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Inhalation and injection studies in rats using dust samples fromchrysotile asbestos prepared by a wet dispersion process

Citation for published version:Davis, JMG, Addison, J, Bolton, RE, Donaldson, K & Jones, AD 1986, 'Inhalation and injection studies inrats using dust samples from chrysotile asbestos prepared by a wet dispersion process', British journal ofexperimental pathology, vol. 67, no. 1, pp. 113-29.

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Br. J. exp. Path. (I986) 67, II3-I29

Inhalation and injection studies in rats using dust.samples from chrysotile asbestos prepared by a wet

dispersion process

J.M.G. Davis, J. Addison, R.E. Bolton, K. Donaldson and A.D. JonesThe Institute of Occupational Medicine, 8 Roxburgh Place, Edinburgh EH8 9SU

Received for publication i 9 April I 98 5Accepted for publication 30 July I985

Summary. Long term inhalation studies and intraperitoneal injection studies in rats wereundertaken with a series of chrysotile asbestos dusts. Three dust samples were generated fromchrysotile modified by the wet dispersion process (WDC) and one was from unmodifiedchrysotile. Following a i year inhalation period, all the chrysotile samples proved extremelyfibrogenic and carcinogenic and there were no significant differences between the WDC dustsand normal chrysotile. In all experimental groups approximately 25% of animals developedpulmonary carcinomas and in the oldest rats advanced interstitial fibrosis occupied on averageio% of all lung tissue. In the injection studies all the dust samples produced mesotheliomas inover 90% of animals. Very little chrysotile remained in the lungs of the animals that survivedlongest following dust inhalation and what there was was present as individual chrysotilefibrils. It is suggested that chrysotile is potentially the most harmful variety of asbestos asshown in these and other animal studies but that it is removed from lung tissue quite rapidly. Inthe long lived human species this may mean that except where exposure levels are very highand of long duration, chrysotile should be less hazardous than other asbestos types.

Keywords: Chrysotile asbestos, wet dispersion process, carcinoma, fibrosis

The industrial use of asbestos has beenshown to result in considerable health risk toasbestos workers (Selikoff & Lee I978).Heavy exposure can result in pulmonaryinterstitial fibrosis (asbestosis), bronchialcarcinoma and mesothelioma.

While the main commercially used asbes-tos varieties differ markedly in their chemicalcomposition, all show considerable potentialto produce pulmonary disease when inhaled.It is now believed that the most importantdust factor in disease development may bethe physical dimension of the fibres, particu-larly length (Stanton 19 72; 19 7 7). Numer-ous experimental studies have been under-

taken in order to examine these effects butmost have used either in vitro or injectiontechniques because these require only smallamounts of dust which can be speciallyprepared. Long-term inhalation studiesunfortunately require large amounts ofasbestos dust and so far it has not beenpossible to obtain sufficient size-selecteddusts of any single asbestos type to examinethe effects of fibre length when dusts areinhaled. As an alternative approach, studiesare continuing in this Institute using suchasbestos materials as are available that arelikely to produce dust clouds of significantlydifferent fibre dimensions. One such type of

II3

J.M.G. Davis et al.material is chrysotile that has been treatedby the 'wet dispersion' process. This processis used commercially with minor variationsthroughout the world and its basic principleshave been described by Heron & Huggett(I97I). In the wet dispersion process bulkchrysotile is treated with a dilute solution ofan anionic surface active agent so that theindividual chrysotile fibrils separate to pro-duce a liquid slurry. The fibrils coagulate intoa film on the application of electrolyte andthis process can be controlled to spin out ayarn in which all the fibrils are intermingledand bound to one another. The yarn is thentreated to remove excess surface active agentalthough a small amount remains bonded tothe fibril surface. This is in contrast to normalchrysotile textile yarn which consists ofseparate chrysotile fibres, mainly parallel toone another and held together only by thespinning process. Smither & Lewinsohn(I973) described wet dispersed chrysotileyarn as being smoother, stronger and moreregular than standard asbestos yarns andsuggested that its use might be without thedust-related health hazards of chrysotile pro-duced by conventional techniques. Becausethe chrysotile fibril arrangement in wetdispersed chrysotile yarn is so different frommaterials previously used in animal experi-mentation, it was considered that an inhala-tion study using this material might producenew information on the way pulmonarytissues react with different types of fibre.Parallel injection studies were conducted forcloser comparison with Stanton's work.

