Document a4m8v5jpE7bBEL2ZM3en2YYaM

&VH,-,;.- PLAINTIFF'S EXHIBIT (YbVcO.OO s CLAIMED * PRIVILEGED X . M . .A BY OCF Archives of Industrial Hygiene and Occupational Medicine Yoi.umi*. 3 JANlMIt Y 1951 Oi'Yim-iiT, IH.'il, my tiik Mmhiui. Avum.utio.s Numiis'.ii 1 EXPERIMENTAL STUDIES OF ASBESTOSIS ARTHUR J. VORWALD, fhOtPath.), M.D. THOMAS M. DURKAN -* AMO PHILIP C. PRATT, M.D. URANAC LAKt, N. T. ' ASHKSTUSIS is a form of pnciuuomicuuiosis resulting front pmlonged inhalation of asbestos dust. The name "asbestos," literally "unbumablc," is not that of a specific mineral but is a term applied to a luuiilier of different minerals whose characteristic feature is a structure com]M>scd of long, parallel, flexible fibers. This structure is unique Itccansc the fibers arc capable of repeated longitudinal subdivision to units of molecular proportions. In length the fibers vary from a few microns to 6 or more incites (15 or more cm.). Some varieties arc stitTer than others, but many are sufficiently flexible to Ite spun into yarn and woven on modified textile machinery. ' The asltestos minerals arc silicates of variable composition and belong to the serpentine and the amphiltole groups. Listed below arc the more common varieties. Aniphibolc group: actinolite, amositc, amphiltole, authophyllitc, crocidolite and tremolile. .. Serpentine group: chrysotilc. The bulk of the asbestos of commerce is chrysotilc, 3Mg0.2Sif),.2H,0, which is mined on (bis continent principally in the Tbctfonl region of the Province of (juebcc, Canada, and in Vermont. Crnciilolitc and ` amosite also arc used commercially but in much smaller amounts. Chrysotile occurs as veins in serpentine, a mineral of similar chemical composition, which exists in massive form and is made up of microscopic fibers without the parallel orientation characteristic of chrysotile. The massive, bluish black scr|icntine. which is smooth and soapy to the touch. ' is traversed by veins of fibrous chrysotilc varying in width from a barely perceptible line to 6 fl5 cm.) or more inches. The fibers run across the vein and not lengthwise with the formation. " i '' . . From the Saranac lLaabboorraattoory onf the Kilward L. Trudeau lK: muiilitiun. This aeries of studies f asttestnsis, initiated at the Saranac I^ttnralory mure titan twenty years ago by the late Dr. l.erny U. Gardner, director iof (lie lahnrntnry, was nearly completed at the time nf his death in October 19-16. Altlioujli partial reports and infnnna! reviews of some nf the experiments had been itivcti from time to time by Dr. Gardner, this paper presents for the first lime a Complete survey of tltc entire experimental investixation. 1 :1r. 1*25 - * m !os 6 cv 5 kV letse Si 2*w Au oc fo5 * *t* PLAINTIFF'S EXHIBIT HUaN'OOO&W __________________ ' Oh ood iiieo mm t>Y\Vr v?*#^ 5#"'i*>* .`r^A -. Attention is ihrccud lo the uuiiiT.il liriKitr. M'i.Ii .ii. uiucli i-> often found in the same formations with scr|icutiiic mu I chrysolite ami may he fibrous in structure, Except for the manufacture of ma^uesium. hrucitc has no commercial value at present liecaiisc its fillers arc not sufficiently flexible to lie used in textiles. Inn they are cajahle of repeated longitudinal sulxlivisiou. Unlike other ashestitorm minerals, hrucite is not a silicate, ami fur this reason it has been a valuable tool in an experimental evaluation of the action of fibrous minerals on lung tissue. KXrEKl MEXTAI. ASIIKSTitSIS For many years studies * have been carried on at the Saranac Laboratory in an investigation of the cause, nature and development of ashestnsis. The present paper is devoted to experimental asbestosis, \x !j CLAIM5I5 privileged ; JBY OCFj I - Oo O ; I ` a " o *> a-a iDr*s :?t 3s4;5c?|s *S/. lik hO a Cv o o U b -G n-* 't*vi \3;h:tl.l 1S.iSS?S-7- ii-cii R^ r'*s:iss'A fevi:- r. iR^sSSS? pH jf'-T* ri <rf f. ifWftVi & .--- VVLr*^" r \j5^V>*i i^g pi Fi(t. 1.--Htiinun asbestosis (P-36-144). The pliotoniii-rugrapli reveals a bronchiole (riitht center I with a smooth muscle bundle at its inieriur margin ami with an extensive rone of collagen deposition largely obliterating the surrounding alveolar structure. The hlack foci are macrophages cuutainiiig incidental pigment. Asbestosis Unties are present but are nut apparent at thin magnification (X 200). and in it arc described the experiments made on animals with various kinds of asbestos dust. Another report, to lie prepared and issued at a future date, will lie concerned with human asbestosis and will cover the health asjiccts of workers who have been exposed to asbestos dust in an industrial environment. Although in man asliestosis is a chronic disease with diffuse pulmo nary fibrosis which requires years to develop, it is i>ossihlc to reproduce 1. (a) Gardner, L. U., and Cummings, D. .: Studio mi Experimental Pneumukoniosis: VI. Initiation of Asbestos Dust: Its Kffcvl ti|*m Primary Tuberculous Infection, J. Indust. Hyg. 13:65 and 97. 1931. (b) Gardner, I.. U.: Chrysolile Asbestos as an Indicator of Subtile Differences in Animal Tissue*, Am. Rev. Tubcrc. 45:762, 1942. t ? * .f * i v .v F f. i-. 006 - .A M hA -- f. \ i' 1061 j fe?S SS te# fcS'-.>-V, 111 nr or nr tie >|Hvii, '' :iiiiiii-iI characteristic tissue changes which arc similar in lilt- lesion- Iiiiiuaii aslicstosis (fig. 1). Since tlic life span of ' tin* c\|HTiiiH'iiia1 .111i111.nl is relatively short, it is not possible to produce the characteristic lesions in animals under conditions identical with the usual industrial environment. Consequently, to obtain a complete evalu ation of the tissue response to inhaled particulate and fibrous material. ay it is necessary to accelerate the reaction by employing higher coneentra- ^ lions ol dust than would ordinarily he encountered in industry. While CLAIMED * conditions of ex|msurc are thus different, the information yielded hv PRIVILEGEl5 animal e.\j>eriments is invaluable in furnishing a liettcr understanding of Bjf OCF the reaction of the human organism to inhaled asbestos dust. Exrr.KiMr.NTAt. Xlmions For investigating the tissue reactions of experimental animals to the various asbestos minerals, two types of technic have been employed, namely, the inhalation method and the injection method.* In inhalation V experiments, grou| of animals--up to 100 or more guinea pigs and sometimes smaller nuniliers of rabbits, cats. dogs, rats nr mice--are kept for eight hours a day in a cubical dust room. 8 ft. (2.5 M.) in dimension, in which a cloud of ashestos dust is maintained by a rotating paddle in a dust hopper.'* At intervals during the experiment a few animals are killed and the tissues examined to determine the nature and the extent of the dust reactioo. Some animals are exposed for periods up to three years. The injection experiments arc used to deter mine in as short a time as possible whether or not a particular dust has a jxttcntial capacity to produce inflammatory reaction when in direct contact with tissues of the body. The method involves injecting the dust, either dry or suspended in fluid, into the animal by the intravenous, the intraperitonea), the intratracheal or another route. * Long term inhalation experiments furnish information on which great reliance is placed when estimating the degree to which a dust might constitute a respiratory hazard to industrial workers. Even though an atmospheric dust may be potentially dangerous, as indicated by injection experiments, only inhalation procedures will reveal whether the dust can he inhaled, pass the natural defense harriers of the body and reach the pulmonary tissue in quantities sufficient to cause damage. Injection methods are useful, however, because they ntakc certain that contact occurs between the dust panicles and tissues and Iwcause they allow accurate estimation of the dosage and of the potential cajiacity of that dose to produce reaction. The intratracheal method is particularly valuable when one is dealing with fibrous minerals like asbestos, since it permits observation of the cfTcct of the filters on pulmonary tissue. Tissue 5usiT.mim.iTv Unlike free silica, asbestos docs not produce specific effects in all organs of all species of animals. The comparative data presented in table 1 are based on completed observations and therefore differ slightly from a preliminary report.,l' Fine quartz introduced into various organs ^ of contact with the tissue. In cuts, rahhits and mice a lew >t the fiber.show an atypical coating after much longer resilience in the lungs. In rats the UKlics are rarely seen, and in dogs none cnnld lx* found. Although the evidence is incomplete, it apftears that the formation of the asltcstosis Itody prevents the fiber front damaging the tissue. Many of the |>oints mentioned aliovc will he elalioratcd on in subsequent para- CLA.TMED PRIVILEGED! BiT OCF oo laaaaoKmm *to>-i*ro* -a: rgsIS* M h - u Sa .its:? r Its m /# A 1-i(T. 2.--./. hiini:in a-ls'-lo-ii ImkIics. This Collection of .i-lic-t<'>is IkmIh-- was found in the iiini* sIhiwii in linurc 1. The usual variations of sire ami cunliguniuun are reprodUcsl (>;4IIU>. B, Kiiinva |iia a-ltvstutis l*ody. This one is similar to some of thusv sliouii in A (X 400>. graphs dealing with the actual ex|x'riments. l-'or presentation our investigation is divided into two sections, one dealing with inhalation experiments and the other with injection experiments. A i. out* io6at VjJ .>4' tSKyfVJ- K5&/5 mm mm t&ftgrii p5S% S fe$y Tv.'&r ;!>">'> PI W$M -SfcM- J*-*ffi I IN II AUCTION KXPKRIMESTS CLAIMED PRIVILEGED BY OCF Four large scale inhalation cx|>erimcnts have lccri conducted in this laboratory with various forms of asbestos dust. In each of these investi gations, more titan KO animals were used, and the experiments were carried on for periods ranging from two to more than five years. The four kinds of nslxstos dust employed are designated as King's floats, short fiber, 100 per cent hall-milled, and long fiber asbestos dust. King's Floats. Asbestos Dust The first inhalation experiment conducted at the Saranac Laboratory with asbestos dust was begun in 192S. Animals inhaled the dust for Table 2.--Chemical Analysis of Asbestos nutting Materials Typ, ot Asbestos Itol-. SlOi r,o> AliOa CrrO. ftloO c.o MtO XstO KtO COs Lbooau ,* Total blot's floats...... .... -J2 BJU , OJw u..v; 12.74 07.IS Sbort fiber......... ... r.17 Loog fiber........ ... SB.40 IJB ft.lt 1.40 0.7ft 0.14 0.00 0M 0.10 0.31 3ftJO 40.11 0.14 0.00 0.50 OJfi 0M ost 14.00 100.11 14.00 99.70 * Not dctermloed. Table 3.--Petrogrophte Analysis of Asbestos Dusting Materials Kifii'i float**: H approximate eompn|tloa. ba*M An partfelrf (except ehry*MHO mutter thao 10 uirrour and reported a* permit ape* obtained from particle count*, it chrysolite 14, arrptMlac 40. magnetite is, rsrbooate* 19, laic It, other tnineraU 4. For ehryrotile, fiber* up to 2U> mierooa loos were included. Short Alter I: The material, l*,ftre Mi; hall luJJta!, eAntiiflnl (reton<Waiter of flhrous rhryaollle and platy (notiflbroue) *erpcntin*. The approximate rtimpn>itiiiD. by permtiaae, wan chryaotilc 17. aerpeotine 4a, magnetite io. Quarts !, brwitc 5, outer nlorralr, laelufiiog dolomite, aetmolU* aod trcmolitc, 11. Long fiber t: Tlie material eonl*t*1 principally of the fibrous asbestn* mineral ehry*otne. bhmls of rmneeparated fiber* ft to 1ft uicroo* In diameter aod up to 90 aileron* fit leogth m-re pmeot. The approximate rompoellloo, by percentage. war ehryeolile 7ft, *erg*btint 1ft, mag* ortile ft, brueite !. other mineral*, amoog vhtrti mere calritt aod ehloritie aod luicarcoua mineral*. ft. Only a trace of quaru vat obwrrrd. The analyst* of Hie King** float* al*to, made by !>r. <*. K. llurltMit .lr., of Harvard University, ha* been reported eiemhrro (UurlUd. C. R., Jr., amt Wlltiiuii>, It.: The Miueralogv of Abe*to Dun, J Inlu*t Hyg. A Toxicol. 17: tie'. t For the short fiber a<>t*toa and the loot filer aboto* the petrographic anal)>il wa* puppleim'uted ailb x-ray tfiffracfioo exaintaatioo. periods up to 33 months. Some guinea pigs with six and nine months' exposure lived for an additional three years after cessation of their ex|>osure. A preliminary report '* presented observations after 29 mouths of exposure. At that time observations covered a period of only 2'/x years and the conclusions as to the ultimate effects of inhaled aslxstos dust were provisional. Those conclusions are substantiated by results of tbe completed study, which is reported as follows. Composition and Atmospheric Conemiration of the Dust.--Tlx (lusting material, a ctimmerrial variety of asbestos known as Kind's floats, was cuni|;>used uf sluirt filters, raiigius in length from 1 mm. to 1 micron or less, ami uf particle* which also varied in size. It was obtained from tlx Thetford, Quebec, plant uf the Asbestos Corporation of America, ami analyses (tables 2 and 3) reveal tliat the amount <>f nitrous ehrysotilc was only 14 per cent, a rather low value. isldto-M* * m- - jv-Sw %ltf8SWN&| r't^aijlsBS1 firT'i-.wM^df' *t.iC3*r*ri?v-., ..* J '.'; 3 .-> 21 tf - a?# .A' re?#: ^<.1* Impingcr samples liken soon after the experiment was sum-d iudivutcd ili.it the dust concentration ua* at first quite low, the average iliot omul hciii 0,,iv ti.O million particles pet cubic foot of air by the standard liglit field teelmic and 0.8 million for particles and fibers greater than 10 microns. After the inhalation experiment had been under way for about two years, the speed of the rotating paddle in llic dusting machine wa> increased and fur the remaining 10 months of the experi ment considerably mure dust was riisperr-cd into the atmosphere. The average dust count of impingcr samples collected after this change was S3.7 million hy the usual light field method and l.fi million for particles and filters larger than 10 micron*. It is probable, however, that the true values of the dust concentration were higher than the counts given in this paragraph. The impingcr samples for the King's JI *aSk prCiLvAilIMeEgEed"aaa.I28alti8oB"8*f* floats e\|>crimcnt were collected in water, hut later studiesT have shown that BY OCF -e *g t> counts of impiiiger samples of asbestos dust taken in water are not reliable. Ethyl s. alenlud instead of water was used as the collecting fluid in all subsequent experi ments. Tamj: A.--Summary of Inhalation Experiment vrith King's Floats Asbestos Dust Katuft of Experiment lhit rxi*oiurc continuous through* out lift lhit !**me fofloacd by pro* InoRni mideuM in normal air Tuliffrultm* kilrcliou ot start ot tlut e.\|HPurc Oiitfu i infection: no dust ex* i*uurc Jlitti* infretioo * after Jl mo. of du*t ftpiMnrr, ibm rrsidtnce In normal air C>mtrnl to Infection: no dust ex* injure Aalaals S4 gulsts pits s rabbits 11 rsts U eulnrs pics 2S guinea pies 1 rabbit SPguia.i pigs S3 (nines P-X* Maximum llaxl- burvlral Bum After Dust Ex Dust Kx- posure. posure. Mo. Mo. Poults Typical p-ribronctiiol*r fibrosis after 1: moothi 10 0 iKon-ign body bronchitis $ 0 Little or no reaction 0 a Nonprwcrf>siTc Ahrol* t ST KooprngrculTC ftbruns 0 19 i Absorption Of foreign body reaction 0 Temporary iirnere*lon ot loleetton, lolloped hr healing with Ahrosis 0 ut Healing by resolution (one exception) cutsee pics 90 u No appreciable increas* io susceptibility to tuberculous infection; healing with Abrosl* U gala,t pigs 0 . ut Mtallog br nsolutloa !--s e> vt lists * Ttir cnin-* pies ,rr bilMtnl with low virulent. Hi (trsla ot tuberdr bsdllus. t Tbis means On; survival period laUowlac lalectloa. Kc.ults of the investigation, briefly summarized in table 4, show that inhalation of King's floats asbestos dust produced a typical peribronchiolar fibrosis in guinea pigs but not in rabbits or rats. Reaction in Romtal Guinea Pigs.--Guinea pigs inhaling this dust for periods up to 33 months had a characteristic fibrosis occurring in conical patches about the respiratory bronchioles. During this exposure the peripheral alveoli were not involved. The particulate elements of the dust were transported through the lymphatic system to the bronchial nodes, causing no significant reaction in either site; the fibrous elements remained fixed at the points of original localization and were seldom detected in the lymph nodes. After exposure of approximately a year a small amount of cellular reaction had been produced about many respiratory bronchioles (fig. 2/1). As more dust was iulialvd, it continued to accumulate in the same location, and later stages of the disease (fig. 3 D) consisted of extensions of the original lesions. Apparently, the inhaled fibers were caught in the pocket-like alveoli that are given off from the lateral walls of the respiratory bronchioles. There they 7. Fulton, \V. B.; Houtz, R. L.; Dooley, A., and Mathews. J. I.: Ash.sto>is: I. The Collection and Counting of Asbestos Dust Encountered in Asbestos Fabri cating Plants, Special Bulletin 37, Pennsylvania Department of Labor and Industry, Harrisburg, 1934.. 'i i 1065 vS^wSWw *> R&t&j irc |iIui!i<>|i>h'<I. ami many of limit were carried into tlie wall by migratory evils. Miwvimuh-.ir Unkisytcs attracted In the area caused an appreciable thicken inc u( the hrinieliinUr wall. Alter Hi months a delicate lilini made its appearance. The prics cvnlvcd gradually, and the niniihcr n( fine intcreelltilar collagenous filter* steadily increased- A* this fibrous deposit contracted, it |iartinlly closed and V>v jJ C3 ^v */'a V iSNv^VVL-* claimed PRIVILEGED BY OCF SsS-f2a-Svs-**> , fJ:2 5. J&P.2W'&*3?d8ito nr.'^T: - ***'* $L-*V -* :^\s S ^:2*$Sa Fig. .1.--King's floats inhalation c.\|>eriiucitl: ImiR of a guinea pig with Inionths' exposure. It includes a respiratory bronchiole, at the left, branching anti becoming an alveolar duct, at the right. Note the accumulation n[ cells in the wall of tlie bronchiole and in adjacent alveoli (X 130). U, lung of a guinea pig with 28 months' exposure. The field includes a bronchiole, at the center, with peribronchial fibrosis extending into the walls of adjacent alveoli. Note the cuhnidal epithelium lining these alveoli. This is the so-called "adenomatoid" appearance (X 200). distorted the alveoli, and with this change the alveoli became lined with cuboidal cells. The result was an adenoma-like appearance 'which frequently accompanies . ;*2jvv.'b4 >*. ,>] - A chronic pulmonary intlaniniatioii resulting from many eanss.. Willis * ilocriU-,1 a similar structure in thr lung* of guinea pigs inhaling silicon cnrlmlc. The longer asbestos exposures resulted only in more thickening uf the walls of the air spaces, largely due to an increase in the amount of fibrosis. The fibrous tissue always remained cellular and failed to sliow the hyaliuization cltaracteristic of silicosis. Fig. 4.--King's floats inhalation experiment: /f. lung of a guinea pig with six mouths' dust exposure followed hy 35 months' inhalation nf norinal air. The reaction is rather slight, hut distinct fibrosis is present (X2M). Note that 28 months of cimtinuous exposure (fig. 3 0) produces much more extensive rcactiuu. B, lung of a guinea pig exposed to the asltestos dust for nine months and living thereafter in normal air fur 37 months. The reaction shown is more than that in A but much less than the reaction in figure 3 It (X 2HU). 8. Willis, H. S., and Brutsacrt, P.: Tumor-like Structures in the I.ungs of Guinna Pigs Artificially Exposed to Silica Dust, Am. Rev. Tulierc. 17:268. 1928. > X- mm *>--a* i IK:- &3fe teM /.I . *y*i ff |Vv.'>|p <&>- V Asl>tu`is Ntdics (tig.2), tir>t seen in the lungs of the guinea pics that had inhaled dust for about two months, hcrante more numerous and more distinctly segmented with increasing ex|assure. The rcactiun produced in guinea pigs exposed for six and nine months did nut progress significantly during a subsequent period of 3S and 37 months when the animals lived in a normal atmosphere (fig. 4). Between eight and 11 months alter exposure reaped, the cellular reaction in the lung had been completely replaced by thin strands otf nfihnrous tissue. At later periods tnhe tsecar tissue was lces*s min QT a TMtrrs t-v.T*>piv5i,**^s * amount, but in the last animal killed, 37 months alter discontinuing dust exposure MED Ji some fibirroossiiss wwaass ssttiilltl vviissiibbllee.. ^^-^lEGEO-S Reaction in Guinea Pigs Infected with Tubercle Hncilli at the Onset nf Dust BY Inhalation.--Of the group of 40 guinea prgs infected with attenuated tubercle bacilli. Ri strain,* at the time that dust exposure was begun. 31 died or were killed before the completion of two years of the exposure and were reported in the paper hy Gardner and Cummings.** Seventeen of these died from intercurrent pneumonia. Briefly, the results were as follows: Ten revealed some evidence of spread of the tuberculous process (fig. 5/1); in 6 of these it was confined to the lungs, and in the other 4 the abdominal viscera also were involved. Extension of tlie infection was first seen after seven months of dust inlialation; during the next 20 months more than half of the animals showed actively spreading tuber culosis, and in 3 of them small cavities had developed. During the last eight months no animals exhibited any evidence of active infection although in half nf them the hepted fihrous Kars of previous spreads were obvious. The Kars were mure extensive tliau is characteristic of either tuberculosis or ashestosis alone. The nine animals which were still alive after two years of dust exposure were killed at intervals during the following year. In four of them the primary foci of infection were healed with fibrosis and even calcification, and there was no evidence of progression (fig. 5B). In the remaining five the tuberculous foci showed evidence of having previously spread locally; in four of than, by the time of autopsy, the foci were healed, with excessive fibrosis; in the fifth animal there was a generalized chronic tuberculous pneumonia in one lohe, and in the other lobes there were isolated primary tubercles, which were still -active but had not spread. MU Reaction in Guinea Pigs Infected with Tubercle Bacilli After Establishment ol Ashestosis.--Twelve guinea pigs, after inhaling King's floats asbestos dust for 2f> months, were infected with tubercle bacilli and that removed to normiil air. Six of these animals died within seven weeks, five from intercurrent nontiil>erculou infection. The remaining six animals were killed at intervals up to 14 nuinths after infection. The subplcural tubercles were no more numerous in the <lu>tcd animals than in the nundusted controls, but a considerable number were found in the depths of the lung about foci ot ashestosis. The tuberculous component nf thr combined reaction stowed only slight local extension about lesions in the lungs and tracheobronchial lymph nodes. Caseation was found in tulicrclcs l'/i months old. but by S`/i months it had completely disappeared, leaving only Kar tissue. Foci of fibrosis still persisted in the last animal, which was killed 14 mouths after infection. Nearlion in Rahhits.--Rabbits exposed to the asbestos dust for' peril d up t IV mouths showed a foreign body ty|>c of reaction of low grade, but no fibrosis. Alltough their lungs contained particulate elements of the dust, filters were not present, indicating that the upper respiratory mechanism of the ralibit is adequate to exclude fibrous foreign bodies. Two rabbits, after inhaling dust for six and 19 9. Slcenken. \V, Jr., and Gardner, L. U.: R, Strain of Tuhvrclc Bacillus: Its Dissociation ami Virulence of Variants in Normal and Silicotic Guinea I'igs, Am. Rev. Tulx-ic. M:5l, 1946. it ji 1068 :.i i l o I r nd fios rt rmi ba tui toen ,d net to eacept be in c o p ie d , accordance l&8 Jrr^'Oj; : m Sfckfgf V<i-'-^si &Vk?; gfegi t;: % months, lived in normal air (nr more than two yeais. At autopsy neither animal thnwed any evidentr of cellular reaction or fibrosis in the tcrmii:al bronchioles. nor were there any ashestosis bodies. Kraetion in While Koti.--All the rats hail aeipiired an infection, resulting in the formation of pulmonary abscesses. before they Caine to autopsy. Apparently. so mueh heavy mucus obstructed their bronchi that very few fibers eoulil liaie entered ,, claimed privileged ' OCF Stti r ^K *rn' ,-*wS3 SfeSrJ - yr i*a .?.* :--VJi *r* *,<. J-ft* M: jawgjMj . [2v&. fip. 5.--King's Hont inhalation experiment: .1, limp nf puinca pip infected with K, tulwrcle bacilli and then.exposed to dust fur 24 mouths. A bronchiole is shon ii just almvc center. Surrounding it is smite collagen deposition, ippelher with typical epithelioid cell infiltration of the wall. Note the kirk of encapsulation and the peripheral epithelioid cell pneumonia, which illustrate a spreading tuber culous prices* ( X 2(J0). B, tulip of a guinea pig infected with K, tulinclc luicilli anil then exposed to dust for .1$ .ipouths. Note the subnlcural distinctly encapsulated caseous focu, the caiMlcuifimcaiitwionn' ati mthe rig| ht border of the lesion and the absence of cells in adjacent . . alveoli, alt of which illustrate a healing tuberculous process (X 2011). ' JiVWt'5r h-,#v VfrSi *.'> 4c*.Vi rrsfiie*-! f t I * . ,*.!t.... t i i^.-. ' .1>%# #:.>.*>V i..**;;.>\ t* their lungs. In a few of the rat*. an occasional asbestosi* body was discovered, but tlw-re ii> no fibrosis. This ph.ise of the experiment war considered unuccessful. Summary and Interpretation of Inhalation Experiment ifilh Kinij's CLAIMED Plants Past.--The findings in the e\|icriincnt with King's floats dust caiPRIVlLEGEO he summarized under two headings: 31T OCF 1. KfTect of the inhaled dust on normal animals. The King's floats dust eaused a characteristic peribronchiolar fibrosis in guinea pigs hut nut in rahhits or rats. .The fibrosis did not increase signifieautly C-is s-a jctiJfrawr&*Lli-a"o*x5 in extent after the dust exposure was discontinued. 2. Effect of the inhaled dust on tuberculosis in guinea pigs. In guinea pigs infected with attenuated tubercle bacilli and then placed in tbe dust room, the results were more variable Ilian is usual in an exjKTiment of this type. A few animals showed no sign of progression of the * J O! infection; in most of them there was evidence of temjxirary progression with subsequent healing; in one animal the tulicrculous process remained active to death. In contrast, when guinea pigs after luring infected are ex]osed to quartz dust instead of asbestos dust, the infectious process continues to progress and eventually causes the death of the animals. On the other hand, infected animals exposed to a harmless dust like iron oxide do not show any progression of the infection.** Guinea pigs infected with attenuated tubercle bacilli after the termination of two years' asbestos dust exposure did not show progressive disease. The only modification of the infection was in its localization, a few Isacilli being retained in the peribronchiolar fibrous tissue, with tulicrcles forming there in addition to the usual tulicrcles liencath the pleura. In view of the variability of the results, the unusual nature of the response and the high projiortiou of deaths due to intercurrent pneu monia, it is felt that only tentative conclusions as to the influence of aslicstos dust on the course of tuberculous infection are justified by this experiment. Siiomt Fiozk Amfstcs Dcst Since hazardous dusts like quartz ate most effective in producing fibrosis when the particles are 3 microns and less in size, an inhalation experiment was performed to determine whether this condition is true for aslicstos dust. It was thought that a short filter asbestos dust consisting almost entirely of fibers and particles smaller than 3 microns would initiate an accelerated tissue response and produce an advanced reaction in a shorter time than did the King's floats dust, which con tained fibers from 1 min. to 1 micron and less in length as well as much particulate matter. '4mm f*. Composition and /Itmospheric Concentration of the Dust.--The dusting material for this experiment was the remains of fibers collected in' dust bins of an asbestosfabricating plant after a carding operation and screened to pass 200 mesh. Since i'JJi.ttfp;.* 10. Vorvvatd. A. J. pPratt, P. C.; Durkan, T. M.; Delaliant. A. B.. and Bailey. D. A.: Sidcr*ls: A I'.cnicii Pneumoconiosis Due to the Inhalation of Iron Dust. Indusl. Med. A Surj;. 19:170, 1950. >: SHr:V jti 3.V-**i'V IS^L 14 t A a .< %* 4 A * . I j uX..* ol.\ / < ** l t / I * -!//.