in vitro diatomaceous · amorphous silica shell, a portion of which on calcining can be converted...

British Journal of Industrial Medicine 1984;41:228-234

In vitro cytotoxicity and quantitative silica analysis ofdiatomaceous earth productsE BYE,' R DAVIES,2 D M GRIFFITHS,2 B GYLSETH,' AND C B MONCRIEFF2

From the Institute ofOccupational Health, ' Oslo, Norway, and the Pneumoconiosis Unit,2 Medical ResearchCouncil, Penarth, S Glamorgan, UK

ABSTRACT Mouse peritoneal macrophages were used to evaluate the relative cytotoxicity of a

series of diatomaceous earth products in vitro. The amorphous and crystalline silica content ofthe products was determined by a combination of infrared spectroscopy and x ray powderdiffraction techniques. The cytotoxicities of the high cristobalite content flux calcined materialswere similar to that of the standard cristobalite; both the natural and straight calcined materialshad significantly greater activities than the flux calcined materials. Thus within the limitations ofthe macrophage cytotoxicity test the hypothesis that crystalline content is the only determinant offibrogenicity of diatomaceous earth is not supported.

Diatomaceous earth, Kieselguhr or diatomite, is amineral found in several parts of the world which,after suitable treatment, may be used as a filler, asthermal insulation, as fine abrasive powder, and asan absorbant of liquids. The deposits of diatomace-ous earth result from the death of diatoms (unicellu-lar algae) over thousands of years in shallow bays orlakes where, as the organic material decomposes,their shells sink to the bottom and are compressedby subsequent sediments. The diatoms have anamorphous silica shell, a portion of which oncalcining can be converted to the crystalline silicacristobalite, which exceeds 50% of some flux cal-cined products.Workers may be exposed to natural and calcined

forms of diatomite. The way the diatomite is cal-cined (straight or with a flux) results in qualitativedifferences in the structural forms of the diatomitesilica. These differences are considered to be impor-tant for the development of pneumoconiosis.Indeed, according to Parkes "the severity [of thisuncommon pneumoconiosis] appears to correlatewith the cristobalite content of the dust and theduration of the exposure."' Before discussing theknown biological effects of diatomite, a review ofthe biological effects of various silica polymorphsmay be worth while.The naturally occurring crystalline silica quartz

can cause silicosis and its presence in the dust fromflint, sandstone, granite, and slate is probablyReceived 4 January 1983Accepted 29 March 1983

responsible for the pneumoconiosis associated withthe use of these minerals. The crystalline silicasquartz, cristobalite, tridymite,2 and coesite3 causepulmonary fibrosis in experimental animal studiesbut stishovite does not.4An extensive range of synthetic amorphous silicas

is manufactured, only a few of which have beenstudied for their biological effects. Nopneumoconiosis was found among workers exposedto precipitated56 or fumed silica7 but evidence ofpulmonary fibrosis8 was found in workers exposed toamorphous silica dust produced as a byproduct ofsilicon metal production. In this process quartz isvaporised in an electric arc furnace but x ray diffrac-tion studies have indicated the presence of crystal-line silica in the dust. Recently, Brambilla et al sug-gested that pneumoconiosis in a silicon factory wasdue to amorphous silica particles.9

Experimental animal studies with fumedsilicas'° " indicated that these materials caused sometissue reaction but there was no progression to col-lagen formation. Similar findings were made with aprecipitated silica in animal inhalation studies.'2Groth et al,'3 however, found early nodular fibrosisin the lungs of monkeys exposed to fumed silica andvitreous silica-that is, fused silica or quartzglass has also been found to be fibrogenic'4 but lessso than quartz.2"

In a major x ray investigation of diatomaceous* earth workers, Cooper and Cralley found that 9%had lung changes interpreted as pneumoconiosis andthat an equal number had doubtful changes.'5 The

