Document 9907bjrx9oq4oKmdgZr5kzmK7

i.imk m artmij ^'IMii * .a in nonbtiinan tush in the dog. L . S3, asi-aoo. in elephant. Arch. log. Asm. .V.y. Acad. 19K3). Bovine arterio- ' roiury atherosclerosis ' changes in sheep and nbosis." Huttcmnrth, utal coronary sclerosis tockrels. Arch, i'athol. rterial diseases I. Spon* D, (19(i(i). Coronary , white curneau pigeon. lode! for experimental is)69). Coronary atheroi> Physiol. Psychol. 68, Jill. i. .il atherosclerosis in osclerosis in the arterial deposition in the cerebral i. Amvr. J. Pathol. 57. id cerebral atherosclerosis WiASWA.MV. V. { 1908). d composite study. F.xp. EXI'MUMK.NT\I. and MOLKCl I.AB 1' \ I H'M.OIV If), -MM--3)5.1 (1 f)71) Experimental Studies on the Effects of Heated Chrysotiie Asbestos and Automobile Brake Lining Dust Injected into the Body Cavities of Mice1 J. M. G. Davis and S. W. Coniam Institute nf Occupational Medicine, Roxburgh Place, Editdstirgh ElIS 9SU, Scotland Received February 20, 1973 A series of experiments with mice involving the intrapleural injection of chrysotiie asbestos dust heated to varying temperatures up to 1000 C suggests that the physical shape of the dust particles is more important than the altered chemistry of the dust. Chrysotiie heated to no inure than K*<> C. produced dust samples containing many long filters, and these samples resulted in large pleural granulomas. Chrysotiie heated to more than 400'C produced dust containing mainly particles of low aspect ratio, and these produced very small graiinluinas. The initial size of the granulomas was very closely related to the final degree of fibrosis found in the animals and, therefore, chrysotiie heated to -100 C or less was much more fibrogenic Ilian when it was heated to higher temperatures. A sample of aul.miohile brake lining dust was found to con tain very little recognizable chrysotiie, am! consisted mainly of particles with a low aspect ratio. When this diet was injccli d into the pleural cavities of mice it produced scry small granulomas and little filiro-iv The discover) In Wagner cl til. t I9R0) that exposure to asbestos dust was often associated with the production of iih sotlielial tumors caused concern about the possible pollution of the normal urban atmospheie In1 asbestos dost. Wagner had shown that very small doses were apparently sufficient to produce tumors in some cases and it was suggested that the use of asbestos products from ironing boards to brake linings might be liberating efficient dust into the atmosphere to cause a health hazard. Since automobile brake linings are constantly being abraded in use and since many tons of asbestos are consumed in this manner each year, the possibility of atmosphere pollution from brake lining dust lias received particular attention. In order to test the level of atmospheric- pollution by asbestos a number of studies have been undertaken. The lungs from unselocted autopsy cases from different parts of the world have been examined and initially, since uncoated asbestos dust is difficult to Hud in tissues, the presence of asbestos bodies was used as a likely index of the presence of asbestos. The figures obtained showed an increase in the percentage of cases containing l>odies with the increasing sophisti cation of the techniques used. The first of these studies was that undertaken hv 1 The dust samples used in this -study were prepared in the laboratories of T.B.A. Industrial Products Ltd. of Rochdale, England. This work was undertaken as part of the research programe organised by the British Ashestaxis Research Council. C'4>yrifht 'ft 197.1 Uy Academic Pres*. Inc. All ngfett cl reproduction is any form reserved 19 * DEFENDANT'S 5? EXHIBIT I 12. HWBUI0006700 .540 DAVIS AND CONIAM Thomson et al. (1963) in Capetown and these workers reported bodies in more than 30' i of cases. Later, Thomson and Graves (1966) found a similar prevalence in Miiuu:; Cauna, Totten, and Gross (1965) found bodies in 40% of a series of lungs from Pittsburg and Anjilvel and Thurlbeck (1966) found them in 45% of their cases in Montreal. Mourman (1966) reported finding bodies in 70% of a group of lungs from Finland and finally, Utidjian, Gross, and De Treville (1968) using a new technique, reported that the lungs of 97 out of 100 consecutive autopsies from Pittsburg contained bodies. However, Davis, Gross, and De Treville (1970) showed that the production of ferruginous bodies was not spe cific for asbestos as had been previously believed and it therefore became neces sary to undertake new studies by which asbestos could be identified by precise methods. This is still a very difficult problem, although Langor et al. (1970) and PooIcy et al. (1970) leave shown that chrysotile asbestos can he positively identi fied in a large proportion of the lungs of town dwellers. The initial concern over generalized atmospheric pollution by asbestos involved the possibility of tumor production but the problem was given a new dimension by the work of Jagatic (1967). Jagatic reported that chrysotile heated to high temperatures was acutely toxic to mice