// . . the material at received contained many lone fibers it was ground in a steel lull mill to reduce practically all the particles to 3 microtis or lc' in sire. When used alone in the standard dilating machine, this finely ground aslieatns tended to pack in the hopper, and it became necessary to mix unc volume of the utigriniud material with three volumes of the ground to generate a saiofatlurr dual rlnutl. It is pertinent to mention here that the addition of the small quantity of ungruuud asbestos was unfortunate, because it confused the interpretation of results. The composition of the short fiber asbestos as received is disclosed bv the chemical and petrographic analyses given in tables - and 3. Samples taken before S .tisS-l*s*Ou and after grinding yielded about the same values on analysis, indicating that there CLAU'EI i*s was no contamination from the mill or loss of water content. PRIVTI Ff* 8se Tlte dust concentration varied during the experiment, the light field counis for ny ^ Si atmospheric samples collected inside the animal cages with the iinpitiger apparatus ranging from 83 million to 183 million. The average of counts was 130 miliiun for the first year of the experiment, 134 million (or the second year and 140 million for the third year. ULc. ; Iola j;O?* *g< J.j iO0It*mIt s-o!S**tIsP*6J I h3 Site-frequency measurements of air-floated dust from inside the cages at a ! W- magnification of 1,300 X revealed a great preponderance of fine particles, nearly ! ?SS|5 Ssils bo oj Table S.--Smmuary of Inhalation Experiment with Short Fiber Albertos Dm! Nature of Kxperimrot Animals Maximum Mail- burvival mum After Duet z Du*t ]2x- poeurv,, potuxt. filo. Mo. RtsuHi : J< Oust exporura continuous through- U fulots pl(t out lift St Halt of rtiflioti itout tht taint it b ext*?l Bifot with Kui(> float* aiu-Mci but f.itfat nn rats I u11 cats of Jnvolv*mtit very nsurb Im t 0 Charictrrmif purlin ol pcrlbroochio!ar flbro- si*; on i*.to>>i* hodie* Subplrurol rrariion oaly * -. I rabbits . No flbroMi wti ffuflOj-; microKopir tvlOcoe* of oiveolar vall tiiirkeaia^ alter i0 mootbs* czpofurt Oust nsanin followed t>y prolosrtd mMeoe* lo oonoal air 11 tulaao pin Beats 1 rabbit s SI u Procreflrioo after removal from dut doubt ful--neither elrarlf established oor dcflaiulf j eluded SI Same ii for cobtiououi exporvre Similar to eomiauoua exposure; ceklrnrc of alicbt recre**(oo * Alter i! month* II* animal* were fZpo*td to 100 per cent hall-milled a*bctOf. film reaction mat probablj due to loo* fibers lo the uafirouod material w&ch waa mixed with the grouad aibeatoi dot to pr<Jure a ftatipfaclor? lu*t cloud. 90 |>er cent of the particles seen being smaller than 3 microns. It was estimated that approximately 1 per cent of the dust was in the form of fibers greater than 10 microns in length. Four sixrcies of animals--guinea pigs, white rats, cats and rabbits--were used in this experiment. The results of the dust exposure, summarized in table 5, arc presented in greater detail below. Reaction in Guinea Pigi.--Eighty guinea pigs were originally placed in the dust room, hut 31 of them were later eliminated from the experiment and killed became of enlargement of the cervical lymph nodes thought to be due to intercurrent infection of the upper respiratory tract. Of the other 59 animals. 46 remained in tlte dust room until they were killed or died at periods up to 34 mniiths. ami 13 animals were transferred to normal air after being exposed to the du.-t (or 30 months. The type of tissue reaction provoked by the inhaled short filter asbestos was essentially die same as that already observed in the experiment with King's fluats asbestos. The rate of reaction also was approximately the same, but the extent of involvement was very much less. After 16 tu 34 months of expo'ure only a very few small foci of reaction, which generally required microscopic exanunatuui for -detection. had been produced in the guinea pigs. Sr W-V'.' PH fe? S<a El wfe?*: #'.;t. .=* rJ fefci&P ^fc'?ys:;a?1g>; ffv^ v--,v-cr* %K&T&fa&ri eg-viC v.jr I !>:.Ii.l> l.l .11. --> i I K./ N cn .tSIll.M U.il.\ Ij Only alter cx|ure hail nmlinurd lor ai>primately one rear wa thrrr an .. . apprcvulilc tendency tm iliKt-rontainiiig phagocytes to gather into clumps. v :;;;VrL^ 16 nmntha phaecyln Iuni collected about the walls of a few of the respiratory Ki. u ILEGcjO brunchhiln which revealed a little proliferation or infiltration of mimnnurlear BY QCE* cells. There urre also sonic niultinuclcated cells, but they were of the inert, foreign lady ty|ie. At 20 to 24 mouths the cellular clumps were sometimes guile prominent, and sometimes changes in the epithelium resulted in the atlenuina-likc or "adenomatoid"' appearance (fig. 3 B) previously described in the sectiun review ing the cx|icrinicnt with the King's floats dust. In most of the subsequent members of the series the reaction remained cellular, but a few exhibited pronounced development of fibrous tissue. In these few memliers of the series the col lagen was pale in color and tenuous, with no appearance of being hvalinircd. Diffuse chronic pleurisy was present in a few animals witltout evidence ot pul- 'WMM& ~'C5 14a Tahi.e 6.--Analytes of Lunas of Guinea Pins After Prolonyrd /dAii/hIium of Short Fiber Asbestos Dust lkpotire to Dint, Mo. 15 10 t4 10 14 90 90 IVrioil In Normal JUr. Mo. Amount of AM. 7*01 I>rk*l Luuc Total Kit>t, % Of Drkd Lunff Total %0t A*lt Dual Exposure I'oottauous Durian Ufe 15.08 4.58 5.15 041 6.46 044 10*3 JuOl 10.34 15.no 4.76 0.49 6tt 043 f.96 9.00 ioo 4.95 < 5.96 0.65 04) 14.46 14.97 i l 6.43 1 5 43 o( \ 5.35 0.76 0.76 046 147 14.48 14.30 II.SO 19.46 < 645 t 606 0.75 19.37 15.11 Duft Exposure Foillo6w4e4d by Prolonyeit Residence la Norma! Air 4 5.16 5.11 6.49 646 9.30 7.16 10 8.11 3.94 o. 6.34 16.91 141 4.77 S.1S 4.77 6.25 O.Si U*3 5.21 5.00 4.00 Ti'ic Itrartioo 4 S+ 14 4+ 44 94 14 94 Tli** yml<ola ivi>ri(inr the rrarilon In *arh proui* of r'tiut-u pi* rq*rpil merely Hip Mulit* *lerr*f of fraction. retiring from ^ (<|ti*'tionatji`') to 4- (tlir maximum for till* txKriinrni). Tht relationship* apply only wlthlo tM tMe aol ninnui k fouipuml aitb yiiiiHiir in otbrr laMcf. nmiury infection. This suggests that pleurisy may he a specific conomiitant of asbestosis, but the evidence is not adequate to establish this i*int. The reaction of the tracheobronchial lymph nodes was more pronounced than in the previous experiment with Ktng*s floats asbestos, probably hccau>c more fine particles had been transported to the nodes in animals inhaling short fiber as1slo*. The imdal reaction wa essentially an increase in reticulum, rather than a fibrosis, with the original evils licisig preserved between the thickened rclicular fillers. In the group removed to normal air after 20 month** inhalation of dust, pngrc<sum of disease was not definitely demonstrated, but neither could it lie al>M*lniely disproxed. owing to .the variability of the response in different animal*. The reaction*, trim* mild to levcre, occurred si*oradtcal!y and bore np ulatiunship to the length of time after cessation of exposure. The differences were attributed to variation in individual susceptibility. This view received support from the chemical analyse* (table 6), hich revealed comparable amounts of ash and silica in lung> with widely different atmHints of tissue change. For example, the ash  it* j i h$.i t i| 4rf* M l I r.t i i. .. i/. .l/M/i / \ /. and silica \ilue* were quite similar (or three animals living in dust >U months and then in normal air (ur 14 months, yet the tissue reaction was severe in one animal mild in another and only doubtful in the third. The formation of ashestosis bodies was at first extremely limited in both croup*. After five months' exposure only a very rare short body could l>c found, usually inside a cell. Some of the finest intracellular particles were surrounded hy yellow deposit* Itavinc the same color as the ashestosis body. One year's eximsurc h<u1 per mitted an accumulation of maoy longer fibers, a number of which were coated and seen as typic.il asliestois bodies. Most of these were still short euuuph to he partially or entirely within phagocytic cells. By the twentieth month and thereafter they !i- ' O o' a8" f 5Ss: Table 7.--Analysts nf Lungs of While Rots Thai Hod Inhaled Short Fiber Asbestos Dust S. 1 Be O Oioii fHiniinti (if Km** rare. Me. o* t I Amt. of of lri1 J.ung 3.9 44 8.9 1.4 *4 1.9 9.1 '33 3.6 3.9 a3.t 9.3 3.6 3.4 Total KH*?. of Driol JjIBf &AI orai O.ro o.no 0.1V ft.m 0.04 0.11 0.00 o.tt 0.04 0.1) 04)7 Total fUlfe. % f Aab 0j0 041 04) 0.0 OO 04> 04) 3.1 M U 14 3.3 9.0 U fhirathio of Esfio* wire* Mo. Amt. nf A*h.a Of Ilrird Lung 1U { 14 |W 1 IJ 1 <* l *.T r 4. J 44 10 1U4.1* TOUl HWr*. <3Of DraU Luos 0.07 04* 04M O.i* o.i? 0.13 v.i: 0.16 0.1? 0.13 0.13 0.1) I*oo Total W(t. atsVsi," 1c j Of AMl 8521" 11 14 privii^gi d IfSM tils:; l.l B* OCF 3.3 3.3 3.4 4.0 4.3 3.0 3.1 JJ 24 * Normal roairols (oo Oust exposure). Table 8.--.(vero^e Values of Ath and Total Siliea for Lungs of White Rats Inhaling Various /lusts for l'orions Periods (Lungs Only, Without Included Lymph A'odes) Ami. of .\ili, % of Prinl )4iS( Total Site. % of Drhxl Lui>k Total SiOs, Sc Of AMi l*ur' tinn tfhort of tf**ure,. 4*U*> Grpfuin- Short Quarts Fihrr gtnou* Mix- ALet* Oirpruui- Short ` Femi* QuarU KiUfr giaoua Mia- AM**- CrP'Uin Ferru ginous Mix Mo. toa Quarts Ch*rt tur tot Quarts Ciiert tufft toa Quarts Chart ture t a.s 4J 14 3 0.0* 041 0.33 aw t.a 11.7 3.9 34 4 3.4 *.i (. U 0.09 0J1 043 0.07 3.3 11.4 34 1.0 0 3.3 7.1 94 3.4 04) 3.04 3.43 0.11 1.6 414 344 34 3.0 4.0 !.n 3.6 0-U 1.41 3.40 0.33 3.9 3*4.4 364 0.1 w 14 7.0 IU 4.1 0.19 4.40 0.00 0.3S 3.3 3C.6 43.3 6.1 were relatively numerous although still rare in comparison with the findings in the King's final* e.\|>eriment. Reaction in White Rots.--Seventy-three white rats were exposed to atnmsphcric slxirl fiber ashestiss dust fur periods up to 32 months. During the first 10 month* animals were killed bimonthly and for the remainder nf the experiment at lc.* frequent interval*. Up to eight months the dust cells were widely scattered and existed in foci nnly sporadically. Kcactiun was limited to occasional slight thicken ing of the septum* about small accumulations of dust cells. At HI month* there was a suggestion of early fibrosis in a few rats, but the change was so. slight that it would prohably have been overlooked without the clump of dust cells which attracted attention to the area. Only 10 animal* were exposed for from 12 tu 32 lungs contained minute foci of welt defined fihm.is but without a*bestu*is bodies. The lesions. ISO diameters or more, consisted nf patches along .V ;t 1073 A.V5- lij. V-. i '* vi*: Wml V i uhll .U.lt 1:1 .11..--a/. HIL.\ 01 AaltLMUSIS 17 alveolar ilm*u in uliirli tin* wall* *if il*c aviciaitil air pacr were very thick, owing CLAIMED to suoflcn collagen framework. Connective lik.-uc and Eout-liirlsshnsssky silvet.-- ....-r FCEO > preparation* revealed oimpleir loss of capillary bed locally. Outvide the collagciT r' v - _p was a thin layer of epithelial cell*. This did not resemble the 'adcinimatoiir' chance cliaracterivtir of guinea pie aslicstnsis. Near the lesions the air spaces were rilled with phagocyte* coniaining gray to vellmv particulate dust and a rare. long, naked asliestn* filter. Carciul search failed to reveal even a suggi-tion of an adteMOii.* hodv. Pleurisy v absent. The tracheohrunchial nodes showed comliact focal collection* of monocytic cell* at 12 month* and. at 20 months. some diffu*e thickeniuc of the reticulum, (u a tew rat* there wav definite fibrosis along the margins of the node, extending into the mediastinal areolar tissue. Results of chemical analyse* made on the white rat* are given in table 7. and the average values have Ihyii recorded in table 8 ior cianiarison with similar values for rats inhaling utlicr dust*. It will be noted lliat the values for asbestos are lower than those for quarts or ehert but approximate those for the gypsuniquartz mixture, in which atmospheric agglutination tended to reduce the amount uf dust inhalctl. This condition prevailed even tlawgh the atitu>s|ilicric concentra tion of asbestos dust was essentially the same as that of the quartz, was one-half tint of the gypsum-quartz mixture and wa* onc-lUtli that of tin- ferruginous ehert. Since thi values for ashc.-tos are Inw. it might be inferred that the total quantity of that dust actually inhaled was small or that it had In-en eliminated from or dissolved within the lungs. Evaluation of these possibilities it not feasible on the basis of the observations derived from this study. RearIinn in Cats.--Twenty cats were used in this inhalation cx|H'riim-nt with the short filler asheslus. Eighteen were kept in the dust rosn continuously until put to death, the exposure period ranging front one month to nearly ?4 mouths. The other two were removed to normal air alter a dust exposure of .11 months: one of these was killed five months, and the other 24 months, later. In general, the tissue response was confined to microscopic foci of fibrosis, which were in the walls of groups of subplcural alveoli rather than in the |>cribronchiolar areas. In one animal the change was extensive enough tn be' visualizrd on gross iiisiKctinu of the section. Only in the animal with the kuigcst exposure-->4 months--did the roentgenogram reveal definitely abnormal sltaduws. A roentgenogram madeafter JO months revealed no abnormality: after 45 months, a taint mottling could be detected throughout both lungs. At autopsy, nine months later, tltcrc was nuly microscopic fibrosis in the suhplvural zone plus heavy lyuiplmcytic infiltration al*>ut smalt bronchioles. Asliestosi* bodies were rare. On prulnnged search a few yellow atypical bodies, smooth and without haustrations, were found in two animals exposed fur more titan a year. Renetivu in Rabbits.-- Eight rabbits were c.\|toscd to (lust for periods extending from one to more than five years; the last animal was removed from the dust ruuin and left in ixinnal air six months before being killed. There was never enough pulmonary fibrosis to Ik detected grossly, and there was no chronic adhe-ive pleurisy. Microscopic evidence of alveolar wall thickening was first detected in me animal after about three years of ex|io*urc and was seen in all five animals examined thereafter, including the one removed to normal air. One animal dial dual of (niralysis after nearly four years of exposure exhibited a reaction visible on gross ins|>cction uf tissue sections. The possibility of pulmonary infection in this animat could nut he excluded. In another animal dying two years later the focal fibrosis was not nearly as obvious or as advanced. Areas of involvement, which were largely visualized because of phagocytic reaction within the air spaces, tended inicrose.spirally to become more fibrous with the passage uf time, but there was never much encroachment on the lumen of air spaers and the structure of the lung wa> preserved. Aslscstnsis Uwlics were not delected in raUiits that died early in the cx|*-riuivm luit were seen in all animals that had Iseen exposed to the dust lor more linn ihrcc years. ' 1 Mil ~'r- ^ * "v--v, i&mm >!rr tf-S-A- Bi VL - `*8$ v-*'4C3 .& ?- STS' 2si &? ?ii 5ls! .':5:.'c-*]$, i - *u.* *-**^v&V L.: S?*" c. -/-...wKjfty rti'vr* >'-%? IA J.W'C itryj.il. illUlJiT* . i.\. <i (17'.; t it .!//:/>/(./A i Summary and interpretation of inhalation Experiment with Short Filler Asbestos Dust.--The original purpose of the cxjicrimcnt was to evaluate the role of short asbestos fibers in the genesis of asbestosis. It was felt also that if the tissues reacted more rapidly and more extensively to short fiber asbestos than to King's floats there would he a basis for Itclievitig that the action of asbestos is in part, at least, a chemical one as postulated for quartz. This experiment, in which the tissue reaction CLAIMED was slower and less extensive than that in the previous expcrinicn^RjyjLjrQjr ) with King's floats dust, indicates tliat the capacity of inhaled asbestos gy qq^ fillers to produce fibrosis is determined primarily by factors not chemical in nature. 5S Of the lour species exposed in this experiment, only the guinea pig and to a lesser extent the white rat responded with characteristic peri bronchiolar fibrosis. The cat reacted With atypical subpleura! fibrosis and the rabbit with only slight parenchymal fibrosis. *! s Ball-Milur Askatos Dust In the inhalation experiment with short fiber asbestos dust a small quantity of unground short fiber asbestos wais mixed with the hallmilled product in order to generate a suitable dust cloud. When that experiment failed to produce an accelerated tissue reaction, in com parison with the response initiated by King's floats, it became apparent that the biologic activity of asbestos is not increased by a reduction of filter size. Thus the possibility arose that the tissue reaction observed was due solely to the relatively few long fibers of the unground asbestos and that the short fibers of asbestos had no more than a very insignificant role in the production of asbestosis, a concept not in accord with previous experiments concerning pneumonoconiosis. Consequently another inhalation experiment was started in which only ball-milled asbestos was used. Composition ontt Atmospheric Concentration of the Dust.--The dusting material was the hall-milled, short fiber asbestos used in the previous inhalation experiment, but ungruund material was not mixed with it. Owing to the tendency of the material to form small spherules which prevented much of the fibrous portion from floating out uf the dusting machine, the dispersal of the dust was not entirely satisfactory. Therefore, after an initial seven months of operation, steel wire brushes were attached to the inside surface of the hopper and to the rotating paddle to disintegrate the spherules and release the fibers. This arrangement gave satis factory results and was used for the remaining 21 months of the experiment. The composition of the raw asbestos used is shown in tables 2 and .1. Petro graphic and x-ray diffraction examination of atmospheric dust, collected in the dust room with an electrostatic precipitator after the installatiun of wire brushes, indicated that about 15 per cent of the air-suspended material was chrysolite, and almut 60 per cent, serpentine: uf the balance, magnetite comprised 10 per cent, brucite 2 per cent, quartz 2 per cent and other minerals 10 per cent. During the seven month period before the wire brushes were used, the chrysolilc content of the atmospheric dust was somewhat lower than 15 per cent, hut reliable values were not obtained. The dust concentration during the first seven months of the ex)>erimcnt was font of air. After the wire hrn-hes were \ 3 .. - j ,.4 m * > -4 1075S I tiKH'.lLU Li AL.--S I UUll.j Oi .ISULS1 USIS IV installed. the dust count* ucre higher, and the over-all average (or the remaining 21 months was about ISO million. Sir.e-frequency studies of atmospheric dust collcctetl inside the animal rage* revealed that nearly V9 |>er rent of the components suspended in the air could l>e classified as dumps or partitles; only ahnut I to 1.5 per cent was fibers. One third to one lull oi the fillers were lunger than 10 microns, indicating a concentration of long Tillers of about 0.8 million. This figure is about one-huli the estimated value of 1.4 million for the short fiber experiment. Guinea pigs, rats and mice were used in the inhalation experiment with the 100 per cent ball-milled asbestos dust. The results are summarised in table 9. Reaction in Guinea Pigs--The experiment was started with 10U guinea pigs. As the dust exposure proceeded, there were 39 accidental deaths, 33 oi these being due to pneumonia in an epidemic. The 61 pigs remaining exposed to the du*t were killed at intervals during exposure, except for 16 guinea pigs transferred to normal air after 23 months of dusting. For the first year oi exposure practically the only reaction to the dust was the presence' of scattered pliagoeytes and an occasional minute asbestnsis body. At 16 and 20 months no gross response teas visible on the tissue section, but microscopically peribronchiolar foci of inflammatory evils T*JIX 9.--Summary of Inhalation Experiment with 100 fee Cent Ball-Milled Asbestos Dust Nature of Experiment Dwt txiKMure coo* Unuoua through* oot nt* DuM expofun fol* k>*n! hjr pro* loscrd rrxideoco la normal air AnJmaU M rulers pl(i 40raU 2< mle* It guinea pica .Maximum Maxi burrival mum After Duxt Duit Expo- Eiio- aura. aura. Wo. Mu. Bttultl 0 No api*rcvial4 pulinonarv reaction 0 No luceMion uf al*io*l It 0 No ucc>:*tioa of ait**to*i Si u Flhroiis tjr|4el of abctoi* wax prmot IS tuo. iltrr rxporure cea*d in ao amomit auftirieot to t vMI4e crotxlf: smaller fori eouM be tarn microtroiiieanT at S mo. aotl mo. aftrr uroiina* tlos of exposure could Ik seen. At 24 months (fig. 6 A) there was still nn change large enough to be seen with a hand lens, although microscopic examination revealed cellular accumulations about terminal bronchioles and many more asbestosis bodies, chiefly within cells. The lungs of animals exposed for the full dusting period of 28 mouths and afterward living in normal air (or two months revealed the changes described above and also very slight peribronchiolar fibrosis. For rx|m*cri animals living eight months in normal air the findings were similar, but at 12 month* three of four animals showed grossly visible cliaractcristic peribronchiolar fibrosis with adenomatoid cluuigc (fig. OB). The tracheobronchial nodes were essentially normal until exposure had licvn continued for more than a year and a half. Animal* killed at 12 iimuth* and at 16 mouths revealed a few minute collections of phagocytes containing particles hut practically no fibers large enough to Ik recognized as such. After 20 months of cx|io*urc many monocyte* filled with yellow granule* were present. At 30 months there had been a slight itKrease in reticulum but no fibrosis. Xo further dtanges occurred in the nodes. Asbestosis bodies were not seen in the nodes of any of the guinea pigs. Minnie a*ln-tnsi bodies were observed in the lungs as early as three months after exposure began, but they did not become numerous until 16 months liad elapsed. The lh<- were short and practically all were intracellular, although at JU mouths soiiir wri.- iom' enough to. project beyond the cell borders. It is WSrff-X ; K&a: &&- >/*&;r::i 7,y: :. j; r-^- ' 4 fesis f&dBg >ff'-*3sr" w* % ic*.*rV:t?u Jv' **l: $'.S.i{ . V^#4v ; 8$&M> ijii.*- *.n' -1) i ,\ Ul t h'l.iL tlYiiih.'i. .-I.\IJ (/i i //'.//ft r.\ .// MIJ'UI.M. important to note that in the later month* .if exposure there ua a distinct increase in the number of long iilicrs, up to *0 microns in length, in the lungs with the formation of rharactcriitic long asbestosis bodies. Oiemical analyse* (table 10) of the lung* revealed that considerable dust had been retained in the lungs. After 24 mouths oi continuous exposure the average > / r * V ' -f r 'v "v~* ..;-a tv *v awJL. ` ^ *4?! jL5r^ CLAIMED PRIVILEGED BY OCF Safe*. *-J?rCc^'3e:o'^s t VVi.' ft V v .^ . . 2b' X. % * "~ j' . A . v ' nv. j&i' ? l:ig. f. -- Dali-milled asliestos inhalation e.\|ieriiiivnt: .4. lung 11f a guinea pig with 24 months' dust cx|x>surc. A bronchiole is sltuwu at tlie center, with a slight accumulation of phagocytic cells but without the formation of cullagcn ( X 2tW). U, lung of a guinea pig with 28 inimths' dust exiinsurc and then 12 mouths' inhalation of normal air. The reaction is much like that shown in ./. but there is a slight deposition of collagen, most apparent at the left (x 200). value fur total silica, per cent of ash, was 25.37. This should lie cmitrastcd with the average value of 14.34 (table 6) for animals exposed 24 mouths tu the short -.fiber asbestos dust.: 006 1077 U: ;i$g f?tiicMi tes $3 **. -aiew-yjgK av^':%*Ss yv>*: .- * ** V JOT't*>.\ pilip I.': ->'>: iulyit.ll.il lit -IL.-- xTL'LHLX Ol rlSOLSIOSlS 21 In view >i( (In- high value* for kilira obtained with the anemalt exposed tn 100 |xr cent lall-millcd dust, it it important to note that their pulmonary res|ui>c t> niurli lt*s than that of animals exposed for 24 months to the short fiber a>heln> in the pres inns experiment. This again indirates that the biologic artivity uf asU'Stos inhaled into the lung it nut increased by a reduction in sire ol the fibers. h'rartinn in ll'hile Kntt and Mire.--In this experiment 40 rats were ex|iose<l fur iH-rimls up to 20 months and 24 mice for periods up to 12 mouths. In neither species did even a suggestion of asbestusis develop, and reaction was limited to phagoeytusis of inhaled particles by widely scattered dust cells which remained free in air spares or were transported to the tracheobronchial lymph nodes. No ashestusis bodies were found in the rats, hut in tlie mice there were a very ieusmall, nonhaustrated forms within phagocytes. Table 10.--.Inalyin pf Lunfts ef Cninca Fill! Hxpuird In Dull in InlmUilinn Experiment with IU0 per Cent Hall-Milled .-fibriluj Dull &f%&- i s: . 'vfesrs ij|l]l ZspfiMirf to Dui, Mo. I t I 1 a is ia u l>riod tu Norm* Air, Mo. Aiut. of A.h, <5-o( lirinJ l.use Total MO:. % ot Drtol Luny Tnl.l KiU:, %Of Ash Durt Kxposur* Coatiouou* Hurinr IJf* f IAS 0 ' I.S0 4.U 0.21 0.9 0.24 4.39 7 Jtt 4J0 | 4-06 0 < 4.IO l 4.04 4.90 0.%| 7.40 0.01 12.01 a.oo 0.70 HIT 0 4.07 0.32 *.t* 4.74 0JG 0.77 4.10 OJ* 7JI 0 ix* 0.3* 7.47 4Of uJb 7.72 0 4JI 0JS lUW \ 466 t Al * s.w 441 1.24 32.70 1.44 33J9 un ISAG mi 31.04 4.04 ! 6.30 1 0JD 1 4Ju 1.34 1.3> 1JU 7.21 22, 3IJ1 39.06 21.70 TMtie Kemcliwu * 0 0 0 0 0 0 A Purl Exi>ourf KolionMt by Prolnoxnl Krrhbnrr lu Norm*! Air ! 3A2 l SAC a 13 1 0.39 f S.IT 1J7 21.0: 3.1V 24*4 4> 0.6* 0.47 14.44 4- 0J4 13.1* 0.04 12.41 t+ CLAIMED "VILEGEO <js%m OCF '^4* k#i >'wm Thf rrtu**l av^raeinr thp rMrtivn In ech tiirrdr llw rrbilvr Uecrrf of rrarlion, ramrinff from u (o * (fiuHtioosliIrt fu iA (Kip matiuiiiin oImtviI in tlii* C\|ierimnti. Tl* Miitton*bi|w i|i|iljr uoly mithin llii laMr suit fainiui iw niii|sami uiili pynit>ot!* in oibrr table*. Summary and Interpretation oj Inhalation Experiment with 10(1 per Cent Hall-Milled jIsheslos Past.--Tlie tissue reactinn ohscrvvd in this experiment was nut as intense as that in file previous investigation with short fiber aslicstos. The reaction was sluwer in development and less extensive even though more dust accumulated in the lungs. Since there were fewer fibers longer titan J microns in tlie material used in this cx|ierimeiit, tlie results tend to confirm tlie interpretation made in tlie summary of tlie previous short fiber experiment that the reaction is not - tion in size of asliestos filters does not increase the biologic activity of r __j i: 1 sg&swg #- S-'Sri-iJK! SS*&~ * *0* -..5 St* Si l.\ l/L J / Al.lt. yf.\// ((. C ll'.ll lll.\ . II. .1lll>l\l\i. The finding of long asbcstosis bodies in animals that hail inhaled the hall-milled material is an example of the difficulty of completely eliminat ing long fibers from a large volume of asbestos as required for an inhalation experiment. In regard to the progression of the tissue reaction after the animals had been removed from the dust, observed in this experiment but not in the others, the following interpretation is offered: When the reaction is well developed at the termination of exposure, the contraction of theCLAIMED .*_flcHirtOE*s> fibrous tissue obscures any progression that may have occurred; in tfilBIVlLEGE] experiment, however, since the reaction observed was less mature, its By OCF subsequent progress was more readily apparent. Loxr, Fite* Asbestos Dost Since inhalation of short fiber and of 100 per cent ball-milled asbestos k OV B VI lih O0S -1I 0*te*iv-1s4-** Mill j V m vE5 ua "ei *UC dust did not result in acceleration of the tissue reaction in comparison with that produced by King's floats, the hypothesis that short fibers of asbestos were of minor importance in the etiology of asbestosis was given added support, and attention was directed to the view that the long fibers were of primary significance in that etiology. The King's floats asbestos used in the first inhalation experiment had a rather low content of fibrous chrysotile and contained considerable serpentine and other impurities. Therefore, it was decided to conduct a new inhalation experiment with a purer form of chrysotile which would be richer in long 1! fibers. r- Composition and .Atmospheric Concentration of the Dust.