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Carcinogenesis vol.6 no.5 pp.667-674. 1985 Inhalation studies on the effects of tremolite and brucite dust in rats 1181 id 1189 J.M.G. Davis. J. Addison, R.E. Bolton, K. Donaldson, A.D. Jones and B.G. Miller talc, chrysotile and vermiculite (2). The identification sample of tremolite as asbestiform depends among other 1195 1201 1207 1211 1217 1227 Institute of Occupational Medicine, Roxburgh Place, Edinburgh EH8 9SU, UK Samples of commercially used asbestos, especially chryso- file, are frequently contaminated by small amounts of other fibrous minerals. Among these are tremolite and brucite although pure tremolite is also produced commercially in relatively small quantities. In order to determine how harmful commercially exploited tremolite might be in comparison with other asbestos types and to explore the possibility that small amounts of tremolite and brucite as contaminants could significantly affect the pathogenicity of industrially used chrysotile, long-term animal inhalation and injection studies using rats were undertaken with what were considered to be mineralogically pure samples ofthese minerals. Rats treated with tremolite developed very high levels of pulmonary fibrosis as well as 16 carcinomas and two mesotheliomas in a group of 39 animals. Tremolite thus proved to be the most dangerous mineral , that we have studied. Animals treated with `brucite' developed moderate levels of pulmonary fibrosis and two carcinomas. Both tremolite and brucite produced mesotheliomas in >90% of animals following i.p. injection. However, it was found that the supposedly pure brucite in fact contained 10% chry sotile, a level of contamination that could well have been responsible for the pathological changes found in both inhalation and intraperitoneal injection studies. The greatest care should be exercised by industry in handling tremolite or materials contaminated with it. upon its length and diameter distributions. Campbell (. Campbell et al. (4) have shown that asbestiform variet minerals have significantly longer and finer size distrib than their non-asbestiform equivalents. Environmental exposure to tremolite in Turkey ha. implicated in the development of human bronchia] care; and mesotheliomas (5). It has also been reported that the of chrysotile miners can contain as much tremolite at autc chrysotile even though only a small percentage of tremol present in the original ore body (6,7). Mesotheiior workers exposed to chrysotile are extremely rare and been disputed whether or not chrysotile alone can produc tumours in humans. A few well-documented cases of theiioma in chrysotile workers do exist, where expos crocidolite or amosite appears to have been excluded b' it would appear that tremolite rather than chrysotile itsel be the cause. Experimental in vitro and in vivo injection so for undertaken (8) confirm that tremolite fibres . carcinogenic in experimental animals. So far there epidemiological evidence to suggest that brucite is imp in human disease but animal injection studies by Pott (9,10) show that brucite fibres can produce mesothelic injected into experimental animals. At the lOM in Edinburgh we have used both inhalatii injection studies to compare the biological effects of a r. of samples of asbestos and other mineral fibres. The : studies were undertaken in order to compare the h potential of tremolite and brucite to that of these other materials and to obtain more information on the likely ; ance of their presence as contaminants in other products 1231 Introduction commercially used talc and chrysotile. The fibrogenic and carcinogenic potential of the main commercially used varieties of asbestos have been well docu Materials and methods 1235 1239 mented (1). Recently, however, attention has been drawn to the fact that asbestos ore bodies, particularly those of chrysotile, are not mineralogically pure. Mixed with the asbestos are other minerals, some of them fibrous, which remain as contaminants Mineralogy The sample of 'tremolite' used in this study was a commercial mater Korea. It contained --V5% by mass of pure fibrous tremolite wr chemically and structurally consistent with published data as conf:; 1243 in the final asbestos products supplied to industry from the mines. Two such minerals are brucite and tremolite. Brucite, Mg(OH)j, is quite distinct from all the asbestos varieties in that