and found that after the injection of heated chrysotile into the peritoneal cavitv, 60'; of mice died within 4S hr. Since chryso tile is the type of asbestos usually used in brake linings and since, during braking, linings are boated to very high temperatures, it hecame necessary to consider possible acute hazards of the inhalation of brake lining dust. In order to study the biological effects of heatf'd chrysotile and brake lining dust in more detail it was decided to undertake a series of experiments in which samples of these dusts were injected into the pleural and peritoneal cavities of mice. In later studies it is hoped to undertake inhalation experiments with these minerals to see whether they should be considered as dangerous atmosphereic pollutants. MATERIALS AN'D METHODS The mineral samples used in those studies were prepared as follows: Samples of high grade Cassiar chrysotile asbestos were heated in an electric tube furnace for 4 hr at temperatures of either 400 C. 600C, SOO'C. or 1.000C. After cooling, the chrysotile was ground in a mechanical mortar and pestle and half of ouch sample was sieved through a 250-mesh copper sieve. With normal chrysotile and chrysotile heated to no more than 400C many long fibers remained in the (hist after grinding and since most of these were removed bv sieving it was puss bio to produce samples both with and without long fibers. With chrysotile heated to more than 800C, however, the dust fibers became extremely brittle and grinding was found to break most of these into relatively short lengths. With these samples, therefore, sieving made little difference to flie size distribution of the dust particles although experiments were conducted with both sieved and i it is rved samples to test their effects on tissues. lie thermal decomposition of chrysotile follows a two-stage sequence of dc^rnulation and breakdown, anil the mechanism has been studied by several Authors including Ball and Taylor (1963) and Brindley and Hayatni (1965). The delivriroxvlation of chrysotile fakes place in the temperature range 600-7S<)''C and CHRYSOTILE AS at 800-850C tho deity to give forstcritc and silt Mg,, Si* O The forsterito persists I ature some enstatite is ft 3Mg, Since the highest tempc decompos.tion of chryst present study. Tbroughi chrysotile for simplicity, ular sample are referred The sample of brake commercial producer of drums of two of their tes designed to simulate a composit.on was similar chrysotile asbestos and oxide and metallic alumi lining dust would cont analysis of the dust samp iron on this occasion. With these samples o experiments were undert Jagatic et al. (1967), dos into the peritoneal caviti single 10 mg doses of n 600 C, 800 C, or 1000( Batches of 25 mice were dust specimens were usi doses of 10 mg of autoi cavities of another bate! suspended in 1 ml ot s these studies were kille samples of tissue from cu e lamination or bufferci necroscopy, sections we Peris' stain for iron. Fi araldite and after sectioi In the first series ot e t on of 2.50 mg of ehryso' 2 f hr and In 48 hr sovi disability lor several das of iliist injection. When HWBUI0006701 cmnsoTiLi: a.shk.vios .wo automobile bhakt; lining oust :mi S1HV-850' C the dehydroxylated noncrystullmc residue is said to rucrystallizc to give forsterite and silic a. The total decontpos.tlon corresponds to Mg,, Si, O10 (OH), = 3Mg2 SiU4 + SiO. + 4H2 O . The forsterite persists together with silica up to 1000C but above this temper* attire some enstatitu is formed 3Mg; SiO, SiOo 2Mgs SiO, + Mg; Si-. O0 Since the highest temperature used in these experiments was 1000sC it was the dccompos.Lon of chrysotile to forsterite and silica that fonnecl tire basis of the present study. Throughout this paper the dust samples are referred to as heated chrysotile for simplicity. Readers interested in the exact chemistry of any partic ular sample arc referred to the information in this paragraph. The sample of brake lining dust used in these experiments was supplied by a commercial producer of -brake linings and had been taken from the rear brake drums of two of their test vehicles. These cars had been put through a test routine designed to simulate a long period of normal usage. The original brake lining composit.on was similar to that described by Lynch in 1968 and in addition to chrysotile asbestos and binding resins the linings had contained both chromium oxide and metallic aluminium granules. It would normally be expected that brake ailing dust would contain metallic iron from the brake drum, but an X-ray analysis of the dust sample performed by A. L. Rickards failed to detect crystalline irmjunn this occasion. Bi these sample's of heated chrysotile and brake lining dusts the following ".incuts were undertaken. In the first series, in order to confirm the results of jagat.c ef al. (1967), doses of 250 mg of chrysotile heated to 1000:C.' were injected nto the peritoneal cavities of 25 Balb/C mice. In the second series of experiments ingle 10 mg doses of normal chrysotile and chrysotile heated to cither 400C, 600 :C. H00C, or 