--The dulling material employed in this investigation was obtained from an asbestos fabricating plane & Samples of several varieties of long fiber asbestos dust were first submitted to the Saranac Laboratory for examination, and one of these, which was low in magnetite and chromite and had a fibrous content estimated to be about 75 per cent, was selected as most suitable. Steel wire brushes, fastened to the inside surface of the hopper and to the rotating paddle as in the preceding inhalation experiment, were used to open up the bundles of asbestos and liberate more fibers into the atmosphere. The com|iotition of the long fiber asbestos used is indicated by the chemical and petrographic analyses given in tables 2 and 3. Analysis ol air-suspended material from the dust room disclosed that shout 60 per cent of the long fiber dust was chrysotile and about 20 per cent serpentine; as already nuled. the composition of a similar air-floated sample of ball-milled, short fiber dust was IS per cent chrysotile and 60 per cent serpentine. Tlie dust concentration as revealed by impinger samples taken inside the animal rages was much lower than the concentration for the ex|icrinients with short fiber or halt-milled dust. For the first year of the experiment with Icing filter a<hestos the average of the light field counts was 32 million particles per cubic foot of air: for the second year, 48 million; for the third year, 3V million,' and for the fourth .) year, 43 million. The siac-frcquency of atmospheric samples of the long fiber asbestos dust and of the ball-milled dust is shown in table 11. Both samples were collected with the electrostatic precipitator. It will be noted that there was far more fibrous material in the long fiber dust. 't Guinea pigs, cats, rats and mice were employed in Ibis inlul.iiion cx|>eriincnt. .The results, summarised in table 12. are described in greater detail Mow. rnhir.ii.n nr .11--sm>ll..s Ol ASUliSTOSIS 21 Kfaflii'ii iii (.Iimru /'ins--Tlu t-x|>crimciil was Marled witli 100 guinea pies. Aiicr cx|siirc hail Iicch carried on (nr a year, a severe epidemic rd piu-iummi.i arose in the du.*t mim and ahnut one third of the animals died or were killed. To replace them, 38 more cuinea pips were added to the surviving group. HiMnlugiral examination revealed lesions in the lungs after eight months of dust e.\|nurc. consisting of cellular connective tissue about the terminal bronchioles (tig. 7.1). ,\t 12 mouths there were adenomatoid changes in the adjacent parenchymal areas, and hy the sixteenth month (fig. 7/() definite fibrosis was present in these areaas well as around the bronchioles. The fihrous lesion could lie seen macro* scopically at 20 mouths. From this time on the reaction increased in extent and in the amount of collagen, and by the thirty-fourth month, it Itad tanned out m* i k ;* ye a8"s KgS&ssijj Tahle II.--Size-Freqneney of Atmospheric Loup Fiber and 100 per Cent BaltMilled Asbestos Duel Collected Inside Canes Crains, % Fiber* Type of Asbestos <> - tticroas Lost flbai................. ... BaDmlllrd ............... ... CS.4 *04 111) Mkroes s.l 14 >10 llicroas 04 0.0 <10 Slirreu 3&.S 0.1 >10 ailerons IT 04 Chimps. % 14 a: Tula) CLAIMED . NX* PRIVILEGED U** BY OCF Tam.f. 12.--Summary of Inhalation Experiment uiili Long Fiber Asbestos Dust Nature of xperltneat Dust exposure coo* ttouous tbrocgbout lift Dust exposure fol io* eJ by proloocrU residence la oormal air Abimals 117 guinea pics 4CbU 3D rota 20 mice IS fulnra pips 0 ffutoca pin Scats Maximum Mailmure Dust F.sposure. Mo. Survival After Dust xpo* sure. Mo. 26 0 45 0 22 0 22 0 Results Definite flbrosi* in 16 ria. &!o*l) devrlopioc flbrosU flr*t s*rn at 21 mo. Marked peribronchiolar flhroais flr>t tffo at 21 mo. limited reaction: oo fibrosis 20 14 Ckarioc of inflammatory r*-itrlloo ansi tjefloltc contraction of fihrous 0 tissue 27 Chariot Of Inflammatory reset iuo aod slight roniranioo of A><nw tlssur 16 24 Similar to continuous e\po*un* croup: surrestinn of procrvaiuo la ont of tbe two moimaU ! considerably into the parenchyma (fig. 8**/). The lesions were rather sharply localized and the extensions from different bronchioles showed no tendency to fuse, even in animals exposed for the maximum period of three years. Although the imrapulmonary reaction sometimes reached the pleura, there was no involve ment of that membrane. Emphysema was not detected at any point. Some thicken ing of the larger bronchi with a chronic inflammatory infiltration was revealed, but it was considered no more than would be produced by a similar |>criiid of inhalation of any dust. In guinea pigs exposed to the dust for 20 months and then removed to normal air. there was a marked tendency for cellular inflammatory reaction to clear. This effect, accompanied by contraction of the fihrous tissue, resulted in a dimiiiiMiing size of the foral lesions. N'onc of these animals, killed at various periods up to 14 month* after exposure, revealed lesions as large as those in the group killed at the cud of the 20 month exposure period or those in animals which remained in the dust rnmn for mure titan 20 mouths. Fourteen months after dust expusurr ceased, tlic foci in four of the six remaining guinea pigs were so small tliat they were visible only with a lumd lens (fig. 8 II). PM - >* W: mm"rJfe1+1 Sv & claimed privilege,, * ,' Bof ocf 24 tXlJUSTKI.il. Ill 11ixn . i.v/f on t r.u h'.x it. .w/;/'/< /.v/ In the (roup ex|*>rJ for 27 months ami then transferred ( a normal amto.plicrr tlie response was quite similar to that indite 20 month ex|ismc .-inimjls mentioned aJiovr. Small (ix'i were always visible on gross ins|>eclion of sections of all guinea pigs of the 27 month series, but in no instance was there evidence of tinreaction. l*S 'irf -s IS aos s Z%i %1Uo4 1*5-* -si iiit , tlcuCogf*teKr ysil^sSi '^.1, . v.1 ^j '*j>' l-ig. 7.--l-otiq filter asliestos inhalation experiment: -I. lung of a guinea pig with eight months' dust exposure. The bronchiole at the center already shows an accumulation of phagocytic cells, and there is a slight deposition of collagen. Com pare with figure 6.1, sitowing the reaction tu ball-milled asln-stos attcr 24 months <X 200). fit, lung of a guinea pig with 16 months' dust exposure. Again note a lironchiule with its surrounding reaction, consisting of fihrusis and adenomatoid change. Col lagen deposition is now seen in the walls of adjacent alveoli, at the right (X -'In)). In the traclteobroncliial lymph nudes reaction was first visible at the third month oi cxposutc. By the eighth numth patches of cellular ennnetite tissue A-irk--- i cl i ./iir. 1/ /i /./ .ii..--sn niir? nr .isni-.sin.sis 25IV'. "* V privilegeo^^^. Itegan it> ;i)tjti;ir in the nn'ilulla. ami I>* the hnirtecnth itmnili ninst ltf the i*mIc 3Y OCF lud ln.cn replaeeil by cellular cniiiicnivc tissue. This picture. whieli rc'Cmlilcd that in early silienM*, iiersintcd to the eml of the experiment. Smiic Miumili nimued. an a variant, heavy sheets n diffusely distributed m*Mucytc* ami large active giant cells, hut there wan never any necrosis or hyaline formation. The M'inillc*sli.nM 59^ , V*-^V ^rK > ->r-isr ,rlIPtS&'i st v'S,s8 0s; iSsSfcJifc*? llpg fkp : fe 1 :Jg^ ^mk Jll? m > Bm fe' -r-.?*qS % ' kS9A p*. -r ,IJi.fi KriS-^WS jva 2,>*i^j. } iKllIk. Fig. 8.--l.ong tiler as|>esto* inhalation e.sieriinetit: .1, lung of a guinea pig with 34 months* dust ex|mMire. A hnmchiolc in seen at the lower center; the large area alsivc* it represent* (he involvement of alveolar wall*. Compare with figure 7 It ami mite the increased extent of reaction ( X 200). /I. lung ui a guinea pig with it) months' Just cx|*o>urc and then 14 months* living in normal air. The reaction is essentially like that shown in figure 7 It: The limmhi"k at the right center in surrounded hy fihrou* tissue with adenomatoid change at the risht. There i* residual scarring in flic walls of ailjafeut alveoli at the left. Jt is apparent that no progression han occurred (X 2UU). new evils were %vllm\ih ir.nn fine pigment granules that Stained for iron. No fiber* or a*h>'ios|. N^|i weie seen. to-: ;**K,JW'5vV4 R^'sg Ry?n:-i-`irfc>r,5?^Si f^ea?rOi#S$Stewfi CLAIMED PRIVILEGED _v BY OCF !b ISULSTKI.IL u veinsll ash occrr.u m\.u. Mi'.nn i\i. Although asKstn.is bother were found in the lime as surly ;u ,,ne month after exposure Itcgan, they were rare and hard to Ami. At live in..ntli, m..rs were visible, chiefly coiled inside giant cells, and at eight months many bodies Tault. 13.--Analysts of Longs of Cniueo Pigs Exposed M Dust in Inhalation Experiment xsith Long Fiber Asbestos Dust F.xi'iijur* to Dust, Mo. Period la Normal Air. Ho. Amt. of Atli. % ot Dried Lurtf Total ttiOs. r. ot PriM I.unj Total SIP;, H ot A.b Reac:. DuM Expoiure Continuou* Purioc Life 44i 0X4 1.10 0 4.3U 0.0? Ml 447 0.04 0*3 0 4.45 0X5 i.n 0 4.41 0.09 1.19 4.99 0.0* 1.40 4.91 0X9 1J0 0 4.49 0X9 1.13 4.91 0.<>6 1.4B 4.77 0X9 1.75 0 4.40 0.1! 3.07 9.04 0.09 1.77 4.71 0.10 131 0 4.91 0X0 l.fll) 4.94 0.07 1X4 * 4.97 0.39 5.30 i: 5.0* 0.10 4.09 9.16 0J1 9.09 + 0 0' 195 IP 9.19 344 s.m 3X5 0.39 13.70 0.1.; 1325 0.31 10.91 3+ 0.41 12.33 0.40 13.01 042 14X0 1+ u0 9.4* X.X 0.35 10.19 0.39 9.29 94- n 9.40 9 | 9.71 0.39 11X1 0.49 13.19 94 : 0 SJJ 9.03 * ; 0-37 0.31 0X4 10.15 11.22 0.10 + S4 J.li 0.70 OJA 0X4 On 12.47 44 IS 8 4.10 s.:t 0.37 9.1t 0.35 13.40 44 Dust F-xporur. Foliosbj PrulonyM Rnidcnct Is Normal Air 1 S.S4 l 9A> 1 jj t !. j 191 0.43 0.(9 0.12 0.31 0.27 12.23 13X1 14.59 7.21 9.M 24 24 t 4.1S 0.34 5.73 l .. 0.22 4 07 24 1 9.01 i S.04 071 410 0.19 3 55 4 1 3.10 | a.Ts \ 1X6 1 2-91 ! 9.19 1 i.ta \ 9.21 ( 175 0.39 11.51 0.49 13.19 94 0.31 4.59 0.19 094 24 0.25 7.W 0.29 1.73 24 0.?i f.!* 0.11 9,7.1 24 titmr rartlon In earh roup r*pr'rnt merely th* rt'Trrr of rrorllf.n, raiiginff from u to it (qii.tionnl.l,.| In I a- (it* utaviimiin (or this fM--riina'til). lli- r.-l.tion.hi|> apply only within this Isblr and rsnnnt I* compared with symbols In other tal.bs- were free in connective tissue. They became fairly ahuv.dant as exposure rnn. tinued, although in some later animals the asbestosis bodies were only moderately numerous. It it important to note from analyses of the lungs (table 13) thut even though the tissue response at any given period of time was much ere.iter in the. guiuvu 0*3 a,j2t I s sa . fSia tCo3s**> ii:si Hai KUoe*tfhotgfl: 00b 1083 I . I t##iSS | Zt-IKIWSTKI.1L iV HYCiV.S'ti ASH Oi l ll'.ll IOX. lt. CLAIMED privileged MIUJU IXI- BY OCF Rtattio* i A/iff.-- Out of JO white mice uscil in this expcrinirnt, tl lived a year or more in duM and died or were killed without showing an appreciable degree of pulmonary infection. The reaction to the inhaled dust was limited t phaRoeytusis by mononuclear cells. Usually these were widely scattered through the air spares: a limited nninlicr were grouped about the terminal hrimrhiidcs. producing tome thickening of their walls. There was no suggestion of fibrosis. Numerous a>bcstosis liodics were observed in animals killed late in the experi ment. Thus these animals exhibited asbestosis bodies without fibrosis. Summary and I ntcrpri-tation oj Inhalation Experiment Mth Long Fihi-r .Isbcstos Dust.--The purpose of this experiment was to evaluate the importance of loop filters in the tissue rrs|>oii.sc to inhalcil asbestos. The results, in comparison with those of previous investigations, indicate strongly that long fibers are chiefly responsible for nsliestosis. Thus, the reaction in guinea pigs developed earlier and became more extensive in this experiment than in previous experiments in spite of a smaller concentration of atmospheric dust and a lower mineral content of the lungs. Furthermore, typical peribronchiolar fibrosis was produced in cats, although in a previous experiment with short fiber dust peribron chiolar fibrosis did not develop in this species. The cause of the cellular fibrosis in the lymph nodes of the guinea pigs is'not clear. It did not occur in other inhalation experiments with asbestos. INJECTION EXPEKIMF.NTS Since the inhalation experiments reported above strongly suggested that long fil>ers of asbestos are the significant factor in the causation of asbestosis, a series of injection experiments was inaugurated wherein the dosage and the length of the filters could be controlled more precisely. Alsu, by the use of controlled dosages, the relative capacities of various asbestos minerals to produce reaction could be compared. In these injection experiments, guinea pigs, rabbits, rats and dogs were used, and' the mineral dust was injected by the intratracheal, the intrapcritonenl and the intravenous technic, but not all the technics were used for each species. For the purpose of simplification the findings in each series of tests, except for dogs, have been condensed and reported in tables, to which reference will he made later. In the case of dogs, only one test was made, and since the findings were negative, no detailed report is included. Fxi'KKISIKXTS Usisc IxrKATUXCIIF.AL Tf.ciixic As the asbestos minerals do not cause typical advanced fibrosis in extrapulmonary tissue, the intratracheal technic is the preferred way of introducing fibrous dust into the experimental animal. In this method the dust suspension is injected by means of a special needle or catheter deep into the trachea, from which it flows into the lungs. Comparison o/ Fibrous and Nonfibrous Dusts.--To demonstrate that the ability of asbestos to produce fibrosis resides in its fibrous character, the series of injection experiments reported in table 14 were performed. .T .V i .T .9 I 8"! "Sqm si - *i . Sr faff .31! r" soil lip -. -IfaJsWe ka*Xr* 'r .4 3 *sji- tvrff -:*** ;* '1' M M. 4 4 -O' *> '.w` -t .% . .i ; -T eb.:r-.:;-i v. fey?S v L,-.?"L' V'\J i "'-.**>^** >?* ?.?*$ - ' * : r*? >y/ vc>..r-v. Jfcs?$K GEifc fe-i&s k;>.vi? iV Tai:i>. U.- ( Kiii/'.ifli<Mi / A\*iirfnnj M Oirwr*JitV oiuj iV^t'nlinf Injected Intrntraeheallv CLAIMED PRIVILEGED by ocf Ih*arc: Kaih animal map firm an Iniritrarkal Inhwtioo of 0.3 re. nf a 3 per cmt tu|a*nion of the dust. To ivrb latrr nmtlrr similar lnj>ftloo u> given. Todl amount of du*t InJiTi^J up me. Aiilinnl* o*ed: bi.\ rroup* of 0 guinea pie* rmrh (one rroup for rarh ty|* nf dii-t>. hri^l* at whirl* animal* mere kilted: Oue or two animal* iu each group al I, 2, 6, tVi and 12 month* after last iajeetinn. l*reparatinn of >lut: t/hryentile (hall milled) uobeatrd: hall milled for 1,170 hr., dra-d aud remind in agate mortar. Chrysolite (hull milled) lenileil: Hall milled ehrysoUle heated for 2 Iir. at about Ha) theo ground in aeate niortar Z or 3 mm. Chrysotiiv (fibrous) unheared: Ground in aeate mortar to pa ?u me*h. Chrysntilo (flbrou*-) ienitrd: 2l)U-meh material heated for 3 hr. at about T0r C. No further grinding. Serpentitie (hall milled) unlenhd: hull milled for 1,1* hr., dried amt r>t'rniiu*l In aeate mortar, fr-nmine (hall milled) ignited: hall inillrd srri*bime lieatnl for 2 hr. al about *(a C., llwo erouud in aeate mortar 5 or 3 min. Mineral Chrysntlle (hall milled; unlH-ated Chrysolite (ball inillrd) ignited Chysotile (flhruu*) uohrated Chrysolite (fllirvu*) Ignited tirru'iillne (liall milled) unbraced |ieri*ntloe (baU milled) Ignite*! Sire of Dut l'ortirlet 2 mieron* aod tees 2 microns and left Results Crloditig destroyed capacity to cause fllirimk At I mo. eondderahle inflammatory edema and cellular pndiirralion and loralUstioit of dut partiele* almut bronchivice; at 2 inn., only a eery alight pndiferative rtartfciu; at f>, kVa tod 13 mo., widely scattered email umnooiieli-ar phagocyte*. At Id mo., a few inirroeopic patchr* of this alveolar wall thirkening with ome adenomatoid change m (portion of air apache abutting ou tliH-keued bronchi. No aihcrtosis bodies rm Reaction limited to large foreign Imdy giant cells without production of fibrous tissue. 20-50 microns approx. 3030 micron* approx. 3 mieron* ami lr** 3 ujlernna aod leas A distinct fihroaie. Reaction louliH in eounrelive tissue about termioal hroochinle*; Uttk within those eui*a. Ountractioo caused adenomatoid appearance ol air spaces given off directly from terminal bronchiole*. Reaction area became smaller with procmi of time; no sew regions involved. No chronic pU*urlsy eveo at (mints abuttiog lotrapulinonary change. At 1 mo. considerable Inflammatory edema anil foci of cellular proliferation; nt t mo. well marked cellular proliferation and flhroii occurring focaily altont mpirumry hrom-huile*. This iractioo dcvttojHtl before a>br*tosi Igwlirs had formed and was as advanced a* that produced hy * yr. inholatloo of asbestos du>t. At 6 nin., reaction ha exleti.*ire thau at 3 mo., apparently due to contrurtion of flhrou* tissue; a*hvstois bodice were abundant. At s+i mo., reaction still less extensive, confined tu the Inmiedmte etrloity of the small terminal bronchiole*, where the rar tissue was quite deu*v ami < bcrumirig hyaline lo baraetcr. Rometiimw it tven ot>litrrut>*l the tirnnrtiiide. AsU>tosis bodir* had ler&me seaire. At 12 mo., the well developed prrihronchiul and intrahrimrluut sdeno* tnatoid areas of fibrosis ha>l prruluetnl roonlrruhic dis tortion. More peripherally were pnlrl*ee of piwuumnilis with eosinophilic infiltration, some nf which wa la-ing traosfonued into fibrous tbsue. Tlie*e seemeil to be pre cursors of the Ivcaluesl, diffuse patriH-s of thin alveolar vail fibrosis seen elsewbere. K**artiun limited to large foreign IhhIjt giant cell* withuiit proliferation. Heating tlw tit>er>, which made them brink-, detroyed their capacity to produrv siKitiflcant reaction. Im-t relatively Inactive. At 1 and 2 mo., simple tdiagoeytosi* without proliferation; at 6 mo., no rhimua eir\*pt podldy lymphoid cell infiltration; at kVa nm., a slight ehroiiie pnntinionhi*; at 12 mo., ouly a little pnrumoultiB without suggestion of tfbrori*. Dut relatively luaetlve. Rcaellua esaentlAlly the same as for unheated arrpcutlne. With Ignited M-rtantluc, iru Irndeocy for dust to be carried to bronchial nodes. .*M *s 0V 8"? am u ;-8S 'mm 3- o3 1_O *h D *e.w,e- ss-.s M*s;* Jilp ,ias:i 3; -- ^4|ij SJ? *$& Sit} ,-* -v**: /.>*?'5.1 <i : r x". " 1 i>vi .*i. t -.4* ; ti*,.-- '*v.--. ><5?* S-asHI V * * ` 1 iV$*-*. ' i1 K\ V.; &* SU-Vrl i js * m:$ *>...i Attt? .*.`.V '' paif rI w* &w*&<!9sSa g; * *;v S'-' :. : irrt'ia _ai X) isuusiki,u. urT.iusn ask tax i r.t i ifx.u. miujicixi: CLAImed .. B1^ -I <JhO .boVu O *a e g> *>"uf !*-l I f!sit o kSJfS^SSi Ml?. 2-1 sip ' k C 0 " Fig. in.--Companion of reactions provoked by injected loin: fiber and ballmilled ail ".'itos dusts: A, lung of a guinea pig which four month* bciorc had received an intratracheal injection of long filler asliestds du*t. Note the peri bronchiolar accumulation of cells with collagen deposition. The bronchiole chiefly involved is in the midst of the reaction (X 200). B, lung of a guinea pig which four months before had received an intratracheal injection of hall-milled asbestos dust. A bronchiole is shown at the right. In contrast with A, note that only a few cells have accumulated about the bronchiole and that collagen deposition is absent (X200). f- ^* ^ V ' . r.f4*.* "../^/j ""-' ef-'r-v^i-'i-Si-A' eg* 8Rg rtiuii.iui nr .u.--srcuius on ,isuf.stosis 'CLAIMED privileged The lots were made with long filter chrysntilr, unbelted. and with chrysotilc that liad Ih-cii ignited to destroy its flexible structure or ball milled to reduce the length of filter to 3 microns and less. At the same time control tests were made with serpentine, which has the same chemical composition as chrysolite but is nonfihrous. A review of the findings reveals that only the unheated, long filter chrysotilc produced typical peribronchiolar fihrosis and that tiall-milled material containing only filters less than 3 microns in length failed to cause fibrosis (figs. 10 and 11). Fibers subjected to ignition also had lost their capacity to cause serious tissue damage. Ignition produced important changes in the chrysotilc fibers, among them liciug loss of water, an alteration from a flexible to a brittle structure and possibly other changes. Experi- OCF Vis!;--* IdR-T-?!. %*n; i 21^*3 s 0T': fesS ft `V ; ' *^VSSfiB fei v-u^tss lilsg ' SL.'L J \.se,*A K\J a.___ __ WWi . _.l. ... * .V x Fig. 11.--Serpentine injection experiment: l.ung uf a Guinea pig that had received an intratracheal injection of this dust four iiK>uth> before. A bronchiole is shown at the left center. The phaencytic cells exhibit little predilection for die bronchiole and culljpcn deposition is absent (X 200). mental studies concerning this observation will be reported in a separate publication. Coiiif>arison of Various Lour/ Fiber Dusts.--Some very interesting findings are disclosed by the results of the experiments recorded in table 13. First, all the long fiber aslicstos minerals tested, with the exception of anthophyllite, produced typical fibrosis. The characteristic peri bronchiolar reaction caused by three representative long filter asbestos minerals--chrysotilc. amositc and crocidolitc--is shown in figures 10 A and 12. Why anthophyllite behaved differently from the other asbestos minerals is not entirely dear. Second, with the mineral brucite, which is not a silicate but is a fibrous form of magnesium hydroxide, a characteristic fibrosis like gft riissw A; .>'*>*i ^gtsSs ms i6 iwT-S sav E&J .1 i*i5fiji *t. iWii * vftgd \ *xr;g WTi3l tt-:\ &v1 _ *<X*: jiS&\, m*. fefttZv _, *r->, rir* f,,-Kw*i !*r; / |_ m /u/ru/rui Itrolly I'm*#!,*-: ,|'ai lujf*r|l n ill U.i n\ il e |KT *vnt *ii-iiI*:i kIifii iw, kn .|......|.>..>..r.i llOM* WM .'* HijJ. Animal* ii**I: Kfum c In H ciiliiia pipa f"* each <Jut. Period* at ehlch anltul acre killed: ('anally at I, 4, a ami I. m*>th itff )a,i iuh<\lna hi** of JuM paMirle*: heparainl o that moat IIU*fa aere irom i to Jt micruna ion*. * Mineral l*hrkoiiir (llirllorill Clrvi il* (Ari/'>na: low Ifuit niiilt ul; O.r.1 >> ;fjj AmOltt CweMollte (llollvia) Croeldolite (S. Africa) Anth(>|hyUitc Itrucltt (SIAM *CM.l ItraulU |NUnrt fll.ro-U. Additional luhrutathft aiveu it>oite rlri*onia (Rt/riiuai unlH-aKM, in table ||. ' *' i Keirlloii virttinlly Itlrmiral mil It llial In Thetford rhry<*tile. Ji(*th RhroiJi ' am! Iron. l<eHo*l* lm*Ue* |*r< m i iu*a * I aim an al^in enfttaialnc vrfjr little ' KilirmU neritrr**tJ a* iilng* aitldu i*rmiunl hrum-hioW* and a* Ru*r iIt|niiIii a! lrfi|!*ry. Hhnini <!**%-|>|*iI nl-*to*i* hcbM ifrii an<| * In cellular aHlailrf a**l!l ............ I al ....... melilft. With sc( fihroit* lUaur r*nifacirl ami tMTupted m;tlr Mf,-n Imt %m more dea*o' Adrtmtnaloid rhan*t*a similar In tli** mil Th<-|fnrd ehryMdilc. J*1*tiriy Itmitnl In tuimnllntc vlrihily il early rai ln.ii >..... . ar-i of rua^Ovr lorallzatjnn. AU*lnai hmile* f,rM*| but a-r<* j,a. Al J iimi, alter Injevtluti, niiiiiila fur! <t( itHim.iiitrbar j.rulib rathm i.fi.riMok, tn*1 hi arra of at*bv|a*l*.: af l% ....... ia*avy (ril^nniliiiilar |>airlw of flliriMr ollru villi |taiiUary WnWlrm- iiartluUv vlu.inc hm.-,, f,( linmrlnfilr; alrmiiiialnbl a|>|*aran*v narkc|; Ofinmfiivi* Hvmu- reaction almanl lnrjr mllam Imt m* liyalini/:iliiMi; al 2 urn., minute Inrl wf aril maltirol Blriii* iUmii hriaH-liin|i'>; al C tint., tuuime aiU|u<|| Ahfo* ailh rviilriM-r of runit*rt*,u: mm!raM<* rlironle t>nruminiUa Villi mnilrali'Mi Ilf |yui|ilMa*ylra ami nlno|ilt(li>. At n m., atnaU lotra- brotirlilolar flliftm* |Mite i!h fia-i of hu*f** ilrliralr Aliri>ala at prrlpiiQr. Tfl'hiil Alidma nnlflrmNliln|iii ami |a*rii*rmrliMiliii aiih furmatlnn of alriiiral iilrHwi |m|ir*. ||aillraliii|i of Unlien Irfurr 41 It mO. * afire iitjA*it<*ri. a*|| h*M*h|a<l hjr *lh m*. IWwIir* |h<ri( after lilli nm. Hfai'liiUi al I nm. Iiraff rmloliCi>itrliNli!U ami |**rj(>rtHichM<lltir already alma ini; Alirmi* chaiiae; ali*Wrtn>i ami IU>riK with idinr mrrorla at lllr of wivr li^alLealbm of ilttrl. Al I nm.. heuvy. aiildy mtlrml ttHlQlinMirHlnUlU ami irriltnmrliiulilik, ui> with rnafkrtl Idol* tnitjr of (rottrhUilra ami ailli an al**itomaloii| a|>|'aramr. At * ami 10*4 liiO.. frarlimi In lima wmillullv lla* mum1 r< al 4 nm. At I? tm*.. loH of Atrmi4 rmlolirotirliluliii* ami |i**ritriMi'hii>llii fill larcr, aiilt imr** arar ti<*iue ami luor** Iflnrmitj' i*f liroiirluol lni's Hit no rati'naiun Into, Of iteltctiftli Of, |a*rl|ihcrl |>nn*nrltyiim. Advanrrnl Atruu* rrnlnl,rtnchlollti< an.| fi>ril>ritu*J(j^!iH. )iralrr! !*. toU hmllra rmln! at * mo. At f mo., ruriy Ahroti rmiolirnnchlnlillv and irrihrnnrliiolllit; many clftnt cell* ami ininr lymnlmcytjp reoctlon. At 4 }o.( rhiftW amt of fmlolnmchioUli> acnllrrrl nirmichiMit the lime frllular Ahnula. Al A ami )f tin*., arra* of lrntrlii<>liii aumtl*r IrmiKC of tvmtrnrtion nt |mr arar at I? mo., markot lympimcytic ii.AJ- tralinn ami admoinairml ap|<>rtiirr. TvP'Col mlvmml Rtrm* roU*l.t`mrliinHtia ami prrihrnnrliloHti* |ira>liiriM by 0.f% aiiaproir>it (| rr. if.t.tl Iim*); ntoai anitiml* wnnM not tnl'rat* ttaual 0% auKtialon. Fibre*'* rfl ilrvfloprl before arr*tnj4 bo<Jlt aero. Al 4 mo., a ell develop,.,1 Abrmu hrntiehidliti* aiilt lyniplMy|e and fTlanf refla ami ailrnomatnM elianr**. Al nto., lyi*'i ;il 4>rxi-lii.liti n,t ifttile at PXtrnaWe or heavily Alinoi a* aim a .'% ii*i<entrm> ndier- al<o the antnr. Many b*i*ly alainnl ^ aiilt a c>****l |tr<.|,nii.r, ,f hauMruted a*letoait laalie*. At 1* "., Iieuvv IU-rnu hr more i*rH<rinrliirljti ami fmlidtronpliiolill*. aitli lympiu^ylr* and riant rrll<; very trkr>l alermmnt>l a>jw*aru*T. I.yni|*liiH`ylle inAltrallon ar<l trianl ii'lb bill no Ahn*i. A very lea ntyi>ieHl au*loU hodler. At I nio., many M attered Im n( ititrol<rfnrlib*l*r c villuMit ina%ivc toeali/nliori: |ynn*lmeylie InAliralimt of a.ilb atl a ('tr Clartt fell?. At A ami 12 tmi.. UtlU* evidence o( tlu*i; a f`*a broite!;n;.-a ami hrooeiil alth ciont ce)j< in O'ljaernt alveoli ptel altb lyiiridiocytic Infillrallon of wall*. FibroRiii about bronchiole*. At 1 um., arm* of dut loe;iii/tion aith rM- Japfe Qf alveoli and InAttratioli ailh acute InllNitumi'.ory nlU. m:tr;o pbaeea and Clant eelW. Within Ha* area were a !* b>ei of . ....... ti.ue ami n*iwmu* area* of bv|ierfr*|*li.v <f ah<*rdr efMfti.Jinm. Many brim* eliiole* paeke*l aitli fll`r. Al I him., f'li'ral Ki>ieriine,* i-f unebaiised: pleura elivbllv thirkvnrd over la*uv.v |iH uli/:it.Jn*t, ,\n orra<innal recm**ntol o<deMoi *N*.v nv:t. At < tun., mnuy f**l n! Iil.*r* In hronrhJnle* and alvrtdar di'ct* ailh erllitbir r*;cti>:i a* u-inr* ; r'm,. nine fori ahoweil ditinet eolla<-ft de|o*iil(.n. At 12 and J mn., T*nair>2 at lH*fore mitti Ahrn*it aliout bmneluub't mnre a|n;irr*ir nf eon- traction and detremvc nf InllammaiUtn. (daut bnnniiniit. lMn;ra markedly Involved. Typical Ahroii* endobronchioliti* and perM>mm*bo1iti* like rmctiMh to aabevtoa minernb. At 1 mo., extensive endobrnn^uuJirit and p>ribrnnehbiliUr vllb yiant <^*ll<'. t*u*e Abrnu* loxp^ ailbiti bmnvhinWt ;tu.| rHitt. lar Abroela alout them: adeunmalnid rbnuire |reni. At " nm.. h**nvy Intrabrnnrtiinlar and i^ribrnuebirdar fibrni |irn>|m <uir m:*rk<-*t *l**firi. ity ailti dielnrlinn of tute> and nblilrratinn of urrMun,itia* air Abroi pale allliout bvalbd/nli'Mi Imt aitli few nurh-i: ie, iiirfo.i*. T>ii eal aU>tOeU bmli*** ?n. Al l and * tun., little rb.itive: Mbrmn |i**n** ennlrarllur. Al iPVs tun., di'ine fll>rm> tiromhioliii. ailh a>lT<*iti*i bnlle. No plnirlfiy. N* exteuion In eurrnunf1i:ii; bu;. No At<roia ailbln a year. At 1 mo., po eeueilon In'i.b* bn.nfli*.*|.4; In |>>riplHrul air *p*m clump* *! tfii'hl <**lb paekxl aiili flue |>ieui,* of ff)aR ailh lyiupboeyrie bdiltrMinu f adjacent and*: m a*tln*i* lo>he*. At 5 nm.. rraelUin le luletiM* tlinn at I mo.; (air i/ed rhimi nf elon- pated pi ant |iltai;i*ey|ea enitlaiuiui; t.|i**nle* ami parlirl*** nf no eiwb>brun**bUt. At I ami a mu., tenet nm *lill *|iuu*hut. At 12 mo, fiH*at ar*a uf i`uemn*mti* ailh |||,ru**i> or emlnbmiirluli*; ini^I.rnte nutulier of tmiHiili lrin*lalnMK flt**i. 1 I* ) .'xtt f ** ' .rKtrii .. . .t'd i ma *eo iSS Io *> X"i "Si H is! .u PPCt^IMED----- iy bPO?CgFTM roinr.u.n hi . //. --X7 ( 7;//:.S* O/- ASMiSTOSlS 33 that produced Iiv the asbestos minerals was obtained (fig. 13/f). Since tlic briu'iic tt>ed contained only 0.90 per cent silica as an impurity, it is obvious tliat a siliceous component is not an essential factor in the development of nsbestosis. `0 fcv&lS&fEa r.'-^n tefc-.'iP'! *r?i:*fv:-Wi ; itei1 .&s3fs$agcg,,| RT^4 3*j . . a :.-.-.$*5 k?S: v7 ><: :. 'r- 2 :***? Fig. 13.--Amositc anil rrneidulitc injection experiments: .4, limp of a guinea pip four iiiotnlis utter an intrat radical injection oi anmsitc. The inflammatory rcactimi exhibits pronounced accumulation oi cells and collagen deposition (x 20flj. if, limp oi a puiuca pip four tuuniht after au intratracheal injection of emcidolite. As in .4. peribronchiolar accumulation oi cells and deposition of collagen are shown (X -'10). Third, no lihrii> resulted from the injection of glass wool fibers (fig. 13 /<), even t!.- :gh glass wool resembles asbestos However, there are iimdamental differences. A glass woo 08 000 1089 *v .w txiu.siki.ii. tncm'iHi .1 \'i> ncci-r.-n mx.ii. in diameter is a M>lid rod which in shnrt hiigilis is f.iirh- ri"iil, while an asbestos fiber of the same diameter is a Iminllr of extremely fine filaments which impart to the fiber a high decree of flexibility. It would seem that this structure and the associated flexibility arc important factors governing the capacity of a mineral to produce peribronchiolar r, CHIMED BV1^050 OCP 1) X V r .Y. *' \ -s # J '-** a " t* V x" v >v ^ .. \w xt >- Br . Fig. 13.