Document YG54xYee1O2bdmE5arxwx6QLV

>1 At Urctilm ol Industrial Hygiene and Occupational Medicine Volume 3 JANUARY 1951 Copyright. 1931, y the Ameriiji.v Meum.u. Asmm i inns Number 1 % EXPERIMENTAL STUDIES OF ASBESTOSIS ARTHUR J. VORWALD, Fh.D.(Poth.), M.D. THOMAS M. OURKAN AND PHILIP C. PRATT, M.D. SARANAC LAKE, N. Y. ASBESTOSIS is a form of pnetnnonoainiosis resulting from pn>aa longed inhalation of asbestos dust. The name "asliestos." literally "unburnable," 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 iiuhpte Ijecause the fibers are capable of repeated longitudinal sulxlivision to units of molecular proportions. In length the filters vary from a few microns to 6 or more inches (15 or more cm.). Some varieties are stiffer than others, but many are sufficiently flexible to lie spun into yarn and woven on modified textile machinery. The asbestos minerals are silicates of variable composition and belong to the serpentine and the amphibole groups. Listed below are the more common varieties. Amphibole group: actinoiite, nmosite. amphibole, anthopbyllitc. crocidolite and tremolite. Serpentine group: chrvsotile. The bulk of the asliestos of commerce is chrvsotile, 3Mg( >.2Si< 2H-0. which is mined on this continent principally in the Thetlord region of the Province of Queliec, Canada, and in Vermont. Crocidolite and amosite also arc used commercially but in much smaller amounts. Chrvsotile occurs as veins in serpentine, a mineral of similar chemical composition, which exists in massive form and is made tip of micrownpic filiers without the |ianillel orientation characteristic of chrvsotile. The massive, bluish black scrjientine. which is smooth and soapy to the touch, is traversed hv veitts of fibrous chrvsotile varying in width from a liarely perceptible line to 6 (15 cm.) or more inches. The fillers run across the vein and not lengthwise with the formation. From the Saranac I.hnratory nf the Edward (.. Trmlrau Foundation. This series of studies of asbestosi*. initiated at the Saranac laboratory inure than twenty years ago by the late Dr. I.croy (J. (iardner. direettir of the lalmratorv. was nearly completed at the time of hi* death in Octohcr 1946. Althotudi |iartial reports and informal reviews of sonic of the experiments lind been given frum time to time by Dr. Gardner, this paper presents for the first time a complete survey of the entire experimental investigation. 1 PLAINTIFF'S EXHIBIT SA-530 2 ixnrsrKi.ii. in'ciExn axp ntrrr.mox.-n. mepicixf. \tteiitum i- diri'ctnl tn tin* mineral hrucite. My* >.li,.( . which is iiiuii found in ihf ..nmt1 formation:. with >crpcutine and chrysotile and max In- tilin*ii- in 'tructurc. Kxrcpt lor tin- mnnufaciurc of magnesium. I.niiitc 1 s.'j- mi i-i'niMH'ivinl valm- at promt lavaii'c it' Ithers are not .tiltiiimth llexihk- l'1 he u-eil m textile'. hut thee arc capable of repeated lutiy imditi.il -uhiln I'imi. l nlike utlier a->ln->tilunii mineral', briicite i- m>t a ilicnie. and inr till' re.i'oii it ha.' Iieeu a ealuahlc tool in an experimental evaluation of tlie action of libruii' mineral.' mi limy ti'-aie. I- VIM-KI M KX I At. X.'lll'loMS For main x ear' 'Indies1 have heeii carried on at the Saranac r.ahoratory in an investigation of the cause, nature ami development of a'hoin.'i,'. The present paper i' devoted to experimental asbestosis. Fig. 1.--Human asbestosis (P-36-144). The photomicrograph reveals a bronchi ole (right center) with a smooth muscle bundle at its inferior margin and with an extensive rone of collagen deposition largely obliterating the surrounding alveolar structure. The black foci are macrophages containing incidental pigment. Asbestosis bodies are present but are not apparent at this magnification (x 200). 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 health 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 nary fibrosis which requires years to develop, it is possible to reproduce 1. (a) Gardner. L. U,, and Cummings, D. E.: Studies on Experimental Pneumokoniosis: VI. Inhalation of Asbestos Dust; Its Effect upon Primary Tuberculous Infection, J. Indust. Hyg. 13:65 and 97, 1931. (b) Gardner, L. U.: Chrysotile Asbestos as an Indicator of Subtile Differences in Animal Tissue*, Am. Rev. Tubere. <3:762, 1942. VORWALD ET AL.--STUDIES OF ASBESTOSIS 3 in one or more species of animal characteristic tissue changes which are similar to the lesions of human asbestosis (fig. 1). Since the life span of the experimental animal is relatively short, it is not possible to produce the characteristic lesions in animals under conditions identical with the isual industrial environment. Consequently, to obtain a complete evalu ation of the tissue response to inhaled particulate and fibrous material, it is necessary to accelerate the reaction by employing higher concentra tions of dust than would ordinarily be encountered in industry. While conditions of exposure are thus different, the information yielded by animal experiments is invaluable in furnishing a better understanding of the reaction of the human organism to inhaled asl>estos dust. 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 or 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 asliestos dust is maintained by a rotating paddle ina 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 years. The injection exjwriments are used to deter mine in as short a time as possible whether or not a particular dust 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 hy the intravenous, the intrajK-ritoneal. 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. Kven though an atmospheric dust may lie jxitentiallv dangerous, its indicated by injection experiments, only inhalation procedures will reveal whether the dust can lie inhaled, pass the natural defense liarriers of the body and reach the pulmonary tissue in quantities sufficient to cause damage, injection methods arc u>efu]. however, because they make certain that contact occurs Ix'tween the dust particles ami tissues and lwcause they allow accurate estimation of the dosage and of the potential capacity of that dose to produce reaction. The intratracheal method is particularly valuahlc when one is dealing with librotis minerals like asbestos, since it permits observation of the effect of the libers on pulmouarv tissue. Tissue Scsi ei-iuiii.itv Unlike free silica, asliestos 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 slightlv from a-preliminary report.,h Fine quartz introduced into various organs 4 IXDLSTKl.U. UYGlEXh AXD OCCri'.lTIOXAL MEPICtXE <>i' various auim;tl> i guinea pi.tr. rabbit, rat. mdii.se, cat. dog. chicken and even tadpole) eventually "'ill pmiliiee silientic nodules but at different Similar inmnlm-ti"n nf lung fiber anbe^tos has resulted in a lilinm^ reactii>n m the lung and. tn a Ic-mt extent. ill the peritoneum 'in imi m ..ilier 'iretm- "i the guinea pig. the rabbit, the cat and the white rat. In mir e\perieuee llie lung' ni the dug and the while turnise tailed in re-piind with li 1 ini-i~. allh"iigli vi lui'ler lia- reported Mich change', in ;t dug that liieil in an a'lio'in- iabruaiing plant. Thi- variatimi in 'pecic. and in nrgau iiicepiihility L jet in be accounted tor'; it i> prc-inued that m the 'it-ceplilile aiiiinaU the greater react inn oi tile hmg lo a-lie'tos. iaf exceeding the reaction oi other organ ti>sue>. in due prmcipallj to the greater mnbilitj oi the lung. I'kCI 1 1 \ll ClI \H.\C*l KHI>Tll > UK .\>IIK>TllN K\perieuce ba> demonstrated that most ni the uoniibrou.s dust particle.- inhaled into the lungs ni man and animal are 10 microns nr less I'.xia.K I.--Rearlinii t<> t.iuhi l-'iber Chrysolite in Littins f Man anil Other .s'/vr/of i'/ ./ilium/ nun....................... tiiiiui'n imr............ Uni.liit................... Cut........................ While rnt.............. Wliilr Iiuiiin-......... Dnir........................ Mnili- o( K.\|'n-ari' Inhalation Inhalation Hint injiYtion Inlinlatiun ainl Injeetlou Inhalatlno amt injection Inhalation ami injection inhalation Injection Klhroria 4+ i+ + + + 0 0 .tiheatoMt Rn<lica Numerous Moderately ouinrnuis Ron and atypical Ran and atypical Very rare Rare aoil atypical Son* * Tl'i* ynihuls n to t- ri'ii'r tu me iieyrre of tissue reaction. in maximum dimension. Larger partieles 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 200 microns in length have heen 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. .V large proportion of nonfibrous particulate dust inhaled into the lung is found in tite 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.* Their2 3 4 2. Schuster, X. H.: Pulmonary Asbestosis in a Dog, J. Path. & Bact. S4 (Pt. 21:751. 1931. 3. Vorwald. A. J.: Variations in Individual Susceptibility to Industrial Dusts Inhaled into the Lungs, Am. Rev. Tuberc. (S: (IB) 13, 1950. 4. Miller. \V. S.: The Lung, Springfield, 111., Charles C Thomas, Publisher, 1937................................. 1 and erent in a teum vhite ailed .nges in in it is lung due dust kss r eras that irge s of tors iave >raiber > Ik- li) ir.st iri heir Justs her. VORWALD ET AL.--STUDIES OF ASBESTOSIS 5 own essential lining is a low cuboidal type of epithelium but, as their name implies, they actually function in respiration througli lateral alveoli distributed along their walls. Either these alveoli or the abrupt change in the character of the lining epithelium, or the small diameter nt the respiratory bronchiole, or the combination of all three factors i< responsible for retention of the fiber at this site. Only after asbestosis is well established are appreciable numbers oi fibers seen in the more peripheral air spaces. Further explanation is required to clarify this observation. Rate of Tissve Reaction to Asbestos Fibers The affected tissues react much more rapidly to asbestos than to quartz dust. For example, in rats receiving asbestos fibers 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. Thus, the development of nodular fibrosis due to inhaled silica lags behind the deposition of dust to a greater extent than does the evolution of the diffuse reaction to asbestos. This results in a difference in the degree of progression which follows termination nt' exposure to dust. For example, on discontinuance of exposure the nodules of silicosis become larger, to a limited extent, for a considerable period of time, whereas the fibrosis of asliestosis increases for only a short time. Subsequently, the ashestotic fibrous tissue contracts; this process often distorts the adjacent pulmonary tissue and may. as a reMilt. progressively interfere with cardiorespiratory function. Asbestosis HlllHKS The peculiar structure known as the asbestosis IkxIv or "curiou.body" is a specific concomitant of asliestosis.5 6 The typical body is a golden yellow, beaded or hattstrated rod. which may he either straight or curved (fig. 2). Often one or both ends are huliious like a dumbbell. The lx>dies vary considerably in length, and dimensions up to 250 microns have lieen recorded. It is lielieved that asliestosis Iwdies are inhaled fillers on which pro tein and iron pigment of tissue origin have been dqxvdtcd." (iUnne-1' observed reproduction of these Iwdies in guinea pigs nine mouths 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 liecause the larger-sized air passages admit fillers of greater dimension. In guinea pigs they form after about 70 davs 5. Gloyuc. S. R.: (ii) The Fnrniatiun of the Asliestosis I tody in the hum*. Tubercle 12:39ft, 1931; (M The Asliestosis I tody, haunt 1:1.151. 1952. in < ianlner ami CuiimiiiiRs.'* 6. I.yncli, K. M.. ami Smith. \V. A.; Asbcstusi- Bodies in Sputum nml I.uiig. J. A. M. A. M:(i59 (Auk. 30) 1930. Simsmi, F. \V,, ami Straclian, A. S.: Asbestosis Bodies in the Sputum: A Study ul Specimens front 50 Workers in an Asbestos Mill, J. Path. & Ilact. 14:1, 1931. Gardner ami Cummmus."* Gardner.16 Gloyiie.-*. a 6 IXPrSTRUL IlYGIEXE AST) OCUTATIOSAL MEPICIXE if contact with the tissue. In cat.-, rabbits and mice a few of the fibers ,,hou an atvpical coating after ntuch longer residence in the lungs. In rats the bodies are rarely seen, and in dogs none could he found. Although the evidence is incomplete, it appears that the formation of the adit-st..i Iiiidy prevents the fiber from damaging the tissue. Many the I'tiim- mentioned above will he elaborated on in subse(|uent para- Fig. 2.--A, human asbestosis bodies. This collection of asbestosis bodies was found in the lung shown in figure 1. The usual variations of size and configuration are represented (.x-WO). B, guinea pig asbestosis body. This one is similar to some of those shown in A (X 400). 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. I'ORIVALD ET AL.--STUDIES OF ASBESTOSIS 7 inhalation experiments Four large scale inhalation experiments have been conducted in this laboratory with various forms of asbestos dust. In each of these investi gations. more than 160 animals were used, and the experiments were carried on for periods ranging from two to more than five 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 Dust 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 Dusting Materials Type of AJbtfto: SlOt FecOa .UiO Cr.Oi MnO CaO King's float!........... 