Materials and methods

Experimental protocol for inhalation studies.Groups of 48 white SPF rats of the AF Hanstrain were exposed by inhalation to one offour different chrysotile preparations. Thesewere: (a) Yarn from wet dispersed chrysotileproduction process. (WDC yarn); (b) Dustcollected from the factory air in a workshopprocessing only this type of WDC (FactoryWDC); (c) A standard chrysotile textile yarnproduced by traditional methods (Chrysotile

yarn); (d) Yarn from an experimental WDCprocess which was never commercialized(Exp. WDC).

Dusting was for 7 h a day, 5 days a weekfor a total of 224 days during a period of I2months. One additional group of rats wasexposed to dust from (d) using a reverseddaylight regimen in which the rats wereexposed during their period of maximumactivity. This experimental variation wasincluded because there was some evidence(Middleton et al. I979) that pulmonarydeposition is increased under these condi-tions and this could affect the development ofpulmonary pathology. This dust treatmentgroup is subsequently referred to as 'WDCReversed Daylight'. Groups of 39 and 25control rats were maintained for their full lifespan during the period of these inhalationstudies.

Dust cloud generation and monitoring. Dustclouds were generated by the methods de-scribed previously by Davis et al. (I 9 8 5a) andsummarized in Table i. The dust generatorswere able to produce satisfactory clouds fromthe factory WDC as supplied but dust gene-ration from WDC yarn and Exp. WDC proveddifficult and these yarns required a vigorouspreliminary milling using a Christie andNorris mill. The sample of chrysotile yarnwas treated in a similar manner to ensurecomparability with the dusts generated fromthe WDC preparations. Dust concentrationsin the chamber were monitored dailythroughout all exposure time for mass con-centration and by approximately ioo shortperiod (snatch) samples at intervals duringthe exposure period for number concentra-tion. The fibre size distributions wereassessed by phase contrast optical micros-copy (PCOM) for fibres > 5 ,m in length andboth fibre length and diameter distributionswere obtained by scanning electron micros-copy (SEM) for fibres >0.4 gm in length and>0. I um in diameter.

Histopathological studies. Four animals fromeach inhalation group were killed at the endof the I 2 month dusting period and four

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more were killed 6 months later. The remain-ing animals were left for their full life spanexcept that the study was terminated whenthe number of survivors in some groupsdropped to six. Estimations of early patho-logical changes were limited to the smallgroups of animals from the first two killingdates. However, for the more advancedlesions occurring in the oldest animals, itwas decided to include all those dying within2 months of the final killing date. In practicethis produced groups of animals varyingfrom 14 to 21 in number. All control animalswere allowed to survive for their full life span.Tissue used for histological examination wasfixed in Karnovsky's fixative. Lungs werefixed by inflation in situ at a standard pres-sure of 30 cm of fixative. Subsequently thetrachea was ligated and the lungs excisedand immersed in fixative before histologicalprocessing and embedding in paraffin wax.Sections were cut in the coronal plane at imm intervals and were stained by eitherhaemotoxilin and eosin, Van Gieson'smethod for collagen or Gordon and Sweet'sstain for reticulin. Tissue samples from someof the oldest animals were prepared fortransmission electron microscopy by postfix-ation in a 2% solution of osmium tetroxidebefore embedding in araldite. Sections werestained with both lead citrate and uranylacetate.Measurement of pulmonary fibrosis was

undertaken by similar methods to thosepreviously published (Davis et al. 1978)except that an electronic image analyser(Graphic Information Systems Limited,GDSi) was available for use in conjunctionwith the light microscope. Single coronallung sections were used with the sectionselected from the central region but avoidingthe major conducting airways and pulmon-ary vasculature. Interstitial fibrosis was esti-mated using a X 2 microscope objective lensand is expressed as a percentage of total lungtissue area (Davis et al. 1978). Peribronchio-lar lesions are more numerous and smallerand so the lung tissue was scanned with ax 4 objective, using an eyepiece graticule

divided into I00 squares which at thismagnification covered a tissue area of 2.9mm2. Peribronchiolar lesions were recordedas a percentage of squares containing lesionsof this type.