--Brucite and glass wool injection experiments: A, lung of a guinea pig which four months before had received an intratracheal injection of brucite. Even with this nousiliccous fibrous mineral there is peribronchiolar accumulation of cells and deposition of collagen similar to that shown in A and H of figure 12 (x 200). B.lung of a guinea pig which four months before had received an intratracheal injection of glass wool. Two bronchioles are shown, one in cross section and the other in longitudinal section. _ Below the latter is a thick-walled blood vessel. The bronchioles are without reaction and can be considered normal for comparison with other figures. Class wool fibers are present in this field but cannot Ik* seen at this magnification (x200).  5r;Afcs.:*^hf j roKn-.u.n nr .u..--stut?ils of asbostosis fibrosis. Experimental studies concerning this observation ' will' be reported in a separate publication. Table 16.--Companion of Reactions Produced by Long Fiber and Short Fiber Dusti Injected Intratrachrally Doact: To iajMtlooi ot 0.5 cc. ot a S per emit lurprarlea firtn two dorr art Jki me. apart. AolrarU uM-d: Six frnup, 0( pulara pier. Parlodf at wblch anlmtli *r killed: 1, 3. t, ftt and 1Z moatha atler talectloa. Total llioeral OirTr"'ilr (Tbetlord) Aaoilte ABthopbyUta TreinoUte Bruclte Sla of Dual Partielri Lone Sher. avso tnicroat Short ni-er, tmleroaa aad left Lode flher, 3050 microaa Short Itlirr, B raicroas aodlara Lone dher. 3050 inlerosi Short liber, So mieroax and leu Long fiber, JO-50 inlerosi Abort fiber, fi microns sad Isas Lone fiber, jo-ro micron* Abort fiber, 30 micron* sad le** Lone fiber, *VSO mlcroos Abort fiber (made by crushing lone fiber* with rublxr polire* man) Rasul ts A disiloet flbroslv BUr to ehrytotila (fibrous) onbetted in tnbk* It, No fibrosi*. Haler to ebrysotUa (bU milled) unbasted la ubio 14. Trulrsl fibrous mdobroachiolitis tad perihroaehiolltis. Refer to table li Reactioo limited to phagocytosis with lymphocytic Infil tration o( adjacent walls. Abort fiber# picked Inside woUca phagocytes: looter ones free: too* coated to fora typical asbestos!* bodies. At I too. after injee* tloo, aleeoil contained goodflml flant etllr; most phagocytes were wit bio air spec*** and had not nitrated to well*. At 4 too., free oxtrecfllular fiber* bad worked themselres Into interstitial ?fs*ue, when tiM^e wst extensive proliferation of lymphoid cell* and monneytts hut no Abrol*. At mo. foreign body reaction with some pneumonitis, no bronchiolitis. Typical a*b**to*i bodies prevent. -- Advanced flbroit* endohrorjehloUtl* and perlhronehlo- lltl*. Refer to table IS. No fibrosis. At I mo., air spaces compressed and larcely filled with gint cells* parked with dull n*e\llea. Walls heavily Infiltrated with monocytes and lymph, old eclif. At < mo., a moderate decree of eeUular Infiltration of walls; small giant evils packed with duff spietiles. At C and .*H uo., mne* of giant calls, coataininf mleeral particles, la small brooehi hut aot tn respiratory bronchioles; sioaHer ones widely seat* fared (o terminal air spaces. Numerous aibestods bodies. No reaction lo eonn^ctive tissue. No eedo* . bronchial proliferation. At it mo., many scattered email monocyte* parked with dust. No eadohroa* chili*. No peripheral fibrosis, la lymph aode. slight retirutosls: no fibrosis. Lymphocytic Inflttrstioo and giant cells bat so definite fibrosis. Refer to table 15. No fibrosis and practically no abcfxtn*ls bodies. At 1 mo., focal eolbctioos of ilun-Alled monocyte* and a few ciant eellf; at 4 tno.. ome adenomatoid epithelial yewetioo: at a iu., simpl** pneumonitis with piiego* cytosis of short filters: at 13 mo.. Isolated aod sharply loeallred collections of dust cells Inside air spam about terminal arterioles. Reaction in wall* limited to IvmphoM cell Infiltration, No flhro*!*. In lymph pode, reaction limited to slight prominence of rctieu. him. Fibrosis about bronchioles. Refer to table IS. Almple foreign body reaction. No acute lofiammaiioo. No accumulation of du*t In or shout terminal hrun* Chlolea. No rodohronchltls. At 1 mo., scattered small (iant cell* and considerable Infiltration of atliec'SH walla with monocyte* and lymphoid cell*. At 4 tno., Uttle change, except more cellular Infiltration of con nective tissue. At t mo., lymphoid infiivratinn and thickening of walls shout aotnc tnul not all terminal bronchiole*. .Typical fibrous endohroneblolitl* und peribronchiolitis like reaction to abcttos mineral*1. Refer to table li. Iaert type of reaction. At 1 mo. ofier Injection, small monocyte* widely scattered through air spam: focus of atelectasis with lymphoid Infiltration of compressed alr-spae* walls. No endobronchial rrsetion at with cbryaollle. At S too., reaction similar to that at 1 mo.; typical asbeatuals bodies seen. At it mo., amah clumps of taactlve dust-filled phngocytes; ao flbrotls. NO reaeiloa la lymph nodes. Comparison of I.ontj Fiber and Short Fiber Dusts.--With quartz dust it has fr'cn demonstrated that the smaller the particles the more. Cf Vdft?? - stf# 'S4s&r r-gpr l *- . Wm erodcd C o rn la o C o a fld a raproduc f n o lokoad tia l do ar Cou ro la a . la fo n r d is tr rt a iab ttluOtoarenldd..o.r n e la B a rt OSDC * 0 ot to bo xcept In P u tM H la a : c o p ie d , accorda n c e w ith th is C o u rt's o rd s r. f5?v m Mf tei P-AVrs*rSn m$m l vnjL M AV.-* * mferj r&V K S*e V-J in(. -< 4K< joyv.*r gfer?s iI EiAJP-. |t &S5?- h&z. jo /xpcsikiml nyafr.xn .ixn out r.iih>\.n. wi.i>ii. i.\n PRCIlVatI1^0 intense is the tissue reaction and that particles larger than 3 miiccrroonnssQQyy nr'^'GEl in diameter cause little reaction. In the case oi asbestos, however, the uCf reverse is true and apparently only long fillers have any specific effect, as was suggested hy the inhalation experiments. This is confirmed by the data of table 1C, in which a scries of tests with fibrous minerals is rqiorted. When the injected dust consisted of fibers 20 to 50 micruns long, all the fibrous minerals tested except anthophyllitr. as noted in the preceding section, produced fibrosis; when the material was prepared hy first grinding the fibrous dust until the length of fibers was reduced to 20 microns and less or, in some cases, to 3 microns and less, none of the injected dusts caused fibrosis. ( These results differ from those of King, Clegg and Rae." who reported the production of reticulosis comparable to the experimental silicotic nodule in.rabbits receiving monthly intratracheal injections of 100 mg. of Rhodesian asbestos fibers, 15 microns long, and the produc tion of diffuse interstitial fibrosis in rabbits receiving similar injections of short fillers, 2.5 microns in length. We lielieve this dose, especially in the long term rabbits, is highly excessive. In our experiments the dosage was kept low in order to minimize untoward reactions which might obscure the peribronchiolar type of fibrosis which characterizes early human asbestosis. 'It'S i-ce8Sa S. -i eO * h 0 0 i e v *> eiiot 1 *E c 89 * I:laiSle2 > ExFUIMEXTS UsiXC INTEAVENOU S TECHNIC The experiments summarized in table 17, in which the intravenous method of injection was employed, show that the asbestos minerals are far different from quartz in their action on tissue. It has been repeatedly demonstrated that intravenous injection of quartz particles 3 microns and less in diameter will cause a typical tissue reaction with the development of hyalinized fibrotic lesions in cxtrapulmonary sites, such as the liver and the spleen. Asbestos minerals, however, on intravenous injection generally produce only an inert type of reaction, as is. revealed by the results given in the table. The reason for the early deaths in the exjierimcnt with chrysotile particles is not clear. Kxfehimexts Using Intuafekitoneai. Technic The results of injection experiments with the intrapcritoncal technic arc given in table 18. It will be noted that the long fiber dusts produced a fibrous reaction while dusts composed of particles 3 microns and less in size caused only an inert typeof response. These experiments indi cate also that the fibrosis initiated by the irritation of aslicstos fillers is not restricted to the lungs, as was formerly assumed, but can be pro duced in the peritoneum as well. OT1IEK EXPERIMENTS WITH ASIIF.STOS MINERALS A number of additional experiments were conducted to throw more light on specific phases of the asbestosis problem. 11. King. E. J.; Clegg. J. W, and liar. V. M.: Elicit of A-lmtu*. anil of Asbestos and Aluminum, on Lungs of Rabbits. Tlmrax I: IPS, IlM*.; alittrarn-d, IndusL Hjrg. Digest, 1947, vol. 11 (Feb.), no. 23-1. 'l I Maimed -T'-ragd .: >r/y*T:-n :.'@S8J ^I m&m 0MpksWiS3 fc-k.>V/ t SjvSKwJ'f! $!&? &*$ isiy*5 v'",V::v k- &XaMk?s*se > : *' < '. "*-: . Kt>*`V^'-! rOKIV.IKI) F.T Ah.--STUDIES OF ASBESTOSJ"S '.:;ciaW;':^^^ PaerrienvE Action Ok Aluminum CoMrouxns PRT'/rLEGEQ When colloidal aluminum hydroxide had been added, to a suspensionBY OCF of long fil*r chrysotile prior to injecting this susjiension intratracheally into rats, the aluniimnn eoni|xnind did not prevent the irritation of tissue due to chrysolite. If anything, the acute inflammatory response evoked by the injected fibrous mineral was accelerated. One month after the last injection of the dust suspension the bronchiolitis was becoming fibrous. King and his associates also found that aluminum failed to protect pulmonary tissue from the irritation caused by asbestos fibers"; in their experiments metallic aluminum was used instead of the hydroxide. Formation or Asbestosis Bowk* The iron in the coating of the aslrestosis body appears to be derived from blood or tissue elements and not, as has been suggested, from the mineral Tiber. After two kinds of chrysotile were injected subcutaneously into the groin of a guinea pig--one kind containing 2 per cent and the other 0.2 per cent ferric oxide--the asbestosis bodies were equally numerous at both sites of injection and showed no difference in their reaction to prussian blue, the reagent which stains iron. This finding is in agreement with that of Giroux." Tissue RtAcno.v to Asuestosis Bowks Asbestosis bodies recovered from human lung tissue and injected intratracheally into guinea pigs failed to produce a fibrous reaction. The materia! for injection was obtained by digesting with sodium hypochlorite solution the lung tissue removed at autopsy from an asbestos worker. The asbestosis bodies could be seen in the guinea pigs for at least a year after injection. This experiment shows that the aslicstosis body has a rather resistant coating which is not destroyed by moderate hypochlorite treatment, which may be maintained in vivo for a year or longer and which renders the fiber inciijiabte of producing fibrosis. It thus appears that the coating is a protective mechanism. This thought was expressed by Beintker as early as 1934." i- .Vi THEORV OF IRRITANT ACTION Two hypotheses have been proposed to explain the tissue irritation and reaction caused by asbestos fibers: the chemical and the mechanical. In the chemical theory, which is based on experience with quartz, it is assumed that the asbestos minerals dissolve in the body fluids and that in this process their bases are leached away to leave silica in a form capable of irritating tissues. According to this hypothesis asbestosis is merely an indirect silicosis. Several facts make the chemical theory untenable: Intratracheal injection of brucitc fibers, which had a silica 12. Giroux, M.: Amiantosc experimental*: valcur pathogo:i\omque du "corps d'amiintc," I.aval roed. 8:239, 1943. 13. Beintker, E.: tiler die Asbestosiskorperchen: Bemerkungen iu der Arbeit von Bcgcr, Virchows Arch. (. path. Anat. 293:527, 1934. ooa ..... CLAIMED IV PRIVILEGED /.xnrsTKML uvc.nisu .ixn out r.iuox.u. Mr.nni.'&Y OCF content of only O.'X) jer cent, caused typical fihn*is like that produced by the asbestos minerals; free silica particles increase in potency as the particle size becomes less, but aslicstos fibers shorter tliau aliout 10 to 20 microns are relatively innocuous; aluminum hydroxide neutralizes the irritating effect of quartz hut not of 'aslicstos; ser|>entine has the same chemical coni|>osiiion as loop fiber chrysotilc. but it produced only an inert type of tissue reaction; there is a wide raupc in the chemical composition of the minerals which do cause asbestosis (table 19). In view of this evidence it seems more likely tliat aslicstosis is caused by an unusual mechanical irritation due to long asbestos fibers, this irritation living related to the peculiar filaineuted structure of the fiber and the associated flexibility, which are jKissessed by no other foreign body studied. Thus, ignition of chrysotile fibers changed their structure and nude them inert, although the same fibers, before being heated, would have produced fibrosis (table 14). Further support for the theory of mechanical irritation is that asbestosis occurs in an organ of high mohility --the lung--and that a fibrous reaction can be produced by injecting Kll*rnu Mineral* AfflO*i|p.......................... Alii||liil,9lr..................... ........ Hrurifr...................... .. OtricMttQi..................... CreridoliU.................... Tmaoliit....................... SiO: n 46.31 SVN 0.% *91.90 Hi. Table 19.--Mnalysri of Fibroin Minerah rOi % 4j00 SAG 047 .7K 949 1047 T41 TtO % aj) ..... ..... Ji ..... 440 AlvOa % 1.00 MB 041 0.4C 0M L01 MB CtO * S.01 is. 0.44 0AM om 0JO 4.44 MtO % 19 040 fojes W.fl0 UM 1545 9045 xo % 0.S3 0.45 043 0.01 04U MS n KsO 040 0.13 0.15 010 0.(6 047 0.fl> I<fflSiOllSoca. Las* > 105 C. %% 0.GS 545 042 5.00 047 4.00 0.53 3145 440 14.00 . 0.05 ISO 0.54 3.70 TAUl 00*7 o?.n Ke.52 W.70 oo.m I uu l liis i * *88 it! t*. |! ii i-i > Mis. i Idlls ;r 1 0%c. ai<tt UqUo u -* ii I !. !i a ;(* II asbestos fibers into the peritoneum, where there is also a degree of mobility, but not by injecting them into other extrapulmonary organs such as the liver, the spleen and subcutaneous tissue. COMPLICATIONS The experimental investigations with asbestos minerals were con cerned primarily with the effect of the dust on normal tissue, but some attention was given to other phases, such as susceptibility to infection. The only experiment in which the effect of asliestos dust on a pulmonary infection was studied was the first inhalation experiment, carried on with King's floats dust. It is unfortunate that, owing to the lack of adiiputc facilities at that time, infection studies could not lie made in the uthcr inhalation exjicriments also. Susceptibility to Ti blacclocs Inkutidx The development of a tuberculous process initiated at the beginning of exposure to dust, and also of a tuberculous infection sti|>criiii|K.cd on an established asbestosis, 'was described in preceding sections of this paper. It may be stated that asbestos when classified according to the effect of a dust on tuberculous infection would be placed below an active % r*mM:--* [W* re IP^sWSSl ivm-.tu* i;r iV AL.-STIDILS or- ASililiiiTUSlii 41 dust like fpiartz hut aliovc an inert dust such as iron oxide. In an'r^3^CjyLrALIMGErDT]| iniected with attenuated tubercle bacilli, quartz causes the infectiously-- process to progress until the animal dies of tuberculosis. Inert dustsC have no ciTcct on the infection, and the lesions usually heal and the disease disappears. Ashcstos dust is in a different category. In the experimental investigation, when the fibrous dust was being inhaled dur ing the evolution of the infection, there was spreading of the tuberculous process for a time, but usually the stimulus for continued proliferation of the tubercle bacilli was not sustained, the progression was arrested and healing followed. In guinea pigs infected .with attenuated tubercle bacilli after being exposed to asbestos dust for slightly more than two years, progressive disease did not develop. The only modification of the infection was one of localization, a few bacilli being retained in the fibrous terminal bronchioles and forming tultcrdcs there, iri addition to the usual foci beneath the pleura. Such tul>erclcs healed in a few months. hr'V-'*. Imm, g'jsijyrL'- Susceptibility to Xoxruimcvuius l.vrrcrtov Th re was no specific experiment concerning the effect of inhaled ashcsto-i dust on nontuberculous infection. Intercurrcnt pneumonia was rather common among animals exposed to asbestos dust, the frequency in guinea pigs exposed in the four inhalation exficriments ranging from 16 to 39 per cent. This incidental evidence suggests the possibility of an effect of asbestos dust on nontuberculous infection. Nevertheless, since such epidemics are not uncommon in inhalation experiments with Other dusts and even in the colony of normal animals, it is felt that the inhalation of asbestos dust does not exert a significant effect on the susceptibility to nontuberculous pulmonary infection. .; COMMENT AND SUMMARY Owing to the vast amount of data included in this investigation, it seems most convenient to summarize and to state as concisely as possible the various observations which emerged from the experiments and to follow each with a brief resume of the evidence. ' A. Various species of animals, including the guinea pig. the rat and the rabbit, hut not the mouse and the dog. develop peribronchiolar fibrosis of the'lung similar to human ashestnsis after 1>cing exposed bv inhalation or intratracheal injection to long chrvsotile asbestos fibers. Both inhalation and injection experiments provide ample support for this statement. Figure 8/1 reveals the cellular fibrosis that occurs in guinea pigs following inhalation of long filler asbestos; figure 9 shows the fibrosis caused in the cat by inlulation of long fiber asbestos dust. Similar but less extensive fibrosis occurred also in rats and rabbits (table 1). Mice and dogs failed to respond. This variation in response of different species to identical dust exposures is still to be accounted for. B. Long a.'liestns fillers arc essential in the production of the peribron chiolar fibrosis; short fibers are incapable of producing this reaction. 08 L 006 ewe 1097$& vf CLAlf-2D privileged *2 txncsihi.iL iV HYCtEXE ASP 0U.Vl`ATlOX.U. MEDIUMS'* CCF Inhalation experiments with aslicatns dust suggest, aud intra tracheal injection experiments coufinn, that pcrihronthiolar fibrosis is produced by asbestos fibers between 20 and 50 microns in length but not by particles shorter than 20 microns (tables 16 and IS). This indicates that the minimum length of fiber possessing the capacity to produce the typical peribronchiolar filiTosis in animals is somewhere between 20 and 50 microns. Pointed studies have not been carried out to determine the tipper limit of effective fiber length. It appears, however, that that limits will be determined by the inhalabilitv of the fiber. , C. The mode of action of the long asbestos filler in the production of ( asbestosis is primarily mechanical rather than chemical in nature.__ ) The evidence for this conclusion has been reviewed in a preceding section, page 39. The flexible filamented structure of asbestos fibers plays an essential part in the irritating action, since the solid, inflexible fibers of glass wool do not produce fibrosis (fig. 13 B). _. D. Typical e.\]<erimental asbestosis was produced by the inhalation of I an atmospheric suspension containing an average of 138 million f asliestos particles per cubic foot of air by light field count, of which I less than 1 per cent consisted of fibers longer than 10 microns. ____ / In the inhalation experiment with 100 per cent ball-milled asbestos dust containing 0.6 per cent of fibers longer than 10 microns (table 11) typical fibrosis was obtained (table 9). The evidence presented shows . at least that an atmospheric concentration of asbestos dust containing less than 1 million (0.0 per cent X 138 million) fibers longer than 10 microns per cubic foot of air is capable of producing experimental asbestosis in guinea pigs. The actual lower limit of concentration of long fibers necessary to produce asbestosis in animals cannot be estab lished from these studies. -- E. The duration of exposure required to develop the pulmonary reaction | to inhaled asbestos dust is inversely proportional to the concentration I of long fibers in the atmosphere; as the concentration is increased, / the reaction develops in shorter time. [ The basis for this statement appears in the data of the inhalation experiment with long filler asbestos. For that experiment the average concentration of the atmospheric dust was about 40 million particles per cubic foot of air, and size-frequency determinations disclosed that 6.7 l>er cent of the air-suspended material consisted of fibers longer than 10 microns (table 11). Thus, by calculation, it is estimated that the con centration of the longer fibers was 2.7 million (6.7 per cent X 40 mil lion). The lungs of animals exposed to the long fiber asbestos dust revealed that the pulmonary reaction develo(>cd in approximately onehalf the exposure time required for its development in animals inhaling the ball-milled product, for which the concentration of the longer fibers was only 0.8 million (0.6 per cent X 133 million). --j F. Established experimental asbestosis ceases to progress on discon tinuance of dust exposure. , -- . The experimental investigation shows, in fact, that on discontinuance of exposure there was an appreciable clearing of the mature pulmonary i'-V-f "H-t am *> -D8! - Ifs sIsit; lit `Is 1 rV j 8 ills its Is tm 610oh *; >V !I i SBKSibi SWiSSi VIHCV* 006 1098 -*> CLAIMED rnUll.lU) KT AL.--STUDIES Or- ASHEST0S1S _PRIVILEGED 43 BY GCF bfei! ilesions, due to contraction of the fibrous tissue. In contrast, sui imma ture tissue re|iunsc, evidenced primarily by cells with little or no fibrosis, continued to progress. It is assumed that, following attainment of fibrotic maturity, the same process of contraction would ensue as was noted for the mature lesion. .9 G. The formation of ashestosis bodies represents a coatiinn;g of the fibers / by blood and tissue elements, which results in loss of ability of the I fiber to produce fibrosis. .as* Intratracheal injection of asltestosis bodies failed to produce the typical asbcstutic tissue reaction in experimental animals. The cessation oi progressive reaction observed soon after exposure terminates may be due to the formation of asltestosis bodies. H. Aluminum hydroxide failed to neutralize the fibrosing action of the long fiber asbestos. Aluminum hydroxide added to the suspension of chrysotilc asbestos prior.to intratracheal injection did not retard or prevent the development of asbestosis in rats. I. Inhalation of asbestos dust did not alter significantly the final outcome of experimental tuberculosis in two series of guinea pigs exposed to the dust. The apparently mild influence of asbestos dust is in distinct contrast to the stimulating effect exerted by inhaled quartz on a tuberculous process in the lung. The interpretation must remain tentative, however, since it is based on an investigation limited to two series of guinea pigs exposed to only one kind of asbestos, namely. King's floats: Table 4 shows that when the infection was coincidental with the onset of dust exposure, there was temporary progression of the infectious process, with subsequent healing; when infection was initiated after 26 months of dust exposure, the course of the tuberculosis was not appreciably altered. The latter finding is quite different from our usual experience with quartz dust or with mixed dusts containing quartz, wherein the adverse influence of quartz on a tuberculous infection is manifested most strikingly when infection is initiated after a period of dust exposure, viz., superimposed bn a background of established silicosis. As indicated above, this more sensitive test, when applied to asbestos dust, failed to demonstrate that the latter had an adverse influence on a tuberculous infection. The inability of asbestos dust in that experiment to aflect unfavorably the tuberculous process furnishes strong support for the interpretation that inhaled asbestos dust has no more titan a mildly unfavorable effect on pulmonary tuberculosis. fo&iflf This investigation was made possible by the generous financial support of a group of companies of the asbestos industry. r; IJ 1' . i iy V V1 *** USE THIS COPY FOR REFERENCE ONLY*** ! I..*- USE THE EXHIBIT THAT IS LOOSE IN THE FOLDER TO MAKE YOUR COPY ftrctiives of lndustria, lijglblib U1IU ULliUyailUIIUI illblllblll& ./ 5 3Volume JA.NTAHY COPY|CHT, rjjl, Y THI AMUIUM MtOKjkL ATIOK Nl.muf.r I CJy', :^.r- \ v; ' ; EXPERIMENTAL STUDIES OF ASBESTOSIS ARTHUR J. VORWAID, Ph.D.fPoth.), M.D. / ,. THOMAS M. DURKAN K AND /jr <* \ PHILIP C. PRATT, M.D. SARANAC LARt, N. T. UK ASBLSTUSIS is a form of pneumonoconiosis resulting from prn- U/ aA. longed inhalation of asbestos dust. The name "asbestos," literally "unburmble," is not that of a specific mineral but is a term applied to a \' number of different minerals whose characteristic feature is a structure composed of long, parallel, flexible fibers. This structure is unique because the fibers are capable of repeated longitudinal subdivision to units of molecular proportions. In length the fibers vary from a few microns to 6 or more incnes (15 or more cm.). Some varieties are stiffer than others, but many are sufficiently flexible to he spun into yam and woven on modified textile machinery. The asbestos minerals are silicates of variable composition and belong to the serpentine and the amnhibolc groups. Listed berow arc the more common varieties. ./. J *i % <\ ,y %\ Amphibole group: actinoiite, amosite, amphiboie, anthopbyiiite. crocidolite and tremolite. Serpentine group: chrysotile. The bulk of the asbestos of commerce is chrysotile, 3Mg0.2?iO..- ^ \..U 2H.O, which is mined on this continent principally in the Thetford region of the Province of Quebec. Canada, and in Vermont. Crocidolite and amosite also are used commercially hut in much smaller amounts. Chrysotile occurs as veins in serpentine, a mineral of similar chemical composition, which exists in massive form and is made up of microscopic fibers without the parallel orientation characteristic of chrysotile. The V,'.massive, bluish black serpentine, which is smooth and soapy to the touch, V >/ v^ I is traversed bv veins of fibrous chrysotile varying in width from a barely perceptible line to 6 (15 cm.) or more inches. The fibers run N1- across the vein and not lengthwise with the formation. From the Saranac Laboratory of the Edward L. Trudeau Foundation. This series of studies of asbestosis. initiated at the Saranac Laboratory more than twenty years ago by the late Dr. Leroy L'. Gardner, director of the laboratory, IT'V A\y vT* u was nearly completed at the time of his death in October 1946. Although partial reports and informal reviews of some of the experiments liad been given from time to time by Dr. Gardner, this paper presents for the first time a complete survey of the entire experimental investigation. i .V' \- ). J * > ?LJ V b-:1 * A 0 /* ' % .. U'' % l ' s 1 JA1-" L / z PLAINTIFF'S EXHIBIT 39*70,0 -; 2 ' IXDUSTRH IHYCIESF. ASD OClII'AHoXAL ^ c/.ve Attention is directed to the mineral hrucite. MgO.lLO, which is often found in tfie same formations with serpentine and chrysotile and may be fibrous in structure. \cept for the manufacture oi magnesium, brucite has no commercial value at present because its fibers are not sufficiently flexible to be used in textiles, but they are capable of repeated longitudinal subdivision. Unlike other asbestiform minerals, brucite is not a silicate, and for this reason it has been a valuable tool in an experimental evaluation of the action of fibrous minerals on lung tissue. EXPERIMENTAL ASHF.STnSlS For many years studies1 have been carried on at the Saranac Laboratory in an investigation of the cause, nature and development of asbestosis. The present paper is devoted to experimental asbestosis. Fig". I.--Human asbestosis f P-36-14-l>. The photomicrograph reveals a bronchi ole (right center) with a smooth muscie bundle at it* interior margin and with an extensive zone of collagen deposition largely obliterating the surrounding alveolar structure. The black loci arc macrophage* o-naming ir:ei'ivn:ii pigment. Asbestosis bodies are present but are not apparent at this magnincation (X 2U0). and In it are described the experiments made on animals with various kinds of asbestos dust. Another report, to be prepared and issued at a future date, will be concerned with human asbestosis and will cover the healtli aspects of workers who have been exposed to asbestos dust in an industrial environment. Although in man asbestosis is a chronic disease with diffuse pulmo-|i nary fibrosis which requires years to develop, it is possible to reproduce || 1. (<j) Gardner, L_ U, and Cummings, D. E.: Studies on Experimental Pneumokoniosis: VI. Inhalation oi Asbestos Dust; Its Effect upon Primary Tuberculous Infection. J. Indust. Hyg. 13:o5 and 97, 1931. (b) Gardner, L. U.: Chrysotile Asbestos as an Indicator oi Subtile Differences in Animal Tissues, Am. Rev. Tuberc. 