6.ft* * 0.07 hort flber............. 9.09 1.40 o.u 0.09 0.83 Lonf fiber......... .. 33.40 5.3t 0.78 0.03 0.31 Mil) XaxO KjO CO; Hi.Vt * * 33,96 0.14 0.20 0.93 40.13 0.06 0.06 0.47 Uni* non Lost Total 12.74 14.09 14.00 97.13 100.11 99.79 Not determined. Table 3.--Petrographic Analysis of Asbestos Dusting Materials Klog'i flotti : The approximate compoeltlon, bawd on particle; (except chryiotllr) .mailer than 10 micron! and reported ai percentages obtained from particle count!, was cbrjrrotlle 14. -erpentloe 40, magnetite 12, carbonate! is. talc 12, other mineral! 4. For chrrsotllr. fibers up to -HO micron! lone were Included. Short flber t: The material, before bring ball milled, coutalnrd a prcpomtcraoce of flhrout chrreotlle and platr (oonflbrou;) serpentine. The approximate competition, by percentage. ' chryaotUe 17, eerpentloe iS. magnetite 10, quart! 2. hruette i, other mineral!, including dolomite, actlnollte and ttamolite, 11. Long flber t: The material eoneleted principally of the flhrotia aibesto; mineral chrreotlle. Shredi of noneeparated flbera S to IS micron; In diameter amt up to SO micron; In trurth were preeeot. The approximate eompoelttoa, hr iierrentaire. wai chrreotlle is. terpentine IS. mag netite S, bruelte 2, other mineral;, among which were ralcltc amt cldorltlc and micaceous mineral!, 2. Onlr a traee of quart! wai obaerred. *The analrala of the King'! float! aebaetoe, made hr Hr. ('. s. Iftirlhiit Jr., of Harvard I'nleertltr. hai been reported eleewhere (llurthiit, (.'. S.. .1 r-. anti Wlllluiue, (\ R.: The Jllueralogr of Aabaatoa Duat. J. Induit. Hrr. A Toxicol. 17: '-"Si. licISI. i For the hort flber aabeeto; and the long fllier a;lK-.n<. the i- trotcrapbic rwmlr*!. a a* .implemented with x-ray dlflractlon exainlnatlou. IHiriods up to 33 months. Some guinea pigs with six and nine mouths' exposure lived for an additional three years after cessation of their i-xposure-. A preliminary rqxirt '* presented observations after months of exposure. At that time observation;, covered a period of only 214 years anti the conclusions as to the ultimate effects of inhaled usIxi.stuH dust were provisional. Those etinclusions are substantiated In results of the completed study, which is reported a> follows. Composition mill Alnuisplierir ( mn niimltmi of the /'./ --The ilti-diim material, a cimimcrcial variety of asliestnx known ax Kind's lloats. u.is conitxised of short liliers, ratiKiiiK in lonatli from 1 mm. to I micron or less, and of iiarticles which also varied.in size. It was nbtanu-d from the Titctfnrd, Queliec, plant of the Asbestos Cnrixiratiun 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 IXDCSTRt.lI. HVi'IEXF. AXD OCCCPATIOXAL MEDICIXE Impingcr .-ample*- taken *'ii after the experiment was started indicated that the dust c'im-emratiii ua- at first i|inte lntv. the average dust count being only 'i0 millimi particles pel culm >''t >'t air he the 'tnuilanl light field technic and 0.8 milium tnr jiartieles and lil*ei' meater than III iniei'eiis. After the inhalation . end tnu nt lial heeli millet way tm aUiin twn vrar**, the spied 'if the rotating pinldlc in 'lie iliisitne nhi. tune w.i** mi 11 .i**i *1 .uni hh the t i-tn.itiinig III niiinllis "I* the cx|H*rimeiil 1't'iisnlei ahle nime ilti'-l w.i. .lispet *eil niln the ,iliti"*.plii*i v. The averaue dust 11 'lint cl itnpmnet -.inipli- mtlnii'l aitii this ih.inne was 5.1.7 ntilliiin hy the usual lieht tii-lil inelliml ami I ( niitfii'ii Im |iai lu les ami liliei s lareer than |(f microns. It is iM'iihahle lii.weni. th:u the true values nf the dust eiiiieetitrattini were IngliiT than the i ntuits mi hi in this |iaraeratih. The iiupmccr samples tnr the King's iln.il' espeiinietit weie mllecieil in water, hut later studies1 have shown that founts "i iinpineer samples of .isU-sins dust taken in water are tint reliable, l-'.thyl ali-nhnl instead of water was used as the collecting fluid in all subsequent experi ments. T.iiiik 4.--Summery of Inhalation Expert mail with Kiwi's Flouts Asbestos Dttsl Nature ot Experiment (ii*' out JlJe continuous through Dum rxt'itjturc Mlimrd Uy pro* (tutiri'il r*-iU`ticv in normal air Ttitii'rt'iilttus Infection * at *tart ot int i*MH<urr i f.otrol' ! inaction: no dust exinjure Tuu-rmimip infection * niter 23 mo. ot ilti-t evpomre, then residence nt itorimtl atr t utirrol* to luici'tion: no dust ex- pus-ure AuiinuU 34 fiilnet pigs 9 rabbits IS rats 13 guinea pigs 23 guinea pigs l rabbit l rabbit 40 guinea pigs 23 guinea pigs ti guinea pigs IS guinea pigs Maximum Uuxi- Survival mum After Hint Kx- Pust Kx- 1, poaure, Mo Mo. Baauft* 33 0 Typical peribronehlnlar flbrosla alter It month19 0 KnrHgti UoitT bronchitis 0 0 I.lttlv or on TcactloQ 6 IS Nonprugressivc fibrosis 9 17 Noupnigresuvc fllirosia 6 | Abiorptlon ot toreUtn body reaction 19 : 0 Temporary progression ot infection, followed hy healinr with Itbrocl* 0 Sit Hrallnt by reeolutlon (on* exnptlum 20 It No apureeiahle Increase In susceptibility to tuberculoua iotcctioo; heeling with llbroeli 0 19 t Healing by retoluttoa - Tin* k'uiin me- were mlerti 'l with tow virulence St rtrclo of tubercle bacillus. This incuiis iln* survival period following Infection. Results of the investigation, briefly summarized in table 4, show that inhalation ot King's floats asbestos dust produced a typical peribronchiolar fibrosis in guinea pigs but not in rabbits or rats. Reaction in Normal Guinea Pigs.--Guinea pigs inhaling this dust for periods up to J5 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 tQ 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. 3 A). As more dust was inhaled, it continued to accumulate in the same location, and later stages of the disease (fig. 3 B) 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 they7 7. Fulton, W. B.; Houtz, R. L.; Dooley, A., and Mathews, J. L.: Asbestosis: 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. VORWALD ET AL.--STUDIES OF ASBESTOSIS 9 ,..rc phagocytosed. and many of them were carried into the wall by migratory ,U. Mononuclear leukocytes attracted to the area caused an appreciable thicken .... r.f the bronchiolar wall. After 16 months a delicate fibrosis made its appearance. e process evolved gradually, and the number oi fine intercellular collagenous crs steadily increased. As this fibrous deposit contracted, it partially closed and a >j ' | I tit. .1.--King's floats inhalation i-x|riiiicnt: .1, lime of a guinea pig with 12 1',mils' exposure. It includes a respiratory liimu liiole. at I lie left, branching ami "lining an alveolar duct, at the right. Note the arrttiitulalion of cells m the a-.11 of the bronchiole and in adjacent alveoli (;< IJD). II, lung of a guinea pig with 28 months' ex|Kisure. The lielil includes a hroiichiule. at the center, with Peribronchial fibrosis extending into the walls of adjacent alveoli. Note the culxiidal Milt helium lining these alveoli. This is the so-called "adenumatnid'' appearance (y, 2(10). distorted the alveoli, and with this change the alveoli became lined with cuboidal cells. The result- was.- an adenotna-like appearance which frequently accompanies io ixin si ki it. nvninxi; .ixn <m rr.i uox.n. .uldic/xf. i'Iii piilini'iun iiiilaiiiinaliixi n-iiltiiiK in mi many cause*. Willis" described a Mind.it -11111 mu- m the limu- a isuiuca mg- mlulnnt mIh'ixi carbide. The lunger i-lif-i.t- r\|i.*-inv- n-iilli-il miiIv in ni*iii* 11 n< l.rimiK *i iltr uall- ,tf tlie air xp.ivo-, '.tieili tliic I** an iin ica-i in tin- .iiii"nn| m| iilnii-,).. J |i(- fiiifnii*. ii--ur aiivav*. I . llul.MIII I I - . . I I I III I I.lllld Im -IlMII till lll.llllll / lltllll I ll.tl.ll l|'||-|l| Ilf -lilt lisl- ' Fig. 4.--King's floats inhalation experiment: A, lung of a guinea pig with six months' dust exposure followed by 35 months' inhalation of normal air. The reaction is rather slight, but distinct fibrosis is present (x 300). Note that 28 months of continuous exposure (fig. 3 B) produces much more extensive reaction. B, lung of a guinea pig exposed to the asbestos dust for nine months and living thereafter in normal air for 37 months. The reaction shown is more than that in A but much less than the reaction in figure 3 B (X 200). 8. Willis, H. S., and Brutsaert, P.: Tumor-like Structures in the Lungs of Guinea Pigs Artificially Exposed to Silica Dust, Am. Rev. Tuberc. 17:268, 1928. Asbestosis t inhaled dust segmented u The rvac pmerest -nt animal- kvr- .liter rxpysur by thin stra auiuunt. but -,4t fibrose, Rcitctu'n InhnLuiun,--( bacilli, R, st killed bciore paper by Oa: pneumonia. . spread of the the lungs, anc ol the infectic next 20 mont culosis, and i months no an them the heal* more extensiv, The nine a killed at inter of infection w evidence of pr showed evident of autopsy, the was a general lobes there we spread. Reaction in Asbestosit.--Tt months, were i of these aniim. infection. The after infection, animals than in the depths of th combined reacts and tracheobror old, but by 554 Foci of fibrosii infection. Reaction m 19 months show Although their present, indicatir to exclude fibroi. 9. Steenken. Its Dissociation Am. Rev. Tuber VORWALD ET AL.--STUDIES OF ASBESTOSIS 11 \sbestosis bodies (fig. 2B), first seen in the lungs of the guinea pigs that had .1 haled dust for about two months, became more numerous and more distinctly -egmented with increasing exposure. The reaction produced in guinea pigs exposed for six and nine months did not .rogress significantly during a subsequent period of 35 and 37 months when the .mmals lived in a normal atmosphere (fig. 4). Between eight and 11 mouth.' nter exposure ceased, the cellular reaction in the lung had been completely replaced liv thin strands of fibrous tissue. At later periods the scar tissue was less in amount, but in the last animal killed, 37 months after discontinuing dust exposure. ..me fibrosis was still visible. Reaction in Guinea Pigs Infected with Tubercle Bacilli at the Onset of Dust Inhalation.--Of the group of 40 guinea pigs intected with attenuated tubercle bacilli. Rt strain,* at the time that dust exposure was begun. 31 died or were killed before the completion of two years oi the exposure and were reported in the paper by Gardner and Cummings.1* Seventeen of these died from imercurrent 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 of dust inhalation: 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 of them the healed fibrous scars of previous spreads were obvious. The scars wenmore extensive than is characteristic of either tuberculosis or asbestosis 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, hv the time of autopsy, the foci were healed, with excessive fibrosis; in the fifth animal therr was a generalized chronic tuberculous pneumonia in one lobe, and in the other lobes there were isolated primary tubercles, which were still active hut had tint spread. Reaction in Guinea Pigs Infected with Tubercle Bacilli After Establishment Asbestosis.--Twelve guinea pigs, after inhaling King's floats asbestos dust for 26 months, were infected with tubercle bacilli and then removed to normal air. Six of these animals died within seven weeks, five from intercurrent nontuberculous infection. The remaining six animals were killed at intervals up to 14 month' after infection. The tubpleural tubercles were no more numerous in the dusted animals than in the nondusted controls, but a considerable numlier were found in die depth* of the lung about fuci of asbestosis. The tuberculous com|ajnvnt of the 'oniliined reaction sltowcd only slight local extension about lesions in the lungs .owl tiac iHxilirnurliial lymph mules. Caseation was found in tulierdcs V/j mouths <ld. lull ly S'/, months it had completely disappeared, leaving only scar tissue, l-nci of fibrosis still persisted in the lust animal, which was killed 14 mouth' aiter infe< tjou. ` Reaction in Ruhhits. -Rabbits tx |* i-td to ttic u'liestiis dust for period' up to Id months slmwcd a foreign Imdy ty|>c ol leactiou of low grade, hut no fibrosis. Ahltough their lungs contained tiartiuilate elements of the dost, libel' were not present, indicating that the upper respiratory mcclianism of the lahbit is adeipiale to exclude fibrous foreign bodies. Two rabbits, after inhaling dust for six ami l`>9 9. Steenken. W.. 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. M:51. 1946. u ixnrsrRi.ii. HYuinxF. axu next patioxal mepicixe m.mtli' liml in normal air fur mure than two years. At autopsy neither animal 'll..m,I am unleme ut eellular reaction nr lihru'is m the terminal bronchioles, nor .\cn iluie am .i'Ih,'I,,'I' U"lii'' iC.'.i. 'i."i in II Ini. litiii Ml the rat' li.ul at.|iiireil an infectiun. resulting in ilit i." hi,in,.u ..I iniliiiiiii.il \ .ill', i"iIn fine Kiev l ame tu auiupsy. Apparently, so iiiiii it `i, .iv \ iinirii' ul*'ii iu't',1 llnai I<itin In tli.it i-i i v lew filters rnuli! have entered Fig. 5.