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i/exp- Path. (1986) 67. 4> S-4 V> The pathogenicity of long versus short fibre samples of amosite asbestos administered to rats by inhalation and intraperitoneal injection .G. Davis, J. Addison, R.E. Bolton, K. Donaldson, A.D. Jones and T. Smith Institute of Occupational Medicine. 8 Roxburgh Place. Edinburgh EH8 9Si' Received for publication 20 September 1985 Accepted for publication 1 1 November 198 s ary. For many years it has been accepted that fibre dimensions are the most important r in the development of asbestos related disease with long fibres being more dangerous fchort for all types of asbestos. This information has been derived from in vitro experiments `Injection or implantation experiments since the kilogramme quantities of specially id dusts that are necessary for long term inhalation have not been available. The lilt study has taken advantage of the availability of a sample of amosite produced so that ist all fibres were less than 5 /an in length. The effects of this dust were compared to dust ed from raw amosite that contained a very high proportion of long fibres. Previous data ^studies with UICC amosite, which was intermediate in length, were also available for prison. At the end of 12 months of dust inhalation, significantly more short fibre amosite (resent in the lung tissue compared to the long but while the long fibre dust caused the opment of widespread pulmonary fibrosis, no fibrosis at all was found in animals treated '(short fibre. One third of animals, treated with long fibre dust developed pulmonary purs or mesotheliomas but no pulmonary neoplasms were found in animals treated with fibre dust. Following intraperitoneal injection, the long fibre amosite produced theliomas in 99% of animals with a mean induction period of approximately soo days, short fibre dust, only a single mesothelioma developed after 8^7 days. In previous 'alation studies with UICC amosite. relatively little pulmonary fibrosis had developed and two benign pulmonary tumours. This would suggest that to produce a significant fnogenic response in rat lung tissue amosite fibres must be longer than those in the UICC aration. Following the injection of UICC amosite. however, mesotheliomas developed in ame proportion of animals and with the same mean induction period as with long fibre m From this it would appear that while very short fibres exhibit little carcinogenicity to ,er lung or mesothelial tissues, mesotheliomas can be produced by dust preparations insisting of shorter fibres than are needed to produce tumours. bywords: amosite asbestos, fibre length, pulmonary fibrosis, inhalation, injection The inhalation of asbestos dust may produce both pulmonary fibrosis and neoplasia in man and experimental animals and man;, experimental studies have been undertaken 4Ki j.M.C. Davis et al. in an attempt to determine the mechanisms by which asbestos fibres produce these pathogenic effects. A number of early studies suggested that the most important factor in tibrogenesis was the silica content of asbestos which stimulated collagen production by chemical action (Beger L934. Kuhn 1941). As early as 1946, however. King et al. (1946) administered chrysotile fibres cut on a special microtome at lengths of 15 pm and 2.5 pm to rabbits by intratracheal injection. They reported a greater tissue reaction from those animals that had recieved the long fibre sample. Later. Vorwald et al. (1951) reported that animals which had inhaled chrysotile fibres in the 20-50 pm range had more pulmonary fibrosis than those brea thing only dust with fibres below 5 pm in length. Scymczykiewicz & Wiccek (1960! obtained similar results when they adminis tered fibrous and amorphous asbestos dust to guinea-pigs by intratracheal injection. They did not. however, give details of the asbestos type employed. Hilscher et al. (197OI extended these studies using both the intratracheal and intraperitoneal injection of finely ground chrysotile and crocidolite. They found these fibres produced little or no fibrosis in either site. In contrast, longer fibres of the same asbestos types resulted in considerable fibro;is in both regions. Davis (1972I conducted a series' of experiments using the intrapleural injection of a number of different mineral types including long and short fibre chryso tile. The short fibre samples were either synthetic chrysotile with a maximum crystal length of 1 pm or chrysotile fragmented by ultrasonic treatment until all fibres were below i pm in length. While the long fibre samples produced extensive fibrosis, the short fibre specimens produced almost no tissue reaction. Wright & Kuschner (1977I reported that following the intratracheal injection of long and short samples of both asbestos and glass fibre all the long fibre samples produced considerable fibrosis while the short samples did not. The fact that fibre dimensions were impor tant in carcinogenesis as well as 111 uenesjj | was demonstrated by the work of m ,iiton < al. (1972. 1977. 