45:762, 1922. m sin the the usv. at ic it! i: tior cor an:: the van nan exp< son i kep; dimpadt aniji and peril mint a p> cont; dust. the i L great cons: atmo expe: be in. pulmi meth' occur accur: dose, valuai it pefr Ur organ.table 1 from a VORWALD ET AlJ^TUDIES OF ASBESTOSIS 3 in one or more species oi animal'characteristic' tissue changes which are ' similar to the lesions of human a>bestosis (fig. 1 ). Fince the life span of the experimental animal is relativelv short, it i* not pos-ihlf i > profiuce the characteristic lesions in animals under conditions identical with the usual industrial environment. Consequently, to obtain a complete evalu- , ation of the tissue response to inhaled particulate and fibrous material. I it is necessary to accelerate the reaction hv employing higher cnncentra-l tions of dust than would ordinarilv he encountered in indu>trv. While . ' t conditions of exposure are thus different, the information yielded by j animal experiments is invaluable in furnishing a better understanding of j the reaction of the human organism to inhaled asbestos dust. I Experimental Methods For investigating the tissue reactions of experimental animals to the various asbestos minerals, two types of technic have been employed, namely, the inhalation method and the injection method. In inhalation experiments, groups of animals--up to 100 or more guinea pigs and sometimes smaller numbers of rabbits, cats. dogs, rats nr mice--are kept for eight hours a day in a cubical dust room. ,S ft. '2.5 M.'i in dimension, in which a cloud of asbestos dust is maintained by a rotating paddle in a dust hopper.At intervals during the experiment a few animals are killed and the tissues examined to determine the nature and the extent of the dust reaction. Some animals are exposed for periods up to three tears. The injection experiments are used to deter mine in as short a time as possible whether nr not a particular du't has a potential capacity to produce inflammatory reaction when in direct contact with tissues of the body. The method involves injecting the dust, either dry or suspended in fluid, into the animal by the intravenous, the intraperiton'-al, the intratracheal or another route. Long term inhalation experiments furnish information on which great reliance is placed when estimating the degree to which a dust might constitute a respiratory hazard to industrial workers. Even though an atmospheric dust may he potentially dangerous, as indicated by injection experiments, only inhalation procedures will reveal whether the du-t can l>e inhaled, pass the natural defense barriers of the body and reach the pulmonary tissue in quantities sufficient to cause damage. Injection methods are useful, however, because they make certain that contact occurs between the dust particles and tissues and because they allow accurate estimation of the dosage and of the potential capacity of that dose to produce reaction. The intratracheal method is particularly valuable when one is dealing with fibrous minerals like asbestos, since it permits observation of the effect of the fibers on pulmonary tissue. Tissue Susceptibility Unlike free silica, asbestos does not produce specific effects in all organs of all species of animals. The comparative data presented in table 1 are based on completed observations and therefore differ slightly from a preliminary report.lh Fine quartz introduced into various organs -tv i-irr 4 IXDUSTRf 'HYC1EXE AXD OCCURATIOXAL MWFtCIXE of various animals (guinea pig, rabbit, rat, mouse, cat. dog. chicken and even tadpole) eventually will produce silicotic nodules but at different rates. Similar introduction <.. long fiber asbestos lias resulted in a fibrous reaction in the lung and, to a lesser extent, in the peritoneum but net in other organs of the guinea pig, the rabbit, the cat and the white rat- In our experience the lungs of the dog and the white mouse failed to respond with fibrosis, although Schuster 5 has reported such changes in a dog that lived in an asbestos-fabricating plant. This variation- in species and in organ susceptibility is yet to be accounted for 5; it is presumed that in the susceptible animals the greater reaction of the hmg to asbestos, far exceeding the reaction of other organ tissues, is due principally to the greater mobility of the lung. PeCVLIAK CHAJLACrnilSTlCS OK ASBF-'TliS Experience has demonstrated that most of the nontibrous dust panicles inhaied into the lungs of man and animal are 10 microns or less Table 1.--Reaction to Long Fiber Chrysotile in {.unns pi Mon anti Other Sfeeirs of Ar.itna! Sr*eln Mu............................ Oolafft pff............... _ Babbit....................... Cat............................. While rat................ Wblt* monaa........... On*............................... Jlodf of Riroturr Inhalation Inhalation mod Injretloo Inhalation and iniernon InhaUUoo and laiwtion Inhalation and Inirrtton Inhalation lajKticn F!t.ro*l 4+t+-f + 0 0 bodlci Nomrro-ji ModtvnMy auin'roii' Rare an<l atypical Rare ae-i myi.ical Vary ranRare an-i .trt'leal None The y:!iUil> } to 4+ rrf. r to the v'srra of il"uc rrariinn. in maximum dimension. Larger particles apparently do not gain access to the lungs, because, first, large particles settle in air so rapidly that few remain suspended in the atmosphere breathed and. second, large particles are more effectively removed by the protective mechanisms of the upper respiratory tract. In the case of fibrous materials these factors have less influence and fillers 100 and even 2(j0 microns in length have been found in the terminal air spaces of human lungs. In small labora tory' animals exposed to asbestos dust the maximum length of fiber found in the lung rarely exceeds 60 microns. A large proportion of nonnbrous particulate dust inhaled into the lung is found in the terminal air spaces (alveolar ducts, atriums, alveoli) in all parts of the organ; in contrast, inhaled asbestos fibers are first discovered in the respiratory bronchioles. These small passages are immediately distal to bronchioles lined by ciliated epithelium.* Their 2. Schuster, N?. H.: Pulmonary Asbcstosis in a Dog, J. Path. & Bact. 34 (pt. 2):751, 1931. 3. .Vorwald, A. J.: Variations in Individual Susceptibility to Industrial Dusts Inhaled into the Lungs, Am. Rev. Tuberc. 62: (IB) 13. 1950. 4. Miller, W. S.: The Lung. Springheld, 111., Charles C Thomas. Publisher, 1937. oW nar alv- cha of ri is; v per: obst quar trad one mor.: in ha.' does dl :t d expo nodu perioshort prod progr T1 body*' golder or cur The l niicror If: tein ah observi subcuu abunda in theI fibers 6 5. C Tubercle ner and ( 6. L} J. A if. Asbcstosi Asbestos Gloj-ne.14- VORWALD ET A TUDIES OF ASBESTOSIS 5 own essential lining is a low cuboidal type of epithelium but. as their name implies, they actually function in respiration through lateral alveoli distributed along their walls. Either these alveoli or the abrupt change in the character of the lining epithelium, or the small diameter of the respiratory bronchiole, or the combination of all three factors is responsible for. retention of the fiber at this site. Only after ashestosis is well established are appreciable numbers of fibers seen in the more peripheral air spaces. Further explanation is required to clarify this observation. Rate of Tissue Reaction to Asbestos Fibers The affected tissues react much, more rapidly to asbestos than to quartz dust. For example, in rats receiving asbestos fillers by intra tracheal injection fibrosis of a characteristic type is visible as early as one month after injection; for quartz dust the latent period is two months or more. Tints, the development of nodular fiLmd? due to inhaled silica lags behind the deposition of dust to a greater extent than does the evolution of the diffuse rea^don to asbestos. This results in a difference in the degree of progression which follows termination of exposure to dust. For example, on discontinuance of exposure the nodules of silicosis become larger, to a limited extent, tor a considerable period of time, whereas the fibrosis of asbestosis increases for only a short time. Subsequently, the asbestotic fibrous tissue commits; this process often distorts tbe adjacent pulmonary tissue and may. as a result, progressively interfere with cardiorespiratory function. Asbf.sttwis SrOIF.S The peculiar structure known as the asbestosis body or "curious body" is a specific concomitant of asbestosis.1 The typical txsdy is a golden yellow, beaded or haustrated rod. 'which may be either strait;!'.: or curved (fig. 2). Often one or both ends are bullious like a dumbbell. The bodies vary considerably in length, and dimensions up to 250 microns have been recorded. It is believed that asbestosis bodies are inhaled fibers on which pro tein and iron pigment of tissue origin have been deposited.*' Gloyne :t> observed reproduction of these bodies in guinea pigs nine muntiis after subcutaneous injection of fibers rendered free of iron. The bodies are abundant in man and in the guinea pig (table 1) but are much larger in the former, probably because the larger-sized air passages admit fibers of greater dimension. In guinea pigs they form.a:\er about 70 days 5. Gloyne. S. R.: (a) The Formation of the Asbestosis Body in the Lung. Tubercle 12:393. 1931; it0 The Asbestosis Body, Lancet 1:1351. 1932. (c) Gard ner and Cummings.1* 6. Lynch, K. M.. and Smith, W. A.: Asbestosis Eodies in Sputum and Lung. J. A. M. A. 95:655 (Aug. 30) 1930. Sims^ru F. W.. and Strachan, A. S.: Asbestosis Eodies in the Sputum: A Study of Specimens irom 50 Workers in an Asbestos Mill, J. Path. & Bact. 54:1, 1931. Gardner and Cummings.1* Gardner.11* Gloyne.5*6- T--' 6 INDUSTRIAL \GIENE AND OCCUPATIONAL ME! P. of contact with the tissue. In cats, rabbits and mice a few of the fibers show an atypical coating after much longer residence in the lungs. In rats the bodies are rarely seen, and in dogs none could l>e found. Although the evidence is incomplete, it appears that the formation of the asbestosis body prevents the fiber from damaging the tissue. Many of the points mentioned above will be elaborated on in subsequent para- fp- n m V'7V Fig. 2.-A. human as!>es:osis todies. This collection of asbestosis bodies was found in the lung shown in figure 1. The usual variations ui size and configuration are represented (X4WJ1. B, guinea pig asbestosis body. This one is similar to some of thuse shown in A (X -blOj. graphs dealing with the actual experiments. For presentation our investigation is divided into two sections, one dealing with inhalation experiments and the other with injection experiments. ;F labor gafic- earn four shdn T with TTpe of A?*Kinx's float Sbori flt-r. Lodz Qber. Not Kir thao 1 AO rr. r.K.rj>c eary*- act;SO. to Shml^ prtKgpc oat it*: miner*. Coirrr er*lo?* i r:r. If* perio- expof expo? mont: 2;4 asbes result Co a cote fibers, also v Asbest unouti VORWALD ET UUIES Of ASBESmSIS 6> INHALATION EXPERIMENTS Four large stale inhalation experiments have l>cen conducted in this laboratory with various forms of asbestos d-ist. In each of these investi gations. more than U>0 animals were ti>cd. and the experiments were carried on tor periods ranging from two to more than live years. The four kinds of asbestos dust employed are designated as King's floats, short fiber, 100 per cent ball-milled, and long fiber asbestos dust. King's Floats Asbestos Dost The first inhalation experiment conducted at the Saranac Laboratory with asbestos dust was begun in 1928. Animals inhaled the dust for Table 2.--Chemical Analysis of Asbestos Dustiny Materials Type of Ajbettoe SK>* KLoc'fl float*........... ... Short fiber............... ... Lobs fiber............... ... 33.32 37.;: 3S.40 PeiOa AlrOi CrrO. MoO BM 9.09 1.40 5.32 0.7 - 0.11 0.09 0.0b co 0.67 0.&j 0.31 MrU Na-0 3J,>. *0.13 0.11 0.06 KsO 0.20 006 CO* 0.01 0-57 leal- UOO Loss Total 12.74 14.00 14.00 97.13 100.11 99.70 Not determined. Table 3.--Petrographic Analysis of Asbestos Dusting Materials I ' ------------ ------- -- - - ----- ------ ~ Ij * ClOf'i float* : The approximate composition, ba. : panicle* (except chrc-ottl-i -mailer | tbao 10 microns and rppr>rifi i**rr*ntai* obtain*-; :rnm porttrle ronar*. w cnry^ii;* II. :r.I serpentine 40. marnftite carbonates i. tale 12, older minerals 4. For chry*otil*. flr*r? up to ?J0 microns lose w*re included. Short filler : Th* material. l-*furr t^iof hull mill**!, contained a pr*'u**nd'ranee of O^rou^ ? eftry*otl!e and p'*.i y (r. or: rtf r`u Thf a* rro vrr.at- c he ?*-' ' w-*. a ehryso: !e ^ rit*. *iuarU 1, LT-:.U J, iniurrul*. aetiaolite and trer o.ite, n. Lonx fiber t: The material eonlitrl principally of the fibrous asbesto* mineral efcryotile. Shmls of ror**rarated.tu-er*.i to micron* in diameter and up to y> mirror.* m leactn tr-r*-present. Tie approximate compuMtion. by percectu^e, cnrysotile 7j. rper.tlr.e '.S, raaicDrtitr 6, bninie 2, :her mineral*. minor whlrti acre caliite and chlorine anti micaceous mineral*, 3. Ooty a trace of quart! was ol**rTr<i. T^e analysis of the Kin* rtoate ar^to. made by Dr. C. ?. Hurlhut Jr., of Harvard Talrenitj, haa be*2 r* portrd rtehere iMurihut. C. S.. At.. and U'iiilam*. l*. K.: The ii;neralory of A?t*eatoa Dust. J. lodtx. He*. JL. Toxicol 17:2'*!. l'o.-.'d. t ror the ej-ert Cher ahto an 1 t::e ioac f.` *r a*r* to the ptroyraphic analysis a.supplemented vttb x-ray diffraction xamloation. periods up to 33 months. Some guinea pigs with six and nine months' exposure lived tor an additional three years after cen.-ation of their exposure. A preliminary reportpresented observations after 29 months of exposure. At that time observations covered a period of only 2Ys years and the conclusions as to the ultimate eilrcts of inhaled asbestos dust were urovisionai. Those conclusions are substantiated by results of the completed study, which is reported as follows. Composition anil Atmospheric Concentration of the Dust.--The dusting material, a commercial variety of asbestos known as King's floats, was composed of short fibers, rancinjr in length from 1 mm. to 1 micron or les*. and of particles which also varied in size. It was ofciained from die Tliettord. Quebec, piant of the Asbestos Corporation of America, and analyses (tables 2 and 3) reveal that the amount of fibrous chrysotile was only 14 per cent, a rather low value. 8 INDUSTRIAL HYGIENE AND OCCUPATIONAL MEDICINE Impinger samples taken soon after the experiment was started indicated that the dust concentration was at first quite low, the average dust ruunt being only 6.0 million particles per cubic toot of air b;. >' .- standard light field technic and 0.8 million for particles and fibers greater than 10 microns. After the inhalation experiment had been under way for about two years, the speed of the rotating paddle in the dusting machine was increased and for the remaining 10 months of the ex|>eriment considerably more dust was dispersed into the atmosphere. The average dust count of impinger samples collected alter this chance was 53.7 million by the usual light field method and 1.6 million for particles and i..>crs larger than 10 microns. It is probable, however, that the true values of the dust concentration were higher than the counts given in this paragraph. The immnger samples for the King's floats experiment were collected in water, but later studies7 have shown that counts of impinger samples of asbestos dust taken in water are not reliable. Ethyl alcohol instead oi water was used as the collecting fluid in all subsequent experi ments. Table 4.--Summary of Inhalation Experiment -with Ring's Floats Asbestos Dust Nature of Experiment Dust exposure continuous itrougb* out Ule Dust exposure followed br pro- louced residence la normal air 'Ttiberrulou* fsfeettoo at start of dust exposure Control** iulrotton: no dust expusure Tul^rrulou* ir?**rf:na ~ no. u( lu>t e'.poM.rc. [!nj :u normal air Cootrois to infection: no dust rx* poaure Animals IA ruic*a p;*s v rabbits 18 rats IS rutnea plr* Zj fuicra pips 1 rabbit i raM-it to guinea pics 22 guinea pi;? tnir.rs p:i> It rulcct pi** Maximum Man- Survival mum \lt. r Dust Ex Di;t >;.r poMjre, P-'-ure. Mo. Mo. !3 0 17 0 60 Ts s !9 6 30 l 19 W 1 Z6 0 0 ut / .Ip', , <" *` c" r * \ ,1 Results ,, r~ Trp'esl p-rlhro-^P^.ijtr Shrols afur )9 tncutr.i >orn;u bo-ly bror.c n:ti Little or no react ion .\'ooprorr*elre Nooproyresstvc nt-ro-i* Absorption of forr.iro t*o<lr reaction Temporary ;>roi:re--ion of infection. !o!.rd br nenurc '.vi'ii ri * :<*. s 11. atm; i > re?ului.>.n toue erccptli.m 13 14 No * In *>t!h ' > tm-rvulou* laf-vtiou; wi-n :: .------- 0 IS t Healinc by resolution `Hie ruinra purs wrrv ta.'fctr-l with low rlrulmce Ri stTiin of tubercle bacillus, t This mesol its* lumral period lotlowiog IclccUon. Results of the investigation, briefly summarized in table 4, show that ir.halaiion of King's fioiis asbestos dust produced a typical peribronchiolar fibrosis.in guinea pigs but not in rabbits or rats. Rccetirn in A'ormdf Guinea Fins.--Guinea pigs inhaling thU dust for ;>criods up to 33 months had a characteristic fibrosis occurring in conical patches about the respiratory bronchioles. During this exposure the peripheral alveoli were not involved. The particulate dements of the dust were transported through the lymphatic system to the bronchial nodes, causing no significant reaction in cither site; the fibrous elements remained fixed at the points of original localization and were seldom detected in the lymph nodes. After exposure 0f approximately a year a small amount of cellular reaction had been produced about many respiratory bronchioles i fig. 3.-1). As more dnst 'was inhaled, it continued to accumulate in the same location, and later stages of the disease (fig. 3 3) consisted of extensions of the original lesions. Apparently, the inhaled fibers were caught in the pocket-like alveoli that are given off from the lateral walls of the respiratory bronchioles. There they ______________ . 7. Fulton, W. B.; H'outz, R. L.; Dooley, A., and Mathews. J. L.: AsbestOMs: I. The Collection ar.d Counting of Asbestos Dust F.ncountererl in Asbestos Fabri cating Plants, Special Bulletin 37. Pennsylvania Department of Labor and Indn.-try, Harrisburg. 1934. ` f j . l L V f were phagoccells. Monor ing of the bre The process fibers steadilv - f. .. : Fig. 3.--Ki months' expos becoming an u wall of the br with 2S rr.onti peribronchial f: epithelium lin: (X 200). distorted the a cells. The re: '* r T - wr.Trc W" v ' VORWALU ET AL.--S1 )IES OF AS8EST0SIS 9 "were phagocytoscd, and many of tliein were carried into the wall hy migratory cells. Mononuclear leukocytes attracted to the area caused an appreciable thicken ing of the broochiolar wall. After 16 months a delicate fibrosis made its appearance. The process evolved gradually, and the number oi t e intercellular collagenous fillers steadily increased. As this fibrous deposit contracted, it partially closed and " .C W'S'-.S . , 'i .'N . w. S.' \ I*SV. r4*''**.*, a. > . A'i- *"t r\ ' .* ^ K fir. S K ry " s-a.' < 'Yi? '' > r- / (]' ** TV - ' 'V V Cv.-VV- . V, ;j X ` < ^^;c.-v.'-: > A-i .>".}-'TS&r*k iy- S : *.v' \i*i;. ; -v&^ . ''W u - tf A iii/' ;V s x v' ''' . X/--.; il ' >* ` ^lsk-->iirCcb'e\, R 'V - . ,, v 'r . . -A L> MW. ___ **_!!{--.-C ' x^- Fic. j.--Kinc's floats inhalation experiment: .-!. lung oi a uuhica ]iig \vi::i 12 months' exposure. It iu.ciujes a respiratory bronchiole, at the i-.it. hrarching and becomiue an alveolar duct, at the right. Note the accumulation oi cells in the wall oi tiie bronchiole and in adjacent alveoli (x 130). 3, lung ot a guinea pig with 23 months' exposure. The field includes a bronchiole, at the center, with peribronchial fibrosis extending into the waiis of adjacent alveoli. Note the cuhoidai epithelium lining these alveoli. This is the so-called "adenomatoid" appearance (x 200). distorted the alveoli, and with this change the alveoli became lined with cuboidal cells. The result was an adenoma-like appearance which frequently accompanies T T-*- - -- --rnT-**ST':<-i.<r VORWALU ET STUDIES OF ASBESTOSIS Asbestosis bodies (fig. ZB), first seen in the lungs of the guinea pigs that had inhaled dust for about two months, became more numerous and more distinctly segmented with increasing exposure. The reaction produced in guinea pigs expose.. for six and nine months did not progress significantly during a subsequent |<riod oi 55 and 37 months when the animals lived in a normal atmosphere (fig. 4). Between eight and 11 mouths after exposure ceased, the cellular reaction in the lung had been completely replaced by thin strands of fibrous tissue. At later periods the scar tissue was less in amount, but in the last animal killed. J7 months alter discontinuing dust e\|>osurc, some fibrosis was still visible. Reaction m Guinea Pigs Infected u-ith Tubercle F.triili at the Onset oi Dust Inhalation.--Of the group of 40 guinea pigs infected with attenuated idivriie bacilli, Rs strain,* at the time that dust exposure was begun. 51 died or were killed before the completion of two years of the exposure and were reported in the paper by Gardner and Cummings.1* Seventeen oi these died from intercurrent pneumonia. Briefly, the results were as follows: Ten revealed some evidence of spread of the tuberculous process (fig. 5 A); in 6 of these it was confined to the lungs, and in the other 4 the abdominal viscera also were involved. Extension of the infection was first seen after seven months vi inhalation : ih:r::u the next 20 months more than half of the animals showed actively spreading tuber culosis. and in 3 of them small cavities had developed. During tnc !a-t right months no animals exhibited any evidence of active infection although in half > i them the heaJed fibrous scars of previous spreads were obvious. The scars wire more extensive than is characteristic of either tuberculosis or a-bcstosis alone. The nine animals which were still alive after two years of dust exposure were killed at intervals during the following year. In four of them the primary foci of infection were healed with fibrosis and even calcification, and there was no evidence of progression (fig. SB). In the remaining five the tuberculous foci showed evidence of having previously spread locally ; in four of them, by the of autopsy, the foci were healed, with excessive fibrosis: in the fifth anna: there was a generalized chronic tuberculous pneumonia in one lobe, and ;:i :: r lobes there were isolated primary tubercles, which were still active but had not spread. Reaction in Guinea Pigs Infected zeith Tubercle Bacilli After Establishment . i Asbcstosis.--Twelve guinea pigs, after inhaling King's floats asbestos dust ior 26 months, were infected with tubercle bacilli and then removed to normal air. Six of these animals died within seven weeks, five from intercurren: nontuhereufjl-s infection. The remaining six animals were killed at intervals up to 14 months after infection. The suhplcuml tubercles were no more remcr u< in the dotted animals than in the nondusted controls, but a considerable number were iour.d in the depths of the lung about foci oi asbestosis. The tuberculous component of the combined reaction showed only slight local extension about lesions in the lungs and tracheobronchial lymph nodes. Caseation was found in tubercles l'j months old. but by S1,* months it had completely disappeared, leaving only scar tissue. Foci of fibrosis still persisted in the last animal, which was killed 14 months after infection. Reaction in Rab!-its.--Rabbits exposed to the asbestos dust fur periods up to 19 months showed a foreign body type oi reaction of low grade, hut no fibrosis Although their lungs contained particulate elements of the dust, fibers were not present, indicating that the upper respiratory mechanism of the rabbit is adequate to exclude fibrous foreign bodies. Two rabbits, alter inhaling dust for sue and 199 9. Steenken. \Y,, Jr, and Gardner, L. U.: Ri Strain of Tubercle Bacillus: Its Dissociation and Virulence of Variants in Normal and Silicotic Guinea Pigs. Am. Rev. Tuberc. 54:51. 1946. ---- 12 IftDUSTFI.lL H Mumcoccupatiox.il months, lived in normal air for more than two years. At autopsy neither animal showed any evidence of cellular reaction or fibrosis in the terminal bronchioles, nor were there any asbestosis bodies. Reaction in White Fats--All the rat* had acquired an infection, resulting in the formation of pulmonary abscesses, before they came to autopsy. Apparently, so much heavy mucus obstructed their bronchi that very few fibers could have entered Fig. 5.-- King's float inhalation experiment: A. lure of guinea pig infected with Ki tubercle baciiii and then exposed to dust tor 2- months. A bronchiole is shown just show center. Surrountimg i: is some collagen deposition, tocether with typicaL cpitheii.i cell infiltration of the wail. Note me lack of encapsulation and the peripheral epithelioid ceil pneumonia, which illiterate a spreading tuber culous process tx2i.O). B, lung of a guinea pig infected with Ri tubercle bacilli and then exposed to dust for 55 months. Note the subplcural distinctly encap'uiatcd caseous focus, the calcification at the right border of the lesion and the an-rnce oi ceils in adjacent alveoli, all of which illustrate a healing tuberculous process f x 200). their h:rg but there cessfuL ; Sutfi Floats f. be surnrr <r 1. E: floats dt pigs but in exter.: 2. E: pigs ir.ft dust TOO ment of infection wit!', ;u; active tb exposed continue On the c oxide dt infectedyears' as only mo beinjj rt Tn vi response monia, i: asbestos experime Since fibrosis v ex pe rime r for asbei consisting would in; reaction : tained fit particula; Compos for this ex fabricating10 10. Vor D. A.: Sid Indust. Me VORWALD ET STUDIES Of ASBESTOSIS their hmgs. In a few of the rats, an occasional asbestosis body was discovered, but there was no fibrosis. This phase of the experiment was considered unsuc cessful. Summary and Interpretation of Inhalation Experiment with King's Floats Dust.--The findings in the experiment with King's lluats dust can be summarized under two headings: 1. Effect of the in haled... dust on normal animals. The King's floats dust caused a characteristic peribronchiolar fibrosis in guinea pigs but not in rabbits or rats. The fibrosis did not increase significantly in extent after the dust exposure was discontinued. 2. Effect of the inhaled dust on tuberculosis in guinea pigs. In guinea pigs infected with attenuated tubercle bacilli and then placed in the dust room, the results were more variable than is usual in an experi ment of this type. A few animals showed no sign of progression of the infection; in most of them there was evidence of temporary progression with subsequent heaiing; in one animal the tuberculous process remained active to death. In contrast,- when guinea pigs after being infected are exposed to quartz dust instead of asbestos dust, the infectious process continues to progress and eventually causes the death of the animals. On the other liand. infected animals exposed to a harmless dust like iron oxide do not show any progression of the infection.10 Guinea pigs infected with attenuated tubercle bacilli after the termination of two years' asbestos dust exposure did not show progressive disease. The only modification of the infection was in its localization, a few bacilli being retained in the peribronchiolar fibrous tissue, with tubercles forming there in addition to the usual tubercles beneath the piettra. In view of the variability < : the results, the unusual nature of the response and the high pro\xmton of. deaths due to intercurrcnt pneu monia, it is felt that only tentative conclusions as to the influence of asbestos dust on the course of tuberculous infection are justified by this experiment. Shoct F:se* Asbestos Dust Since hazardous dusts like quartz are most effective in producing fibrosis when the particles are 3 microns and less in size, an inhalation experiment was performed to determine whether this condition is true for asbestos dust. It was thought that a snort fiber asbestos dust consisting almost entirely of fibers and particles smaller than 3 microns would initiate an accelerated tissue respon-e and produce an advanced reaction in a shorter time titan did the King's floats dust, which con tained fibers from 1 mm. to 1 micron and less in length as well as much particulate matter. Composition and Atmospheric Concentration of ltie Dust.