmacrophages. This stimulation of cells responsiblefor defence and removal gives rise to several harm-ful sequelae-namely, (1) worsening of lung clear-ance, normally between about 97 and 99%10 11; (2)scarring of lung tissue as a result of the chronicintra-alveolar inflammation12; (3) disintegration ofthe respiring lung tissue with consecutive emphyse-ma'3; (4) response of the pneumocyte type II sur-factant system with deterioration of lung-function.In this case, specific reactions of a lung epithelialcell type to silica are involved."'-'6 To check forpathogenesis of pneumoconioses, animal studiesshould test both the acute intra-alveolar processesas well as the chronic effects of dusts on pulmonaryhealth. For methodological reasons, the experi-ments should be carried out after inhalation of thetest substances to determine the intra-alveolarmechanisms involved. The often used method ofintratracheal injection interferes with the sensitiveprimary pulmonary defence system due to terough method of application so that subsequenteffects may not be testable. By contrast the chroniceffects can be examined by giving a sufficiently highdose via intratracheal injection and assuming theworst case after a sufficiently long dust exposuretime (usually between six and 12 months). Thispaper deals with the inhalation studies.Experiments with the intratracheal application ofSiC are reported in a companion paper.'7

Materials and methodsDUST SAMPLESThe dust samples under study were SiC (WackerGmbH batch No D Mikro-F1200 M678), corun-dum (fused alumina; Wacker GmbH batch No DF1200/3 M74375), kaolinite, tempered and groundclay, and quartz (DQ12, DMT, Essen). The aver-age grain size was below 3 gm.

ANIMAS AND EXPOSURE SCHEMES: STUDY DESIGNFemale Wistar rats, initial body weight, 180-220 g(Lippische Versuchstierzucht, Charles River Co)were used. The rats were exposed oronasallyin a modified Kimmerle inhalation chamber.Conditions in the chamber were in accordance withthe Organisation for Economic Cooperation andDevelopment guidelines for testing of chemicals.Dust concentration was measured gravimetricallywith a Cassella instrument (JohannesburgConvention for Fine Dusts) and monitored contin-uously by a Tyndallometer (Hundt-,u Digital,Wetzlar, Germany).The animals were exposed for five hours a day

on five consecutive days, followed by a rest periodof two days and a re-exposure period of five consec-utive days. Total exposure time was 50 hours. Theinhalation schedules, including two sets of indepen-dent inhalation series; were:

First inhalation series (50 animals in each group)(1) Exposure to a constant concentration of 20 mgSiC/m3 respirable air for five hours a day.(2) Exposure to a constant concentration of 20 mgquartz/m3 respirable air for one hour a day followedby an exposure of 20 mg SiC/m3 respirable air forfour hours a day.(3) Exposure to a constant concentration of 20 mgkaolinite/m3 respirable air for one hour a day fol-lowed by an exposure of 20 mg SiC/m3 respirableair for four hours a day.(4) Exposure to a constant concentration of 20 mgcorundum/m3 respirable air for five hours a day (15animals).

Second inhalation series (42 in animals each group)(1) Exposure to a constant concentration of 20 mgSiC/mi3 respirable air for five hours a day.(2) Exposure to a constant concentration of 20 mgquartz/m3 respirable air for five hours a day.(3) Exposure to a constant concentration of 20 mgtempered, ground clay dust/m3 respirable air forfive hours a day.(4) Sham exposure (to air).

INVESTIGATION OF BRONCHOALVEOLAR LAVAGEFLUIDThree and 90 days after exposure in the first series,and three, 21, and 90 days after exposure in the

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Toxicological investigations on silicon carbide. 1. Inhalation studies

siC Corundum140 a

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Time of investigationFigure 1 First inhalation series; comparison ofwet weights of mediastinal lymph nodes. Values are mean (SD) from eightanimals per group per investigation date. A significant increase in weight until the end of the investigation occurred only in theSiC + quartz group.

second inhalation series, seven rats per group andseven control rats exposed to air were killed with anoverdose of pentobarbital and exsanguinated; thetrachea was cannulated, and the lungs weredegassed and lavaged in situ five times through thetrachea, each time with 5 ml physiological saline.The lavage fluid was centrifuged (300 g for 10 min-utes at 4°C) to sediment the cells. The cells wereresuspended in Hanks balanced salt solution andtotal cell counts were made with a cell counter(Coulter Electronics). Differential cell counts weremade from cytocentrifuge smears treated withPappenheim stain.