1000C were injected into the pleural eavities of similar mice. Batches of 25 mice were used for each dust sample and both sieved and nnsieved lust specimens were used in each case. In the final scries of experiments single doses of 10 mg of automobile brake lining dust were injected into the pleural cavities of another batch of 25 Balb/C mice. In all eases the dust samples were uspended in 1 ml of sterile distilled water before injection. Animals from all these studies were killed at intervals from 7 days to 1 yr after injection and samples of tissue from each were fixed in either fortnol saline for light microscopy .'animation or buffered osmium tetroxide for electron microscopy. For light m croxcopy, sections were stained with either hematoxylin and cosin, E.P.S., or Peris' stain for iron. For electron microscopy, the tissues were embedded in raldite and after sectioning were stained with lead citrate. OBSERVATIONS In the first scries of experiments those animals given an intraabdominal inject on of 250 mg of ehrysotile heated to 1000C showed signs of toxic effects within \ hr and by 48 hr seven had died. The remainder showed continuing signs of isub.litv for several days Imt all eventually recovered and none died within 6 mo injection. When those mice that had died w.thin 48 hr of injection were , ....................................... wmfflw1 7 M J* 4- * ri ' VS *V-j? A'il i. l: v HWBUI0006702 iUl M2 DAVIS AND OONLAM examined it was found that little or no cellular response had developed around the dust which remained .scattered and loose in the peritoneal cavity. A histo logical examination of the viscera of those animals showed no signs of damage with the exception of some patchy hydropic change in the kidney tubules. When mice from this series were killed 7 days after dust injection it was found that the dust in the peritoneal cavity had hv now become compacted into large gran ulomas. Most of these remained free in the peritoneal cavity, supported by strands of connective tissues, but some became attached to the surface of the omentum. It was only occasionally however that adhesions were formed between the loops of the intestine. Within the granulomas the cellular reaction to the dust consisted largely of macrophages with a few giant cells, fibroblasts, lymphocytes, and plasma cells and with the smaller lesions cells were able to penetrate throughout the dust mass. With a -dose of 250 mg, however, some of the dust lesions were so large that complete penetration did not occur and the central regions of these granulomas remained completely acellular. By 2 wk alter dust injection most of the granulomas were surrounded by a thin but clearly defined capsule of fibrous tissue and some collagen had been produced among the cells of the granuloma itself. A gradual increase in collagen was seen within the lesions between 7 days and 1 \t alter injection hut within tin's period the granulomas still contained many cells. jUfcJn the experiments using 10 mg doses of cither heated chrysotile or brake lining HBt no animals died within the first 2 wk after injection and none showed any .o.xic effects. Granulomas were produced in response to all the injected samples but cellular reaction to the dust was found to vary according to the dost type, the temperature to which it had berm heated, ami its method of preparation. Animals injected with either normal chrysotile or chrysotile heated to 400'C were found after 2 wk to have cellular granulomas with complete cellular penetration throughout the dust masses, hi these lesions cells masked the dust to such an extent that it was extremely difficult to see with the lieht microscope. In the animals injected with chrysotile ground and sieved the granulomas were relatively small and formed few adhesions, but in those animals injected with unsieved dust the granulomas were much larger for the same dust dose and frequently formed adhesions between the contents of the chest cavity. In animals injected with dust heated to 600C cellular granulomas were still produced but there appeared to be loss dilFercocc between the sieved and the unsieved dust groups, and both produced only small granulomas without adhesions. In the groups of animals injected with chrysotile heated to either 800 or 1000'C the resulting granulomas were always small compared with the dust dose used and no adhesions were found in cither the sieved or the unsieved group. In these experiments, however, it was found that the chrysotile dust was now' clearly visible in die tissues and in the light microscope appeared to consist of cylindrical or rectangular particles which ruimed from i to 10/nn in diameter and which could he up to 50/im in length. Normally cells were still able to penetrate throughout the dust masses but fewer cells were present than in granulomas produced by normal chrysotile. By 2 wk alter dost injection a thin capsule of fibrous tissue was usually formed and those granulomas remaining free in the pleural cavity. Those that formed Lesions, however, did not develop any specific lav cr of fibrous