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In vitro cytotoxicity and quantitative silica analysis of diatomaceous earth products

prevalence of abnormal chest films was especiallyhigh in employees in the mills, where the dust con-tained a high percentage of cristobalite. Exposure inthe quarries was associated with a lower proportionof abnormal films, none of 25 employees who hadworked there exclusively for over five years had a

positive film but 40% showed doubtful linear no-dular changes. The dust in the quarries was essen-tially amorphous silica, the quartz content of thecrude diatomite being 2%. The authors pointed outthat this evidence is in agreement with earlier obser-vations indicating that the risk of pneumoconiosis isrelatively low in workers whose exposure is confinedto crude diatomite as compared with those exposedto calcined material. Nevertheless, it may be unwar-ranted to regard the natural material as innocuous asthe dust concentrations in the quarries were notnecessarily of the same order of magnitude as thosein the mill. Franzinelli et al have made similar obser-vations.'6 Beskow and Omura et al have alsoreported cases of diatomaceous earth pneumo-coniosis but the exact nature of the dust was notindicated.'7 18

In experimental animal studies Tebbens andBeard found that inhalation of raw naturaldiatomaceous earth (containing no detectable crys-talline silica) produced no fibrosis in guinea pigs'9and Swensson found that intratracheal injections ofKieselguhr into rats had an appreciably less pro-nounced reaction than quartz." A heated Kiesel-guhr (800°C for 24 hours), however, produced astronger tissue reaction than the natural Kieselguhrmaterial, although x ray diffraction showed theabsence of crystalline silica in both materials. Asimilar result was obtained by Klosterkotter'° usingfumed silica. This material caused some tissue reac-tion after intratracheal instillation but after beingheated for eight hours at 800°C, while still amorph-ous (by x ray diffraction), it was highly fibrogenic.Wagner et al in an inhalation study using calcined

diatomaceous earth (containing 61% cristobalite)

found no parenchymal fibrosis in rats or guinea pigsbut some hyalinised fibrotic nodules in the hilarlymph nodes of dogs.20Few in vitro studies have been conducted on

diatomaceous earths. Dusts cytotoxic towards mac-rophages in vitro usually cause fibrosis in animalstudies2' -23; thus the crystalline silicas quartz,tridymite, and cristobalite are cytotoxic.2' Daviesfound that several synthetic amorphous silicas werecytotoxic towards macrophages.24 Their (general)lack of fibrogenicity in animal studies may be due inpart to synthetic amorphous silicas being eliminatedfrom the lung more readily than quartz.25

Since the factors determining the biologicaleffects of diatomite are unclear, we examined theircytotoxicity towards macrophages in vitro. As thecomposition of diatomite may be critical, it was con-sidered essential to characterise the materials understudy by x ray diffraction and infrared spectroscopicanalysis given the changes in composition that ariseduring various calcining processes.

Materials and methods

MINERAL DUSTSStandard cristobalite was supplied by Arbetars-kyddsstyrelsen, Stockholm, Swedqn. The material isprepared by heating a-quartz (Fyle quartz) and asuitably fine fraction (<5 gm) obtained by sedimen-tation. DQ12 standard quartz26 was supplied by theInstitute of Occupational Medicine, Edinburgh, andmagnetite from BDH Chemicals Ltd, Poole, Dorset.Seven diatomaceous earth products (B-H in the

table) were examined. The natural material, thecrude diatomaceous earth, would have been dried attemperatures between 150 and 400°C, the calcinedmaterial heated to 870-1100°C, and the flux cal-cined material prepared by mixing the crude mater-ial with a flux, usually disodium carbonate, and heat-ing to about 1 1000C.'5 Suitable fine fractions wereobtained by sedimentation in distilled water. The

Silica content and biological activity. The amount ofsilica is determined by combined infrared spectroscopy and x raydiffraction, and the biological activity is evaluated as dust cytotoxicity towards mouse peritoneal macrophages

Code Name Type* a-Quartz Cristobalite Amorphous SiO2 Relative biological(%o) (%o) (%o) activityt