Lung surfactant factor (LSF) phospholipids wereextracted by the method of Folch et al (1957).Supernatant lavage fluid (10 ml) was added to 30ml chloroform:methanol (2:1) and the mixture wasstirred for five minutes and centrifuged (1100 g for5 minutes at 4°C) to separate the methanol andchloroform layers. The chloroform layer wasremoved, dried under nitrogen, and redissolved in200,u chloroform:methanol (2:1) for analysis byhigh perform-ance liquid chromatography (HPLC).The LSF phospholipids were separated accordingto the method of Pison et al. 8 The equipment(pump with gradient former, spectrophotometer,

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and computing software) was purchased fromGynotek (Munich, Germany). The chromatograph-ic analysis was performed at 30°C at a flow rate of2 ml/min on a column (250 x 4 mm internaldiameter) prepacked with Lichrosorb diol (5 ,um).A guard column (75 x 4-6 mm internal diameter)was prepacked with Si 100 (30 pm) (Serva). Themobile phase consisted of acetonitrile:water (80:20;pH 6) and pure acetonitrile. A linear gradient from87'5% to 25% for pure acetonitrile was formedbetween six and 15 minutes. Detection wavelengthwas set at 200 nm. Phospholipid composition wascalculated by comparison with standard phospho-lipid mixtures (phosphatidyl glycerol (PG), phos-phatidyl inositol, (PI), phosphatidyl ethanolamine,phosphatidyl choline, lysophosphatidyl choline, andsphingomyelin) obtained from Sigma, Munich,Germany.

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LUNG FUNCTIONA lung function test was carried on eight animals ofthe first inhalation series, which included exposureto SiC, kaolinite, quartz, and corundum. Eightlungs of each exposure group were excised 90 daysafter finishing the inhalation and tested for peakflow.

ELIMINATION OF DUST FROM THE LUNGSSeven rats per group were killed with an overdoseof pentobarbital 3, 11, 21, and 90 days after expo-sure in the first inhalation series, and 3, 21, and 90days after exposure in the second series. The lungswere removed and stored in acetone for measuringthe dust content in the tissue. Dust content wasdetermined gravimetrically with a formic aciddigestion method.

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Time of investigationFigure 2 Second inhalation series; comparison ofwet weights of mediastinal lymph nodes. Values are mean (SD) from eightanimals per group per investigation date. A significant increase in weight until the end of the investigation occurred only in thequartz group.

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ResultsORGAN WEIGHTS AND HISTOLOGYIn both inhalation series, the rats showed normalbehaviour and normal development after theinhalation and during the observation period of 90days.

In the first series a comparison of weight devel-opment of the mediastinal lymph nodes showed no

significant deviations from normal after exposure toSiC or corundum (fig 1). Additional exposure toquartz resulted in a significant weight increase inthe lymph nodes as an expression of the pathogenic

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* SiC* SiC + quartzO SiC + kaolinite

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Figure 4 (A) Total phospholipids (mean SD) in the firstinhalation series. Concentration was significandy increasedat 90 days in the quartz group. (B) Ratio ofLSF subfractionsat day 90 after exposure. Ratio PG:PI is roughly 2:1 forcontrol and SiC groups and 1:2 for SiC + quartz andSiC + kaolinite groups.

response of the lymphatic tissue.The second series involved a group of eight ani-

mals subjected to SiC inhalation at a concentrationof 20 mg/m3, as in the first series. Quartz DQ12served as a positive control dust but, contrary to thefirst series, this was applied at a dose of 20 mg/m3for five hours daily on 2 x 5 days as was a clay dust(tempered, ground) also given at a concentration of20 mg/m3. All animals exposed to dust showedincreased lymph node weights; however, theweights for the quartz group were clearly greaterthan those for the SiC and clay dust groups as earlyas day 3 after ending inhalation (fig 2).None of the groups showed any significant

changes in lung weights compared with the con-

trols.