tissue between the main granuloma and the organ to which they were attached. By 2 wk, also I ( -- -Nn mmi fiHHYSOTILE Ail Fig. 1. A section of a mat sotile asbestos heated to 60 within tin* cell, and the sti appears loosely arranged ir stage contracted closely arm some collagen was usua main part of die lesions duction was more rapit to S00C or more, than or chrysotile heated to this distinction was no I in any given area of gi similarly prepared clirv attires. The physical sh. effect on fibrosis. The t dose was closely related samples heated to no in more fibrogcnic than s higher temperatures. Al Klrctnm microscope samples heated at 600 t giant cells and low e HWBUI0006703 CHinSOTILE ASIU.STOS AND AUTOMOBILE BRAKE LINING DUST 343 Ftt.. 1. A srction of a mucrnpham' Irom a iihiiim' arumilninu 2 k after an inject inn nf ell i ysotile asbestos heated to fiOO'C. Croups of clirysotile crystals are '< '-n in two piiauosomes ( P) within the cell, and tin1 structure of these enstals appears tiunn.il. In lmth cxm-s, the dust ippears loosely arranged in the pliagnsnines anil the phaunMiine membrane has nut at this tage contracted closely around the dust. (>3.000. ome collagen was usually present among the plugoeytic cells that made up the nain part of the lesions. During the first few weeks after injection collagen pro duction was more rapid in those granulomas induced by c lirysotile dust heated o 800' C or mure, than in granulomas resulting from either unheated clirysotile r clirysotile heated to no mure than 600'C. However, by 6 mo after injection this distinction was no longer apparent, and the long-term production of collagen ;n any given area of granulation tissue did not appear to differ much between imilai Iv prepared chrvsotile samples that had been heated to different temper atures. The physical shape of the dust particles did. however, have considerable effect on fibrosis. The total amount of collagen produced in response to a 10-mg lose was closely related to the size of the resulting granulomas and since unsieved samples heated to no more than 4CXDC produced large lesions these samples were more fibrogenic than sieved samples heated to 400C or to samples heated to lighcr temperatures. All these produced very small granulomas. EJ^jtrori microscope studies showed that almost all the injected dust from heated at 600 C or less was pliagoevfused by macrophages and occasional ..ATtclIs and few extracellular dust particles were found. The process of HWBUI0006704 ;U4 DAVIS AND CONIAM CliBYSOTIU-; AS chrysotile crystals had h crystals in the bundle {I the crystals together dur: large masses rather than each. When the tissues ' original outline of the c detected (Fig. 2), but se was no longer visible. It dissolution within the ti.v by an irregular honeya. masses were much thicke no greater length, and macrophages. Very larg although these cells are ; more frequently the la macrophages. Inside the behavior of the heated normal chrysotile are tak< fluid and the phagosome Fig. 2. A section of a moiiv uramilonias ! uk after the intrapleural tnji-cfiim of ciirysotile dust heated to 800JC A law bundle of fused ciirysotile crystals has heeu piiacocytosed by a uiacrophui'e (Mi. ami the pluutiiMmif uu-mliratu- is in close e< intact u ith the dust t urnmod). Although the ciirysotile crystals are partially fused nitli one aiiutlier, the uriitinnl er\ stul outline is still discernible. A 65,000. phagocytosis and the subsequent behavior uf the dust within ceils appeared identical to that reported previously (Davis, 196S, IViTOa). Dust particles were initially taken up into large vacuolated phagosomes (Fig. i), but the membranes of these eventually contracted and became closely opposed to the dust particles. Usually the dust remained oil its own inside the phagosome membrane, but occasionally it was found mixed with ferruginous granules and membrane debris in the dense structures that are known as phagosome residual Ixidies. Most of the dust remained in those structures, but occasionally an odd fiber was found that appeared to have escaped into the cell cytoplasm. The macrophages from these experimental groups showed no signs of damage even when they contained large amounts of dust. No differences could be detected in the structural appearance of crystals of normal chrysotile and ciirysotile crystals that had been heated to 400JC. With dust heated to fiOOhowever, it was noticeable that the phagocytosed dust, whether sieved or unsieved, contained more individual chrysotile crystals than crystal bundles, and many phagosomes were found closely [lacked with indi al crystals. Electron microscope examination of lesions produced by chrysotile eel tu bOO (,' or KHXJ f.' showed why the dust was clearly visible in these Tsnms even with the light microscope. During the heating process the individual Fig. 3. A section of a inucrop mile Bust lieali'd to MOO'C. Tlu partii-k- uf fused vhvyxntlif (C mmii.il crystal outline li.es been \ TO.tXXJ. HWBUI0006705 |:HHYSOTII.F. ASHKSTOS AND AUTOMOBILE BRAKE 1.IVINC DUST .tile crystals liacl lost their