19811. Stanton n grouj implanted numerous samples of careful^! sized fibrous dust into pleural cavities of rats! and reported that fibres > 8 pm in length and <1.5 pm in diameter appeared most effec tive in producing mesotheliomas. These find ings have been confirmed by Pott & Pried, richs (1972) and Pott et al. (19761 using intraperitoneal rather than intrapleural implantation of fibrous dusts. Until recently it was difficult to examine the importance of fibre length on pulmonary pathology in long term inhalation tudies because large amounts of specially 7 -pared dusts are required for such work, h 1981, however, the Manviile Corporation 111 the USA prepared several kilograms ofshort fibre amosite and supplied the Institute of Occupa tional Medicine in Edinburgh with sufficient of this material to undertake the present study. Materials and methods Amosite samples used for dust cloud generation. Both the long and short amosite dusts used in these, studies were prepared from the same batch of South African amosite. The short fibre amosite sample was prepared and char acterized by the Manviile Corporation ; the USA by grinding in a ceramic ball milled system followed by sedimentation in >.\ a ter. The resulting materials was so fine that only 57% of particles had an aspect ratio >5:1 and were therefore regarded as fibres. The mean length of these fibres was 2.68 uni while the mean length of all particles was 1.42 pm. The crystallinity of both the fibrous and non-tibrous particles was checked by transmission electron microscopy (TEM |. All particles examined showed a high degree of crystallinity as indicated by the presence of characteristic spot patterns seen in electron diffraction and well ordered periodic lattice fringes seen in high resolution imageevidence of damage to the crystal lath >r loss of crystallinity induced during the ; n- Fibre length on process could be found. In at psis of individual particles was t in the TEM by energy dispersiw *oscopy. The elemental compos- particle was consistent with ence amosite samples from a. The long fibre amosite samp rj:ed at this Institute from the |h of amosite that was used to prep t fibre material. ktcloudgeneration and monitoring. T` jiplation phase of this study was ^n in inhalation chambers of t! cribed previously by Beckett 119 7 ials were housed, fed and waterc :iber throughout the exposure p for the generation of the long fibs i cloud, the modified Timbrell dusi [(Beckett 1975) proved satisifao: jst clouds generated from the sh< Eiterial with this apparatus were : atain aggregates. Por this dust, ih Itiidized bed generator (Marple et a SI model 3400I was used. In . ^uce the risk of dust aggregaric pier both dust clouds were expoillium 204 source of /{-particles telectrostatic charge on the ftpes before entry into the ahmbers (Liu & Pui 1974). The j|ques produced dust clouds <. r aost entirely of individual fibres '-.'itjles. The mass concentration of the d-.; chambers was monitored daily by throughout the 7 h of exposure u-an open filter holder facing verticil wards and a Casella MRP.i 1 ',.\ a - pling instrument (Dunmore c( al. 1 former sampler monitored the t fconcentration and the latter mon/ respirable dust concentration. I: mean respirable dust airborne cmi. of ro mg/nri was achieved hv ad; generators in responses to cave days measurement, Libre number concentratii hin n distributions Ibr the expcnii:-." n in carcinogenesis as well as ( (>gen Fibre length and pathogenicity of asbestos 417 Wl iiij process could be found. In addition clouds were assessed from membrane filter is demonstrated by the work pis of individual particles was under- snatch samples collected on 90 separate i <-)7. 11)77. t <-)811. Status.; s gr( the TEM by energy dispersive X-ray days. Fibres were counted from all these planted numerous samples ol earefi. Jjoscopy. The elemental composition of samples using phase contrast optical micros " 'brous dust into pleural cavities of Ipartide was consistent with that of copy (PCOM) at a magnification of <6o<. iorted that fibres > H /tm in length Ittce amosite samples from South and fibre sizing was undertaken by scanning , urn in diameter appeared most ei . The long fibre amosite sample was electron microscopy (SEMI at a magnifica in producing mesotheliomas. Theses jjjfcted at this Institute from the same tion of x 10 000. With PCOM only fibres " have been confirmed by Pott & Ptfi frbfamosite that was used to prepare the with a length > 5 urn. a diameter < 3 urn :s U972) and Pott et al. (19761 uJU-- ji'flbre material. and an aspect ratio of greater than 3:1 were aperitoneal rather than intrapleurj considered (ARC 1971, A1A 19791. With dantation of fibrous dusts, pcloud generation and monitoring. The dust SEM examination all fibres longer than 0.2 ntil recently it was difficult to examine Ration phase of this study was under- iim were measured if their aspect ratio was importance of fibre length on pulmonsuy! ,, in inhalation chambers of the type greater than 3:1. uilogy in long term inhalation studltj jibed previously by Beckett (1975). The mse large amounts of specially -'wparai ils were housed, fed and watered in the Animal inhalation studies. For the inhalation *s are required for such work. : . 1981,-! jiber throughout the exposure phase, studies groups of 48 SPF male U'istar rats m ever, the iVlanville Corporation m tf^ ,2 the generation of the long fibre amo- the AF.-Han strain were exposed to dust prepared several kilograms of short fibre, ijoud. the modified Timbrell dust genera - clouds of either long or short amosite for ~ h me and supplied the Institute of Occupa-I Beckett 1975) proved satisifactorv but each day. five days a week for a total of 22.. d Medicine in Edinburgh with sufficient! I'clouds generated from the short fibre days during a period of 12 monihs. 