CELLS IN BRONCHOALVEOLAR LAVAGE FLUIDIn the first series bronchoalveolar lavages (BALs)permitted conclusions to be drawn about the intra-alveolar processes prevailing at the time of investi-

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DaysFigure 5 (A) Totalphospholipids (mean (SD)) in thesecond inhalation series. Concentration was significantlyincreased at 90 days in the quartz group. (B) Ratio ofLSFsubfractions at day 90 after exposure. All PG:PI ratios above1-5 exceptfor quartz group at day 90 (< 0-5).

gation. Of interest in the present study are theresults of the cell numbers and the involvement ofvarious cell types in the pulmonary response to theeffects of dust. High total cell numbers as well as

alveolar macrophages were found three days afterthe end of inhalation in the SiC group (fig 3A).These conditions were reversed after 90 days, whenincreased cell numbers were found in the quartztreated group. These values must be regarded asthe result of an adaptive adjustment to dust expo-sure in association with the toxic effects of the indi-vidual samples. The number of granulocytesreflects the pathological inflammatory stimulus.The results in fig 3A show a strong inflammatorystimulation by quartz dusts, whereas SiC and kaoli-nite apparently produce no specific stimulation ofgranulocytes.

Similar results were found in the second series ofstudies. Here, a sham control was used. The num-ber of cells in lavage fluid was similar in the shamcontrol and SiC group over the entire experimental

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SiC SiC + SiC + Corundum Controlquartz kaolinite

Figure 6 Maximum peak flow (mean (SD) values at 90days after exposure). Values for SiC + quartz and SiC +kaolinite groups were significandy lower than controls.

period (fig 3B). More cells were found in the BALfluid of groups exposed to clay and quartz. Thenumbers in animals exposed to clay mineral werenormal by day 90.

Exposure to SiC did not result in a noticeableshift in the numbers of alveolar macrophages, lym-phocytes, and granulocytes in the total cell number.Figure 3B shows that the number of lymphocytesand granulocytes constitute less than 10% of thetotal cell number.The situation was different in the case of quartz.

As the dust concentration was fivefold higher com-pared with the first series, a correspondingly highergranulocytic response was seen in the BAL fluid.The findings indicate that at this concentrationthere was an increase in the amounts of granulo-cytes even up to day 90 of observation. Up to 70%of cells found in the lungs were granulocytes.

LUNG SURFACTANT PHOSPHOLIPIDS INBRONCHOALVEOLAR LAVAGE FLUIDThe LSF phospholipids are a component of thepneumocyte type U-LSF system. Both the totalamount of LSF phospholipids as well as the com-position of the subfractions need to be considered.Pronounced responses are to be expected based onclinical and experimental studies of intra-alveolarinflammatory processes, particularly in the case ofinitiation of fibrotic processes of different origin.

In both series the total amount of LSF phospho-lipids was only increased in animals exposed toquartz (figs 4A and 5A).The relation between subfractions of LSF is a

particularly sensitive response to the inflammatoryor fibrogenic effects of dusts. In particular the rela-tion between PG and PI reflects the changes in theintra-alveolar environment. According to earlierstudies, the normal ratios range from 2:1 to 3:1. Onexposure to quartz and kaolinite abnormal ratios of< 1:1 were obtained. By contrast, SiC induced nosuch alterations in the LSF subfractions, with val-ues corresponding to those of the untreated control

animals (fig 4B). In the second series of studies,there were indications of a slight but significantdecrease below the level of 2:1 after exposure toclay as well as a slight toxic effect at day 21 afterinhalation (fig 5B). This finding is in agreementwith the increase in granulocytes in the BAL fluidalso seen at this time.

LUNG FUNCTIONFigure 6 presents the results of the testing of thefive exposure groups. Control animals as well asanimals exposed to SiC showed simular maximumflow values (>8-5 mlIs), whereas exposure to quartzand to a lesser degree kaolinite gave lower flowrates (<8.0 mlIs).

ELIMINATION OF DUST FROM THE LUNGElimination studies show a differential response toSiC and quartz. Two inhalation series with SiCwere carried out: SiC alone and quartz in combi-nation with SiC (first series) as well as SiC alonecompared with quartz alone (second series). Both

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Figure 7 Dust deposits in lungs. (A) first inhalation series(o SiC, OSiC+ quartz); (B) second inhalation series(-SiC, Oquartz). Dust deposits at day 3 after inhalationwere higher in SiC group. Subsequently SiC was eliminatedmore effectively.

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inhalation series produced similar results (fig 7Aand B). The initial amount of dust directly deter-mined on day 3 showed the lowest and highest lungdust burden in the groups exposed to quartz andSiC alone respectively. The experimental times ofinvestigation allowed the elimination rate to bedetermined from these different starting points.Quartz elimination was clearly inhibited up to 90days after completion of elimination. In the case ofSiC only 23% of the initial amount was found inthe lungs, but 60% of quartz was still retained. Inour opinion, the differing situation at day 3 is dueto an initially enhanced elimination during expo-

sure to quartz dust; clear evidence of this was

obtained from previous studies. On the other hand,the present data cannot definitely rule out that a

different primary deposition based on non-uniformgrain size distribution had taken place.