normal tubular structure and fused with the other stals in the bundle (Rickards, 1967). This fusion was complete enough to hold I he crystals together during grinding so that the dust was broken up into relatively 1 a :e masses rather than fine fibrous bundles often containing only a few crystals i ii. When the tissues were examined only a few weeks after dust injection the iriginal outline of the ehrysotile crystals within the fused masses could still be [ie'-'ctcd (Fig. 2), but several months after dust injection this crystalline structure no longer visible. It appeared that the dust had undergone some degree of liissolntion within the tissues, and the neat crystal arrangement had been replaced ly an irregular honeycomb structure (Fig. 3), Although the fused ehrysotile n. ses were much thicker than normal ehrysotile dust particles, most of them had jo greater length, and the majority could still be phagocytosed by single jiacrophages. Very large particles were occasionally seen inside giant cells jit )ugh these cells are not common in mouse granulomas (Davis, 1970b), and jit 3 frequently the larger particles were closely surrounded by unfused jiacrophages. Inside the cells there were some notable differences between the jt rvior of the heated ehrysotile masses and normal ehrysotile. Particles of b ial ehrysotile are taken up into large phagosomes which presumably contain liid and the phagosome membrane is otten well clear of the contained dust, at t&r* 4 --r i^ *i 4 .r. -t.** -t i -r: " .S'- |-t~ ' - ? 1 -- ^> 1 - * 1 s'* *' .v- ^ - s vl .. "V *.* ft t: i. A v rtiim of a macrophage from a mouse aramilotna 3 mo after the injection of ehrysJ ist heater! to HO(V(l. The m;i( ropiuiUr is surrounded by collagen fibers < K). but a large of fused t hrysotile f C*) is present within the? cytoplasm. In this case, however, the Lout fine lias hern lost and has hern replaced by an irregular iimieynmih pattern. ppp *gm*m HWBUI0006706 ...........ini r. ;uf> DAVIS AND CONIAM mill ill -nnh*fcl IMQI .mi * wii chrysoth.i: asuks granulomas never formed a< by thin strands of connectiv could be seen to consist diameter, but a number of from 2 to 25 ^m in diameh I were able to penetrate dee] always comparatively few quickly surrounded by a t injection, and some collugci within 2 wk. After this timi lesions became less ccllula been bound together by a granulomas were so small, f T to the dust was never very g I Fio. 4. Particles of rhrysolilr dust williin .t macrophage hum a mouse pleural eranulotna, 3 mo after dust injection. The dust had lieeu healed to fttlt) (' before injection, and by this stage the ehrysotile crystal pattern lias hri-n replaced by an irreutdai honexcuml) structure. The largest dust particle (LM is assneiated \v ith masses ot dense crannies ( K) that probably represent ferritin, nr haemosiderin. and in some areas these have penetrated deeply into the dust. X 33,000. least in the early stages. With the masses of heated fused ehrysotile however, al though they were taken up into membrane bounded vesicles the membrane was always seen in close contact with the dust. Many of the small dust masses could still be taken up by single macrophages which usually showed no signs of damage. In these granulomas, however, it was not uncommon to find dead macrophages containing dust although such dead macrophages base only rarely been found in lesions produced by normal ehrysotile. In animals injected with either unsieved normal ehrysotile, or ehrysotile heated to 400C some typical ferruginous bodies (Davis, 1970b) were found in the gran ulomas from about 2 wk after dust injection. Sieved samples of normal ehrysotile and ehrysotile heated to 400C did not, however, result in the production of any ferruginous bodies. This was also true of both sieved and unsieved samples of ehrysotile heated to 600C. In animals treated with ehrysotile heated to either MM) C or 1000'C no true asbestos l>odics were found in'which the dust particles were surrounded by a distinct coating of ferruginous material. However, with some of the larger ehrysotile fragments, especially those showing the greatest Ujrgrw of dissolution, it appeared that dense granules similar to ferritin nr Ptcmosidcrin had penetrated deep into the dust ( Mg. 4). Brake lining dust when injeeted into Hie pleural cavity of mice produced smaller granulomas than any nl the asbestos t) pes used in this study and those t'lc. 3. A particle oi axitouie! Kraiuilmu.i 2 \\l> .ittcr ilust it meinbrjinc. but this is oh>scl\ mainly of dense amorphous u are, lumx-x cr. xxitluo the ilasl aluimtiiiiii l A V ' 1*0,000. HWBUI0006707 UVSOTII.E asbestos AM) automobile bhake limnc dust :m7 It* mas never formed adhesions, being mainly suspended in the pleural cavity lin strands of connective tissue. In the light microscope the brake lining dust uld be seen to consist mainly of irregularly shaped particles 5-25 pm in a meter, but a number of elongated particles were also present which ranged i 2 to 25 pm in diameter and which could be up to 50 pm in length. Cells re able to penetrate deeply into the dust masses but the number of cells was l.vays comparatively few compared to the dust load. The granulomas were I i :ly .surrounded hv a thin capsule of fibrous tissue within a few days of -`ion. and some collagen had been produced among the dust containing cells Stliin 2 wk. Alter this time collagen production increased quite rapidly and the |i is became less cellular so that 1 yr after injection the dust particles had bound together by a network of old acellular collagen. Since the initial l.imlomas were so small, however, the amount of collagen produced in response |t' dust was never very great. s\ S] ty. -... J*. *" * . . ^5*. * yyir L %., Sr{, j *-4"\VTb' ia -.'XT >*>, i--sa w5^5rS,&l 1 ''k&SK * ' ` ^ AaJ 'w.