it does not contain silicon but it is commonly found in chry sotile and other serpentines since it often forms in similar geo scanning electron microscopy (SEM)* and X-ray diffraction (XRD) However, a few XRD peaks were present which could not be account, tremolite. Some of these impurities had a layer structure with a 7 3.6 A spacing typical of minerals such as kaolinite. serpentine, or ch The presence of talc was also suspected. The tremolite itself was ch pure, being composed almost entirely ofcalcium, magnesium and silic logical conditions. It can occur in the form of solid fibres, as tubular crystals, or in large foliated masses. Tremolite, Ca2MgsSijO:,(OH),, is a commonly occurring amphibole mineral which is only rarely found as a pure fibrous asbesti- minor amounts of iron, well within the limits of the composition permitted for the mineral. The brucite used in this study was chemically and structurally consts published data. The specimen was 85 --90% by mass of pure bruc' firmed by SEM and XRD analysis. In addition to brucite, however, form variety in quantities large enough for commercial exploi tation. It is found more frequently in both the asbestiform and non-fibrous states as a contaminant in other materials such as material contained Pyraurite (Mg,FejCO)(OH),,4H.O). This appmottled brown segments on a proportion of the brucite fibres under dr microscope. The Pyraurite probably comprised 5--10% of the bulk Chrysotile was also present as confirmed by XRD. Since, however. _ `Abbreviations: SEM, scanning electron microscopy; XRD, X-ray diffraction. of analysis gives poor quantitative results for chrysotiie when small are mixed with other minerals, the bulk sample was treated with glac: acid to remove the brucite and the chrysotile content was estimate^ IRL Press Ltd., Oxford. England. J.M.G. Davis et al. 99 90 J 1 80 2 70 ((/S) 60 r 40 2 30. 5 5 20 s 10 Z ^ 55 2. 1. 0.5. 5 TREMOUTE O BRUCITE o o o o 30 40 50 100 fibre Length (pm) 200 500 I. Length distributions of fibres of treraolite and brucite >5 /un in Rgth obtained by phase contrast fight microscopy. analysis. This technique showed that chrysotile comprised -10% of the `brucite' sample. Dust generation anti monitoring The planned dust concentrations was 10 mg/m1 of respirable dust. The dust clouds were generated using a Timbrel! dust generator and inhalation chambers as described by Timbrell et al. (11) but modified as described by Beckett (12). Selecting the airborne dust by a cyclone system prior to injecting it into the chamber airstream ensured a high proportion of respirable dust in the clouds. The dust generators were able to produce suitable clouds of tremolite dust from the bulk sample as it was received. Generation of a respirable dust cloud from the brucite sample required some preliminary breakage and dispersion by pass ing it through square-toothed intermeshed steel rollers prior to loading into the dust generator. The mass concentration of dust in the chambers was measured daily by sampling throughout the 7 h of exposure using both an open filter holder facing vertically downwards and a Casella MRE113A dust sampling instrument. The former sampler monitored the total dust concentration and the latter monitored the respirable dust mass concentration. The target mean respirable dust con centration of 10 mg/m1 was achieved by adjusting the equipment in response to each daily measurement. The fibre number concentration was estimated from short period membrane filter samples taken on - 100 separate days. The number concentration for these samples was taken as being proportional to the mass concentration for that day. thus giving a mass/number ratio which was then averaged over 100 days. The membrane filler samples were examined by phase-contrast optical micro scopy, following the normal practice for occupational hygiene measurements (13). This routine technique defines fibres to be included in the count by criteria of size: length >5 microns, diameter <3 microns, and aspect ratio >3:1. The size distribution of fibres was examined by both phase-contrast optical ^nicroscopy and SEM. The optical studies were performed by a single Observer. but all SEM studies were undertaken by two observers. Samples for jpoetron microscopy were collected on Nucleopore filters. A portion of each filter was subsequently attached to an electron microscope stub with a conduc tive adhesive (carbon paste) and sputter coated with a thin layer of gold. The samples were examined by SEM (Cambridge Instruments S600) at 10 OOOx magnification and measurements taken on the length and diameter of those fibres with aspect ratio >3:1. length >0.4 microns and diameter >3 microns. 