Lung dust content. Lung dust estimationswere performed on animals from the first twokilling dates. Only the left lung was used sothat the right lung was available for histolo-gical studies. Dust retained in the lungs wasrecovered by a low temperature plasmaashing process using a Nanotech Piooapparatus and estimated by infrared spectro-photometry (Dodgson & Whittaker 1973).Studies in this laboratory have shown thatthe dust content ratio between left and rightlungs following experimental inhalation offibrous dust such as asbestos in rats is o.6: Iand this correction factor was therefore usedto estimate the pulmonary dust burden ofeach animal.

Animal injection studies. In addition to theinhalation studies, the ability of the fourchrysotile samples to produce mesotheliomawas examined using the intraperitonealinjection assay. For the WDC yarn, thefactory WDC and the chrysotile yarn, groupsof 24 rats received a dose of 25 mg of dust.For the exp. WDC, the group size was 32animals. The dust samples were suspendedin 2 ml of Dulbecco's phosphate bufferedsaline and were injected into the peritonealcavities of the rats under ether anaesthetic.The dust was collected from the animalinhalation chambers using an elutriationprocess in an attempt to simulate the respir-able fraction of the dust clouds used in theinhalation studies (Bolton et al. I 982).

Results

Dust cloud generation and measurement

During development work on dust gene-ration procedures it was found to be ex-tremely difficult to generate high densityclouds of respirable fibres from bulk prep-

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arations of wet dispersed chrysotile (WDC).This applied particularly to the exp. WDCsample. Even after treatment with a Christieand Norris Mill it was found that the maxi-mum concentration of respirable dust thatcould be generated was approximately 4 mg/m3 of air. It was therefore decided to under-take the present study at this dust concentra-tion even though the results would not bedirectly comparable with previous long-terminhalation studies with asbestos from thisInstitute which had standardized on anexposure concentration of I0 mg/m3 (Daviset al. I978, I980, i985a, b).The average respirable dust mass concen-

trations that were attained for all five expo-sures were close to the planned level of4 mg/mi3. These figures together with total dustmass concentrations and fibre number con-centrations are given in Table 2. The respir-able mass concentration of chrysotile esti-mated by infrared absorption spectroscopyfalls within the range 3.3-3.7 mg/m3 forfour experiments but is 2.8 mg/m3 for thefactory WDC. This sample was, however,known to contain some non chrysotile con-, ,, .,, I ,9590 ,

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Studies in rats with dust from wet dispersed chrysotiletaminants from the factory environment. Forthree of the dusts used in this study, WDCyarn, factory WDC and chrysotile yarn, therespirable mass concentrations were about80% of the total dust concentration. For theexp. WDC, however, the equivalent valuewas only 60%. This indicates that the dustcloud generated from this material containedcoarser fibres and this is confirmed by thenumber concentration and the size distribu-tion data illustrated in Table 2 and Figs I, 2and 3. The number concentrations (fibres> 5 ,um in length) obtained by PCOM wereapproximately I00 fibres per ml for the exp.WDC and in the range 400-700 fibres per mlfor the other clouds. Fibre length distribu-tions from the experimental dust cloudsobtained by both light microscopy (magnifi-cation x 6oo) and SEM (magnificationx io ooo) are illustrated in Figs I and 2 andSEM diameter distributions are given in Fig.

3. These size distributions confirm that theexp. WDC consisted of fibres that on averagewere much longer and slightly thicker thanthe other materials in the study. The fibrelength distributions obtained by light micros-copy suggested that the dusts from WDCyarn, factory WDC and chrysotile yarn werevery similar but SEM examination showedthat the WDC yarn had fibres slightly longerthan the factory WDC or the chrysotile yarn.