-atr.b. i I >. A |)ai tide n{ aiitmnoliile brake liiiimi dust within u macrophuKe from u mouse pleural I luma 2 nl( after <J*i>.r injection. The dost is separated from the cell cytoplasma by a irarx^ Imt this is closely opposed to the dust at al points (arrowed). The dust consists of dense .iinnrphous materia!, and contains no recognizable clirysotile asbestos. There eInn the dust some dense crystalline particles that prohaliis represent metallic / riO (hxi. DAVIS AND CONIAM Electron microscope studies showed that the brake lining dust consisted mainly of dark masses showing no internal structure, and containing no recognizable chrysotile. In most cases the only contaminants within the dust masses were smooth crystalline particles usually about 0.05 of a micron in diameter (Figs. 5 and 6). Since metallic iron was not present in the original dust sample these were probably particles of metallic aluminum added to the original brake lining mix ture as an abrasive. Very occasionally dust particles were found that consisted of a lighter and more homogenous material which contained in addition to the crystalline particles already mentioned elongated crystals of apparently normal chrysotile (Fig. 7). Tins type of dust, however, made up much less than \c/< of the total. Small dust particles were phagocytoscd in large numbers by single macrophages but as with chrysotile heated to high temperatures normal phago some vacuoles were not seen even in specimens examined only a few days after dust injection. The phagosome membrane contracted quickly around these smaller dust particles to form phagosome residual bodies, and these often contained some ferruginous granules and membrane debris as well as dust (Fig. 6). The larger dust fragments were sometimes found within small giant cells, but as with heated I ---e, fi. A mntruphuun from a immsf pb'urul gianuluma 4 wk alter the iiijt-i-titm of ht.Ae |iniri'4 ilusl. J)n\t launch's M)) arc present, with inemlir.inc elements and Mime line er.m "nl.'ir material, in irregular stnietnres that pmhuhly represent phagosome residual lhe / dft 000. Fir.. 7. A particle of brake nl this dust. In this instance t are embedded recognizable ci particles that probably repre chrysotile many of the 1 extracellular. Dead ntacr granulomas at all stages granulomas produced by The present study has uf asbestos partich's is in in issues. Several workei and Webster (19651 ha\ to lung tissue than short fi to dust injected into the j samples of a mimiter of fiber tlust. In the studies i heated to no more than itig large proportion 01 the original cvxstal bund and theretore 'erv long t HWBUI0006709 ntRYSUTII.K ASUKSTOS \\D AUTOMOBILE BRAKE UMNO DUST M-IP $ a r Mmm^h f Ui*. ' ^ ^ pi ,>-,. r *,-* F F^i * 1HV j ; \ ,^kv. mji:... v\ :> -* - V. ii*"*** ,4!l^a - *JwC1*`.'s/ v /*- */.<-r*/ :*...' n . 'v'*-;! Ir* y.-v .,*:. . < ;*<%...MV>- *j,ma-' !V M' * . .v,47 |Ei<.........V particle <>f brake liiiiiiu cluU u uliin .i iiwhim- in.icrophauc 2 \\k after the injection |rMs dn>t. In this instance the dust consists ni Imv density ainorphons material within which mbcddcd recoanizable crystals i>1 i lir\ sutile jsIm'sIos i (!) and l.u c'ei iionHhrrms cr\ sfalline :ies that probably represent metallic aluminum i A ). fi'l.fMKt. sotile many of the larger particles although surrounded by cells, remained cellular. Dead macrophages containing brake lining dost were found in the Imulomas at all stages ot the experiment, bnt they were less frequent than in uloma-s produced by chrvsotile heated to either S(XJC or 1000 C. DISCUSSION e present study has confirmed previous suggestions that the physical shape bestos particles is important in determining the degree of fibrosis produced |t ssucs, Several workers including King et at. (1946), Vorwald et ai. (1951), Webster (1965) have suggested that long fiber asbestos is more fibrogenic tg tissue than short fil>cr dust and Davis in 1972 showed that this also applied lust injected into the pleural cavity. It was found in this work that long fiber les of a number of mineral dusts produced much more fibrosis than short dust, in the studies using heated chrysotile it was found that if*the dust was [ted to no more than 4(X)C mechanical grinding still produced dust eontain large proportion of long fibers. When normal chrysotile is finely ground riginal crystal bundles tend to split longitudinally rather than transversely re very long thin fibers are produced. Sieving this dust removes most m yi.J .