1 lie 11s material to undertake the presenfj jsjrial with this apparatus were found to animals were lo weeks old at the stun a ,,ain aggregates. For this dust, therefore, dusting. Two batches of 36 and 2 9 unduste.: jjdized bed generator (Marple et al. 19781 animals were maintained within the same rials and methods itc samples used for dust cloud generation}) v_. el 34001 was used, (n order to Be the risk of dust aggregation even |er both dust clouds were exposed to a unit as controls during the same overall time period. Four animals from each experiment.): the long and short amosite dusts used Inf Bum 204 source of /(-particles to reduce group were killed at the end of the 1 2 month' studies were prepared from the same welectrostatic charge on the airborne dusting period and four more were killed o of South African amosite. The short , before entry into the exposure months later. The remaining animals were mosite sample was prepared and char jabbers (Liu & Pui 1974). These tech- left for their full life" span except that the ' by the Manville Corporation m the jjies produced dust clouds consisting study was terminated when the number a grinding in a ceramic bin milled |ost entirely of individual fibres or parti- survivors in one group (the long fibre treat lollowed by sedimentation in water, '-tilting materials was so fine that only i. 'he mass concentration of the dust in the ment group) had dropped to six. Estimation' of early librotic lesions were limited to the ( particles had an aspect ratio >3:1 ibers was monitored daily by sampling small groups of animals from the first nw ere therefore regarded as fibres. The mghout the 7 h of exposure using both killing dates. However, for the more length of these fibres was 2.68 pm bopen filter holder facing vertically down- advanced alveolar interstitial fibrosis occur 'he mean length of all particles was 1s and a Casella MRKi 1 \A dust sam- ring in the oldest animals, till iho.se ,iv::v m. The crystallinity of both the fibrous | -;j ig instrument (Dumnore et al. 1 9641. The within 2 months of the final killing date w n-fibrous particles was checked by ter sampler monitored the 'total' dust included. In practice this produced group' 1 ission electron microscopy (TPM I. All g icentration and the latter monitored the 18 animals treated with long fibre amorne ' examined showed a high degree of | '\i lirable dust concentration. The target and 23 animals treated with short fibre nuy as indicated by the presence of | |tean respirable dust airborne concentration amosite. Groups of 9 and 1 3 control anima - eristic spot patterns seen in electron of ro mg.'m * was achieved by adjusting the of similar age were included m tins eMim : m and well ordered periodic lattice generators in responses to each previous lion of iritersitial fibrosis. 1 tings ir.un -7 wen in high resolution imng- - No days measurement. animals in this study were examined lii'i- . of damage to the crystal lai e nr Fibre number concentrations ami fibre size gically for the presence of neoplasms, '.rr v -lalliuity induced during th- rep- distributions for the experimental dust ;iies were also taken lor hi'tolow. 'r- >i>; . other organs showing macroscopic abnor malities. Tables 3 and 4 include only animals surviving 1X months or more after the end of dusting since we have found that few neo plasms. and particularly pulmonary neo plasms. develop in the AF HAN rats before this lime. Tissue used for histological examination was fixed with Karnovsky's fixative and embedded in paraffin wax. Lungs were fixed by inflation at a standard pressure of 30 cm total pulmonary dust burden for . ch ang | mal). Dust retained in the lungs is recoi vered by a low temperature plasi: ashing j process using a Nanotech Proo apparatus; Following tissue ashing the amosite residue*j were washed in 0.2 .viHCl at room temped ture before estimations of the amounts off retained fibre were made using the infra-red i spectroscopy technique described by Bolton et al. (198 3). jjnparisons of levels of pulnumai "id lung dust burdens were mac neralized linear models facilitiiical package GENSTAT <Alu H). Differences between the ir for pulmonary and non-pur juts were analysed using tlv. ley U-test (Siegal 19961. Sts '& of fixative. Subsequently, the tracheas were ligated and the lungs excised and immersed in fixative. Sections were cut in the coronal plain at t mm intervals and were stained by either haematoxilin and eosin, Van Giesen'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. (1978) except that an electronic image analyser (Graphic Information Systems l.imited, GDSi) was available for use in conjunction with the light microscope (Davis etal. 1985). Single lung sections were examined and the sections selected to contain the maximum area of lung parenchyma. As previously described, interstitial fibrosis was estimated Animal injection studies. In addition to the inhalation studies the ability of the long and i short amosite preparations to produce L mesotheliomas was examined using thee* intraperitoneal injection assay. A dose of11 either 25 nig or to mg of dust suspended jn j ml of Dulbecco's phosphate buffet ' : salineij was injected under ether anaestheiu mo the| peritoneal cavities of groups of 24 rats of the{| AF/Han strain. The dust was collected from(i the animals inhalation chambers by an| elutriation process chosen to select the res-'a pirable fraction of the dust cloud (Bolton ctal, IT 1982). The short fibre dust was dispersed In iia the saline by ultrasonic treatment. All anl.il mals were allowed to live out almost all of(| their dull life span and were killed when\!