--King's float inhalation experiment: A, lung of guinea pig infected with Ri tubercle bacilli and then exposed to dust for 24 months. A bronchiole is shown just above center. Surrounding it is some collagen deposition, together with typical epithelioid cell infiltration of the wall. Note the lack of encapsulation and the peripheral epithelioid cell pneumonia, which illustrate a spreading tuber culous process (x200). B, lung oi a guinea pig infected with Ri tubercle bacilli and then exposed to dust for 35 months. Note the subpleural distinctly encapsulated caseous focus, the calcification at the right border of the lesion and the absence of cells in adjacent alveoli, all of which illustrate a healing tuberculous process (x 200). animal I. nor ting in lltljr, so entered leted le is ither ition tber- VORWALD ET AL.--STUDIES OP ASUP.Mtnls 1.1 their lungs. In a few of the rats, an occasional asbestosis body ua-> discovered, but there was no fibrosis. This phase of the experiment was considered unMiccessful. Summary and Interpretation of Inhalation Experiment with Kinri's Floats Dust.--The findings in the experiment with King's floats dust can be summarized under two headings: 1. Effect of the inhaled 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 healing; in one animal the tuberculous process remained active to death, in contrast, when guinea pigs after being infected are exposed to quartz dust instead oi asbestos dust, the infectious prooescontinues to progress and eventually causes the death of the animal.'. On the other hand, infected animals exposed to a harmless dust like iron oxide do not show any progression of the infection.1" Guinea pigs infected with attenuated tubercle Iracilli 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 tulierdes lteneath the pleura. In view of the variability of the results, the unusual nature of the response and the high pro|Xirtion of deaths due to intercurrent pneu monia, it is felt that only tentative conclusions as to the influence of asbestos dust on the course of tulierculous infection tire justified by thi> experiment. Short Imhkr Asiik-to- Hi st Since hazardous dusts like quartz are most effective in producing fibrosis when the particles are 3 microns and less in si/e. an inhalation experiment was performed to determine whether this condition is true for asltestos dust. It was thought that a short fi1>cr asliestos dust consisting almost entirely of fillers and particles smaller than 3 micron.' would initiate an accelerated tissue response anil produce an advanced reaction in a shorter time than 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 Concernrat iou of the Dust.--The dustinc material for this experiment waj the remain.'; of fibers collected in dn>t bins of nil asbestos fabricating plant after a carding operation and screened to pass JfX) inusli. Since10 10. Vorwald. A. J.; Pratt, P. C.; Durkan, T. M.: Dclahant, A. R.. ami Bailey. O. A.: Siderosis: A Benign Pneumoconiosis Due to the Inhalation of Iron Dust. Imfust. Med. k Surg. 1*:170, 1950.  . ^ , , -, i , ! I_ the material as received contained many lung fibers, it was ground iu a steel ball mill tu reduce practically all tlte particles to 3 microns or less in size. When used alone in the standard dusting machine, this finely ground asbestos tended to pack in the linppcr. and it iicvumc iare--ary t<> mix one volume ni the unground material null three volume* <> tin urmunl to pencr.Hc a satisfactory dust cloud It i* pertiutni to mention luat that the addition oi the small quantity of utigruuild islu'stos n.is nnloitmiate. Ii.ui*i- it uiiii'i-il the inteepietation o| results. I he composition oi die *liiit IiIm-i .ishesp.s ,i. iixrieed is diselnscd lie the eheitueal and |*vtrtigr.i|ilm' .-iit.il*.,* e.txin in lal.li s J .mil .1 X.tnqiic* taken lieinre and alter ymuting yielded alwaU llir '.1111' \ .1 liu s ..n .nulls sis Hutu .it Ilie that there was no eoiitamiiiatton from the mill .n loss oi h.ipi nuiintl The dust concentration sailed dm my the \|hi nnrnl. Ilie l<s;lit held omit* for attno*pheric samples collected inside die annual races with the inipuitpT apparatus ranynm from S3 million to 1R2 imllion. The aveiaue of ioiiiii* teas t.ltl million for die first year of the experiment. 134 million for the second year and 1411 million for the third year. Size-frequency measurements of air-floated dust from inside the cages at a maRiiitication of 1.300 x revealed a great prc|Kiuderauce of flue particles, nearly Table 5.--Summary of Inhalation Experiment with Short fiber Asbestos Dust Nature of Experiment Oust expoitire continuous througti- out lit* Animals tu guinea pigs 73 rata IS cata 7 rabbits Dust exposure followed by proloogad residence in normal air 13 guise* pigs rats 1 rabbit Maximum Maxi Survival mum After Du*t Ex Dux Ex* poaure. IKJlUff, Mo. Mo. Zt 0 sx 0 54* 0 47* 0 *0 u 31 24 Results Rats ot naetton about the taint aa In cxptn mvnt mtta Elm's floats asbestos but txteat ot lntolTsment very much less t Characteristic patches at peribronchiolar fibro sis; no asbestosls bodies 8ubpleutal reaction only No flbroal* aaaa (rossty; mlcroaeopte tvMenet of alveolar iraU thickening alter 0 months' exposure - Progression ttter removsl tram dust doubt tui--neither ttearty tsUbUthtd nor definitely excluded Same as for continuous exposure atmllar to continuous exposure; eektenee ol lUcbt retreesloo * Alter XI months the animals were exposed to 100 per cent ball-milted aabettoe. The reaction sat probably due to long fibers In the uoground material which was mixed with the ground asbestos dun produce a satisfactory dust cloud. 90 per 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 species of animals--guinea pigs, white rats, cats and rabbits--were used in this experiment. The results of the dust exposure, summarized in table S, are presented iu greater detail below. Reaction in Guinea Pigs.--Eighty guinea pigs were originally placed in the dust - room, but 21 of them were later eliminated from the experiment and killed because 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 the dust room until they were killed or died at periods up to 34 months, and 13 animals were transferred to normal air after being exposed to the dust for 20 months. The type of tissue reaction provoked by the inhaled short fiber asbestos was essentially the same as that already observed in the experiment with King's floats asbestos. The rate of reaction also was approximately the same, but thq 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. teei ball When nded to tground t cloud, .ground by tlte before >t there at* for laratus ion for million Bkti i la exptrii bat *steal blolir fbro- *gle evidence ft* 46 months' JBbtn duet doubtbo nor delaltel; mdeoee of ! *beetoe duel VORWALD ET AL.--STCDIES OF ASBESTOSIS Only after exposures had continued for approximately one year was there an appreciable tendency for dust-containing phagocytes to gather into clumps. ById months phagocytes had collected about the walls of a few of the respiratory bronchioles which revealed a little proliferation or infiltration of mononuclear cells. There were also some multinucleated cells, but they were of the inert, foreign body type. At 20 to 24 months the cellular clumps were sometimes quite prominent, and sometimes changes in the epithelium resulted in the adenoma-like or "adenomatoid-1 appearance (fig. 3 B) previously described in the section 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 few exhibited pronounced development of fibrous tissue. In these few members of the series the col lagen was pale in color and tenuous, with no appearance of being hvaiinized. Diffuse chronic pleurisy was present in a few animals without evidence of pul- Table 6.--Analyses of Lunas of Guinea Pigs sifter Prolonged Inhalation of Short Fiber Asbestos Dust Exposure to Dust, Mo. 3) Period In Normal Air. Mo. Amount of Ash, % ot Dried Lum Total SIOs. ?o Of Dried Lunr Total StOt, ~c u( Ash Dust Exposure Continuous Durum Life ( 5.M 0.51 10.2J 1 4.38 0.48 10.08 { 3.10 0-34 10.54 { 5.00 0.49 9 j 4.70 0.43 9.00 l. QSi 10.00 ( 3.58 0.55 14.40 (e.3 0.90 14.07 \ 5.If 0.7< 14.48 1 5.30 0.78 14.20 t 3.35 0.08 17.59 ( 0.33 1.27 10.40 | Owl 0.73 12.37 j fl.:l3 0.1*0 15.11 bllowcrf by I'mlongett Re*UU`nc<' in Norn J 5.10 ) 5.11 0.48 0.:l V50 7.10 ( 0.11 I 3.96 0.tif 10.2! V.: S.31 ( 4.77 5.18 14.77 0.23 O.fli 0.22 5.31 3.no 4.00 T:ue Hi ai tloD ' The vmbola avrraflnir the tlur reunion In .-m-li arum, of Kulne* pins represent merely llie relative decree of rearilun. ranaiuc from s: (iuetluiinMe) lo 1+ ttin- maximum for tlil rxpertmemi. The relationships apple only wiililn tlu tul.le nml raiihot lie eompared with yioliois In other tallies. limitary infection. This suggests that pleurisy may In- a sttccifie cnurnniitaitt of .ixItcMnsis, hut the evidence is not adequate to establish this twint. The reactinn nf the traclieeihrnnchial lymph nodes was more pronounced than in the previous i-x|KTiimiit with King's floats nslieslns. prnliahlv ltccauc inure fine particles had been lr:ius|Hirtcd In the nudes in animals inhaling short filler asliestos. The nodal reaction was essentially an increase in reticulum, ratliee than a lihrosis. with the original cells Iicing preserved Ih-Iwccii the Ihiekeiied reticular liliers. til the group removed to iioimal ail after 20 months' inhalation of dust, proutessiun of disease was not definitely demonstrated, hut ueiiher could it In- absolutely disproved, owing to the variability of the res|Niiisr in dilTcrcnt animals. The reactions, from mild to severe, occurred ,s|Miradically and Ixirc no relationship 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 analyses (table 6), which revealed comparable amounts of ash and silica hi lungs with widely different amounts of tissue change. For example, the ash 16 IXDUSTRUL HVGIEXE mJXD OCCCPATIOXAL MEPlCfXE ami Mlica values were quite similar ior three animals living in dust 20 months .niri then in normal air ior 14 months, yet the tissue reaction was severe in one animal, mild in another and only doubtful in the third. The lormatinu ni Iwlic* was at lirst extremely limited in both group*. \tuv i\c numhx i\pnMiw only wry rare 'hurt ImmIv multi In* usually 'ii'Mt .i -1 il S"im *i the tino't inti.u rilul.tr parti l< p'.-ii- liaviru 11*i ..iine i "lu as die ImnI Wfiv Mim >uiuUa<l ly yell** in* \c;ir`s <*\pMsitr<.* hail pm 1' Ml W iiirli uric mah*d .ut -111 * 1 i tumuli l* 1*i' pal hall .r iiilnelv wnhin plMumtht irlU iim nll i ami lltrrt*.ifIrr ili' r on i hutivXtS !/' Iflull* VViii/ 1 hut /if/iii/oi/ \/i<#7 /*i/vr . I.v/vv/m Puxt l`iir;M.t|i ' I MHl -i,r<, Mu \mt. of \%h.\ mi hnt lime | :i*. t :< Tolal Sill;. ii hrivtl {.mu: .(:< a .1 l Totul S0-. of Ash on ill! (Ml 0.0 oo oo 3.2 Uf l..i ii.Oo 2.3 : 0 0.11 3.0 3.4 0.07 2.2 * Niiriiiul < c!Hroi mo diirt expoiure). (iiimlitoi of -iifr, Mm. tut. of Mi. * r UniM l.tuttf .U 3.4 3.7 :\c, i.i '44 1.6 #4 .4.7 ToUl Mu:, of Dnist Limit IH>7 0U3 0.M 0.1* U- 0.15 U.l 7 0.16 0.1* 0.13 0.15 0.(3 Total SHI;, *1 of Anh 2.1 1.5 1.1 2.2 3.4 40 4.2 34 U 24 2.3 T vblf. .Iverage l tilth's of Ash and Total Silica for Lungs of White Rats Inhaling Various l>nsts far Various Periods (Lungs Only, Without Included Lymph S'odcs) - Amu. oi A-h.1; uf Driml (.unit Total SIO>. Tc of Dried Lung Total SlOj.rtof Aeh Ptira* tion Miort iityitsuin* Short Gypsum* Short Grpnuin- nf Kx Fiber Ferru Quirt/. Fiber Ferro- Quart* Fiber Fami- Quartz luvmre. Asbes ginous Mix Aabea* flQoue Mix Afhea- traoua Mix- Mo. tos* quartz Chert ture tos Quartz Chert ture tot Quartz Chart tura 2 3.:: U 34 24 0.00 0JI 0.3S o.os 3.S I1.T 3.9 24 l :s.4 '.5 3.9 34 0.00 041 0.32 0.07 3.5 11.4 3.6 2.0 6 3.5 7.1 9.0 3.4 0.06 2.04 3.45 0.11 1.6 <1.5 34.4 i.< 5 3.5 4.6 1>.0 34 0.15 1.44 2.40 0.32 3.9 20.4 SU 0.1 :o 14 74 14.1 4.1 0.15 4.40 6.00 0.23 3.2 56.* <3.3 .T were relatively numerous although still rare in comparison with the findings in the King's floats experiment. Reaction in White Rats.--Seventy-three white rats were exposed to atmospheric short fiber asbestos dust for periods up to 32 months. During the first 10 months animals were killed bimonthly and for the remainder of the experiment at less frequent intervals. L'p to eight months the dust cells were widely scattered and existed in foci only sporadically. Reaction was limited to occasional slight thicken ing of the septums about small accumulations of dust celts. At 10 months there was a suggestion of early fibrosis in a few rats, but the change was so slight that it would probably have been overlooked without the dump of dust cells which attracted attention to the area. Only 10 animals were exposed for from 12 to 32 months. In each of them the lungs contained minute foci of well defined fibrosis distributed like that of asbestosis but without asbestosis bodies. The lesions, visible only at a magnification of 150 diameters or more, consisted of patches along I l VORIVALD ET AL.