DiscussionThe toxicological studies on the biological effects ofSiC were carried out with the aid of inhalationexperiments. The standard reference dust quartzDQ12 and corundum served as comparative duststogether with various clay mineral dusts. The

inhalation experiments were carried out accordingto the standard model in Essen, which allows theinvestigation of both the initial inflammatoryprocesses in the alveoli (alveolitis) directly afterdust exposure (three days postinhalation) and pos-sible long term pulmonary damage (21-90 dayspostinhalation). Based on other studies it is knownthat distinct fibrotic responses can be detected inthe lungs and in the mediastinal lymph nodes even

with colliery dusts containing less than 1% ofquartz at the doses used in the same experimentaldesign.19 In these studies, however, less emphasiswas placed on the question as to what extent thedusts studied caused chronic pulmonary diseasesand more on the testing of decisive primary eventsafter dust deposition which contribute to the typeand extent of fibrogenic pneumoniotic diseases.These events are primarily associated with the alve-oli and result from the effects arising from the inter-action between alveolar macrophages and dust.These effects include the liberation of mediators, tocause inflammatory processes as well as stimulationof the lung surfactant pneumocyte type II system.The indirect effects of these primary actions are

alterations to clearance efficiency of the lungs andan enhanced lung dust burden. This results in thedecisive lung dust dose that, in conjunction withthe specific fibroblastic effects of the particles, isfinally responsible for the overall effect of the dusts.To prevent non-specific side effects by unphysio-

logical dust application-for instance, intratrachealinstillation-inhalation experiments have been

used. Thus the qualitative onset of the questionablepathogenic event chain induced by the test sub-stances can be investigated. Non-specific epiphe-nomena by intratracheal injection are possiblyinflammation to dust overload and a non-specificoutcome to lung elimination and dust retention.

Valuable information can be obtained from theresults of inhalation experiments. These aredesigned to test the acute effects of dust as well asto study those events leading to chronic and termi-nal transition of dust effects. Initially, organ weightscan be used as the simplest variable and those ofthe mediastinal lymph nodes are of particular valuein the case of fibrogenic and cytopathogenic dusts.These simple variables reflect the three divisionaltoxicity of dusts in the samples studied. Roughlythe dusts can be subdivided into three classes:

(1) Dusts without recognisable effects in relationto air controls-namely, SiC, corundum, and kaoli-nite in one experiment.

(2) Dusts with small and only transitory effects-namely, tempered clay as well as low doses of thepositive reference dust, quartz DQ12.

(3) The toxic quartz DQ12, which producedeffects in all variables studied at the standard dose.

Quartz showed the highest effects whereby theweight of the lungs and also the lymph nodes hadincreased soon after ending inhalation. There wasalso a progressive increase over the entire observa-tion period of 90 days. The dusts of intermediatetoxicity-for example, kaolinite colliery dust-onlygave a temporary increase in lymph node weights.By contrast, SiC produced no significant changes inorgan weights at any time of observation.

Further variables were cell populations deter-mined from the BAL fluid, analysed for cell num-ber and composition. Normally, about 90% of cellsin BAL fluid are alveolar macrophages and theremainder are made up of lymphocytes and a fewgranulocytes. The number of macrophages increas-es on exposure to toxic dusts, a certain amount ofwhich is attributable to socalled inert dusts result-ing in a physiological response. The amount ofgranulocytes in BAL fluid is a sensitive index ofpossible toxic dust effects.2024 In principle, the pre-sent data show three response patterns: firstly, noreactions, secondly, a transient increase in granulo-cytes and a decrease in- the period between the 21stand the 90th day after inhalation, and thirdly adefinitive increase and steady state at a high leveleven at day 90 after completing inhalation. Thegranulocytes precisely reflect the situations alreadyfound in in vitro tests, which, on the one hand,allow SiC to be classified as the least stimulatorydust and, on the other, quartz as the most stimula-tory. The other samples studied characteristicallyonly showed a transient increase in the amount ofgranulocytes in the BAL fluid directly after inhala-

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tion. An important activity of alveolar macrophagesis that of scavenger function. Lung purging wasclearly affected within the limits of the test model;thus an initial phase of lung purging seems to bestimulated by toxic dusts. This is in agreement withthe results from other investigations on collierydusts.2526 After 90 days, these circumstances werereversed, because SiC is much better eliminatedthan the toxic dust at this time. This continuousprogressive improved elimination must be rankedas favourable in terms of health assessment.