| moribund. |jcharacterization |i respirable and total dust jived for both dusts in this si in Table 1 as are the fibre : ; and > to fim seen by PCO\ short fibre amosite preparai I very few fibres > 5 /an in leu the long fibre amosite du ned a far higher proportion than any other amosite piv |busly examined in this ia i et al. 1978; i 982) with ov pres > lo /mi in length and 1 Qgth as seen by SEM. The Buttons of the two dusts wer . be expected since the same |te was used in their prepar using a x 2 microscope objective lens and is expressed as a percentage of total lung tissue area. Peribronchiolar lesions are more numerous and smaller and so the lung tissue was scanned with an eyepiece graticule covering a tissue area of 2.92 mm2 and divided into too squares. A x 4 objective lens was used. Peribronchiolar lesions were recorded as a percentage of squares contain ing lesions of this type. 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 histological studies. (Studies in this laboratory have shown that the ratio of dust content between left and right lungs following experimental inhala tion of fibrous dust such as asbestos in rats is approximately 0.6:1 and this correction factor was therefore used to estimate the '.I Statistical techniques. Survival functions for 'j each experimental group were estimated using the product-limit method (Kaplan & '!j Meier 1998b The survival curves lor the j different series were tested for homogeneity j ft were not identical. The T ration in general consisted r fibres. Figs. 2 and 3 illustra; listributions obtained by SH.\I hges of fibres exceeding a 21 \ llameter) are plotted against if. using the Generalized Wilcoxon iBreslowj statistic in the statistical package BMDP (Dixon et al. 1983). In order to compare the 1 r. Figures for dust mass and sir he long and short fibre amosite 2 overall mortality of the four groups of ani mals. the death of an animal as part of a planned kill was treated as control, all other Long libr-.' amosite deaths being treated as responses. Survival curves were also estimated for the response of developing a non-pulmonary tumour. All dust mass [fag/mJ of airi lirabie dust mass m deaths of animals without non-pulmonary tumours were recorded as censoring events, whilst non-pulmonary tumours were treated as responses irrespective of whethei the death was planned or unplanned. Mjjjmg/eH of airi Moires > i (<m 'n length |F.gg.(per ml of air1 )l||]}res > ro /tin in length '^''(per ml of airi i ' 2"e<, 11 1 1. uilmonary dust burden for e h ari)_ Dust retained in (he lungs u reco_ by a low temperature plasma ashing ' ring a Nanolcch P1 no apparatus, issue ashing the amosite residue* .led in 0.2 mHCI at room tempera, ei'ore estimations of the amounts of d fibre were made using the infra-red iscopy technique described by Bolton 1985). ' injection studies. In addition to the (ion studies the ability of the long and amosite preparations to produce '.eliomas was examined using the ritoneal injection assay. A ase of 13 mg or tomgofdustsuspem edint kilbeceo's phosphate buffered saline .cted under ether anaesthetic into the eal cavities of groups of 24 rats of the 1 strain. The dust was collected from unals inhalation chambers by an i on process chosen to select the res'raction of the dust cloud (Bolton elal 1'he short fibre dust was dispersed in 5 no by ultrasonic treatment. All anl- i re allowed to live out almost all of^j ill life span and were killed when 1 :d. ' ' hmques. Survival functions for "" . ncntal group were estimated product-limit method (Kaplan & 138). The survival curves lor the series were tested for homogeneity e Generalized Wilcoxon (Breslowl m the statistical package BMDP el. 198 3 |. In order to compare the mortality of the four groups of ani death of an animal as part of a nil was treated as control, all other mg treated as responses. Survival .re also estimated for the response vng a non-pulmonary tumour. All animals without non-pulmonary yere recorded as censoring e\ eats. - --pulmonary tumours were sited nc.s irrespective of wheth the planned or unplanned. Fibre length and pathogenicity of asbestos 419 Knaparisons of levels of pulmonary fibropd lung dust burdens were made using generalized linear models facilities in the deal package GENSTAT (Alvey et al. . Differences between the induction for pulmonary and non-pulmonary purs were analysed using the Mann hey U-test (Siegal 1956). Ills characterization / respirable and total dust masses Ifeved for both dusts in this study are in Table 1 as are the fibre numbers |jjan and > to pm seen by FCOM. ae short fibre amosite preparation con . very few fibres > s pea in length (Fig. the long fibre amosite dust cloud ined a far higher proportion of long than any other amosite preparation Jously examined in this laboratory jis et al. 1978; L982) with over 1 1% of pres > to pm in length and 3% > 25 pm lgth as seen by SEM. The diameter jjutions of the two dusts were close as be expected since the same batch of ilte was used in their preparation but were not identical. The long fibre ration in general consisted of slightly ker fibres. Figs. 2 and 3 illustrate the fibre ^distributions obtained by SEM. The perSitages of fibres exceeding a given length ((diameter) are plotted against that length. e 1. Figures for dust mass and fibre number `the long and short fibre amosite dust clouds Long iibre Short fibre amosite amosite dust mass ofairl pirable dust mass '.V|r;(mg/rn3 ofairl !i$jj}jres > 3 /an in length 7$'(per ml of airi .Fibres > 1 o [tin in length ' (per ml of air I t 1.9 20(10 1 1.6 When plotted on log-probability axes, the data are close to straight lines, indicating