668 The minimum length required arises from the combination of the aspect ratio definition and the resolution limit, which for the SEM corresponds to a fibre of - 0.1 micron diameter. Some fibres of <0.1 micron diameter were detected and reported and measured as closer to 0.05 microns but generally fibre dia meters were measured to the nearest 0.1 micron. ] Animal inhalation studies For the inhalation studies groups of 48 SPF male Wistar rats of the AF/Han strain were exposed to either tremolite or brucite dust for 7 h each day, 5 days a week, for a total of 224 days, during a period of 12 months. The animals were 10 weeks old at the start of dusting. A batch of 36 undusted animals was maintabled within the same unit as controls during the same overall time period. Four animals from each group were killed at the end of the 12 months dusting period and four more were killed 6 months later. The remaining animals were left for their full life span except that the study was terminated when the number of survivors in one group dropped to six. Estimations of early pathological lesions were limited to the small groups of animals from the first two lulling dates. However, for the more advanced lesions occurring in the oldest animals, it was decided to include all those dying within 2 months of the final killing date. In practice this produced groups of 12 animals for each dust treatment. Tissue used for histological examination was fixed with 10% formol saline solution and embedded in paraffin wax. Lungs were fixed by inflation at a standard pressure of 30 cm of fixative. Subsequently the tracheas were ligated and the lungs excised and immersed in fixative. Sections were cut in the coronal plane at 1 mm intervals and were stained by either haematoxyiin and eosin. Van Gcison's method for collagen or Gordon and Sweet's stain for reticulin. Measurement of pulmonary fibrosis was undertaken by similar methods to those previously published by Davis et al (14) except that an electronic image analyser (Graphic Information Systems Limited. GDS1) was available for use in conjunction with the light microscope. Single lung sections were examined with the section selected to contain the maximum area of lung parenchyma. As previously described, interstitial fibrosis was estimated using a 2x microscope objective lens and is expressed as a percentage of total lung-tissue area. Peribronchiotar lesions are more numerous and smaller and so the lung tissue was scanned wtth an eye-piece graticule covering an area of 2.91 mm and divided into 100. squares. A 4x objective lens was used. Peribronchiolar lesions were recorded as a percentage of squares containing lesions of this type. Small areas of irregular alveolar wall damage were measured by the same techniques. However, since these lesions were difficult in all cases to distinguish from frag ments of peribronchiolar fibrosis included in the sections an overall measure ment was undertaken initially to include both lesions, followed by a second one involving only definite peribronchiolar fibrosis. An estimate of lung tissue with irregular alveolar wall damage was obtained by subtraction. j / i ' , 1* j ; 1 Lung dust content Lung dust estimations were performed on animals from the first two killing dates. Only the left lung was used so that the right lung was available for histo logical studies. Dust retained in the lungs was recovered by a low-temperature j plasma ashing process using a Nanotech P100 apparatus. Studies in this laboratory have shown that the dust content ratio between left artd right lungs following experimental inhalation of fibrous dusts such as asbestos in rats is 0.6:1 and this correction factor was therefore used to estimate the total pulmonary dust burden of each animal. The tremolite residues were washed in 0.2 m HC1 at room temperature before estimations of the amounts of retained fibre were made using the i.r. spectrophotometer techniques described by Middleton etal. (15). Since brucite is known to be highly soluble in acids, the brucite residues were washed only with distilled water initially. However, because the original dust cloud had | contained