Histopathological findings

All five groups of rats dusted with thechrysotile preparations developed the samepattern of pathological change previouslyreported in similar studies from this Institute.At the end of the I2 months dusting periodthe main lesions present were deposits ofgranulation tissue around the terminal andrespiratory bronchioles (Figure 4). This gra-

Fig 4. An area of fibrotic granulation tissue close to the terminal bronchiole of a rat treated by inhalationwith dust from wet dispersed chrysotile for twelve months. x 320.

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Studies in rats with dust from wet dispersed chrysotilenulation tissue consisted mainly of macro-phages and fibroblasts but foreign body giantcells were also present. At 12 months afterthe start of dusting there was marked reticu-lin staining in the peribronchiolar depositsalthough relatively little collagen could bedemonstrated by Van Gieson's stain. Withincreasing time after dust exposure, how-ever, collagen staining became progressivelymore marked. All treatment groups showeddeposits of this peribronchial fibrosis aroundmany of the smallest airways at i2 months(Table 3) but levels in the two groups ofanimals treated with exp. WDC were signifi-cantly higher than for the groups treatedwith WDC yarn, factory WDC or chrysotileyarn. (P= <0.05). By i8 months, four out ofthe five treatment groups showed a reduc-tion in peribronchiolar fibrosis and takenoverall, this reduction was significant(P= <0.05). After i8 months from the start

of dusting, widespread alveolar interstitialfibrosis developed and this tended to obscurethe earlier fibrotic deposits. For this reason,estimations of peribronchiolar fibrosis werelimited to the first two killing dates.From about i 8 months onwards, areas of

lung tissue in some animals showed a pro-gressive thickening of alveola septa. (Fig. 5).In its earliest form this thickening wascaused almost entirely by hyperplasia ofalveolar lining cells, but later there wasconsiderable deposition of reticulin andeventually collagen in the septal walls. Asshown in Table 3, areas of alveolar intersti-tial fibrosis became more widespread in alltreatment groups with increasing time afterthe end of the dusting period. While the areasmeasured varied from a mean of 8.8% oflung tissue in animals treated with chrysotileyarn to 12.8% in animals treated with WDCyarn, these differences were not statistically

Fig 5. Alveolar interstitial fibrosis in a rat treated by inhalation with dust from wet dispersed chrysotile for24 months. x i88.

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Studies in rats with dust from wet dispersed chrysotilesignificant. In many areas of lung tissue theinterstitial fibrotic element of these lesionsremained predominant throughout thestudy, but in others the hyperplasia of alveo-lar epithelial cells became progressively moremarked to produce a pattern of adenomato-sis. Some definite adenomas could be seen tohave developed from the central regions ofthese areas and it is likely that this was alsothe site of origin of some carcinomasalthough by the time most of these werediscovered they were too widespread to becertain.

All five groups of dusted rats in this studydeveloped primary pulmonary tumours inover 35% of animals with no significantdifferences between treatment groups. (Table4). While carcinomas predominated in fourof the groups, the group treated with factoryWDC, which had the highest overall figuresdid have i i adenomas to i o carcinomas.One adenoma and one carcinoma were

found in control animals. The carcinomawas a small lesion (2 mm diameter) whichshowed only early signs of invasion and wasnot responsible for the death of the animal.As with other animal studies in which

asbestos has been administered by inhala-tion, relatively few mesotheliomas developed(Table 4) but the groups of rats treated withthe various chrysotile preparations allshowed areas of vesicular pleural meta-plasia. Histological examination showedthese areas only occurred where patches ofalveolar interstitial fibrosis had reached thelung surface although this pleural involve-ment did not always result in metaplasia.Areas of metaplasia consisted of loose fibroustissue containing large vesicular spaces linedwith flattened cells. (Fig. 6). Electron micro-scope studies showed that these cells were ofmesothelial type and that occasionally thewalls between vesicular spaces were so thinthat they consisted of two closely apposed

Fig, 6. Vesicular metaplasia on the pleural surface of a rat treated by inhalation with dust from wetdispersed chrysotile. The pulmonary parenchymal tissue close to the pleura shows alveolar interstitialfibrosis with the alveolar spaces lined with prominent rounded epithelial cells. x 320.