--STUDIES OP ASBESTOSIS 17 alveolar ducts in which the walls oi the associated air spaces were very thick, owing to swollen collagen framework. Connective tissue and Foot-Rielschowskv silver preparations revealed complete loss oi capillary bed locally. Outside the collagen was a thin layer oi epithelial cells. This did not resemble the "adenomatoid" change characteristic of guinea pig asbestosis. Xear the lesions the air spaces were filled with phagocytes containing gray to yellow particulate dust and a rare, lung, 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 it) months, some diffuse thickening of the reticulum. In a tew rats there was definite fibrosis along the margins of the node, extending into the mediastinal areolar tissue. Results of chemical analyses made on the white rats are given in table 7. and the average values have been recorded in table 8 for comparison with similar values for rats inhaling other dusts. It will be noted that the values for asbestos are lower than those for quartz or chert but approximate those ior the gypsumquartz mixture, in which atmospheric agglutination tended to reduce the amount of dust inhaled. This condition prevailed even though the atmospheric concentra tion of asbestos dust was essentially the same as that of the quartz, was oite-half that of the gypsum-quartz mixture and was one-fifth that oi the ferruginous chert. Since the values for asbestos are low. it might he inferred that the total quantity of that dust actually inhaled was small or that it had been eliminated from or dissolved within the lungs. Evaluation of these possibilities is not feasible on the basis of the observations derived front this study. Reaction in Cats.--Twenty cats were used in this inhalation experiment with the short fiber asbestos. Eighteen were kept in the dust room coutmuiiusly until put to death, the exposure period ranging from one month to nearly 54 months. The other two were removed to normal air after a dust extmsure of .11 mouths: 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 subpletira! alveoli rather than in the iierihrouchiolar area-. In one animal the change was extensive enough to be visualized on gro-s inspection if the section. Only in the animal with the longest ex|>osure--54 month---did the roentgenogram reveal definitely abnormal shadows. A roentgenogram made after .10 mouths revealed no abnormality: after 45 months, a faint mottling could la: detected throughout both ltmgs. At autopsy, nine mouths later, there was only tnirrnscopir fibrosis in the suhpleural zone plus heavy lymphocytic infiltration almut -mall hrmirhioles. Asbestosis bodies were rare. On prolonged -careli a few yellow atypic al Imdics, smooth ami without haustrations. were found in two animals xfin-d for more than a year. Kmctinn in h'nlihils. -Eight rabbits were exposed to dust for period* extending from mir to inorr Ilian five years: the last animal wa- removed from the dust loom and left in normal air six mouths before heing killed. There was never enough pulmonary lilcru-i- in Ik- detected grossly, and there was no ehrnnie adhesive pleurisy. \fi< to-.iopij rvideme of alveolar wall thickening was first detected iti me animal alter ahont liner yeai-. of e-po-tiir and was -eeti in all live animalexaniined Ihereafler. in* hiding .............. leimmil to itomial air. One animal that died of paralysis aflei ue.niv foot ve.o- oi espo-.tiie eshthin-l a reaction visible on gross ius|xliim of tissue - lions. Tice |<--iliihtv oi poloioitait infection in this animal could not lie excluded. In auoiliet animal dying two u-.n- later the focal fibrosis was not nearly as ohvioii- as adv.uued \iea- of involvement, which were largely visualized ltccan.se of phagocytic reaction within the air spaces, tended microscopically to become more fibrous with the passage of time, hut there was never much encroachment on the lumen of air spaces and the structure of the lung was preserved. Asbestosis bodies were not detected in rabbits tliat died early in the exiicriment hut were seen in all animals that had been exposed to the dust for more than three years. 18 IXM'STRUL HYGlliS'F. AND OCCtPATlOXAL MEDICINE Summary and Interpretation of Inhalation Experiment with Short Eiher . Isbeslos Past.--The original purpose of the experiment was to evaluate the role of short asliestos fibers in the genesis of asbestosis. It na. felt ul-o that if the ti-Mte- reacted more rapidly and more extensively in 'hurt tilier aliestii than to King's floats there would lie a basis for lulu ime that the action ot nolicstii' i> in part, at least, a chemical one a* pii'tulatcd for ipiuri/. This r\|KTiiiietit. in which the tissue reaction a> 'loner and Ic'S cstcti'ivc than that ill the picvioits ex|ierinieiit with Kills; ' lloal' dii't. nulicalc' that the eapaiilv of inhaled asbestos libel'' to produce lihni'is i determined primarily hv tailor' not ebemieal in nature. Of the four species exjHi.'Ctl in this exjierinient. only the guinea pig and to a loser extent the white rat responded with characteristic peri bronchiolar tihrosis. The cat reacted with atypical siilipiciiral fibrosis and the rabbit with only slight parenchymal fibrosis. I5ai.i.-Mim.ki. .\shkstus Dcst In the inhalation experiment with short filter asbestos dust a small |uautit> of unground short filter asliestos 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 coni1 wrist m with the response initiated by King's floats, it became apparent that the biologic activity of asliestos is not increased by a reduction of tilier size. Thus the possibility arose that the tissue reaction observed was flue solely to the relatively few long fillers of the unground asbestos and that the short fillers of asbestos had no more than a very insignificant rule in the production of asliestosis. a concept not in accord with previous experiments concerning pneuntonoconiosis. Consequently another inhalation experiment was started in which only hall-milled asbestos was used. Composition and Atmospheric Concentration of the Dust.--The dusting material a> the hall-milled, short fiber asbestos used in the previous inhalation experiment, hut unground 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 Roaring out of the dusting machine, the dispersal of the dust was not entirely satisfactory- Therefore, after an initial seven months of operation, steel wire finishes 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 3. Petro graphic and x-ray diffraction examination of atmospheric dust, collected in the dust room with an electrostatic precipitator after the installation of wire brushes, indicated that about 15 per cent of the air-suspended material was chrysotile, and 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. During the seven month period before the wire brushes were used, the chrysotile content of tltc 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 100 million particles per cubic foot of air. After the wire brushes were CISE ~with Short ent was to estosis. It extensively a basis for emical one lie reaction experiment ;d asbestos it chemical guinea pig ristic periral fibrosis wt a small t the ballWhen that , in com e apparent thiction of * observed asbestos ignificant xord with nsequently bail-milled inf material experiment, ency of the >ortion from not entirely steel wire ating paddle nave satisjritnctlt. 1 3. T*etr, ctetl in tin ire brushes. ryotile. ami 10 per cent. During the c content of liable values eriment was rushes were VORWALD ET AL.--STUDIES OF ASBESTOSIS 19 installed, the dust counts were higher, and the over-all average for the remaining 21 months was about ISO million. Size-frequency studies of atmospheric dust collected inside the animal cages revealed that nearly 99 per cent of the components suspended in the air could he classified as clumps or particles: only about 1 to 1.3 per cent was 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 Pigs.--The experiment was started with 100 guinea pigs. As the dust exposure proceeded, there were 39 accidental deaths. 32 of these being 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 foci oi inflammatory cell- Table 9.--Summary of Inhalation Experiment with 100 per Cent Ball-Milled Asbestos Dust Nature ot Experiment Dust sxposurs con tinuous tbiougbout lilt Dust exposure tottowed by pro longed residence In normal air Animals M guinea pigs 40 rata 24 mice IS guinea pigs Maximum Maxi Survival mum After Duit Dual Expo* Expo* jure. pure* Mo. Mo. ' Result; U 0 No apprtrlnblc pulimmiirv rcnrl ion 20 0 No suftrstiun of Ion* 12 0 No suggestion ol asbestosi* 28 12 Fibrosis typical ot asbesto-is was present li mo. alter exposure ceased In an amount sufficient to be visible grossly: smaller tori rould be seen microscopically nt t mo. and S mo. alter termina tion ot exposure could be seen. At 24 months (fig. fi.H there was still mi change large enough to be seen with a hand lens, although microscopic examination revealed cellular accumulations about terminal hronchioles and many mure asliestosis bodies, chiefly within cells. The lungs of animals exposed for the full dusting period of 28 mouths and afterward living in normal air for two mouths revealed the change* dexcriltcd almrc and also very slight peribronchiolar fibrosis. For exposed animats living eight iitniiths in normal air the finding- were similar, but at 12 months three f four animal* xlmwnl grossly visible churaricri-tiv peribronchiolar fibrosis with .'leiionuiloid change (fig. ft It). The IraelieoliroiK-hial males uetc essentially normal until exposure had ticeu onlinueil for more than a vrur and a lull'. \ititii.ils killed at 12 mouths and at IP moullis revealed a leu tmniile i ..Heelnms ,.i pli.igis ries containing particles liul practically mi IiIk-i- laige eiioupli I., lie .e>..ein/e.l .is such, \ftcr 20 months of exposure many monoeyles Idled mill icll.m gi.tmdes v\ere present. At .10 months there had been a slight increase in icliviihiui hut no lilim-i-. No further changes occurred in the nixie*. Aslx-siosis lslies weie not seen in the nodes of any of the guinea pigs. Minute asliestosis bodies were observed in the lungs as early as three months after exposure liegan, hut they did not become numerous until 16 months had elapsed. The Imdies were short and practically all were intracellular, although at . -20 -muntliv sonic were long enough to project beyond the cell borders. It is ycmsn .txn narr.trins.It. Mumas-F. .11 /vnrmri.it. nv i,it- tluii in ilic later niiiittltj. ..I exposure then* was a distinct increase i it*taut t" ""*............... .......... icruiii in length . '* -t*hc 1lu--,,-- wi,h #,h|iea m ihr iuuiiImm ci liiu; iitwiHP t*` /(* tilicrmi iiimikmi hi i iui.u It f i*ltr I"1H! lttlic*. i 'umti.il (t.ililt l<* tin- liniti* rcvi:iin{ (hat cwtMcIcraWe dual had ...... iti.tfvl mi tin iimc* \n * JJ mnlu T rniiitiiiiiD> i\|NMirr the average I r ** -*\x S '** s^y-tSri-. .-A t c, *, > .V 0l Fig. 6.--Ball-milled asbestos inhalation experiment: A. lung o( a guinea pig with .'4 months' dust exposure. A bronchiole is shown at the center, with a slight accumulation of phagocytic cells but without the formation of collagen (X 200). B, lung of a guinea pig with 26 months' dust exposure and then 12 months* inltalation of normal air. The reaction is much like that shown in A, but there it a slight deposition of collagen, most apparent at the left (x 200). value for total silica, per cent of ash. was 25.37. This should be contrasted with the average value of 14.34 (table 6) for animats exposed 24 months to the short ci.we -tinct increase mgs with the <ble dust had the average J* i {tunica jug with a -light (X 200). 12 months' but there is Itrasted with to the short VORWALD ET AL.--STL'DIES OF ASBESTOSIS '! In view of the high values for silica ubtained with the animals exposed tu 100 per cent ball-milled dust, it is important to note that their pulmonary response was much less than that of animals exposed for 24 months to the short tiber asbestos in the previous experiment. This again indicates that the biologic activity of asbestos inhaled into the lung is not increased by a reduction in size of the libers. Reaction in ll'hite Rats and Mice.--In this experiment 40 rats were exposed for periods up to 20 months and 24 mice for periods up to 12 months. In neither species did even a suggestion of asbestosis develop, and reaction was limited tu phagocytosis of inhaled particles by widely scattered dust cells which remained tree 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 few small, nonhaustrated forms within phagocytes. Tabus 10.--Analyses of Lungs of Guinea Pigs Exposed to Dust in Inhalation Experiment unlit 100 per Cent Rail-Milled Asbestos Dust Period Exposure to Dust, JIo. toANir.oMrmoa. l Amt. ot Asti, Dried Euont r siUTio, tTaclot Dried Lung T%otoafl SiO.N Afh Tissue RencCion * Duet Expoeure Continuous Duiint Life 1 4.45 0.21 4.2S 00 4. 0.30 T.05 4.35 0.24 5.00 4.00 0.23 4.90 1 0 4.U) 0..I4 7.40 l 5.05 0.6t 12.01 0 5.00 0.70 12.4? a 0 3.0? 3.74 0.32 0.50 00..37?? 0 8 0 5.10 5.0? . 5.02 0Q.J3&8 0.39 77?...34?1?2 0 l! 0 4.35 _ \ 5.65 0.24 0.52 n.?-i 1.25 1.45 22.`2i..21S0 0 0 U i) 5.40 5.01 11..0121 211?..0950 0 5.65 3.20 11..22?? 22.00 24.01 24 w t S0..otl 11..2<15 29.05 21.70 Dttt Kxintirc Kollo**! h* Hrolitritrc in Normal Air 28 aX l 7.25 f M.iiT 1.37 2J.0.1 2.19 25.21 r l 5.25 l 5.!*0 n.oa ' I2. .*7 I4.V. - 28 12 1 0.: 1 5.17 0.84 I.S.li'* 0.