Valuable information can be derived from theresults on lung surfactant. Here, a distinct grada-tion of the effects between the unexposed controlsand quartz treated animals was registered. Theamount of surfactant is an important index. Quartzgave a pronounced and lasting increase in surfac-tant; this agrees with the results of many otherexperiments on silica.'4 16 27 28 The cause of theexcessive surfactant production by silica is due tothe pathogenic stimulation of the type II pneumo-cytes by factors released from the alveolarmacrophages.'5 A corresponding increase was notfound with the control dusts (clay mineral dusts,kaolinite) or with SiC. In our opinion compositionof LSF subfractions provides a sensitive, graduatedindication of minor effects from dusts not able tobe detected by other variables.29 30 In this case, theabsolute amounts of PG and PI as well as the rela-tion between the two substances have proved to beparticularly sensitive indices. Figure 4B shows thatSiC responds similarly to the control both withrespect to the total amount as well as the relation ofPG to PI. The situation is different with quartz aswell as kaolinite where the relation of PG to PI isaltered. In other experiments the inversion of theratio PG:PI has also been found with silica andother substances that induce lung fibrosis.3132 Ourresults indicate that this ratio is sensitive to subtlechanges in intra-alveolar physiology. Worsening oflung function after quartz and kaolinite exposurecan be related to the changes in composition of[SF; our results are comparable with those afterbleomycin induced pulmonary fibrosis.33An assessment of the changes in relation to

primary elimination remains an open question,because according to our clinical experience, thismay possibly be considered as a physiological cura-tive response to burdensome situations in the lung.In general the role of surfactant in the context offibrotic lung diseases is not well understood. Thereare also clinical data showing that an increase in theamount of surfactant in the BAL fluid is associatedwith diffuse interstitial fibrosis after exposure toasbestos.34 Disturbances in fractional compositionare reported from patients with acute respiratorydistress syndrome35 and idiopathic pulmonaryfibrosis.36 The present results on the effect of the

dust on the primary phase of elimination are notconclusive. The low lung dust values at day 3 afterinhalation of quartz dust can, on the one hand, becaused by a lower primary penetration and deposi-tion or, on the other, by an already increased elimi-nation stimulated during inhalation. The resultsfrom particle size distribution seem to support this,as the quartz DQ 12 penetrates rat lung to a higherdegree than SiC dust based on their finer grain sizedistribution. From this it can be inferred that theprimary elimination is initially increased throughthe general stimulation processes in the alveoli witha greatly enhanced cell recruitment. These resultsare in agreement with our own studies on mixeddusts, in which dusts of varying cytotoxicity fromvarious areas of work in the colliery were tested andcompared with respect to their elimination. Heretoo a strongly enhanced primary elimination on thepart of the more cytotoxic dusts was seen, bothduring inhalation itself as well as up to 10 daysafter.By contrast, the results for long term deposition

are clear as can be seen from the data for 90 daysafter inhalation. Thus cytotoxic quartz led to a dis-tinct long term worsening of elimination comparedwith SiC. This is in agreement with other resultswhere experiments were designed to study such along term effect; an increased retention was alwaysnoticed in the case of cytotoxic quartz comparedwith other dusts studied. The causes of theincreased retention lie in the enhanced transitionrate of the quartz dust into the intraseptal and lym-phatic regions of the lung. This agrees with the factthat after inhalation of quartz dust the lymph nodeweights were noticeably increased in comparisonwith those seen with other dusts (lymphotropism ofquartz dusts according to Klosterkotter). It isremarkable that as well as the high elimination ofSiC practically no lymphatic penetration by thedust had taken place. From a hygienic point ofview, it is relevant that even this index shows nonegative response under the influence of SiC. It canbe concluded that a burdensome situation necessi-tating a physiological curative response is not givenfor SiC. The significance of these results in respectto the medical hygienic evaluation for SiC isdiscussed in the accompanying paper.'7

Summary and conclusionsThe hygienic, occupational medical rating of themajor test substance SiC is based on experimentalprocedures related to primary pulmonary reactionsfollowing deposition. The present results wereobtained with several independent assays and twodifferent inhalation experiments.