Estimate 95% Limits

A Cristobalite 0 100 0 0-13 0-12 0-15B Damolin GM-I n 2-7 0 45 0-20 0-19 0-22C Filter Cel n 1-0 0 109 0-55 0-50 0-61D Damolin KM-W c 2-4 0 72 0-16 0-15 0-18E Standard Super Cel c 0-9 6 100 0-18 0-16 0-19F Ceca Clarcel CBIJ2 c 0-7 11 94 0-59 0-54 0-64G Dicalite White Filler f <0-5 77 15 0-13 0-12 0-14H Ceca Clarcel DIV/2 f <0-5 85 0 0-11 0-10 0-12

*n= Natural; c = Calcined; f = Flux calcined.tDilution factors, on this scale - DQ12 quartz = 1-00 and magnetite = 0-02 (0-00-0-04).

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samples examined are representative of commer-

cially available products.DUST CHARACTERISATIONThe samples were examined by transmission elec-tron microscopy. Quantitative assessment of thesilica content was carried out by a combination ofinfrared spectroscopy and x ray powder diffrac-tion.27 Infrared analyses were carried out using a

JASCO IR-A2 spectrometer and the potassiumbromide disc procedure. Diffractometric measure-

ments were carried out with a Philips diffractometerequipped with a broad focus copper anode x ray

tube and a graphite crystal monochromator.The infrared techniques provide information of

the total silica content by integration with 10 cm-'intervals of the silica absorption region at 700-900cm-'. The x ray diffraction technique yields the crys-

talline silica content. One milligram samples are

suspended in water and filtrated with Nucleporefilters (pore size 0-8 ,um). A diffraction line from a

silver filter placed under the Nuclepore filter in thesample holder is used as an external standard forabsorption correction.28 The amorphous silica con-

tent may be obtained by subtraction of the crystal-line component from the total silica content. Thecorresponding calibration constants for the materi-als are established with Fyle quartz and our laborat-ory standards of cristobalite and Kieselguhr.

CULTURE OF MACROPHAGES WITH DUSTSUnstimulated mouse peritoneal macrophages were

obtained by lavage of 22-27 g female TO mice(Tuck and Son Ltd, Battlesbridge, Essex) with threeml Medium 199 (Flow Laboratories, Irvine, Scot-land) containing 5 IU heparin, 100 U benzylpenicil-lin, and 100 ,ug streptomycin/ml. Approximately 1-2x 106 cells (in 2 cm3 of the medium) were added toeach well of Linbro tissue culture multi-well plates(Flow Laboratories, Irvine, well diameter of24 mm), and left for one hour at 37°C in a 5%COJ/95% air atmosphere.At the end of the period the non-adherent cells

were removed by washing with phosphate bufferedsaline (PBS) and 2 ml Medium 199 containing theantibiotics and 10% newborn calf serum (FlowLaboratories, Irvine) was added. This serum hadpreviously been heat inactivated (56°C for 30minutes) and acid treated.29 Cultures were then leftfor 24 hours at 37°C in a 5% COJ95% air atmos-phere before the addition of fresh dust containingmedium.

Stock dust solutions were made up in PBS atapproximate concentrations, ultrasonication beingused to disperse the dust. The dust was added to themedium containing 10% serum at various concen-

Bye, Davies, Griffiths, Gylseth, and Moncrieff

trations (see results) and left for two to three hoursat 370C before being added to the macrophage cul-tures. Two ml " dusted" medium were added to eachof three culture dishes that were then incubated foranother 18 hours.At the end of this period the medium was col-

lected and the adherent cells disrupted by the addi-tion of 2 ml saline containing 0 1% Triton X100 andOl% bovine serum albumin, and by rubbing thedish with a sterile siliconised rubber bung. Bothmedium and cell lysates were centrifuged at 500 gfor 10 minutes and the supernatants assayed for lac-tate dehydrogenase (LDH) by a continous flowfluorimetric method30 using a Perkin Elmer model3000 fluorescence spectrometer.The release of LDH from the cells into the culture

medium is an indicator of dust cytotoxicity and iscalculated as:% enzyme released into culture medium =

MM+-C x 100

where M = enzyme activity of medium and C =enzyme activity of cell lysate.