a significant proportion of chrysotile asbestos, the analysis of these lung dust residues was repeated after washing with glacial acetic acid. Animal injection studies In addition to the inhalation studies, the ability of the tremolite and brucite to produce mesotheliomas was examined using the i.p. injection assay. A dose of 25 mg of dust suspended in 2 ml of Dulbecco's phosphate buffered saline was injected under ether anaesthetic into the peritoneal cavities of two groups of 32 rats of the AF/Han strain. The dust was collected from the animal inhalation chamber by an elutriation process and represented the respirable fraction of the dust cloud (16). Results Dust characterisation The planned average mass dust concentration of both tremolite and brucite was achieved during the dusting period with >40% of the daily dust concentrations within 3 mg/mJ of the target concentration. oct ratio i fibre of detected AH/Han .5 days mis were as mainperiod. dusting lals were ' number notogical o killing animals, al killing eatment. 10I saline ition at a re ligated .e coronal osin. Van ulin. icthods to nic image le for use examined hvma. As iicroscope irea. Peritissue was id divided .ions were mall areas Inhalation of tremolite and brucite dust i: .econd one tissue with Fig. 2. SHM of the tremolite dust used in the inhalation and injection studies. While the tremolite fibres are of uniform type the brucite is a mixture of relatively thick straight brucite fibres and thin curly ones which are chrysotile asbestos. (10 OOOx magnification) wo killing e for histomperature its in this The fibre number concentrations of the materials as deter mined by phase-contrast optical microscopy for fibres longer than 5 microns was 1600/ml for tremolite and 230/ml for right lungs brucite. This substantial difference in fibre number for equal s in rats is e the total mass concentrations of respirable dust was partly due to the large amount of non-fibrous dust in the brucite cloud as well as emperature mg the i.r. to differences in the fibre dimensions. The length distribution of 1000 fibres longer than 5 microns, as determined by one nee brucite ashed only cloud had sis of these icid. observer using phase-contrast optical microscopy, is shown in Figure 1. The tremolite sample contained more fibres between 10 pm and 100 pm in length but the differences were small. Typical SEM photographs ofthe tremolite and brucite dusts are illustrated in Figures 2 and 3. The tremolite is seen to consist d brucite to i. A dose of almost entirely of straight fibres of similar type, many of them extremely thin. The brucite sample, however, is more complex. 1 saline was :aiups of 32 il inhalation iction of the Apart from particulate material the bulk of the sample is made up of straight fibres with a relatively large diameter. These are identifiable by EDXA analysis as brucite. There are also large numbers of very thin fibres, the longest of which are distinctly curly. EDXA analysis showed that these are chrysotile. Analy sis by i.r. subsequent to washing with glacial acetic acid showed that the proportion of chrysotile was '10% by mass. elite Almost all the fibres seen with the resolution of the light micro t40% scope counts would have been brucite but the majority counted by SEM were probably chrysotile. the target The cumulative fibre size distributions obtained from the SEM at 10 OOOx magnification are produced in Figures 4. These showed that the tremolite sample contained a a proportion of long fibres than the brucite sample, and th tremolite fibres on average were also thinner unless they extremely short. The distributions were obtained b;observer using the SEM, although a second observer conr" that the tremolite samples tended to be longer and thinne: the brucite. However, there were differences betwee absolute fibre size estimates obtained by the two obse: confirming the recent observations of Cherrie etal.