--The ducting material for experiment was the remains of fibers collected in dust bins ni an asbestosfabricaring plant alter a carding operation and screened to pass 200 mesh. Since10 10. Vorwald. A. J.: Pratt, P. C.: Durlean, T. M.; Delahant. A. B.. and Bailey, D. A.: Sidcrosis: A Bcnicn Pneumoconiosis Due to the Inhalation of Iron Dust. Indust. Med. & Surg. 19:170, 1950. r^c14 IXDUSTRIAT HYG1ESE AXD OCCUPATIOSAL UEVICIS'E the material as received contained many long fibers, it was ground in a jteel ball mill to reduce practically all l!-.e particles to 3 microns or less in size. When tiled alone in the standard dusti:.'.' machine, this finely ground asbestos tended ti. pack in th.e hopper, and it became necessary to mix one volume nf the ungmund I marerial with three volumes ot the ground to generate a satisfactory dust cloud, ? I (t fs pertinent to mention here that the addition of the small quantity of unground i 4 asbestos was unfortunate, because it confused the interpretation of results. The composition of the short liber asbestos as received is disclosed by the I * chemical and petrographic analyses given in tables 2 and 3. Samples taken before 4 and after grinding yielded about the same values on analysis, indicating that there was r>o contamination from the miil or loss ot water content. The dust concentration varied during the experiment, the light fh.d counts for atmospheric samples collected inside the animal cages with the impinger apparatus i 1 ranging from dj million to ldJ nuilion. The average of counts was 130 million for iJ the first year of the experiment, 134 million for the second year and 140 million it ] (or the third year. :l Siie-frequency measurements of air-floated dust from inside the cages at a magnification of 1.300 X revealed a great preponderance of tire particles, nearly ,j Tam r S.--Summary of Inhalation Experiment with Short Fiber .Asbestos Dint Satan ot Zxptrimrst Animali Maximum Maxi- iurviral in Itfr Dual fcx Du.u Ex posure, posure. Mo. Mo. HnulU Dost expofun eoattsuous through- 46 cxuoea plra 24 o. Hale oi reaction about ibe Mm** tn rl out lit* tn*nt with Ki te * Hoars asf*-s'o but rvfct .Of lOTOlT^rr. :;: srfy inucu ie** * .1 T3 nil C 0 Cbaracterrtv later*** of >-ribroncnlolor Qhroto a-i- IS rata ? rabbits >4 47 * 0 Subp>ura: r<*sct: *"- oaiy 0 So ~.-ji mirro^or-ir of a.\-'v sr 1 ' spo,,.r? kfr.::*.* ef>r w :n Dust rrporarf foDowed by proloared U ruioea plaf 14 afr rrniocal from *fi;M .foui.i reuUtoce la aormal air iul-- r.eltftfr rl* *rlr eataDlMiM Dor UcrtniMy :i exclu-ifl S cats tl l Sas.e as for rort:auous e^po*ure 1 rabolt e Similar to rontihuou? exposure: evuicac'- of -i aiii'bi refTfMioa !i Atter S3 eioclhs the snlmil* w<re exposed to ICO per eot hill r-illel i>heto. * The rvietiac ru prot iMr due to iuoc flhers ip the aocrounJ icxtfrial epirh * mixed -rith the rroued i'Pestos du>: ji to produce a aaujlactory dust cloud. 90 per cent of the particles seen being smaller than 3 microns. It was estimated j- that approximately 1 per cent oi the dust was in th.e iorm of fibers greater than A 10 microns in length. `1 Four species of animals--guinea pigs, white rats, cats and rabbits--were used in this experiment. The results oi the dust exposure, summarixed . in table 5, are presented in greater detail below. >. hu Reaction in Guinea Pigs.--Eighty guinea pigs were originaiiy planed in the dust room, but 3! oi them were later eliminated from the experiment and killed because of enlargement of the cervical lymph nodes thought to be due to iniercurrent i infection of the upper respiratory tract. Of the u'.her 59 animals. 46 remained in il- the dust room until they were killed or died at periods up to 34 months, and 13 A . animals were transferred to normal air after being exposed to the dust tor 20 months'. The type of tissue reaction provoked by the inhaled short fiber asbestos was essentially the same a; that already observed in the experiment with King's floats 1 asbestos. The rate of reaction aiso was approximately the same, but the extent of involvement was very' much less. After 16 to 24 months of exposure only a very few small foci of reaction, which generally required microscopic examination for detection, had been produced in the guinea pigs. b appre 16 rr. brohe cell's, foreif prom: or "a. ing th of th devch lagen DiffU: Ta! Eip- Du. * 1?' thr r*la ext+r.n* monarv asbestoi of the experiin been tn reaction original In th sion of disprove reaction, the lerip to Tana chemical it: lungs I'ORWALD ET AL.--ST^JIES OP ASBESTOSIS 15 Only after exposures had continued for approximately one year was there an appreciable tendency for dust-containing phagocytes to gather into clumps. By 16 months phagocytes had collected about the walls of a few of the rv.-piratorv bronchioles which revealed a little proliferation or infiltration of nn.unntii lenr cells. There were also some nuiitinucleated cells. 1 . they were of the inert, foreign body type. At 20 to 2-1 months the cellular clumps were <<<nietinies quite prominent, and sometimes chances in the epithelium resulted in the admnma-like or "adenomatoid'' appearance (fig. 3 B) previously described in the seciimi review ing the experiment with the King's floats dust. In most of the subsequent members of the series the reaction remained cellular, but a iew exhibited p-oiiuiinred development of fibrous tissue. In these few memlicrs of the series the col lagen was pale in color and tenuous, with no ap|>earancc of being hynlini/ed. Diffuse chronic pleurisy was present in a iew animals without evidence ..t pul- TaBLE 6.--Analyses of Lunas of Guinea Pigs After Prolonged Inhalation of Short Fiber Asbestos Dust Expomrr 10 Dust. Mo. Period In Normal Air. Mo. Amount of Aah.of l>rtrii l.unff Total SlUs. % of DrW'd Lun* Total mo?. % of A*I| Du 't Exi'f*urc Coat.nuou* I'ur i:;k* Lift- Ti.ue R> arltOQ * | 50? 0.51 10.:i If ft 1 4* 0.46 10.06 l 5.16 0-54 20.54 X 15 | $ 00 0 1 4.1-3 049 0.43 0-53 9. & nr> lu 00 so 0 ( 5.SC t 61: o.ss o.vo 14 40 it.or r X4 0 j J.)f j 5.SO 0.78 0.78 14. SO 3 + . An 0 !j j-# Si 0.96 u: 17 9 19.40 4* 34 A9 1 r.-. . ( 9.3i 0.73 QJj6 !*.37 15.11 4 Dui Exposure Folio vu-.t hy Prolonged H* *i Jenff 12 Normal Air 4 j i:s 1 ;u r 4$ o>. 'j.ZO 10 i c: * 1 :> 0.54 5.51 3 4* I t.n 0.23 9 11 j s.is o.zc U-C JJl ro 4.00 2 4- * Th* arrrir'r.r th* m*tloo Ln earn rrotif. of gemra *?r r*pr***r.t tlv rHalu* Arrrpr of rfii'>n. farm* from -- i q*jp*t!oraf-n i to a- mm* naviMur. :>v npfrlfnenn. TNe rWarloQftip.* appir only vitmo tni toMe and raccoi ttmipifM 7mtoi? la other titles. T * monarv infection. This suggests that pleurisy rr.av he a neeifie concomitant of asbestosis. but the evidence is not adequate to eslabh-h this ;jir.t. The reaction of the tracheobronchial lymph nodes was more pronounced than in trie previous experiment with King's floats asbestos, probab'.v because more fine particles had been transported to the nodes in animals iniialirg short fiber asbestos. The nodal reaction was essentially an increase in reticuium. rather than a fibrosis, with the original cells being preserved between the thickened reticular fibers. In the group removed to normal air after 20 months' inhalation of du't. progres sion of disease was not definitely demonstrated, but neither could it be absolutely disproved, owing to the variability r.f the response in different animals. The reactions, from mild to severe, occurred sporadically ami bore no relationship to the length of time after cessation of exposure. The diiTereiices were attributed to variation in individual susceptibility. This view received support from the chemical analyses ftable 6). which revealed comparable amounts oi ash and silica in hmgs with widely different amounts of tissue change. For example, the ash 16 INDUSTRIAL\ YGIEXE AND OCCUPATIONAL MU NE and silica values were quite similar for three animals living' in dust 20 months and then in normal air ior 14 months, yet the tissue reaction was severe in one animal, mild in another and only doubtful in die third. The formation of a'hcslosis bodies was at first extremely limited in Ixjtli groups. After five months' exposure only a very rare short body could be found, usually inside a ceil. Some of the finest intracellular particles were surrnumlcd by yellowdeposits having the same color as the asbestosis body. One year's exposure had per mitted an accumulation of many longer fibers, a number of which were coated and seen as typical asbestosis bodies. Most of these were soil short enough to be partially or ortirely within phagocytic cells. Ry the twentieth month and thereafter they Table 7.--Analyses of Lunas of White Rats That Had Inhaled Short Fiber Asbestos Dust Ztorattoa of hxpoUR. SlO. 0* 4 Amt. of .\*h. of Print Ludc 19 43 2.9 ZM 3.3 30 3.4 '3.5 3.6 2 :j 13.2 3.3 3.9 1 ** Total SIO*. *9 of Pried Lung 0.00 0.00 H.ftl U.00 o.no 0.00 0.00 0.06 o.n 0.C9 Q.'je 0.08 0.11 0.07 Total M<>. `v Ot ASO 0.0 0.0 4' . 0.0 0.0 00 0.0 3.1 .3 5 a.i 1-5 2.3 3.0 u XormaJ control* too dust exposure). Purailoa of hu*lure. Mo. 6 Amt. of A*h. <?o < f Prl-.l Lung S.3 J.4 a.: 3.0 36 9 5.6 4.4 l 3.: f 4.3 10 46 1 5.3 U.7 Total 'r ot Prl. -( Luou 0 07 0.03 U.M* 0.06 A 0.15 o i; 0.3 5 A I** 0.;: 0.15 0.12 Total sit >1. of A-h 2.3 1-5 M 3.4 4.0 42 3a i3 !J i.3 Tabu 8.--Average Values of Ash and Total Siliea for Lungs oj White Rots Inhaling Various Dusts for I'ari.us Periods fl.t:n-~s Only. it Included nigh 'A-desl Sat ot Ash. **. or Dried Lust Total SlOt, `Sc ot Dried Lone TOtal SiM;, % ot Asrt Dura- -----------------------. ------------------------ *-------------------- coo Short of Ex- Fiber pocure, A-?L*eJ- Mo. 104 Quarts GypsumFerru- Quarts tmous Mix CtXTt ture Short >ii-*r tos Quartz iii)P`i:n- snort Frrru- Quartz > !>*r ri-ous Mix A9t-- Chert ture tos Quartz ''lyi-uiri. Ferru Qnnriz ginous M.XChert ture t U 4J 5.9 U> 0.00 0*51 oja O.W 2.8 11.7 2.9 f, 4 Li u .9 !J o.eo 0-51 0.72 0.07 2.5 UA 3.6 2.0 U> 7-1 9-9 3.4 0.06 2.94 3.43 o.n L6 41.5 34.4 '..4 8 3J U 9.0 3.6 0.13 1.44 2.40 o.r 3.9 233 J. 1 10 iS To 14.1 4.1 0.15 4.40 8.C0 o.cs a iO 0 43.2 8.7 were relatively numerous although still rare in comparison with the findings in the King's floats experiment. Reaction in (Vhite Rats.--Seventy-three white nits were exposed to atmospheric short fiber asbestos dust for periods up to 32 norths. During the first 10 months animals were killed bimonthly and for the remainder of the experiment at less frequent intervals. Up to eight months the dust cells were widely scattered and existed m foci oniv sporadically. Reaction was limited to occasional slight thicken ing of the septums about smail accumulations of dust cells. At 10 months there was a suggestion oi early fibrosis in a few rats, but the change was so slight that it would probably have been overlooked without the clump of dust cells which attracted attention to the area. Only 10 animals were exposed ior from 12 to 32 months. In each of them the lungs contained minute foci of well defined fibrosis distributed like that oi asbestosis but without ashestosis bodies. The lesions, visible only at a magnification of 1~0 diameters or mure, consisted of patches along f a/veoja to SWC prepar. was a : charac filled: v naked asbestc pact diffuse the ma Res the avi values are lov quart! of dust tion of of thg Since;t of that dissolvi basis o: Rea the she pnt to The at or.e o: ti'.e t:Ys U'dll) U t il t the roe after j-' be dete* microsf smaJi yellow . expo;-: p- from o: room a enough pleurisy one an: examirt died of on grd; this auii focaJ fi: which v tended i was nr. lung w: early ir the dus: YORIVALD ET AL.- VDIES or ASBESTOSIS 17 alveolar ducts in which.tlic walls ofthc associated air spaces were very thick, owing to swollen collagen framework. Connective tissue and Fi>t-Biclscln>w>kv silver preparations revealed complete loss ot capillary Ud I--cally. Outside the collagen was a thin layer id epithelial cell'. This did not rvse-ddc the ''adenomatoid' change characteristic of guinea pig asbestosis. Near the le-iuns the air spaces were filled with phagocytes containing gray to yellow particuiatc dust and a rare, long, naked asbestos fiber. Careful search failed to reveal even a suggestion of an asbestosis body. Pleurisy was absent. The tracheobronchial nodes showed com pact focal collections of monocytic cells at 12 months and, at 2d months, some diffuse thickening oi the reticulum. In a few rats there was definite fibrosis along the margins of the node, extending into the mediastinal areolar tissue. Results of chemical analyses made on the while rats are given in table 7. and the average values have been recorded in table S fur cuinparison with similar values for rats inhaling other dusts. It will be noted :hat the values f<.r asbestos are lower than tlwse for quartz or chert but approximate those for the gypsumquartz mixture, in which atmospheric agglutination tended to reduce the amount of dust inhaled. This condition ; revailed even though the atmospheric concentra tion of asbestos dust was essentially the <ame as that of thr nnartz. was im-li.-ili dint of the gypstim-ijuartz mixture and \\a> ouc-hitli that of tiie ferruginous eiiert. Since the values tor asbestos are low, it might be inferred that the total quantity of that dust actually inhaled was mall or that it had been eliminated frr.ni or dissolved within the lungs. Evaluation of these piiihil:'ties is not feasible on the basis of the observations derived frum this study. Reaction in Cats.--Twenty cats were used in this inlialation experiment with the short fiber asbestos. Eighteen were kept in the dust room continuously until put to death, the exposure period ranging fmm one month to nearly 54 months. The other two were removed to normal air after a dust exposure of 31 months ; one of these was killed five months, and the other 24 months, later. In general, the tissue response was confined to microscopic foci of fibrosis, which were in the walls of groups oi suhplcural alveoli rather than in the j>crihroncliiol:ir areas. In one animal the chanre was extensive enough to he si'uahzcd "n gr-- ix-picti'Vi of the section. Only in t'nc animal with the longest exposure--i4 months--did the roentgenogram reveal definitely ahnormal shadows. A roentgcn'-cram marie alter 3D months revealed no abnormality; after 45 months, a taint mottling couid be detected throughout both lungs. At autopsy, nine months later, there wn> only microscopic fibrosis in the subplcural zone plus heavy lymphocytic infiltration about small bronchioles. Asbestosis bodies were rare. On prolonged search a fewyellow atypical bodies, smooth and without frustrations, were found in two animals exposed for more than a year. Reaction in R.ihhils.-- Eight rabbits were exposed to d;;?t for periods extending from one to more than five years; the last animal wa removed from tiie dust room and left in normal air six montits before being killed. There was never enough pulmonary fibrosis to be detected grossly, and there was no chronic adhesive pleurisy. Microscopic evidence of alveolar wall thickening was first detected in one animal after about three years of exposure and was seen in all live animals examined thereafter, including the one removed to normal air. Otic ~-;r-.al that died of. paralysis after nearly tour years of exposure exhibited a react, n visible on gross ins|>eetion of tissue sections. The possibility of pulmonary infection in this animal could not be excluded. In another animal dying two years later the focal fibrosis was not nearly as obvious or as advanced. Areas of involvement, which were largely visualized because of phagocytic reaction within the air spaces, tended microscopically to*become more fibrous with the passage oi time, but there was never much encroachment on the lumen of air spaci and the structure of the lung was preservetL Asbestosis bodies were not detected in rabbits that died early in the experiment but were seen in all animals that had been exposed to the dust for more tlian three years. 18 ixdustri.il 71EXE A XP OCCVTATIOXAL MED! ft Summary ar.d Interfrelation of Inhalation Experiment :.-ith Short Fiber Asbestos Dust.--The original purpose of the experiment was to evaluate the role of short asbestos fibers in the genesis of asbestosis. It was felt also that if the tissues reacted umre rapidly and more extensively to short fiber asbestos than to King's floats there would be a basis for believing that the action of asbestos is in part, at least, a chemical one as postulated for quartz. This experiment, in which the tissue reaction was slower and less extensive than that in the previous experiment with King's floats dust, indicates that the capacity of inhaled asbestos filers to produce fibrosis is determined primarily by factors not chemical in nature. Of the four species exposed in this experiment, only the guinea pig and to a lesser extent the white rat responded with characteristic peri bronchiolar fibrosis. The cat reacted with atypical suhpleural fibrosis 11 and the rabbit with only slight parenchymal fibrosis. 1 Ball-Milled A>b*>tos Dlst In the inhalation experiment with short filler asbestos dust a small quantity of onground short fiber asbestos was mixed with the ballmilled product in order to generate a suitable dust cloud. When that experiment failed to produce an accelerated tissue reaction, in com parison with the response initiated by King's floats, it became apparent that the biologic activity of asbestos is not increased by a reduction oi fiber size. Thus the possibility arose that the tissue reaction observed was due solely to the reiativeiv few lo-.g fibers of the unground asbestos and that the short fiber- of asbestos had no more titan a very in-ignificant role in the production of asbestosis. a concept not in accord with . previous experiments concerning pneumonoconiosis. Consequently another inhalation experiment was started in which onlv ball-milled asbestos was used. Composition and Atmospheric Concentration oj the Dust.--The dusting material was the ball-milled, short fiber asbestos used in the previous inhalation experiment, but unground material was not mixed with it. Owing to the tendency of the material t form small sohcrules which prcvmtrl much of the fihrnu- ; onion from floating out of the dusting machine, tiie dispersal oi the dust was not entirely satisfactory. Therefore, after an initial seven months of operation, steel wire brushes were attached to the inside surface of the hopper and to the rotating paddle to disintegrate the spherules and release the fibers. This arrangement gave satis H factory results and wtu used for the remaining 21 months of the experiment. 'ri - The composition of the raw asbestos used is shown in .tallies 2 and 2. Pctru- , graphic and x-ray diffraction examination o; atmospheric dust, collected in the dust room with an electrostatic precipitator after the installation of wire brushes. 1 ' f indicated that about 15 per cent of the air-suspended material was chrysotile. and_ jJ-> about 60 per cent, serpentine: of the balance, magnetite comprised 10 per cent. ^ " brucite 3 per cent, quartz 2 per cent and other minerals 10 per cent. Earing the Vi seven month period before the wire brushes were used, the chrysotile content of the atmospheric dust was somewhat lower than 15 per cent, but reliable values were not obtained. The dust concentration during the first seven months of the experiment was about 1O0 million particles per cubic foot oi air. After the wire brushes were installed 21 morn Size- revealcid classificc to one :h of long value of Guine per cent React As the c due to pr killed at air after reaction minute a: the tisstin Table Xrc Expil Dust espc tiruort cut Ute. Dust e.-rr'- low-.; t* lo: g..q r In aorta. could be s- be seen * accumulati within cei months ar described living eigFr of four art adenomatoi The ttl continued f 16 months but practic of exposur months the: changes oo any oi the Minute after expos elapsed. T 20 months " * T" ii.iy mvTm:r VORWALD ET AL.-J^JDIES Of ASDESTOSIS 19 installed, the dust counts were higher, and the over-all average for the remaining 21 months was about 150 million. Size-frequency studies oi atmospheric dust collected inidc the animal cages revealed that nearly 99 per cent ot the components suspended in the air could be classified as clumps or particles; only about 1 to 1.5 per cent wa% fibers. One third to one half of the fibers were longer than 10 microns, indicating a concentration of long fibers of about 0.8 million. This figure is about one-half the estimated value of 1.4 million for the short fiber experiment. Guinea pigs, rats and mice were used in the inhalation experiment with the 100 per cent ball-milled asbestos dust. The results are summarized in table 9. Reaction in Guinea Pins.--The experiment was started with It>J guinea pics. As the dust exposure proceeded, there were 39 accidental death'. 32 of'these luting due to pneumonia in an epidemic. The 61 pigs remaining exposed to the dust were killed at intervals during exposure, except for 16 guinea pigs transferred to normal air after 28 months of dusting. For the first year of exposure practically the only reaction to the dust was the presence of scattered phagocytes and an occasional minute asbestosis body. At 16 and 20 months no gross response was visible on the tissue section, but microscopically peribronchiolar loci of iuilaiuiuatory cells Table 9.--5mmory of Inhalation Experiment with 100 per Cent Ball-Milled Asbestos Dust Niturf of Experiment Dust txpo'ure coo* tmiiTj tbroufo* oat Ute Dust (ol* lovd by pro- lonprd rr>:rV-r* in uortr.ai bit 54 ralaei pus 40 rats U mice 16 c^iQe* pixs Maximum Maxi Survival mum After Dust Dust Expo Expo sure, sure. Mo. Mo. U 0' no' 0 i: 0 a 11 V...' t/ Ke Results No appreciable pulmonary reartloo No suRge.-tion of a?c*'tcis No susrc'-.-iion of Ss0*--K'`:s Fibrosis typical o( i?!>ctois a* prefer.? :2 m'v f.l'.-t *ro;'.r* i in an mvivr.: :rr.: vi*;U-: er.'.ai'.er could L* seen rr.;cro*opicallf at 1 mo. and S mo. alter terrsma* Con ol zxpoiure could be seen. At 24 months (fig. 6.-1) there was still no change large enough to be seen with a hand lens, although microscopic examination revealed cellular accumulations about terminal bronchioles and many more asbestosis bodies, chiefly within cells. The lungs of animals exposed for the full dusting period of 23 months and afterward living in normal air tor two months revealed the changes described above and also very slight peribronchiolar fibrosis. For exposed animais living eight months in normal air the findings were similar, hm is H months three of four animals showed grossly visible characteristic peribronchiolar fibrosis with anenomatoid change (fig. 6 5). The tracheobronchial nodes were essentially normal until exposure had been continued for more than a year and a haif. Animals killed at 12 months and at 16 months revealed a few minute collections of phagocytes containing particles but practically no fibers large enough to be reenpnired as such. After 20 months of exposure many monocytes filled with yellow granules were present. At 30 months there had been a slight increase in reticuium but no fibrosis. No further changes occurred in the nodes. Asbestosis bodies were not seen in the nodes of any of the guinea pigs. Minute asbestosis bodies were observed in the lungs as early as three months after exposure began, but they did not become numerous until 16 months had elapsed. The bodies were short and practically all were intracellular, although at 20 months some were long enough to project beyond tiie cell borders. It is 20 ISDUSTRIAL IESE ASD OCCURA TIOSAL MEDI important to note tint in the later months of exposure there was a distinct increase in the number oi Ions; fibers, up to 70 microns in length, in the lungs with the formation of characteristic long asbestosis Julies. Chemical analyses (table 10) of the lungs revealed that considerable dust had been retained in the lungs. After 24 months of continuous exposure the average In vie|w 100 per ct-t was much asbestos in of asbesto's Reactin' for periods species dnl plugocywoi free in air asbotusis 1 small, n ,::h Table 10i- xpot:r* Lust. M< i ! ! 3* r i 3 .: f 3: 14 1 u tS a a r decree c*. rvir Mpenmeat). , t nut)ola in ot. \ i Summa Cent Ball-. Fig. 6.--Ball-milled asbestos inhalation experiment: lung ni a g-.iir.ca pig I experiment short fiber!, with 24 mc-Pths' dust exposure. A bronciuoie i* shown at the cirtcr. with, a slight accumulation of nhngucytic ceils but without ti;.- f<>rmat:ost oi ccilacen (X 2;). B, lung of a guinea pig with 23 months' du-t exposure and then 12 months' extensive e were fewer inhalation of menial air. The reaction is much hisc that shown in A. tut there is r experiment, a slight deposition of coiiagen, most apparent at the-lei: (X 200). t summary o value for total silica, per cent of ash, was 22.37. This should be contra.-ted with i i primarily c: the average value oi 14.24 (tabie 6) for animals exposed 24 months to the short tion in size fiber asbestos dust. asbestos in! w VORIVALD ET AL.--ST^^ES OP ASBEST0S1S 21 In vit of the high, values for silica obtained with the animals expciv. ! to 100 per cent ball-milled dust, it is imi>ortant to note that their pulmonary icsponse was much less than that of animals exposed for 24 mnrtitj to the short fiber asbestos in the'previous-experiment. This again indicates that the biologic activity of asbestos inhaled into tlie lung is not increased by a reduction in site oi the tibers. Reaction in ll'hile Rati and Mice.--In this experiment 40 rats were exposed for periods up to 2i> months and 24 mice for periods up to 12 months. In neither species did even a suggestion of a.'bcstosis develop, and reaction was limited to phagocytosis oi inhaled particles by widely scattered dust cells which remained free in air spaces or were transported to the tracheobronchial lymph nodes. No asbestosis bodies were found in the rats, but in the mice there were a very tew small, nunhaustrated forms within phagocytes. Taale 10.--Analyses of Lunas of Guinea Pins Exposed to Dust in Inhalation Experiment teith luO per Cent Ball-Milled Asbestos Ihist Zxpomv to Pun. J<o. Period to Normal Air, ilo. Amt. of aid. To of Dried Lupc Total SIO., Si at Dried Luac Total SIO;, of Ah T1ue Reaction I>uil Exposure Cnnilouou* During Life 4.5ft 0.31 4.3 0 4.JO / 0.30 7.06 4.3ft 0.24 6.00 0 4.66 0.3 4.90 t0 0.34 :.*o 5. lift 0.61 12.ul 0 5.60 0.70 12.47 3 0 ft.o; 0.K 6.35 5.74 0.56 ft 5.10 0.38 7J1 0 4.OS - 0.15 7.47 5.02 0J0 7.72 0 8 0 4.35 0.52 11.36 0 s.u 1.3 22.16 0ii c.:i 1.4ft 3.13 0 0Ifl 0 5.40 5.01 1.02 le.W Ml 2: r-3 0 3. Gft s..v 1.2$ 1.3 Ct 6J U 6.30 5.ii 135 20.06 1.21 21.70 * Dual Expoture followed by Prolonged Residence la Normal Air . , i ii t-a e.o7 si; S.98 6.3* 5.1T u: 2.19 0.63 O.ffT O.M 0.64 21.63 25.21 <? oo 14.5ft 13.08 12.41 + + 2+ TT^e rr-.V.N averse::.r reaction in each group rrpreont uteriir th- rlativ* decree of pe*ctmn. ranr;ng from ( in ~ fi]u^'f:opai>i 10 2- lhe n:a*;:r.uin ot-^rr^l is tr.r* experiment). The r!stioajbip* apply only within this table acd ear.sor be compar.-d with tymbols jc otLer tables. Summary and Interpretation of Inhalation Experiment zeith 100 per Cent Ball-Milled Asbestos Dust.--The tissue reaction observed in this experiment was not as intense as that in the previous investigation with short fiber asbestos. The reaction v.as slower in development and less extensive even though more dust accumulated in the lungs. Since there were fewcT fibers longer than 3 microns in the material used in this experiment, the results tend to confirm the interpretation made in the summary of the previous short fiber experiment that the reaction is not primarily chemicai in nature, and to support the impression that reduc tion in size of asbestos fibers does not increase the biologic activity of asbestos inhaled into the lung. 22 ISDUSTRI f!HYCIESE AXD OCCL'PATJOS'AL WDICIXE The finding of long asbestosis bodies in animals that had inhaled the ball-milled material is an example of the difficulty of completely eliminat ing .long fibers from a large volume oi asbestos as required tor an inhalation experiment. ^uln regard to the progression of the tissue reaction after the animals ,| ^ had been removed from die dust, observed in this experiment but not in I, the others, the following interpretation is ottered: W hen the reaction is well developed at the termination of exposure, the contraction of the fibrous tissue obscures ariv procres.