I23

Fig 7. Transmission electron microscope photograph of the wall of one vesicle in an area of pleuralmetaplasia. In this case the extremely thin wall consists only oftwo flattened cells of mesothelial type withno basement membrane visible between them. x 32 300.

Fig 8. Transmission electron microscope photograph of an area of pulmonary fibrosis from the lung of arat twenty seven months after the start of treatment with dust from wet dispersed chrysotile. Numerousindividual chrysotile fibrils are visible among the collagen fibres, the longest chrysotile fibril in thisphotograph being over 5 ,um in length. x 36 ooo.

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layers of extremely extended and flattenedcells with no basement membrane. (Fig. 7).Where cells were supported by areas offibrosis a basement membrane was present.

Electron microscope examination of speci-mens of lung parenchyma from some of theanimals in the present study, several monthsafter the end of dusting showed that most ofthe original chrysotile fibre bundles hadseparated into individual fibrils. This was

particularly marked with the preparations ofWDC (Fig. 8) but was also apparent in tissuesfrom animals treated with dust from thestandard chrysotile yarn. In the animals thatsurvived longest after the end of dust expo-

sure, only small amounts of chrysotileremained in the lung tissue and this was

almost entirely confined to two tissue sites.These were firstly, areas of fibrosis, wherechrysotile fibrils could be found amongst thecollagen fibres and secondly, in the alveolarbasement membranes, which were oftengreatly thickened. Almost no chrysotile wasfound in alveolar macrophages in the oldestrats from the present study.

Apart from the pulmonary tissue both thefive treatment groups of rats and the twocontrol groups progressively showed thenormal types of pathological change asso-

ciated with advanced age. The most commonlesion in this category was chronic progres-sive nephrosis which was found in over 5o%of animals. The numbers of non-pulmonarytumours found in the present study are listed

in Table 5. There were no significant differ-ences between the five dust exposure groups.

One batch of controls did have noticeablymore tumours than chrysotile treated ani-mals but since this was not apparent in theother control group it is likely to have beendue to chance.

Lung dust burden

The retained lung burden of chrysotile dustat the end of a dusting period and six monthslater is illustrated in Table 6 for all treatmentgroups. At the end of dusting the mean

figures ranged between 250 pg and 500 pg

for the five groups while 6 months laterbetween 15% and 60% of this dust had beencleared. The lung dust samples from one

group of animals killed at i8 months aftertreatment with the exp. WDC were acciden-tally destroyed during analysis.

Injection studies

All the four samples of chrysotile underconsideration produced mesotheliomas inover 90% of rats following the intra-perito-neal injection of 25 mg of dust. A differencewas apparent, however, in the mean tumourinduction period. While the WDC yarn,

factory WDC and exp. WDC all producedmesotheliomas in an average of 310-340days, the standard chrysotile yarn requiredan average of 400.

Table 6. Mean masses of chrysotile dust receovered from rat lung tissue

Time after the end of dusting

Dust type 3 days 6 months

WDC yarn 420ug (I20) I50 Pg (55)Factory WDC 240 ug (8) I20 pg (33)Chrysotile yarn 400 Pg (94) 230 Pg (49)Exp. WDC 490 pg (78)Exp. WDC (Reversed daylight) 280 pg (25) 240 pg (68)

Figures in brackets are standard deviations.