(14 12.41 2+ The symbols aTerifln* the tl*ur renrtlnn in i*arh *rinn n*pr***4'Ut tm*M\ ti* Mumi degnt of reaction, rtrurlnr from o to + t|ueMlonnWri in j* (Uh maximum iMrvrti in thi* experiment). Th# rHsttonrhlp* apply only within thin tahk* mul cannot - ciminiirfii with ymltoif la oilier tablet. Stiutiitltry and Interpretation of /ulwhition pertinent with 7DO per Cent fhilt-Milleil .Isheslns Pttsl. The Iissue reaction observed in tlitex|ieritncnl \v:i.s not as intense as lli.il in the previous investigation with short filter aslicslos. The reaction was shiui-r in ileveln|mient ami !e. extensive even though mure ilnsi nccnnmlnlcd in llie lungs. Since there were fewer fillers longer than .1 mirnns m the material nseil in this experiment, the results tend Ui confirm the interpretation made in the summary of the previous short filler ex|ierimettl that the reaction is not primarily chemical in nature, and to sup|mrt the impression that reduc tion in size of asbestos fillers does not increase the biologic activity of -asljestos-inhaled into the lung. jj ixnc.trKi.il. iivcinxE oci rr.tiiox.ti. vr.niawF. The linditiy of U>u^ :i>lK--tn>i.- bodies in animals that had inhaled the ball-milled material i- an evample nl the difficulty of completely eliminattti!: Ions; fibers irmii a laruc volume of n-ltr-ins as re<[itired for an inhalation experiment. In rreard i" the projjre'Mon oi ibr ii--nr tr:ntiimi after the animalli.id In i n u iiihvi iI irum the dtt-l. nh-erveil in lln- experimetil hilt not ill ihr iither-. the inflowing iiiirrprrtaliim is nllrrrd: When the reaelion ivv ell developed at the tet'imnalion of exposure, the emit rat lion of the fibrous tiue obscure- am pmt:rr--ion that may have uvurrrtl; m this espeiimeiit. however, -uue the te.ution observed was le-- mature, il-uh-eipient progress vva- more leiuhlv a|ipareut. Ijivi. hi in- k \-nr-iii- |ii -i Since inhalation of short fiber and of 100 per cent halt-milled aslieslo.s ihist did not result in acceleration of the tissue reaction in coui|iarisuu with that produced by King's floats, the hypothesis that short fillers 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 float- asbestos used in the first inhalation experiment had a rather low content of fibrous chrysolite and contained considerable serpentine' and other impurities. Therefore, it was decided to conduct a new inhalation experiment with a purer form of ehrysotile which would be richer in long filters. ii'wpositimi tiinf . Itmospheric I'niici-ntriitiuii of the Dust.--The dusting material employed in this investigation was obtained front an asbestos fabricating plant. Samples of several varieties of long tiher asbestos dust were first submitted to the Saranac Laboratory for examination, and one oi these, which was low in magnetite ami chromite and had a fibrous content estimated to be about 75 per cent, was -elected 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 composition of the long fiber asbestos used is indicated by the chemical and petrographic analyses given in tables 2 and 3. Analysis of air-suspended material from the dust room disclosed that about 60 per cent of the long fiber dust was ehrysotile and about 20 per cent serpentine; as already noted, the composition oi a similar air-floated sample of ball-milled, short fiber dust was 15 per cent ehrysotile and 60 per cent serpentine. The dust concentration, as revealed by impinger samples taken inside the animal cages was much lower than the concentration for the experiments with short fiber or ball-milled dust. For the first year of the experiment with long fiber asbestos the average of the tight field counts was 32 million particles per cubic foot of air; tor the second year, 48 million; for the third year, 39 million, and for the fourth year. 43 million. The size-frequency 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 :!:e electrostatic precipitator. It will be noted that there was far more rhrou- material in the long fiber dust. Guinea pigs. cats, rats and mice were employed in this inhalation experiment. The r*-u!>-. -nmmarized in table 12, are described in greater detail below. fc Reortwit i. After exposur arose in the d; replace them. . examination r consisting of c 12 mouths t!" and hy the si.' as well as a tropically ,\t _ hi the amnui: TiViii.h 11--j- Tjp* ot Asfc Lunt atm... IiiU-mflM .. T.vi.e 12.--.91 Xatora ot ExMitsui Cutusom i tlnuoux tarot out lift nmi axpoun lo*d or pro lontfd mlrta is Bonsai an considerably i localized and fuse, even in the intrapulnu ment of that ti inf of the lai but it was cc inhalation of In guinea air, there was effect, accomp size of the fo 14 months aft the end of the dust room for the foci in fo visible only w VORWALD ET AL.--STUDIES OF ASBESTOSIS 23 Reaction in Guinea Pigs.--The experiment was started with 100 guinea pigs. Alter exposure had been carried on tor a year, a severe epidemic of pneumonia irose in the dust room and about one third of the animals died or were killed. To replace them, 38 more guinea pigs were added to the surviving group. Histological examination revealed lesions in the lungs after eight months of dust exposure, consisting of cellular connective tissue about the terminal bronchioles (fig. 7'A). At 12 months there were adenomatoid changes in the adjacent parenchymal areas, and by the sixteenth month (fig. 7 B) definite fibrosis was present in these areas as well as around the bronchioles. The fibrous lesion could be seen rnacroscopically 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.--Size-Frequency of Atmospheric Long Fiber and 100 per Cent BallMilled Asbestos Dust Collected Inside Cages Grains. % Type of Asbestos ' <3 Microns Looy flber............. 63.4 Ball-milled ..................... 90.6 3-10 Microns 1.1 4.6 >10 1 Microns 0.0 0.0 Fibers, To <10 Microns SS.S 0.3 > 10 Microns 6.7 0.G Clumpe, % 1.0 3.5 Total 100 100 Table 12.--Summary of Inhalation Experiment tcith Long Fiber Asbestos Dust Nature of Experiment Dust exposure eontlnnaos throngsout lift Hint exposure fol lowed by-prolonfcri residence In normal air Animals 117 guinea pits 4 Cits 3} rats 50 mice 1! guinea pigs 9 guinea pigs fats Maximum Maxi Surdra] mum After Dust Dust Expo Expo sure. sure, Mo. Mo. Results 30 0 Definite fll>rai in tfi mo. 43 0 Slowly developing fll*ro.l flrf at 34 mo. S3 0 Marked peribronchiolar flhroei* flrt seen at 34 mo. S3 0 7.imitcd reaction: no flhron* SO 11 Olcarlne of intlfttmimtory reaction aoti definite contraction of nitron* tissue ST Oariof of inflammatory reaction and slight contraction of rti-rou* tissue 18 St Similar to continuous expmnngroup: jurscMlon of progrrsln In one of the two animals < "iisiderabfy into the parenchyma (fig- 8.1). The lesion* were rather sharply 'ocalized ami the extensiuns from different bronchioles showed no tendency to iiisc. even in animals exposed fnr the maximum period of three years. Although the intrapulmonary reaction sometimes reached the pleura, there was no involvemeat of that membrane. -Emphysema was not detected at any point. Some thicken ing of the larger hronchi with a chronic inflammatory infiltration was revealed, hut it was considered tm more than would lie produced hv a similar period of '"halation of any dust. In guinea pigs cx|mscd to the dust for 20 muiiths and then removed to normal dr, there, was a marked tendency for cellular inflammatory reaction to clear. This 'fleet, accompanied by contraction of the lilumi- li-Mic. resulted in a rlumui-hiue size of the focal lesions. None of these animal', hilled at various periods itp to 14 months after exposure, revaalcd lesions as large as those in the grnuii hilled at the end of the 20 month exposure period or those in animals which remained in the dttst room for more than 20 months. Fourteen mouths alter 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. 8 B). 24 lynrsrKi.ii- nvniiixn and occrr.iriox.-iL medicine lit tin- group i,ir mnnth's and then tran>tcrred to a nonnal atmosphere the icspmisc wa> i|ititt similar to that in the JO month exposure animals mentioned ahovt. Small hi were always visihlt mi iri'o" inspection of sections nt all ennii-a pits ..i tin- 27 iinnilh 'mV*. hot in no iustaiict was there evidence { the i > .H'limi Fig. 7.--Long fiber asbestos inhalation experiment: A, 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, showing the reaction to ball-milled asbestos after 24 months (x 200). B. lung of a guinea pig with 16 months' dust exposure. Again note a bronchiole with its surrounding reaction, consisting of fibrosis and adenomatoid change. Col lagen deposition is now seen in the walls of adjacent alveoli, at the right (x 200). In the tracheobronchial lymph nodes reaction was first visible at the third month of exposure. By the eighth month patches of cellular connective tissue EDICINE mat atmosphere nimals mentioned : sections oi ail evidence of the rORWALU ET AL.--STUDIES OF ASBESTOSIS began to appear in the medulla, and by the fourteenth month mo-t of tin; node had been replaced by cellular connective tissue. This picture, which resembled that in early silicosis, persisted to the end of the experiment. Some animals showed, as a variant, heavy sheets of diffusely distributed monocytes and large active giant cells, but there was never any necrosis or hyaline formation. The spindle-shaped i a guinea pig I'eady shows an oollageu. Cum.liter 24 months ote a bronchiole id change. Col- right t X 200) ile at the third pnnective tissue hill. X.-- I.miik IiIh i .isln-stos oiliiil.ilnm experiment' .1. lone .i minim |'ie with .14 mouths' ilnsl r\|N.suu', A liiniirliiule is seen at the lowet center: llic large area nUivc n ii'|nesi>nis tlit- imuhifiiuii ot alwxdar walls. t mup.nv with figure 7 II and mite the UK leased extent ttl l ent lion I x 2001. II, lung of a guinea pig with 20 imnitlix' ilnsl cx|msitte and then 14 months' living in normal air. file reaitimi i' essentially like that 'lunin in liguie 7 / The bronchiole at the tight center is surrounded by hhroiis tissue with adctinuiutnid change at the right. There is residual scarring in the walls of adjacent alveoli at the left. It is apparent that no progression has occurred ( V 200). new cells were yellowish from fine pigment granules that stained for iron. Xu fibers or. aslicstosis bodies were seen. IW W P W W ""I.M UI IU |' n j,, ixprsTRi.il- lireiEXr. .txn nurr.mox.ti. mf.puixf. Utliuugli nlH`t.->is taxlii'S were (mind in llii- Uinit :is early aa .Hie ni.Hitl. l.x|,,,s,,r(- tu-uan. they were rare mu I hard to find. At five iii-iitha mure w.r. m.iI.I., I'liieilv euiled mdde aiaut eella. and at ellil innntlia many laulica r .m i i.. .iihtlvu t it/ / Min/v </ I'hts lit past'd to Oust m inhalation / r/vniuruf with I *nj / i/'it .Oust I- xjH-itr** u I >ttl, Mo. lVrlt'l iit Normal Ur. Mo \ml ! V*h. **'( hfhM I uutf I* Total SUN. I mot Tout HUN. %of \Hi ItntMiiiK (Miring I ICi* TlMUf Rcertlim ' | I .15 4..C . 1.31 n oi u IKI tf.lM im ti ut 4.43 4.4S 4.33 Ii.u5 O.U'i u.mi l.ttl Lift 1.40 | 4.33 l l`:.i 0.(43 0 0* U.Ui 1.20 1.13 1.43 l 4.:: j 4.tfl l 5.00 (t.Uft 0 \'i Q.ift 1.75 2.07 I.77 i aI 4.72 0.10 O.UU 2.21 J.1KI 0.07 LO* nos 5.20 12 0 { ;,.oi t 3.16 0.20 Q.::l 4.09 3.99 * | 2.9s 0-18 12.70 IS it 1 S3 1 :t.l0 ft.X'i 22.23 U.3t 10.91 2+ 20 . | 3.54 u 1 -U. u.43 12.22 0.49 13.03 002 14.59 1+ 24 \ .5.42 0 l 3.52 0.35 1001 0.29 809 1+ 7 \ :Un 0 i .5.74 0.39 not 0.19 13.15 3+ y30 j1 SA5M2 0.37 10.56 0.31 0.24 11.22 non 4+ 34 S 5.35 0 l tl.70 0.30 8.60 0.64 12.47 4+ 36 0 \ 4.10 t 2.74 0.37 901 0.35 12.80 4+ Dust Exposure Followed by Prolonged Bealdeoce In Normal Air 20 Q 1[ 33..5640 \ 3.33 0.43 1202 0.49 13.63 0.32 14.89 *+ 20 4 l 2.92 l 2.81 0.21 0.27 70S 9.80 2+ \ 4.18 20 10 \ 4.30 0.24 0.22 5.75 5.07 *+ 20 14 \ 5.01 l 3.04 0.21 008 4.19 308 + 27 j 3.40 0 ( 3.74 0.39 11.61 0.49 13.15 3+ 17 3 ) 306 ( 2.54 OOl 003 8.80 604 2+ 2T 7. ( 3.19 | 301 0.25 0.28 7.99 8.72 t+ r \ 3.21 \ 2.75 0.29 0.23 8.96 101 2+ The symbols smiting the tissue reaction In etch rroup represent merely the derm of retetlon. ringing lrom o to (questlonsble) to + (the mixtmnm tor tmi oxpen* ment). The relstionshlps apply only irtthin thl table and cannot be compand with ymfiOU in other tables. were free in connective tissue. They became fairly abundant as exposure con tinued. although in some later animals the asbestosis bodies were only moderately numerous. It is important to note from analyses of the lungs (table 13) that even though the tissue response at any given period of time was much greater in the guinea pigs of thi asbestos, tb Rsactur 14. 25, 33 cats, after an addittor cellular acarterioles t lymph nodi pointed, ye months, rea ncuive tiss and arteriol *aUs (tig. A < A Fir. 9.raontht' dui and collate: an occasion: reaction wa. lower in d 25, 33 and lesion*. Reaction from pneum exposed for and offered was just be peribronchic bodies were animal. Tlr accompanied VORWALD ET AL.--STUDIES OF ASBESTOSIS 27 pip of this experiment than in those exposed to either short fiber or ball-milled asbestos, the amount of mineral matter in the lung ash was much less. Reaction in Cats.--Four cats inhaled the long fiber asbestos dust for periods of 14, 25, 33 and 42 months, respectively, and were immediately killed. Two other cats, after being exposed to dust for 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 bronchioles and peripheral arterioles together with compact collections of similar cells in the tracheobronchial lymph nodes. At that time there were no typical asbcstosis 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 the respiratory bronchioles and arterioles, marked lymphoid hyperplasia and lymphoid infiltration of bronchiolar walls (fig. 9). Typical asbcstosis bodies were not formed, although there was Fig. 9.