-ion that may have occurred: in this ' U experiment, however, since the reaction observed was less mature, its subsequent progress was more readily apparent. I :I L Dong Fiber Asbestos i st . f Since inhalation of short fiber and of 100 j>er cent ball-milled asbestos dust did not result in acceleration of t!m tissue reaction in comparison with that produced by King's floats, the hypothesis that short fibers of asbestos were of minor importance in the etiology of asbestosis was given added support, and attention was directed to the view that the long fibers were of primary significance in that etiology. The King'- floats asbestos used in the first iniiaiation experiment had a rather low content of fibrous chrvsotile and contained considerable scTjseriune_aiKl : I other impurities. - Thereiore. it was decided to conduct a new iniiaiation t t: experiment with a purer form of chrysotile which wot; 1 he richer in h ::g fibers. Covtfpsili-Jn and Atmi-srhrric l'ci.vufM.'i"ii the I>:i<t.--The duslim: material empio)ed in this iiwestica::>>n wa> sr->m an a-bot-n fabricating -lam. Samples of several varieties oi long ni/cr asbestos duit were lira! submitted to the Saranac Laboratory for examination, and one of these. which ias low in magnetite /I (l and chromite and had a fibrous content estimated to be about per cent, iva. selected as most suitable. Steel wire brushes, fastened to the inside surface of the hopper and to the rotating paddle as in the preceding inhalation experiment, were used to open up the bundles oi aslestus ami liherate more fibers into the atmosphere. The composition of the lone filer a`bestos used is indicated by the chemical and petrographic analyses given in tables 2 and 3. Analysis oi air-su.-pended II material from the dust room disclosed that about t*i per cent of the long fiber dust was chrysotile and about 20 per cent serpentine: as already noted, the composition : of a similar air-floated sample oi haii-milled. short fiber dust was 13 per cent chrysoliie and 60 per cent serpentine. The dust concentration as revealed by itnpincer samples taken inside the animal cages was much lower than the concentration tor the experiments with short fiber or bail-milled dust. For the first year of the experiment with long fiber a.-bestos the average of the light field counts was 32 million panicles per cubic foot of air: for the second year, 4d miiiicn ; for'the third year, 39 million, and for the fourth year, 43 miiiiuii. - The sire-frequency of atmospheric samples of the long fiber asbestos dust and of the bail-milled dust is shown in table 11. Both samples were collected with the electrostatic precipitator. It will be noted that there was far more fibrous material in the long fiber dust. Guinea pigs. cats, rats and nticc were employed in this iniiaiation experiment. The results, summarized in tahie 12, are described in greater detail below. *" m a. -S-^SW "l --l K--SITS- ... > -rr Aft arcrep: exa con 121 and 'as; SCO; in : Tae Tab Du' PtJP ic K> f cons local fuse,' the L ment mg o but i inhaL In air. th effect, size c 14 m* the er dust r the t'c visible VORWALD ET Ai tidies nr asbestosis 23 Reaction in Guinea Pigs.--The experiment was started with 100 guinea pigs. After exposure had been carried on for a year, a severe epidemic of pneumonia arose in the dust Piom and about one third of the animals died or were killed. To replace them. 38 more guinea pigs were added to !. surviving group. Histological examination revealed lcsiuiis in the lungs aiter eight months oi dust exposure, consisting of cellular connective tissue about the tcrnvual bronchioles (fig. 7.41. At 12 months there were adenomatoid changes in the adjacent parenchymal areas, and by the sixteenth month (fig. 7 3) definite fibrosis was present in these areas as well as around the bronchioles. The fibrous lesion could he semi macroscopically at 20 months. From this time on the reaction increased in extent and in the amount of collagen, and by the thirty-fourth month, it had fanned out Table 11.--Sise-Frequency of Atmospheric Lana Fiber and ICO per Cent BallMilled Asbestos Diut Collected Inside Cages Grata!, % Fibers, % Typ* of Asbestos Long fiber......................... Ball n-.lUfJ .................... <3 Microns C5.4 90.8 3-10 Mtcroas 1.1 4.8 >10 Microns 00 0.0 <10 Microns ZZ.i Os >10 Clumps, Microns 6.7 1.9 0.6 2:2 Total IP' 100 T>8LE 12.--Summary of Inhalation Extcriir.ent rtfr/i Long Fiber Asbestos Dust Nature of Experiment Dust exposure coattauous throughout Ufa Dust exposure followed by pro* loor1 residence tn normal air Animal! 117 ruiara pigs 4 C-tx 20 rau Maximum Maxi- Survival mum Alter Dust Dust ,. Expo- Expo- , " r:re, ure. Mo. Mo. / M /s - Result! * .* o ; P-fleit# 8>rn* !:i rrn. a o >!( u;y c'v- j.fc- nrt errn at 11 no. 0 Marked prihronenio:sr flhro:* flr*t 20 mice :: culara plfi 23 20 9 ruiara pin r eats u 0. II1/ * u reaction; i.o rthrc.ns Cleariox of inflammatory r'etln and d*flaSte eor.triuUon of fir*rous tissue C'lrar of Inflammatory ruction and lUght eoatra' tloa of ri!<ruu ti#me Similar to continuous exposure gTOUp: r'jrre.'ticn oi progression La ooe of tbe two animals considerably into the parenchyma (fig. SA). Trie lesiens were rather sharply localized and the extensions irom different br r.chioles showed no tendency to fuse, even in animals exposed for the maximum period oi three years. Although the intrapulmonary reaction sometimes reached the pleura, there was no involve ment of that membrane. Emphysema was not detected at any point. Some thicken ing of the larger bronchi with a chronic inflammatory infiltration was revealed, but it was considered no more than would be produced by a similar period of inhalation of any dust. In guinea pies exposed to the dust for 20 months and then removed to normal air, there was a marked tendency for cellular inflammatory reaction to clear. This effect, accompanied by contraction of the fibrous ti.-sue. resulted in a diminishing size of the focal lesions. None of these animals, killed at various periods up to 14 months after exposure, revealed lesions as large as those in the group killed at the end of the 20 month exposure period or those in animals which remained in the dust room for more than 20 months. Fourteen months after dust exposure ceased, the foci in four of the six remaining guinea pigs were so small that they were visible only with a hand lens (fig. SB). * O-W - . . 24 INDUSTRIALIST,! EX F. AXD OCCL'PA TIOXAL MEI^^XF. / In the group exposed for 27 months and then transferred to a normal atmosphere f began to : the response was quite similar to that in the 20 month exposure animals mentioned above. Small foci were always visible on gross in-pection of sections of ail *. 4 1 had beer. that in ear guinea pigs of the 27 month series, hut in no instance was there evidence of the j as a earia: reaction. J cells, but : i :) \ \. i i f- i -sA* yp-'-r.'vV- h r i , I* ,` . 'sr-r ; >2. \ r w.-v i y> ?*.**A ..........w* e .... T T ; - y r \ - y I n: ? j s ., -- ~~?r '.y-' y \ i I* f 'l I fcV " UC `I- ^5% X F 'i w, b i Tr- ' %'f . 1 ri r.* A'- > :; 'f-2 .i >^ -7 *v * >; Fig. --Lung fiber asbestos inr.aiuiiun experiment: A, lung of a guinea p:g Fig. S.-- i with eight moutits' dust exposure. Ti br*-r.cnioic at the center already sr.ows an accumi:!atiu:i oi 'piiap'jcvtic coils, and tiicro^is a siisht (L-puMiiun ct o-llaircit. Com with 24 me large area7 pare with f.gurc 6 A, showing the reaction to bail-milled asbestos alter 24 months figure 7 B .a (X 200). B, h.mg B. lung of a. guinea pig with 16 months' dust exposure. Again note a bronchiole living kt tv. with its-surrounuing reaction, corisistine of r.hrosis ar.ci adenomatoid change. C'l- The broreh lager. deposition is now seen in the wail* :i adjacent alveoli, at the right (x -3>J). change a: t the left. It In the tracheobronchial lymph nodes reaction was first visible at the third month of exposure. By the eighth munth patches of cellular connective tissue new cells w fibers or as I "TT "V-TTgpy y sf n>. i v >ja).w ?s ... I'ORWALD ET . TCDIES OP ASBESTOSlS > began to appear in the medulla, ami by the fourteenth month moi of tin- m-lc had been replaced by cellular connective tissue. This picture, which resembled that in early silicosis, persisted to the end of the cx| ruiiriit. Some animals showed, as a variant, heavy sheets oi diffu-rly distributed i... :i' c> tes and In w active giant cells, but there was never any necrosis or hyaline formation. The spindle-shaped Fig. 8.--Long fiber a.-bcstos inhalation experiment : A. hang of a guinea pig with 34 mouths' dust exposure. A bronchiole is seen at the lower center; the large area above i: represents the involvement oi alveolar wails. Compare with figure 7 B and note the increased extent of reaction { x 3'"< A. B. lung of a guinea pig with 30 months' dust exposure and then U months' living in normal air. Tiie reaction is essentially like that shown in figure 7 B: The bronchiole at the rigist center is surrounded by fi: rous o--ue with adenomatoid change at the right. There is residual scarring in the walls oi adjacent alveoli at the left. It is apparent that no progression has occurred ly. 300). new cells were yellowish from fine pigment granules that stained for iron. No fibers or asbestosis bodies were seen. ^ee.s,--v. -s - i 26 1XDUSTRI.- HYGIEXE AXD OCCUrATIOX.il. UWAciXE Although asbestosis bodies were found in the lung as early as one month after explore began, they were rare and hard to find. At five month', i:. .re were- visible, chiefly coiled inside ^iant cells, and a; eight months many bodies Tabus 11--Analysts of Luiujs of (>ui>i.j I'ios Eeposed to Dust in Inhalation Experiment t:ith Lsnij lril<er Asi'cstos Dust Expoture to Dut, Mo. rerhvt la Normal Air, Mo. Amt ot Asn, *cOf Drir.t Luce Totil SiO-. of Dri*d Ltinj Totml SlOi of Arn Dust Exposure LoDtiDuoui r*unn( Life Tissue Reaction * 4.35 0.04 1.10 ] 0 i.Zi o.cu 2.11 4.37 0.04 0.93 0 4.15 0.06 1.33 S 0 4.44 0.06 1.18 4.15 0.06 1.46 4.38 0.06 1.20 t 0 4.Si 0j06 1.13 4-il - . . 0.U6 l.ii 4.77 0.0*4 1.75 5 0 4 *V. 5.Cc o \i c,.(n 2 *7 80 n0 10 9 r9 14 0 IT 0 SO u S4 0 sa 9 4.n s.9r S.JI 4-?7 5 .0 i.15 .*< I.S3 1.15 3.54' 3. Si 3.12 Sit 3 40 r:i , ft. !' ;.m 6.70 4.10 0.10 0.09 0.07 0.25 o.:> 0.31 0.15 0J 0 43 0.4? 0.i2 0.33 0-3 C.*2> 0.49 r. *7 . :i 6 *.'4 0.50 0.S-4 0.37 0.33 :.rt !.) l-ii 5.20 4.C*` b:0 12 70 12.25 :o: 12*2 ::.ca 14.5* 10.19 5.2? \.j 1 10.13 10 :*S 8.no 12.47 9.11 12.rO t+ 3+ Jr 4-r 44. t* Doit Exposure Followed by Prolonged Residence tfl Normal Air ! U 0.43 12 22 0 s.w 5^3 0.4? 23.ui 0.52 14.50 1+ 0.21 7.23 JO 4 . :.si 0.27 9.57 2 + \ 4.:? n **{ e --v 20 . 10 <.ao 0.22 5.07 2+ 20 14 5.01 0.71 4.1? S.04 0.18 3.Co + 3.40 0.39 11.51 n 0 3.7* 0.49 23.15 J+ ) 3J6 0.31 . <50 7 1 - 0.13 6.54 i+ < 3.1? 0.3 7.99 3.-.S 0.23 8.72 2 + ar 9 3.TS 2:5 c V, 8.31 2+ TN yfrbol* areraeicif f!-*v.e rractle^ !r. rarh rrruip n*rrrc'*r`t r**rely the relative o*. ri'a^Mcn. rsrgir.j? ircri .* to -- to <-- (IN' n.3\iim:rr. tor tnl? f\r*-r.- xnectv rei;:'.lonyrtips apply only *ith:n tnl ia:*le n1 cannot be compared wttb aymooi* la other table*. were free in connective tissue. They became fairly abundant as exposure con tinued, although in some later animals the asbestosis bodies were only modernteiv numerous. It is important to note from analyse* of the lungs (table 13) that even though the tissue response at any given period of time was much greater in the guinea pips ;o: asbes'tc Rfe 14, 25. cats, :a: an a<lc cellular arteric lymph poir.re: month' nective and an wails -1 Fig.months'' a- *..a. .C-^t.rr.. an occas. reaction slower L25, 33 ar lesions. React: from pnp exposed i and offer, was just peribronc. bodies w( animal, accomparr: VORWALD ET AL.--ST ,<r or- AsnEsrosis 27 pigs of this experiment than in thoe exposed to either short fiber or ball-milled asbestos, the amount of mineral matter in the lung a>h was muchjcsi--_ Reaction in Cats.--Four cats itihaitd the long fiber ashes' dust for periods of 14 25, 33 and 42 months, respectively, and were immediately killed. Two other cats, after being exposed to dust iur 18 months, lived in a normal atmosphere for an additional 24 months. Fourteen months' exposure was sufficient to produce cellular accumulations of phagocytes around terminal bronrhioles and peripheral arterioles together with compact collections of similar cells in the tracheobronchial lymph nodes. At that time there were no typical adicstosis bodies, but smooth, pointed, yellow fibers were seen very rarely. With continued exposure, up to 42 months, reaction in the locations noted progressed to the formation of cellular con nective tissue which made well defined sheaths about * tic respiratory bronchioles and arterioles, marked lymphoid hyperplasia and lymphoid infiltration of bronchiolar walls (fig. 9). Typical asbestosis bodies were not formed, although there was Fig. 9.--Long fiber asbestos inhalation experiment: Lung oi a cat with 42 months' dust ext>osure. Two bronchioles are shown with adjacent cellular reaction and collagen deposition (x203). an occasional fiber, smooth, yellow and pointed. Pleurisy was not present. The reaction was similar in location to that in the guinea pigs, but fibrosis was much slower in development. Roentgenograms of cats made after exposure periods of 25, 33 and 42 month-, respectively, faded to demonstrate evidence of pulmonary lesions. Reaction in Rats--Although 20 rats were placed in the dust room, many died from pneumonia ana were not suitable for study. Five animals, of which one was exposed for 19 months ana four tor 25 months, were tree from pulmonary iniection and offered a basis for tentative conclusion;. In the 19 month animal, the reaction was just beginning. All four animals killed at 25 months showed a well marked perihroucltiular fibrosis. After, a long search, only two small, smooth asbestosis bodies were found in the 19 month animal and none was iViuna in the 25 month animal. Thus these animals exhibited fibrosis without asbestosis bodies or fibrosis accompanied by only a very' mirequent asbestosis body. ''viA ~ 2& IXDUSTR1AL HYGIEXE AXD OCCVPATIOSAL MEDIC1XE Reaction in Mice.--Out of 20 white mice used in this experiment. 11 lived a year or more in dust ami 'Lied or were killed without showing an appreciable decree of pulmonary infection. The reaction to the inhaled dust was limited to phago cytosis by mononuclear cells. Usually these were widely scattered through the air spaces: a limited number were grouped about the terminal brou.hh lc.. producing some thickening of their vails. There was no suggestion of fibrosis. Numerous asbestosis bodies were observed in animals killed late in the experi ment. Thus these animals exhibited asbestosis bodies without tihrosis. Summary and Interpretation of Inhalation Experiment zAth Lout7 Fiber Asbestos Dust.--The purpose of this experiment was to evaluate the importance <<i lung fibers i:i tltc tis-'ic response to inhaled .''.i-be.-tos. The results, in comparison with those of previous investigations, indicate strong!}' that long fibers are chiefiv re'ponsible for asbestosis. Thus, the reaction n. guinea pigs dcveln:>cd earlier and became more extensive in this experiment titan in previous experiments in spite of a smaller concentration of atmospheric dust and a lower mineral content of the lungs. Furthermore, typicr.i peribronchiolar fibrosis was produced in cats, although in a previous experiment with short fiber dust peribron chiolar fibrosis did not develop in this species. The cause of the ceilular fibrosis in the lymph nudes of tltc guinea pigs is m.t I: did not occur in other inhalation experiment' with asbestos. INJECTION EXPERIMENTS Since the inhalation experiments reported above strongly sugge-ted that long fibers of asbestos are the significant factor in the causation of asbestosis, a series of injectiun experiments was inaugurated wherein the dosage and the length of the fibers could be controlled mere precisely. Also, by the use or' controlled dosages, the relative capacities of various asbestos minerals to produce reaction couid be compared. In these injection experiments, guinea pigs, rabbits, rats and dogs were used, and the mineral dust was injected by the intratracheal, the intraperitoneal and the intravenous technic, but not ail the technics were used tor each species. For the purpose of simplification the findings in each series of tests, except for dogs, have l>cen condensed and reported in tallies, to which reference will be made later. In the case of dogs, only one test was made, and since the findings were negative, no detailed report is included. Experiments Using Intratracheal Technic As the asbestos minerals do not cause typical advanced fibrosis in cxtrapulmonary tissue, the intratracheal technic is the preferred way of introducing fibrous dust into the experimental animal. In this method the dust suspension is injected by means of a 'pedal needle or catheter deep into the trachea, from which it flows into the lungs. Comparison of Fibrous and Xonf.brous Dusts.--To demonstrate that the abilitv of asbestos to produce fibrosis resides in its fibrous character, the series of injection experiments reported in table 14 were performed. y- 5 I g w * V* ff *a l >- Table 14.--Comparison cj Reactions to Chrysolite ami Serpentine Injectei Intrairacheally Dottier F.tch t:v.io*l wa* mm an intratracheal Inpyiion of 0.3 ee. ot a 3 per cent tup-n?lon of the dust. Two wi***l* !('-r another similar lujcvitou i given. Total amount of dust Injected vat jo m( .Animals used: >.\ gr.n]<* <' : pic* each (on** croup for each type of dustj. Periods at which w::`rr:ul- w.re kbled: Doe or two anisiml* 13 earb group at 1, 2, ft. v.j and 12 months after last li^ccuco. Preparation of oust: * '.rytoiile (ball in1 Uni) ufiheaud: HU milled for 1,IV br.. dried and rr-trourul Id scale ruortar. Chrysotlle (ball milled) vc-fd: lull milled ebrysotile h*atnJ for 2 hr. at about 7.0 C., theo ground lo acate mortor ,, i<r \> nun. L'hrysoillc (Anro*i) uchratr-i: (.round la agate mortar to pwa 200 me^h. Chrysolite (fibrous) Iguitoj: jUM:n!n material heated tor & hr. at about Ttrt C. No further frfndlor. ''rp' Pr ;re (foil ji- Lj--i v; Jj !*;. und.I 'or ].;* br.. I and rueround :u scalp muriar. Serpentine (ball milieu iirru^l: hall milled arrtycuce Itcatni for 1 hr. at about 7ou C.. men (round (a agate mortar 1 >r a min. Mineral 1/hrysutUe (hall rallied) unhealed Chryaotilr (ball mlll.-d) Ignite.] Cbysotite (fibrous) unhealed Chrysolite (fibrous) l(nlted Serpentine (ball milWl) coheated 8rrpentlne (baQ milled) IgnUni Sir* of T>ut Part mirror.' a:d It^i 1 microns anti R cults P,finding d*rtroy**1 rvwrsrv t ru%# fit.At \ mu. cuoaiuerahle infio.'irr.oiufT ('Inna aod rciiular pr I<f-ra- tioo sud ioealuation of dust particles about r.r<*r.lr-1 - oles; at 2 mo , on:y a **ry eiuitit tTouf' taue* r,a'`imt>: at 9, SS aod If ruo., wt.;,-iy m altered maU toononm 1. r phagocytes. At L: mo. a f*w rn:cr<.e(.p:r picrm* <! thlo alveolar wail tb.rkrr.snr e-itn some adenomatoid chance m [onmr. n* mf ar.ntribv.* on tr.:rkn-(*| bronchi. No r1-: *'*o. heaotU.n l!rr.:tfd tc 'ar^-e fr *irn b.Mr krsa.-.t re.i* :tr.>.ut pr- -(uruoo ol 20 30 uiirroos approx. CU-U) micron' approx. 2 tnlrroof aod less 3 micron* aod less A dlsUoet ftbroaa. R^aetloo loeai.ned to rooneelite about terminal bronrf ioie*; !:t:.e (".o'* Oootraeuoa eaused .tOorsatoid appeannr* r.t sir spaces (ivea off d;r*rtiy from ternsnsi : mzrr.-.clc*. Reaction area b^rarnc % ,i!rr aitn prr-k-r'* of n*> Dew rritioDS lOTtilTr.1, N rrr^D'e ri**ufi?y even ai abuttmc totrspolraor.ary rn-tr.ge. At 1 no. cvr.`-;> rr-l* LoSarnmatory nj^fn.'t and foes f c*.ivJar :-ro..:rrat.cr.: at mo. *eil mirwwi -:!ul.r prri;firat:oo aod 2-.*n*i occumog foca^y af*ou: respiratory rron<*:m.>. Th e r*ar! lo*1 ! I '.r* 1 <... r . .*. !: j 1 d and we* a> adranc<l a tnaf t:u*ij'd l-f . yr. mt-a.i- tloa of aahcitos eliter. At 6 mo., rac::on k -strr.`-ee than at Z mo . apparently due to eontraetiuo of if-ro'^ U*?ue; asoertusts bod:e? re afc:^to0t. At ^ mo., reactioo still U^s exur.siv*. rnrffrM t*- th* itrrt.**liaie Tielo;iy of the cn.ail trronnal brocrniolvv *n*r* me ar tissue Taj qu;ir deae ar.d was t erommg hyaline lo mar- after. Sometimes It *v*n obliterat'd t.V l-ronrh-.o.e. .Abe<to*.s bodiT- ha-J t<-.'u:ur scare*. At 12 no., t:: Tell rteveto:e*l per:rrur.rr::l ood ntrebron.-nial aura- matold ar^na of flhro?i had produ:rt considerable di?* toruoa. More periph.-raHy w^r* patri.rs of prp.uiiior.ltls with eocloopnilic Iriffitration, som^ of wnich n l-:n( transformer Into Pd<rr*:a ti'-ue. They -*e:n*d to t* pr^ cursors of the Jocail/ol. d.iffuae patenei of tnln alveolar TaU fibrosis wvo elswhtre. Reartloo limited to lanre foreign b>dy giant cell* without proliferation. Hralin? t:e fibers. wni<*fi made them brittle, destroyed thfir rapacity to produce ngoiffcaot react ion. Dust relatively loan Ire. At 1 and 2 mo., simple pfiaeocytosis without prniife-ation; at v mo., no chance except possibly lymphoid c*-LJ InflliratJoo: at ?*- mo., a Ucht rhroole pneurn'.n-.ti': at VI mo, oniy a Lille pDvumonUU without *ucr`''Hon of fibro>is. Dust relatively Inactive. R^nctloo rentlally the earns as for unheated s*r[*nf:r.e. With lehlbd rrntf::si li-jj t- ..'.envy lor dust to be c.trned to brunchial nodes. 29 1 i 1 tj j 1 m i 1 30 INDUSTRIAL HYGIENE AND OCCUPATIONAL MEDICINE I! :A L 1 . >*{ -'-'a T > *: A ( i ! i i :i ,t ri \ n. it i, *. Fie. 10.--Comparison oi reactions provoked by injected '.one hber and ballmilled asr.estos cuats : A. iune of a guinea pig which four months before had received an intratracheal injection oi long r.ber ashestus dust. Note the peribronciiiolar accumulation of cells with collagen deposition. The bronchiole chiefly involved is in the midst of the reaction (X J00). B, lung of a guinea pig which four months before had received an intratracheal injection of bali-milied asbestos dust. A bronchiole is shown at the right. In contrast with A, note that only a few cells have accumulated about the bronchiole and that collagen deposition is absent (x200). 1 : * { ( I I'ORli'ALD ET AL.--S^^)IES Of ASBEST0S1S 31 The tests were made with long fiber chrvsotile. unheated. and witli chrysotile that had been ignited to destroy its flexible structure or ball milled to reduce the length ot fiber to 3 micron' and !e<. . At the same time control tests were made with serpentine, which has the same chemical composition as chrvsotile but is nonnhroe.s. A review of the findings reveals that only the unheated. long fiber chrysotile produced typical peribronchiolar fibrosis and that ball-nulled material containing only fibers less than 3 microns in length failed to cause fibrosis (figs. 10 and 11). Fillers,subjected.to ignition also had lost their capacity to cause serious tissue damage. Ignition produced important changes in the chrysotile fibers, among them being loss of water, an alteration from a flexible to a brittle structure and possibly other changes. Experi- Fig. 11.--Serpentine injection experiment: Lung ot a Guinea pig that had received an intratracheal injection oi this dust four months betnrt. A bronchiole is shown a: the lert center. The phacocytic ceils exhibit iittie predilection tor the bronchiole and collagen deposition is absent (x 300). mental studies concerning this observation will be reported in a separate publication. Comparison oj J'arioits Loup Fiber Dusts.-- Some very interesting findings are disclosed by the results of the experiments recorded itt table 15. First, all the long fiber asbestos minerals tested, with the exception of anthopnvilite. produced typical fibrosis. The characteristic neri" bronchiolar reaction caused bv three representative long fiber asbestos minerals--chrysotile. amosite and crocidolite--is shown in figures 10 A and 12. Why amhophyilite behaved differently from the other asbestos minerals is not entirely dear. Second, with the mineral brucite, which is not a silicate but is a fibrous form of magnesium hydroxide, a characteristic fibrosis like . :-Trr t'-'-----*t-- .1^7. v . 11 .t*; Table IS.--Comparison of AYta Carious f.oiiij Fiber lutratrtiihtaii} Injected Doaat*: To infnrUoaa of 0-6 cr. of > prr cent lUM^H'ivu iirca two %evfc* apart. Total dowe vat jO mg. ApJmal* UM: from C to 9 fulnr* piga tor c*r& dual. ftrloda at vhirn animal* vary *it 1. 4. *nii 1.' month* aft*r |**t Injprttun. Elar ot Uut patiuita*. bfparatrO to f Riot w^tv from to y> micrpoo loo*. Mineral Cbryaottle lT^*Uonl> Ch(A/yrnizoof nliea: lr iroo ruatfnt; tXifV 6Vsi> AiDoaUe CroeirioHt* (Bolivia) CrorMnliea (S. Africa) ABthopi> UUe TrrrroUfr Bruclta Gift** too) KesuiU D>t:uft {V>roit. A<M:tJufliJ Information fives) opposite ehryaocik (fthrous) to tuM U. virtually i.t'Mk-al villi that to Itutfoot fhttioiil". Both afM a.*hr**r<i*i- i-hih* |r'Kjc:*fxt T<fjs ni> f*riuii:nr< ttt Jrna. Kbroh> oti".:?! a* j.*uc> within terminal hrf>r ^q,j * m># at i*rrr*fc*-r^ih*v ; .j f--?.* w.f* !^*a ao*J a.* m rt.A.Inr -rate writ : . r in--l a| ,,h month. \\iih,a*<\ fit'fOu* ti*uc ri;trniMi^J and orrupw**) *o?n}f*-r urm l.nt u,i utun- A<W>nnmatoM rha.' u"* *i:n*!ar to 7,v*tfnM rftrtotii-. rir:r>v flttrttM to timti'N' ;.t* v.r.iv.ty of -r*y i.-ri *?.* of tr.*'-.nV tm-ali/ation. A*-'........... . m*hv: i-i.i , -r f Jj;,, :i-r in;i*'iiu{(, intQM*- t* i <it mti:orur)* ar ir*-i.P*r:it:.r v.i.nt ronrhini* as.} In a**a of *(-*<'ui: :( IS too.. !.<-\ y i-nhr*nftuo:.-ir patim-x ol Dtro*ta oftr-n with p-ipillary j.rmrrt'i*:.* partially do-mf lymrn M t>raarnlol<: ailcv.r.mnti,).! arpearaunv niHrL^J; rMiia*rt!TP tu-ur fraction ho**J h^ary rolifu;*n t*ut no hylo#^mr,` *t rto.. (mtmt# (n^i .r wf!| maturrl fit-ro**..** at>ut hronchio). ; *< i no r tnatnrr at^.ri*..* fShrovjv with ^wit. r: of r.>ntrorllon: t on%-.l<*raM* rhrocitf pnfunu.r:::* lt;i tcAttrat-oh ot JyiT-j-hm yt*-* anrt #.-i;*.*(* i-. \\ o m., mtr.v t)rorifni|r fit>rou t-n:c< w:tn tort o! iiMir** .j.iirMP at i^rm;#ry, flhroviv ap-t c*0" ' .* with Vrrrr*' atyj-:,p*i .. . 1 'j11iriii f < '<'* i;;, r.. aflt *r\)wil*n, .! \*y s'h Int u< p-r*jt *ft*r f.rh *:n Rrartion at l nu. t**4vy n`lotronrrto;nnf nrirr.nf`uojitalrra-:; Toowinr rtlirou* *ii:i w4'. at t tv.f.-i- %;t}i ............... ( lir* ol uias,*M> |o< a ./* i . ol At i um> . ntr-r-- * mitnhrorw huilsti* sr.i j r*-ri*.irn:ontS. t-.-vv ???., a.'*-* rrnly if f*rn.tinn *: mn-l * ri*,arat r*. \< n1.**. *. ;:.i. r*ai*tton in l* *.*. u\ i iiiv thi* .' * i * v.m. At i f *.no . x-e\ nr fit>ryu ^Sit.triin. ii~'i prtf.rnn<*i' i;*i* *i>ii pear aril im.f-- ! 'orrmiy t>i oro/i'i:al tnim'* tint ao -ttriirmn itiiu, or atp>etais ol, pern/rx-rul pairsrnyion. AdracwrO flt*ron ^nihif'rnncfHrtUft* anl pfit.mnfhiollM juaifn,i to.ta NvIIpi nofr.j ar v rro. ,t i mo.. riv rtr-r**i* rdrifiDnrniui.'tlv an-1 pffihrxiorhk'Jit).*: jrtnry r.'ast p^tli anl *'W> tjtni>'*ocvr:r rmrtfivi U 4 tso., vroail ar^a* -n-1, .r.rrrrhioht:* -ratwr^i thr.. 'ch'**-c t'\.' ^Itu'ar rtNrov* .M 4 b*>) tno., ar * nl of coDtrarlion of !w r^*r st 'f xio , It: :prn>rftr tr.u.na ai;tt a m:o..i <*. Tvp.-;4! trapr^l r,frti,. n i!i pr..t i.-riMf-'iM-'in r: t-r A J-f .::;! K - /? !*)- * " U*1^* -J. .: ......... ./*. r i r:.o., 1 * ,*r - u. i !'.' : * . : p. ** * -. *-? *. w ? o t X' ........ .. t - - , kfiani ril a:;*i ruar*". t * rm... f > t- I r.r.inp,,....;,ti* Oiiita a* ikiri*nMv<* or a* n*atly n?roi: *iih a ^ r*(-n-.on. o:'.-* vs th^ *nr M-.inv ! *.'* /!'"' vi*n a *! {.rij..if:..,*. haintpTtrrJ a>^`*tO'f' At VI tno , fTT M rou o.,,o.nrf:, Dorr r^rfbror.riv.nMin :: rnifr)>ronrhio;.tt<. Ttcn .'mrt:orrcw ac.i crU^: Terr marki^l lon-nowiuoM avt- atv-'ri4. I.yno.'tnvyttr infljtratror ort fftart r^ll* lnt r< A r*fT fv tfy;...-a! tt<rt04t feo*H>*v \t \ ::;u . :;:auy *v*i frrfrif.roprMolsp .:j-: without nupif lorni:/v!o: lrnut:oom* irniir*t:on of * ? ar j a **v fit*'.! .Vt * a''l V.` u-o^ I'.ttlr ivi.i-i rr m * * m rr*-u.i.-* a.n.f t.ronriil tih CKOt ^ ^.tfan of tUmti rv-l with ly:ninr j t:r ir.mtra:>oa of wnii. atifiijf t ' r.'U , tr-:-4 ff i'.-'I ',i*r*L^i ion w;f\ n*l. of airrr,;j ; r fV f m * ion with t.t.ji- ir'luti.'nauity Tv.*rro. phar*'? aod tisr.t ti-'.l* TT.ttr;p tr?n ar. nrr# % v* !*< of * *mi r./1 ar*`:i- r.' I:\^nropny of .i-r^nlte rp;fhrjnjpn. jlar.y cnlni** paritM u tt- r.\r < rr.n.. as-t<raranr of l^vo:; OSrhaP.p^t: p>itfd !r Mi.rWmrd Otr .bj*3rr .'ori:/af ton^ of *luf :t O^Tfi^.onwl * *-*-1 Nofr if r*.i* . !r..**iy fo^f of tt.k. r* tn t?onrofoV g-.-l fi.o.inr with, .. :: i:: w r r-nrr.nn fotnr to<*1 ynowr.*t ii.*t vrt <lrfw>*.ttnn. .U U and rr.'O., rrarilorj a* r^forw with f*.Vrn. vronph.ou* ;i<trr;jf t*rai;* of '-in. trirffor. an<1 <*! .r.f.nn>)nat50i. *'.ant r* ii> vfor.mrnt. Hl^irra marnMlr tnrolvr-I. Ty7>:.rv.f CVrouJ 1 T- lohr-' ! tv*A '-iwnroiifj rrarffnn to miavrM*. \` l <i.. * .'!