ANALYSIS OF RESULTSIt is supposed that each mineral acts as a dilution ofquartz and the factor relating the concentration ofquartz equivalent to a concentration of a mineral iscalled the dilution factor. These factors were esti-mated by calculating regression equations for M/(M+C) in terms of dose with the common constraintthat the effects should be identical at zero concent-ration; 95% confidence limits were obtained usingFiellers theorem. The dilution factors so obtainedare mutually dependent through their common useof the estimated slope for quartz and the commonintercept.

Results

DUST MORPHOLOGYFigure 1 shows transmission electron micrographs ofstandard cristobalite and the seven diatomaceousearth materials under study. The samples are notrespirable fractions but each contains a high propor-tion of materials less than 10 ,um in diameter. Theparticles present in the diatomaceous earth sampleshave a wide variety of shapes consisting of fragmentsof diatom skeletons, the flux calcined samples (G,H) having an altered appearance.

SILICA CONTENTFigure 2 shows that the infrared absorption of theamorphous and crystalline silica overlap almostcompletely at 800 cm-'. Typical x ray powder dif-



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Fig 1 Transmission electron micrographs of diatomite materials. A-cristobalite; B-H--diatomite (see table foridentification).

fraction patterns are shown in fig 3; the infrared andx ray diffraction spectra of the other correspondingproducts are similar. The a-quartz content of thediatomaceous earth materials was low (table). Nocristobalite was found in the natural uncalcinedmaterial or in one of the calcined materials (D)which had been heated up to 650°C. In the calcinedmaterials (F & E) 11% and 6% cristobalite was pre-

sent and the flux calcined materials (G & H) con-tained 77% and 85% cristobalite, respectively. Boththe natural and calcined materials contain amorph-ous silica, which is significantly reduced in the fluxcalcined materials. It will be seen from the table thatthe total silica content of some samples issignificantly above 100%. This is mainly due to vari-ations in material properties, such as particle size,

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1200 Cf1800 400

Fig 2 Infrared spectra ofdiatomaceous earth, a-quartz,and cristobalite. Block letters refer to products (see table foridentification).

between different mineral qualities when only oneKieselguhr is used as a calibration standard.

MACROPHAGE CYTOTOXICITYThe release of the cytoplasmic enzyme lactatedehydrogenase from the macrophages (an indicationof cytotoxicity) after treatment with quartz, magne-tite, and the diatomaceous earth products are shownin fig 4; their relative biological activity is shown inthe table. The activities of the diatomite productsare dose related and are significantly higher thanmagnetite, a non-fibrogenic3' dust. The activity oftwo samples (C,F) was high, about 50% of theactivity of standard DQ12 quartz, which is one ofthe most cytotoxic of standard quartzes.32The activities of the high cristobalite flux calcined

materials (G, H) were similar to the standard cris-tobalite (A). Both the natural and calcined materialshad significantly greater activities than the flux cal-cined materials but this was not related to the quan-tity of crystalline or amorphous silica present.

Discussion

It is clear from this work, and an earlier study on

Degrees

Fig 3 X ray diffraction pattern ofdiatomaceous earthproducts, a-quartz, and cristobalite. The scan range forquantitative determinations are: a-quartz: 2600-27-68, 20(0); Cristobalite: 21 10-22-76, 20 (°); Silver: 37-20-38-87,20 (). The silver line (111) is used as an externalstandard.27 Block letters refer to products (see table foridentification).