(ll). it was found that inter-observer differences were a source of variation in fibre size estimations obtained : Cambridge Instruments S600 SEM. In the same study, shown that any single observer tended to produce con. fibre size estimates from repeated assessments, and a reason the data presented in Figures 4 and 5 were cons appropriate to illustrate the relative differences in the si: the tremolite and brucite dust samples. Histopathological findings Both groups of dusted animals developed the same pattepathological change previously reported in similar studies the Institute but to varying degrees. At the end of the 12- dusting period the main lesions present were deposits of i lation tissue around the terminal and respiratory bronc(Figure 6). This granulation tissue consisted mainly of rr Fig. 3. SEM of the brucite dust used in the inhalation and injection Studies. While the tremolite fibres are of uniform type the brucite is a mixture of relatively thick straight brucite fibres and thin curly ones which are chrysotile asbestos. (10 OOOx magnification) phages and fibroblasts .but a few foreign giant cells were also present. The deposits of granulation tissue tended to be both larger and more frequent in animals treated with tremolite than in those that had inhaled brucite. When the degree of involve ment of the lung parenchyma was assessed (Table I), the differ ence between the two dust treatments at both 12 and 18 months after the start of dusting was statistically significant (p<0.01). During this period, the lesions did not increase in size and fre quency and indeed in the tremolite-treated animals the 18-month group produced lower figures than those found at 12 months. With the small groups of animals examined, how ever, this reduction was not statistically significant. After 18 months from the start of dusting widespread interstitial fibrosis developed and this tended to obscure the earlier fibrotic deposits. For this reason, estimations of peribronchiolar fibro sis were limited to the first two killing dates. At 12 months after the start of dusting there was marked reticulin staining in the peribronchiolar deposits although relatively little collagen - be demonstrated by Van Geison's stain. As the animals agev.. aowever, collagen staining became progressively more marked. Within the areas of peribronchiolar fibrosis, many ktremolite fibres were clearly visible with the light microscope. 'Brucite fibres could not be seen, however, at any stage. In addition to the peribronchiolar lesions, macrophages con taining dust were visible in most alveoli at the end of the dusting period although they were more frequent close to the terminal bronchioles. In addition, however, there were small irregular patches of alveolar wail damage and thickening (Figure 6). It is possible that these small areas of damage represent precursors of the more widespread and distinct areas of interstitial fibrosis that developed in the older animals although this has not yet been proved. As shown in Table I, however, the extent of this type of pathological change in the individuals of any group of animals bears a close relationship to the amount of peri bronchiolar fibrosis present. In the present study, animals treated with tremolite had significantly more of this alveolar damage than those treated with brucite (p<0.0l). From about 18 months onwards, areas of lung tissue in some animals showed a progressive thickening of alveolar septa. In its earliest form this thickening was caused almost entirely by hyperplasia of alveolar lining cells but later there was consider able deposition of reticulin and eventually collagen in the septal walls (Figure 7). Dust deposits were frequently visible among the fibrous tissues in the thickened septa in animals treated with tremolite dust. As shown in Table I, areas of interstitial fibrosis became more widespread in both treatment groups as the animals aged but far more was found in the tremolite-treated group than in the animals that had inhaled brucite (/K0.01). In some areas the interstitial fibrotic element of these lesions remained predominant throughout the study but in others the hyperplasia of alveolar epithelial cells became progressively more marked to produce a pattern of adenomatosis. Some is so S | 4> aW I ", i: S 30 20 TRCMOUTE O eaucire # ' ;:i 1s io q 70. g 50 VJ 550 *40 ! 20 3 2 to 5 * * 2. f. inhalation of tremolite and brucite dust in t mtMoun Omocm o Hativeiy' regular 6). It is cursors fibrosis not yet t of this roup of if perianimals tlveolar in some epta. In irely by onsiderte septal ; among ted with fibrosis t as the .