*li.;.ror:rhf<i!iti npif prrmron- fhioi.U* v.th u:sht rff-rnu i*>op in!',": t*rirtrhiojr* gnil rr>iu* lar v-V <**>f>*,i** p'r^-nt. At J rno.. h^ary Vifrahrn.n^vcjlar a:*-! t . r r*: : h;n!nr nt n-,- tri*tu :rT marine deforn^tf wi.'h ifortiori of v.f-- n l o.`.,t.rtu.--r ot vi.r.'>un*hnv' air par**: fihrovi* pair *;rnm:r )i\ :.-arloft tn:t ^i!!i rnrX* no pcroi., TTpi- ral ho.f-.-4 %,-!. At 4 nn-i > inn . )jll!r rharifr: ro.nfrartlr.r. At M':r it'-., -!rn* nf-fon* kor.n'ul'tU i:n wrbotoft^ toAlrf. .Vo pVurif. S\j \frr.Mon to aurrounjii.r !-.:njr. >'o ft? ro*ln wfthln ft r*tr. At l mo., no n**rion lrMi hmorhlolw: lr. fv,rifih*'rl air of ff.ar.t *.U- (isrtof wffh dr.*- *prn>4 of gia wjfh IrmpfiOP) f:* :: ** trufioo of adliurn; wall*: no a*f**'?of hodir*. At I nir.. Tract ..n 1- tt>an at J n<o : rn.inpr of 'iop. pit-A riant phr< . ' rti.iot.roPrhitin, At 4 '.nr.i.-.g f.<.;. at-.-l part of rJa.*: "O % ? **.. riBMiin '.i .11 VrttrniW.ipf. At 12 fno.. f.*-i( Rr*g< of >- hi mo flhro-i* it *ndoti:onctutl^; moderate nutnh^r of vmuocii iron ftUvr?. thac j the ;b obvio 1 dcvel> j II I I > 3s r. i ( w i Fig. 1Z pig four cr reaction ex. B, Jur.g lite. As. ic shown (X Third, (fig. 13 B However.: voRWALix -iu .4Dins or isasbestos 33 that produced bv the asbestos minerals was obtained (fig. 13 --I). Since the bmcite used contained only 0.90 per cent silica as an impurity, it is obvious that a siliceous comjjoncnt is not -a essential factor in the development of asiiotosis. Fig. 12.--Amosite and crocidolite injectl'm experiments: A. lunc nf a guinea pig lour momhs a:ur a:i intratracheal injection nt au.oe.tc. The inflammatory reaction exhibits pronounced accumulation oi ceils ami collagen deposition tx 2'J)). B. lung ot a guinea the four montiis after an intratracheal injection of crocidolite. As in A, peribronchio.ar accumulation of cells and deposition oi collagen are shown (x 200). Third, no fibrosis resulted from the injection of glass wool fibers (fig. 13 5), even though glass wool resembles axbestos in many ways. However, there are fundamental differences. A glass wool liber 3 microns >< 4 ft rxnusrmKL hygiexe asd occvpatioxal DIC1S F. in diameter is a solid rod which in short lengths is fairly rigid, while an asbestos fiber of the snnie diameter is a bundle of extremely fine filaments which impart to the fiber a high decree nf flexibility. It would seem that this structure and the associated flexibility are important factors governing the capacity of a mineral to produce peribronchiolar - * r^v.- i 4 ' K`*V'V'' ^ ' - --' - w wI - -- --O'r.W-.i*; .- \\ ..V? . - `7 I*?* c,-1 h-\ ; .- v ~\c j ^|- * \"VOS- - . v v N s ,, :-v "' f */*S -.-V.v ij K. 'id ;_ w* ` -V '* * V; ?* *'*'** * * yr.- 'V a. V -i >. \. >4 4 v ^ * <% a. ' ^-v-'- j -s J f ^ f " ** * 'W ; V mk. > * oSr'-'r, r'-k>- 0~ --i V *' - >* ^ 1 1 Sv V< B' Fig. 23.-- Erucite and class wcsjI injection experiments: A, lung- of a guinea pig which tour months be:-'re hau received an tntratraciical injection oi brucite. Even with this nonsilicecus hbrous mineral there is peribronchiolar accnmuintion . of cells and deposition of coilagen similar to iha: shown in .1 and B of figure 12 1X2). B, lung of a guinea pie which four months before had received an intratracheal injection of glass wool. Two bronchioles are shown, one in cross section and the other in longitudinal section. P.-.low the latter is a thick-walletl blood vessel. The bronchioles aie without reaction and can be considered normal for comparison with other ficurcs. Glass wool libers are present in this ficid but cannot be seen at this magnification (X 200). t -T VORH'ALD ET AL.--S^^IES Of ASBESTOSIS 35 fibrosis. Experimental studies concerning this observation will be reported in a separate publication. Table 16.--Comparison of Reactions Produced by ..mo Fiber and Short Fiber Dusts Injected Intralrachcaliy Dosage: Two injection* of 0.5 cc. of a 5 per eeoi iujpen Hon gives two week* apart. dote wa* 60 tng. Animal* aw'd: Slit group* of guiara pir*. Period* at wfilcn animals were iilJid: 2, 6, SH *J>d 22 mouths after Injection. Total MloeraJ Cbryeotlle (Tbetford) Amoalta CrocJdoffte (Hoi)via) A&thopbylllte fllxe of Dust partlcfc* Loor Z'-+t. microns Short fif-er, t mkTOOA ao-2 Locr fiber, 2>50 OkCTOCJ Short fiber, SO axrocs and Long fib<-r. j>5> gictods Short fiber. SO micron* and lew* Locr fifi^r. 2>U> morons Short crwr. 3 meres* and Iraa Result* A d>*tinct f|hrn*iv R*'lr to chrysolite (fibnm*) un beaten In tabU* \\, No libroals. Keter to chrysotile (ball milled) uobeated lu table U. Typical fibrous erdobronehlMm* and perlbronchlolJtl*. Refer to laMe 15. lU'8-Mioo limited to rh*cr*yieii? with lymph*.-yifc infil tration of adjaecet *aiiv ^ho;t fibers packed lntoe awollco hagocyce*; lorm-t ones fr**e: ome cant'd to form teptcal \ ,.U} n't-r ..; e. liui. ai\ OU rv : 1 c * ; >;/,*] giant n.lv. mu>t pbrgucytes were wutrn air 'pares and hut sot migrated to walla. At 4 !., free liular fi'vrs ha.t worked themeiw* into l-t# ai t!<!ie. where there a* exter*ir* '?f!nn of Jjir.pno:.! r:; and rnoru'cylit hut no n: r.- \i mo. for* w b.ir rrari^-n nirh i urinr.ni, ao Lro:.e?tio;:tu. 1"y; :cal *kbetn*4* hn.l t. A lranr^l fihroue en.lo'.ronrh.rujtli and pcrihronehlo* I *i-. HrfeT to table 15. No fibrosis. At 1 mo., air space* eomprf(?*d and JarL-rly fUJol with giant r*i; pnrfcoi with di:f t:r*na. 'Vails hrarlly Infiltrate! w-fjj monocyte* an-s lymph- old eell*. At 4 mo., a moderate derree of eriiu'ar Infiltration of walls: sicnii guot cHl porke.i a ip dU't spir;:le. At C ar.-l ruo., rrae^c* of gicrt r*n*. eootaia.Tr mineral pm--'.-, ir --r.ii Iror.r'n > :ni Ic r'^f-irutorp hronf::;.;-: rrrail-r one* w:.!*ly raf- r*ro*t :u t.-rmlcal a:r bodi>w >io reaer:or \m. N:;rrerm:5 ti.**'je. >t : Imr t-r.-:.: rut :: :-o t mr.ny s-Mi-rr-! si.:ni' nronocyte* p-rs * eitp No erM>. roa- rhitir. No peripheral fii la lymph nod\ flight reurulhs;*: do flbrosi*. Lympnorytlr infiltration and giant eeU* but ao definite Refer to taM* 16. No fi-rnit and praetleallr no vtosl* bodle*. At 1 mo., focal roiwtioaa of .f-.m-i'.l'rr) monoryte* and a few glart cells: at 4 rr.o.. atl-nomatoid ejhr.e!:a! reweiion: at 3 mo., ^tr.p;.- i-nr^vnomtia with p iaco- tyto-i* of >hort fiber*: at mo I'Oland and shr.rply local.led eoiiectlor* of .1 r*-,> mide air ppaer^ about terminal arter'i -:'v !>-.rt*.oa in wai I'.iv.l'.ed to Icmi'ho'.'l cell Ipfiitrati-'-i. No fir.rosis. ir. lvtnpn node, reaction limited t-* * -ht pror.-.bvnrr of rvtlcu* Tremollte Locr fiber, ?>50 tr.icron* Short fiter. 30 microas ud less Fl* ro?;s about bronchioles. RefeT to table 15. Simple forcirn body react op. \o acute laflamnation. No accut>iulation of dut l:t o* about terrr.lnai broc- chlol<-. No cndohror.r*-:f .\i \ rr.o . rcattered small giant cei` and con*!d* rn M.* r'.fi'Tp.t-n-i of acjhf*st walls a itli monocytes a::j 'v*- .uni ceils. At 4 mo., UtfM chance except rr.or* "uinr `-fiitretlon cf con- crrtire 11->u*. At S mo.. ,r.:.j :r.filL.*a::on and tnick-epfrc of walla about but r.ot all termir.aj Brucfte Locr fiber. yyy* micros* Short fiber (tnade hy cruh!r.e Jong fiber? :th rubber poLcemao) Trr-:< 2l ..fi* roil. cortohro''',*-,i',:,,4 and r^rfbror.cMohtia ;. r.-a^tinn to a-t^to* r.:::\ raf*. R*fcr-to l*'!e 15. Inert tyr^ of reaction. '! ' * a^'er injection. rnaU nj^ < y;, * w -:y scatfe*- * y -r: m air fo< t:a cf c't* with lym; : " 'stfon of rotr,prv*-d s:r-:a-* wails. No * -i**-, tl reaction as with c.nry-citAt * mo.. \ r.iinr to that at 1 mo.: !>p:caj ar^^toN r-- * - <'*. At J2 mo.. rrall ci'utnr? of luactiTe duat-fi.iv.i phacr-cyte; no fif rosi*. No renctios in lymph cod. Comparison of Long Fiber end Short Fiber Pusts.--With quartz dust it has been demonstrated that the smaller the particles the more T.wu.r, I?---- 'Sm*. r.,<ir:yi o f l,,j> r ,i,, n l l ^ c r i n w r l , by hxtmv,hiiu T tth n ic :3 1 I ;I I 'll I, * 1 I I 1 I ' i ) i I; 36 ' ISDUSTRI^JHYCIESE .-TVP OCCL F.ITIOSML .t^^C/.VE intense is the tissue reaction and that particles larger than 3 microns in diameter cause little reaction. In the case of asfsestos, however, the reverse is true and apparently only long filers have any specific effect, as was suggested by the inhalation ex\>erinicnts. This is confirmed by the data of table 16, in which a series of tests with fibrous minerals is reported. When the injected dust consisted of fibers 20 to 50 microns long, all the fibrous minerals tested except anthophyllitc. as noted in the preceding section, produced fibrosis; when the material was prepared by first grinding the fibrous dust until the length of fibers was reduced to 20 microns ami less or. in some cases, to 3 microns and less, none of the injected dusts caused fibrosis. These results differ from those of King. Gegg and Rae." who reported the production of reticulosis comparable to the experimental silicotic nodule in rabbits receiving monthly intratracheal injections of 100 mg. of Rhodesian asbestos fillers. 15 micruns long, and. the produc tion of diffuse interstitial nbrn-is in rabbits receiving similar injections of short fibers, 2.5 microns in length. We believe this dose, especially in the long term rabbits, is highly excessive. In our experiments the dosage was kept low in order to minimire untoward reavtinns which might obscure the peribronchiolar type oi fibrosis which characterizes early human asbestosis. Experiments Using Intravenous Technic The experiments summarized in table 17, in which the intravenous method oi injection was employed, show' that the asbestos minerals arc far different from quartz in their action on ti.-Mie.. Tt has fxen rejxratedly demonstrated the: tutraveii'dii injection c : quartz i articles 3 micron- ami less in diameter will cause a typical tissue reaction with the development of hyalinized fibrutic lesions in extrapuimonary sites, such as the liver and the spleen. Asbestos minerals, however, on intravenous injection generally produce only an inert type oi reaction, as is revealed hy the. results given in the table. The reason for the early deaths in the experiment with chrysotile particles is not clear. Experiments Using Intrapejutoneal Technic h The results of injection experiments with the intraperitoneal technic 11 are given in table IS. It will be noted that the long fiber dusts produced h a fibrous reaction while dusts composed of panicies 3 microns and less (/ in size caused only an inert type of response. These experiments indiI cate also that tire fibrosis initiated by the irritation of asbestos fibers is I not restricted to the lungs, a- was formerly assumed, but can be pro duced in the peritoneum as well. OTHER EXPERIMENTS WITH ASttESTOS MINERALS A number of additional experiments were conducted to throw more light on specific phases of the asbestosis problem. ( II. King. E. J.: CIrgg, J. W.. anil Rae, V. M.: Effect of Asbestos, and of i, Asbestos and Aluminum, on Uur.g? of Rabbits, Thorax 1:!SS, l'J44; abstracted, j Indust. Hyg. Digest. 194vol. 11 tFeb.), no. 23-1. i c ) L f l, t I I * , r, .*, T a iii.e 17.-- 'u rn m o ry uf h ijr ilio n lix p tr im c n ll l<y h ilr n n 'n o u i T rc h tiici imf ill= = *i Ill=~T l-e to -m*!!:=liissl Hi i!`nil =:= i _== 1 ssili iJ=Ia! i = -5 = 5 -=-4 3 r-"i ai 3 1 *> 1 iPl 3aiSf--2 2 Ih- ;r = s=- i = a 111?: lilt: *_ ! = |j: iiUil =i -i jilill! 1-5 Jz T .3 e * i. -- a 11^1 UJi tin Mil lin=U III;-5i- : ':t lili f a i !: iii7 ,. u - -i: = a ajw ^=a i=S- l3-I=i a 3 > s I ;---- c v Jj^a ItH* i svi^i IIt-:- *ff J 2 --- :5f=i Hils: I : f i I: Hal = SHrniB!iit -- r :!S1 ;. = *a ia = i *:a ;-a = J" 1 2 i "Z T :IHI = --a- . i = 0 * z Z i1 * -a i i -?"=_ii=i=a ?* ^- a! C liiiIrU 4li- := i c ?: = a = =5 = - = rii i " >.; = ill a a lilts;! ri: ` uKii - ir~ = * Zi iaia? 3^ ? = Ia ~ C = T = - - "= -if a ; =i = ss _-- i =. - -`J- := : I i!=t lilii7:r r"r :]> 'ii Hi!Z. * - z = .a T -- -- raa:i UlUll S -t-o----. r s = 3*! c a *-= a = ztzt-. zi- = I m = | ii 9 : i: 1 c !_ -iij- af afz aa: - - s'; s-: i;: Uill: ill .it i=\ *i I- i -^ 4 l5i 5 d" i5 ai-al^ :at rii rs " rs x i r - :' ja: ias 2 i? 111 r* ~- .17 =* Ii ! i r- f 0 I 'wrr.T*'" **** -1 I. \ )\ :9 M u. t if21 till nil 11 ilM -ii w _ K M i * -=_"<= =--7 Ills 4-1- 1W I 2e Im s t =3 m 1? 1 .r =: m *--* % i z V<? SiS " tiii-;j=i I^li rmt if HI UlU !II=: _T f i; ?* -C a ; ; I- 3 ;1 g ?= j;-iiir c = i- = = = = Hi !'? = : * = - M- 2 2 = = - v.- i = ; z = i - I?- =' = ;iH irif; 5= 1? = **r- c C a = i Isi >. C -- f Hi 38 .i . | S =-=i nn! I= i = _ if if es Wlh of loc into ratdue to; c by the> i last rc-jt fibrous, protect : in their hydroxk The : from bio into the other 0 nurner reaction in agree Asbf intrated maierkj soltittoi The asi.aftcr in; rather rt treattner which rt that the: bv Eeiri: Two and re2C In the c: assumed in this p capable r is merely untenable 12. Gir: d'aniiacir." 13. Bek. von Bcrer; VORWALD ET AL, .DIES OF ASBESTOSIS 39 Protective Actios or All-hincm Compounds When colloidal aluminum hydroxide had been added to a suspension of long fiber chrysotile prior to injecting this s..-pension intrarrnclu-.illy into rats, the aluminum compound did not prevent the irritation of tissue due to chrysotile. If anything, the acute inflammatory resi>onsc evoked by the injected fibrous mineral was accelerated. One month after the last injection of the dust suspension the bronchiolitis was becoming fibrous. King and his associates also found that aluminum failed to protect pulmonary tissue from the irritation caused by asbestos fibers 11: in their experiments metallic aluminum wa> used instead of the hydroxide. orFormation Asbestosis Bodies The iron in the coating of the asbestosis body apj'ears to be derived from blood or tissue elements and not, as has been suggested, from the mineral fiber. After two kinds of chry'otilc were infected stibctnanconslv into the groin of a guinea pig--one kind containing 2.per ant and the other 0.2 per cent ferric oxide--the asbestosis bodies were equally numerous at both sites of injection and showed ms difference in their reaction to prussiau blue, the reagent which stains iron. This finding is in agreement with that of Giroux.15 Tissue Reaction to Asbestosis Bodies Asbestosis bodies recovered from human lung tissue and injected intratracheally into guinea pigs failed to produce a fibrous reaction. The material for injection was obtained by digesting with sodium hypochlorite solution tlie lung tissue remove-.! at autopsv from an asbestos wcrkcr. The asbestosis bodies could be seen in tiie guinea pigs for at least a year after injection. This experiment shows that the asbestosis body has a rather resistant coating which is not destroyed by moderate hypochlorite treatment, which may be maintained in vivo for a year or longer anti which renders the fiber incapable of producing fibrosis. It thus appears that the coating is a protective mechanism. This thought was expressed by Beintker as early as 1934.'1 THEORY OF IRRITANT ACTION Two hypotheses have been proposed to explain the tissue irritation and reaction caused by asbestos fibers: the chemical and the mechanical. In the chemical theory, which is based on experience with quartz, it is assumed that the asbestos minerals dissolve in the body fluids and that in this process their bases are leached away to leave silica in a form capable of irritating tissues. According to this hypothesis asbestosis is merely an indirect silicosis. Several facts make the chemical theory untenable: Intratracheal injection of brucite fibers, which had a silica12 13 * 12. Giroux, M.: Amiantose experimentalc: valeur patiiugnomoniquj du "corps d'amiante." Laval mi-d. 8:239, 19-43. 13. Beintker, E.: Cbcr die A = bestosisk6mcrchen: Bemerkungen zu der Arbeit von Beger, Virchows Arch. f. pa-ii. Anat. 2C3:;27, 1934. 40 INDUSTRIAL HVCHEXE AM) OCCUPATIONAL MEDICI* content of only 0.90 per cent, caused typical fibrosis like that produced by the asbestos minerals; free silica, p' rticlcs increa-e in potency as the particle size becomes less, but a-botus fillers shorter than about 10 to 20 microns arc relatively innocuous; aluminum hydroxide neutralizes the irritating effect of quartz but not of asbestos; serpentine has the same chemical composition as long fiber chrysotile, but it produced only an inert type of tissue reaction; tit-, re is a wide range in the chemical composition of the minerals which dn cause asbestosis (table 19). In view of this evidence it seems more likely that asbestosis is caused by an unusual mechanical irritation due to long asbestos fibers, this irritation being related to the peculiar fiiamcmcd structure of the fiber and the associated flexibility, which arc possessed by no other foreign body studied. Thus, ignition of chrysotile fibers changed their structure and made them inert, although the same nbersL before being heated, would have produced fibrosis (table H). Further support for the theory of mechanical irritation is that asbestosis occurs in an organ of high mobility --the lung--and that a fibrous reaction can be produced by injecting ; ' j .' > Tault 19.--Analyst's oj Fibrous Minerals FlOrous Mlnenb SlOi ^ AmOfltr.......................... 46.3 AmphlhOlf.......................... ii.04 Aolhophyllilr.................. i>.sO UruHtf.............................. 0. 0 Chnruoni*..................... as.v. Crocidoi'i*.................... s< '/t TTtmulitr...................... FhO. % < 06 3.00 0.17 * 7< :..ia r.'t- Ml FtO % AlrOi % 1.09 1.69 9.16 0.3 0.40 ;o os< . loi ....... OX C0 i S OI 11.3 044 O0t o.oi o.> 4.11 M*0 % SS 3.19 3.37 S3 99 :i.m t;.i> SO.S3 NtrO 0.3 0.43 O.S2 0.0) o.-js s ee '1.7- EiO Sfc 0. 0.11 0.15 0.1S o.o> o.57 0.:" Ignition < lUu C. > 106 C. SV % 0,{ S.3 0.3 1.80 0.17 nM 4.08 S.M .so o.i?e ii. s.ju O.'t 2.7? Tm, T. Hr 90:: 99.7 asbestos fibers into the peritoneum, where there is also a degree of mobility, but not by injecting them into other extrapiilmonary organs such as the liver, the spleen and subcutaneous tissue. COMPLICATIONS The experimental investigations with asbestos minerals were con cerned primarily with the effect of the dust on normal tissue, but some attention was given to other phases, such as susceptibility to infection. The only experiment in which the effect of asbestos dust on a pulmonary infection was studied was the first inhalation experiment, carried on with King's floats dust. It is unfortunate that, owing to the lack of adequate facilities at that time, infecti-m studies could not be marie in the other inhalation experiments ai:o. ScscEpnmurr to Tcsijicilocs Instctiov The development of a tuberculous process initiated at the beginning of exposure to dust, and also of a tuberculous infection superimposed on an established asbestosis, was described in preceding sections of this paper. It may be stated that asbestos when classified according to the effect of a dust on tuberculous imection would be placed below an active I v t VC dust like qu: infected wit process to p have no eft: disease disa: experimental mg the evolu process lor a the tubercle healing tolli bacilli after years, progr the infection fibrous term; the usual iotr. There \yr asbestos dust rather comm in guinea pig 16 to 39 per an effect or since such ep other casts c inhalation of susceptibility Owing to seems most c< the various c follow each w A. Various s rabbit, it fibrosis o; by inhalat fibers. Both inha for this staterin grnirea pig1; the fibrosis cr Similar but "I (table 1). M of different sp B. Long asbe chiolar fibr I'OKWALD ET AL.- lEE or Asnr.sTOSiE 41 dust like quartz but above an inert dust such as iron oxide. In animals infected with attenuated tubercle bacilli, quartz causes the infectious process to progress until the animal dies of tuberculosis. Inert dusts have no effect on the infection, and the lesions usually heal and the disease disappears. Ashestos dust is in a ditTerent category. In the experimental investigation, when the fibrous dust was being inhaled dur ing the evolution of the infection, there was spreading of the tuberculous process for a time, but usually the stimulus for continued proliferation of the tubercle bacilli was not sustained, the progression was arrested and healing followed. In guinea pigs infected with attenuated tubercle bacilli after being exposed to asbestos dust for slightiv more than two years, progressive disease did not develop. Tlie only modification of the infection was one of localization, a few bacilli being retained in the fibrous terminal bronchioles and forming tubercles there, in addition to the usual foci beneath the pleura. Such tubercles healed in a few months. Sc'SCXPTtB'.UTV TT N'oNTVHK.RCCUiV- I \ mT'.liS There was no specific exj'eriment concerning the effect of inhaled asbestos dust on nontuherculnus infection. Intercurrent pneumonia was rather common among animals exposed to asbestos dust, the frequency in guinea pigs exposed in the four inhalation experiments ranging from 16 to 39 per cent. This incidental evidence suggests the possibility of an effect of asl>estos dust on nontuherculous infection. Nevertheless, since such epidemics are not uncommon in inhalation experiments with other dusts and even in the colony of normal animals, it is feit that the inhalation of ashestos dust does not exert a significant effect on the susceptibility to nontuberculrms pulmonary infection. COMMENT AND SUMMARY Owing to the vast amount of data included in this investigation, it seems most convenient to summarize and to state as concisely as possible the various observations which emerged from the experiments and to follow each with a brie: resume of the evidence. A. Various species of animals, including the guinea pig. the rat and the rabbit, but not the mou.-e ami the dog. devci.-.i> peribronchiolar fibrosis of the lung similar to human asbe.-iosis aiter being exposed by inhalation or intratracheal injection to long chrysotile asl>estos fibers. 1 Both inhalation and injection experiments provide ampie support for this statement. Figure S A reveals the cellular fibrosis that occurs in guinea pigs following inhalation of long fiber asbestos; figure 9 shows the fibrosis caused in the cat by inhalation of long fiber asbestos dust. Similar but less extensive fibrosis occurred also in rats and rabbits (table 1). Mice and dogs failed to respond. This variation in response of different species to identical dust exposures is still to be accounted for. B. Long asbestos fibers are essential in the production of the peribron chiolar fibrosis; short fibers are incapable of producing this reaction. 42 INDUSTRIAL HYGIENE AND OCCUPATIONAL MEDICINE Inhalation experiments with asbestos dust surest, and intra tracheal injection experiments confirm, that peribronchiolar fibrosis is \ produced by asbestos libers between 20 and 50 microns in length but not x 1 by particles shorter tlun 20 microns (tables 16 and IS). Tin's indicates that the minimum length of filler possessing the capacity to produce the typical peribronchiolar fibrosis in animals is somewhere between 20 and 50 microns. Pointed studies have not been carried out to determine the upper limit of effective filler length. It appears, however, that that limit will l>e determined hv the irhalability of the fiber. C. The mode of action of the long asbestos fiber in the production of asbestosis is primarily mechanical rather than chemical in nature. The evidence for this conclusion has been reviewed in a preceding section, page 39. The flexible filamented structure of asliestos fibers plays an essential part in the irritating action, since the solid, inflexible__ _ fibers of glass wool do not produce nluo-is (t y. in !>). D. Typical experimental asbestosis was produced by the inhalation of an atmospheric suspension containing an average of Pits million asbestos particles per cubic font of :dr by light field count, of which less than 1 per cent consisted of nhers longer than l(j microns. In the inhalation experiment with 100 per cent l*all-mil!ed asbestos dust containing 0.6 pier cent of fibers longer than 10 microns (table 11) (typical fibrosis was obtained (table 9). The evidence presented shows at least that an atmospheric concentration of asbestos dust containing less than 1 million f0.6 per cent X 13S million) filters longer than 10 microns per cubic foot of air is capable of producing experimental asbestosis in guinea pigs. The rici.i.-.i low er, limit oi m.. duration o: long fibers nerecc.iry to produce asbestosis in animals cannot be estab lished from these..studios. E. The duration of exposure required to develop the pulmonary reaction to inhaled asbestos dust is inversely proportional to the concentration of long fibers in the atmosphere; as the concentration is increased, the reaction develops in shorter time. The basis for this statement annears in the data of the inhalation experiment with long fiber asbestos. For that experiment the average concentration of the atmospheric dust was about 40 million particles per cubic foot of air, and size-frequenev deter:.filiations disclosed that 6.7 per cent of the air-suspended material consisted of fibers longer than 10 microns <table 11). Thus, hy calculation, it is estimated that the con centration ci the longer fibers was 2.7 million f 0.7 per cent y 40 mil lion). The lungs of aniiuais exposed to the long filler asbestos dust revealed that the pulmonary reaction developed in approximately onehalf the exposure time required for its development in animals inhaling the ball-miiied product, for which the concentration of the longer fibers was only 0.S million (0.6 per cent X 13S million;. F. Established experimental asbestosis ceases to progress on discon tinuance of du.-t exposure. The experimental investigation shows, in fact, that on discontinuance of exposure there was an appreciable clearing of the mature pulmonary' I I. * 5 I ( c f1 l I t lesion; ture. t fibrosi. of fibr noted ; G. Th by fib In;: typical of prog due to H. Alu Ion A lb prior f : of as'.-c- I. Inna, of e.' the c The to the; prorccs since it ex; - -f:: shows t. exposur: with sub dust exp The latte dust or u of quarar infectic-ri on a bad sensitive the latte: inability tubercu.T inhaled k. pulmonar This ii group of c VORWALD ET AL.--ST ies or ashestosis 43 lesions, due to contraction of the fibrous tissue. In contrast, an imma.ture tissue response, evidenced primarily l>\ ccIN with little nr no fibrosis, continued to progress. It is assumed that, following attainment t of fibrotic maturity, th.e same process of contraction would ensue as was noted for the mature lesion. G. The formation of ashestosis bodies represents a coating of the fibers by blood and tissue elements, which results in loss of ability of tlie fiber to produce fibrosis. Intratracheal injection of ashestosis !>odie failed to produce the typical asbestotic tissue reaction in experimental ammais. The conation of progressive reaction observed scon after expu.-uri- terminates may he due to the formation of ashestosis bodies. H. Aluminum hydroxide failed to neutralize the fibrosing action of the long fiber asbestos. Aluminum hydroxide added to the suspension of chrysotilc asbestos prior to intratracheal injection did not retard nr prevent the devr'-.mimt of ashestosis in rats. I. Inhalation of asbestos dust did not alter significantly the final outcome of experimental tuberculosis in two series of guinea pigs esp ied to the dust. The apparently mild influence of asbestos dust is in distinct c'-ntrast to the stimulating effect exerted by inhaled quartz on a tuberculous processjn the lung. The interpretation must remain tentative, however, since it is based on an investigation limited to two series of guinea tugs exposed to onlv one kind of asbestos, nameiy, King's flea's: Ta: !e 4 shows that when the infection was coincidental with the or.^et of dust exposure, there was tctn:*ornry progression cf the .r.tccttous t r. . with subsequent heaiing; witen infection was initiated after 26 mouths of dust exposure, the course of the tuberculosis, was r.ot appreciably altered. The latter finding is quite different from our usual experience with quartz dust or with mixed dusts containing quartz, wherein the adverse influence of quartz on a tuberculous infection is manifested mo- strikingly when infection is initiated after a period of dust exposure, viz., superimposed on a background of established silicosis. As indicated above, this more sensitive test, when applied to asbestos dust, faded to demonstrate that the latter had an adverse influence on a tuberculous infection. The inability of asbestos dust in that experiment to affect unfavorably the tuberculous process furnishes strong support for the interpretation that inhaled asbestos dust has no more than a mildly unfavorable effect on pulmonary tuberculosis. This investigation was made possible by the generous nn.'.ncial sup[>or: of a group of companies of the asbestos industry.