that the size distributions are approximately log-normal. Whilst there is a large difference in the length distributions of the two fibre samples (here indicated by the large sepa ration between the two lines), the diameters of the short and of the long fibres are very similar. The slopes of the lines for short amosite are slightly steeper than those for the long-fibre amosite, indicating that the vari ances in the (logarithmically transformed) lengths and diameters for short amosite are slightly less than the corresponding vari ances for long amosite. Pathological findings There were no significant differences between the overall survival of animals treated with either long or short fibre amosite or the two groups of controls. Animals treated with the long fibre amosite prep aration developed the same types of patho logical change previously reported in similar inhalation studies from this Institute (Davis et al. 1978: 1982; 1985). At the end or the t2 month dusting period the main lesions present were deposits of granulation tissue around the terminal and respiratory bron chioles (Fig. 4). This granulation tissue con sisted mainly of macrophages and fibroblasts but some foreign body giant cells were also present. At the end of the dusting period there was marked reticulin staining in the perobroncial deposits although relatively lit tle collagen could be demonstrated by Van Gieson's stain. In lesions from older animals, however, collagen staining increased in intensity, in animals killed after the end of dusting, the overall area of peribronchiolar fibrosis per section was similar but the proportion of lung tissue occupied b\ these lesions was significantly less (Table 21 iP <0.01 variance ratio test). However, the number of lesions per lung section remained the same. The differences was partly due in the increase in size of the rat lungs during this period but also to the facr that there 4-u J.M.U. Dims cl ai I-ibre m Mian ifioqw J Ek$,' Fihiv length ilisinlni'.;.'i,fibre amosile dusi .inist:-. B'po magnification n.d .-.si:: ; t ratio ^ \ to i. .\ slier. ;>' ebijjcg.i'sized I; A Iona tihiv or. !f)t Fig. i. Scanning electron microscope photograph of the hulk sample of shod (ibre amosile used in (ho!'i| mhalalion and infection studies. (Soon. appeared to be some contraction of the loose granulation tissue originally formed. As' these librotic lesions aged, the epithelial cells lining adjacent alveoli frequently hecame cuboidal in shape (Fig. s'- Animals treated with the short (ibre amosile preparation showed no evidence of peribronchiolar fibro sis at tiny stage. At the end of the dusting period the lungs contained very large numbers of pulmonary macrophages packed wit It dust particles hut these cells remained almost entirely free in the alveolar spaces, sometimes large groups of these cells were aggregated in atveoli close to respirutorv nroncm.iies 1 lie; was i! i lormanon of granulation tissue or thickening ot alveo lar septa at these points. In general from about tS months onwards, areas of lung tissue in animals exposed to long lihre amosile showed a progressive (hiekening of alveolar septa. In its earliest form this thickening was caused almost entirely by hyperplasia of type II pneumocytes but later there was consider able deposition of retieuiin and eventually collagen in the septal walls Fig. 71. '.. cumu P-3- lain lations Ot dust were iVcuuemlv \ isihw among the lihrnus tissue m die thickener, eta. As .... 6U lib's l:,:|'06oo m )); aspec shown in lahle 4 these areas ' '.eolar sfo6 Sbr-- nterstiria! lihr.wK he. wore - as / fel). i `vle.i- Fibre length anil pathogenicitii of asbestos 441 10 Mean length (|j.ml 100 fipibre length distributions of the long and Ifibre amosite dust clouds. SKM sizing at 1 magnification 0.4 pm; diameter < 1 pm; l^atio > 3 to 1. O, short fibre amosite (700 fezed): &. long fibre amosite (700 fibres I sample of short fibre.* amositc used m the dilation fissile or thickening ol alveo la at these points. o general from about 18 months Is. areas of lung tissue in animals .1 to long fibre amosite shmved a (?}'><! 0O 0% -sice thickening of alveolar septa. In 1^1 $? est form this thickening was caused Pw-' entirely by hyperplasia of type II icyles but later there was considcrposition of reticulin and eventually 0.5 1 0 23 Mean diameter (pmi 10 1 in the septal wallslFig. 71. Accutnu>l dust were frequently visible among us tissue in the thickened sepia. As n Table 4 these areas of a alar ml lihrnsis became more exten ea< vraj. 3. Fibre diameter distributions of 1 he long and Sport fibre amosite dust clouds. KKM -17.111 a at wooo magnification, '-oa am diameter 1 S|m; aspect ratio > \ to 1. short fibre amosite; iyoo fibres sized i; o. long fibre amosilc **> fibres Sffiedl. ' the animals aged. In the oldest animals a mean ol r 1.0% of lung tissue was involved with interstitial fibrosis in the long fibre treatment group. Tile corresponding figure for animals treated with short fibre amosite was 0.1 3% which is comparable to figures seen in old control animals. In some areas the interstitial fibrotic element became pro gressively more marked with time but in some lesions hyperplasia of the epithelial cells lining the tissue spaces became more pronounced to produce a pattern of adeno matosis. Three pulmonary adenomas, eight pul monary carcinomas and two pleural mesotheliomas were found in the group of animals treated with long fibre amosite but no pulmonary tumours developed in the short fibre treatment group (Table ft. One adenoma