-VN-. ....` -o*. iv. ^. *eV. V >' HWBUI0006710 ;<50 DAVIS AMD CONIAM of these long fibers and it was therefore possible by using both sieved and unsieved dust samples to gain some idea of the effect of fiber length on the tissues. It was found that the long fiber samples produced larger granulomas than the short fiber dust and since the amount of fibrous tissues produced depended on the initial size of the lesions (Davis 1972) long fiber dust was more fibrogenic than short. When dust was heated to 600C the structure of the chrvsotile crystals appeared unchanged in the electron microscope but the ground dust samples contained fenv long fibers. It appeared that the crystals were more brittle than normal chrysotile and that in most cases the bonding between the crystals had been weakened. This was probably associated with the gradual dehydration that occurs when crysotile is heated from between 100C and 600C. Grinding this type of dust tended to break it into individual short crystals and there was little differ ence between sieved and unsieved dusts. Since few long fibers were present both samples resulted in the production of small granulolnas and little fibrous tissue was produced compared to the size of the dust dost-. When chrysotile was heated to 800C or 1000C the individual crystals fused with one another and the fused bundles remained brittle. When these samples were ground the original large bundles of chrysotile crystals fractured transversely as one unit and the resulting dust consisted of thick, but short particles. Sieving this dust did not change the particle size distribution and lnith samples produced very small granulomas resulting in relatively little fibrosis. With the hiak<- lining dust the particle size Blistri button was very similar to chnsutile heated to 800 Tl or more. Some eolongated partich-s were present but these always had a very low aspect ratio. Since very little uf this dust could be recognized as chrysotile even with the elec tron microscope, the results arc not directly comparable with heated chrysotile but very small granulomas were produced in response to brake lining dust and little fibrosis resulted from its injection. The first group of experiments in this study confirmed the suggestions of Jagatic et al. (1967) that chrysotile asbestos heated to high temperatures is far more toxic than normal chysotile. It would appear, however, that very high doses are needed to produce a lethal effect and after a 10 mg dose none of the mice showed any signs of disability. This 10 mg dose tor mice corresponds to one of approximately 30 g for a 12 stone man and this is an impossible figure for anyone to take in hv inhalation over a short period even under the worst industrial conditions. It is very unlikely, therefore, that this toxic effect of heated chrysotile need be considered seriously when dealing with the problems of general atmos phere pollution by this type of asbestos. From the experimental point of view, however, the reasons for this toxicity are of considerable interest. It has been suggested that when chrysotile is heated to more than 800C free silica is liberated in the amorphous state, and several workers htive shown that this can he lethal. Gye and Purdy (1922) demonstrated that colloidal silica was rapidly fatal to experimental animals when injected intravenously and Dale and King ! i `>53) produced similar results. These-workers found that colloidal silica was ten times more toxic than crystalline quartz in these conditions. Klosterkotter and ^ittcti (1953) also showed that tnolecularly dispersed silica, whether administered "nitraehealiy or intraperitonesilly, was lethal to rats. It therefore seems possible that CHRYSOTILK ASBh the deaths of animals gh acute silica poisoning. Regardless of general sj heated to 800`C or more toxicity for individual ma< using the intrapleural inj 1970a) it was noted that it With heated chrysotile or quite frequent although percent of the total. Even distinct differences from t utile. When normal chry> for some time in a large p ture contracts around the c: (Davis, 1968) that this p phagocytosis but this mus macrophages with vacuola granluonias for several wet and brake lining dust, vacu membrane was always clo it would appear that then sible for quicker contract) residual bodies, and it is ii to the raised content of am Marks et al. (1956) shot est degree of systemic toxf to macrophages, it may bt cause the rupture of phago able to increase the pen reduce phagosome contrac The finding that chrvso' normal chrysotile yet rest 1964 finding that inneroph from silica dust. In fact. 1 iu these findings. It has l resulting from the injectiot initial cellular response to length. Since the heated i uiomas were very small an dust cytotoxicity probablv the total collagen product! The finding that autou is encouraging, hut the it brake lining dust corner mesotheliomas, and the pr. tumors were found in an lining'dust hut the