126

Studies in rats with dust from wet dispersed chrysotile

Discussion

The three samples ofwet dispersed chrysotileand one sample of dust from normal chryso-tile textile yarn examined in the presentstudy all proved to be highly fibrogenic andcarcinogenic. Even at mass dose levels of4 mg/m3 these materials were more patho-genic to rats than UICC Rhodesian chrysotileat Io mg/m3 (Davis et al. 19 78). The studieswere undertaken at this low dust concentra-tion because it had proved extremely difficultto generate clouds of respirable dust fromany of the WDC samples. In order to generatesuitable clouds with these materials it wasnecessary to subject the yarn to a prelimin-ary milling. Because of the low dust concen-

tration, the high levels of pulmonary diseasefound in all experimental groups were unex-

pected. This raises the question of whether or

not the reduced hazard suggested for theindustrial use ofWDC products because theygenerate little dust (Smither & LewinsohnI 9 73) is offset to some degree by an increasein the harmfulness of the dust that is pro-

duced.The results from this study do not, how-

ever, prove that the wet dispersed form ofchrysotile is any more harmful to rats thanother chrysotile materials, since normalchrysotile yarn subjected to a similar preli-minary milling was found to be equallydangerous. It is interesting that UICC chryso-tile has proved less active than these otherchrysotile dusts but this may result from thepreparation techniques used for all the UICCsamples (Rendall I980). The UICC standardreference samples of respirable asbestosfibres have amply fulfilled the purpose forwhich they were prepared and have provedextremely useful for many research purposesbut there have been criticisms that they weretoo thoroughly milled and are too short toproduce the maximum tissue response. Stan-ton et al. (I972, 1977) concluded that themost important factor in fibre pathogenesiswas the number of long thin fibres in dustsand it would appear that the UICC samples

have relatively few fibres of the most danger-ous size.The present study has confirmed that most

chrysotile dusts produce very severe effects inrats. This has previously been noted in anumber of publications where chrysotile hasappeared more harmful than the amphiboles(Wagner et al. I973, I974; Davis et al.1978). Some of this increased pathogenicitymay be due to the fact that the chrysotilepreparations had more long fibres than theamphiboles. However, some recent work inour Institute with a long amosite dust cloudindicates that other factors may be involved(Davis et al. 198sb). This amosite dust cloudhad more fibres > 5 ,m in length (2000/mlby light microscopy) than the chrysotile andWDC products reported in the present paper,was used at a higher airborne mass concen-tration (io mg/m3) but produced fewerpulmonary tumours and less pulmonaryfibrosis than these chrysotile preparations.The additional factor found with chrysotiledust may relate to the separation of inhaledfibres into their individual fibrils particularlyin lung tissue where there are high concen-trations of surfactant. This would result in atissue dose ofdangerous fibre units far higherthan indicated by counts of airborne dusts.The separation of chrysotile fibrils in tissue

could explain the major anomaly that hasbeen found between animal experimentalstudies and human epidemiology. Whileexperimental studies almost without excep-tion have reported chrysotile to be the mostdangerous fibre tested, studies of asbestosworkers have indicated that amphiboledusts, particularly crocidolite, are the mostharmful to humans (Wagner et al. I960,Weill et al. 1979). Studies of lung dustcontent in humans have shown that whilechrysotile has usually formed the major partof any dust exposure, amphibole fibres pre-dominate in the lungs at autopsy (Gylseth etal. I983). It would appear very likely thatwhile chrysotile can persist long enough in ashort lived species like the rat to provoke aneoplastic response or pulmonary fibrosis, itmay be removed from human lungs before

I27

128 J.M.G. Davis et al.disease can develop. The separation of chry-sotile bundles into individual fibrils is likelyto be important in this process since theselong but extremely thin structures might beexpected to be more susceptible to dissolu-tion.The separation of WDC products into

individual chrysotile fibrils within the tissuesmight explain the findings from the presentinhalation studies using the sample of experi-mental WDC. The 4 mg/m3 dust clouds ofthis material contained far fewer fibres thanthe other dust clouds studied but thosepresent were longer and thicker. If all inhaledfibres that were deposited in the lung tissueseparated into individual fibrils, then thenumber of these subunits would probably besimilar with all the products tested and thesimilarity in the pathological response wouldnot have been surprising.

Acknowledgements

This study was undertaken as part of theresearch programme sponsored by the Bri-tish Asbestosis Research Council.

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