and one carcinoma were found m control animals. The carcinoma was the first spontaneous malignant primary pulmonun tumour that we have ever found in our strain of rats was a small lesion (4 mm in diameter which was not responsible for the death ot the animal. One peritoneal mesothelioma was found in each of the group treated with amosilc dust. However, both of these tumours were found in association with a testis and appear to have developed from the epithelial covering of the'tunica vaginalis areported from untreated populations of nits 1 Clou Id 1977). it is probable therefore that neither of these mesotheliomas was asso ciated with amosite treatment. The numbers of tumours occurring in other tissue sites for the two groups of rats treated with amosite and the two control groups is illustrated in Table 4. Differences n; the proportions of animals developing nor pulmonary tumours were not statistician significant when the time at risk was taken into account. The relatively high number ! tumours in control group one appears 10 |v refitted to the lack of planned kills ;u com;'' groups' which allows a higher proportion : animals to reach advanced age and incret.svtheir chance of developing a tumour One difference between the dc\ elopm.*;;: ` Fibre Fig. 4. Foci of loose granulation tissue in the walls of respiratory bronchioles and alveolar dun lungs of a rat killed at the end of 12 months inhalation of long fibre amosite. pulmonary and non-puimonary tumours was noticeable in this study. Non-pulmonary tumours began to be recorded after only 1 year of the study and in the animals treated with long fibre amosite the mean period of these tumours was 694 days from the start of dusting. In contrast, the mean induction period of pulmonary tumours was 891 days. This difference is statistically significant at the P< 0.002 level. I.mifl dust burden The retained dust burden of amosite in animals treated with either the long or short preparations is illustrated in Table v Less dust was present in the lung parenchyma of I animals treated with long fibre amosite than j| in those treated with short fibre at both the first and second killing dates. Analysis of variance shows that this difference is signifl* 5 cant (P < 0.01). The figures suggest propor- jj tionately greater clearance of short fibres ' than long during the 6 months between the J first and second killing dates. However, withlj the small group sizes, this difference was not significant. inieetion studies The results from experiments m v. of either 2=; mg or to mg ol the i'y bronchioles and alveolar ducts from thejj nu fibre amosite. * 4.50. j present in the lung parenchyma ofjj Is (reated with long fibre amosite than (! .c treated with short fibre at both the 9 id second killing dates. Analysis of j ce shows that this difference is signifl'<0.01). The figures suggest proporly greater clearance of short fibres j ng during the 6 months between the i J second killing dates. However, with 1 dl group sizes, this difference was not f ml. 'indies fits from experiments in win as mg or 1 n mg of the Fibre length and pathogenicity of asbestos <N C3Ol* MI < IN. C 6 0 1l WJ 1 ONN 1 ! NIN. ON cO CJ I OMN vj T3VJi O E3 j fcN| * - ! -t o V oi w *o55 SCO 2 ON' p> OI vC 4 15. ocon ,> (t sC ,, i_/~ t1r- See text for units. Figures in brackets are ranges. Oa. O .3>S T3 3v 3 ri JJ _C qj o - 424 -4 > c^N J.M.C. Davis et al. '-it *S 'M Fibre letu/i mr I*l;i.O: Hr & J2 Fig. v Section of terminal and respiratory bronchioles from the lungs of a-rat killed16 months after thee of 11 months inhalation of long fibre amosite. Fibrotic foci are less marked than in Fig. 4. bin now son alveoli are lined with cuboidal epithelium, x 5 so. short amosite dusts were injected into the peritoneal cavity of rats are illustrated in Table 6. Data from previously published work with TICC amosite (Holton et al. 1482) is included for comparison. The long fibre amosite produced mesotheliomas in almost till animals with a mean tumour induction period of S20- s p> days. These figures are very similar 10 results obtained with UICC amosite. The short fibre amosite prepartition produced on.lv a single mesothelioma with an induction period of S ty days. Discussion The results from the long term inhalation studies reported in this paper are in agi ment with findings in many previous publl cations which showed the relatively Id pathogenicity of short asbestos fibres. short fibre amosite dust cloud used in thi present study contained only t % of fibres > /tin in length and o. rt> > 1 o um and we thus the shortest amosite preparation tht has been available for long term inhalatfi experiments. After a 1 year period of d inhalation at to mg mJ this short lib amosite failed to produce either pulmonai fibrosis or pulmonary tumours min thdliJ lifetime of the laboratory rat. i I: og fibrd'JI amosite sample with fo",, of lii" S u&M and 1 1 of fibres to uni was . ..