All results obtained were consistent and corre-spond to the present day knowledge on the initia-

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tion of chronic pneumopathies from events primari-ly induced in the alveolus. The role of alveolarmacrophages in lung purging as well as the sensitivechanges in the LSF PII cell system are in agree-ment with the results of the differential cell patternin BAL fluid as well as those of TNF-a productionfrom the various exposure situations (see compan-ion paper).'7

SiC showed no divergent results from those ofcontrols in all the tests studied. By contrast, quartzalways behaved differently from the control andSiC even at small doses. Various other dust samplescotested were considered to be of intermediate toxi-city in several indices. Here, the testing of LSF sub-fractions proved to be particularly sensitive. A finalranking of substances from an occupational medi-cine-hygienic viewpoint should be undertaken inconjunction with the results from long term studies.

1 Gardner LU. Studies on the relation of mineral dusts to tuber-culosis. Im. The relatively early lesions in experimentalpneumoconiosis produced by carborundum inhalation andtheir influence on pulmonary tuberculosis. American Reviewof Tuberculosis 1923;7:344-57.

2 Bruusgaard A. Abrasives. Encyclopaedia of occupational healthand safety, 1983;1:4-5.

3 Funahashi A, Schlueter DP, Pintar K, et al. Pneumoconiosisin workers exposed to silicon carbide. Am Rev Respir Dis1984;129:635-40.

4 Peters JM, Smith TJ, Bernstein L, Wright WE, HammondSK. Pulmonary effects of exposures in silicon carbide manu-facturing. BrJ Ind Med 1984;41:109-15.

5 Smith TJ, Hammond SK, Laidlaw F, Fine S. Respiratoryexposures associated with silicon carbide production: esti-mation of cumulative exposures for an epidemiologicalstudy. BrJ Ind Med 1984;41:100-8.

6 Bruch J. Personal observation and discussion with hygienic inspec-tion of the ESD Delfzui, Holland: 1989.

7 Osterman JW, Greaves IA, Smith TJ, et al. Respiratory symp-toms associated with low level sulphur dioxide exposure insilicon carbide production workers. Br J Ind Med 1989;46:629-35.

8 Durand P, Begin R, Samson L, et al. Silicon carbide pneumo-coniosis: a radiographic assessment. Am J Ind Med 1991;20:37-47.

9 Miller JW, Sayers RR. The physiological response of peri-toneal tissue to certain industrial and pure mineral dusts.Public Health Rep 1936;51:1677-88.

10 Gross P. The processes involved in biological aspects of pul-monary deposition, clearence and retention of insolubleaerosols. Health Phys 1964;10:995-1002.

11 Klosterkotter W, Gono E. Long term storage, migration andelimination of dust in the lungs of animals, with special respect tothe influence of polyvinyl-pyridine-n-oxide. Oxford: UnwinBrothers, 1971.

12 Davis GS, Hemenway DR, Evans JN, Lapenas DJ et al.Alveolar macrophage stimulation and population changes insilica-exposed rats. Chest 1981;80:85-105.

13 Bruch J, de Beer E, Kraus R, Breining H. Morphometriemenschlicher Silikoselungen. Ergebnisse von Untersuchungenauf dem Gebiet der Staub und Silikosebekampfung imSteinkohlenbergbau. Steinkohlenbergbauverein (Hrsg) 1989;17:227-31.

14 Bruch J. Die Vermehrung der Pneumozyten Typ II unter demEinflul3 von Quarz. In: Ergebnisse von Untersuchungen aufdemGebiet der Staub-und Silikosebekampfung im Steinkohlebergbau.Essen: 1973: 99-112.

15 Bruch J, Schlosser JW. Veranderung der Zellpopulation inden Alveolen der Rattenlunge nach Inhalation von Quarzund ihre Beeinflussung durch PVNO. In: Ergebnissevon Untersuchungen auf dem Gebiet der Staub-und

Silikosebekampfung im Steinkohlebergbau. Essen: 1977:175-84.

16 Miller BE, Dethloff LA, Hook GER. Silica-induced hypertro-phy of type II cells in the lungs of rats. Lab Invest1986;55:153-63.

17 Bruch J, Rehn B, Song W, Gono E, Malkusch W.Toxicological investigations on silicon carbide. 2 In vitro celltests and long term injection tests. Br J Ind Med 1993;50:807-13.