--Long fiber asbestos inhalation experiment: Lung of a cat with 42 months' dust exposure. Two bronchioles are shown with adjacent cellular reaction and collagen deposition ( X 200). 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. KocntgcnuKrams of cats made after exposure periods of 25, 33 and 42 mouths, rcsjKX'tively, failed to demonstrate evidence of pulmonary lesions. Reaction in Rats.--Although 20 rats were placed in the dust room, many died from pneumonia and were not suitable for study. l;ivc animals, of which one was exposed for 19 months and four for 25 mouths, were five irom pulmonary infection and offered a basis for tentative conclusions. In the IQ mouth animal, the reaction was just beginning. All four animals killed at 25 months showed a well marked peribronchiolar fibrosis. After a long search, only two small, smooth asbcstosis bodies were found in the 19 month animal and none was found in the 25 month animal. Thus these animals exhibited fibrosis without asbcstosis bodies or fibrosis accompanied by only a very infrequent asbestosis body. js ixnixix/.u. uu'ir.sn ,-ixn ocu iwtiuxm. mf.dicixe krih'imii in 1/nv -Out "i -<> white mice nseti in tlii- experiment. U lived a year >r more in <lu;t anil diol were killed without slimiiHg an appreciable degree ..i pH Inn'iiar; miVrtiim. The reaction to the inhaled dust was limited to phago- iM..si> 11\ iii.iiiiiiiinlt.ir nil- l-ailly tlice were widely -eattcrcd through the air ,i.Ui> .1 linnint ntinilii r u.u er"U|icd alniut tile terminal hruuchiiihs. iinxlucmg v. nu iliukittinc "i il'i-tt a dl' Thin- u.i* no MiKitv'timi <if fihri">n>. \ imin "ii- .I'lii'i..'!' tin ' mu- "liM-iml m .mmial' killed late in the experi- 11 It - l till!' lltt't i- lin II' t itlllillitl .i')M-'lt'l' ImmIm ' Wllllflll Illll-Iisi' N *rI I lint1 ; i- '. . .M/i.'ii ,.| I iilinl,ili,'ii / if, mm lit xX'lh i / on#/ i i /i,v.t-.'\ ','it I In* jirii'iim' i*i tins r\|ti-iinn-lit c\,in tu I'walnati' tin- iiii|>iift.tin r t>l I*nt" liliiM s in tin- lissiir ii's|niiisi- |n iiiliali ii .is|m's|iii,. The results, in yimipat'isuii w till tiiuse nt |ii'e\iuiis imi-stiyaiiinis, jiuhratr strunwlv that lung lihers are chiefly res|iini.silile iur u.nIk*m<ims. Thus, the react it in in guinea pig.- ileve!n|>eil earlier anti heeanie inure extensive in this experiment than in previous exjteriutents in spite of a' smaller eoneentratiou nf atmospheric dtiM anti a lower mineral content of the lungs. Furthermore, topical perihroitchiolar fibrosis was produced in cats, although in a previous experiment with short filler dust peribron chiolar fibrosis tlitl 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 EXPERIMENTS Since the inhalation experiments reported above strongly suggested that long fibers of asbestos are the significant factor in the causation of asliestosis, a series of injection experiments was inaugurated wherein the dosage and the length of the fillers could be controlled more precisely. Also, by the use of controlled dosages, the relative capacities of various asbestos minerals to produce reaction could he 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 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 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 extrapulmonarv 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 of Fibrous and Sonfibrous 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. jicise 11 lived a year eciable degree ited to phagobrough the air lei. producing s. in the experi)ii: t with Long a to evaluate tied asbestos, ions, indicate tosis. Thus, ore extensive of a smaller tant of the produced in list peribron- ei the guinea eiments with 4 l' .. V suggested causation of toted wherein wre precisely, ties of various lid. In these sere used, and mtraperitonea! used for each each series of d in tables, to . only one test ailed report is iced lilirnMN in eferred wav of In this method edle or catheter t. emonstrate that brous character, were performed. r Table 14.--Comparison of Reactions to Chrysolite and serpentine Injected Introtrachcally Dosage: Each animal was riven an intratracheal Injection ot v..' cc. of a j per cent suspension of tha duat. Two weeks later another similar iujevtioo wn< riven. Total amount of dust injected vac 30 mg. Ammals uced: Six group? of 0 guinea piv? each tone group .'or each tvpe of dust). Periods at which animal? were killed: One or two animals in each group at i, i, 6, S4 and 12 month* alter laat injection. Preparation of duat: Chry*ottlo (hall milled) unheated: Ball milled for 1,1*6 hr., dried hdJ retrount) to agate mortar. Chrysotile (ball milled) ignited: Ball milled chrysotiie heated lor 2 hr. at about tup e . then ground in agate mortar 2 or 3 min. Cbrytotlle (flbroua) unbeated: (.round in agate mortar to pass 200 me?n. L'hrysoih# (fibrous) ignited: 300-mcab material heated (or 2 hr. at about TOO C. No further grinding. Serpentine (ball milled > unbeated: Ball milled tor 1.4S8 hr., dried and reground In agate mortar. Serpentina (ball milled) Ignited: Bail milted serpentine heated tor 2 hr. at about <00 then ground In agate mortar 2 or i nun. Mineral Chrysotile (hall milled) unheated l/hrysotile (hall milled) Ignited ChysotUa (fibrous) unheated ('hryaollta llltiniiai Ignited Kerpefilllte fhall milled i uniMated Serpentina (baU milled) Ignited Size of Dust Particles 3 micron* and less 3 micron* and lest Results Grinding destroyed capacity to cause fibrosis. At 2 nto. considerable inflammatory edema tod cellular prolifera tion and localization of dust particles about bronchi oles: at i mo., only a very slight proliferative reaction: at o. 64 and 12 mo., widely scattered inull mononuclear phagocytes. At 12 mo., a few microscopic patches **f thin alveolar wall thickening with some adeuuuiatoid change in portion of air spaces abutting ou tha-kemd bronchi. No asbeatoslt bodies seen. Reaction limited to large foreign body giant evil* without production of fibrous tisuc. tu-jo microns approx. Si-fill mtrnm* approx a tult-riiii* mol 3 microns and less A distinct fibrosis. Reaction loridl/ed to connective ti^oc about terminal bronchioles: little within those tui*-*. Contraction caused adenomatoid uppearance of mr spaces given off directly from tmniual bronchiole'. Reaction area became smaller with proaresa of time: no new regions involved. No chrome pleurisy even at points abutting IntrapulRionary rhsnre. At ) tuo. considerable Inflammatory edema ami foci of cellular proliferation; at 2 mo. well marked cellular proliferation nm! ftbrowu occurring focatfy about respiratory bronchioles. Thru reaction developed before asbestos!* bodies had formed and was as advamvd us that produced hv 2 yr. Itihalatton of aslieatoa dust. At tf mo., reaction (<* extensive than at 2 mo,, apparently due to i-ontractloo of fibrous tls*ue; asbestosls holies were abundant. At *4 mo., reaction still less extensive, confined to the immediate rldolty of (he small terminal bronrhloks, where the scar tissue was quite dense amt was t'ccomlng hyaline In char acter. Sometime* It even obliterated the bronchiole. InnIhh bad lieromr M-umv At 1? mo.. I lie well ilevcloprd ivrlhrnnehial and hitrahronrhlal atkuomatnht an-** of fthmsi* had produce! considerable dis tortion. Moo* peripherally were patrlw* i>( pneiimoiutis with eodmiphUic iiiflitrmtlon, some of whlrli was twine transformed into fltirous tissue. The*e ermed to tw pre cursors of (be localised, dlffuft* patches of thin alveolar wall fihrosf* seen elsewhere. ttenrlhm limited to (urge lumen IhI giant cell* wiihont prollfcratloit. Ifcathtg ttwv fibers, which inttili* them bflltle, dmiron*d Ihelr enparily lo pr*hm- mgtitllemU rvnrihm. thinl relatively Inactive. At 1 nmt 2 mo., dmple pltago- i-ylimi* wlttuml prollU-rnifnii; al <i tin* , im elmiiae except {Mimdldy lymphoid irll lufilirattoft: at mo., a 'light elirotdi- puetiuiotillU: at 12 mo., oulv a little puvtmioultla without stiggeftiuti of fibrosis. Dust relatively inactive. Rcacttoo essentially the same as tor unheated serpentine. With ignited terpentine, Ini tendency lor duat to be canted to bronetilal nodes. 29 30 IXDLSTR1.IL HYGIENE AND OCCLPATIOXAL MEDICINE LORWALD The tests were made chrysotile that had beer milled to reduce the fen time control tests wer chemical composition a.findings reveals that or typical peribronchiolar ottlv libers less than 3 H) ami 11). Fillers si to cause serious tissue in the chrysotile fillers, from a flexible to a britt Fig. 10.--Comparison of reactions provoked by injected long fiber and ballmilled asbestos dusts: A. lung of a guinea pig which four months before had received an intratracheal injection of long fiber asbestos dust. 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 ball-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). Fig. 11.--Serpentine ir received an intratracheal i is shown at the left cent the bronchiole and collage mental studies concern: publication. Comparison of Vat findings are disclosed t 15. First, all the long of anthophyllite, prod bronchiolar reaction ci minerals--chrysotile, a and 12. Why anthopb minerals is not entirely Second, with the ' fibrous form of mag: MEDICINE -I VORWALD ET AL.--STl'DIES OF ASBESTOSIS 31 The tests were made with long fiber chrysotile. unheated, and witlj_ chrvsotile that had been ignited to destroy its flexible structure or balNt milled to reduce the length of fiber 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 nonfibrous. A review oi the findings reveals that only the unheated, long fiber chrvsntile produced typical peribronchiolar fibrosis and that ball-milled material containing only fibers less than 3 microns in length failed to cause fibrosis (figs. 10 and II). Fibers 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. E.xperi- fiber and balliths before had Note the- perionchiole chiefly an intratracheal t the right. In it the bronchiole Fig. 11.--Serpentine injection experiment ' I.ung uf a (iniiica nig that had received an intratracheal injection of this dust four months heiorc. A bronchiole is shown at the left center. The phagocytic cell*, exhibit little predilection for the bronchiole and collagen deposition is absent (X 30(1). mental studies concerning this observation will be reported in a separate publication. Comparison oj Various f.uuii Fiber Posts.--Some very interesting findings are disclosed by the results of the e.\]icrimeuts recorded in table 15. First, all the long filler nslicstos minerals tested, with the exception of anthoplivllitc, produced typical fibrosis. The characteristic peri bronchiolar reaction laiiscd by three representative long Tiber aslicstos minerals - chrysotile, ninosiic and crncidulite -is shown in figures 10 .-I and 12. Why antlmpliyllitc lieliavt-d tlillen-iillv from the other aslicstos minerals is not entirely clear. Second, with the mineral lintcitc. which is not a silicate but is a fibrous form of magnesium hydroxide, a characteristic fibrosis like t rorMolit* (Uoiivin* Tin (S. .vtnra) An(lm|lh\ Hit- TmiKiJiic wool .iilv i*l imii ju u mi iii.ii i.. Tifi liiiiit nt<n*U ............................... >ti .hi inniiiiiiiiiir %-ry mil- Hiiiun tiriiiniHi linnichliiii tnt l-. - * 5 |* : (<! t \ i * ** .iii.- u.ii `5 ' * '*** * ,,,t *! .t*l .i. i U*f.ir<* nl*ln*|* !.<.!( *r* ni * mi.mil With UK**. r an h t*ti w m- nmr** .................. ........ iii..*.* ith Tlt'-tmril IM*un*y " * l ........ mil \ . "Mt . tt *\ |. nrlu.it iiImhO im-ft* ( iim*ivt* " v*i>. *;..*. I ulir* .......... . I.<|{ urn* Iru \i t mo. after ftniu- , jiiim.i.' ! ' i ium..mn Imi i*rln.rnli*ti tilitmt liniliflmiji** *n<t |J .iMt* .*3 ,il < rltt*i* :H tin*. lii:u |h Ml.roitrlUMlMr |MM<'tH*< of ntr*ii with c.ipill.irv cl*** . imu* imrilnlh liimm of |.ri'i*rli`..|. ; :i**11 iil*>1 .*..11 : u* -tnrks|: .`.uttirrli v tt*M* ft'SCUOn >Im.u.*.