synthetic amorphous silica,24 that the cytotoxicity ofsilica polymorphs towards macrophages is notdependent on the presence of a crystalline silicacomponent. The in vitro cytotoxicity test suggeststhat diatomite should be fibrogenic in vivo althoughno fibrosis was reported in the one publishedstudy.'8 Possibly amorphous silicas are a group ofmaterials that give positive results with the in vitrotechnique applied in the present study, but may havelow or no fibrogenicity in vivo. More animal inhala-tion data seems necessary to clarify this issue.Swensson and Klosterkotter found fibrogenic

effect with certain heated amorphous silicas whichdid not contain any crystalline components,'°1' andit would be useful to evaluate the changes thatamorphous silicas undergo during heating.No explanation can be given for the cytotoxic

effects of the diatomite products but it is possible

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that the surface hydroxyl groups33 or surfacecharge34 could play a part in membrane disruption ashas been suggested for quartz. The morphology ofthe particles could also play a part, some of the mat-erials had fibrous particles and it has been shownthat the cytotoxic effects of fibrous asbestos mineralsare related to their morphology.35The lower biological activity of the flux calcined

materials is not easily explained but could be relatedto alterations in particle morphology during thefluxing process or the effect the fluxing agent has onthe surface properties of the mineral.

We thank Dr J C Wagner for reading and MrsRosemary Hill and Miss Anne Eide for typing themanuscript. Mrs Sissel Olaisen is gratefully acknow-ledged for excellent technical help.

Product C 0 Product F O

Requests for reprints to: Dr E Bye, Institute ofOccupational Health, PO Box 8149 Dep, Oslo 1,Norway.

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2 King EJ, Mohanty GP, Harrison CV, Nagelschmidt G. Theaction of different forms of pure silica on the lungs of rats. Br JInd Med 1953;10:9-17.

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Plunkett ER, Dewitt BS. Occupational exposure to Hi-Sil andSilene. Arch Environ Health 1962;5:469-72.

6Wilson RK, Stevens PM, Lovejoy HB, Beil ZG, Richie RC.Respiratory effects of inhaled amorphous silica. In: DunnomDD, ed. Health effects ofsynthetic silica pariculates. American

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Society for Testing and Materials, 1981:185-98. (ASTM STP732.)

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21 Marks J, Nagelschmidt G. Study of the toxicity of dust in use ofthe in vitro dehydrogenase techniques. AMA Arch Ind Health1959;20:383-9.

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23 Miller K, Harington GS. Some biological effects of asbestos onmacrophages. Br J Exp Pathol 1972;53:397-405.

24 Davies R. Effects of synthetic silicas on mouse peritoneal mac-

rophages in vitro. In: Dunnom DD, ed. Health effects ofsynth-etic silica particulates. American Society for Testing and Mater-ials, 1981:67-81. (ASTM STP 732.)

25 Klosterkotter W, Bunemann G. Animal experiments on theelimination of inhaled dust. In: Davies CN, ed. Inhaled parti-cles and vapours. Oxford: Pergamon Press, 1961:327-41.

26 Robock K. Standard quartz DQ12 <5 gsm for experimentalpneumoconiosis research projects in the Federal Republic ofGermany. Ann Occup Hyg 1973;16:63-6.

27 Bye E, Edholm G, Gylseth B, Nicholson DN. On the determina-tion of crystalline silica in the presence of amorphous silica.Ann Occup Hyg 1980;23:329-34.

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29 Gordon S, Werb Z, Cohn ZA. In: Bloom BR, David JR, eds. Invitro methods in cell mediated and tumour immunity. London:Academic Press, 1976:344.

30 Morgan DML, Vint S, Rideout JM. Continuous flow fluorimetricassay of lysosomal enzymes. Med Lab Sci 1978;35:335-41.

31 Davis JMG. The fibrogenic effects of mineral dusts injected intothe pleural cavity of mice. Br J Exp Pathol 1972;53: 190-201.

32 Wagner MMF, Wagner JC, Davies R, Griffiths DM. Silica-induced malignant histiocytic lymphoma. Incidence linkedwith strain of rat and type of silica. Br J Cancer 1980;41:908-17.

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34 Nolan RP, Langer AM, Harington JS, Oster G, Selikoff LJ.Quartz hemolysis as related to its surface functionalities. Envi-ron Res 1981;26:503-20.

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