-treated 1.01). In ressively s. Some 3 4 5 7 t tO FIBRE LENGTH (jjm) Fig. 4. Length distributions of fibres of tremolite and brucite >0.4 jim in length obtained by SEM at a magnification of 10 OOOx. definite adenomas could be seen to have developed from the central regions of these areas and it is likely that this was also' the site of origin of some carcinomas although by the time most of these were discovered they were too widespread to be certain. Two pulmonary adenomas were found in the group of rats treated with tremolite and three in the Brucite group. As shown in Table II. however, a total of 16 carcinomas and 2 mesothelio mas developed in the tremolite group while only 2 carcinomas occurred with brucite. This difference in the number of malignant tumours is statistically significant (p<0.001). No pulmonary tumours were found in the lungs of control animals. The numbers of tumours occurring in other tissue sites for the three groups is illustrated in Table III. While the control group produced the highest numbers of both benign and malignant tumours these numbers were not significandy higher than in the groups treated with either tremolite or brucite. Lung dust burden The retained dust burden of tremolite in the lungs of a group of four rats killed at the end of the dusting period showed a mean value of 10 800 jig. After a further 6 months the mean value for the second group of four rats to be killed was 6210 jig. The extracted dust samples for the four brucite-treated animals killed at the end of the dusdng period were destroyed during analysis. In the group killed 6 months after the end of dusting, the mean lung dust content was 64 fig when measured using the i.r. absorbance figures for brucite. However, since it was knu . .hat the original brucite dust clouds had been contami nated wun --10% of chrysotile, the lung residues were treated with glacial acetic acid to dissolve brucite and then re-analysed using the i.r. absorbance figures for chrysotile. No significant loss of lung dust mass was found and it was concluded that 6 months after the end of dusting almost all the remaining lung dust was chrysotile. 0.5 1.0 2 0 FIBRE DIAMETER (|im) 3.0 50 Fig. 5. Diameter distributions of fibres of tremolite and brucite >0.4 jut length obtained by SEM at a magnification of 10 OOOx. Injection studies In the i.p. injection studies a very high proportion of me theliomas was produced by both tremolite and brucite. In tremolite study 27 animals out of 29 available for considers (93%) developed tumours and in the brucite study 27 anir out of 28 (96%); However, tremolite dust induced mtheliomas faster than brucite. The mean survival time animals with tumours was 352 days in animals injected tremolite and 418 for animals injected with brucite. Discussion The present study has shown that both the tremolite and bn dusts used were fibrogenic and carcinogenic to rats. While amounts of fibrosis and the number of pulmonary tumours duced by brucite were relatively low, tremolite prodi significandy more of these lesions than UICC chrys (/K0.01) which previously had been the most carcinogenic treated by our Unit (using the same exposure facilities) (U these previous studies all amphibole dusts tested had sh only low levels of pathogenicity. The reasons for the activity in tremolite. which is also an amphibole are, there of great interest. Stanton et al. (18,19) suggested thacarcinogenicity of mineral fibre samples was most ck related to the number of fibres present >8 microns in lengd <1.5 microns in diameter. The fibre sizing of tremolite ur taken by SEM in the present study indicated that the propo of fibres >5 jtm and >10 /im in length was approxim double that found for either UICC crocidolite or amosit Davis etal. (14) although the tremolite figures were simii those reported for a factory amosite sample in 1980 (20). tremolite fibres were, however, rather thinner than the am and crocidolite samples examined and the number of f >5 microns in length and visible by phase-contrast light m scopy was more than double that found with any of these. J.M.G. Davis et al. Fig. 6. A light microscope section of lung tissue from a rat that had inhaied tremoiite dust for 12 months. One respiratory bronchiole is surrounded by masses of fibrous tissue (F). in addition, the alveoli in two areas (outlined) show irregular thickening. f200x magnification) Table I. Levels of pulmonary fibrosis and irregular alveolar wall thickening produced by tremoiite and brucite dusts Time after start of exposure (mo.nths) Peribronchiolar fibrosis Irregular alveolar wall" thickening Interstitial fibrosis Number of rats examined Tremoiite 12 23.0 (21.4-24.2)b 35.2 (27.7-41.0) 0 3 18 13.4 (9.7-18.9) 27.7 f20.8-35.4l 3.0 (0-5.6) . 