| |m*hi\ u'II.iwii i ,i. In ;m ni im. , mtuiiir (( of wifi aiaiur*'*! nifn * .ii u * r*m *. at inn . iiiiihm* NNtNato|M w it it Vnlriti m; *iim Mir 'Mti*hli ruble rimmlt* imruinoulti" with infi:irnMfn m lvin|tii<Hvit** Hint uiuMpliii. At : um., small mtra* |irM"riii>i:tr rthfnn* pbur* with tow of more delicate fibrosis at periphery. Tvpu-Iil iH-roii* rii.hihri.i'rhiiiUii* aiui peribronchiolitis wit It formation of aivpirnl ttU'*rt.i* bode** lliiii*trnliin of l*o.lir* itcgan Itcfofv 4lli tui. Htl'T llltrrllti*'. Wr|| irVr|o|a>| hy Mil HIM. ttndle*. pef*i|t Wftl'f Uth 1UO. Ifrarliiiit til ! inn. hraw 'iidobronehioHti* ttnd tterihronchlullti* already -imwiiic llhrtMt* rhaner*: nlrhTtn*i* nnl (throni with some necrosis at Ur f m:i**tv<* 1nr:iii/:ilit|t m du*l, At 4 tun.. heavy. Widely scattered r!at|tii.rittirttmiiti* ami |M<rihrnnrhmiiti*. now* rthrou*. with marked <lefor* min **f hrtou'hiolr* witii an al'iioimloitl appearance. At 9 and 10H him . rriiriioti m hmif -**rmiallv th** *,itm* n at 4 mo. At 15 mo., loel of floruit* rtiiiMf.roiti'hiohti* noil |> rihrottehiollii* still lane, with more .tens* *ur ti**ii* ii'iil more itrformitv or r.runehial tube* hut no cxtcnslou into, or atelectasis of. peripheral purvnrhyiiiK. \tlvnnrtsl firrnt rii.lot.rnncluolitU end prrihrourhlniltU. Beaded athea* tt<i Ihh|{i* i*oi*n| at ** urn. At l mo., early fibrous endohronctdoMtia and iN-rihrmirhioilti*: many yiant cells end some Irmphocytle reaction. At 4 nm.. small areas of emtohronehioUtt* spattered throughout the lung: tviiuiar fli>ro*>i. At a unit ]> mo., an*as of lironchiolltla smaller beeausa of eontraetinn of dene rnr tissue: at 12 mo., marked lymphocytic lofU* tration an*l ntlenomutoiil appearance. Typical ndvat'etHi hhroits emlohronrhiotltls and peribronehlolltla produced hv n.v. *it*ji*n*loit (1 cr. total dose): most animals vould not tolerate usiml -'"c- <itiM*nston. Flhroi< well derelopetl before ashestosls bodies M-ni. At l nm., welt dcrelopiil flhrotts bronchiolitis vlth lymphocytes and uintir c."H* and adenomatoid change. At mo., typical hronchioUtlf not imte n* *-\t`n*vp nr lieavih* fibrous as with a suspension, other* wt*e the *itme Many deeply tainei filters with a good proportion of buiKirated nste*rot At 12 mo., heavy fibrous bronchiolitis, more peribronchiolitis and endobronchmiitls, with lymphocytes and giant cvit<: very marked ndcoomatokl appearaoce. f vtiipbocvtie infiltration and riant ceils but no fibrosis. A very few atypical atiwstod* bi*die< At 1 mo., many scattered foci of intrabronchiolar dust without iiia*ive locali/ation: lympbocytie infiltration of valla and a tew riant cell* At ? and 12 mo.. Httte evidence of dust: a few bronchioles and brourbi with giant cells in adjacent alveoli and with lymphocytic Infiltration of walls. FlhrosU nhmit bronchioles. At 1 mo., areas of dust localisation with cob lapse of alveoli and infiltration with acute inflammatory celts, macro* phorc and giant cells. Within the area were a few foci of fibrous tissue and iiumcrous areas of hypertrophy of alveolar epithelium. Many bron* chink** parked with fiber*. At 4 mo., general appearance of lesion unrhanted: pleura slightly thickened over heavy localisations of dust. An occasional segmented asbestosis body seen. At * mo., many foci of fibers in bronchioles and alveolar duet* with cellular reaction as before: also, some foci showed distinct collagen deposition. At 12 and 18 mo., reaction as before with fibrosis about bronchioles more apparent because of eon* traction and decrease of inflammation. Giant cells prominent. Pleura markedly involved. Typical flhrous endobroschlolltis and peribmnrhiolltls like reaction to asbestos minerals. At 1 mo., extensire endohronchloHtis and peribron chiolitis with giant cells: dense fibrous loops within bronchioles and cellu lar fibrosis about them: adenomatoid change present. At 2 mo., heavy Intrabronchiolar and peribronchiolar fibrosis producing marked deformity with distortion of tubes and obliteration of surrounding air spaces: fibrosis pale without hyaMntxatlon but with few nuclei: no necrosis. Typi* cal asbestosis bodies seen. At 4 aod 8 mo.. little change: fibrous ttssua contracting. At 10tt mo., dene flhrous bronchiolitis with asbestosis bodies. Xo pleurisy. Xo ertension to surrounding tunr. Xo fibrosis within a year. At 1 mo., no tvaetfon Inside bronebiolat: In peripheral air spares dumps of giant calls peeked with fine (pteules of glass with lymphocytic Infiltration of adjacent waits: no asbestosis bodies. At 2 mo., reaction less intense than at I mo.: fair*tised damps of don* gated giant phagocytes containing spicules nd particles of glass: no winhronrbtti*. At 4 and a mo., reaction stUI diminishing. At IS mo., fitrnl areak of nneumomtls with no fibrosis or endobroncbitls; moderate ntitiiier of smooth lron*staining fibers. 32 rORH\ that produced bv the brucite used obvious that a development or * Fig. 12.--Amosit pig four months ah reaction exhibits pro B, lung of a guir lite. As in A, penb shown (x200). Third, no fibi (fig. 13i?)t even However, there ar * UORIUALD ET AL.--STUDIES OF ASBESTOSIS 33 that produced by the asbestos minerals was obtained (fig. 13 A). Since the brucite used contained only 0.90 per cent silica as an impurity, it is obvious that a siliceous component is not an essential factor in the development of asbestosis. 18o;* vTT.w / r. " r. V>^ >* W*7 1 * >S!*' * 4t. - iSS X his* *>vj 'Tut: r4 W'_. B E*. m*.KSi- . Fid. 12.--Amusitc anil erocitlulitr injection experiment-.: 1. lime ol a uuima |iif( four mouths after an intratracheal injection ( .uim-atc. The inllnnmintnry reaction exhibits primotiuccil accumulation of cells ami collagen ile|>oiliun tx 2IKI1. H, lung of a guinea pig four moutlix after an intratracheal injection of cvocitlolitc. A in .1, peribronchiolar accmuuiatiou of icll- ami ilepo-atioii of collat-'cn are shown (y 20(1). Thinl. nn lilirusix resulted from lltc inject ion of glass wool filters (fig. 1311), even though glass wool rescntbles asbestos in many ways. O However, there are fitinlameiilal ililVerenees. A glass wool liber 3 microtis i- 34 INDUSTRIAL HYGIF.XH AND <H CtTATlOXAL MEDICINE in diameter i- a solid rod which in short length:* is fairly rigid, I while an asbestos fiber of the same diameter is a bundle of extremely fine filaments which impart to the fiber a high degree of flexibility. It would seem that this structure and the associated flexibility are important factors governing the capaciiv hi a mineral to produce peribronchiolar ' Fif. 13.--Brucite and glass wool injection experiments: A, lung of a guinea pig winch four months before had received an intratracheal injection of brucite. Even with this nonsiliceaus fibrous mineral there is peribronchiolar accumulation of cells and deposition of collagen similar to that shown in A and B 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. Glass wool fibers are present in this field but cannot be seen at this magnification (x200). I lung of a guinea Section of brucite. tolar accumulation ind B of figure 12 :d an intratracheal jection and the blood vessel. The |r comparison with cannot be seen at VOR.WALD ET AL.--STUDIES OF ASBESTOSIS 35 fibrosis. Experimental studies concerning this observation will be reported in a separate publication. Table 16.--Comparison of Reactions Produced by Long Fiber and Short Fiber Dusts Injected Intratracheally Dosa*e: Two lnlKtlons ot 0.5 ee. of a 5 per cent suspension fires two weeks spsrt. dose was 50 me. Animals used: Six croups ot mines Pits. Periods at wdteti animals were killed: l, 2, 0, Stt and 12 months alter Injection. Total Mineral CbrysotUe (Thettord) Amoelta Crocldollta (Bolivia) Anthopbytllt* TmnoUte llrurlte Slat ot Dust Particles Lonr fiber. 20-50 microns Short fiber, 3 microns and leu Lone fiber, 2000 microns Short fiber. 20 microns andlsss Lone fiber, 20-50 microns Short fiber, 20 microns and less Lon* fiber. 20-50 microns Short fiber, 3 microns and less Lon* fiber, 20-so microns Short fiber. 20 microns and less f.uiiir IIImt. VO Hi inlrrtins Miurl Alter (made by erusliltt* tniti: llls-rs with rubber fadh-e. man) Results A distinct fibrosis. Refer to chrysotllc (fibrous) un heated in table It. No fibrosis. Refer to chrysotlle (ball milled) unhealed In table IS. Typical fibrous endobronchlolltis and peribronchiolitis. Refer to table 15. Reaction limited to phafocytosls with lymphocytic infil tration of adjacent walls. Short fibers packed Inside swollen gbatocytes; longer ones tree: some costed to form typical ssbestosis bodies. At 1 mo. after injec tion, alveoli contained food-sited flant cells: most pbffocytet were within air spaces and had not tolerated to walls. At 4 mo., free extracellular fibers bad worked themselves into interstitial tissue, where there was extensive proliferation of lymphoid cells and monocytes but no fibrosis. At 3 mo. (orvicn body reaction with some pneumonitis, no bronchiolitis. Trplcal asbestosis bodies present. Advanced fibrous endohronchiolltls and peribronchio litis. Refer to table 15. No fibrosis. At l mo., air spaees compressed and larirely filled with oant veils packed with dust m-edlrs. Walls heavllr infiltrated with inonorytes and lymph oid cells. At < mo., a moderate decree of cellular Infiltration ot walls: small alant cells packed with dust spicules. At 0 and SHi mo., masses ol ciant cells, contalnln* mineral particles, in small bronchi but not in respiratory hronehlotcs: smaller ones widely scat tered In terminal air spaces. Numerous asbestosis bodies. No reaction in connective tissue. No endo bronchial proliferation. At 12 mo., many seartered small monocytes packed with dust. -No rndobronchltls. No peripheral fibrosis. In lymph node, sliclit reticulosis: no fibrosis. I.ymphocyttr Infiltration and slant cells but no definite fibrosis. Refer to table 15. No flhrosls and practically no asbestosis bodies. At I mo., focal rallectlooa of dust-filled monocytes and a tew slant cells: at 4 mo., some adenomatoid epithelial reaction: at a mo., simple pneumonitis with pha*ocytosls of short fibers: at 12 mu.. Isolated and sharply liK-allzed collections of dust cells inside air spares about terminal arterioles. Reaction In walls limited to lymphoid veil Infiltration. No fihrosls. In Ivinph nmle, reaction limited to -ll*ht prominence of reticu lum. Fibrosis about bronchioles. Refer to table IS. Simple fnrelcn body reaction. No aeirte Inflammation. No areumulatlon of dust In or about terminal brnoehloh-s. No rodohronchltH. At t mo., scattered small (iaut cells and considerable ludltrntion of adiaeent walls with iiionoeyii-s and lymphoid cells. At 4 mu.. Iltlh- chance except more cellular Infiltration of con nective tissue. At 3 mo.. Ivtnphold infiltration nnd thlekrnln* of walla shout some tint not all terminal bronchioles. Typlral fibrous rndohrnnrhlnlltls and prrthronrhln||tls lllo* reaction to asbrstos minerals. Refer to table IS. Inert tvpc of reaction. U 1 inn. after Injection, small iiiouia-yles widely scattered thmiiah air spaces; focus of ali-leelasls with Ivniphold Infiltration of compressed air spare walls. No endobronchial reaction as with rfirvsoiife. it 2 mo., renrrhin similar to that at 1 tun.: typical nale-strisle Imdlrs seen. At 12 mo., small clumps of Inactlyr iltiat-nilrd phaaorytes; no flhrosls. No reaction In Ivinpli nodes. Comparison of Long Fiber and Short Fiber Dusts.--With quartz dust it has been demonstrated that the smaller the particles the more 3o /v/irw/v'/.//. in'uinxn .ixn nn-i i`.utown, .unniaxn intense is the tissue reaction anil that particles larger than 3 microns in diameter cause little reaction. In the ca>e of asliestos, however, the reverse is true and apparently only long filters have any specific effect, as was suggested hy the inhalation experiments. This is confirmed hv the data of table 1(>. in which a series of tests u ith fibrous minerals is reported. \\ ben the injected dust consisted ..1 libers JO to Ml microns long, all the fibrous muiei.tls tested csiipt .utlboplu llite, as noted in the preceding section, produced lihiosis; ben the maleiial w.i- prepared bl first grinding the lihimis dllsl linlll llie length ol libel . u.i. ledlieed to JO microns and lcs or. in some cases, to .> niuiote. and li~... none of the injected dusts caused fibrosis. These results dili'er from those of King. Clegg and Iv'ae." who reported the production of reticulosis coni|>aralile In the e.\|ieruMeiitul silicotic nodule in rahhits receiving monthli intratracheal injections uf WO mg. of Rhodesian asbestos fillers. 15 microns long, and the produc tion of diffuse interstitial fibrosis in rabbits receiving similar injections of short libers. 2.5 microns in length. We believe this dose. es{icciaily in the long term rabbits, is highly excessive. Fn ottr ex]ierimeuts the dosage was kept low itt order to minimize untoward reactions which might obscure the peribronchiolar type of fibrosis which characterizes early human ashestosis. * *\ Experiments Using Intravenous Technic The experiments summarized in table 17. in which the intravenous method of injection was employed, show that the asliestos minerals are i far different from quartz in their action on tissue. It has lieen repeatedly demonstrated that intravenous injection of quartz particles 3 microns and less in diameter will cause a typical tissue reaction with the development of hvalinixed fibrotic lesions in extrapulmonary sites, such as the liver and the spleen. Asliestos 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 experiment with chrysotile particles is not clear. Experiments Using Intraperitoneal Technic The results of injection experiments with the intraperitoneal technic are 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 type of response. These experiments indi cate also that the fibrosis initiated by the irritation of asbestos fibers is not restricted to the lungs, as was formerly assumed, but can be pro duced in the peritoneum as well. OTHER EXPERIMENTS WITH ASBESTOS 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. \V., and Rae. V. M.: Effect of Asbestos, and of Asbestos and Aluminum, on Lungs of Rabbits, Thorax 1:188, 1946; abstracted. Indust. Hyg. Digest, 1947, rol. 11 (Feb.), no. 234. S It 9 T-* f ?i r =. ii 1 cI sv Qi QC i 3 So : * 3a 9 as ca O I EDIC1XE -.nan 3 microns s, however, the specific effect, is confirmed by ous minerals is ) to 50 microns te. as noted in li was prepared rs was reduced and less, none ad Rae,11 who c experimental 11 injections of ad the producifflar injections Jose, especially xperiments the actions which it characterizes jla intravenous minerals are -n repeatedly & microns and * development A as the liver nous injection f revealed by deaths in the itoneal technic lusts produced Tons and less friments indii:stos fibers is can he pro- ji.VLS to throw more Ksbestos. and of |!M6; abstracted. .17 Tn-iiH'ht* in '-ron* I ! I . I' \u jm r i lu n if t i lu n ly rriti lio n ith no thnmn- in *21 mo. >l-- rvallou'< im o tf n l fi, 12 m ol 21 mo. T able 18.