-r thafitj Jj-jSection of lung tissue from a ra SLpulmonary macrophages pack ""'(the respiratory bronchioles. A 1 normal, x 400. gipther amosite preparation led and produced pulmonar 30% of animals and large bfronchiolar fibrosis and alvco: brosis. The 1'ICC standaiv, (jle of amosite which was > 3y in our laboratory ilhivis fibre length distribution '-> |between the present long aiu: rations, ft was found in Iv fibrogenic compared m mn we have examined at '!: concentration mo mg :r only two benign punnoin a group of j.i > rats. : t lungs of a rat killed (> months after the end 's marked than in Fig. 4. bin mm-some Ky. Section of lung tissue from a rat killed at the end of 14 months inhalation of short fibre* am. s. 7 pulmonary macrophages packed, with dust are present and these are particularly .iggreg.r u Qd the respiratory bronchioles. Apart from the presence of these cells, however, the lung tri. * ears normal. - 400. s reported in this paper arc 111 agree- with findings in many previous publi- is which showed the relatively low tonicity of short asbestos fibres. The fibre amositc dust cloud used in the :t study contained only 1 of fibres > s length and 0.1% > to am and was lie shortest antosite preparation that ett available for long term inhalation nents. After a 1 vear period of dust 'ton at to mg,in' this short fibre failed to produce either pulmonary >r pulmonary tumours wttlr.n the d the laboratory rat. The Ion fibre simple with ',o '., of tifires .an of fibres 1 o kiii vs as ionic hat' other amositc preparation we have lined and produced pulmonary tumours 30% of animals and large amounts of Ibronchiolar fibrosis and alveolar iniersti- fibrosis. The CRT standard reference . tple of amositc which was tested preIbusly in our laboratory (Davis ri til. 107s. m a fibre length distribution roughly rnid- between the present long and short fibre `;;$reparatkms. It was found to be only moder'.ittely fibrogenic compared to other ashesto' -dusts wc have examined at the same air 'borne concentration mu mg m't and pro duccd only two benign pulmonary adeno mas in a group of j'i ratv finis it would appear that to produce a significant care genic response in rat lung tissue in studf this size, amositc libres must he longer ' those in the i'lt'C preparation, fhese : mgs. however, contrast wgh 'h. experimental injection 'indies, hi 'ive long fibre amositc prepttrtiuon mvu; more than no',';, of mesotheliomas m 1 4: of rats while the short dust produce.! a single tumour. Results from :niecti<*n 'K with l*ICC amositc 'Rollon c: .i. t -.'4 m thisctise almost idenltea: :o those long libre anuisite dust. I-Vom '.hi- tppetir tltat while \er. 'i'-.-r tier-*title , arcinogemctr. - utter tic. .Mkz. j.M.G. Davis cl al. Fibre Icntji t'4. Numbers of tumours occurring of rats examined System ye/peritoneal ienital Jo. skeletal and ;umentary !o-endothelial :ular I ong um> 4-' H -- Tig. 7- Section of lung tissue from a rat killed 20 months after the end of twelve months inhalation oflo libre amosite. The normal lung architecture has been replaced by a lesio.n similar to hums! 'honeycombing' where the lung spaces no longer corresponde to the original alveoli. The honeycon space are lined by cuhoidal epithelium and the thickened septa between them are libro--.. hut contain many cells, 400. Table 5. Numbers of pulmonary tumours and mesotheliomas found in rats treated with three different? preparations of amosite asbestos and in two control groups of these animals Tumour tvpe Long Short LICC Control Control amosite cimosile cimosile* iil'OUp I croup * No of rats J.O 4-: -1 i \denomu \deiio-carcmoma - Squamous-carcinoma ndilTerenuated-carcmnma 5 '1 Pleural mesothelioma >l Tentoneal ouesoihehoma ! ib C N 1 () 11 11 () l 1 J fj&enign: M. Malignant, `b it . Estimates of the lung Just con with long and short libre asbestos. ir end ting Long amosite hi a 557<>/<g< 1 S9o` =502 s 5080^15701 j.47 figures quoted are the meuiu ' ,ts in each case. es in brackets are standard lit jthelial tissues at the doses ijtiieliomas can be produca: ationsconsisting ol'shortc! ' [6. Mesotheliomti production 1 .es of amosite dust Number of animals developing tnesnihe!.' |3;'' Mean tumour mducuoi: period in davs " Fibre length and pathogenicity oj asbestos 4. Numbers of tumours occurring at sites other than lung 427 jerofrats examined 1 System live/peritonea! genital grille iilo, skeletal and jgumentary jlo-endothelial icular bong amosite 4<> BM -- -- --- I \-- 14 --5 4 i( Short amosite 42 B \1 Control group 1 B .U Control group 2 2s BM -- ^I $l I 1-- 2 2 ,,-- h 2i 1 7 ~~~ n 2t 1 -- \ --; 2 1i / 1s s 4 feenign: M. Malignant. V end of twelve months inhalation of long eplaced by a lesion similar to human *- the original alveoli. The honevcomb letween them are fibrosed ' if also found in rats treated with three different ! these animals ncc Control Control imusuc* group 1 group 2 41 ib is 0 (1 0 ts O Ou ) <t i j 5. Estimates of the lung dust content of rats with long and short fibre samples of iite asbestos. are needed to produce pulmonary tumours. Taken together these findings may explain both the comparative rarity of mesothelio mas in both rats and humans following dust after end 'idusting hong amosite Short amosite inhalation and also their characteristically long latent period between first exposure and jSyo/igdsyo) Sf>4Pg(J70l 5080 /<g (370! 4470/(g(s<Sol :e figures quoted are the means of groups of rats in each case. :gures in brackets are standard deviations. lothelial tissues at the doses examined, ratheliomas can be produced by dust tumour development. Fibres >2S /an in length are unlikely to be transported through lung tissues to reach the pleural or peritoneal mesothelium. if this length were necessary to transform mesothelial cells then mesotheliomas might not occur at all even in heavily exposed individuals. Medium length fibres (8-1 q fim) probably represent a size that is difficult but not impossible to move. After a long period of time enough of these parations consisting of shorter fibres than inay reach mesothelial tissues to produce SSj!V ri(f|ble 6. Mesothelioma production in rats following the mtraperitoneal injection of three /pj^iples of amosite dust _ 4M. mi !