4 27-29 " - 14.5 (3.8-26.9) 12 Pi rr, 1 Brucite 12 1.6 (0.5--2.7) 5.8 (3.1-7.5) 0.8 4 18 1.7 (1.0--2.9) 7.6 (3.6-V.y) 0 4 27-29 ~~ - 2.9 10.2-8.4) 12 'Estimates obtained by subtraction (see text). hFigures in brackets are standardldeviations. mass of tremoiite retained in the rat lungs was almost identical to that previously found with other amphibole dusts at both 12 and 18 months after the s: f dusting so that the number of tremoiite fibres present in ti,, .ng tissue would have been correspondingly high. Stanton's work was undertaken using hc technique of intrapleural implantation and the injection I tudies reported in the present paper have confirmed that tremoiite is very highly carcinogenic in this type of experiment. There is, however, no reason why the same criteria should not apply to the lung parenchyma and the carcinogenic potential of the tremoiite dust used in our inhalation study is likely to have I been due to its high content of long thin fibres. These studies .j would suggest that tremoiite samples containing thin fibres ! should be treated with appropriate caution if used by industry. ( In addition, samples of chrysotile and talc contaminated with ; tremoiite are more likely to be more harmful than uncontami- | nated material. ; The results of the brucite studies indicate more complex patterns. The bulk brucite sample used for this work was con- j sidered to be as pure as can be obtained. However, both the j 672 Inhalation of tremolite and brucite dust in masses -29 .9 2-8.9) to have studies a fibres uiustry. ed with ontami- lex vas conboth the Fig. 7. A light microscope section of lung tissue from a rat treated with tremolite that died 29 months alter the start of dusting. The normal alveolar stuc has been replaced by spaces lined by cuboidal epithelium which are separated by deposits of interstitial fibrosis. <400x magnification) Table If. Pulmonary tumours produced by tremolite and brucite clouds Tremolite Brucite Control Number of rats examined Adenomas Total carcinomas Adenocarcinomas Squamous carcinomas Mesotheliomas 39 2 16 8 8 2 38 36 30 20 i0 t0 00 original sample and the respirable dust cloud generated from this material were found to contain ~ 10% of chrysotile by weight. Since most of the `respirable' brucite fibres in the dust cloud were of much larger diameter than the chrysotile present, chrysotile is likely to have formed >10% of the total fibre number and may even have been more numerous than brucite. Only very small amounts of mineral remained in the rat lungs 6 months after the end of dustinn and analysis indicated that most of this was chrysotile. It is t :ble, therefore, that this residual burden of chrysotile was responsible for the observed lung damage, with brucite producing little effect. In the present inhalation study, the rats were effectively exposed to a `brucite' dust cloud containing a chrysotile mass concentration of slightly more than 1 mg/mx. They developed similar numbers of pulmonary tumours and similar levels of fibrosis to animals Table ffl. Sites of tumours other than lung Tremolite Number of rats examined Organ system Digestive/peritoneal Urinogemtal Endocrine Musculo, skeletal and integumentary ReticuioendotheliaUvascuIar Totals 39 B* Mb 12 "t 4i -i 3 -1 78 Brucite 38 BM 1 41 4 -t 56 Coi B 1 T x 11 `B - benign. bM = malignant. previously exposed at this Institute to a dust cloud of Rhodesian chrysotile at a dust concentration of 2 mg/m Similarly in the injection studies the rats injected with 25 dust effectively received doses of -2.5 mg of chry Mesotheliomas were produced in similar numbers to previously reported at a dose level of 2.5 mg of Rhea chrysotile in dose response studies, reported by Bolton (21). In conclusion, these studies suggest that pure tremoli any materials contaminated with it should be treateu J.M.G. Davis et al. appropriate care by industry. In addition, the idea that the pathogenicity of the 'brucite' sample examined couid be due to its chrysotile content highlights the problem of attributing ,erved pathological effects to individual components of a &ed dust sample. Acknowledgement This study was undertaken as part of the research programme funded by the Asbestosis Research Council. References l.Selikoff.I.J. and Lee.D.H.K. (1978), Asbestos and Disease, Academic Press, NY, 2. DementJ.H. (1977), Asbestiform Minerals in Industrial Talcs: Commer cial Definitions versus Industrial Hygiene Reality, National Bureau of Standards, Special Publication 506. Proceedings of the Workshop on Asbestos: Definition and Measurement Methods held at NBS Gaithersberg MD. (Issue 1980), 513-323. 