18 Pison U, Joka T, Obertacke U, Bruch J. Surfactant system andthe development of pneumonia during artificial ventilation,1987 Annual Meeting of the American Thoracic Society,New Orleans. Am Rev Respir Dis 1987;135:A 200.

19 Bruch J, Rehn B. Influence of peak exposure on high and lowamount containing mine dust. Luxembourg: EEC researchreport 1991.

20 Donaldson K, Bolton RE, Jones A, et al. Kinetics of the bron-choalveolar leucocyte response in rats during exposure toequal airborne mass concentrations of quartz, chrysotileasbestos, or titanium dioxide. Thorax 1988;43:525-33.

21 Donaldson K, Brown GM. Assessment of mineral dust cyto-toxicity toward rat alveolar macrophages using a "Cr releaseassay. Fundam Appl Toxicol 1988;10:365-6.

22 Donaldson K, Slight J, Brown GM, Bolton RE. The ability ofinflammatory bronchoalveolar leucocyte populations elicitedwith microbes or mineral dust to injure alveolar epithelialcells and degrade extracellular matrix in vitro. British JournalofExperimental Pathology 1988;69:327-38.

23 Beck BD, Feldman HA, Brain JD, et al. The pulmonary toxic-ity of talc and granite dust as estimated from an in vivohamster bioassay. ToxicolAppl Pharmacol 1987; 87:222-34.

24 Begin R, Dufresne A, Cantin A, Possmayer F, Sebastien P.Quartz exposure, retention, and early silicosis in sheep. ExpLung Res 1989;15:409-28.

25 Bruch J, Li C, Gono E, Song H. Differences in the retentionand elimination after inhalation of a mineral poor and amineral rich mine dust. In: Ergebnisse von Untersuchungenauf dem Gebiet der Staub-und Siikose-bekdmpfung imSteinkohlebergbau. Essen: 1987: 229-36.

26 Li C, Bruch J. Factors controlling particle elimination andpenetration in the lung effect of particle properties.International conference on inhalation toxicology: the design andinterpretation of inhalation studies and their use in risk assess-ment. Hanover, Germany, 23-27 March, 1987; 23-7.

27 Grunspan M, Antweiler H, Dehnen W. Effect of silica onphospholipids in the rat lung. BrJInd Med 1973;30:74-7.

28 Miller K, Webster I, Handfield R, Skikne MI. Ultrastructureof the lung in the rat following exposure to crocidoliteasbestos and quartz. JPathol 1978;124:39-44.

29 Rehn B, Gono E, Pison U, et al. Der Einflu1i derStaubbelastung auf die Zusammensetzung des Surfactants.In: Norpoth K, ed. 27. Fahrestagung der DeutschenGeselschaftfuirArbeitsmedizin 1987. Stuttgart: 1987:531-4.

30 Li C, Bruch J, Pison U. Phospholipid lung profile in rats afterdifferent dust exposure. Am Rev RespirDis 1987;135:A 447.

31 Kawada H, Horiuchi T, Shannon JM, et al. Alveolar type IIcells, surfactant protein A (SP-A), and the phospholipidcomponents of surfactant in acute silicosis in the rat. AmRev Respir Dis 1989;140:460-70.

32 Liau DF, Barret CR, Bell ALL, Ryan SF. Functional abnor-malities of lung surfactant in experimental acute alveolarinjury in the dog. Am Rev Respir Dis 1987;136:395-401.

33 Horiuchi T, Mason RJ, Kuroki Y, Cherniak M. Surface andtissue forces, surfactant protein A, and the phospholipidcomponents of pulmonary surfactant in bleomycin-inducedpulmonary fibrosis in the rat. Am Rev Respir Dis 1990;141:1006-13.

34 Klein B. Anderung des Lungensurfactants bei asbeststaub-exponierten Patienten [dissertation] Essen: University ofEssen. 1992.

35 Hallmann M, Spragg R, Harrel JM, Moser KM. Evidence oflung surfactant abnormality in respiratory failure. Study ofbronchoalveolar lavage phospholipids, surface activity, andplasmamyoinositol. J Clin Invest 1982;70:673-83.

36 Robinson PC, Watters LC, King TE, Mason RJ. Idiopathicpulmonary fibrosis. Abnormalities in bronchoalveolar lavagefluid phospholipids. Am Rev RespirDis 1988;137:585-91.

Accepted 19 October 1992

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