%$ bong amosite Short amosite blCC amositi 2^ mg u mg 2^ mg to mg 2> mg Number of animals developing mesotheliomas Mean tumour induction period in days 20 ^2" 2 ; 'SS S^ 1 14.. 1 n S 17 \ A ' m j. \ * I..U.U. LAU LS ft ill. mesotheliomas in a few individuals. Short fibres (shorter than 5 /tm) are relatively easily moved (Morgan etal. 1978) but do not appear to be very carcinogenic. While almost all workers, who have reported experimental studies on fibrous dusts, agree that 'short fibre' preparations are at least relatively harmless, a series of papers by Kolev (1976: 1982: 1984) has reported that crocidolite or anthophyllite ground until all dust particles were isometric could produce mesotheliomas in rats and the isometric crocidolite sample apparently pro duced more mesotheliomas than a fibrous control sample of crocidolite. These results are difficult to evaluate since detailed size distributions of the injected dust samples were not given but one possible explanation comes from the photographs and histological descriptions of some of the tumours pro duced. These are described as a giant cell type and a sarcoid type. This type of histological pattern was found by Wagner and Wagner (1972) and Wagner (1976) following the intrapleural injection of isometric quartz particles and the tumours were classified as thymomas. Tumours of similar giant cell and sarcoid type (classified as malignant histiocy tomas) were also found in the pleural cavities and lungs of rats treated ceramic fibre dust (Davis et al. 1984). Although this dust did contain some long fibres the majority of dust particles were isometric. From these results it seems possible that rats respond to a number of isometric dusts with a formation of tumours that may look superficially like mesotheliomas but are in fact of reticulo endothelial origin. From a very large amount of published experimental work there is now a general concensus that fibre dimensions are the major factor in disease production and recently Pott & Ziem (1983) suggested that because of this attempts to produce less dangerous asbestos materials by modifying the fibre surfaces would not prove successful. Bignon & Jaurand (198 5 I. however, reported that acid treatment of chrvsotile caused a reduction in iibrogenicity and carcinogeni city and suggested that while iibr iirneasions were important the surface 1 mistry of fibres does play a part in disease -roduc. tion. As the authors point out, howcuer, the findings of Bignon's group need not be taken as proof that the chemical composition of fibres is a factor in carcinogenicity. It is likely that changes in fibre chemistry could also effect fibre length and durability and that these factors may influence the experimental results. Acknowledgments This study was undertaken as pari of the '<1 research programme sponsored by the Brf- i| tish Research Council. The autl .ts are1 grateful to the Manville Corporation of the J! USA for supplying the short fibre amoslte dust used in this study and to the Quebec Asbestos Mining Association for a grant to purchase a fluidized bed dust generator. References Ai.vi-v N.G., BANr11a.11 C.F.. Baxter R.l. el al.ijj (1977) GENSTAT. A general statistical pro* gramme. Rothamsted Experimental Station. .. Asbestos International Association (1979)! Reference method for the determination off airborne asbestos fibre concentrations at work places by fight microscopy (membrane filter method). Recommended technical met1 si no. 1. I.ondon: Asbestos International Assr. i.nion. Asbestos Research Council ( i 97 ii The Measure ment of Airborne Asbestos Dust by the Mem brane Filter Method. Rochdale Ibancsl ARC Technical Note 1. BeckErr S.T. (r979) The generation and evalu ation of IIICC asbestos clouds in animal expo sure chambers. Annuls of Occupational Hygiene 18, 1 87-198. Bhgek I'.). (1 9 54). I'berdie Asbesiosiskorperchen. Virchows Arch. Patlwl. Anal. Physiol. Klin. Med. 290. 280-595. Bignon |. & Iahrand M.C. (198 51 Significant dela composition elinique des fibres par rapport 3 leur puissance eanccrogene. In nl'ieus Ousis. Report of a conference. Stashourg. >n 225 254. 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([9771 Tht influence of varying lengths of glass and asbes tos fibres on tissue response in guinea pigs. |n ed. Inhaled Particles IV. W.H. Walton pp. 4^-. 477. Oxford: Pergamon Press. Bp, /. exp. Path. (1986I 67. 431-4 .<! Enzyme activities and a heart muscle and sk Michael Krog, Stc tents ofSurgery. University Hospitu, Received Accepted for nary. A prominent feature of an nth muscle cells, fn this study il; nses appear was investigated. Th. i serum creatinine 165 /miol l< 1 of adenine nucleotides were me nation disclosed no changes ir icoktnase, an important glycol-, itile and aorta, whereas the hex fdrogenase remained unchanged muscle. Fat metabolism w., xyacyl-CoA-dehydrogenase. Ac Wed markedly increased activities jKlevels were found in the heart n; |pool of adenosine phosphates r s animal model described offers ild be useful for studying the off yords: adenine nucleotides, aor If'- ,increased incidence of cardie.-, es has been reported in ier et al. 1974I. In experiment arterial lesions, eharacte'r necroses and calcification ; Ejerblad et al. 19 79cI as we aulation of glycosaminoglycai Jl 1979a) and collagen <Ejerbl;. |gc), have been described. A simi. jjidisease has also been seen in ents (Ejerblad et of. i9 79<h iiv 5)- A myocardial disease. ch.ir orrespondence: M. Krog. \l!> ' den.