3. Campbell.WJ. (1977), Identification of Selected Silicate Minerals and their Abestiform Varieties. National Bureau of Standards. Special Publi cation 506. Proceedings of the Workshop on Asbestos: Definition and Measurement Methods held at NBS Gaithersberg MD. (Issue 1980), 201-221. 4. Campbell. W.J., Higgins.C.W. and Wylie,A.G. (1980), Chemical and Physical Characterisation of Amosite, Chrysotile. Crocidolite and nonfibrous Tremolite for Oral Ingestion Studies by the National Institute of Environmental Health Sciences, Bureau of Mines Report of Investigation Rt.8452. United States Department of the Interior. 5. Yozicioglu.S., Otayto.R.. Bacli.K., Sayli.B.S. and Yorulmaz.B. (1980), Pleural calcification, pleural mesotheliomas and bronchial cancers caused by tremolite. Thorax, 3S, 564. 6. Pooiey.F.D. (1976), An examination of the fibrous mineral content of asbestos lung tissue from the Canadian Chrysotile Mining Industry. Environ. Res., 12, 281. 71,1R10o'wlands.N.. Gibbs.G.B. and McDonald'.A.D. (1982), Asbestos fibres in ^kth,thee lungs of chrysotile miners and millers - A preliminary report Ann. r cup. Hyg.. 26, Nos. 1-4.411. ^^WTWaagnerJ.C., Chamberiain.M.. Brown,R.C.. Berry.G.. Pooiey.F.D., Davies.R- and Griffiths.D.M. (1982). Biological effects of tremolite Br. J. Cancer. 45, 352-360. 9. Pott.F. and Friedrichs.K.H. (1972). Tumoren der Ratte nach i.p. Injektion faserformiger Staube, Natunvissenschaften, 59. 318. 10. Pott.F., Friedrichs.K.H. and Huth.F. (1976), Results of animal experi ments concerning the carcinogenic effect of fibrous dusts and their inter pretation with regard to the carcinogenesis in humans, Zeniraibt. Bakteriol. Microbiol. Hvg. Abt. I, 162. 467-505. 11.Timbrel!.V.." Skidmore,./.W,, Hyett,A.W. and Wagner. J.C. (1970). Exposure chambers for inhalation experiments with standard reference samples of asbestos of the international Union Against Cancer (U1CC), J. Aerosol Sci., 1.215-223. 12-Beckett.S.T. (1975). The generation and evaluation of UICC asbestos clouds tn animal exposure chambers. Ann. Occup. Hyg.. 18. 187-198. 13. Asbestosis Research Council (1971), The Measurement of Airborne Asbestos Dust by the Membrane Filter Method. Rochdale (Lancs) ARC Technical Note i. 14. Davis.i.M.G., Beckett,S.T., Bolton.R.E.. Collings.P. and Middleton. A.P. (1978). Mass and number of fibres in the pathogenesis of asbestosrelated lung disease in rats. Br. J. Cancer, 37. 673-688. 15. Middleston.A.P., Beckett.S.T. and Davis.J.M.G. (1977). A study of the short-term retention and clearance of inhaled asbestos by rats using UICC standard reference samples, in Walton.W.H. (ed.). Inhaled Particles IV, Pergamon Press. 247-257 16. Bolton.R.E.. Davis.J.M.G.. DonaldsonJC. and Wright.A. (1982). Vari ations in the carcinogenicity of mineral fibres. In Walton.W.H. (ed.). Inhaled Particles V. Pergamon Press, 569-580. 17.Cherrie.J. (1983), An investigation of the reproducibility of counting an^ suing of asbestos fibres by SEM. Report of the Fourth Internationa. Colloquium on Dust Measuring Technique and Strategy. Edinburgh 1982. Asbestos International Association, London. 369-378. ft.Stanton.M.F. and Wrench.C. (1972). Mechanisms of mesothelioma ik induction with asbestos and fibrous glass. J. Natl. Cancer Inst.. 48. f 797-821. (9. Stanton.M.F., Layard.M.. Tegeris.A., Miller.E.. May.M. and Kent.E. (1977). Carcinogenicity of fibrous glass. J. Natl. Cancer Inst.. 58. 587-603. 674 20. DavisJ.M.G.. Beckett,S.T., Bolton.R.E. and Donaldson.K. (19801. A comparison of the pathological effects in rats of the UICC reference samples of amosite and chrysotile with those of amosite and chrysotile collected from the factory environment, in WagnerJ.C. (ed.). Biological Effects of Mineral Fibres. International Agency for Research on Cancer. Lyon. i.A.R.C. Scientific Publication 30, pp. 285-292. 21. Bolton.R.E.. Davis.J.M.G.. Milier.B.G.. Donaldson.K. and Wright.A. 11984), The effect of dose of asbestos on mesothelioma production in the laboratory rat. in Report of the Vlth International Pneumoconiosis Con ference. Bochum 1983. Published by the International Labour Organi sation, pp. 1028-1035. Received on It May 1984; accepted on 23 January 1985