esptu response to the dust and ' HWBUI0006711 ciihvsotiu: \sri:sios and adtomoiiile rhaki: lining dust :if>i taths of animals given large injections of heated ehrysotile were due to silica poisoning. .egardlcss of general .systemic toxicity, however, it was found that ehrysotile eated to 800C or more and brake lining dust both showed much more cyto toxicity for individual macrophages than normal ehrysotile. In a previous study "sing the intrapleural injection of ehrysotile in experimental animals (Davis, )70a) it was noted that it was very rare to find dead macrophages in the lesions. With heated ehrysotile or brake lining dust, however, macrophage death was `mite frequent although dead macrophages never made up more than a few ercent of the total. Even the living macrophages from these granulomas showed iiistinct differences from those in lesions produced in response to normal chrysotile. When normal ehrysotile is phagocytosed by macrophages it is retained >r some time in a large phagosome vacuole before the membrane of this strucire contracts around the dust particle. It was shown in organ culture experiments ( Davis, 1968) that this process could be completed in as little' as 12 hr after hagoeytosis but this must be rare and after intrapleural injection of ehrysotile incrophages with vacuolated dust containing phagosomes could be found in the granluomas for several weeks. With ehrysotile heated to 800C or more, however, id brake lining dust, vacuolated phagosomes were never seen and the phagosome icmbrnne was always closely opposed to the dust particle. From these findings it would appear that chemical differences between the dust samples arc responcible for quicker contraction of the phagosome membrane and the formation of 'sidual bodies, and it is interesting to consider whether this property was related ro the raised content of antorpliit.s silica in the heated samples. N^jks et al. (1956) showed that whereas amorphous silica produces the great^f'ee nf systemic to\icit\. the crystalline varieties are mure rapidly cytotoxic Terophages. It may lie. however, that while nonerystalline silica is unable to cause the rupture of phagosome membranes (Allison ct al.. 1965) it is nonetheless hie to increase the permeability of most phagosome membranes enough to ?ducc phagosome contraction time'. The finding that ehrysotile heated to more than 600C is more cytotoxic than ormal ehrysotile set results in less lihrosis appears to contradict Hepplestons 964 finding that macrophage death is an essential precursor to fibrosis resulting from silica dust. In fact, however, two conflicting factors arc probably involved i these findings. It has been Found (Davis, 1972) that the amount of fibrosis suiting from the injection of mineral dusts closely corresponds to the size of the initial cellular response to the dust and this in turn depends largely on the fiber ,nn2th. Since the heated ehrysotile contained no long fibers the resulting granlomas were very small and although macrophage death resulting from increased oust cytotoxicity probably accelerated the production of collagen in these lesions the total collagen production was never very large. The finding that automobile brake lining dust has a very low fibrogenicity is encouraging, but the initial concern over the pollution of the atmosphere by brake lining dust concerned its possible association with the production of lesothr-Iionias, and the present study has not settled this point with certainty. No .nmors were found in animals injected with either heated ehrysotile or brake lining dust hut the experiments were mainly concerned with the initial tissue expense to the dust and were terminated after 1 \t. It is now known, however. .HI. HWBUI0006712 r DAVIS AND CO MAM ( CHRYSOTILE ASL that whereas intrapleural injection of asbestos in rats produces large numbers of mesotheliomas (Wagner, 1960) few arc produced by similar injection in mice ( Davis, unpublished) and so no significant results would have been produced irotn prolonged experiments of this type using brake lining dust. It is planned to undertake new series of experiments that will test the long-term effects of brake lining dust in conditions where normal chrvsotile is known to produce tumors. At this point, however, it may be pointed out that electron microscope studies of lesions produced in response to brake lining dust have shown that this dust contains extremely little recognizable chrysotile and what there is is mostly embedded in the plastic bonding material. No computation of urban atmospheric pollution by chrysotile from this source can therefore be made by calculating brake drum wear. REFERENCES Allison, A. C., Harincton, J. S. Bihsec.k, M,, and \ ami. T. ( info). Observations on the cytotoxic action of silica on macrophages. Inhaled Partichand Vapours. II. In "Proceedings of the British Occupational Hygiene Society Symposium. (.'ainbridge' ( Davies. C. V.. ed.), pp. 121-131. Published by Pergamon Press. Anjilvel, L., and Thurldeck. W. M. ( 1968). 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