--Nummary o/ Injection bxperintenls by Iiilraperitoncal Technic ~3 * 9 -n a =" js ?i 2 n 5* rZ --** s3 * l-Sl -III *- --S* ^5. &?* 3W> MV -5 . ?"? | r ^ * s J> i m5 t _ w --* t22: t>B3^<3j -*5 to S4 v*a 52 ftffi J 5 *X avft ---- -* 3-- S-&2; jI 1 11 1 . r *z ? im i -s-;Ti tf .. 3. 7 T 1 ; 3 : j*3 | 2. &rji;v??; I ?a 5 1 ^o~ ** r? =:;= r * A , -- 2 si*3t eagi f-5 iof-?pv i ! ^*$ii 3 2--2 sM i~ =_ r T!^ a * o S; <ZiZi %t .0*0 053 v 71 . f * a * . i?" * - Z/.3 -? 3 I 2 1" t UM!lZ :aa iiii tIUfl*l*iiiii s o" z " 15 1r I; il 52.-*--4sl --72I .*2r*3 ; v= C Slfuisa" ll -a -- * *>asa*1*1 8 . |l!I f1|i:||/ 1. * -- a5 7 ? if2 IP fc? S& 5 to 2 a ** s H** Uz-.-.t 3 a.'Z f 'mSS alS2saa f3:fi si3S 1 aao- -=; . 7 ^ nS-2 :* 1 ei*- S'? |S*S5 1*^| si2 iftil !Iii | sisilf|55| !5i** ii Ia uaj^jJ-S* *it ;2 2v? S- ^3 7 .aS^c a T to ___i . i|?JI? "s SgdH ; s--aaU5pi$33?I9-ia-| "toa^ ^ *3 ill *S5o-- a s-^= rpS i it zt66z: S=5 i 5 5J = f iti sir-tc a ie is f3SSS 5 sfS=o 3 3?== js a IS -3 -I S3 s!s -* 312 to S* l8e3S- I 1 5I | si ii, ul JJ- <c S S 5 S=g**l 2J5 fisf2 3 3 3| a PJ3 ^ ssl 11 11 I ?l!& < 38 * VORIVAL Pbote When colloidal of long tiher chrysc into rats, the alumir due to chrvsotile. liy the injectctl fihr last injection of tf filirnn-.. King and protect pulmonary * in their experimei hydroxide. The iron in the from blood or tissu mineral fiber. After into the groin of a other 0.2 per cent numerous at both : reaction to Prussian in agreement with Tu Asbestosis bodic intratracheally into | material for injectioi solution the lung ti The asbestosis bodi< after injection. Th rather resistant coat treatment, which m. which renders the fi that the coating is a by Beintker as earh Two hypotheses and reaction caused In the chemical thet assumed that the as in this process their capable of irritating is merely an indirec untenable: Intratra 12. Giroux, If.: At <fandante," Laval mid. 13. Bei&tlcer, E. : t) von Beter, Virchow* t a-0,00! IfHi! Ifslif isSSs'! TsS# m2S=3 saaAl-* Iflli! *5**55 afiJaf *11*3i 2?ii*l 2 o* 85 P*ai JJ M M M si *ss s a*e ss-2 g allsgjfifl | a_a| --72 2T 2> g* *a-a*39mS3* | Si I tfisS ^ >*- c< a..--aR. -- VORIVALD ET AL.--STUDIES OF ASBESTOSJS 39 Protectot Actios of Aluminum Compounds When colloidal aluminum hydroxide had been added to a suspension of long fiber chrvsotile prior to injecting this susjiension intratracheally into rats, the aluminum compound did not prevent the irritation of tissue due to chrvsotile. 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 u : in their experiments metallic aluminum was used instead of the hydroxide. Formation or Asbestosis Bodies The iron in the coating of the asbestosis body appears to be derived from blood or tissue elements and not, as has been suggested, from the mineral fiber. After two kinds of chrvsotile 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.1* 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 the lung tissue removed at autopsy from an asbestos worker. The asbestosis bodies could he seen in the guinea pigs for-at least a year after injection. This experiment shows that the asliestosis 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 incapable of producing fibrosis. It thus appears that the coating is a protective mechanism. This thought was expressed by Beintker as early as 1934.** TIIFORV OF IRRITANT ACTION Two hypotheses have liven proposed to explain the tissue irritation and reaction caused by asliestos libers: the chemical and the mechanical. In the chemical theory, which is based on experience with quartz, it is assumed that the asliestos 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 asliestosis is merely an indirect silicosis. Several facts make the chemical theory untenable: Intratracheal injection of brucite fillers, which had a silica 12. Giroux, M.: Aniiantnsc expcrimentalc: valour patlKigmimomiiiic du "corps d'anuante," Laval tried. 9:239, 1943. . 13. Beintker, E.: t)ber die Asbestosiskorperchen: Bemerkungen zu der Arbeit von Beger, Virchow* Arch. f. path. Anat. M3:527, 1934. t\ir^n<i.iL uvuh-m: .iM' <klii'atiox.u. ur.t>tci\T. o-iucrit *.<[" uiiiv O.'X) per 1*(11. cau.-ed typical tihrnM. like that produced '>y the :iibc't'/ mineral': free silica particles increase in potency as the ;iaru-!i> -i/c l>vo>mr> le>;.. lutt a-liv't'" lilier- -hnrter than aliout 10 to JO :i:vr"M' -in* rvi.uhih mr.iK-tn'ii'. .ihiminnm ludrnxiilc neutralizes tin- :rri> itir.e I'Vct *! |n.irshni << < .1 -1 -ti<- : 'crprtuine ha< the .,in le v.I:< " " ii.ii; :tl u i. In > ,. ! 11.-. I ml il* I'lniliiccil miU in nert i : i .ivl h m . 'Iiri. i. i :>lr t.itic m the clu-imcal o>im-'iU"ii 'he iiuiuT.il' vihlih !" . n i ilu-ii*'i' i l.ihU PM in ui' 'ill' V. ii'.uu- i! vein. ni"lc likt h th.il .I'lu-'lii'i-. i' iMii'i'il 11\ .hi iii'i'ii.il iiu i iui'ical irntatum <liu- t.i !i"i" .'hiii,' tihers, this irritatiim heine rrlateil the peculiar tilaiiicittcd -tr net lire of the IiIht and the aS'<viatrd Hc\ihility. uliich are |in>se."ed h\ im other ioreitjii liodv stmlied. Tin-.', 'tuition ni chrysotile tihers changed their structure and made them ;iu-rt. altlumch' tile same tihers. before lieing heated, would luue produced tilini' i table 141. Further support tor the theory of mechanical irritation is that ashe-tosi-; occurs in an organ of high mobility --the luri"--and that a lilirous reaction can lie produced by injecting Table l*J-- ln.i/v.>vv ft t'ibr,<us Minerals e* .<: K.*t> 1LO1 co MrfO N'iO wl* * ' o 'i \t 110*11 ....... .. .imphtlHtl'i ,,, .. . Ar.tlmpi.vllir* .. .x . Mrum*.......... .' V. *4 '**> < 4' 106 ` : *6 *? :* 1.0 2.01 i.uo ii.;Sj 0.44 . 6 >.w 1.S ;:.jr iti.JT u.il 0.4.1 ivu < . , ... * * j. 0.20 ... -*4 '* \M\ 0.** IJ.i' 6.02 TVmohr-*...... **o 7 9.36 4.44 20.32 G.7* ttuition Lon K*t) < lose. > 105C. Totnl % <-, % `e 0.20 o. . 0.12 0.32 MO to.;; 0.15 o.r *.<* 06.32 0.16 0.53 st.m 99.79 0.06 4JO 14.90 90*9 0-57 0.02 t.56 90.64 0.90 0.34 2.75 99.*2 asbestos fibers into the peritoneum, where there is'also a degree of mobility, but not by injecting them into other extrapulmonary organs such as die liver, the spleen and subcutaneous tissue. COMPLICATION'S 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, infection studies could not be made in the other inhalation experiments also. ScscrmsiLrrv to Tcbescclocs Intection' 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 infection would be placed below an active VORWALD ET AL.--STUDIES OP ASBESTOSIS -II 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 tul>erculosis. Inert dusts have no effect on the infection, and the lesions usually heal and the disease disappears. Asbestos 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 tubercles there, in addition to the usual foci beneath the pleura. Such tubercles healed in a few months. SCSCF.PTUItUTY TO NuNTCBEKCCUICS IsFRCTIUV ` There was no specific experiment concerning the effect of inhaled asbestos dust on nontuljerculous 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 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 nonttiberatlotts pulmonary infection. COMMK.VT AMI St'M MAttV 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 .sjHi-ies of animals, including the guinea pig. the rat and the rabbit, but tint the mouse and the dog. develop iwrihronchiolar fibrosis of tbe lung similar to human asliestosis after Ixfing excised by inhalation or intratracheal injection to long chrysolite aslteslns Jilior.s. Both iuhalafion and iiiiccfinii e\|HTinients pnniile ample support for this stalemeiil. Figure S. I meals the cellular fihrosis that incurs in guinea pigs billowing inhalation of long filler aslieslnx; figure 11 shows the fibrosis caused in the cat by inhalation of long filler aslicstos dust. Similar but less extensive librosjs ix-curred also in rats and rabbits I table I). Mice and dogs failed to rescind. This variation in response of different species to identical dust ex|Hisurcs is still to be accounted for. 11. I.oiig aslirxiii-. fillers are essential in the production of the peribron chiolar fibrosis; short filters are iueafcible of producing this reaction. 42 IXDL'STRIAL HYGIEXE AXP OCITPATIOX.IL MEDICINE Inhalation experiments with asbestos dust surest, and intra tracheal injection experiments confirm, that peribronchiolar fibrosis is produced by asbestos filters between 20 and SO microns in length but not bv particle', ,-horter than 2<> microns f table- 16 and 181. This indicates that the minimum length of fiber )><>--c--ing the capacity to produce the tvpicai |H-ribnuicbinlar lihru-is m animal-, t- -nmewhriT between 20 anti 30 microns. I'uiim-d -todies have nut been carried nut i. determine the upper limit of ctieciiw liber length. Ii appear-, however, that that limit will be detcriuiiieil by the inbalabibn .n the IiIht, I C. Tile mode ol action of the lone a-be-tu- IiIht in the prndin lum of asbestosis is prunariit meclianical rather Ilian chemical ui nature. The evidence lor this conclusion has been reviewed in a preceding section, page 3l). The flexible filaineuted structure of asln'-tos liliers plays an essential |>art in the irritating action, since the solid, inflexible fibers of gla.-.s wool do not produce fibrosis (fig. Id />). 3 D. Typical experimental asliestosis was produced by the inhalation of an atmospheric susjiensiou containing an average of 138 million asbestos particles per cubic foot of air by light field count, of which less than 1 per cent consisted of filters longer than 10 microns. Tit the inhalation exficriment with 100 |ier cent liall-milled asbestos dust containing 0.6 (ter cent of libers longer than 10 microns (table 11) typical fibrosis was obtained (table 0). The evidence presented shows at least that an atmospheric concentration of asbestos dust containing less than 1 million (0.6 per cent X 138 million) fibers longer than 10 microns per cubic foot of air is capable of producing experimental a 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 of long fibers in the atmosphere; as the concentration is increased, the reaction develops in shorter time. The basis tor this statement appears 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-frequency determinations disclosed that 6.7 per 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 developed 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 138 million). 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 EDICINE st, and intraiolar fibrosis is i length but not This indicates to produce the >etween 20 and it to determine `'ever, that that ; production of si in nature. m a preceding asbestos fibers solid, inflexible 9 inhalation of 138 million aunt, of which ) microns. Billed asbestos xu (table 11) eaented shows Mt containing eager than 10 xperimental .-entration of mot be estab- ary reaction concentration i is increased, the inhalation t the average i particles per osed that 6.7 s longer than 1 that the con:it X 40 milasbestos dust ornately onemals inhaling longer fibers ' s on discon- iscontinuance re pulmonary 1 I ` - VORWALD ET AL.--STUDIES OF ASBESTOSIS 43 lesions, due to contraction of the fibrous tissue. In contrast, an immature tissue response, evidenced primarily by cells with little or ttti 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. G. The formation of asbestosis bodies represents a coating of the fiber.by blood and tissue elements, which results in loss of ability of the fiber to produce fibrosis. Intratracheal injection of asbestosis bodies failed to produce the typical asbestotic tissue reaction in experimental animals. The cessation of progressive reaction observed soon after exposure terminates may be due to the formation of asbestosis bodies. H. Aluminum hydroxide failed to neutralize the fibrosing action of the long fiber asbestos. Aluminum hydroxide added to the suspension of chrvsotile asittstos 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 |wriod of dust exposure, viz., superimposed on a background of established silicosis. As indicated aliovc. this more sensitive test, when applied to asliestns dust, failed to demonstrate that the latter had an adverse influence on a lulieroulous infection. The inability of asbestos dust in that cxjHTinicnt to a fleet unfavorably the tuberculous process furnishes strung sup|jrt for the interpretation that inlialed aslicstos dust has no more than a mildly unfavorable elVect on pulmonary tnlterculosis. This investigation was iitaile 1* '--iliir |ty tlic generous financial su|']Kirt ut a group n( ctmt|>aiiirs ( tin- a-lieslm imln-try. ----- -